the effects of dpp-4 inhibitors on vascular endothelial...

The effects of DPP-4 inhibitors on

vascular endothelial function in

diabetes

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Salheen Madani T. Salheen, BSc, MSc

School of Medical Sciences

College of Science Engineering and Health

RMIT University

July 2015

i

Declaration

I certify that except where due acknowledgement has been made, the work is that

of the author alone; the work has not been submitted previously, in whole or in part, to

qualify for any other academic award; the content of the thesis/project is the result of work

which has been carried out since the official commencement date of the approved research

program; any editorial work, paid or unpaid, carried out by a third party is acknowledged;

and, ethics procedures and guidelines have been followed.

Salheen Madani T. Salheen

Date: 28.07.2015

iii

Acknowledgement

A PhD project is a journey with ups and downs and also involves a lot of

hardwork, commitment, mental stress, frustrations and a sense of achievement on

successful completion. This acknowledgement is devoted to all those wonderful people

who were a part of this journey and who contributed in their own capacity to make it

happen and be presented in the present form.

First and foremost, I would like to express my sincerest and greatest gratitude to

my supervisor Professor Owen Woodman for offering me a wonderful opportunity to do

my doctoral research in his laboratory. His wide excellent knowledge and logical way of

thinking have been of great value to me. His understanding, encouraging and personal

guidance have provided a good basis for the present thesis. One simply could not wish for

a better or supportive supervisor. Thank you!

I am deeply grateful to my co-supervisor Dr Simon Potocnik for his help, support

and technical assistance which made my research simple and easy. We had fascinating

discussions and cheerful moments in the lab. It has been a privilege and a pleasure to work

with him.

I take this opportunity to thank all the academic, technical and the current (Kai and

Saher) staff in vascular research group at the Department of Cell Biology & Anatomy,

RMIT University, it has been a great pleasure working with each of you in one way or

another in the laboratory. To Kai, thank you for your technical assistance making the

laboratory an excellent environment to work in. I am also grateful to Saher for sharing and

providing animal tissue for experimentation and it has been a pleasure working with you

guys. I would also like to thank the Libyan Government represented by University of

iv

Tripoli and Ministry of Higher Education for awarding me scholarship to do my PhD

study.

This thesis is dedicated to my parents and in memory of them. Thank you both for

giving me strength to reach for the stars and chase my dreams. I would also like to express

my heartfelt thanks to my family to whom I owe a great deal. My brothers and sisters in

particular to my brothers Mussa Madani, Mohamed Madani, Dr Yousef Madani, and Fathi

Madani as well as my sisters Tibra Madani, Suaad Madani, Najia Madani, and Hanan

Madani for their morale support and encouragement.

Finally, my wonderful wife, Faten Laswad, deserve special mention who believed

that I can do this. Without your understanding, endless patience, support and

encouragement, it would have been impossible for me to finish this work. Thank you for

providing me with constant support, encouragement and understanding which is the pillar

behind my achievements. I owe every bit of my achievements to you.

vi

Publications

S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2015). The DPP-4 inhibitor

linagliptin and the GLP-1 receptor agonist exendin-4 improve endothelium-dependent

relaxation of rat mesenteric arteries in the presence of high glucose. Pharmacol Res, 94,

26-33.

S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2015). The Dipeptidyl

peptidase-4 inhibitor linagliptin preserves endothelial function in mesenteric arteries from

type 1 diabetic rats without decreasing plasma glucose. Manuscript submitted to Int J

Cardiol.

S.M. Salheen, J.CD. Nguyen, T.A. Jenkins, U. Panchapakesan, C. Pollock, O.L.

Woodman (2015). The DPP-4 inhibitor linagliptan restores endothelium-dependent

relaxation in mesenteric arteries from rats fed a western diet. Manuscript is in preparation.

vii

Conference abstracts

S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2012) The DPP-4 inhibitor

linagliptan improves endothelium-dependent relaxation of rat mesenteric arteries in the

presence of high glucose. Proceeding of Australian Society of Clinical and Experimental

Pharmacologists and Toxicologists, Sydney, Australia, Poster 201, Abstract 428.


linagliptan improves endothelium-dependent relaxation of rat mesenteric arteries in the

presence of high glucose and hyperglycaemia in STZ-induced diabetic rats. Proceeding of

Australian Society of Clinical and Experimental Pharmacologists and Toxicologists,

Melbourne, Australia, Poster 325, Abstract 130.

S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2014) DPP-4 inhibitor

linagliptin restores endothelium-dependent relaxation in small mesenteric artery from type-

1 diabetic rats. Proceeding of Australian Society of Clinical and Experimental

Pharmacologists and Toxicologists, San Diego, USA, Poster 402, Abstract 1652.

S.M. Salheen, J.CD. Nguyen, T.A. Jenkins, U. Panchapakesan, C. Pollock, O.L.

Woodman (2014) The DPP-4 inhibitor linagliptan restores endothelial dysfunction of

mesenteric arteries in western diet fed rats. Proceeding of Australian Society of Clinical

and Experimental Pharmacologists and Toxicologists, Toronto, Canada, Poster 525,

Abstract 211.

viii


linagliptin and the GLP-1 receptor agonist exendin-4 prevent high glucose-induced

impairment of endothelial function in rat mesenteric arteries. Proceeding of Australian

Society of Clinical and Experimental Pharmacologists and Toxicologists, Melbourne,

Australia, Poster 549, Abstract 323 and oral presentation.

Salheen M. Salheen, A. Mather, U. Panchapakesan, C. Pollock, O. L. Woodman (2014)

DPP-4 inhibitor linagliptin restores endothelium-dependent relaxation in small mesenteric

artery from type-1 diabetic rats. Proceeding of College of Science, Engineering and Health

Higher Degree by Research Student Conference, RMIT University, Melbourne, Australia,

Poster 104 and oral presentation.


linagliptan and GLP-1 agonist exendin-4 improves endothelium-dependent relaxation of

rat mesenteric arteries in the presence of high glucose Proceeding of the International

Society of Cardiovascular Pharmacotherapy, Adelaide, Australia, Poster 549

Salheen S., Woodman O., Mather A., Panchapakesan U., Pollock C. (2014). DPP-4

inhibitor linagliptin restores endothelium-dependent relaxation in small mesenteric artery

from type-1 diabetic rats. The FASEB Journal, 28 (1 Supplement), 1051-4.

ix

Salheen, S. M., Nguyen, J. C., Jenkins, T. A., & Woodman, O. L. (2014). The DPP-4

Inhibitor Linagliptin Reverses Endothelial Dysfunction of Mesenteric Arteries From Rats

Fed a Western Diet. Arteriosclerosis, Thrombosis, and Vascular Biology, 34 (Suppl 1),

A211-A211.

x

Table of contents

Declaration ...................................................................................................................................... i

Acknowledgement ....................................................................................................................... iii

Publications ................................................................................................................................... vi

Conference abstracts ................................................................................................................. vii

Table of contents ............................................................................................................................x

List of figures ................................................................................................................................xx

List of tables ............................................................................................................................... xxii

Summary .........................................................................................................................................1

Chapter 1 Literature review .......................................................................................................5

1.1 Introduction ............................................................................................................................5

1.2 Biological action of vascular endothelium ........................................................................7

1.2.1 Nitric oxide (NO) as EDRF ..........................................................................................9

1.2.2 Prostacyclin (PGI2) as EDRF .....................................................................................10

1.2.3 Endothelium-dependent hyperpolarization (EDH) as EDRF .................................12

1.2.3.1 Classical form of EDH response ........................................................................14

1.2.3.2 Non-classical form of EDH response ................................................................16

1.3 Generation of reactive oxygen and nitrogen species in diabetes ..................................18

1.3.1 Categories of reactive oxygen species and nitrogen species ..................................19

1.3.1.1 Superoxide anion ..................................................................................................20

xi

1.3.1.2 Hydroxyl radical ...................................................................................................20

1.3.1.3 Peroxynitrite ..........................................................................................................20

1.3.1.4 Hydrogen peroxide ...............................................................................................21

1.3.2 Diabetic sources of reactive oxygen and nitrogen species .....................................21

1.3.2.1 Mitochondria as a source of superoxide ............................................................22

1.3.2.2 Polyol pathway......................................................................................................26

1.3.2.3 Overproduction of Advanced glycation end-product (AGEs) ........................27

1.3.2.4 Activation of protein kinase C (PKC) ................................................................27

1.3.2.5 Increased hexosamine pathway activity ............................................................28

1.3.3 Sources of reactive oxygen and nitrogen species in vascular endothelial cells ...29

1.3.3.1 Xanthine oxidase ..................................................................................................29

1.3.3.2 NADPH oxidase ...................................................................................................29

1.3.3.3 Endothelial nitric oxide synthase ........................................................................30

1.3.4 Sources of endogenous antioxidants in diabetes .....................................................33

1.3.4.1 Catalase ..................................................................................................................33

1.3.4.2 Superoxide dismutase enzyme (SOD) ...............................................................33

1.3.4.3 Glutathione peroxidase (GPx).............................................................................34

1.3.4.4 Thioredoxin ...........................................................................................................34

1.4 Endothelial dysfunction in diabetes ..................................................................................35

1.4.1 Reduction of NO bioavailability ................................................................................36

1.4.1.1 Expression and phosphorylation of eNOS ........................................................37

xii

1.4.1.2 Direct reduction of NO bioactivity ..................................................................37

1.4.1.3 Augmented arginase activity ...............................................................................38

1.4.1.4 Amplified levels of ADMA.................................................................................38

1.4.1.5 Uncoupling of eNOS and BH4 ............................................................................39

1.4.3 Diminished EDH responses ........................................................................................41

1.4.3.1 Alteration in the expression or activity of endothelial KCa channels .............41

1.4.3.2 Transformed expression or activity of MEGJs .................................................43

1.5 The incretins and the dipeptidylpeptidase-4 inhibitors ..................................................44

1.5.1 Historical overview of the incretin concept .............................................................44

1.5.2 General description of incretin hormones and exendin-4 .......................................46

1.5.2.1 Glucose-dependent insulinotropic peptide (GIP) .............................................46

1.5.2.2 Glucagon-like peptide-1 (GLP-1).......................................................................47

1.5.2.3 The incretin mimetic exenatide (exendin-4) .....................................................48

1.5.3 Dipeptidylpeptidase and dipeptidylpeptidase inhibitors .........................................50

1.5.3.1 Dipeptidylpeptidase (DPP-4) ..............................................................................50

1.5.3.2 Dipeptidylpeptidase inhibitors ............................................................................51

1.5.4.2.1 The DPP-4 inhibitor linagliptin ...................................................................54

1.5.5 Biological actions of the gliptins as potential treatment for diabetic vascular

complications .........................................................................................................................55

1.5.5.1 Modulation of glucose homeostasis by the incretin system ............................55

1.5.5.2 Effects of DPP-4 inhibitors on vascular endothelial cells and NO ................57

xiii

1.5.5.3 Effects of DPP-4 inhibitors on blood pressure .................................................58

1.5.5.5 Effects of DPP-4 inhibitors on inflammation and atherosclerosis .................59

1.5.5.4 Cardiovascular effects of DPP-4 inhibitors .......................................................60

1.6 Hypothesis ............................................................................................................................64

1.6.1 Specific aims of the project ........................................................................................64

Chapter 2 General Methods ......................................................................................................66

2.1 Animal experiments ............................................................................................................66

2.1.1 Induction of diabetes ...................................................................................................66

2.1.2 Blood glucose analysis ................................................................................................67

2.1.3 Estimation of glycated haemoglobin .........................................................................67

2.2 Isolation of vascular tissue .................................................................................................68

2.2.1 Preparation of mesenteric arteries .............................................................................68

2.3 Vascular functional experiments .......................................................................................71

2.3.1 Mesenteric arteries relaxation and contraction assays ............................................71

2.3.2 Estimation of basal NO level assay ...........................................................................74

2.4 Estimation of reactive oxygen species .............................................................................76

2.4.1 L-012 assay ...................................................................................................................76

2.4.2 Lucigenin assay ............................................................................................................76

2.5 Western Blot ........................................................................................................................77

2.5.1 Extraction of protein from tissue samples ................................................................77

2.5.2 Bradford protein assay ................................................................................................77

xiv

2.5.3 Gel preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) ..........................................................................................................................78

2.5.4 Preparation of SDS-PAGE .........................................................................................79

2.5.5 Performance of low temperature SDS-PAGE ..........................................................80

2.5.6 Procedures of immunoblotting ...................................................................................80

2.6 Chemicals and Reagents ....................................................................................................81

2.7 Statistical analysis ...............................................................................................................81

Chapter 3 The DPP-4 inhibitor linagliptin and the GLP-1 receptor agonist exendin-4

improve endothelium-dependent relaxation of rat mesenteric arteries in the presence

of high glucose ..............................................................................................................................83

3.1 Introduction ..........................................................................................................................83

3.2 Methods ................................................................................................................................86

3.2.1 Isolation of mesenteric arteries ..................................................................................86

3.2.2 Vascular experiments ..................................................................................................86

3.2.3 Superoxide measurement in mesenteric artery ........................................................88

3.2.4 Reagents ........................................................................................................................88

3.2.5 Statistical analysis ........................................................................................................89

3.3 Results ..................................................................................................................................90

3.3.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide production ..90

3.3.2 Effect of high glucose, DPP-4 inhibitors, and exendin-4 on endothelium-

dependent relaxation .............................................................................................................92

3.3.3 Effect of pyrogallol on endothelium-dependent relaxation ....................................97

xv

3.3.4 Effect of linagliptin on NO-mediated relaxation ...................................................101

3.3.5 Effect of high glucose on NOS and Nox2 related protein expression ................101

3.3.6 Effects of linagliptin on EDH-mediated relaxation ...............................................104

3.4 Discussion ..........................................................................................................................107

3.4.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide generation 107

3.4.2 Effects of DPP-4 inhibitors and exendin-4 on vascular endothelial function ....108


3.4.4 Effect of linagliptin on EDH-mediated relaxation ................................................110

3.4.5 Conclusion ..................................................................................................................110

Chapter 4 Acute treatment with the DPP-4 inhibitor linagliptan improves

endothelium-dependent relaxation of rat mesenteric arteries from STZ-induced type

1 diabetic rats .............................................................................................................................113

4.1 Introduction ........................................................................................................................113

4.2 Materials and Methods .....................................................................................................114

4.2.1 Experimental animals and induction of type 1 diabetes .......................................115

4.2.2 Vascular function assay ............................................................................................115

4.2.3 Assessment of ROS in mesenteric artery................................................................115

4.2.4 Reagents ......................................................................................................................116

4.2.5 Statistical analysis ......................................................................................................116

4.3 Results ................................................................................................................................116

4.3.1 Body weights and blood glucose .............................................................................116

xvi

4.3.2 Effect of diabetes and linagliptin on superoxide production ...............................117

4.3.3 Effect of diabetes and linagliptin on vascular function ........................................120

4.3.4 Role of NO in normal and diabetic mesenteric arteries ........................................122

4.3.5 Role of EDH-type relaxation in normal and diabetic mesenteric arteries ..........122


4.3.6 Effect of linagliptin on EDH-type relaxation .........................................................126

4.4 Discussion ..........................................................................................................................128

4.4.1 Effect of diabetes on superoxide and endothelium-dependent relaxation ..........128

4.4.2 Effect of diabetes on NO and EDH-type response ................................................129

4.4.1 Effect of linagliptin on vascular superoxide and relaxation .................................130


4.4.3 Effect of linagliptin on EDH-mediated relaxation ................................................131

4.4.4 Conclusion. .................................................................................................................131

Chapter 5 The DPP-4 inhibitor linagliptan restores endothelial function of mesenteric

arteries from rats fed a western diet .....................................................................................134

5.1 introduction ........................................................................................................................134


5.2.1 Experimental animals ................................................................................................135

5.2.2 Isolation of mesenteric arteries ................................................................................136

5.2.3 Vascular function experiments .................................................................................136

5.2.4 Measurement of superoxide generation in mesenteric arteries ............................137

xvii

5.2.5 Western Blot ...............................................................................................................137

5.2.6 Reagents ......................................................................................................................137


5.3 Results ................................................................................................................................138

5.3.1 Effect of WD on body weights ................................................................................138

5.3.2 Effect of WD and linagliptin on oxidative stress in mesenteric arteries ............138

5.3.3 Effect of linagliptin on endothelial function in mesenteric arteries from WD fed

rats .........................................................................................................................................138

5.3.4 Investigation of the contribution of NO and EDH-mediated relaxation in SD and

WD ........................................................................................................................................144

5.3.5 Effect of linagliptin on NO-mediated relaxation in WD ......................................144

5.3.6 Effect of WD on the expression of NOS and Nox2 ..............................................145

5.3.7 Effect of linagliptin on EDH-mediated relaxation in WD ....................................149

5.4 Discussion ..........................................................................................................................151

5.4.1 Effects of WD and linagliptin on oxidative stress .................................................151

5.4.2 Effect of WD diet on endothelial function .............................................................152

5.4.3 Effect of linagliptin on endothelial function in WD .............................................152

5.4.4 Effect of linagliptin on NO-mediated relaxation in WD fed rats ........................153

5.4.5 Effect of linagliptin on EDH-mediated relaxation in WD fed rats ......................153

5.5.5 Conclusion ..................................................................................................................154

Chapter 6 The DPP-4 inhibitor linagliptin preserves endothelial cell function in

mesenteric arteries from type-1 diabetic rats without decreasing plasma glucose ....156

xviii

6.1 introduction ........................................................................................................................156


6.2.1 Induction of type 1 diabetes .....................................................................................159

6.2.2 Linagliptin treatment .................................................................................................159

6.2.3 Isolation of mesenteric arteries ................................................................................160

6.2.4 Investigation of vascular function and reactivity ..................................................160

6.2.5 Estimation of basal release of NO in mesenteric arteries .....................................161

6.2.6 Western Blot ...............................................................................................................161

6.2.7 Measurement of superoxide release by mesenteric arteries .................................162

6.2.8 Materials......................................................................................................................163


6.3 Results ................................................................................................................................164

6.3.1 Body weights and blood glucose .............................................................................164

6.3.2 Effect of linagliptin on vascular superoxide production .......................................164

6.3.3 Effect of diabetes and linagliptin on vascular function ........................................166

6.3.4 Determination of the relative contribution of NO and EDH to endothelium-

dependent relaxation ...........................................................................................................170

6.3.5 Effect of linagliptin on NO- and EDH-mediated relaxation in the mesenteric

arteries ...................................................................................................................................170

6.3.6 Effect of linagliptin on Nox2, total-eNOS and eNOS monomer/dimer .............175

6.4 Discussion ..........................................................................................................................177

xix

6.4.1. Effect of linagliptin on vascular endothelial function ..........................................177

6.4.2. Effects of linagliptin on NO-mediated relaxation in rats mesenteric arteries ...178

6.4.3 Effect of linagliptin on EDH-mediate relaxation in rats mesenteric arteries .....180

6.4.4 Conclusion ..................................................................................................................181

Chapter 7 General Discussion ................................................................................................183

7.1 Effects of diabetes and WD on vascular function.........................................................183

7.2 Protection of diabetes or WD-induced endothelial dysfunction by the DPP-4

inhibitors ...................................................................................................................................185

7.3 Future directions ................................................................................................................188

7.4 Conclusion .........................................................................................................................189

Chapter 8 References ................................................................................................................191

xx

List of figures

Figure 1.1 .......................................................................................................... 8

Figure 1.2 ........................................................................................................ 11

Figure 1.3. ....................................................................................................... 13

Figure 1.4 ........................................................................................................ 24

Figure 1.5 ........................................................................................................ 25

Figure 1.6 ........................................................................................................ 32

Figure 1.7. ....................................................................................................... 53

Figure 1.8. ....................................................................................................... 56

Figure 2.1 ........................................................................................................ 70

Figure 2.2 ........................................................................................................ 72

Figure 2.3 ........................................................................................................ 75

Figure 3.1 ........................................................................................................ 91

Figure 3.2 ........................................................................................................ 93

Figure 3.3 ........................................................................................................ 94

Figure 3.4 ........................................................................................................ 96

Figure 3.5 ........................................................................................................ 99

Figure 3.6 ...................................................................................................... 100

Figure 3.7 ...................................................................................................... 102

Figure 3.8 ...................................................................................................... 103

xxi

Figure 3.9 ...................................................................................................... 106

Figure 4.1 ...................................................................................................... 119

Figure 4.2 ...................................................................................................... 121

Figure 4.3 ...................................................................................................... 125

Figure 4.4 ...................................................................................................... 127

Figure 5.1 ...................................................................................................... 140

Figure 5.2 ...................................................................................................... 141

Figure 5.3 ...................................................................................................... 142

Figure 5.4 ...................................................................................................... 143

Figure 5.5 ...................................................................................................... 147

Figure 5.6 ...................................................................................................... 148

Figure 5.7 ...................................................................................................... 150

Figure 6.1 ...................................................................................................... 167

Figure 6.2 ...................................................................................................... 168

Figure 6.3 ...................................................................................................... 169

Figure 6.4 ...................................................................................................... 173

Figure 6.5 ...................................................................................................... 174

Figure 6.6 ...................................................................................................... 176

xxii

List of tables

Table 2.1 ......................................................................................................... 73

Table 3.1 ......................................................................................................... 95

Table 3.2 ......................................................................................................... 98

Table 3.3 ....................................................................................................... 105

Table 4.1 ....................................................................................................... 118

Table 4.2 ....................................................................................................... 123

Table 5.1 ....................................................................................................... 146

Table 6.1 ....................................................................................................... 165

Table 6.2 ....................................................................................................... 172

1

Summary

Diabetes mellitus is an independent risk factor for cardiovascular disease. It is well

known that hyperglycaemia causes overproduction of reactive oxygen species that leads to

vascular endothelial dysfunction. Endothelial cells, lining all blood vessels, modulate the

tone of the underlying smooth muscle by responding to mechanical forces and

neurohumoral mediators with the release of a variety of contracting and relaxing factors.

Endothelium-dependent relaxing factors include nitric oxide (NO), prostacyclin and an

unidentified cause of endothelium-dependent hyperpolarization (EDH). It is well

established that endothelial dysfunction plays a critical role in the initiation and

development of cardiovascular complications. The aim of this thesis was to investigate the

effects of a high concentration of glucose, hyperglycaemia in diabetes, and consumption of

a western, high fat, diet on the mechanism(s) of endothelium-dependent relaxation in rat

mesenteric artery. Further, the effect of acute and chronic treatment with dipeptidyl

peptidase-4 (DPP-4) inhibitor linagliptin was investigated on endothelium-dependent

relaxation in normal and diabetic rat mesenteric artery. As oxidative stress plays a critical

role in the initiation of endothelial dysfunction, it was hypothesised that drugs with

antioxidant activity might be helpful in maintaining normal vascular function in diabetes.

In the first study, the aim was to investigate the effects of DPP-4 inhibitors and a

glucagon-like peptide 1 receptor (GLP-1R) agonist, exendin-4, on the mechanism(s) of

endothelium-dependent relaxation in rat mesenteric arteries exposed to a high

concentration of glucose (40 mM). Organ bath techniques were employed to investigate

vascular endothelial function in rat mesenteric arteries in the presence of normal (11 mM)

or high (40 mM) glucose concentrations. Incubation of mesenteric artery rings with high

2

glucose for 2 h caused a significant increase in superoxide anion generation and a

significant impairment of endothelium-dependent relaxation. Exendin-4 and the DPP-4

inhibitor linagliptin, but not sitagliptin or vildagliptin, significantly reduced vascular

superoxide levels and improved endothelium-dependent relaxation in the presence of high

glucose. The beneficial actions of exendin-4, but not linagliptin, were attenuated by the

GLP-1R antagonist exendin fragment (9-39). Further experiments demonstrated that the

presence of high glucose impaired the contribution of both nitric oxide and endothelium-

dependent hyperpolarization to relaxation and that linagliptin improved both mechanisms

involved in endothelium-dependent relaxation. These findings demonstrate that high

glucose impaired endothelium-dependent relaxation can be improved by exendin-4 and

linagliptin, likely due to their antioxidant activity and independently of any glucose

lowering effect.

In the second study, the aim was to investigate the effect of the acute presence of

linagliptin, a DPP-4 inhibitor, in vitro on the mechanism(s) of endothelium-dependent

relaxation in rat mesenteric arteries isolated from streptozotocin (STZ)-induced diabetic

rats after 10 weeks of diabetes. The study demonstrated that endothelial dysfunction was

due to a decreased contribution of both NO- and EDH to endothelium-dependent

relaxation. Furthermore, endothelial dysfunction was associated with elevated oxidative

stress due the increased activity of NADPH-oxidase. This study demonstrated that acute

treatment with linagliptin reduced the levels of oxidative stress and improved endothelial

function by improving of the contribution of NO and EDH to endothelium-dependent

relaxation in mesenteric artery from the diabetic rats.

3

A high-fat ‘western’ diet (WD), a trigger for the development of type 2 diabetes,

may cause endothelial dysfunction as one of the earliest events in atherogenesis. In the

third study, the aim was to examine whether consumption of a WD affected endothelium-

dependent relaxation of rat mesenteric arteries and whether the DPP-4 inhibitor linagliptin

improves endothelium-dependent relaxation. Wistar Hooded rats were fed a standard diet

(SD, 7% total fat) or WD (21% total fat) for 17 weeks. Consumption of the WD

significantly increased superoxide release from mesenteric arteries assayed by lucigenin

chemiluminescence and linagliptin significantly reduced the vascular superoxide

production. WD significantly reduced the sensitivity to acetylcholine (ACh) and acute

treatment with linagliptin improved endothelial function. The contribution of EDH to

ACh-mediated relaxation was determined in the presence of L-NNA and ODQ to block

NOS and guanylate cyclase respectively. The study demonstrated that EDH-type

relaxation was improved by linagliptin. Furthermore, acute treatment with linagliptin also

significantly improved the contribution of NO (determined in the presence of TRAM-34 +

apamin to block IKCa and SKCa) to relaxation. The results demonstrated that acute

treatment with linagliptin in vitro significantly reduced vascular superoxide levels and

improved the contribution of both NO and EDH to preserve endothelium-dependent

relaxation in small mesenteric arteries isolated from rats fed a high fat diet.

In the fourth study, we hypothesized that long-term chronic treatment with DPP-4

inhibitor linagliptin in vivo (4 weeks, 2 mg/kg per day oral gavage), would improve

relaxation in mesenteric arteries from STZ-induced diabetic rats. The lucigenin-enhanced

chemiluminescence assay was used to measure superoxide production in mesenteric

arteries from normal and diabetic rats. The study demonstrated that chronic treatment with

linagliptin did not affect plasma glucose but markedly reduced the levels of superoxide

4

production and improved endothelium-dependent relaxation in mesenteric arteries from

type1 STZ-induced diabetic rats. Treatment with linagliptin selectively improved NO-

mediated relaxation in rats associated with an increased expression of eNOS, reduced

eNOS uncoupling and down regulation of the NADPH-oxidase subunit Nox2 expression.

In conclusion, this thesis extends our understanding of the pathological process

underlying endothelial dysfunction in the microvasculature caused by exposure to high

glucose concentrations in vitro, the consumption of a high fat diet, and sustained

hyperglycaemia. All of these factors caused oxidative stress via increased

activation/expression of NADPH-oxidase and eNOS uncoupling and subsequently

endothelial dysfunction. Long-term chronic treatment with linagliptin in STZ-induced

diabetic rats had no effect on plasma glucose levels, indicating that linagliptin exerted its

improvement in vascular function via a mechanism independent of glucose lowering. The

beneficial protective actions of linagliptin likely occurs via at least two mechanisms; a

GLP-1 receptor independent pathway in which the linagliptin is able to rapidly scavenge

ROS and inhibit NADPH oxidase as a source of superoxide production and reduced eNOS

uncoupling to preserve NO bioavailability or through a GLP-1 receptor dependent

pathway. These beneficial actions make linagliptin a potential adjunctive treatment for

diabetic vascular complications in patients with both type 1 and type 2 diabetes

importantly expanding the range of patients able to benefit from the use of this drug.

5

Chapter 1

Literature review

1.1 Introduction

Diabetes mellitus, a metabolic disorder characterized by hyperglycaemia, is a

disease that has been rapidly increasing worldwide. According to the fact sheet of the

World Health Organization (WHO), there are more than 346 million people worldwide

suffering from diabetes which can lead to complications such as atherosclerosis,

nephropathy, and retinopathy (WHO, 2011). In 2004, an estimated 3.4 million people died

from the consequences of high blood glucose (WHO, 2011). It has been reported that by

2030, about 438 million people worldwide are expected to have diabetes (Donald et al.,

2012). In 2010, the prevalence of diabetes mellitus in adults in the United Kingdom and

the United States was 7% and 11% respectively (Noble et al., 2011). In Australia, more

than 3.5 million Australians have diabetes. Diabetes mellitus in Australia is the cause of a

considerable portion of the total burden of diseases, and significant costs. For example,

Type 2 diabetes costs Australia $3 billion a year (Australia Diabetes, 2011).

Hyperglycaemia can result as a consequence of a deficiency in the release of

insulin or result from insulin resistance. Patients with diabetes are classified into two

groups; patients having type 1 (insulin-dependent) or type 2 (insulin-independent) diabetes

mellitus. Prolonged periods of type 1 or type 2 diabetes mellitus, and thus an extended

exposure to hyperglycaemia, can have severe effects on both the macro- and

microvasculature. This is as an outcome of disturbances in several metabolic and

6

haemodynamic mechanisms that occur as a result of abnormal blood glucose homeostasis.

Pathological conditions associated with these complications may develop in the nervous

system (neuropathy), the eye (retinopathy), kidney (nephropathy), and cardiovascular

system, all of which lead to the increased mortality and morbidity rates linked to advanced

cases of diabetes mellitus (Australia Diabetes, 2011).

The principle of current medicines used in the treatment of diabetes is based on the

idea of insulin replacement for type 1 diabetes, since insulin is not created by β-cells in the

pancreas, or to enhance the insulin sensitivity or secretion for patients who are suffering

from type 2 diabetes. These anti-hyperglycaemic treatments attempt to provide tight

control of glucose levels and decrease detrimental macro- and microvascular risks and

dysfunction caused by hyperglycaemia. But, large scale prospective studies for the two

types of diabetes 1 and 2 such as the Diabetes Control and Complications Trial

(DCCT/EDIC) and the UK Prospective Diabetes Study (UKPDS), demonstrated that tight

control and adjustment of glucose level may slow, but cannot prevent the development of

cardiovascular complications (David et al., 2005, Holman et al., 2008). Accumulating

evidence suggests that reactive oxygen species (ROS), provoked by hyperglycaemia is the

primary cause of the initiation and progression of diabetic cardiovascular complications

including impairment of the modulatory role of the vascular endothelium that could initiate

the development of these diabetic complications (Brownlee, 2001, De Vriese et al., 2000,

Pieper, 1998). As a consequence, it is desirable to obtain alternative therapies that directly

address the cardiovascular complications in order to minimize the high prevalence of

morbidity and mortality related to diabetes.

Incretin based therapies are a pharmacological class of glucose lowering agents

used as a new strategy in the treatment of patients with type 2 diabetes mellitus (Drucker,

2003). These compounds have less risk of hypoglycaemia and positively impact on the

7

obesity observed in this patient population (Amori et al., 2007). Dipeptidyl peptidase-4

(DPP-4) inhibitors, by inhibiting the action of the DPP-4 enzyme and increasing the

availability of glucagon-like peptide-1 (GLP-1) and/or increasing its receptor (GLP-1R)

stimulation, act to stimulate insulin secretion and inhibit glucagon secretion.

This review describes the critical role of high glucose, high fat diet and

hyperglycaemia in diabetes-induced ROS production and their detrimental effects on the

vascular endothelial cells. Furthermore, the evidence to suggest that DPP-4 inhibitors may

be a promising therapy for treating cardiovascular complications in diabetes independently

of their glucose lowering effect is described.

1.2 Biological action of vascular endothelium

The vascular endothelium, a simple monolayer of cells that covers the interior

surface of all blood vessels, has been recognized to have several important physiological

functions and is now considered as a purposefully placed, multifunctional organ (Epstein

et al., 1990). The endothelium is located as a barrier between the circulating blood and the

vascular smooth muscle and dynamically participates in the maintenance and control of

vascular function (Behrendt and Ganz, 2002). The vascular endothelium play an important

biological role in the regulation of vascular tone by producing endothelium-derived

relaxing factors (EDRF) which include nitric oxide (NO), prostacyclin (PGI2), and

endothelium-dependent hyperpolarisation (EDH) (Figure 1.1).

8

Figure 1.1 Diagram representing the endothelium-derived vasoactive substances. Nitric

oxide synthase (eNOS), stimulated by shear stress and/or acetylcholine, results in synthesis

and release of nitric oxide (NO) which exerts its action via relaxation of vascular smooth

muscle. The diagram also shows other endothelium-derived factors such as prostacyclin

(PGI2) and endothelium-dependent hyperpolarization (EDH). Adapted from (Vanhoutte et

al., 2009).

9

1.2.1 Nitric oxide (NO) as EDRF

In 1980 Furchgott and Zawadzki, reported that the endothelium was necessary to

mediate the ability of acetylcholine (ACh), and other agonists of muscarinic receptors, to

induce relaxation of rabbit isolated aorta (Furchgott and Zawadzki, 1980). This effect of

acetylcholine is mediated by an endothelium-derived relaxing factor that later was

identified as nitric oxide (NO) (Palmer et al., 1987, Moncada et al., 1991). NO is a small

molecule which readily diffuses to underlying smooth muscle and is generated, along

with L-citrulline, from the cationic amino acid L-arginine by the enzyme nitric oxide

synthase (eNOS) (Palmer et al., 1988). The activity of eNOS requires cofactors such as

nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide,

flavin dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin (BH4) and

calmodulin (Bayraktutan, 2002, Hevel and Marletta, 1992) (Figure 1.2).

Besides the action of ACh, NO is synthesized and released from the endothelial

cells in response to a large variety of chemical, physical and humoral stimuli including

shear stress, platelet products (serotonin, adenosine diphosphate (ADP)), circulating

hormones (catecholamines), receptor-independent agonists (e.g. calcium ionophores) and

autocoids (histamine, bradykinin) which leads to stimulation of the eNOS enzyme and

release of NO.

NO is a very unstable molecule with a half-life of 4-50 seconds before it is

scavenged by oxygenated haemoglobin, superoxide anions or molecular oxygen (Rao,

1997, Nakagawa and Yokozawa, 2002). NO is recognized as a very strong vasodilator

which, in addition, possesses antiatherogenic properties such as reducing the adhesion of

platelets and leuckocytes to the endothelium, and inhibition of VSMC migration

(Radomski et al., 1987, Kubes et al., 1991). These actions are mainly mediated by the

activation of soluble guanylate cyclase (sGC) which increases formation of cyclic

10

guanosine monophosphate (cGMP) inside smooth muscle cells or platelets causing

relaxation and reduced aggregation respectively (Moncada et al., 1991, Ignarro et al.,

1981).

1.2.2 Prostacyclin (PGI2) as EDRF

Prostacyclin is thought to be one of the most important compounds in the

prostanoids, a family of bioactive lipid mediators that are produced by cyclooxygenase

(COX) from arachidonic acid (AA) present in the membrane of all cells of the body

(McAdam et al., 1999). Under normal physiological conditions, prostacyclin acts as an

effective vasodilator and contributes to the inhibition of platelet aggregation, adhesion of

leukocytes, and proliferation of vascular smooth muscle cells (VSMC) (Gryglewski et al.,

1976). These effects of PGI2 are controlled by the stimulation and activation of PGI2

receptors (IP) causing activation of adenylate cyclase and as a result, production of cyclic

adenosine monophosphate (cAMP) is increased (Narumiya et al., 1999).

11

Agonists

Figure 1.2 Shows the suggested mechanisms that regulate the creation of nitric oxide

(NO) in endothelial cells. Activation of receptors leads to an increase in the concentration

of Ca2+

. NO is synthesised by the conversion of L-arginine to L-citrulline through the

action of eNOS; endothelial nitric oxide synthase. Some cofactors are required to

synthesise NO such as BH4; tetrahydrobiopterin, calmodulin, NADPH; nicotinamide

adenine dinucleotide phosphate, FMN; flavin mononucleotide, and FAD; flavin adenine

dinucleotide. EC; endothelial cell. Adapted from (Vanhoutte et al., 2009).

GTP

[Ca2+]

Nitric oxide

L-arginine

L-citrulline

Nitric Oxide Synthase

(eNOS)

FAD Calmodulin

NADPH

BH4

FMN

EC

12

1.2.3 Endothelium-dependent hyperpolarization (EDH) as EDRF

EDH may be caused by an as yet chemically unidentified factor that is responsible

for causing hyperpolarization of the VSMC that is not due to the action of NO or PGI2

(Garland et al., 1995). EDH acts to regulate vasodilatation and mediates hyperpolarization

of vascular smooth muscles (McGuire et al., 2001, Luksha et al., 2009, Chen et al., 1988,

Edwards et al., 1998). The ability of EDH to contribute to endothelium- dependent

relaxation is mainly noted in resistance vessels (Shimokawa et al., 1996, Busse et al.,

2002) which are essential in maintaining blood pressure and regulating organ perfusion.

EDH induced relaxation can be classified into two general classes, that is, a classical form

and a non-classical form of EDH responses (Figure 1.3).

13

Figure 1.3 An illustration of the suggested EDH pathways associated with endothelial and

smooth muscle ion channel opening. EETs; epoxyeicosatrienoic acids, HNO; nitroxyl,

BKCa; large-conductance Ca2+

-activated K+ channel, SKCa; small-conductance Ca

2+-

activated K+ channel subtype 3, IKCa; intermediate-conductance Ca

2+-activated K-channel,

KIR; inwardly-rectifying K-channels, [Ca2+

]; intracellular calcium concentration, EDH;

endothelium-dependent hyperpolarization; MEGJ, myoendothelial gap-junction, EC;

endothelial cell, VSMC; vascular smooth muscle cell. Adapted from (Edwards et al.,

2010).

14

1.2.3.1 Classical form of EDH response

Endothelial cell activation by endothelium-dependent agonists such as ACh, leads

to increased intracellular Ca2+

which causes the beginning of the EDH response. Classical

EDH responses basically involve the opening of Ca2+

-activated K+

-channels (KCa) both

small conductance Ca2+–activated K

+ channels (SKCa) and intermediate conductance Ca

2+-

activated K+

channels (IKCa) (Edwards et al., 1998, Komori and Vanhoutte, 1990).

Application of a combination of apamin (selective SKCa blocker) and charybdotoxin (non-

selective inhibitor of large-conductance Ca2+

-activated K+

-channels (BKCa), and (IKCa)

abolished EDH responses, indicating the vital role of these channels since iberiotoxin, a

selective BKCa blocker, was unable to substitute for charybdotoxin (Waldron, 1994). The

SKCa and IKCa channels are located in the endothelium and are organized in endothelial

microdomains, especially within projections close to the adjacent smooth muscle cells and

nearby the inter-endothelial gap junctions. Hyperpolarization of the endothelial cells by the

stimulation and activation of either SKCa or IKCa channels leads to K+ efflux through them

which can act as a diffusible EDH which stimulates vascular smooth muscle Na+/K

+-

ATPase and inwardly-rectifying K-channels (KIR) respectively (Edwards et al., 1998,

Sandow et al., 2006, Absi et al., 2007), resulting in smooth muscle hyperpolarization and

relaxation.

The SKCa and IKCa channels are located in the endothelial cells in different

endothelial microdomains and they facilitate the hyperpolarization process in the vascular

endothelial cells through different downstream pathways (Sandow et al., 2006, Absi et al.,

2007, Dora et al., 2008). SKCa channels are located in caveolar partitions and preferentially

stimulate vascular smooth muscle cell KIR channels (Absi et al., 2007). This has been

supported by observations which demonstrated that the hyperpolarization of the VSMC,

because of the opening of endothelial SKCa channels by CyPPA, a selective opener of

15

SKCa, are specifically abolished not only by apamin, but also by a KIR selective blocker,

barium (Weston et al., 2010). In contrast, IKCa channels are non-caveolar (Weston et al.,

2008) and largely exist within the endothelial cell projections that cross the internal elastic

lamina. Hyperpolarization of VSMC, particularly by the activation of Na+/K

+-ATPase, is

due to the opening of IKCa channels (Sandow et al., 2009, Dora et al., 2008).

It is well recognized that K+

efflux is a mediator of some EDH responses

(Chrissobolis et al., 2000, Edwards et al., 1998, McNeish et al., 2005). The identification

of specialized microdomains that are located on both endothelial cells and vascular cells

has provided an enhanced understanding of how hyperpolarization can be enabled through

electrical coupling via projections for myoendothelial gap junctions (MEGJs), with the

probability of contribution from the changes in extracellular K+

which works as a chemical

mediator (Sandow et al., 2006). A number of experimental observations using rat

mesenteric artery demonstrate that there is a coupling between adjacent endothelial cells

by connexins such as CX37, CX40, and CX43 that are located in MEGJs which are

spatially close to zones where there are clusters of SKCa channels (Sandow et al., 2006).

On the other hand, densities of IKCa in the same blood vessels are established to be

spatially connected with CX37 and CX40 MEGJs. This is consistent with supporting

electrophysiological experiments which also suggested that IKCa were mainly expected to

be linked with microdomains that are close to MEGJs in the endothelial cells (Crane et al.,

2003). The significance of endothelial-VSMC communication and the presence of

specialised microdomains is also supported by data which demonstrate the possibility to

attenuate the electrical coupling of endothelial cells and VSMC through MEGJs by the

application of inhibitors to block cell-cell coupling, resulting in impairment of the

hyperpolarization and relaxation of VSMC by EDH (Edwards et al., 1999, Yamamoto et

al., 1999, Little et al., 1995, Mather et al., 2005). In general, K+ and myeoendothelial

16

electrical coupling as possible candidates for EDH seem equally attractive interpretations

of classical EDH responses, however perhaps not in all cases. Dora and colleagues, based

on data from small mesenteric artery of rat, suggested a model of EDH responses wherein

activation of endothelial cells by agonists such as ACh leads to increase Ca2+

in

endothelial cells, consequently activating SKCa with subsequent spread via MEGJs, and the

following activation of IKCa is dependent on the concentrations of Ca2+

in the area of

endothelial cell projections (Dora, 2010, Dora et al., 2008). By the activation of IKCa,

which seems to be limited to the endothelial cell projections, leads to hyperpolarization of

VSMC due to sufficient endothelial cell K+ efflux causing activation of VSMC Na

+/K

+-

ATPase and possibly KIR (Dora et al., 2008). Taken together, the mechanism of action of

these two classical EDH pathways may act in a separate, parallel or even synergistic way.

At any rate, activation of endothelial KCa channels as a starting point is essential in the

action of both pathways.

1.2.3.2 Non-classical form of EDH response

Whereas the classical EDH response works via the opening of endothelial KCa

channels, the non-classical EDH response causes hyperpolarization and relaxation of the

VSMC independently of channels on the endothelium. It is suggested that when the

endothelial cells are stimulated by shear stress or by local hormones such as bradykinin or

substance P, elements such as nitrogen oxides (NO and nitroxyl (HNO)), hydrogen

peroxide (H2O2), and epoxyeicosatrienoic acids (EETs) are released from the endothelial

cells and diffuse to VSMCs, causing hyperpolarization and relaxation (Bolotina et al.,

1994, Edwards et al., 2000, Andrews et al., 2009).

It is widely documented that NO is an endothelium-derived relaxing factor, causing

relaxation of the blood vessels through the activation of soluble guanylate cyclase (sGC)

17

(Palmer et al., 1987, Rees et al., 1989), but there is also evidence that NO causes VSMC

hyperpolarization through the opening of BKCa channels (Bolotina et al., 1994, Tare et al.,

1990). In addition, the one electron reduced and protonated form of NO, HNO is

synthesised endogenously and causes hyperpolarization and relaxation (Andrews et al.,

2009, Ellis et al., 2000, Wanstall et al., 2001). The hyperpolarizing effect of HNO can be

partially attenuated by iberiotoxin or 4-aminopyridine, demonstrating the involvement of

both BKCa and Kv channels (voltage-gated channels) respectively (Andrews et al., 2009,

Favaloro and Kemp-Harper, 2009, Yuill et al., 2011, Bullen et al., 2011).

Epoxyeicosatrienoic acids (EETs) are synthesised by the vascular endothelium as

cytochrome P450 metabolites of AA. EET production occurs in response to agonists such

as ACh and bradykinin or shear stress (Fisslthaler et al., 1999, Campbell et al., 1996) and

there is evidence to suggest that KCa channels on the VSMC and the endothelium are

activated by EETs producing hyperpolarization and relaxation (Baron et al., 1997, Hoebel

et al., 1997, Earley et al., 2005, Vriens et al., 2005, Edwards et al., 2000). Further

observations indicate that EETs are able to activate BKCa channels in the VSMC (Baron et

al., 1997, Edwards et al., 2000). It has been reported that bradykinin stimulates the

endothelial release of 11, 12 EET and 14, 15 EET which causes EDH responses in porcine

coronary arteries via activation of endothelial SKCa and IKCa, as well as maxi KCa-

dependent VSMC hyperpolarization. This effect was blocked by the EET antagonist 14,

15-EEZE in addition to iberiotoxin (Edwards et al., 2000, Weston et al., 2005).

Additionally, experimental results have shown the capacity of EETs to stimulate BKCa

channels in VSMC (Baron et al., 1997, Edwards et al., 2000). The mechanism of

activation of BKCa channels by EETs is complicated and isoform-dependent but the most

physiologically applicable pathway is through the activation of transient receptor potential

(TRP) channels in order to promote the influx of Ca2+

into the VSMC which then stimulate

18

the release of Ca2+

from ryanodine sensitive Ca2+

stores and subsequently, the local

increase of Ca2+

will activate BKCa which then leads to hyperpolarization and relaxation of

the VSMC (Earley et al., 2005, Vriens et al., 2005, Watanabe et al., 2003, Grgic et al.,

2009). In addition to activation of TRP channels in VSMC, TRP channels on the

endothelium can be activated by EETs to generate endothelial hyperpolarization and

consequently VSMC hyperpolarization (Earley et al., 2009, Fleming et al., 2007). Overall,

these different actions indicate that EETs display both classical and non-classical EDH

responses but, conclusions in regard to the mechanism of action of EETs differs depending

on the type of arteries examined (Baron et al., 1997, Earley et al., 2009, Edwards et al.,

2000, Fleming et al., 2007, Weston et al., 2005, Watanabe et al., 2003).

H2O2 is synthesized through the reduction of superoxide anions that occur either

spontaneously or by the enzyme Cu-Zn superoxide dismutase. Studies on the mice

mesenteric artery have demonstrated that H2O2 was able to cause relaxation and

hyperpolarization of VSMC via the opening of KCa channels and production of H2O2 is in

response to ACh stimulation (Fujiki et al., 2005, Shimokawa, 2010). This EDH-mediated

hyperpolarisation and relaxation in wild type and eNOS-/-

mice was inhibited by catalase,

which metabolises H2O2 (Fujiki et al., 2005, Shimokawa, 2010). In addition, there is

evidence that H2O2 -induced hyperpolarization is mediated, at least in part, by the opening

of BKCa (Barlow and White, 1998) and KATP channels (Wei et al., 1996).

1.3 Generation of reactive oxygen and nitrogen species in diabetes

Diabetes-induced hyperglycaemia is now recognized to result in oxidative stress

(Flammer and Luscher, 2010). “The term oxidative stress refers to a condition in which

cells are subjected to excessive levels of molecular oxygen or its chemical derivatives

19

called reactive oxygen species (ROS)”(Bayraktutan, 2002). Reactive oxygen species are

byproducts of normal cellular metabolism which can be valuable or detrimental to human

health. Oxidative stress describes a condition in which intracellular production of ROS

challenges the capability of cellular antioxidant systems to neutralise them, consequently

causing serious cellular damage and complications such as endothelial dysfunction (Cai

and Harrison, 2000). ROS can have either a useful or harmful effect to the body. The

beneficial effects of ROS are exerted at low physiological concentrations and are used in

host defence processes and also in many cellular signalling pathways such as responses to

growth factors and stress (Yoon et al., 2002). The detrimental effect of ROS occurs when

there is over production of ROS and/or impairment of endogenous antioxidant mechanisms

(Flammer and Luscher, 2010). Overproduction of ROS in diabetes may cause damage to

cellular DNA, lipids, and proteins through denaturation, thus impairing normal cellular

function. For example, during diabetes, overproduction of ROS in the vascular

endothelium may be the cause of endothelial dysfunction that could contribute to

cardiovascular complications. Cellular enzyme systems that act as important sources of

ROS include NADPH oxidase, endothelial nitric oxide synthase (eNOS), xanthine oxidase

and the mitochondrial respiratory chain. These systems are discussed in detail below.

1.3.1 Categories of reactive oxygen species and nitrogen species

Reactive oxygen and nitrogen species may be present either in the form of free

radicals or non-radicals. The free radicals are molecules that contain one or more unpaired

electrons and have a high level of reactivity. In mammalian cells, molecular oxygen acts as

a terminal electron acceptor in order to metabolize organic carbon and produce energy,

therefore, free radicals obtained from molecular oxygen are the most important type of

20

radical species. The main types of ROS are superoxide anion (O2.-), hydroxyl radicals

(˙OH), peroxynitrite (OONO-), and non-radicals such as H2O2.

1.3.1.1 Superoxide anion

Superoxide anions are a crucial source of ROS and have the ability to interact with

other molecules either directly or indirectly via enzymatic reactions or metal catalyzed

processes to increase the formation of secondary ROS. For example, in normal conditions,

superoxide is short-lived and either rapidly metabolized to hydrogen peroxide (H2O2)

through superoxide dismutase (SOD) or to peroxynitrite as a result of reaction with NO

(Reiter et al., 2000, Beckman, 2003).

1.3.1.2 Hydroxyl radical

The hydroxyl radical is considered as one of the most reactive free radicals.

Hydroxyl radicals have a very short half-life and have the ability to randomly oxidize

many of the close targets within cells such as DNA (mutation), lipids (peroxidation) and

proteins (oxidation) (Kehrer, 2000). The hydroxyl radical can be formed chemically either

through the reaction between superoxide and hydrogen peroxide (Haber–Weiss reaction;

H2O2 + O2˙-→O2 + OH

- + ˙OH) (Kehrer, 2000), or as a result of the reaction of hydrogen

peroxide in the presence of reduced metals (Fenton reaction; H2O2 + Fe2+

/Cu+→ Fe

3+/Cu

2+

+ OH- + ˙OH) (Thomas et al., 2009).

1.3.1.3 Peroxynitrite

The reaction between NO and superoxide produces a reactive oxidant form

peroxynitrite (Squadrito and Pryor, 1995, Szabo, 2003). Peroxynitrite is documented to act

21

as an influential oxidizing mediator causing nitration and hydroxylation of phenolic

compounds, particularly tyrosine residues (Reiter et al., 2000, Jourd'heuil et al., 2001). In

addition, hydroxyl radicals are generated by the protonation of peroxynitrite through the

following chemical reactions (Hogg et al., 1992):

NO. + O2

.- → ONOO

-

ONOO- + H

+ →

.OH +

.NO2

1.3.1.4 Hydrogen peroxide

Hydrogen peroxide is a small, stable, non-radical ROS that has the ability to readily

diffuse through both cellular and nuclear membranes. Physiologically, H2O2 is produced

by several pathways. For example, H2O2 can be produced either by peroxisomes from

molecular oxygen or as a consequence of superoxide anion dismutation by SOD. Within

the cells there are a number of enzymes that act to reduce H2O2 to H2O such as catalase,

glutathione peroxidase (GPX), and thioredoxin (Yeldandi et al., 2000, Carter et al., 1994).

1.3.2 Diabetic sources of reactive oxygen and nitrogen species

Under diabetic conditions, it is thought that high glucose levels (hypergylcaemia)

cause damage to tissues via four main pathways: (1) glucose and other sugars being highly

metabolised through the polyol pathway; (2) overproduction of advanced glycation

endproducts (AGEs) intracellularly and also increased coupling to its receptor; (3)

increased activity of the hexosamine pathway; and (4) activation of protein kinase C

(PKC) isoforms. The findings of clinical studies in which one of these pathways was

inhibited are unsatisfactory, as hyperglycaemia-induced tissue injury was not significantly

22

changed (Geraldes and King, 2010, Ramasamy and Goldberg, 2010), however, there is

evidence that all of these pathways are activated as a result of a single stimulus, that is

overproduction of ROS from mitochondria. Furthermore, by controlling mitochondrial

ROS, the tissue damage due to hyperglycaemia is minimized (Brownlee, 2001, Nishikawa

et al., 2000).

1.3.2.1 Mitochondria as a source of superoxide

Mitochondria play an important role in oxidative phosphorylation via the electron-

transport chain. In the mitochondria, the system involved in oxidative phosphorylation

consists of five major multi-enzyme complexes, called, complexes I, II, III, and IV and

ATP synthase and the mobile electron carrier ubiquinone (coenzyme Q), and cytochrome c

(Figure 1.4). Under normal conditions, when a molecule of glucose is taken up into the

cells, glycolysis causes production of pyruvate from glucose and the pyruvate then enters

the mitochondria. The pyruvate is then oxidized by the action of tricarboxylic acid (TCA)

cycle to produce nicotinamide adenine dinucleotide (NADH) together with flavin adenine

dinucleotide (FADH2). Transformation of electrons derived from oxidative reaction of

NADH and FADH2 activated through the electron transport chain involves complexes I-

IV to finally reach the electron acceptor, molecular oxygen. Throughout the course of

electron transfer through the chain, protons are driven across the mitochondrial membrane

yielding a voltage gradient. This voltage is then collapsed and finally yields ATP through

ATP synthase. During the electron transport procedure, and as a result of the escape of an

electron before it is added to an oxygen molecule, the mitochondria generate superoxide

radicals. The escape of electrons occurs at 2 prime locations ie complex I (NADH

dehydrogenase) and the interface between complex II and coenzyme Q (Turrens, 1980,

Turrens et al., 1985). Furthermore, the reduction of ROS occurs due to uncoupling protein-

23

1 (UCP-1) which leads to a collapse of the proton gradient across the mitochondrial

membrane. Under normal physiological settings these processes are firmly controlled

making this system an important source of ROS. It is estimated that around 1% of oxygen

is condensed to superoxide instead of entirely to water under normal physiological

conditions.

In diabetes, hyperglycaemia causes more pyruvate to be oxidized in the TCA cycle

and this acts to drive more electron donors (NADH and FADH2) into the electron

transport chain. This process increases the voltage gradient across the mitochondrial

membrane until a critical threshold is reached as a result of the increase of electron efflux

to the point at which the electron inside complex III is unsettled (Korshunov et al., 1997),

leading to generation of superoxide as a result of accumulation of electrons at coenzyme Q

which donates the electrons one at a time to molecular oxygen, thus producing superoxide

(Nishikawa et al., 2000). Overproduction of ROS by the mitochondria is considered as a

key initiating factor in the stimulation of four main fundamental pathways: (1) increased

flux of glucose and other sugars into the cells via the polyol pathway (Lee et al., 1995); (2)

increased production of intracellular glucose-derived advanced glycation endproducts

(AGEs) and binding to its receptor for AGEs (Brownlee, 1995); (3) stimulation of protein

kinase C (PKC) isoforms and (4) activation of hexosamine pathway (Koya and King,

1998) (Figure 1.5).

24

Figure 1.4 The generation of superoxide through the electron-transport chain in

mitochondria. Hyperglycaemia causes high mitochondrial membrane potential (ΔµH+) by

deriving electron donors from the TCA cycle (NADH and FADH2) and pumping protons

through the inner membrane of the mitochondria. Consequently this prevents electron

transport at complex III leading to an increase in the half-life of free radical intermediates

of coenzyme Q (ubiquinone) that produce superoxide from reduction of O2. Figure

obtained from (Brownlee, 2001).

25

Figure 1.5 A summary of signalling pathways causing diabetic tissue impairment due to

hyperglycaemia. Adapted from (Nishikawa et al., 2000).

26

1.3.2.2 Polyol pathway

The polyol pathway is essentially related to the family of aldo-keto enzymes that

are utilized as carbonyl substrates. NADPH acts to reduce the carbonyl compounds to their

particular sugar alcohols (polyol). The aldose-reductase enzyme acts to metabolise glucose

to sorbitol, thus, in diabetic states there is a high concentration of intracellular glucose

which is changed to sorbitol by aldose-reductase mediated by NADPH as a cofactor. It

seems that during hyperglycaemia, utilization of NADPH by this reaction prevents the

replacement of glutathione, which is needed to maintain glutathione peroxidase (GPx)

activity. This would eventually reduce cellular antioxidant activity and also because

NADPH is a cofactor required to reproduce reduced glutathione which is an important

scavenger of ROS, this could initiate or intensify intracellular oxidative stress (Ii et al.,

2004, Lee and Chung, 1999, Chung et al., 2003). It has been reported that in diabetic mice,

atherosclerosis is increased due to the overexpression of human aldose-reductase and

decreased gene expression that plays an important role in the regulation of glutathione

regeneration (Vikramadithyan et al., 2005).

Once the glucose is transformed to sorbitol, then the sorbitol is oxidized to fructose

in the presence of sorbitol deydrogenase. This process is accompanied by reduction of

NAD+ to NADH, providing more substrate to complex I of the mitochondrial respiratory

chain. As described above, in diabetes, the mitochondrial respiratory chain is recognized to

be a major supplier of ROS.

27

1.3.2.3 Overproduction of Advanced glycation end-product (AGEs)

In normal physiological situations, AGEs are produced from the non-enzymatic

reaction of glucose with other glycating compounds that are obtained from both glucose

and over-oxidation of fatty acids with proteins. In diabetic conditions, the production of

AGEs and its receptor is increased as a result of the constant action of hyperglycaemia and

oxidative stress (Fu et al., 1994, Thornalley et al., 1999). The binding of AGEs to its

receptors may lead to up-regulation of the transcription factor NF-κB via the activation of

cytokines which eventually causes an increase in the production of ROS. Therefore,

diabetes results in the damaging series of ROS formation, overproduction of AGE and its

receptor, activation of NF-κB, and cytokine production and excessive free radical

formation (Bayraktutan, 2002).

1.3.2.4 Activation of protein kinase C (PKC)

PKCs are a family of around eleven isoforms, with the ability to phosphorylate

various protein targets. Both Ca2+

ions and phosphatidyl serine are recognized as activators

of classical isoforms of PKC. The activity of these classic isoforms is also largely

enhanced by diacylglycerol (DAG). In addition, hyperglycaemia causes de novo synthesis

of DAG, leading to increased activity of mostly β- and δ- isoforms of PKC (Inoguchi et

al., 1992). This results in amplifying stress signaling pathways such as Jak/STAT,

p38MAPK, and NF-κB (Haneda et al., 1997). It is documented that activation of PKC has

been involved when the production of NO is suppressed (Derubertis and Craven, 1994),

and that the insulin-stimulated expression of eNOS in cultured endothelial cells has been

inhibited due to the activation of PKC (Kim et al., 2006). Furthermore, PKC activation

may enhance the activity of NADPH oxidase which acts as a ROS generating enzyme,

28

causing dysfunction of endothelial cells that can be ameliorated by using a PKC inhibitor

(Hink et al., 2001).

1.3.2.5 Increased hexosamine pathway activity

The hexosamine pathway has been demonstrated to be involved in the pathogenesis

of diabetic complications (Federici et al., 2002). In diabetes, hyperglycaemia has been

reported to reduce activity of glyceraldehyde-3-phosphate dehydrogenase in bovine aortic

endothelial cells through increased production of mitochondrial superoxide which in turn

causes increased activity of the hexosamine pathway (Du et al., 2000). In diabetes there is

high intracellular glucose and most of the glucose is metabolized firstly to glucose-6

phosphate via glycolysis and then subsequently to fructose-6 phosphate and then on

through the rest of the glycolytic pathway. However, some of fructose-6 phosphate is

diverted to act as a signalling pathway in which an enzyme GFAT (glutamine: fructose-6

phosphate amido-transferase) transforms fructose-6 phosphate into glucosamine-6

phosphate and finally forms UDP (uridine diphosphate) N-acetyl glucosamine. After that,

N-acetyl glucosamine uses serine and threonine amino acids residues as transcription

factors in a similar way to the normal phosphorylation process. The elevated modification

by this glucose amine causes pathological changes in gene expression (Sayeski and

Kudlow, 1996, Kolm-Litty et al., 1998, Wells and Hart, 2003). For example, elevated-

modification of the transcription factor Sp1 causes increased expression of transforming

growth factor-β1 and plasminogen activator inhibitor-1. Both have harmful effect on the

vasculature in diabetes (Du et al., 2000). Overall, increased glucose flux through the

hexosamine pathway has been documented as a factor in the pathogenesis of diabetic

complications and plays an important role both in hyperglycaemia-induced abnormalities

of glomerular cell gene expression (Kolm-Litty et al., 1998) and in cardiomyocyte

29

dysfunction in cell culture induced by hyperglycaemia (Clark et al., 2003). Furthermore, in

diabetes associated carotid artery plaque, modification of proteins in endothelial cells via

the hexosamine pathway is markedly increased (Federici et al., 2002).

1.3.3 Sources of reactive oxygen and nitrogen species in vascular endothelial cells

As discussed previously, vascular health is maintained by the appropriate function

of the endothelial cells. Mitochondria have been recognized as a major source of ROS

production in diabetes, but there are additional enzymes such as xanthine oxidase, NADPH

oxidase, and eNOS in the vascular endothelial cells that are thought to have a significant

role in increased ROS production.

1.3.3.1 Xanthine oxidase

In the endothelial cells, the enzyme xanthine oxidase acts to catalyze the oxidation

of hypoxanthine to urate by utilizing oxygen molecules as electron acceptors and liberating

ROS such as superoxide and hydrogen peroxide (Figure 1.6). Immunohistochemistry

studies demonstrated that xanthine oxidase is situated in endothelial cells (Jarasch et al.,

1981) and observations from experiments using electron spin resonance technologies

confirmed that xanthine oxidase is a crucial source of formation of vascular superoxide in

type I diabetes (Matsumoto et al., 2003).

1.3.3.2 NADPH oxidase

NADPH oxidase is a superoxide-producing enzyme primarily recognized as a

cytosolic enzyme complex in neutrophils where it contributes to the nonspecific defense

30

mechanism against pathogens via the formation of low quantities of superoxide by the

mechanism of electron transport (Babior et al., 2002). The NADPH oxidase complex

consists of five subunits involving a membrane-associated p22phox

and a gp91phox

subunit

and around four cytosolic subunits: p47phox

, p67phox

, p40phox

, and GTPase rac1 or rac2

(Babior, 2004). NADPH oxidase is also present at physiological levels in vascular tissues

(Valko et al., 2007). In addition to the control of vascular cell growth, proliferation,

activation, and migration, the NADPH oxidase-derived superoxide is also involved in

modulation of vascular tone (Feng et al., 2010). Conversely, in many of cardiovascular

diseases, including diabetes, NADPH activity and expression is up-regulated to form large

quantities of ROS causing oxidative stress (Figure 1.6) (Guzik et al., 2002, Takayama et

al., 2004, Ray and Shah, 2005, Drummond et al., 2011, Paravicini et al., 2004).

1.3.3.3 Endothelial nitric oxide synthase

The isoforms of NOS can be classified into three categories, namely, inducible

(iNOS), neural (nNOS), and endothelial (eNOS). The common action of all of these

isoforms is to convert L-arginine to L-citrulline and thus to liberate NO. This reaction is

catalyzed by a number of cofactors such as calmodulin, BH4, flavin mononucleotide, and

adenine dinucleotide. The second two isoforms produce low quantities of NO in a short

period of time and are mainly present in endothelial and neuronal tissue. In diabetic

conditions, where there is an increase of oxidative stress an unbalanced production of

nitric oxide and superoxide leads to further elevated formation of superoxide by the

NADPH oxidase, xanthine oxidase, and the mitochondria. The reaction between

superoxide and NO leads to the formation of a highly reactive intermediate called

peroxynitrite and lower NO bioavailability in the vascular regions, causing vascular

dysfunction (Figure 1.6). Peroxynitrite causes eNOS uncoupling, where the eNOS is

31

transformed from a dimer to a monomer to produce superoxide instead of NO. This also

occurs under conditions of diminishing levels of L-arginine and/or BH4 oxidation (Cai et

al., 2002, Hink et al., 2001, Ryoo et al., 2008, Münzel et al., 2010, Cai et al., 2005).

32

Figure 1.6 Scheme shows the suggested mechanism, sources, and pathways of the

production of ROS in endothelial cells. EC, endothelial cell; O2.-, superoxide anion; O2,

oxygen molecule; H2O2, hydrogen peroxide; NADPH, nicotinamide adenine dinucleotide

phosphate; NO, nitric oxide; OONO-

, peroxynitrite. Adapted from (Sugamura and

Keaney, 2011).

33

1.3.4 Sources of endogenous antioxidants in diabetes

Under diabetic conditions, the phenomenon of oxidative stress arises when the

cellular redox balance favours pro-oxidant production over endogenous antioxidant

defences (Rains and Jain, 2011). This is usually observed when there is an overproduction

of ROS (as described above) and/or in case of compromised function of endogenous

antioxidant systems. There are several sources of antioxidant systems involved

endogenously such as catalase, superoxide dismutase (SOD), glutathione peroxidase

(GPx), and thioredoxin.

1.3.4.1 Catalase

Catalase enhances the degradation of hydrogen peroxide to water and oxygen. In

diabetes, although there is increased expression of catalase, the activity was reduced due to

overproduction of hydroxyl radicals, thereby contributing to oxidative stress (Shi et al.,

2007, Heales, 2001, Góth et al., 2001, Góth, 2008).

1.3.4.2 Superoxide dismutase enzyme (SOD)

SOD catalyses the dismutation of superoxide producing oxygen and hydrogen

peroxide, having a vital role as an antioxidant agent to prevent cellular damage that may be

induced by superoxide radicals (Scandalios, 1993). Within the cells there are isoforms of

SOD situated in different compartments: (1) copper-zinc SOD (SOD 1) is located in the

cytoplasm, (2) manganese SOD (SOD 2) is positioned in the mitochondria, and also there

is extracellular copper-zinc SOD (SOD 3) (Faraci and Didion, 2004).

34

1.3.4.3 Glutathione peroxidase (GPx)

GPx reduces lipid hydroperoxides to lipid alcohol and reduces free hydrogen

peroxide to water (McCay et al., 1976). GPx1 is present in the cytoplasm of almost all

mammalian tissue and is the most abundant isoform of GPx. Under diabetic conditions, the

level of GPx expression has been reported to be diminished (Sindhu et al., 2004).

Additionally, studies show that GPx plays a role in the prevention of diabetes associated

atherosclerosis. For example, a study using ApoE knockout mouse demonstrated that the

extra deletion of GPx1 caused acceleration in the development of atherosclerosis by

upregulation of profibrotic and proinflammatory pathways (Lewis et al., 2007, Torzewski

et al., 2007). This accelerated progression of atherosclerosis was reduced by treatment of

knockout mice with ebselen, a mimetic of GPx1 (Chew et al., 2010).

1.3.4.4 Thioredoxin

The thioredoxin system is a fundamental system for preserving the balance of

cellular redox in endothelial cells and vascular smooth muscle (Yamawaki et al., 2003,

Yamawaki and Berk, 2005). The thioredoxin system includes; thioredoxin, thioredoxin

reductase, and NADPH that, through the interaction with redox-active centre of

thioredoxin (Cys, Gly, Pro, Cys), reduces the oxidized cystein groups on protein. This

forms a disulfide bond, which subsequently causes a reduction of thioredoxin reductase

and NADPH (Yamawaki et al., 2003). Thioredoxin peroxidase plays a role in the

antioxidant properties of thioredoxin by using SH groups as reducing equivalents. The

reduction of the oxidized form of thioredoxin peroxidase by the action of thioredoxin,

causes scavenging of ROS like hydrogen peroxide (Yamawaki et al., 2003). The

thiorodoxin-interacting protein, an endogenous inhibitor of thioredoxin, modulates the

antioxidant activity of thioredoxin. Under diabetic conditions, it has been demonstrated

35

that reduced activity of thioredoxin contributes to oxidative stress, where diabetes

increases the expression of thioredoxin-interacting protein (TXNIP) and reduces the

antioxidant activity of thioredoxin (Qi et al., 2009, Tan et al., 2011).

1.4 Endothelial dysfunction in diabetes

Endothelial dysfunction is defined as an impairment of the balance between

vasodilating and vasoconstricting substances that are produced by the endothelium (De

Vriese et al., 2000). It is considered an important contributor to the pathogenesis of

vascular diseases and the complications of diabetes mellitus (Hadi and Suwaidi, 2007).

Indeed, under physiological conditions there is a delicate balance of the release of

endothelium-derived relaxing factors and endothelium-derived contracting factors,

however, this sensitive balance is altered adversely in cardiovascular diseases, thus,

contributing to the detrimental effect on the vascular tissues and ending with damage to

organs (Tan et al., 2002). Endothelial dysfunction is recognized as an early and

independent predictor of the pathological cardiovascular outcomes of diabetes mellitus and

plays a major role in the initiation of cellular events and complications associated with

diabetic conditions (Hink et al., 2001). Endothelial dysfunction is associated with a

reduction in the relaxation of blood vessels in response to endothelium- dependent

vasodilators, such as ACh or bradykinin, or to flow-mediated vasodilatation. In humans,

studies to assess endothelial function by both invasive and non-invasive techniques have

been used in order to measure both agonist-induced and flow-mediated vasodilatation

(Barac et al., 2007, Buscemi et al., 2009). In addition to reduced production of

endothelium-derived relaxing factors such as NO, EDH, and PGI2, there are also changes

in the presence of proinflammatory molecules, the expression of adhesion molecules, and

36

enhanced production of vasoconstrictor molecules such as COX-derived metabolites of

arachidonic acid (AA), ROS, and endothelin-1 (ET1) that all have pathological

consequences. In diabetes, the impairment of endothelium-dependent relaxation is usually

associated with the production of ROS and increased expression of proinflammatory

molecules (Brownlee, 2001). Collectively, it is the combination of proinflammatory and

proliferative conditions and the impairment of endothelium-dependent responses to stimuli

that underlies endothelial dysfunction and offers the basis for the development of micro-

and macro-vascular disease. Although the aetiology of vascular endothelial dysfunction

under conditions of diabetes has been extensively examined in studies using humans or

animal models, the pathway which could be the primary mechanism to explain the

connection between diabetes and its associated vascular disease complications remains

unclear. Therefore, in the next section the possible mechanisms that play a role in the

initiation of endothelial dysfunction in diabetic vascular disease are described.

1.4.1 Reduction of NO bioavailability

Endothelium-derived NO bioavailability, as estimated by excess of NO over ROS

levels, is a one of the most important indices for determining vascular endothelial function.

Any condition in which there is reduced eNOS activity or where the generation of ROS is

elevated would lead to a reduction of NO bioavailability and consequent impairment of

endothelium-dependent relaxation. In diabetes there are many mechanisms involved in the

reduction of NO-mediated relaxation include: (1) over-generation of superoxide anions

leading to scavenging of NO and consequently increased levels of peroxynitrite; (2)

reduced synthesis of NO by eNOS enzyme due to eNOS uncoupling; (3) increased activity

of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS; (4) increased

37

uncoupling of eNOS and decreased bioavailability of BH4 which will be discussed in

detail in the following sectors.

1.4.1.1 Expression and phosphorylation of eNOS

In addition to inhibition of NO by superoxide, NO synthesis can be inhibited at an

enzymatic level. An alteration of the levels of eNOS expression may play a role in

endothelial dysfunction associated with diabetes. Using cultured endothelial cells and/or

isolated blood vessels from diabetic animals some studies have shown that the expression

of eNOS may be decreased (Okon et al., 2005, Srinivasan et al., 2004, Salt et al., 2003),

whereas there are other reports that the expression of eNOS is unchanged (Matsumoto et

al., 2006, Pannirselvam et al., 2006) or increased (Ding et al., 2007). It has been reported

that reduction of NO production may be attributed to the mechanism of phosphorylation of

eNOS at Ser1177 which activates the enzyme (Michell et al., 1999). Studies demonstrated

that in aortae from diabetic mice, phosphorylation of eNOS at Ser1177 was reduced

(Zhang et al., 2009, Zhong et al., 2007b, Leo et al., 2011b), as well as in renal arteries

(Zhong et al., 2007a), resulting in decreased synthesis of NO.

1.4.1.2 Direct reduction of NO bioactivity

In diabetes, the direct inactivation of NO has been attributed to the effect of

overproduction of ROS in the vasculature (Bitar et al., 2005). Indeed, several reports show

that diabetes causes imbalance of bioactive radicals by which the expression and/or

activity of pro-oxidant enzymes such as lipoxygenases increased, therefore, the generation

of superoxide anions increased and impairing endothelium-derived NO-mediated

38

relaxation (Woodman et al., 2008, Woodman, 2009, Malakul et al., 2008, Leo et al.,

2010).

1.4.1.3 Augmented arginase activity

One of the main roles of arginase is to catalyze the hydrolysis of L-arginine to

ornithine and urea, therefore, causing competitive inhibition of eNOS to utilize its

common substrate, L-arginine to synthesize NO. In diabetes, it has been reported that there

is an increase in the expression of arginase and as a consequent increase in its biological

activity causing impairment of endothelium-dependent relaxation which could be

ameliorated by acute application of an arginase inhibitor (Romero et al., 2008).

Furthermore, arginase activity is increased and NO synthesis is significantly reduced by

exposing endothelial cells to a medium containing a high glucose concentration (25

mmol/L, 24 hour) (Romero et al., 2008). The activity of arginase in cells exposed to high

glucose can be prevented by transfection of endothelial cells with arginase I small

interfering RNA which also normalizes NO production. Thus in diabetes, the activity of

arginase may have a role in reducing NO production (Romero et al., 2008, White et al.,

2006).

1.4.1.4 Amplified levels of ADMA

ADMA is an endogenous inhibitor of NOS. Under diabetic conditions, it has been

reported that the levels of ADMA increased causing impairment of the response to ACh in

endothelium-dependent relaxation in aortae (Xiong et al., 2003). In diabetes, the activity of

dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA, is

reduced causing accumulation of ADMA resulting in impairment of NOS activity (Xiong

39

et al., 2003). The levels of ADMA are significantly reduced by restoration of DDAH

activity through two mechanism, either by drug intervention or gene transfer which

increases the expression of DDAH and consequently increases NO synthesis and improves

endothelium-dependent relaxation in diabetic aortae (Lu et al., 2010, Xiao et al., 2009, Yin

and Xiong, 2005).

1.4.1.5 Uncoupling of eNOS and BH4

BH4 is a cofactor which plays an essential role in the modulation of eNOS activity.

BH4 is produced by guanosine 5-triphosphate cyclohydrolase I (GTPCH) (Li et al., 2010).

More than 60% of BH4 is expressed in the wall of blood vessels and associated with

endothelium and modulated by shear stress (Harrison et al., 2010, Tsutsui et al., 1996,

Widder et al., 2007, Li et al., 2010, De Bono and Channon, 2007). In order to be stabilized

as a dimer, eNOS requires appropriate levels of BH4, or the eNOS will act as an NADPH

oxidase if it is in a monomeric state, where the molecular oxygen becomes an electron

acceptor rather than L-arginine (Cai et al., 2005). In the monomeric state of eNOS,

superoxide anions are produced rather than NO (Cai et al., 2005). More important than the

level of BH4, it seems that the maintenance of optimal endothelial function depends

critically on the ratio of BH4 to oxidized biopterins. Indeed, it has been reported that the

levels of BH4 in small mesenteric arteries from db/db diabetic mice were not significantly

reduced in comparison to mesenteric arteries from normal animals, rather, the levels of

oxidized biopterin products such as 7,8-dihydrobiopterin (BH2) were increased, therefore,

the BH4/BH2 ratio was reduced (Pannirselvam et al., 2003). Furthermore, it has also been

reported that BH2 has the same affinity as BH4 to bind to eNOS, thus, BH2 competitively

displaces bound BH4 from its site in eNOS to impair NO production by forming an

uncoupled eNOS-BH2 complex, indicating the importance of BH4/BH2 ratio in order to

40

maintain the function of endothelial cells (Crabtree et al., 2008, Crabtree et al., 2009a).

Several studies showed that a reduced ratio of BH4/BH2 leads to a reduction of NO

bioavailability via uncoupling of eNOS causing endothelial dysfunction. This reduction of

ratio can be reversed by dietary provision of the BH4 precursors such as sepiapterin or

supplementation of BH4 (Cai et al., 2002, Cai et al., 2005, Hink et al., 2001). In addition to

overproduction of superoxide induced by hyperglycaemia and consequent decrease of the

ratio of BH4/BH2, it has also been demonstrated that the reduction of the BH4/ BH2 ratio is

modulated by the production of peroxynitrite. In diabetes which initiates the mechanism of

ubiquitination and 26S proteasome-dependent degradation of GTPCH which causes further

decrease in BH4/BH2 ratio (Wang et al., 2009, Wang et al., 2010, Xu et al., 2007). All

these effects induced by hyperglycaemia are produced by increased expression of SOD or

inhibition of eNOS or by the effects of inhibitors of 26S proteasome such as MG132 or

PR-11 (Xu et al., 2007). Similarly, the reduction of GTPCH and BH4 in STZ-induced

diabetic mice can be reversed by the SOD mimetic, tempol (Xu et al., 2007). In addition,

the BH4 biosynthetic pathway plays a role in maintenance of eNOS by BH4 recycling via

dihydrofolate reductase (DHFR) which has been identified as a critical modulatory factor

in the regeneration of BH4 from BH2 (Crabtree et al., 2011, Sugiyama et al., 2009,

Crabtree et al., 2009b). In diabetes, expression levels of DHFR could also participate in

the contribution to uncoupling of eNOS, but, the data in this regard are not consistent.

Several studies showed increases in the expression of DHFR in diabetes (Oak and Cai,

2007, Ren et al., 2008, Yamamoto et al., 2010). On the other hand, it has been reported

that in diabetic aorta the expression of DHFR was decreased (Wenzel et al., 2008). Despite

these inconsistencies of the reported data in regards to DHFR expression, all studies have

shown that in diabetes the ratio of BH4/BH2 was diminished and the endothelial

41

dysfunction was concomitant to uncoupling of eNOS (Oak and Cai, 2007, Ren et al., 2008,

Wenzel et al., 2008, Yamamoto et al., 2010, Channon, 2004).

1.4.3 Diminished EDH responses

The reports about the relative contribution of NO and EDH to endothelium-

dependent relaxation in mesenteric arteries from diabetic arteries are inconsistent. For

instance, it has been reported that the contribution of EDH to endothelium-dependent

relaxation is increased in diabetes to offer some compensation for the reduction of

bioavailability of NO (Shi et al., 2006). In contrast, another study showed that the action of

NO is not affected by diabetes and the contribution of EDH is selectively decreased (Wigg

et al., 2001). Generally, the EDH is complex in its nature and its contribution depends on

the vascular beds investigated. Several studies have endeavored to investigate the

impairment of EDH responses in arteries under pathological conditions such as diabetes

via investigating the underlying cellular mechanisms responsible for this impairment

(Burnham et al., 2006b, Young et al., 2008, Leo et al., 2011b). There are probable

pathways attributed to be responsible factors causes decrease in EDH responses in diabetes

include either modification of the expression or activity of KCa channel or impairment of

MEGJ expression or activity.

1.4.3.1 Alteration in the expression or activity of endothelial KCa channels

Changes in the expression or the activity of KCa channels could alter the

contribution of EDH to endothelium-dependent relaxation. In diabetes, the impairment of

EDH responses has been extensively investigated and its correlation to the expression level

of KCa channels, investigated but there is a lack of consistent data in this regard (Brondum

et al., 2010, Ding et al., 2005, Pannirselvam et al., 2006). For instance, it has been

42

reported that in STZ-induced diabetic ApoE-deficient mice, the expression of SKCa2 and

SKCa3 channels was reduced causing an impairment of EDH-mediated relaxation (Ding et

al., 2005). In contrast, another study showed that in insulin resistant Zucker fatty rats, even

though there is an impairment in EDH responses, the expression of SKCa3 channels was

augmented in small mesenteric arteries (Burnham et al., 2006a). Moreover, several other

reports showed that even where there is impaired EDH-mediated relaxation there is no

difference in the expression levels of KCa channels in diabetic arteries compared to normal

arteries (Pannirselvam et al., 2006, Brondum et al., 2010). It is important to mention that

the measurement of the expression levels of either protein or gene of the endothelial KCa

channels has the common problem that its commonly suggested that the channel function

reflected by the molecular weight of the native single channels reflects that of the

functional channel (Brondum et al., 2010, Hilgers and Webb, 2007). To some extent this is

not the case, as the functional channel in normal intact tissue has been reported to be

constituted by high molecular weight complexes of these channels (Chadha et al., 2010).

The deficiency of EDH-mediated relaxation response in mesenteric arteries from obese

animals is modulated by the decrease in the expression and/or activity of the KIR channels,

which is a downstream effect on SKCa-mediate the EDH responses (Haddock et al., 2011).

Furthermore, it has also been reported that the activity of KIR channel and/or the activity of

Na+-K

+-ATPase is diminished in diabetes (Makino et al., 2000). Overall, despite the lack

of consistent data in regard to the expression levels of KCa channels in the endothelial cells,

all the studies demonstrated that EDH-mediated relaxation is impaired in diabetic arteries,

indicating that the functional/bioactivity of the endothelial KCa channels.is a more essential

indicator of EDH type responses than endothelial KCa channel expression level.

43

1.4.3.2 Transformed expression or activity of MEGJs

Under diabetic conditions, myoendothelial cell communication can be directly

affected to reduce EDH-mediated relaxation via transforming the distribution and/or

expression of key vascular connexins of MEGJs (De Wit and Griffith, 2010). It has been

reported that the contribution of EDH to endothelium-dependent relaxation is reduced in

mesenteric arteries from db/db type 2 diabetic mice, although there is no change in mRNA

expression level for the key vascular connexins, Cx37, Cx40, Cx43, Cx45 (Pannirselvam

et al., 2006). On the other hand, a study in STZ-induced type 1 diabetic ApoE-deficient

mice showed that Cx37 expression was reduced in small mesenteric arteries (Ding et al.,

2005). Likewise, in aortic arteries from STZ-induced diabetic animals, the protein

expression of the connexins Cx37 and Cx40 was diminished (Hou et al., 2008) and also in

coronary arteries (Makino et al., 2008), suggesting that there is a connection between the

reduction in EDH-mediated relaxation in diabetes and the expression of connexins.

Furthermore, it has been reported that in small mesenteric arteries there is a general

reduction in mRNA and protein levels of Cx40 as well as EDH responses (Young et al.,

2008). The myoendothelial cell communication could be disturbed by the alteration of the

expression of the vascular connexin. A positive correlation has been demonstrated between

the levels of vascular connexin expression of Cx40 and Cx37 in the cremaster arterioles

from Cx40-deficient mice (De Wit, 2010). In Cx40-deficient mice, the conduction of

activated endothelium-dependent vasodilatation in cremaster arterioles is impaired due to a

decrease in the expression of Cx37 (Figueroa et al., 2003, De Wit, 2010). Therefore, in

diabetes, the impairment of EDH-mediated relaxation may be a result of the reduction of

Cx37 expression which also resulted from the reduction of the expression of Cx40.

Furthermore, it has been reported that in diabetes, the expression of Cx37, Cx40, and Cx43

can also be affected when high glucose levels inhibit the intercellular communications

44

through gap junction in VSMC in bovine aorta via PKC-mediated phosphorylation of

Cx43 (Kuroki et al., 1998). Similarly, Sato et al. (2002) reported that the activity of gap

junction communication in rat microvascular endothelial cells was reduced due to high

glucose and this reduction was concomitant with a reduction in Cx43 mRNA and protein

expression (Sato et al., 2002). Therefore, in diabetes, changes in the expression and/or

regulation of the connexins that make up MEGJs could play a critical role in the

endothelial dysfunction.

1.5 The incretins and the dipeptidylpeptidase-4 inhibitors

1.5.1 Historical overview of the incretin concept

The incretins are hormones that are secreted from enteroendocrine cells and

released from the gut into the blood stream within minutes of the ingestion of food (Baggio

and Drucker, 2007). The incretin hormones have a physiological role in the modulation of

the insulin secretory response to food nutrients (Baggio and Drucker, 2007). The “incretin

effect” is a term referring to the insulin secretory response due to the stimulation of

pancreatic β-cells by incretin hormones which have insulinotropic effect and account for at

least 50% of the total insulin secreted after oral glucose administration (Jose and Inzucchi,

2012). In 1902, Bayliss and Starling described the mechanism of pancreatic secretion,

where they found that the pancreas secretes juices via the pancreatic duct after acid

infusion into the digestive system, and this continued even when the innervation of the

intestine was inhibited (Bayliss and Starling, 1902). They conclude that the nature of the

signal that stimulates the pancreas was most likely a chemical, rather than nervous,

stimulus. After the interstinal wall had been stimulated by acid, they removed the extracts

and injected it into the blood stream and once again, they saw juices coming from

pancreatic duct of the injected animal, and they proved that the stimulation of the flow of

45

pancreatic juice was due to the stimulation of the pancreas by the extracts that must

contain a substance that was usually secreted from the intestinal wall into the blood stream

to stimulate the pancreas, and they called this substance “secretin” (Bayliss and Starling,

1902). Moor (1906) reported achieving some success in some experiments he applied on

young diabetic patients who received by mouth extracts of intestinal mucosa (Moore,

1906). This was the first trial based on incretin therapies in order to treat diabetes;

however, these experiments were essentially doomed, because the chemical nature of

peptides which would be degraded when given orally. In 1921, Banting and Best extracted

insulin from the pancreas to further investigate the probability of food entering the gut

causing release of an excitant into the blood stream to finally stimulate insulin secretion

and lower blood glucose (Banting et al., 1922). La Barre (1932) used the term “incretin” to

refer to an extract secreted in the upper part of the gut mucosa to cause hypoglycaemia (La

Barre and Ledrut, 1934). The concept of the incretin system progressed when

radioimmunoassays techniques for insulin become accessible. Between 1964 and 1976

there were at least three studies which independently showed that glucose administered

orally causes a greater insulin response when compared with intravenous glucose injection

although the glucose level obtained by intravenous injection was the same (Elrick et al.,

1964, McIntyre et al., 1964, Perley and Kipnis, 1967). Indeed, the three groups knew that

the oral intake of glucose leads to enhanced incretin release into the blood stream and

subsequently causes an increase in insulin secretion rather than did plasma glucose itself.

In 1971, Brown and Dryburgh isolated and identified the structure of amino acid from a

peptide that was obtained from intestinal mucosa (Brown and Dryburgh, 1971). It has been

reported that the exogenous administration of peptides causes inhibition of gastric acid

secretion in dogs and this peptide was called gastric inhibitory polypeptide (GIP) (Brown

and Dryburgh, 1971). Furthermore, Brown and colleagues identified that GIP had

46

insulinotropic properties (Dupre et al., 1973). GIP was deduced as the first incretin been

isolated and its properties identified. By the end of 1985, glucagon-like peptide-1 (GLP-1)

(7-36) was isolated as a peptide secreted in the gut and reported as a potent insulinotropic

factor (Schmidt et al., 1985). Subsequently clinical investigations demonstrated that GLP-

1 taken intravenously to increase plasma levels of GLP-1 to supraphysiological levels

could have the ability to increase insulin secretion and reduce glucose levels in patients

with type 2 diabetes mellitus. Thus incretin based therapy was proposed as a new approach

to treat type 2 diabetes mellitus.

1.5.2 General description of incretin hormones and exendin-4

1.5.2.1 Glucose-dependent insulinotropic peptide (GIP)

GIP, the first incretin hormone to be identified, consists of a single 42-amino acid

peptide chain derived from a 153-amino acid precursor (Takeda et al., 1987). It is a

member of the structurally related family of hormones including secretin, glucagon, and

vasoactive intestinal peptide. GIP was initially observed to inhibit gastric acid secretion as

well as gastrointestinal motility in dogs receiving supraphysiological dosages (Brown et

al., 1974). It has been reported that GIP at physiological plasma levels exerts glucose-

dependent stimulatory effects on the process of insulin secretion and enhances glucose

uptake by tissues via an insulin-mediated process (Dupre et al., 1973). Many reports

demonstrated that GIP production occurred in enterendocrine cells (called K-cells) which

are located in the proximal small intestine (duodenum and jejunum) and GIP is released as

a response to nutrients containing glucose or fat, and then subsequently released into the

blood-stream in an amount higher than the amount produced in the fasted state (Dupre et

al., 1973, Pederson et al., 1975, Elliott et al., 1993). It has been found in a study carried

out in fasting glucose concentration state that the effects of GIP strongly depends on the

47

glucose level in the blood, where administration of oral fat alone, such as oral corn oil,

without carbohydrate leads to stimulation of GIP release, however, this is not sufficient to

enhance insulin secretion, indicating that GIP-mediated insulin secretion is a glucose-

dependent process; i.e. it is necessary for glucose to be high in plasma in order to enhance

GIP to act on stimulation of insulin secretion (Ross and Dupre, 1978). The insulinotropic

effects of GIP can be achieved through the binding to its specific receptor (GIPR) causing

increased levels of intracellular cAMP and Ca2+

in pancreatic β-cells, and promoting cell

proliferation and cell survival in β-cells (Tru mper et al., 2001, Trumper et al., 2002). The

biological half-life of GIP is very short due to the rapid degradation of GIP by the action of

dipeptidyl peptidase 4 (DPP4). It has been reported that plasma GIP concentrations are

normal or increased in type 2 diabetes, however, the insulinotropic effects are lacking

(Ross et al., 1977, Vilsbøll et al., 2001). Although the mechanisms by which β-cells

respond to GIP remains unclear, some studies suggest that the decrease in physiological

response of β-cell to GIP was due to the effects of hyperglycaemia which causes down

regulation of GIPR expression/activity (Lynn et al., 2001, Zhou et al., 2007).

1.5.2.2 Glucagon-like peptide-1 (GLP-1)

GLP-1 was discovered through the cloning process and identification of the

proglucagon gene (Bell et al., 1983). GLP-1 is a product of post-translational cleavage of

the proglucagon gene via the prohormone convertase enzyme PC1/3 (Zhu et al., 2002,

Ugleholdt et al., 2004). GLP-1 has been considered as the second peptide that has incretin

activity and potentially stimulates insulin secretion from β-cells of the pancreas (Schmidt

et al., 1985). GLP-1 is primarily produced in enteroendocrine cells (called L-cells) that are

located within the enterocytes across the small bowel and ascending colon (Doyle and

Egan, 2007, Jang et al., 2007). There are multiple forms of GLP-1 hormone that have been

48

identified in humans and the COOH-terminally amidated form, GLP-1 (7-36) amide

represents the major circulating, biologically active form (at least 80%) with lesser amount

of the minor glucine extended form GLP-1 (7-37) (Ørskov et al., 1986). Similar to GIP,

GLP-1 is positively coupled to a specific receptor (GLP-1R) in order to achieve its

insulinotropic effects by increasing intracellular cAMP and Ca2+

levels in β-cells.

Furthermore, beside its insulinotropic effects, GLP-1 plays a role in inhibiting gastric

emptying and decreasing food intake (Willms et al., 1996), prevents secretion of glucagon

(Komatsu et al., 1989), thus minimizing the rate of the production of endogenous glucose

(Prigeon et al., 2003). Collectively, all of these actions should lower blood glucose levels

in type 2 diabetes. GLP-1 has also been reported to play a role in protecting β-cells from

apoptosis (Farilla et al., 2002) and to enhance the proliferation of β-cells via upregulation

of the transcription factor in β-cells, pancreatic duodenal homobox-1 protein (PDX-1)

(Perfetti et al., 2000, Stoffers et al., 2000), that recognizes increased transcription of the

insulin gene and up-regulates glucokinase and glucose transporter 2 (GLUT2) (Wang et

al., 1999). Several studies reported that the continuous treatment of type 2 diabetic patients

with GLP-1 can normalize blood glucose levels, enhance the function of β-cells, and

restore the first-phase of insulin secretion in β-cells (Holz et al., 1993, Zander et al., 2002),

hence, recently GLP-1/GLP-1R have become important therapeutic targets for treating

type 2 diabetes. GLP-1 has a short half-life (1.5-2 min) due to its degradation by the DPP-

4 enzyme.

1.5.2.3 The incretin mimetic exenatide (exendin-4)

Exenatide (exendin-4) is a 39–amino acid incretin mimetic, purified from the

salivary secretions of the Gila monster (Heloderma suspectum), which exerts its

glucoregulatory activities in a similar manner to mammalian incretin hormone glucagon

49

like peptide 1 (GLP-1) (Kolterman et al., 2003, Fineman et al., 2003, Nielsen and Baron,

2003, Nielsen et al., 2004). Exendin-4 causes glucose-dependent promotion of insulin

secretion and inhibition of high secretion of glucagon, and slows gastric emptying. The

process of glucose-dependent enhancement of insulin secretion by exendin-4 is mediated

by binding to the pancreatic GLP-1 receptor (Göke et al., 1993). In animal experimental

models of diabetes and in insulin secreting cell lines, both exendin-4 and GLP-1 improve

pancreatic β-cell function by increasing the expression of key genes responsible for insulin

biosynthesis and secretion (Nielsen et al., 2004). Several studies reported that exenatide

and GLP-1 are able to reduce food intake, enhance weight loss, and have an insulin-

sensitizing effect (Nielsen and Baron, 2003, Young et al., 1999, Nielsen et al., 2004,

Szayna et al., 2000, Gedulin et al., 2005). Investigators conclude that exendin-4 markedly

promotes the formation of new β-cells (Xu et al., 1999).

Besides the beneficial insulinotropic effects of exendin-4, it may have favorable

direct effects on endothelial cell function. It has been reported that exendin-4 reduces

macrophage adhesion to the endothelium, an early step in the formation of atherosclerotic

plaques and subsequent endothelial dysfunction (Arakawa et al., 2010). Findings in human

clinical trials showed that the acute administration of exendin-4 counteracts endothelial

dysfunction induced by ischemia and reperfusion in the heart (Ha et al., 2012) or after a

high fat meal (Koska et al., 2010). It has also been reported that the GLP-1 receptor

agonist liraglutide has a potential role in the inhibition of vascular disease progression via

the prevention of atherogenesis, stabilization of plaque and endothelial function together

with potential protection against major cardiovascular events in an ApoE−/−

mouse model

of atherosclerosis (Gaspari et al., 2013).

Both GLP-1 and exendin-4 have been shown to reduce the vulnerability of cells to

apoptotic signaling under conditions of oxidative stress (Chen et al., 2006, Tews et al.,

50

2009). Several mechanisms have been proposed to interpret this cytoprotective effect

including, counter-regulation in the reduction in proteins electron transport chain (Tews et

al., 2009), decrease the expression of pro-apoptotic thioredoxin-interacting protein

(TXNIP) implicated in cell glucose toxicity process (Chen et al., 2006), reduction of

endoplasmic reticulum stress (Tsunekawa et al., 2007), and down-regulation of NF-KB

which acts as a promoter to activate β-cells and stimulate NO synthase and NO pathway

(Liu et al., 2008).

1.5.3 Dipeptidylpeptidase and dipeptidylpeptidase inhibitors

1.5.3.1 Dipeptidylpeptidase (DPP-4)

DPP-4 is a complex enzyme consisting of 766 amino acids, expressed on the

surface of several cell types including vascular endothelial cells (Fadini and Avogaro,

2011, Augustyns et al., 1999). It is a serine aminopeptidase that cleaves GLP-1 and GIP

via dipeptide cleavages with alanine, proline, or hydroxyproline in the N-terminal amino

acids (Drucker, 2007). It has been reported that in vitro, DPP-4 cleaves many chemokines

and peptide hormones, however, in vivo, there are comparatively fewer peptides have been

identified as endogenous physiological substrates for DPP-4. Both GIP and GLP-1 are

endogenous physiological substrates for DPP-4. DPP-4 knockout mice studies showed

improved glucose tolerance concomitant with increased bioavailability of GIP and GLP-1

and enhanced secretion of insulin subsequent to oral glucose challenge. This was

accompanied by decreased accumulation of fat and diminished intake of food as well as

increased resistance to diet-induced obesity (Marguet et al., 2000, Conarello et al., 2003).

In vivo studies on mice which had their incretin receptors genetically inactivated showed

that the mechanisms transducing the glucoregulatory effects of DPP-4 inhibitors were

dominated by GIP and GLP-1 receptor-dependent pathways (Marguet et al., 2000). DPP-4

51

is expressed in the endothelial cells, particularly in the microvascular circulation

(Matheeussen et al., 2011), but it has been reported that the expression and the activity of

DPP-4 is only increased in microvascular endothelial cells in the presence of high glucose

(Pala et al., 2010). This suggests that DPP-4 inhibitors may reduce the detrimental effects

of hyperglycaemia on endothelial cells.

1.5.3.2 Dipeptidylpeptidase inhibitors

DPP-4 inhibitors are pharmacological glucose lowering compounds that have been

introduced as a new strategy for treatment of type 2 diabetes mellitus (Barnett, 2006,

Richter et al., 2008, Palalau et al., 2009, Hollander and Kushner, 2010, Scheen, 2011, Jose

and Inzucchi, 2012). In recent years, several structurally dissimilar DPP-4 inhibitors such

as (1) sitagliptin, (2) vildagliptin, (3) saxagliptin, (4) alogliptin, and (5) linagliptin (Figure

1.7), have been developed and are now being used clinically. The structural dissimilarities

of DPP-4 inhibitors could affect their beneficial pharmacological action. For example,

Kröller-Schön and colleagues (2012) reported that the DPP-4 inhibitor linagliptin

significantly inhibited the oxidative burst in human leucocytes but this was not shared with

other DPP-4 inhibitors such as sitagliptin, vildagliptin, alogliptin and saxagliptin (Kroller-

Schon et al., 2012). DPP-4 inhibitors act as incretin hormone enhancers which control

postprandial elevated levels of blood glucose by increasing insulin secretion and

decreasing glucagon secretion via glucose-dependent manner. Thus, in presence of high

plasma glucose levels, the incretin hormones signal to the liver to reduce glucose

production (Jose and Inzucchi, 2012). The DPP-4 inhibitors are able to reduce both fasting

plasma glucose (FPG) and post prandial glucose (PPG) levels (Aschner et al., 2006). It has

been reported that DPP-4 inhibitors do not reduce elevated blood glucose levels to a

greater extent than existing therapies (Fakhoury et al., 2009, Monami et al., 2010), but

52

they provide many potential benefits including the ability to induce a sustainable decrease

in glycated HbA1c that is accompanied by a low risk of hypoglycaemia and is not

associated with weight gain (Barnett, 2006).

53

Figure 1.7 DPP-4 inhibitors show dissimilarities in their chemical structure. Obtained

from (Lin et al., 2013).

54

1.5.4.2.1 The DPP-4 inhibitor linagliptin

Liangliptin (originally: BI 1356), developed by Boehringer Ingelheim

Pharmaceuticals (Ingelheim, Germany), inhibits the enzyme DPP-4 in a competitive and

reversible manner. It prevents the degradation of GLP-1 and GIP hormones and increases

insulin secretion from pancreatic β-cells (Gallwitz, 2012). Linagliptin has a unique chemical

structure based upon a xanthine scaffold structure (BI 1356; 8-(3-(R)-aminopiperidin-1-yl)-

7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7 dihydropurine-2, 6-Dione)

(Figure 1.7) (Deacon and Holst, 2010). Analysis of the X-ray crystal structure of linagliptin

in complex with human DPP-4 showed that the butynyl substituent of linagliptin occupies

the S1 hydrophilic pocket of the DPP-4 enzyme and the aminopiperidine substitute in the

xanthine scaffold occupies the S2 substitute and the primary amine of linagliptin interacts

with the key amino acid residues, (Deacon and Holst, 2010). Linagliptin is characterized by

its high potency and excellent selectivity for the DPP-4 enzyme (Thomas et al., 2008), with

a long duration of action which is advantageous for once-daily dosing (Thomas et al., 2008,

Eckhardt et al., 2007).

55

1.5.5 Biological actions of the gliptins as potential treatment for diabetic vascular

complications

1.5.5.1 Modulation of glucose homeostasis by the incretin system

In normal physiological conditions, the gut-derived incretin hormones GLP-1 and

GIP are released from the large and small intestine in response to food ingestion. Both of

these hormones stimulate insulin secretion and release from the pancreatic β-cells in

proportion to blood glucose levels (Jose and Inzucchi, 2012). In addition, GLP-1 decreases

glucagon secretion, slows gastric emptying, and increases satiety (Jose and Inzucchi, 2012,

Scheen, 2011, Drucker and Nauck, 2006). The actions of GLP-1 and GIP are rapidly

terminated by dipeptidyl peptidase-4 (DPP-4) (Figure 1.8). Thus, the compounds that

inhibit the biological action of DPP-4 enzyme such as DPP-4 inhibitors will prolong the

half-life and increase the bioavailability of the incretin hormones and consequently

improve their biological effect to maintain glucose homeostasis in individuals with type 2

diabetes.

56

Figure 1.8 The physiological effect of the incretin system. The DPP-4 enzyme is activated

after meal ingestion. As blood glucose levels increase in the blood, incretin hormones such

as GLP-1 and GIP are released from neuroendocrine cells in the intestinal tract. These

hormones cause insulin secretion from beta-cells in the pancreas. Additionally, in a

glucose dependent manner, GLP-1 decreases glucagon secretion from pancreatic alpha-

cells, increases the feeling of satiety, and slows gastric emptying. Through the action of

DPP-4, the incretin hormones (GLP-1 and GIP) are degraded rapidly into their inactive

forms. Blocking the action of DPP-4 increases the biological half-life of incretin hormones

and improves their physiological actions. Obtained from (Jose and Inzucchi, 2012).

57

1.5.5.2 Effects of DPP-4 inhibitors on vascular endothelial cells and NO

In diabetes, endothelial dysfunction plays an important role in the initiation and

development of vascular complications such as atherosclerosis, hypertension, and coronary

artery disease (Liu et al., 2012, Matsubara et al., 2012a). The risk of developing coronary

artery disease in type 2 diabetic patients is 2-4 fold higher compared to individuals without

diabetes (Grundy et al., 1999). In type 2 diabetes mellitus, treatment with gliptins showed

a potential vascular protective effect via GLP-1-dependent mechanisms (Hu et al., 2013).

Gliptin stimulate endothelium-dependent GLP-1 receptor (GLP-1R)-induced vasodilation

involving the cyclic adenosine monophosphate-dependent kinase A (PKA) pathway (Seino

et al., 2010). Involvement of PKA pathway sequentially activates several downstream

mediators which stimulate eNOS and increased production of NO (Liu et al., 2012). In

addition to engagement of GLP-1R-mediated signalling pathway, there is some gliptin

compounds that potentially exhibit improvement in endothelial function and acts against

pathological vascular developments via GLP-1R- independent signalling pathways (Shah

et al., 2011b, Kroller-Schon et al., 2012, Ding and Zhang, 2012). The gliptins vascular

protective effects are well documented in animal models. In study using mouse model of

hind limb ischemia, the DPP-4 inhibitor MK-0626 increased eNOS expression in vascular

tissue and showed potential improvement in blood circulation (Shih et al., 2014). Also, the

DPP-4 inhibitors sitagliptin and saxagliptin increased bioavailability of NO and improved

the endothelial function in spontaneously hypertensive rat models (Aroor et al., 2013, Liu

et al., 2012, Mason et al., 2012, Mason et al., 2011). The DPP-4 inhibitor linagliptin

improved endothelial function and reduced oxidative stress and inflammation in rat model

of lipopolysaccharide-induced septic shock (Kroller-Schon et al., 2012). It has also been

reported that linagliptin significantly improved the function of endothelial cells in a rat

model of renovascular hypertension (Chaykovska et al., 2013). Taken together, data from

58

these animal models under pathological challenges demonstrate that gliptins exhibit

enhancement in vasorelaxation and facilitate the increase in blood flow. Additionally,

gliptins also showed direct endothelium-dependent vasorelaxation in rat aortae ex vivo and

the DPP-4 inhibitor linagliptin was the most potent vasorelaxant and has antioxidant effect

which was not shared with other gliptins involved in the study such as alogliptin and

vildgaliptin (Kroller-Schon et al., 2012). In a study using human umbilical vein

endothelial cell culture, the acute presence of either alogliptin or its mediator, GLP-1,

caused eNOS upregulation and significant increases in activity and expression of eNOS

causing an increased in bioavailability of NO (Zhong et al., 2013, Ding and Zhang, 2012,

Shah et al., 2011b, Shah et al., 2011a). In several human clinical trials, gliptins exhibit

improvement in endothelial function with vasoprotective effects (Matsubara et al., 2012a,

Kubota et al., 2012, Van poppel et al., 2011, Noda et al., 2013, Suzuki et al., 2012, Fadini

et al., 2010). Potential vascular benefits have also been identified with gliptins such as

vildagliptin and alogliptin. These benefits may be due to different pathways eventually

causing an upregulation of NO-mediated relaxation (Van poppel et al., 2011, Noda et al.,

2013).

1.5.5.3 Effects of DPP-4 inhibitors on blood pressure

There is limited evidence that DPP-4 inhibitors affect blood pressure (BP). Recent

trials show that BP was significantly reduced with DPP-4 inhibition (Liu et al., 2012, Lee

et al., 2013, Chaykovska et al., 2013, Mistry et al., 2008, Ogawa et al., 2011). The BP in

Zucker diabetic fatty (ZDF) rats and SHRs was significantly reduced after less than 2-4

weeks of treatment with sitagliptin, saxagliptin, and linagliptin (Liu et al., 2012, Kroller-

Schon et al., 2012, Mason et al., 2012). Indeed, because GLP-1 is known to reduce salt

intake and increase urinary salt excretion, thus gliptins may show a reduction in BP in salt

59

sensitive hypertension (Ogawa et al., 2011). This beneficial effect was found in a trial in

patients having uncontrolled type 2 diabetes mellitus currently treated with

antihyperglycaemia and antihypertensive medications and the DPP-4 inhibitor sitagliptin

was introduced to their treatment programme (Ogawa et al., 2011). In contrast, other

studies report that the gliptins have little effect on BP (Jackson et al., 2008, Koren et al.,

2012, Van poppel et al., 2011).

1.5.5.5 Effects of DPP-4 inhibitors on inflammation and atherosclerosis

Atherosclerosis is an outcome of complex pathologic proinflammatory events in

the blood vessels and it is linked with activation of leukocytes with an increased

production of inflammatory cytokines (Hansson, 2005). The formation of atherosclerosis

plaques is due to the accumulation of inflammatory mediators in the blood vessels

(Barbieri et al., 2013, Ervinna et al., 2013). Hyperglycaemia is a major risk action for

atherosclerosis, due to increased oxidative stress and inflammation in addition to a

negative action on function of platelets (Barbieri et al., 2013). Taken together, all of these

risk factors promote the risk of atherosclerosis among type 2 diabetic patients.

Several animal studies demonstrated that gliptins have the capacity to delay the

development of atherosclerosis both in diabetic and nondiabetic conditions (Shah et al.,

2011a, Chaykovska et al., 2013, Ervinna et al., 2013, Terasaki et al., 2013, Ta et al.,

2011). Vildagliptin normalized the oxidative stress and inflammatory markers in the aortae

of Long-Evans Tokushima fatty rats (Matsui et al., 2011). Studies using apoE-null mouse

models showed that gliptins, such as alogliptin, anagliptin, linagliptin, and des-fluoro-

sitagliptin, markedly diminished the expression of proinflammatory cytokines and reduced

inflammation by inhibiting monocyte/macrophage activation and chemotaxis (Zhong et al.,

60

2013, Matsubara et al., 2012b). Furthermore, sitagliptin significantly decreased

postprandial plasma LDL and vLDL cholesterols and delayed the progression of

atherosclerosis in a hamster model of insulin-resistant fructose-induced hyperlipidaemia by

increasing incretin action and this was consistent with other findings of sitagliptin-treated

mice and also with apoE-deficient mice treated with alogliptin and anagliptin (Ervinna et

al., 2013, Ta et al., 2011, Hsieh et al., 2010). The beneficial effects of gliptins on

postprandial lipid profile may reduce the risk of cardiovascular pathology for individuals

with type 2 diabetes (Eliasson et al., 2012). Moreover, it has been reported that the DPP-4

enzyme itself may be inherently atherogenic, thus there is some speculation that may act as

a risk factor for atherosclerosis, because it stimulates cell proliferation in human coronary

smooth muscle and enhances monocyte migration, inflammatory reactions mediated by

macrophages, and tissue remodelling. Overall, it is still unclear whether the gliptins have

the ability to reduce hyperlipidaemia and cardiovascular consequences. Therefore new

investigations are needed to understand their potential to prevent the initiation and

development of atherosclerosis associated with diabetes.

1.5.5.4 Cardiovascular effects of DPP-4 inhibitors

Studies assessing the beneficial effects of DPP-4 inhibitors on actual

cardiovascular outcomes are limited, and their conclusions should be considered as highly

preliminary. Accumulating evidence suggests that the incretin-based therapy has

favourable cardiovascular outcomes, where the incretin has the potential to be used as

cardiovascular protective therapy in experimental models of myocardial infarction (MI)

and heart failure (Bose et al., 2005, Anagnostis et al., 2011, Jose and Inzucchi, 2012). In

human clinical trial, it has been reported that the DPP-4 inhibition by sitagliptin by

increasing the plasma concentrations of GLP-1 could protect the heart from ischaemic left

61

ventricular dysfunction during dobutamine stress echocardiography (DSE) in patients with

ischaemic heart disease (Khan et al., 2010).

In addition to the cardiac beneficial effects of DPP-4 inhibition which are possibly

mediated by GLP-1, DPP-4 inhibitors could also have direct effects on the risk factors

affecting the heart or vasculature, independently of the incretin system pathway. For

example, the mechanism which involves an enhanced signalling via the bone marrow

chemokine identified as stromal cell-derived factor (SDF)-1alpha. Once there is damage to

the blood vessels, local growth factors and cytokines are released and signal the bone

marrow to release endothelial progenitor cells directed to the injured places of the blood

vessels and differentiated into mature endothelial cells and contributes to the

reconstruction of the vasculature. Interestingly, one of the regulators of the endothelial

progenitor cells which enhance its mobilisation is SDF-1alpha which is known to be a

substrate for the DPP-4 enzyme. Thus the inhibition of DPP-4 will increase SDF-1alpha

concentrations to eventually promote the delivery of endothelial progenitor cells to the site

of injured blood vessels (Heissig et al., 2002). This beneficial effect of DPP-4 inhibition

on this chemokine was demonstrated by Fadini et al., who reported that after four weeks of

treatment of type 2 diabetic patients with sitagliptin, there was an increase in endothelial

progenitor cells, as well as an increase in SDF-1alpha levels, in addition to a reduction in

the proinflammatory chemokine monocyte chemotactic protein (Fadini et al., 2010).

Mistry and colleagues reported that non-diabetic patients with mild to moderate

hypertension treated with sitagliptin showed a small but significant reduction in their

systolic blood pressure (Mistry et al., 2008). In a clinical trial, treatment of individuals

with type 2 diabetes with sitagliptin, taken as a monotherapy was not associated with an

increased risk of major adverse cardiovascular events and was generally well tolerated in

clinical trials of up to 2 years in duration (Williams-Herman et al., 2010). Frederich et al.

62

evaluated the relative risk of cardiovascular events in trials assessing saxagliptin in

patients with type 2 diabetes mellitus and conclude that saxagliptin had a potential to

reduce cardiovascular events in diabetic patients (Frederich et al., 2010). A clinical trial

examined the relationship between linagliptin and cardiovascular outcomes in type 2

diabetic patients compared to other diabetic patients receiving a placebo with treatment

duration of at least 12 months. Linagliptin does not increase cardiovascular risk and, also,

to exhibit a potential reduction of adverse cardiovascular diabetic events such as

cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, or hospitalisation

for unstable angina compared with placebo or other treatment (Johansen et al., 2011).

White et al. in a randomized clinical trial examined the effects of alogliptin on

cardiovascular end points such as cardiovascular death, non-fatal myocardial infarction

and non-fatal stroke in individuals with type 2 diabetes. The findings demonstrated that the

risk of cardiovascular events was lower in diabetic patients treated with alogliptin

compared with diabetic individuals receiving a placebo (White et al., 2010). Indeed,

several large-scale clinical trials planned specifically to evaluate the effects of DPP-4

inhibitors on cardiovascular events are currently proceeding including, TECOS

(Randomised, Placebo Controlled Clinical Trial to Evaluate Cardiovascular Outcomes

After Treatment With Sitagliptin in Patients With Type 2 Diabetes Mellitus and Inadequate

Glycaemic Control), EXAMINE (Cardiovascular outcomes Study of Alogliptin in

Subjects with Type 2 Diabetes and Acute Coronary Syndrome), SAVOR-TIMI 53

(Multicenter, Randomised, Doubleblinded, Placebo-Controlled Phase IV Trial to Evaluate

the Effect of Saxagliptin on the Incidence of Cardiovascular Death, Myocardial Infarction

or Ischaemic Stroke in Patients with Type 2 Diabetes) and CAROLINA (Cardiovascular

Outcome Study of Linagliptin Versus Glimepiride in Patients with Type 2 Diabetes).

Overall, small clinical studies suggesting that there are beneficial effects of DPP-4

63

inhibitors on diabetic cardiovascular risk. However, the relationship between the DPP-4

inhibitors and the actual cardiovascular outcomes remains poorly understood. To address

this doubt, several large scale clinical trials using different DPP-4 inhibitors are now

underway. Together, these large scale clinical trials could improve our understanding of

the potential effects of these glucose-lowering drugs on cardiovascular events in diabetes.

64

1.6 Hypothesis

The broad aim of the thesis is to investigate the pathological changes associated

with vascular dysfunction in the acute presence of high glucose or in small arteries from

diabetic rats or from high fat western diet (WD) fed rats. Based on our understanding of

the biological action and pharmacological activity of DPP-4 inhibitors, as discussed above,

we hypothesize that DPP-4 inhibitors may improve vascular endothelial function in

diabetes or western diet via GLP-1 dependent and/or independent pathways.

1.6.1 Specific aims of the project

(1) This project aims to investigate whether high glucose increases ROS production

and causes endothelial dysfunction after incubation of 2 hours. Further to investigate

whether the acute presence in vitro of DPP-4 inhibitors (linagliptin, sitagliptin,

vildagliptin) or the GLP-1R agonist, exendin-4 prevents the impairment of endothelial

function in small mesenteric arteries exposed to 40 mM glucose and to compare their

antioxidant effects and also to investigate the effects of linagliptin on the relative

contribution of NO-mediated and EDH-mediated relaxation to endothelium-dependent

responses in vessels exposed to high glucose.

(2) It is well documented that STZ-induced hyperglycaemia causes increases in

oxidative stress and impairment of endothelial function after a duration of 10 weeks. This

project aimed to determine the acute effects in vitro of the DPP-4 inhibitor (linagliptin) on

the mechanism(s) of endothelium-dependent relaxation in small mesenteric arteries from

STZ-induced type 1 diabetic and normal rats, where any benefit will occur independently

of any change in glucose levels where it is not possible for glucose concentration to

influence the action of linagliptin.

65

(3) It is well established that small arteries and arterioles are primarily involved in

determination of circulatory resistance and tissue perfusion. However, few if any studies

have explored the effect of WD on the change of microvascular function. This project

aimed to examine the effects of the acute presence of the DPP-4 inhibitor (linagliptin) in

vitro on the mechanism(s) of endothelium-dependent relaxation in small mesenteric

arteries from rats fed a high fat WD. Additionally, to explore the beneficial effects of

linagliptin on the contribution of NO and/or EDH-mediated relaxation in small resistance

arteries from WD fed rats.

(4) Based on the findings from the earlier studies, it was show that high glucose,

STZ-induced diabetes and western high fat diet causes endothelial dysfunction due to

impairment of the contribution of both NO-mediated and EDH-type relaxation, this was

associated with increased activity of NADPH-oxidase-derived superoxide production and

eNOS uncoupling in the mesenteric artery. Also based on the results from (1), (2), (3), it

was demonstrated that acute treatment with linagliptin in vitro reduced oxidative stress and

improved endothelial function in small mesenteric artery. The project aimed to investigate

the effects of chronic treatment in vivo with the DPP-4 inhibitor (linagliptin) on the

mechanism(s) of endothelium-dependent relaxation in small mesenteric arteries from STZ-

induced diabetic and normal rats, and if so, whether it acts via direct scavenging of ROS

and/or by inhibiting the sources of ROS production in the diabetic microvasculature, where

any benefit will occur independently of changes in glucose levels in STZ-induced type 1

diabetes. .

66

Chapter 2

General Methods

2.1 Animal experiments

Male Wistar rats were purchased from Animal Resource Centre (Perth, WA,

Australia). All the experimental animals were kept in the Research Animal Facility at

RMIT University. Animal welfare conditions were taken in account where all animal were

kept under controlled environments of illumination (12 h light/12 h darkness) and

temperature (20-25C). All animals received free access to food (standard pellet diet) and

water ad libitum. All the procedures were approved by the Animal Experimentation Ethics

committee of RMIT University and complied with the Australian National Health and

Medical Research Council code of practice for the care and use the animal for scientific

purposes (AEC approval numbers 1121 or 1309).

2.1.1 Induction of diabetes

Streptozotocin (STZ) which causes pancreatic islet cell destruction was used to

reduce insulin release thus generating an experimental model of type 1 diabetes. Sterile 0.1 M

citrate buffer at pH 4.5 was used to dissolve STZ and prepared to a stock concentration of 100

mg/ml. A single-dose of STZ was used to induce type 1 diabetes at 6-8 weeks of age (~200

g) after overnight fasting. The rats were temporarily exposed to heat lamps (~3-5 min) to

raise blood flow to the tail vein directly prior to the injection. To restrain the movement of

the rats, they were wrapped in a towel with the tail exposed for injection. Ethanol (70%)

was used to clean the tail to reduce the risk of infection and about 0.1 ml of STZ (50

67

mg/kg of body weight) was injected into the tail vein (26G/27G needle). Age-matched rats

used as a control group were injected with the same volume of vehicle (0.1 M citrate

buffer). After the injection of the STZ-treated and vehicle, the two groups were housed

separately and received free access to food and water.

2.1.2 Blood glucose analysis

One week after STZ injection, rats blood glucose levels were measured using a

glucometer (Accu-chek Advantage, Roche, U.S.A). Briefly, the rats were wrapped by

towel allowing access to the tail which was pre-heated under a lamp. Ethanol (70%) was

used to clean the tail to diminish the risk of infection. Applying a 26G needle, the tail vein

was lanced to let bleeding occur and one drop of blood was collected onto a blood glucose

strip fitted to the blood glucometer. The rats were considered diabetic if the blood

concentration was higher than 20 mM. The rats received a low dose of insulin (4-5 IU,

Protaphane, Novo Nordisk, NSW, Australia) to keep glucose <30 mM and to promote

weight gain and reduce mortality, without achieving euglycaemia. All high glucose

animals received insulin treatment on Mondays, Wednesday, and Fridays in the evening

(4-6 pm). The blood glucose for these rats was monitored weekly on Friday mornings (10-

11 am). At the time of tissue collection ie the end of experimental period, blood samples

were collected from the left ventricle using a heparinized 18G needle and 3 ml syringes,

and the glucose concentration was determined using the same procedure.

2.1.3 Estimation of glycated haemoglobin

Left ventricular blood was also transferred into vacuum blood collection tubes to

be used for subsequent analysis of glycated haemoglobin (HbA1c). The blood was diluted

68

1:1 with milliQ water in an eppendorf tube. HbA1C was measured using a Micromat

HbA1c analyser (Biorad, Sydney, NSW, Australia) following the manufacturer instruction.

2.2 Isolation of vascular tissue

At the time point of tissue collection with the end of experimental period, the rats

were killed by asphyxiation with CO2 followed by exsanguination. The mesenteric arteries

were immediately placed in ice-cold Krebs bicarbonate solution (4.7 mM KCl, 118 mM

NaCl, 1.18 mM MgSO4, 25 mM NaHCO3, 11.1 mM D-glucose and 1.6 mM CaCl2), which

were used for additional functional experiments and ROS measurement. The remaining

arteries were snap frozen in liquid nitrogen and stored at -80°C for subsequent western blot

analysis.

2.2.1 Preparation of mesenteric arteries

After the tissue collection, the entire tissue mass of mesenteric arteries was moved

onto a large sylgard coated Petri dish, having ice-cold Krebs biocarbonate solution. The

whole tissue of mesenteric arteries was pinned to the sylgard Petri dish using 26G needles

in order to expose the branches of mesenteric arteries. Small mesenteric arteries (the third

order branch of the superior mesenteric artery, internal diameter ~300 µm) were isolated,

cleared of fat and connective tissue using a dissection microscope and cut into small rings

of about 2 mm in length. Stainless steel wire (40-µm) in diameter was used and inserted

through the lumen of the mesenteric artery in a gentle approach and great care was taken in

inserting the wires to avoid any damage of the lumen of the artery (endothelium). The

rings of the mesenteric arteries after inserting of the wires were then mounted on a

Mulvany-Halpern myograph (model 610M, Danish Myo Technology, Aarhus, Denmark)

by fixing the wire onto the jaws of the myograph to the isometric force transducer side.

69

The first wire was used a guide to locate the lumen of the arteries, the second wire was

inserted in the same manner with great care and fixed to the jaws of the myography to the

micrometer side. The micrometer reading was increased thus that both the wires in the

lumen of the artery were slightly separated and were in close contact with the wall of the

artery (Figure 2.1). After the rings of the mesenteric arteries were mounted and fixed to the

myography, the vessels were allowed to stabilize at zero tension for around 15 min before

normalization. The standard optimization method was used to set the optimal value of

resting tension for each ring of the mesenteric arteries and was measured by applying the

resting tension-internal circumference relationship (Mulvany and Halpern, 1977;

McPherson, 1992). In summary, the rings of mesenteric arteries were stretched in slight

boosts (2 mN) with equilibration period was allowed between stretches. The passive

tension in the rings was recorded and the vessels stretched till the passive tension was

above 90 mm Hg (mean arterial blood pressure in rats). The rings of the mesenteric

arteries were then fixed to an internal circumference equal to 90% of the calculated

circumference at a passive transmural pressure of 13.3 kPa for the rest of experiment, the

maximum active wall tension is achieved as at this level of stretching is reached.

Therefore, the stretching of the vessels determine the passive tension-internal

circumference and achieves an internal circumference equal to 90% of that of the entire

blood vessels under transmural pressure of 100 mmHg. All the experiments were made in

equal criteria and at 37°C and bubbled with carbogen (95% O2 and 5% CO2). After the

normalization was performed, the mesenteric rings were fixed at the optimal tension for 30

minutes to stabilize and recorded on a chart recorder (model 3721; Yokogawa, Tokyo,

Japan).

70

Mulvany-Halpern myograph

Figure 2.1 Diagrammatic representation of the Mulvany-Halpern myography chamber

used to mount small mesenteric arteries. The ring segment of mesenteric artery was

mounted on jaws, one wire fixed to the force transducer and the other wire to the

micrometer in chamber of 5 ml volume containing Krebs buffer which continually aerated

with 5% CO2/ 95% O2.

71

2.3 Vascular functional experiments

After the process of stabilization was finished, the vessels were subjected to a

maximum contraction with an isotonic high K+-containing physiological saline solution

(KPSS), in which the Na+ ions were replaced by K

+ ions ([K

+]KPSS=123 mM). The tension

was resumed to basal tension, after a series of washouts with normal Krebs solution. In

order to evaluate the integrity of the endothelium, the arteries were precontracted with

phenylephrine (PE, 0.01-1 μM) to a level equal to (approximately 50%) of the KPSS

response and then relaxed with a high concentration of ACh (10 µM). If the relaxation

induced by ACh was larger than 80% of the precontraction, the endothelium was

considered intact and functionally fit to run the experiments. Mesenteric arteries with

endothelium that was not intact were excluded from the experimental studies.

2.3.1 Mesenteric arteries relaxation and contraction assays

After several washouts with normal Krebs, the mesenteric arteries were

precontracted again with PE (0.01-1 µM) to 50% of the KPSS response. Relaxant

responses to cumulative concentration additions of ACh (0.1 nM-10 µM) and sodium

nitroprusside (SNP, 0.01 nM-10 μM) were determined. All experiments were conducted in

the presence of indomethacin (10 µM) to inhibit the production of prostanoids and to block

any contribution of prostanoids to the vascular relaxation.Furthermore, cumulative

concentration-response curves to ACh and SNP were investigated subsequent to

incubation with pharmacological inhibitors (Table 2.1). The different combination of the

inhibitors is discussed in detail in each chapter. An example of a trace of the experimental

protocol is illustrated in figure 2.2.

72

Figure 2.2 A trace of the vascular relaxation protocol. Maximum contraction of the

mesenteric arteries was achieved with 123 mM KPSS. PE was used (black dots) to

precontract arteries and the endothelium was considered intact when the maximum

relaxation exceeded 80% relaxation. The cumulative concentration response curve to ACh

(10-10

-10-5

M) or SNP (10-11

-10-5

M) (red dots) were evaluated either in the absence or

presence of pharmacological inhibitors (Table 2.1).

73

Table 2.1 List of drugs used in vascular functional experiments

Drug Inhibitory activity Concentration

Indomethacin COX 10 µM

N-nitro-L-arginine (L-NNA) NOS 100 μM

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

(ODQ)

sGC 10 μM

1-[(2 chlorophenyl)(diphenyl)methyl]1H pyrazole

(TRAM-34)

IKCa 1 µM

Apamin SKCa 1 µM

Iberiotoxin (Ibtx) BKCa 0.1 µM

Tempol SOD mimetic 100 µM

Pyrogallol Superoxide

generator

30 µM

Linagliptin DPP-4 inhibitor 1 µM

Sitagliptin DPP-4 inhibitor 1 µM

Vildagliptin DPP-4 inhibitor 1 µM

Exendin-4 GLP-1R agonist 1nM-1µM

Exendin-Fragment (9-39) GLP-1R antagonist 1 µM

74

2.3.2 Estimation of basal NO level assay

After the endothelium was investigated and considered to be intact, the vessels

were precontracted again with PE (10-100 nM) to approximately 20% KPSS. The addition

of the NOS inhibitor, L-NNA (100 µM), would induce additional contraction to the

vessels. Due to removal of the basal level of NO that was acting against PE contraction.

The basal level of NO was assessed by the extent of L-NNA-induced contraction (Figure

2.3).

75

Figure 2.3 Pattern of the protocol to assess basal NO activity. The mesenteric arteries

reached maximum contraction induced by 123 mM K+. ACh (10

-5 M) was added to induce

relaxation and if the relaxation was greater than 80%, the endothelium was considered

intact. The addition of the NOS inhibitor, L-NNA (100 μM) to the arteries precontracted to

approximately 20% of KPSS, produced further contraction of the vessels and was used as

an indicator of the basal activity of NO.

76

2.4 Estimation of reactive oxygen species

The level of ROS was measured by the estimation of the superoxide production by

L-012 or lucigenin-enhanced chemiluminescence assays.

2.4.1 L-012 assay

L-012 is a chemiluminescent probe used to measure superoxide levels. L-012

reacts with superoxide, to produce photons which could be quantified by a photon counter.

The rings of rat mesenteric arteries were incubated in high glucose for 2 h and then

incubated at 37°C for 30 minutes in Krebs-HEPES buffer (composition (mM): NaCl 99.90,

KCl 4.7, KH2PO4 1.0, MgSO4·7H2O 1.2, D-glucose 11.0, NaHCO3 25.0, CaCl2·2H2O 2.5,

Na HEPES 20.0, pH 7.4) either alone, in the presence of tempol, a cell permeable SOD

mimetic (100 µM), linagliptin, DPP-4 inhibitor (1 µM), sitagliptin, DPP-4 inhibitor (1

µM), vildagliptin, DPP-4 inhibitor (1 µM), exendin-4, GLP-1 agonist (1 µM), and

exendin-fragment (9-39), (1 µM). 300 µL of Krebs-HEPES buffer, containing L-012 (100

µM, Wako Pure Chemicals, Osaka, Japan) and the appropriate treatments were placed into

a 96-well Optiplate, which was loaded into a Polarstar Optima photon counter (BMG

Labtech, Melbourne, VIC, Australia) to measure background photon emission at 37°C.

After background counting was completed, a single ring segment of mesenteric artery was

added to each well and photon emission was re-counted. Background counts were

subtracted from superoxide counts and normalized with dry tissue weight.

2.4.2 Lucigenin assay

The superior mesenteric artery was isolated, cleared of fat and connective tissue

and incubated in Krebs solution. The arteries were cut into small rings (2- to 3- mm-long

77

segments) in Krebs-HEPES buffer. The mesenteric arteries were incubated for 45 min at

37ºC in Krebs-HEPES buffer either alone or in the presence of NADPH (100 μM) as a

substrate for NADPH oxidase, diphenyliodonium (DPI, 5 µM), a flavoprotein inhibitor

that inhibits NADPH oxidase, and linagliptin (1 µM). 300 µl of Krebs-HEPES buffer,

containing lucigenin (5 µM) and the suitable treatment were placed into a 96-well

Optiplate. The Optiplate was loaded into a Polastar Optima photon counter (BMG

Labtech, Melbourne, VIC, Australia) to measure background photon emission at 37ºC.

After the background reading was recorded, a single ring section of mesenteric artery was

placed into each well and photon emission was recounted to measure the production of

NADPH oxidase- driven superoxide.

2.5 Western Blot

2.5.1 Extraction of protein from tissue samples

The mesenteric arteries from 2-3 rats of the same treatment groups were pooled,

snap frozen and considered as n=1. Using a 1 ml glass homogenizer, the mesenteric

arteries (~ 200 mg) were homogenized in lysis buffer solution consisting of (100 mM

NaCl, 10 mM Tris, 2 mM EDTA, 0.5% w/v sodium deoxycholate, 1% vol/vol triton X-

100, pH7.4, protease and phosphatase inhibitor cocktails (Roche, Sydney, NSW,Australia).

After 20 min of incubation with lysis buffer at 4ºC, the mesenteric arteries tissues were

homogenized and tissue samples were centrifuged for 10 min at 4ºC and 13,200 RPM. The

supernatant of tissue samples were collected and kept at -80ºC to be used when required.

2.5.2 Bradford protein assay

The Bradford assay was used to measure the protein concentration of the tissue

samples. Phosphate buffered saline (PBS) (100 μL) in a test tube was used to dilute the

78

tissue samples 1:100 and 100 μL of 0.2 M NaOH was added to the test tube. After an

incubation period of 15 minutes, milliQ water (600 μL) and 200 μL of red protein assay

reagent dye (Biorad, Sydney, NSW, Australia), were added to the test tube. The solution

which turned blue with the addition of protein was vortexed and 300 μL of the solution

was loaded to a 96-well plate and in a plate reader the absorbance (590 nM) of the samples

was measure. In addition, in the same 96-well plate, a bovine serum albumin (BSA)

standard curve was generated (0-20 μg/mL). Each of the standard and the protein samples

were applied in duplicate and if the absorbance of the tested samples were not compatible

with the range of absorbance in the standard curve, the unknown samples were further

diluted and the procedures were repeated as defined above. The standard curve was used to

calculate the protein concentration of the protein in the unknown samples. The amount of

protein in each sample was equal (30 µg) and the protein samples were aliquoted and PBS

was used to top up the samples so they contained identical amount of protein (30 μg ) in

the same total volume (10 μL or 15 μL) of 2X Sample buffer (20% w/v glycerol, 2% SDS,

62.5 mM Tris, 0.05% bromophenol blue and 5% -mercaptoethanol, pH 6.8) and was added

to each of the samples and stored at -80°C until required.

2.5.3 Gel preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE)

According to the manufacturers’ instructions, the plates were collected (Biorad,

Sydney, NSW, Australia). The 7.5%, 10%, 12% and 15% resolving gel (30% acrylamide,

distilled water, 1.5 M Tris, pH 8.8, 10% SDS, 10% ammonium persulfate and TEMED)

was arranged and moved into the glass plates by pipette; isopropyl alcohol (~100 μL) was

added to remove bubbles. Acrylamide-resolving gel was formed after 1-1.5 hours at room

temperature by the polymerization of the solution. The alcohol was removed once the

79

resolving gel had set by dabbing using filter paper and 4% stacking solution (30%

acrylamide, distilled water, 0.5 M Tris, pH 6.8, 10% SDS, 10% ammonium persulfate and

TEMED) was added on to the top of the resolving gel, the 15-well comb was inserted, and

the gel was left to polymerize at room temperature for 0.5-1 hour.

2.5.4 Preparation of SDS-PAGE

The method of SDS-PAGE was carried out by a mini-PROTEAN device (Biorad,

Sydney, NSW, Australia). Denaturation of protein samples (30 µg) in sample buffer was

completed by heating the samples at 95°C for 5 minutes. The prestained kaleidoscope

protein ladder (7 μL, Biorad, Sydney, NSW, Australia) and the denatured protein were

loaded into wells. The running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3)

was used to perform the electrophoresis at 100 V until the separation was complete (2

hours). Subsequent to the process of electrophoresis the gels were carefully detached from

the gasket and equilibrated in ice-cold transfer buffer (25 mM Tris, 192 mM glycine, 20%

methanol, 0.037% SDS, pH 8.3) which works to eliminate additional salt and detergents

from the running buffer and leads to increase the conductivity of the transfer buffer and

reduce the amount of heat produced during the transfer. In the same transfer buffer, the

Hybond nitrocellulose membranes, filter papers and sponges were also equilibrated.

According to the manual for Biorad wet transfer cell, the sponges, filter papers,

nitrocellulose membranes and gels were assembled, and gels were transferred at 350 mA

for 2-3 hours. At the end of the transfer process, ponceau stain was used to confirm the

transfer of protein was successful.

80

2.5.5 Performance of low temperature SDS-PAGE

To investigate the expression of eNOS monomers and dimers, we used a low

temperature SDS-PAGE technique. As described in section 2.6.2, a 6% resolving gel was

prepared. In low temperature SDS-PAGE, an ice cold resolving and stacking gel were

used. Non-denatured protein sample (60 μg) and protein ladder (7 μL) were loaded into the

wells of 6% resolving gel and ice cold running buffer used to perform electrophoresis at 30

V overnight at 4°C. After the end of the electrophoresis stage, the proteins were transferred

from the gel to the nitrocellulose membrane at 30 V overnight at 4°C. Likewise, the

membrane was stained with ponceau stain to confirm the transferred of protein was

successfully performed.

2.5.6 Procedures of immunoblotting

To perform immunoblotting of eNOS, Nox2 and caveolin-1 protein levels (all BD

Transduction Laboratories, Lexington, KY, USA), calmodulin (Millipore, Billerica, MA,

USA), either by 5% w/v skim milk in Tris Buffered Saline plus 0.1% Tween-20 (TBST)

for 1 hour at room temperature or 5% BSA/TBST (phosphorylated proteins) for 1 hour at

4°C were used in order to block the nitro-cellulose membranes before the addition of the

primary antibodies (in 5 mL 3% BSA/TBST). Subsequently, the nitro-cellulose

membranes were incubated with primary antibodies (1:1000) overnight at 4°C. After that

the membranes were washed (3 x 10 minute washes with 10 mL TBST), and then

incubated with secondary antibody (sheep anti-mouse) (Millipore, Billerica, MA, USA) (in

5 mL, 5% skim milk/TBST) (1:2000) at room temperature for 1 hour. After the incubation,

the membranes were washed with 10 mL TBST (3 x 10 minute washes). The detection of

the secondary antibody which conjugated with horseradish peroxidase was performed after

the incubation of the membrane with either enhanced chemiluminescence reagents

81

(Amersham, GE Healthcare, Sydney, NSW, Australia) or Supersignal West Femto

(Thermo Scientific, Rockford, IL, USA) for 1 minute, and the detection of the

chemiluminescence signals on the membrane were achieved by digital image scanner

(Biorad Chemidoc). Densitometry used to quantify the detected protein bands. After

blocking non nonspecific binding with 5% w/v skim milk, nitro-cellulose membranes were

re-investigated with a loading control antibody (actin), diluted 1:1000 in 5 mL 1%

BSA/TBST. Followed by 3 x 10 minute washes of the membranes with 10 mL TBST

TBST, and once more explored with secondary antibody (Millipore, Billerica, MA, USA)

diluted 1:2000 in 5 mL 5% skim milk/TBST for 1 hour, and the bands were imagined and

qualified as described previously.

2.6 Chemicals and Reagents

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except

for linagliptin (Boehringer Ingelheim, Germany), acetylcholine perchlorate (BDH

Chemicals, Poole, Dorset, UK), and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All

chemicals were dissolved in distilled water, with the exception of L-NNA, which was

dissolved in 0.1 M sodium bicarbonate, indomethacin, which was dissolved in 0.1 M

sodium carbonate, ODQ, and TRAM-34 which was dissolved in dimethyl sulfoxide

(DMSO). Linagliptin, vildagliptin, sitagliptin, exendin-4, and exendin-fragment (9-39)

which were dissolved in distilled water.

2.7 Statistical analysis

All results are presented as the mean ± SEM, n indicates the number of

experiments. The concentration response data from rat isolated mesenteric arteries were

fitted to a sigmoidal curve using nonlinear regression (Prism version 6.0, GraphPad

82

Software, San Diego, CA, USA) to calculate the pEC50. The maximum relaxation (Rmax) to

ACh or SNP was determined as a percentage of the phenylpherine pre-contraction. pEC50

and Rmax values were compared and analysed using one way ANOVA or two way

ANOVA with post hoc analysis using Bonferroni’s test or Student’s unpaired t-test as

appropriate. p<0.05 was considered statistically significant.

83

Chapter 3

The DPP-4 inhibitor linagliptin and the GLP-

1 receptor agonist exendin-4 improve

endothelium-dependent relaxation of rat

mesenteric arteries in the presence of high

glucose

3.1 Introduction

Vascular disease is the main cause of morbidity and mortality in diabetes and

impairment of endothelial function is considered to be a principle factor in the

development of vascular disease (Grundy et al., 1999, De Vriese et al., 2000).

Hyperglycaemia is thought to be a critical cause of endothelial dysfunction resulting in

reduced NO release, generation of oxygen free radicals and impairment of endothelium-

dependent relaxation (Calles-Escandon and Cipolla, 2001, Rodriguez-Manas et al., 2003).

Acute exposure to high glucose reduces the sensitivity to acetylcholine-induced

endothelium-dependent relaxation (Guo et al., 2000) with a reduced NO release (Reyes-

Toso et al., 2002) associated with an increase in reactive oxygen species (Guo et al.,

2000).

Incretin-based therapies, including inhibitors of dipeptidyl peptidase-4 (DPP-4) and

glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are effective in the treatment

of type 2 diabetes (T2DM) by increasing insulin secretion (Ussher and Drucker, 2014,

Drucker and Nauck, 2006). The incretin hormones, GLP-1 and glucose-dependent

insulinotropic polypeptide (GIP), are released by intestinal endocrine cells in response to a

84

meal (Kim and Egan, 2008). These hormones have a key role in the modulation of

pancreatic islet insulin release and, thus, of glucose homeostasis (Kim and Egan, 2008).

Drugs that inhibit the effect of DPP-4, which cleaves both GLP-1 and GIP, extend the half-

lives of these hormones and increase islet function and glycaemic regulation in T2DM

(Pratley and Salsali, 2007, Ahren et al., 2004).

In addition to their ability to lower glucose there is increasing evidence that DPP-4

inhibitors or GLP-1R agonists improve endothelial function in T2DM patients (J Motta et

al., 2012, Shah et al., 2011b) and in type 1 diabetic rats (Özyazgan et al., 2005). It has

been reported that GLP-1 improves endothelial function and exerts protective actions in

type-2 diabetic patients with coronary artery disease (Nystrom et al., 2004) and it has been

reported that linagliptin and sitagliptin improve endothelium-dependent relaxation in type

2 diabetic rats (Takai et al., 2014)

Diabetes is associated with overproduction of reactive oxygen species (ROS)

which is in turn associated with impaired NO release and/or activity causing endothelial

dysfunction (De Vriese et al., 2000, Leo et al., 2011b). There is growing evidence that

GLP-1R agonists and DPP-4 inhibitors may improve endothelial function. GLP-1R

agonists are reported to increase eNOS activity in isolated endothelial cells (Hattori et al.,

2010, Erdogdu et al., 2010) and to enhance endothelium-dependent dilatation in human

subjects (Basu et al., 2007). Similarly DPP-4 inhibitors increase eNOS activity (Shah et

al., 2011b) to increase eNOS expression in zucker obese rats (Aroor et al., 2013) and to

improve endothelium-dependent relaxation in spontaneously hypertensive rats (Liu et al.,

2012). By contrast, there is also a report that sitagliptin and alogliptin impair endothelium-

dependent dilatation in patients with type 2 diabetes (Ayaori et al., 2013). A contributing

factor to improved endothelial function may result from antioxidant effects of both GLP-

1R agonists and DPP-4 inhibitors (Kroller-Schon et al., 2012, Ávila et al., 2013, Erdogdu

85

et al., 2013, Shiraki et al., 2012, Ishibashi et al., 2010, Hendarto et al., 2012) which could

decrease NO inactivation by ROS.

As high glucose induced impairment of endothelium-dependent relaxation in

mesenteric arteries is associated with oxidative stress, thus, the aim of this study was to

investigate whether the acute presence of DPP-4 inhibitors (linagliptin, sitagliptin,

vildagliptin) or GLP-1R agonist, exendin-4 prevents the impairment of endothelial

function in small mesenteric arteries exposed to 40 mM glucose and compare the

antioxidant effects of linagliptin with those of sitagliptin, vildagiptin, and exendin-4 in

vitro. Furthermore, we sought to investigate the effects of linagliptin on the relative

contribution of NO-mediated and EDH-mediated relaxation to endothelium-dependent

responses in vessels exposed to high glucose. The interest in linagliptin was due to results

obtained in earlier part of study where linagliptin, but not the other DPP-4 inhibitors

reduced superoxide production in mesenteric arteries exposed to high glucose solution.

86

3.2 Methods

All procedures were approved by the Animal Experimentation Ethics committee of

RMIT University and complied with the Australian National Health and Medical Research

Council code of practice for the care and use of animals for scientific purposes (AEC

approval number 1121).

3.2.1 Isolation of mesenteric arteries

Male Wistar rats (~8-weeks of age, ~250g) were used. The mesenteric arcade was

isolated after rats were killed by asphyxiation with CO2 followed by exsanguination. The

mesenteric arteries were immediately placed in ice-cold Krebs bicarbonate solution (4.7

mM KCl, 118 mM NaCl, 1.18 mM MgSO4, 25 mM NaHCO3, 11.1 mM D-glucose and 1.6

mM CaCl2) containing the cyclooxygenase (COX) inhibitor indomethacin (10 µM), to

inhibit the production of prostanoids. The third order branch of the superior mesenteric

artery (internal diameter ~300 µm) was isolated, cleared of fat and connective tissue and

cut into small rings (~2 mm). The arteries were mounted on a Mulvany-Halpern myograph

(Danish Myo Technology, Aarhus, Denmark). The artery rings were stabilized at zero

tension for 15 min in Krebs solution bubbled with 95% O2/5% CO2 at 37ºC. Passive

tension of the blood vessels was identified through stretching to obtain an internal

circumference comparable to 90% of that in blood vessels under transmural pressure of

100 mmHg (Mulvany and Halpern, 1977).

3.2.2 Vascular experiments

The maximum contraction of the mesenteric arteries was assessed by addition of an

isotonic physiological saline solution containing a high concentration of K+

(123 mM,

87

KPSS). The basal tension was restored after 3-4 washes using Krebs solution. The

integrity of the endothelium was examined by precontraction of the mesenteric arteries

with phenylephrine (0.1-3 µM) to ~50% of the KPSS response and relaxed with a high

concentration of acetylcholine (ACh, 10 µM). ACh-induced relaxation of the precontracted

rings was more than 80% in all cases indicating that the endothelium was functionally

intact. The mesenteric arteries were incubated for 2 hours with 11 mM glucose or 40 mM

glucose and again precontracted with phenylephrine (0.1-3 µM) to ~50% of the KPSS

response. The high concentration of glucose (40 mM) was based on a previous reports

demonstrating that short incubations (4-6 h) with a similar concentration (44 mM)

impaired endothelial function in isolated blood vessels (Tesfamariam et al., 1991, Qian et

al., 2006). Cumulative concentration-response curves to the endothelium-dependent

relaxant, ACh (0.1 nM-10 µM) and the endothelium-independent relaxant, sodium

nitroprusside (SNP, 0.01 nM-10 µm) were determined after 20 min incubation with

linagliptin (1 µM), sitagliptin (1 µM), vildagliptin (1 µM) or exendin-4 (1 µM). The

concentration of linagliptin was based on the observations of Kroller-Schon et al. (Kroller-

Schon et al., 2012) that 1 µM was the lowest concentration to inhibit the respiratory burst

in polymorphonuclear leucocytes and in preliminary experiments we found that this

concentration had significant antioxidant activity and the same concentration was chosen

for other compounds. Exendin-4 was found to be equally effective to improve endothelial

function at a concentration of 1 µM (supplementary Figure 1). Relaxant responses were

also assessed in the presence of different combinations of N-nitro-L-arginine (L-NNA, 100

μM), an inhibitor of nitric oxide synthase (NOS), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-

1-one (ODQ, 10 µM), an inhibitor of soluble guanylate cyclase (sGC), apamin (1 µM), a

selective blocker of the small conductance calcium-activated K+

channel (SKCa), 1-[(2-

chlorophenyl) (diphenyl)methyl]-1H-pyrazole (TRAM-34, 1 µM), a blocker of

88

intermediate conductance calcium-activated K+

channel (IKCa), iberiotoxin (Ibtx, 100 nM),

a blocker of the large conductance calcium-activated K+

channel (BKCa), superoxide

dismutase (SOD, 50 U/mL) used to reduce superoxide, and pyrogallol (30 µM) used to

generate superoxide.

3.2.3 Superoxide measurement in mesenteric artery

Superoxide production in the mesenteric artery was measured by using L-012 and

lucigenin enhanced-chemiluminescence assays. Segments of mesenteric arteries were

incubated in 40 mM glucose for 2 h and then incubated at 37°C for 30 minutes in Krebs-

HEPES buffer (composition (mM): NaCl 99.90, KCl 4.7, KH2PO4 1.0, MgSO4.7H2O 1.2,

D-glucose 11.0, NaHCO3 25.0, CaCl2.2H2O 2.5, Na HEPES 20.0, pH 7.4) either alone or

in the presence of tempol, a SOD mimetic (100 µM) or linagliptin (1 µM). 300 µL of

Krebs-HEPES buffer, containing L-012 (100 µM, Wako Pure Chemicals, Osaka, Japan)

and the appropriate treatments were placed into a 96-well Optiplate, which was loaded into

a photon counter (Polarstar, BMG Labtech, Melbourne, VIC, Australia) to measure

background photon emission at 37°C. After background counting was completed, a single

ring segment of mesenteric artery was added to each well and photon emission was re-

counted. Background counts were subtracted from superoxide counts and normalized with

dry tissue weight. Artery-derived superoxide was also measured using lucigenin as

previously described (Leo et al., 2011b)

3.2.4 Reagents

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except

for linagliptin (Boehringer Ingelheim, Germany), acetylcholine perchlorate (BDH

89

Chemicals, Poole, Dorset, UK), and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All

chemicals were dissolved in distilled water with the exception of L-NNA, which was

dissolved in 0.1 M sodium bicarbonate, and indomethacin, which was dissolved in 0.1 M

sodium carbonate.

3.2.5 Statistical analysis

Results are presented as the mean±SEM, n indicates the number of experiments.

The concentration response data from isolated mesenteric arteries were fitted to a

sigmoidal curve using nonlinear regression (Prism version 6.0, GraphPad Software, San

Diego, CA, USA) to calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP

was determined as a percentage of the phenylephrine precontraction. pEC50 and Rmax

values were compared using one way ANOVA with post hoc analysis using Bonferroni’s

test. p<0.05 was considered statistically significant.

90

3.3 Results

3.3.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide production

Using L-012 and lucigenin chemiluminescence assays, the level of superoxide in

mesenteric arteries incubated in 40 mM glucose was demonstrated to be significantly

higher than arteries incubated in 11 mM glucose (Figure 3.1). Linagliptin (1 µM)

significantly reduced superoxide production in the presence of normal and high glucose

whereas exendin-4 (1 µM) only reduced ROS levels in the presence of high glucose

(Figure 3.1a, b). In contrast, sitagliptin (1 µM) and vildagliptin (1 µM) had no significant

effect on the superoxide levels in the mesenteric rings under either condition (Figure 3.1a).

The ability of linagliptin to reduce superoxide from arteries exposed to normal or high

glucose was also confirmed using lucigenin enhanced chemiluminescence (Figure 3.1b).

91

a)

b)

Figure 3.1 ROS measurement in mesenteric arteries using L-012 (a) and lucigenin (b) enhanced-

chemiluminesence assays. Superoxide levels were increased in mesenteric rings exposed to 40 mM

glucose. Superoxide levels detected in the presence of normal (11 mM) glucose were significantly

reduced by the SOD mimetic tempol (100 μM) or DPI (5 µM), a flavoprotein inhibitor that inhibits

NADPH oxidase and linagliptin (1 µM) but not by any of the other treatments. The elevation of

superoxide caused by high glucose was significantly attenuated by exendin-4 (1 µM) and

linagliptin but not by the other DPP-4 inhibitors. Results are shown as mean±SEM. n=6

experiments. *p<0.05 vs 11 mM glucose untreated, #

p<0.05 vs 40 mM glucose untreated,

Bonferroni’s test.

92

3.3.2 Effect of high glucose, DPP-4 inhibitors, and exendin-4 on endothelium-

dependent relaxation

High glucose (40 mM) significantly reduced the sensitivity and maximum

relaxation to ACh, in the mesenteric arteries (Figure 3.2a, Table 3.3). The sensitivity and

maximum relaxation to ACh in mesenteric arteries exposed to mannitol (29 mM

mannitol+11 mM glucose) was similar to that determined in mesenteric arteries exposed to

11 mM glucose, indicating that the high glucose-induced impairment of the endothelium-

dependent relaxation was not due to a hyperosmotic effect (Figure 3.3). SNP responses

were unaffected by the high glucose (Figure 3.2b). Linagliptin (1 µM) significantly

increased the sensitivity to ACh and the maximum relaxation in arteries exposed to 40 mM

glucose (Figure 3.4, Table 3.1). The other DPP-4 inhibitors, sitagliptin (1 µM) and

vildagliptin (1 µM), showed a significant increase in the maximum relaxation, but no

change in the sensitivity to ACh-induced relaxation of mesenteric arteries exposed to 40

mM glucose whereas the GLP-1R agonist exendin-4 (1 µM), also caused a significant

increase in the maximum relaxation and the sensitivity to ACh in the presence of high

glucose (Figure 3.4, Table 3.1). To investigate the role of the GLP-1 receptor in the

beneficial effects of linagliptin and exendin-4, responses to ACh were tested during co-

incubation with the GLP-1 receptor antagonist exendin-fragment (9-39) (1 µM). The

presence of exendin-fragment (9-39) had no effect on ACh-induced relaxation of

mesenteric arteries exposed to 11 or 40 mM glucose (Table 3.2). In the presence of high

glucose exendin-fragment (9-39) prevented the ability of exendin-4 to improve ACh-

induced relaxation (Figure 3.5B) but did not affect the capacity of linagliptin to improve

the endothelium-dependent relaxation (Figure 3.5A, Table 3.2).

93

Figure 3.2 Vascular function in mesenteric arteries. Cumulative concentration-response curves to

ACh (a) and SNP (b) in endothelium- undamaged mesenteric arteries. In each group of

experiments (a, b), mesenteric arteries were precontracted with PE to similar levels: (a) 11

mM glucose 65±3, 11 mM glucose+linagliptin 63±2, 40 mM glucose 61±3, 40 mM

glucose+linagliptin 60±1, (b) 11 mM glucose 60±2, 11 mM glucose+linagliptin 61±3, 40

mM glucose 62±3, 40 mM glucose+linagliptin 60±4 %KPSS, n=5-11 experiments. Results

are shown as mean±SEM. *pEC50 significantly different from 40 mM glucose

(Bonferroni’s test, p< 0.05).

94

Figure 3.3 The addition of mannitol (29 mM) to glucose (11 mM) had no effect on the

endothelium-dependent relaxation indicating that the glucose-induced dysfunction was not due to

hyperosmosis. *pEC50 significantly different from 11 mM glucose and Mannitol (Bonferroni’s test,

p<0.05), #pEC50 signifiacantly different from 40 mM glucose (Bonferroni’s test, p<0.05), n=6-9.

Results are shown as mean±SEM.

95

Table 3.1 The effects of linagliptin, sitagliptin, vildagliptin and exendin-4, on endothelium-

dependent relaxation to ACh in mesenteric arteries in the presence of normal (11 mM) and high

(40 mM) glucose concentrations.

Glucose (11 mM) Glucose (40 mM)

ACh n pEC50 Rmax n pEC50 Rmax

Control 11 6.96±0.10 98±1 9 6.36±0.12* 91±2

*

Linagliptin 7 7.21±0.05 99±1 6 7.31±0.03# 97±1

#

Sitagliptin 8 6.59±0.05 98±1 9 6.31±0.05 98±1#

Vildagliptin 8 6.58±0.02 97±1 9 6.32±0.04 97±1#

Exendin-4 8 6.61±0.04 99±1 9 6.80±0.05b 97±1

#

All experiments were conducted in the presence of indomethacin (10 µM). n=the number

of experiments. *Significantly different to 11 mM glucose (p<0.05), ANOVA plus

Bonferroni’s multiple comparison test. #Significantly different to control within each

group (p<0.05), ANOVA plus Bonferroni’s multiple comparison test.

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Figure 3.4 Cumulative concentration-response curves to ACh in endothelium-intact mesenteric

arteries exposed to 11 and 40 mM glucose. In each group of experiments, mesenteric arteries were

precontracted with phenylephrine to a similar level. All experiments were performed in the

presence of indomethacin. Results are shown as mean±SEM of 6-11 experiments. *pEC50

significantly different from 11 mM glucose (Bonferroni’s test, p<0.05). #pEC50 significantly

different from 40 mM glucose (Bonferroni’s test, p<0.05). See Table 3.1 for pEC50 and Rmax values

derived from these curves.

97

3.3.3 Effect of pyrogallol on endothelium-dependent relaxation

The effect of the superoxide generator, pyrogallol on ACh-induced relaxation is

shown in (Figure 3.6). Pyrogallol (30 µM) significantly reduced the sensitivity to ACh

(pEC50 control 7.66±0.06, pyrogallol 7.07±0.06, p<0.05, Bonferroni’s multiple comparison

test) but the maximum relaxation to ACh was not affected (Rmax control 96±1%, pyrogallol

95±3%). The addition of superoxide dismutase (SOD, 50 U/mL) as a scavenger of

superoxide, improved the sensitivity to ACh in the presence of pyrogallol (7.64±0.07,

p<0.05, Bonferroni’s multiple comparison test) (Figure 3.6). The sensitivity to ACh in the

presence of pyrogallol was increased by linagliptin (7.64±0.14, p<0.05) or exendin-4

(7.17±0.12, p<0.05) demonstrating that linagliptin and exendin-4 effectively scavenged

superoxide ions produced by pyrogallol.

98

Table 3.2 The effect of the GLP-1 receptor antagonist exendin-fragment (9-39) on the ability of

linagliptin and exendin-4 to preserve ACh-induced endothelium-dependent relaxation in

mesenteric arteries.

Glucose (11 mM) Glucose (40 mM)

ACh n pEC50 Rmax n pEC50 Rmax

Control 5 7.03±0.18 97±1 5 5.88±0.16* 86±3

Exendin-fragment (9-39) 5 6.51±0.06 94±2 5 6.37±0.06 88±2

Linagliptin+exendin-frag. ND ND 5 6.80±0.10# 97±2

Exendin-4+exendin-frag. ND ND 5 5.85±0.08 93±3

All experiments were conducted in the presence of indomethacin (10 µM).

n = the number of experiments. Results are shown as mean±SEM of 5 experiments

*Significantly different to 11 mM glucose (p<0.05), ANOVA plus Bonferroni’s multiple

comparison test. #Significantly different to 40 mM glucose (p<0.05), ANOVA plus Bonferroni’s multiple

comparison test, ND= Not determined.

99

Figure 3.5 Demonstration of the beneficial effects of linagliptin (panel A) and exendin-4

(panel B), on responses to ACh in the presence of high glucose. The additional presence of

the GLP-1 receptor antagonist exendin fragment (9-39) did not affect the protective actions

of linagliptin but prevented the beneficial effect of exendin-4. In each group of

experiments, mesenteric arteries were precontracted with phenylephrine to a similar level.

All the experiments were conducted in the presence of indomethacin. Results are shown as

mean±SEM of 5 experiments. *pEC50 significantly different from 11 mM glucose

(Bonferroni’s test, p<0.05). #pEC50 significantly different from 40 mM glucose

(Bonferroni’s test, p<0.05). See Table 3.2 for pEC50 and Rmax values derived from these

curves.

100

Figure 3.6 Cumulative concentration-response curves to ACh in the absence (control) or presence

of pyrogallol (30 μM). Pyrogallol significantly decreased the sensitivity to ACh and this effect was

prevented by the presence of linagliptin or exendin-4. Mesenteric arteries were precontracted with

phenylephrine to a similar level. All the experiments were conducted in the presence of

indomethacin. Results are shown as mean±SEM of 5 experiments. *pEC50 significantly different

from control (Bonferroni’s test, p<0.05). The values for the pEC50 under different conditions are

given in the text.

101

3.3.4 Effect of linagliptin on NO-mediated relaxation

To investigate the NO-mediated component of relaxation in mesenteric arteries

responses to ACh were assessed in the presence of the combination of TRAM-34 and

apamin. The addition of TRAM-34+apamin to vessel rings exposed to 11 mM glucose

reduced the sensitivity, but not the maximum relaxation, to ACh in comparison to the

control (Figure 3.7A, Table 3.3). By contrast, in arteries exposed to high glucose, the

addition of IKCa and SKCa blockers did not change the sensitivity to ACh, however, the

maximum relaxation was significantly diminished in comparison to the control (Figure

3.7B, Table 3.3). When responses to ACh were compared between arteries (Figure 3.5b)

high glucose reduced the maximum relaxation to ACh in the presence of TRAM-

34+apamin (Table 3.3), indicating that high glucose impaired the contribution of NO to

endothelium-dependent relaxation. The additional presence of linagliptin had no effect on

arteries exposed to 11 mM glucose but significantly increased the maximum relaxation

induced by ACh in arteries exposed to 40 mM glucose in the presence of TRAM-

34+apamin (Table 3.3) indicating that linagliptin enhanced the contribution of NO to

relaxation when the glucose level was elevated.

3.3.5 Effect of high glucose on NOS and Nox2 related protein expression

Incubation of the rings of mesenteric arteries in 11 mM glucose concentration did

not change the expression of total eNOS, whereas, 40 mM glucose concentration

significantly decreased the expression of total eNOS (Figure 3.8a). The expression of

Nox2 was significantly increased in the mesenteric arteries which incubated in 40 mM

glucose compared to 11 mM glucose (Figure 3.8b).

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Figure 3.7 Examination of the effect of high glucose on the contribution of NO to endothelium-

dependent relaxation in mesenteric arteries. Cumulative concentration-response curves to ACh in

the absence (control) or in the presence of TRAM-34+apamin in endothelium-intact mesenteric

arteries exposed to 11 mM (A) or 40 mM glucose (B). In each group of experiments, mesenteric

arteries were precontracted with phenylephrine to a similar level. All the experiments were

conducted in the presence of indomethacin. Results are shown as mean±SEM of 5-9 experiments.

*pEC50 (A) or Rmax (B) significantly different from control (Bonferroni’s test, p<0.05). #pEC50

significantly different from TRAM-34 + apamin (Bonferroni’s test, p<0.05). See Table 3.3 for

pEC50 and Rmax values.

103

Figure 3.8 Protein expression of eNOS (a) and Nox2 (b) in mesenteric arteries exposed to 11 mM

and 40 mM glucose determined by western blot analysis. High glucose (40 mM) significantly

reduced the expression of eNOS and increased the expression of Nox2. n=6 experiments.

Representative blots are shown on each the corresponding graphs. Results are shown as

mean±SEM. *Significantly different either from 11 mM glucose. (Student’s unpaired t-test,

p<0.05).

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3.3.6 Effects of linagliptin on EDH-mediated relaxation

To investigate the EDH-mediated relaxation, the contribution of NO was

eliminated by the presence of L-NNA+ODQ to inhibit eNOS and soluble guanylate

cyclase respectively. L-NNA+ODQ reduced the sensitivity to ACh in arteries exposed to

11 mM glucose (Figure 3.9A, Table 3.3). The sensitivity to ACh in arteries exposed to 40

mM glucose was also significantly reduced in comparison to arteries exposed to 11 mM

glucose in the presence of L-NNA+ODQ (Table 3.3), suggesting that high glucose

impaired the non-NO, non-prostanoid component of endothelium-dependent relaxation.

Linagliptin had no effect on EDH-mediated relaxation in mesenteric arteries exposed to 11

mM glucose (Figure 3.9A, Table 3.3) but improved the sensitivity to ACh-mediated EDH-

mediated relaxation in arteries exposed to high glucose (Figure 3.9B, Table 3.3). When the

contribution of NO was eliminated by L-NNA+ODQ, the maximum relaxation to ACh

was unimpaired in mesenteric arteries exposed to either normal or high glucose

concentration. The addition of TRAM-34 and apamin in the presence of L-NNA+ODQ

significantly reduced ACh-induced relaxation in arteries exposed to 40 mM glucose (Table

3.3). When Ibtx was added to the combination of L-NNA+ODQ+TRAM-34+apamin, the

relaxation to ACh in arteries exposed to 40 mM glucose was abolished (Table 3.3). In

artery rings exposed to 11 mM glucose, ACh continued to cause a maximum relaxation of

15% in the presence of combination of L-NNA+ODQ+TRAM-34+apamin and the

addition of Ibtx caused no further inhibition (Table 3.3). The presence of linagliptin had no

effect on the maximum relaxation induced by ACh in the presence of L-

NNA+ODQ+TRAM-34+apamin or with the addition of Ibtx in mesenteric arteries

exposed to 11 mM glucose (Table 3.3), in contrast, acute treatment with linagliptin

improved the ACh-induced relaxation in arteries exposed to 40 mM glucose in the

presence of L-NNA+ODQ+TRAM-34+apamin (Table 3.3).

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Table 3.3 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of rat mesenteric arteries exposed to normal (11 mM) and high

(40 mM) glucose in the absence or presence of linagliptin (1 µM).

Glucose (11 mM) Glucose (11 mM)+linagliptin Glucose (40 mM) Glucose (40 mM)+linagliptin

ACh n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax

Control 10 7.31±0.07 95±1 7 7.15±0.08 98±0 9 6.32±0.2# 91±2 6 7.25±0.06 97±1

TRAM34+apamin 6 6.55±0.0* 93±1 7 7.04±0.14 98±1 7 6.46±0.14 44±8

*,# 5 6.74±0.1

Ω 94±1

Ω

L-NNA+ODQ 9 6.64±0.12* 93±1 8 6.67±0.09

* 96±0 8 6.35±0.02 89±1 7 6.91±0.12

Ω 96±2

LNNA+ODQ+

TRAM-34+apamin

7 5.71±0.39* 15±2

* 5 5.52±0.2

* 32±10

* 9 ND 7±0

* 7 4.94±0.6

* 28±7

*

LNNA+ODQ+

TRAM-34

+apamin+Ibtx

4 ND 15±1* 5 ND 25±4

* 7 ND 1±0

* 6 ND 10±1

*

A comparison of the sensitivity (pEC50) and maximum relaxation (Rmax) to ACh in the absence (control), or the presence of TRAM-34 (1

µM)+apamin (1 µM), L-NNA (100 µM)+ODQ(10 µM), L-NNA (100 µM)+ODQ(10 µM)+TRAM-34 (1 µM)+apamin (1 µM) or L-NNA (100

µM)+ODQ(10 µM)+TRAM-34 (1 µM)+apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments were

conducted in the presence of indomethacin (10 µM).

n=the number of experiments.

*Significantly different to control within each group (p<0.05), Dunnet’s test, #Significantly different to 11 mM glucose within inhibitor group (p<0.05), Bonferroni’s test,

ΩSignificantly different to 40 mM glucose within inhibitor group (p<0.05), Bonferroni’s test,

ND, not determined.

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Figure 3.9 Examination of the effect of high glucose on the contribution of EDH to endothelium-

dependent relaxation in mesenteric arteries. Cumulative concentration-response curves to ACh in the

absence (control) or in the presence of L-NNA+ODQ in endothelium-intact mesenteric arteries

exposed to 11 mM (A) or 40 mM (B) glucose. In each group of experiments, mesenteric arteries were

precontracted with phenylephrine to a similar level. All the experiments were conducted in the

presence of indomethacin. Results are shown as mean±SEM of 7-9 experiments. *pEC50 significantly

different from control (Bonferroni’s test, p<0.05). #pEC50 significantly different from L-NNA+ODQ

(Bonferroni’s test, p<0.05). See Table 3.3 for pEC50 and Rmax values.

107

3.4 Discussion

This study demonstrated that impairment of endothelial function caused by high (40

mM) glucose in rat mesenteric arteries was prevented by the presence of the DPP-4 inhibitor

linagliptin or by exendin-4, a GLP-1R agonist. Both agents reduced the increase in vascular

ROS caused by the high glucose concentration whereas the other DPP-4 inhibitors sitagliptin

and vildagliptin failed to either reduce oxidative stress or to improve endothelial function.

Importantly, whereas the protective action of exendin-4 involved activation of the GLP-1R

the antagonist exendin fragment (9-39) had no effect on the beneficial action of linagliptin.

Thus linagliptin acted independently of GLP-1 as expected in this isolated tissue and rather

has direct antioxidant activity, perhaps by radical scavenging. High glucose impaired the

contribution of both NO and EDH to endothelium-dependent relaxation and linagliptin

improved the contribution of both of these relaxing factors.

3.4.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide generation

The findings in this study indicate that incubation of mesenteric arteries in high

glucose for 2 hours significantly increased generation of superoxide. This increase in

oxidative stress is consistent with other observations made in blood vessels isolated from rats

with either type 1 or type 2 diabetes (Leo et al., 2011b), which is considered a key cause of

endothelial dysfunction and consequent vascular disease (Fatehi-Hassanabad et al., 2010).

We investigated the ability of the DPP-4 inhibitors, linagliptin, sitagliptin, and vildagliptin as

well as the GLP-1R agonist, exendin-4 to reduce vascular superoxide levels and found that,

whereas linagliptin and exendin-4 showed a significant reduction of superoxide production,

the other DPP-4 inhibitors, sitagliptin and vildgliptin, had no significant effect. Takai et al.

(Takai et al., 2014) reported that linagliptin and sitagliptin improve endothelial function in

type 2 diabetic rats but it is likely that effect is, at least in part, secondary to the lowering of

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plasma glucose. Our observations are similar to that of Kroller-Schon et al. (Kroller-Schon et

al., 2012) who reported that whereas linagliptin significantly inhibited the oxidative burst in

human leucocytes sitagliptin, vildagliptin, alogliptin and saxogliptin were ineffective. The

difference in the antioxidant activity of the DPP-4 inhibitors may be explained by their

structural dissimilarities. Linagliptin has been reported to have antioxidant activity by

inhibiting xanthine oxidase (Yamagishi et al., 2014) but this seems unlikely to explain the

observations in isolated vascular tissue. Perhaps linagliptin acts as a radical scavenger in a

manner similar to polyphenols such as flavonoids (Woodman and Chan, 2004), supported by

its ability to prevent pyrogallol-induced impairment of endothelium-dependent relaxation.

There are reports that vildagliptin can reduce oxidative stress after chronic treatment in rats

with type 1 diabetes (Ávila et al., 2013) and after renal ischaemia and reperfusion (Glorie et

al., 2012) but the mechanism of action has not been established. Reports that exendin-4 has

antioxidant activity (Erdogdu et al., 2013, Padmasekar et al., 2013) and that GLP-1 decreased

ROS production from cardiac microvascular endothelial cells cultured in a high glucose

medium accompanied by a decreased expression of NADPH oxidase subunits (Wang et al.,

2013) indicate a role for activation of GLP-1R in vivo. Thus linigliptin, when administered in

vivo may have 2 mechanisms by which it acts as an antioxidant, direct radical scavenging and

increased activation of GLP-1R secondary to DPP-4 inhibition.

3.4.2 Effects of DPP-4 inhibitors and exendin-4 on vascular endothelial function

In this study, 40 mM glucose significantly reduced the sensitivity and maximum

response to the endothelium-dependent relaxant ACh without affecting endothelium-

independent relaxation as previously reported (Ceriello et al., 1996, De Vriese et al., 2000,

Cosentino et al., 1997, Cosentino et al., 2003). We confirmed that the impairment was not

due to hyperosmosis as the presence of mannitol (29 mM) in addition to normal glucose

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concentration had no effect on endothelium-dependent relaxation. To test whether DPP-4

inhibitors or activation of the GLP-1R improved endothelial function after exposure to high

glucose ACh responses were assessed in the presence of linagliptin, sitagliptin, vildagliptin

and exendin-4. Linagliptin and exendin-4 improved endothelium-dependent relaxation such

that the sensitivity and maximum response to ACh in the presence of high glucose was

restored to the control levels. By contrast sitagliptin and vildagliptin had no effect. The ability

to improve endothelial function parallels the capacity of linagliptin and exendin-4 to reduce

vascular superoxide in the presence of high glucose. To determine whether these beneficial

effects involved activation of the GLP-1R we repeated the experiments in the presence of the

antagonist exendin fragment (9-39) revealing that, whereas the protective actions of exendin-

4 were prevented by the antagonist, the actions of linagliptin were maintained demonstrating

that exendin-4 had GLP-1R-dependent actions but linagliptin did not. Data in this study is

consistent with reports showing that exendin-4 inhibited ROS-induced injury to endothelial

cells in a GLP-1R-dependent manner (Oeseburg et al., 2010, Erdogdu et al., 2013). Together

these observations suggest that when administered in vivo linagliptin has the capacity to exert

an antioxidant effect through 2 mechanisms, direct radical scavenging and by increasing the

activity of GLP-1 at its receptor.


To further examine the mechanism of the improved response to ACh in the presence

of linagliptin we separately investigated the contribution of NO and EDH to relaxation. To

examine the role of NO-mediated relaxation, EDH-mediated relaxation was abolished by the

endothelial KCa channel blockers TRAM-34 and apamin. Under those conditions high

glucose reduced the maximum response to ACh indicating an impaired contribution of NO to

relaxation as has been reported previously (Cosentino et al., 1997) and as observed in animal

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models of diabetes (Leo et al., 2011b). Interestingly, acute treatment with linagliptin in vitro

significantly increased the maximum relaxation to ACh in high glucose exposed arteries in

comparison to normal glucose. The restoration of NO activity by linagliptin treatment is

consistent with its antioxidant effect reducing vascular superoxide and increasing the

bioavailability of NO.

3.4.4 Effect of linagliptin on EDH-mediated relaxation

In order to evaluate the relative contribution of EDH to endothelium-dependent

relaxation, we examined ACh-induced relaxation in the presence of L-NNA and ODQ to

prevent the effect of NO synthesis and sGC activity respectively. The sensitivity to ACh in

mesenteric arteries exposed to high glucose was significantly reduced in the presence of L-

NNA and ODQ in comparison to normal glucose, indicating that high glucose impaired the

contribution of EDH to endothelium-dependent relaxation, as previously reported (Ozkan and

Uma, 2005, Liu and Gutterman, 2002, Clark and Fuchs, 1997). Acute treatment with

linagliptin of the mesenteric arteries exposed to high glucose showed enhanced EDH-

mediated relaxation which might be due to the reduction of superoxide levels by acute

linagliptin treatment.

3.4.5 Conclusion

Acute treatment of rat mesenteric arteries exposed to high glucose in vitro with the

DPP-4 inhibitor linagliptin or the GLP-1R agonist exendin-4 reduces superoxide generation

and preserves the contribution of both NO and EDH to endothelium-dependent relaxation.

Interestingly, whilst the protective actions of exendin-4 were GLP-1R dependent, linagliptin-

induced improvement of endothelium-dependent relaxation was not affected by the GLP-1R

111

antagonist consistent with a capacity to directly scavenge ROS. Linagliptin enhanced both

NO and EDH activity to improve endothelial function in the mesenteric arteries. Importantly,

under the conditions of these experiments, linagliptin and exendin-4 improved endothelial

function in the absence of any change in glucose levels. This suggests that it may be

worthwhile to explore the potential for these agents to improve vascular function in

pathologies beyond their current use in type 2 diabetes.

113

Chapter 4

Acute treatment with the DPP-4 inhibitor

linagliptan improves endothelium-

dependent relaxation of rat mesenteric

arteries from STZ-induced type 1 diabetic

rats

4.1 Introduction

The impairment of endothelium-dependent relaxation has been identified as a critical

factor in the initiation and development of diabetes-induced vascular complications (De

Vriese et al., 2000, Fatehi-Hassanabad et al., 2010). Diabetes mellitus type 2 is characterised

by insulin resistance and beta-cell dysfunction, causing hyperglycaemia and secondary

mirco-macrovascular complications (Bailey, 2008, Kimura et al., 2003). Hypoglycaemic

agents and insulin when used in the management of diabetes do not prevent the development

of parallel vascular complications (Brown et al., 2010, Holman et al., 2008). Under diabetic

conditions, generation of oxygen radicals, primarily superoxide anion as a consequence of

hyperglycaemia, has a key role in the pathogenesis of vascular complications (Brownlee,

2001, Forstermann, 2008, Li et al., 2013, Nishikawa et al., 2000). Potential targets for

pharmacological therapies include eNOS, NADPH-oxidase and mitochondria which all have

been reported as sources of ROS generation in diabetes (Fatehi-Hassanabad et al., 2010, Hink

et al., 2001).

Dipeptidyl peptidase-4 (DPP-4) inhibitors are compounds used in the management of

type 2 diabetes mellitus (Drucker and Nauck, 2006). We (Chapter 3) and other have

114

demonstrated that in addition to their glucose lowering action, the DPP-4 inhibitors might

exhibit a variety of other biological actions such as antioxidant and vasorelaxant effects.

(Kroller-Schon et al., 2012). Furthermore, a number of studies have revealed that DPP-4

inhibitors improve the activity of vascular eNOS (Shah et al., 2011b) or increased the

expression of eNOS, for example in Zucker obese rats (Aroor et al., 2013). In the previous

chapter, the acute antioxidant effect of the DPP-4 inhibitor linagliptin was demonstrated to

restore endothelial function in small mesenteric arteries exposed to a high glucose

concentration (Chapter 3). It was therefore hypothesised that acute treatment with linagliptin

would have the ability to reduce the oxidative stress and inhibit the source of ROS generation

in diabetic vasculature and consequently to improve endothelial function.

Endothelial dysfunction induced by hyperglycaemia in diabetes arises due to

impairment of both NO and/or EDH-mediated relaxation and vascular oxidative stress

(Heitzer et al., 2000, Mather et al., 2001, Antoniades et al., 2004, Leo et al., 2011b). Thus,

the aim of the present study was to explore whether acute treatment of small mesenteric

arteries from type 1 diabetic rats in vitro with the DPP-4 inhibitor linagliptin restores

microvascular endothelial function, and if so, whether the effects are through direct

scavenging of ROS. Additionally, we investigated whether linagliptin improved contribution

of NO and/or EDH to endothelium-dependent relaxation in diabetic arteries.

4.2 Materials and Methods

The experiments were approved by the Animal Experimentation Ethics Committee of

RMIT University (AEC approval number 1121) and complied with the Australian National

Health and Medical Research Council code of practice for the care and use of animals for

scientific purposes.

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4.2.1 Experimental animals and induction of type 1 diabetes

Diabetes was induced according to the methods described in Chapter 2.1. Briefly,

male ~8 week old Wistar rats, weighing approximately 200 g, were randomly divided into

two groups: normal and diabetic. Type-1 diabetes was induced by a single injection of

streptozotocin (STZ, 50 mg/kg) into the tail vein after overnight fasting. The normal group

received an equivalent volume of the vehicle (0.1 M citrate buffer, pH 4.5). Blood glucose

was measured at the time of experiment for all groups of rats using ACCU-CHECK

Advantage II monitor (Roche, Mannheim, Germany). Rats with a blood glucose of >20

mmol/L were considered to be diabetic.

4.2.2 Vascular function assay

The vascular function experiments were performed according to methods described in

Chapter 2.3. Briefly, mesenteric arteries were precontracted to a similar level using PE (0.1-3

µM), and cumulative concentration-response curves to ACh (0.1 nM/L-10 µM) and SNP

(0.01 nM-10 µM) were investigated. In addition, responses to ACh and SNP were examined

after 20 minutes incubation with different combinations of L-NNA (100 µM), a non-selective

eNOS inhibitor, ODQ (10 µM), a sGC inhibitor, TRAM-34 (1 µM), a selective blocker of

IKCa, Ibtx (100 nM), a selective blocker of BKCa and apamin (1 µM), a SKCa inhibitor.

4.2.3 Assessment of ROS in mesenteric artery

ROS in the mesenteric artery was measured using either L-012 or lucigenin as

described in Chapter 2.4.1.

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4.2.4 Reagents

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except for

ODQ (Cayman Chemical, Ann Arbor, MI, USA), and acetylcholine perchlorate (BDH

Chemicals, Poole, Dorset, UK). Linagliptin was a gift from (Boehringer Ingelheim,

Germany). All drugs were dissolved in distilled water with the exception of indomethacin,

which was dissolved in 0.1 M sodium carbonate, and L-NNA, which was dissolved in 0.1 M

sodium bicarbonate.


Data were expressed as mean ± SEM, n indicates the number of experiments. The

concentration response data from rat isolated mesenteric arteries were fitted to a sigmoidal

curve using nonlinear regression (Prism version 6.0, GraphPad Software, San Diego, CA,

USA) to calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP was determined

as a percentage of the phenylephrine precontraction. Differences between groups of pEC50

and Rmax values were compared and analysed using one way ANOVA with post hoc analysis

using Bonferroni’s test or Student’s unpaired t-test as appropriate. p<0.05 was considered

statistically significant.

4.3 Results

4.3.1 Body weights and blood glucose

Table 4.1 shows the levels of body weight gained, blood glucose, and HbA1c. Ten

weeks after treatment with STZ, the body weight gained was significantly higher in the

control than in diabetic group (Table 4.1). In diabetic rats, the blood glucose and HbA1c

levels were significantly greater compared to normal rats (Table 4.1).

117

4.3.2 Effect of diabetes and linagliptin on superoxide production

The superoxide level in mesenteric arteries was measured by L-012 and lucigenin

chemiluminescence assays. The level of superoxide generation in intact mesenteric arteries

from diabetic rats was significantly greater than in normal rats. Treatment with tempol, a cell

permeable SOD mimetic or DPI, a flavoprotein inhibitor that inhibits NADPH-oxidase,

reduced the production of superoxide anions to equivalent levels in normal and diabetic rats

(Figure 4.1a, b). The acute presence of linagliptin (1 µM) attenuated the generation of

superoxide anions in mesenteric arteries from normal and diabetic rats (Figure 4.1a, b).

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Table 4.1 Mean body weight gained, blood glucose, and HbA1c levels at the end of experiment of

normal and diabetic rats.

n= the number of rats.

*Significantly different from normal group (Student’s unpaired t-test, p<0.05).

Results are shown as mean±SEM.

10 weeks after vehicle or STZ treatment

n Normal n Diabetic

Body weight (g) 10 524±17 9 372±16*

Blood glucose (mM) 8 5.3±0.2 9 28.5±1.0*

HbA1c (%) 9 5.2±0.1 9 13.0±0.2*

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Figure 4.1 Measurement of ROS in intact mesenteric arteries. In diabetic arteries, superoxide (a),

NADPH-oxidase activity (b) was increased and were decreased by acute presence of linagliptin or by

the presence of DPI, a flavoprotein inhibitor that inhibits NADPH oxidase, or tempol, a cell

permeable SOD mimetic. Results are shown as mean±SEM. n=5-9 experiments. *p<0.0001 vs

normal, #p<0.0001 vs diabetic.

120

4.3.3 Effect of diabetes and linagliptin on vascular function

The mesenteric arteries from diabetic rats showed a significant impairment of their

relaxation response to ACh with a reduction in sensitivity, but not the maximum relaxation

(Figure 4.2a, Table 4.2). In contrast, the sensitivity (diabetic, 7.56±0 vs. normal, 7.47±0, n=

4-5, p<0.05) and maximum relaxation (diabetic, 97±16% vs. normal, 97±7%, n= 4-5,

p<0.05) to SNP were not affected (Figure 4.2b). The acute presence of linagliptin in arteries

from normal rats did not affect the sensitivity and response to ACh, but, in mesenteric arteries

from diabetic rats linagliptin, caused a significant increase in the sensitivity (Figure 4.2a,

Table 4.2). The presence of linagliptin did not affect the sensitivity or maximum response to

SNP in normal or diabetic rats (Figure 4.2).

121

Figure 4.2 Cumulative concentration-response curves to ACh (a), SNP (b) in endothelium-intact

mesenteric arteries. In each group of experiments (a, b), mesenteric arteries were precontracted with

PE to similar levels: (a) normal 60±1, normal+linagliptin 62±3, diabetic 59±1, diabetic+linagliptin

63±1, (b) normal 58±2, normal+linagliptin 57±3, diabetic 61±3, diabetic+linagliptin 59±4 %KPSS,

n=5-12 experiments. Results are shown as mean±s.e.m. *p<0.05 vs. diabetic. See Table 4.2 for pEC50

and Rmax values for ACh.

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4.3.4 Role of NO in normal and diabetic mesenteric arteries

The response to ACh-induced relaxation in mesenteric arteries from normal rats was

reduced by the presence of TRAM-34 and apamin to block the IKCa and SKCa channels

(Figure 4.3a, Table 4.2). In contrast, in mesenteric arteries from diabetic rats, the sensitivity

to ACh was unaffected by the presence of a combination of TRAM-34 and apamin (Figure

4.3b, Table 4.2), but the maximum relaxation was significantly reduced (Figure 4.3b, Table

4.2). The presence of a combination of TRAM-34, apamin and Ibtx inhibited the maximum

relaxation of vessels from both normal and diabetic rats (Table 4.2). This is indicating that

NO contributes to endothelium-dependent relaxation which is impaired due to diabetes.

4.3.5 Role of EDH-type relaxation in normal and diabetic mesenteric arteries

The sensitivity to ACh in both normal and diabetic mesenteric arteries were

significantly reduced by the presence of NO synthase inhibitor, L-NNA and a sGC inhibitor,

ODQ (Figure 4.4a, b, Table 4.2). When the contribution of NO was eliminated by L-

NNA+ODQ, the sensitivity to ACh was significantly lower in the diabetic compared to the

normal arteries (Table 4.2), indicating that diabetes impairs the contribution of a non-NO and

non-prostanoid factor to endothelium-dependent relaxation. The maximum relaxation to ACh

was unaffected by the presence of L-NNA+ODQ in normal or diabetic arteries.

123

Table 4.2 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of mesenteric arteries from normal and diabetic rats in the

absence or presence of linagliptin (1 µM).

A comparison of the sensitivity (pEC50) and maximum relaxation (Rmax) to ACh in the absence (control), or the presence of TRAM-34 (1 µM)

+apamin (1 µM), L-NNA (100 µM)+ODQ (10 µM), L-NNA (100 µM)+ODQ (10 µM)+TRAM-34 (1 µM)+apamin (1 µM) or L-NNA (100 µM)

+ODQ (10 µM)+TRAM-34 (1 µM)+apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments were performed in

the presence of indomethacin (10 µM). n = the number of experiments.

*Significantly different to control within each group, p<0.05, Bonferroni’s test,

ФSignificantly different to normal within inhibitor group, p<0.05, Bonferroni’s test,

¥Significantly different to diabetic within inhibitor group, p<0.05, Bonferroni’s test. Results are shown as mean±SEM,

ND= not determined.

Normal Normal+linagliptin Diabetic Diabetic+linagliptin

ACh n pEC50 Rmax (%) n pEC50 Rmax (%) n pEC50 Rmax (%) n pEC50 Rmax (%)

Control 10 7.20±0.11 98±0 6 7.06±0.09 95±1 9 6.12±0.1Ф 96±1 8 7.03±0.09 96±1

TRAM34+apamin 6 6.62±0.12* 93±1 7 7.07±0.20 94±2 7 6.40±0.20 54±8

*Ф 6 6.71±0.3 93±2¥

L-NNA+ODQ 10 6.65±0.12* 92±2 10 6.49±0.20 96±2 8 5.66±0.06

*Ф 78±7 8 6.41±0.17¥ 91±7

LNNA+ODQ+

TRAM-34+apa+Ibtx

4 ND 6±1*

6 ND 6±0* 4 ND 4±0

* 4 ND 3±1

*

124


In order to investigate the role of the NO-mediated component of the relaxation,

responses to ACh were assessed in the presence of the KCa blockers, TRAM-34, apamin and

Iberiotoxin.. When the responses were compared between mesenteric arteries from normal

and diabetic rats, it is apparent that diabetes significantly reduced the maximum relaxation

induced by ACh in the presence of TRAM-34+apamin, indicating that NO-mediated

relaxation was impaired by diabetes. Acute treatment with linagliptin had no effect in normal

mesenteric arteries (Figure 4.3c, Table 4.2), but significantly increased the maximum

relaxation in mesenteric arteries from diabetic rats (Figure 4.3d, Table 4.2). A similar finding

was observed in linagliptin treated diabetic mesenteric arteries in the presence of combination

of TRAM-34+apamin+Ibtx, indicating that linagliptin was able to improve the contribution

of NO to endothelium-dependent relaxation in mesenteric arteries from diabetic rats (Table

4.2).

125

Figure 4.3 Contribution of NO to endothelium-dependent relaxation in mesenteric arteries.

Investigation of NO-mediated relaxation in mesenteric arteries isolated from normal (a), diabetic (b)

in the absence or presence of linagliptin, normal+linagliptin (c), diabetic+linagliptin (d) rats. In each

group of experiments, arteries were precontracted with PE to similar level. n=6-7 experiments.

Results are shown as mean±s.e.m. *p<0.05 vs diabetic. See Table 4.2 for values.

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4.3.6 Effect of linagliptin on EDH-type relaxation

To identify the role of EDH-mediated relaxation, the action of NO-mediated

relaxation was abolished via the addition of L-NNA and ODQ. In the presence of L-

NNA+ODQ, the sensitivity but not the maximum response to ACh was significantly reduced

in mesenteric arteries from diabetic rats compared to normal rats, suggesting that diabetes

impaired the EDH type response. The presence of linagliptin in vitro had no significant effect

on ACh-induced EDH-mediated relaxation in mesenteric arteries from normal rats (Figure

4.4a, Table 4.2), but significantly increased the sensitivity to ACh in mesenteric arteries from

diabetic rats (Figure 4.4b, Table 4.2), indicating that linagliptin improved the contribution of

EDH-type relaxation in diabetic rats. The addition of the combination of L-

NNA+ODQ+TRAM-34+apamin+Ibtx, abolished ACh-induced relaxation in either untreated

diabetic or linagliptin-treated diabetic as well as untreated normal or linagliptin-treated

normal mesenteric arteries (Table 4.2).

127

Figure 4.4 Contribution of EDH to endothelium-dependent relaxation in mesenteric arteries.

Examination of EDH-type relaxation in mesenteric arteries isolated from normal and diabetic rats in

the absence or presence of linagliptin, (a), normal+linagliptin, (b) diabetic+linagliptin. In each group

of experiments, arteries were precontracted with PE to similar level. n=8-10 experiments. Results are

shown as mean±s.e.m. See Table 4.2 for values of pEC50 and Rmax derived from these data.

128

4.4 Discussion

This study demonstrated that, after 10 weeks of diabetes, endothelial dysfunction was

evident in the rat small mesenteric artery. This was due to a decreased contribution of both

NO-mediated and EDH-type relaxation to endothelium-dependent relaxation which

associated with increase in superoxide production. This study also demonstrated that acute

treatment of mesenteric arteries from type 1 STZ-induced diabetic rats attenuates the high

levels of oxidative stress caused by hyperglycaemia and ameliorates endothelial dysfunction

in mesenteric arteries from diabetic rats. Interestingly, linagliptin acted independently of

glucose lowering action and rather has direct antioxidant activity, possibly via radical

scavenging as demonstrated in (Chapter 3). Hyperglycaemia in type 1 STZ-induced diabetic

rats impaired the contribution of NO and EDH to endothelium-dependent relaxation and

acute treatment in vitro with linagliptin enhanced the contribution of both these mediated-

relaxation factors.

4.4.1 Effect of diabetes on superoxide and endothelium-dependent relaxation

In this study, the results indicate that type 1 STZ-induced diabetes augmented the

level of ROS production in mesenteric arteries which is consistent with previous reports (Leo

et al., 2010, Leo et al., 2011b, Inoguchi et al., 2000, Guzik et al., 2002). The increase in

superoxide was concomitant with a selective impairment of endothelium-dependent

relaxation in the mesenteric arteries. The level of superoxide production and the degree of

impairment of endothelial function is consistent with the opportunity that hyperglycaemia-

induced oxidative stress was responsible for the impairment of endothelium-dependent

relaxation in the mesenteric arteries. The overproduction of superoxide by the mechanism of

mitochondrial electron transport chain due to hyperglycaemia-induced oxidative stress

129

becomes an important interpretation for the pathogenesis of diabetic complications

(Brownlee, 2001). This mechanism is followed by the activation of four downstream

pathways: increased polyol pathway flux, increased formation of advanced glycosylation end

products, activation of protein kinase C, and increased hexosamine pathway flux (Nishikawa

et al., 2000).

4.4.2 Effect of diabetes on NO and EDH-type response

In the resistance vessels, such as the rat mesenteric artery, endothelium-dependent

relaxation is mediated by multiple factors including NO and EDH (Edwards et al., 2010).

High glucose concentration-induced oxidative stress has been associated with the impairment

of both NO- and EDH-mediated relaxation (Chapter 3) (De Vriese et al., 2000). In diabetes,

the increase in superoxide anions production can cause impairment in endothelium-dependent

relaxation via a number of mechanisms. Superoxide reacts with NO immediately, to form

peroxynitrite which simultaneously causes reduction in the availability of NO for relaxation

and increases peroxynitrite-induced toxicity. For example, peroxynitrite has been

demonstrated to uncouple endothelial NO synthase due to tetrahydrobiopterin (BH4)

oxidation, therefore additional stimulating the synthesis of superoxide (Münzel et al., 2010),

and leads to protein nitration of the calcium-activated potassium channels which are

responsible for EDH-type responses (Liu and Gutterman, 2002). Furthermore, it has been

reported that the peroxynitrite alone can cause impairment to EDH-type responses in the

mesenteric arteries, which might be improved by the existence of antioxidant treatment (Li et

al., 2008).

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4.4.1 Effect of linagliptin on vascular superoxide and relaxation

As we described early, in diabetes, increases in the generation of superoxide caused

an impairment in eNOS activity, increased NO inactivation, increased eNOS uncoupling via

oxidation of BH4, further increased activity of NADPH oxidase, and increased formation of

peroxynitrate which may promote the initiation of endothelial dysfunction. The presence of

linagliptin in vitro significantly reduced increased level of superoxide generation detected,

indicating that the linagliptin may reduce the oxidative stress, possibly by direct scavenging

of superoxide and improved endothelium-dependent relaxation in mesenteric arteries from

type 1 diabetic rats without affecting arteries from normal rats. Our findings in this study are

consistent with our findings in (Chapter 3) and other observations demonstrated that

treatment with linagliptin improves injury to cardiovascular tissue induced by salt-sensitive

hypertension which seem to be attributed to the reduction of oxidative (Koibuchi et al., 2014)

or enhancement of endothelial function and reduced vascular stress in aortic artery challenged

to experimental sepsis (Kroller-Schon et al., 2012).


To investigate NO-mediated relaxation, ACh-induced relaxation was assessed in the

presence of endothelial KCa blockers to inhibit the EDH-mediated relaxation. This inhibition

of the EDH-type response, revealed significant decrease in the sensitivity to ACh and the

maximum relaxation in diabetic arteries compared to normal arteries. Acute treatment with

linagliptin significantly increased the maximum relaxation mediated by ACh in mesenteric

arteries from diabetic rats in comparison to untreated mesenteric arteries from diabetic rats,

indicating that linagliptin improved the contribution of NO to endothelium-dependent

relaxation in diabetic mesenteric arteries. The prevention of peroxynitrite formation and the

reduction of ROS activity in diabetic vasculature in rats might be at least in one part via rapid

131

free radical scavenging effect of linagliptin as we demonstrated in (Chapter 3), in order to

preserve the bioavailability of NO in vascular endothelial cells as we also observed in this

study. The response to SNP-induced endothelium-independent relaxation in this study was

comparable in both normal and diabetic rats, demonstrating that in both normal and diabetic

rats, the responses to exogenous NO-induced relaxation in smooth muscle cells was not

impaired.

4.4.3 Effect of linagliptin on EDH-mediated relaxation

In order to investigate the contribution of EDH-type relaxation to endothelium-

dependent relaxation, the NO-mediated relaxation was inhibited by the application of L-NNA

and ODQ to inhibit NO synthesis and sGC activity respectively. In the presence of L-

NNA+ODQ, the sensitivity to ACh was decreased in diabetic arteries when compared to

normal arteries, showing that diabetes impaired the contribution of EDH to endothelium-

dependent relaxation as previously reported (Leo et al., 2011b, Makino et al., 2000, Wigg et

al., 2001). It has been reported that superoxide generation by auto-oxidation of pyrogallol

might impair EDH type responses in rat mesenteric arteries (Ma et al., 2008). Furthermore, it

has been suggested that K+ as an one of EDH factor and the vasodilatation response to K

+ is

impaired in the mesenteric arteries in diabetic rats (Makino et al., 2000). Acute treatment

with linagliptin of mesenteric arteries from diabetic arteries improved EDH-mediated

relaxation which likely due to the antioxidant effect of linagliptin (Chapter 3).

4.4.4 Conclusion.

In the present study we have demonstrated that acute treatment of mesenteric arteries

from diabetic rats in vitro with the DPP-4 inhibitor linagliptin significantly reduces the

132

generation of superoxide and preserves the contribution of both NO and EDH to

endothelium-dependent relaxation. As we demonstrated in (Chapter 3), the attributed

beneficial protective effect of linagliptin in this study, at least in part, by rapidly scavenge

ROS and reduce oxidative stress to increase NO bioavailability and improve EDH-type

response in vascular endothelial cells. Interestingly, the beneficial effect of linagliptin to

protect NO and EDH activity observed in this study shows that it has potential as therapeutic

agent for use in the reduction and/or prevention of microvascular complications associated

with diabetes beyond its current use in type 2 diabetes.

134

Chapter 5

The DPP-4 inhibitor linagliptan restores

endothelial function of mesenteric arteries

from rats fed a western diet

5.1 introduction

Cardiovascular morbidity and mortality have been reported to be associated with

metabolic syndrome (Isomaa et al., 2001). Endothelial dysfunction is known to be linked to

obesity, atherogenesis, hypertension and insulin resistance that enhances the risk of

cardiovascular diseases (Caballero, 2003). It has been observed that obesity, particularly in

viscera, is correlated with the early stages of development of endothelial dysfunction (Arcaro

et al., 1999, Caballero, 2003). Excessive considerations have been devoted to high-fat content

of diet which is proposed to be a key event and responsible for the development of obesity

and subsequent vascular dysfunction, even before progress to type 2 diabetes mellitus

(Cuevas et al., 2000, Woo et al., 2004, Hajer et al., 2008) These observations demonstrate

that the consumption of high-fat diet is associated with the development of the impairment of

endothelium-dependent relaxation of large arteries due to transformed NO signaling

mechanisms (Molnar et al., 2005, Roberts et al., 2000).

It is well established that small arteries and arterioles are primarily involved in

determination of circulatory resistance and tissue perfusion. Nevertheless, few if any studies

have explored the effect of western diet (WD) on the change of microvascular function. Also,

it is of specific interest to investigate whether the DPP-4 inhibitor linagliptin which recently

we (Chapter 3 and 4) demonstrated was able to act as an antioxidant compound that can

135

ameliorate endothelial dysfunction in small mesenteric arteries isolated from STZ-treated

rats. Therefore, the aim of the studies presented in this chapter was to investigate whether

acute in vitro treatment with linagliptin reverses endothelial dysfunction in small resistance

mesenteric arteries from WD fed rat and, if so, whether it acts via direct scavenging of

superoxide anions and/or prevention of the sources of ROS generation in endothelial cells in

mesenteric arteries. Additionally, we sought to explore the beneficial effects of linagliptin on

the contribution of NO and/or EDH to endothelium-dependent relaxation in small resistant

arteries from WD fed rats.


All procedures were approved by the Animal Experimentation Ethics committee of

RMIT University and complied with the Australian National Health and Medical Research

Council code of practice for the care and use of animals for scientific purposes (AEC

approval number 1245).

5.2.1 Experimental animals

Male Wistar Hooded rats (Monash University, Australia) weighing 180–200 g at the

beginning of the study were used. At least one week acclimation was allowed prior to any

testing and during this period rats were handled daily to reduce stress. At the start of the

feeding regime, rats were housed in groups of four under a light/dark cycle (12 h/12 h), room

temperature (21±1°C) and humidity (30–70%) kept constant with water available ad libitum

in the home cage. Animals were randomly assigned to either a control (SD, Standard

AIN93G rodent diet, 7% total fat including 1.05% total saturated fatty acids; Specialty Feeds,

Perth, Australia) or western diet (WD SF00-219, 21% total fat including 1.80% total

136

saturated fats and 0.15% cholesterol; Specialty Feeds, Perth, Australia). Animals were

allowed ad lib access to diets for 7 weeks after which they were placed on a food restricted

diet where animals received 70% of their normal daily food consumption for a period of 10

weeks. Food intake and body weight were measured twice weekly. The rats were killed 10

weeks after being placed on their restricted diet by asphyxiation by CO2 inhalation, followed

by decapitation, and their abdomen opened to isolate the mesenteric arteries. Blood samples

were obtained from the carotid arteries and the blood glucose levels were measured using a

one-touch glucometer (Roche, Sydney, New South Wales). Glycated haemoglobin (HbA1c)

was measured using the in2itTM

(II) analyser (Bio-Rad, Hercules, CA, USA).


After rats were killed, the mesenteric arteries were isolated and immediately kept in

ice cold Krebs bicarbonate solution. The third-order branch mesenteric arteries (internal

diameter ~200-300 µm) were isolated and prepared as described in Chapter 2.2.

5.2.3 Vascular function experiments

Vascular reactivity was evaluated as previously described (Chapter 2.3). Briefly, after

maximum contraction of the vessels and the assessment of the integrity of the endothelium,

the mesenteric arteries were precontracted with PE (0.1-3 µM) to attain comparable active

tension and cumulative concentration-response curves to ACh (0.1 nM – 10 µM) and SNP

(0.1 nM–10 µM) were obtained. In some experiments, cumulative concentration- responses

to ACh were assessed after 20 minutes incubation with different combinations of L-NNA

(100 μM), a non-selective NOS inhibitor, ODQ (10 μM), a sGC inhibitor, TRAM-34 (1 μM),

a selective blocker of IKCa and apamin (1 μM), a SKCa inhibitor.

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5.2.4 Measurement of superoxide generation in mesenteric arteries

Superoxide anion generation in the mesenteric arteries was measured using the

lucigenin-enhanced chemiluminescence assay as described in Chapter 2.5.1.

5.2.5 Western Blot

Tissue samples of first order, second order, and third order mesenteric arteries from 2

rats from the same treatment group were pooled and considered as n=1. Western blots were

accomplished as described in Chapter 2.7. To explore eNOS mono/dimer development in

tissue sample, a non-boiled sample was resolved by 6% SDS-PAGE at 4ºC overnight

(Chapter 2.7.5).

5.2.6 Reagents

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except for

linagliptin (Boehringer Ingelheim, Germany), acetylcholine perchlorate (BDH Chemicals,

Poole, Dorset, UK), and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All chemicals

were dissolved in distilled water with the exception of L-NNA, which was dissolved in 0.1 M

sodium bicarbonate, and indomethacin, which was dissolved in 0.1 M sodium carbonate.


Results are presented as the mean±SEM, n indicates the number of experiments. The

concentration response data from isolated mesenteric arteries were fitted to a sigmoidal curve

using nonlinear regression (Prism version 6.0, GraphPad Software, San Diego, CA, USA) to

calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP was determined as a

percentage of the phenylephrine precontraction. pEC50 and Rmax values were compared using

138

one way ANOVA with post hoc analysis using Bonferroni’s test or Student’s unpaired t-test

as appropriate. p<0.05 was considered statistically significant.

5.3 Results

5.3.1 Effect of WD on body weights

The body weights of SD and WD fed rats is shown in Figure 5.1. The final body

weight in WD fed rats was significantly higher than in SD fed rats.

5.3.2 Effect of WD and linagliptin on oxidative stress in mesenteric arteries

The level of superoxide generation in mesenteric arteries from SD or WD fed rats was

measured by using lucigenin-enhanced chemiluminesence. In WD fed rats, the level of

superoxide induced fluorescence was significantly higher than that in SD fed rats (Figure

5.2). Acute treatment with linagliptin (1 µM) significantly reduced the level of superoxide

generation detected in mesenteric arteries from WD fed rats (Figure 5.2).

5.3.3 Effect of linagliptin on endothelial function in mesenteric arteries from WD fed

rats

The level of contraction to the high K+

physiological saline solution (KPSS, 123

mmol/L) was similar in arteries from SD and WD rats (Figure 5.3). The endothelium-

dependent relaxation agonist ACh and the endothelium-independent relaxation agonist SNP

were used to assess the effect of linagliptin on the vascular function in mesenteric arteries

from SD and WD fed rats. In the mesenteric arteries from WD fed rats, the sensitivity to

ACh, but not the maximum relaxation was significantly decreased compared with SD fed rats

139

(Figure 5.4a), whereas, the sensitivity and the maximum relaxation to SNP were not altered

(Figure 5.4b). Acute treatment with linagliptin had no effect on the response of the

mesenteric arteries to ACh in SD fed rats, but in WD fed rats, linagliptin significantly

increased the sensitivity to ACh (Figure 5.4a).

140

Figure 5.1 Body weight gain over 10 wk of rats receiving a WD and SD. n=8-15. Results are shown

as mean±SEM. n=8-15 experiments. *p<0.0001 vs SD.

141

Figure 5.2 Superoxide levels in intact mesenteric arteries isolated from SD and WD fed rats. NADPH

activity was raised in WD mesenteric arteries which were attenuated with linagliptin treatment.

Results are demonstrated as mean±SEM. n=6-8 experiments. *p<0.05 vs SD #p<0.05 vs WD.

142

Figure 5.3 Mesenteric arteries isolated from SD and WD fed rats induced to maximum contraction by

KPSS (123 mM). KPSS caused a similar level of contraction in arteries from both groups of rats.

143


ACh (a), SNP (b) in endothelium-intact mesenteric arteries. In each group (a, b), mesenteric arteries

were prcontracted with PE to a similar level: (a) SD 66±2, SD+linagliptin 64±1, WD 65±1,

WD+linagliptin 66±1, (b) SD 64±1, SD+linagliptin 64±1, WD 61±6, WD+linagliptin 66±1 %KPSS,

n=7-10 experiments. Results are shown as mean±SEM. *p<0.05 vs SD.

144

5.3.4 Investigation of the contribution of NO and EDH-mediated relaxation in SD and

WD

In mesenteric arteries from SD fed rats, the response to ACh was partially decreased

by either the combination of N-nitro-L-arginine (L-NNA), a NO synthase inhibitor and 1H-

[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor or

inhibitors of intermediate-conductance calcium-activated K+ channel (IKCa), small-

conductance calcium-activated K+ channel (SKCa) with 1-[(2

chlorophenyl)(diphenyl)methyl]1H pyrazole (TRAM-34), apamin and iberiotoxin (Ibtx)

respectively, indicating that both NO and EDH contribute to endothelium-dependent

relaxation. Moreover, the response of mesenteric arteries from WD fed rats to ACh-induced

relaxation was decreased in the presence of either NO inhibitors or EDH inhibitors compared

with SD fed rats suggesting that both NO and EDH-mediated relaxation were impaired by

consumption of WD by the rats.

5.3.5 Effect of linagliptin on NO-mediated relaxation in WD

In order to investigate the contribution of NO to endothelium-dependent relaxation, a

combination of IKCa and SKCa channel inhibitors (TRAM-34+apamin) were used. The

addition of TRAM-34+apamin to the mesenteric arteries from SD fed rats reduced the

sensitivity but not the maximum relaxation to ACh, compared to the control (Figure 5.5a,

Table 5.1). On the other hand, in mesenteric arteries from WD fed rats, the addition of IKCa

and SKCa inhibitors significantly reduced both the sensitivity and the maximum relaxation in

comparison to the control (Figure 5.5b, Table 5.1). In mesenteric arteries from WD fed rats,

the reduction in the responses to ACh (the sensitivity and the maximum relaxation) in the

presence of TRAM-34+apamin indicate that WD consumption impaired the contribution of

NO to endothelium-dependent relaxation. Acute treatment with linagliptin significantly

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increased the response to ACh in the presence of TRAM-34+apamin by increasing the

sensitivity and the maximum relaxation in the arteries from WD fed rats (Table 5.1) indicating

that linagliptin has the ability to enhance the contribution of NO to endothelium-dependent

relaxation.

5.3.6 Effect of WD on the expression of NOS and Nox2

In mesenteric arteries from WD fed rats, the expression of eNOS significantly

decreased in comparison to mesenteric arteries from SD fed rats (Figure 5.6a). In WD fed

rats, the proportion of eNOS expressed as monomer was significantly higher than that in SD

fed rats (Figure 5.6b). Nox2 expression was significantly increased in mesenteric arteries

isolated from WD fed rats (Figure 5.6c).

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Table 5.1 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of rat mesenteric arteries isolated from SD and WD fed rats in

the absence or presence of linagliptin (1 µM).

SD SD+linagliptin WD WD+lingaliptin

ACh n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax

Control 8 7.56±0.08 Ω

94±2 8 7.44±0.09 Ω

94±0 8 6.97±0.06# 96±2 6 7.46±0.09

Ω 95±1

TRAM34+apamin 6 6.97±0.0* Ω

90±4 6 7.25±0.09 Ω

90±3 6 6.46±0.12*#

65±9 6 6.22±0.08 Ω

91±2

L-NNA+ODQ 5 6.71±0.10* Ω

97±1 5 6.88±0.09* Ω

96±0 5 6.24±0.06*#

95±1 5 6.86±0.05 Ω

93±0

LNNA+ODQ+

TRAM34+apamin+

Ibtx

5 ND 10±1* 5 ND 7±1

* 9 ND 6±1

* 7 ND 7±0

*


µM)+apamin (1 µM), L-NNA (100 µM)+ODQ(10 µM), L-NNA (100 µM)+ODQ(10 µM)+TRAM-34 (1 µM)+apamin (1 µM) or L-NNA

(100 µM)+ODQ(10 µM)+ TRAM-34 (1 µM) +apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments

were conducted in the presence of indomethacin (10 µM).

n=the number of experiments.

*Significantly different to control within each group (p<0.05), Bonferroni’s test, #Significantly different to SD within inhibitor group (p<0.05), Bonferroni’s test,

ΩSignificantly different to WD within inhibitor group (p<0.05), Bonferroni’s test,

ND, not determined.

147

Figure 5.5 Contribution of NO to endothelium-dependent relaxation. Mesenteric arteries isolated

from both SD and WD fed rats to examine NO-mediated relaxation in the presence of TRAM-

34+apamin were isolated from SD (a), WD (b). In each group of experiments, arteries were

precontracted with PE to similar levels: 63±2 (a), 65±0.6 (b) %KPSS, n=8-9 experiments. Results are

shown as mean±SEM. #p<0.05 vs SD (control),*p<0.05 vs WD (control). See Table 5.1 for values.

148

Figure 5.6 Analysis of protein expression of eNOS (a, 130 kDa), eNOS dimer and monomer

measured in on representative WB (b, 260 kDa) and Nox2 (c, 58 kDa) in SD and WD mesenteric

arteries. In WD mesenteric arteries, the expression of eNOS significantly reduced and the proportion

of eNOS was expressed as the dimer decreased, and the expression of Nox2 increased. n=6

experiments. Results are shown as mean±SEM. *p<0.05 compared to SD.

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5.3.7 Effect of linagliptin on EDH-mediated relaxation in WD

To investigate the contribution of EDH to endothelium-dependent relaxation, NO-

mediated relaxation was abolished by the addition of L-NNA+ODQ to block eNOS and

soluble guanylate cycalse respectively. The sensitivity to ACh-enhanced relaxation reduced in

the presence of L-NNA+ODQ in mesenteric arteries from SD fed rats (Figure 5.7a, Table

5.1). Also, the sensitivity to ACh, but not the maximum relaxation, in mesenteric arteries

from WD fed rats was significantly reduced in the presence of L-NNA+ODQ (Figure 5.7b,

Table 5.1), indicating that WD impaired the non-NO, non-prostanoid component in

endothelium-dependent relaxation. Acute treatment with linagliptin did not increase the

sensitivity to ACh and had no effect on EDH-mediated relaxation in arteries from SD fed rats

(Figure 5.7a, Table 5.1), however, linagliptin increased the sensitivity to ACh-induced EDH-

mediated relaxation in mesenteric arteries from WD fed rats (Figure 5.7b, Table 5.1). The

addition of TRAM-34, apamin, and Ibtx in the presence of L-NNA+ODQ significantly

reduced ACh-enhanced relaxation in mesenteric arteries from both SD and WD fed rats

(Table 5.1). Acute treatment with linagliptin did not improve the relaxation induced by ACh

in mesenteric arteries from SD or WD fed rats under those conditions.

150

Figure 5.7 Contribution of EDH to endothelium-dependent relaxation. Mesenteric arteries isolated

from both SD (a) and WD (b) fed rats to investigate EDH-mediated relaxation in the presence of L-

NNA+ODQ. In each group of experiments, arteries were precontracted with PE to similar levels:

62±0.7 (a), 65±0.8 (b) %KPSS, n=8-9 experiments. Results are shown as mean±SEM. See Table 1 for

values of pEC50 and Rmax.

151

5.4 Discussion

This study showed that endothelial dysfunction in mesenteric arteries caused by

consumption of WD (high fat diet) was prevented by acute treatment with the DPP-4 inhibitor

linagliptin. Linagliptin reduced the increased production in vascular ROS caused by WD

cosumption. Consumption of WD impaired the action of both NO and EDH and acute

treatment with linagliptin improved the contribution of both of these relaxing factors.

5.4.1 Effects of WD and linagliptin on oxidative stress

To investigate the effect of WD on oxidative stress, we measured the levels of

superoxide generation in mesenteric arteries from SD and WD fed rats. The findings in this

study indicate that the level of superoxide generation significantly increased in WD

mesenteric arteries compared to SD fed rats. These findings suggest that WD promotes the

development of oxidative stress and endothelial dysfunction primarily because of an

increased NADPH oxidase-derived superoxide anion production which may react with NO,

thereby enhancing the generation of the NO/superoxide reaction endproduct peroxynitrite

which in turn causes uncoupling of eNOS and inactivation of NO as noted in this study.

Furthermore, the findings also showed that the expression of Nox2 was increased in the

mesenteric arteries of WD fed rats, indicating that the impairment of endothelial-dependent

relaxation may be in part due to the overproduction of superoxide anions caused by increased

expression of the enzymatic source of superoxide generation. We inspected the ability of the

DPP-4 inhibitor linagliptin to decrease the levels of vascular superoxide and found that

linagliptin expressed a significant decline in superoxide generation in WD mesenteric

arteries by its ability to act as antioxidant compound as we have demonstrated in chapter 3.

152

5.4.2 Effect of WD diet on endothelial function

ACh was used to evaluate the effect of diet (SD and WD) on endothelium-dependent

relaxation. The findings of this study indicate that the consumption of WD causes impairment

of endothelial function in small resistant mesenteric arteries from these rats, and this is in

agreement with some reports which demonstrated that consumption of high fat diet induced

endothelial cell dysfunction in male and female rat aorta (Roberts et al., 2005, Reil et al.,

1999) or obesity-induced insulin resistance, hyperinsulinemia, and hypercholesterolemia that

have been anticipated to be involved in the development of endothelial dysfunction (Ohara et

al., 1993, Arcaro et al., 1999, Caballero, 2003, Busija et al., 2005). Thompson and colleagues

using coronary arteries and Woodman and colleagues using brachial arteries demonstrated

that the consumption of high fat diet caused impairment of endothelial cell function in male

and female pigs, respectively (Thompson et al., 2004, Woodman et al., 2003). The results in

this study demonstrated that the impairment of endothelial function may be attributed, in part,

to a reduced expression of eNOS, since WD consumption reduced the total protein content of

eNOS and reduced dimerization of eNOS in mesenteric arteries and this is consistent with the

report by Woodman et al, who noted that the consumption of high-fat diet induce a reduction

in brachial artery eNOS using immunohistochemistry (Woodman et al., 2003). Additionally,

SNP was used in order to investigate the effect of WD on vascular smooth muscle relaxation,

SNP-induced relaxation in this study was similar in both SD and WD fed rats, indicating that

the relaxation of smooth muscle in response to exogenous NO was not impaired in WD fed

rats.

5.4.3 Effect of linagliptin on endothelial function in WD

The findings in this study showed that rats fed a WD had a significant reduction in the

response to ACh-induced endothelium- dependent relaxation in comparison to SD fed rats.

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Acute treatment with linagliptin in vitro improved endothelial function and increased

endothelium-dependent relaxation of mesenteric arteries isolated from WD fed rats.

Therefore we investigated whether linagliptin improved the endothelial function by

increasing the contribution of NO and/or EDH to the endothelium-dependent relaxation.

5.4.4 Effect of linagliptin on NO-mediated relaxation in WD fed rats

ACh-induced relaxation was used in order to investigate the contribution of NO to

endothelium-dependent relaxation in the presence of the combination of TRAM-34 and

apamin an IKCa and SKCa inhibitors respectively, in mesenteric arteries isolated from SD and

WD fed rats. Under the conditions of blocking the action of EDH-mediated relaxation, the

maximum relaxation response to ACh was significantly reduced in mesenteric arteries from

WD fed rats. Acute treatment with linagliptin in vitro, significantly improved the response to

ACh-induced relaxation and increased the maximum relaxation in WD mesenteric arteries in

comparison to the control. This is likely due to lingliptin reducing the oxidative stress by the

antioxidant effect which in turn increased the bioavailability of NO in mesenteric endothelial

cells by preventing the formation of peroxynitrite as a product of the reaction of superoxide

and NO via rapid free radical scavenging action of linagliptin (Chapter 3, 4). Overall, acute

treatment with linagliptin preserved the bioavailability of NO in mesenteric arteries of WD

fed rats.

5.4.5 Effect of linagliptin on EDH-mediated relaxation in WD fed rats

In addition to NO-mediated relaxation, the endothelium-dependent relaxation in rat

mesenteric arteries is mediated by both classical and non-classical EDH type responses

(Edwards et al., 2010, Leo et al., 2011b, Garland et al., 2011). To investigate the contribution

154

of EDH to endothelium-dependent relaxation in mesenteric arteries from SD and WD fed

rats, L-NNA+ODQ were added to prevent NO formation and sGC activity respectively. The

sensitivity to ACh-induced relaxation in WD mesenteric arteries was significantly decreased

in comparison to SD mesenteric arteries in the presence of L-NNA+ODQ, indicating that

feeding rats with WD for 10 weeks impaired the contribution of EDH to endothelium-

dependent relaxation. Acute treatment with linagliptin in vitro significantly improved the

sensitivity to ACh-induced relaxation in mesenteric arteries from WD fed rats, indicating that

linagliptin preserved the contribution of EDH to endothelium-dependent relaxation by

reducing the oxidative stress under the conditions of feeding animals with WD.

5.5.5 Conclusion

The findings in this study demonstrated that consumption of WD induced oxidative

stress, downregulation of vascular NOS, and reduced NO production to promote endothelial

dysfunction. The acute treatment in vitro with the DPP-4 inhibitor linagliptin improves the

endothelial function in mesenteric arteries developed in WD fed rats. This is probably via

conserving the beneficial effects of both NO and EDH-mediated relaxation. In this study we

showed that the DPP-4 inhibitor linagliptin has the ability to decrease the production of

superoxide anions which is perhaps, at least in part, via direct radical scavenging action. The

beneficial effect of DPP-4 inhibitor linagliptin by improving the vascular endothelial function

in small resistance mesenteric arteries in WD fed rats demonstrated in this study shows it

possesses the capability to act as a pharmacological intervention that may be anticipated to

improve the NO and EDH-mediated relaxation and could be valuable in the prevention of the

pathological role of WD in the development of endothelial dysfunction in the vasculature.

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Chapter 6

The DPP-4 inhibitor linagliptin preserves

endothelial cell function in mesenteric

arteries from type-1 diabetic rats without

decreasing plasma glucose

6.1 introduction

Cardiovascular disease starts with risk factors such as hyperglycaemia in diabetes,

hypertension, and dyslipidaemia (Dzau et al., 2006). Endothelial dysfunction is considered a

critical factor in the initiation and development of vascular complications induced by diabetes

(De Vriese et al., 2000, Fatehi-Hassanabad et al., 2010). Macro- and microvascular

complications are presently the main causes of morbidity and mortality amongst diabetic

patients in both type 1 and type 2 diabetes mellitus (De Vriese et al., 2000). Endothelial cells

play an active role to regulate the basal vascular tone and reactivity of blood vessels in both

physiological and pathological conditions, by releasing a diversity of contracting and relaxing

factors in responding to stimulating factors such as mechanical forces and neurohumoral

mediators (Furchgott and Vanhoutte, 1989, Ferrara, 2002). The most important endothelium-

derived relaxing factors (EDRFs) are nitric oxide (NO), prostacyclin and endothelium-

dependent hyperpolarization (EDH) (Feletou and Vanhoutte, 1999, Woodman et al., 2000). It

has been demonstrated that the endothelial dyfunction caused by the impairment of eNOS

expression/activity and the reduction of NO production could be promoted by some risk

factors such as hypercholesterolemia and diabetes, thus, the endothelium-dependent

vasodilatation used as a parameter to investigate endothelial function in both physiological

and pathological circumstances (Arnal et al., 1999, Leo et al., 2011b).

157

Dipeptidyl peptidase-4 (DPP-4) is a glycoprotein peptidase broadly expressed in

various cell types which displays complex biological actions and has multifunctional

properties (Drucker, 2006, Drucker, 2007, Cordero et al., 2009, Yazbeck et al., 2009).

Inhibition of the DPP-4 system by DPP-4 inhibitors, which are class of blood glucose-

lowering drug, signifies a new approach in order to treat type 2 diabetes and take advantage

of their neutral effect on body weight and low risk of the occurrence of hypoglycaemia

(Panchapakesan et al., 2013, Zhong et al., 2013). DPP-4 inhibitors prolong the half-life of

incretins such as glucagon-like-peptide-1 (GLP-1) and glucagon induced peptide (GIP) by

inhibiting the degradation of these incretins and thus consequently lower blood glucose via

improved insulin secretion (Drucker, 2006). Interestingly, previous studies show beneficial

effects of GLP-1 in situations such as in the regulation of endothelial function and cardiac

remodeling (Zhao et al., 2006, Nikolaidis et al., 2004, Basu et al., 2007, Green et al., 2008).

The DPP-4 inhibitors have the ability to prevent the impairment of cardiac diastolic function

(Aroor et al., 2013), improve glomerulopathy (Nistala et al., 2014), ameliorate dysfunction in

rat aortic artery in experimental sepsis (Kroller-Schon et al., 2012) and reduce oxidative

stress in vascular endothelial cells (Ishibashi et al., 2013). We have recently found that acute

treatment with linagliptin ameliorate vascular dysfunction in mesenteric arteries exposed to

high concentration of glucose (40 mM) independently of glucose lowering effect.

We have previously demonstrated that in small arteries from STZ-treated rats with

diabetes-induced endothelial dysfunction results from the impairment of both NO-mediated

and EDH-mediated relaxation, associated with eNOS uncoupling and an increase in Nox2-

derived superoxide generation (Leo et al., 2011b). We have also shown that treatment with

3’,4’-dihydroxy flavonol reduces oxidant stress and improves endothelium-dependent

relaxation in type 1 diabetic rats (Leo et al., 2011a). Therefore, it is of particular interest to

examine whether linagliptin, a DPP-4 inhibitor which we have recently demonstrated is able

158

to also act as an antioxidant, can alleviate endothelial dysfunction in diabetic vasculature

independently of the glucose lowering properties. Thus, the aim of the present study was to

examine whether chronic in vivo treatment with linagliptin, a DPP-4 inhibitor with unique

xanthine-based structure, preserves endothelial function in small resistance mesenteric

arteries from type 1 STZ-induced diabetic rats and, if so, whether it acts via the mechanism of

direct scavenging of ROS and/or through the inhibition of the sources of ROS generation in

microvascular endothelial cells in diabetic mesenteric arteries independently of reducing

blood glucose effects.

159


Ethics statement: All the procedures were approved by the Animal Experimentation

Ethics committee of RMIT University (AEC approval number 1309) and complied with the

Australian National Health and Medical Research Council code of practice for the care and

use of animals for scientific purposes.

6.2.1 Induction of type 1 diabetes

Male Wistar rats (~ 8-weeks old and weighing approximately 200g) were randomly

divided into two groups: normal and diabetic. A single injection of streptozotocin (STZ, 50

mg/kg IV) after the rats were fasted overnight was used to induce type 1 diabetes, whereas,

rats in the control group received vehicle of a comparable volume (0.1 mol/L citrate buffer,

pH 4.5). Blood glucose was monitored and when greater than 25 mmol/L, rats were treated

with insulin (3-4 IU, s.c., 3 injections/ week, Protaphane, Novo Nordisk, NSW, Australia).

Blood glucose concentration was measured by a glucometer (Roche, Sydney, NSW,

Australia) and glycated haemoglobin (HbA1c) was measured using a Micromat HbA1c

analyser (Biorad, Sydney, NSW, Australia). Blood samples were collected from the left

ventricle at the end of the experimental period.

6.2.2 Linagliptin treatment

Six weeks after induction of diabetes, the two groups of rats were further divided into

4 subgroups (normal, normal+linagliptin, diabetic, diabetic+linagliptin) administering either

vehicle (1% carboxy methylcellulose, CMC) or linagliptin (2 mg/kg) by daily oral gavage for

a period of 4 weeks. The rats were administered the last dose of linagliptin at least 24 hours

prior to the collection of tissue for experimentation.

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After ten weeks of STZ treatment, the rats were killed by asphyxiation with CO2

followed by exsanguination. The mesenteric arcade was isolated and collected and

immediately placed in ice-cold Krebs bicarbonate solution (4.7 mmol/L KCl, 118 mmol/L

NaCl, 1.18 mmol/L MgSO4, 25 mmol/L NaHCO3, 11.1 mmol/L D-glucose and 1.6 mmol/L

CaCl2) containing the non-selective cyclooxygenase (COX) inhibitor indomethacin (10

µmol/L), to inhibit the production of prostanoids. Small mesenteric arteries (third order

branch of the superior mesenteric artery, internal diameter ~300 µm) were isolated, cleared of

fat and connective tissue and dissected into rings of about 2 mm in length and then mounted

on a Mulvany-Halpern myograph (model 610M, Danish Myo Technology, Aarhus,

Denmark). After the rings were mounted, they were allowed to stabilize at zero tension for 15

min before normalization. The blood vessels were stretched to attain an internal pressure

comparable to 90% of that of the blood vessel under a transmural pressure of 100 mmHg

(McPherson, 1992, Mulvany and Halpern, 1977) to determine the passive tension-internal

circumference. All experiments were conducted at 37ºC in the Krebs solution bubbled with

carbogen (95% O2 and 5% CO2).

6.2.4 Investigation of vascular function and reactivity

After equilibration for 30 minutes, blood vessels were subjected to maximum

contraction by the addition of an isotonic physiological saline solution containing a high

concentration of K+

(123 mmol/L, KPSS). Basal tension of vessels was restored after several

washouts using Krebs solution. In order to investigate the integrity of the endothelium, the

vessels were precontracted with phenylephrine (0.1-3 µmol/L) to ~50% of the KPSS response

and relaxed with a high single dose of acetylcholine (ACh, 10 µmol/L). ACh-induced

relaxation of the precontracted rings was more than 80% in all cases indicating that the

161

endothelium was functionally intact. After several washouts, the mesenteric arteries were

precontracted again with phenylephrine (0.1-3 µmol/L) and cumulative concentration-

response curves to the endothelium-dependent relaxant agonist, ACh (0.1 nmol/L-10 µmol/L)

and the endothelium-independent relaxant agonist, sodium nitroprusside (SNP, 0.01 nmoL/l-

10 µmol/L) were examined. Furthermore, after 20 minutes, responses to ACh and SNP were

investigated with different combinations of N-nitro-L-arginine (L-NNA, 100 μmol/L), an

inhibitor of nitric oxide synthase (NOS), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

(ODQ, 10 µmol/L), an inhibitor of soluble guanylate cyclase (sGC), apamin (1 µmol/L), a

selective blocker of the small calcium-activated K+

channel (SKCa), 1-[(2-chlorophenyl)

(diphenyl)methyl]-1H-pyrazole (TRAM-34, 1 µmol/L), a blocker of the intermediate

calcium-activated K+

channel (IKCa), and iberiotoxin (Ibtx, 100 nmol/L), a blocker of the

large calcium-activated K+

channel (BKCa).

6.2.5 Estimation of basal release of NO in mesenteric arteries

In order to investigate the effect of diabetes on the basal level of NO release,

endothelium- intact blood vessels were exposed to L-NNA (100 µmol/L) after precontraction

with phenylephrine (10-100 nmol/L) to ~ 20% KPSS. , A contractile response to L-NNA was

considered to indicate inhibition of basal release of NO (Leo et al., 2011b).

6.2.6 Western Blot

Western blots were performed by collecting the mesenteric arteries from normal and

diabetic rats. From the same treatment group, the endothelium-intact small mesenteric

arteries from 2 animals were pooled and considered as n =1. The same amount of protein

homogenate of the treatment animal groups were exposed to SDS-PAGE and analyzed

162

through western blot processes with mouse/rabbit primary antibodies (all 1:1000, overnight,

4oC) includes endothelial NO synthase (eNOS), Nox2 (all BD Transduction Laboratories,

Lexington, KY, USA). The membranes were reinvestigated with a loading control antibody

(actin), in order to normalize for the amount of protein. After 1 hour incubation at room

temperature with anti-mouse/rabbit secondary antibody (1:2000) (Millipore, Billerica, MA,

USA), all proteins were identified by enhanced chemiluminescence reagents (Amersham, GE

Healthcare, Sydney, NSW, Australia). Densitometry (Biorad Chemidoc, Sydney, NSW,

Australia) was used to quantify the protein bands expressed as a ratio of the loading control.

To examine tissue eNOS ratio of monomer/dimer formation, 6% SDS-PAGE at 4ºC used to

resolve a non-boiled sample and the membranes were examined and imaged as described

above.

6.2.7 Measurement of superoxide release by mesenteric arteries

To measure superoxide generation in mesenteric arteries, lucigenin-enhanced

chemiluminescence was used. The superior mesenteric artery was isolated, cleared of fat and

connective tissue and incubated for 45 minutes at 37ºC in Krebs- HEPES buffer solution

containing NADPH (100 µmol/L) as a substrate for NADPH-oxidase in the presence or

absence of diphenyliodonium (DPI, 5 µmol/L), a flavoprotein inhibitor that inhibits NADPH

oxidase. 300 µL of Krebs-HEPES buffer, containing lucigenin (5 µM) and the suitable

treatment were placed into a 96-well Optiplate. The Optiplate was loaded into a Polarstar

Optima photon counter (BMG Labtech, Melbourne, VIC, Australia) to measure background

photon emission at 37ºC. After the background reading was recorded, a single ring section of

mesenteric artery was placed into each well and photon emission was recounted. The

mesenteric arteries were placed in Krebs-HEPES buffer that contain either lucigenin (5 µM)

alone or with the presence of NADPH (100 μM) to measure the production of NADPH

163

oxidase- driven superoxide. The signal stimulated by NADPH was measured in the presence

or absence of DPI (5 µM). The background counts were subtracted from superoxide counts

and normalized with dry tissue weight.

6.2.8 Materials

Linagliptin was a gift from (Boehringer Ingelheim, Germany). All other chemicals

were purchased from Sigma-Aldrich (St Louis, MO, USA), except for ODQ (Cayman

Chemical, Ann Arbor, MI, USA), and acetylcholine perchlorate (BDH Chemicals, Poole,

Dorset, UK). All drugs were dissolved in distilled water with the exception of indomethacin,

which was dissolved in 0.1 M sodium carbonate, and L-NNA, which was dissolved in 0.1 M

sodium bicarbonate.


Data were expressed as mean ± SEM, n indicates the number of experiments. The

concentration response data from rat isolated mesenteric arteries were fitted to a sigmoidal

curve using nonlinear regression (Prism version 6.0, GraphPad Software, San Diego, CA,

USA) to calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP was determined

as a percentage of the phenylephrine precontraction. Differences between groups of pEC50

and Rmax values were compared and analysed using two way ANOVA with post hoc analysis

using Bonferroni’s test. p<0.05 was considered statistically significant.

164

6.3 Results

6.3.1 Body weights and blood glucose

The body weight, blood glucose and HbA1c levels of rats in the 4 groups are shown in

Table 1. Ten weeks after treatment with streptozotocin or vehicle, the final body weight in

normal rats was significantly higher than in diabetic rats (Table 6.1). Furthermore, in diabetic

rats, the blood glucose and HbA1c levels were significantly greater than in normal rats. Four

weeks treatment with linagliptin had no significant effect on body weight, HbA1c or blood

glucose levels in either normal or diabetic rats.

6.3.2 Effect of linagliptin on vascular superoxide production

The superoxide level in mesenteric arteries was measured by lucigenin-enhanced

chemiluminescence. In diabetic rats, the level of superoxide-induced fluorescence was

significantly higher than that in normal rats (Figure 6.1). After 4 weeks of treatment with

linagliptin, the level of NADPH oxidase-driven superoxide was significantly attenuated in

diabetic rats, but there was no effect in normal rats (Figure 6.1). In all groups,

diphenyliodonium was able to inhibit NADPH oxidase-driven superoxide production in the

mesenteric arteries (Figure 6.1).

165

Table 6.1 Mean body weight gained, blood glucose and HbA1c levels at the end of the experiment of

normal and diabetic rats with or without linagliptin (2 mg/kg oral gavage daily for 4 weeks) treatment.

10 weeks after vehicle or STZ treatment

n Normal n Normal+linagliptin n Diabetic n Diabetic+linagliptin

Body weight

(g)

8

498±18Ф

9

538±21Ф

11

346±11*¥

11

379±25*¥

Blood glucose

(mM)

8

5.1±0.3Ф

8

4.5±0.2Ф

11

26.4±2*¥

12

27.8±1.7*¥

HbA1c

(%)

7

6.2±0.2Ф

8

5.8±0.2Ф

10

14.8±0.6*¥

12

13.9±0.4*¥

n= the number of rats. *Significantly different from normal group (Bonferroni’s test, p<0.05), Ф

Significantly different to diabetic group, p<0.05, Bonferroni’s test. ¥

Significantly different to

Normal+linagliptin group, p<0.05, Bonferroni’s test. Results are shown as mean±SEM.

166

6.3.3 Effect of diabetes and linagliptin on vascular function

Both diabetes and linagliptin treatment had no effect on the level of contraction to the

high K+ physiological saline solution (KPSS, 123 mmol/L) (Figure 6.2). The sensitivity to

ACh but not the maximum relaxation was significantly reduced in mesenteric arteries from

diabetic rats (Figure 6.3a, Table 6.2), whereas the sensitivity and the maximum relaxation to

sodium nitroprusside (SNP) were not affected (Figure 6.3b). 4 weeks of linagliptin treatment

(2 mg/kg per daily, oral gavage) did not affect the response to ACh in mesenteric arteries

from normal rats, but in diabetic rats treated with linagliptin, the sensitivity to ACh was

significantly increased in comparison to the sensitivity and response to ACh in mesenteric

arteries from diabetic untreated rats (Figure 6.3a, Table 6.2). The treatment with linagliptin

had no effect on the response to SNP in normal rats or diabetic rats (Figure 6.3b).

The basal level of NO release was investigated by assessing the level of contraction

induced by L-NNA in mesenteric arteries precontracted with PE to 20% of the KPSS

response In normal mesenteric arteries the L-NNA- induced contraction was significantly

higher than that with diabetic mesenteric arteries (Figure 6.3c) showing that diabetes reduced

the basal release of NO. Treatment with linagliptin significantly increased the L-NNA-

induced contraction in diabetic mesenteric arteries compared to untreated diabetic arteries but

had no effect on L-NNA-induced contraction in normal mesenteric arteries (Figure 6.3c).

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Figure 6.1 ROS measurement in intact mesenteric arteries. NADPH activity was raised in diabetic

mesenteric arteries which were reduced with linagliptin treatment. Results are shown as mean±SEM.

n=7-10 experiments. *p<0.05 vs normal, #p<0.05 vs diabetic.

168

Figure 6.2 Mesenteric arteries were induced to maximum contraction by KPSS. Exposure of

mesenteric arteries from both normal and diabetic rats with or without linagliptin (2 mg/kg per day

orally for 4 weeks) treatment to KPSS (123 mM). Diabetes or linagliptin treatment did not affect the

level of contraction of the mesenteric arteries.

169


ACh (a), SNP (b), and basal NO release (c) in endothelium-intact mesenteric arteries. In each group

(a, b), mesenteric arteries were pre-contracted with PE to a similar level: (a) normal 66±2,

normal+linagliptin 64±1, diabetic 65±1, diabetic+linagliptin 66±1, (b) normal 64±1,

normal+linagliptin 64±1, diabetic 61±6, diabetic+linagliptin 66±1 %KPSS, n=7-10 experiments.

Results are shown as mean±SEM. **p<0.01, ***p<0.001. See Table 2 or results section for pEC50

and Rmax values.

170

6.3.4 Determination of the relative contribution of NO and EDH to endothelium-

dependent relaxation

In mesenteric arteries from normal rats, the ACh-induced relaxation was partially

reduced by either the combination of N-nitro-L-arginine (L-NNA), a NO synthase inhibitor

and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase

inhibitor or inhibitors of intermediate-conductance calcium-activated K+ channel (IKCa),

small-conductance calcium-activated K+ channel (SKCa) with 1-[(2

chlorophenyl)(diphenyl)methyl]1H pyrazole (TRAM-34), apamin and iberiotoxin (Ibtx)

respectively, confirming that both NO and EDH were involved in endothelium-dependent

relaxation (Figure 6.4, Table 6.2). Further, the response to ACh-induced relaxation in

mesenteric arteries from diabetic rats was significantly further reduced in the presence of

either NO inhibitors or EDH inhibitors in comparison to normal rats, indicating that the

contribution of both NO and EDH to endothelium-dependent relaxation were impaired by

diabetes.

6.3.5 Effect of linagliptin on NO- and EDH-mediated relaxation in the mesenteric

arteries

The response to ACh was assessed in the presence of TRAM-34+apamin in order to

investigate the contribution of NO to endothelium- dependent relaxation. Treatment with

linagliptin for 4 weeks (2 mg/kg, daily oral gavage), significantly increased the maximum

relaxation to ACh in mesenteric arteries from diabetic rats but had no effect on responses in

normal rat arteries (Figure 6.4, Table 6.2) indicating that linagliptin could restore the

contribution of NO to endothelium-dependent relaxation in diabetes.

171

To investigate the contribution of EDH-mediated relaxation, the actions of NO were

inhibited by the addition of L-NNA and ODQ. Treatment of normal and diabetic rats for 4

weeks with linagliptin (2 mg/kg, daily oral gavage) significantly increased on ACh-induced

EDH mediated relaxation diabetic arteries but not in arteries from normal rats (Figure 6.5,

Table 6.2).

172

Table 6.2 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of mesenteric arteries from normal and diabetic rats with or

without linagliptin (2 mg/kg oral gavage daily for 4 weeks) treatment in the presence of indomethacin.

Normal Normal+linagliptin Diabetic Diabetic+linagliptin

ACh

n

pEC50 Rmax (%)

n

pEC50 Rmax (%)

n

pEC50 Rmax (%)

n

pEC50 Rmax(%)

Control

8

7.26±0.13

99±0

9

7.02±0.12

94±3

10

6.14±0.11Ф¥

96±1

10

6.84±0.13¥

97±0

TRAM34+apamin

7

6.52±0.06*

91±2

7

6.29±0.10*

95±1

9

6.08±0.14

35±9*Ф¥

9

6.59±0.18

96±2¥£

L-NNA+ODQ

7

6.31±0.09*

95±0

7

6.45±0.09*

94±0

8

5.59±0.05* Ф¥

81±7

8

6.38±0.10¥£

95±0

LNNA+ODQ+

TRAM-34+apamin

7 5.62±0.29* 27±8

* 4 5.35±0.21

* 45±7

* 4 5.45±0.06

*

4±0* Ф¥

7 5.35±0.11* 2±1

* Ф

LNNA+ODQ+

TRAM-34+apa+Ibtx

4

5.27±0.17*

14±2*

4

5.31±0.07*

25±8*

ND

ND


µM)+apamin (1 µM), L-NNA (100 µM)+ODQ(10 µM), L-NNA (100 µM)+ODQ(10 µM)+ TRAM-34 (1 µM) +apamin (1 µM) or L-NNA (100

µM)+ODQ(10 µM)+ TRAM-34 (1 µM) +apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments were performed

in the presence of indomethacin (10 µM). n = the number of experiments.

*Significantly different to control within each group (p<0.05), Bonferroni’s test, Ф

Significantly different to normal within inhibitor group, p<0.05, Bonferroni’s test.

¥Significantly different to normal+linagliptin within inhibitor group, p<0.05, Bonferroni’s test.

£Significantly different to diabetic within inhibitor group, p<0.05, Bonferroni’s test.

Results are shown as mean±SEM, ND= Not determined.

173

Figure 6.4 Relative contribution of NO to endothelium-dependent relaxation. Mesenteric arteries to

examine NO-mediated relaxation in the presence of TRAM-34+apamin were isolated from normal (a),

diabetic (b), normal+linagliptin (c), diabetic+linagliptin (d) rats. In each group of experiments, arteries

were precontracted with PE to similar levels: 63±2 (a), 65±0.6 (b), 65±1 (c), 63±1 (d) %KPSS, n=8-9

experiments. Results are shown as mean±SEM. See Table 6.2 for values.

174

Figure 6.5 Contribution of EDH to endothelium-dependent relaxation. Mesenteric arteries to examine

EDH-mediated relaxation in the presence of L-NNA+ODQ were isolated from normal (a), diabetic (b),

normal+linagliptin (c), diabetic+linagliptin (d) rats. In each group of experiments, arteries were

precontracted with PE to similar levels: 62±0.7 (a), 65±0.8 (b), 62±2 (c), 64±0.8 (d) %KPSS, n=8-9

experiments. Results are shown as mean±SEM. See Table 2 for values.

175

6.3.6 Effect of linagliptin on Nox2, total-eNOS and eNOS monomer/dimer

In diabetic rats, the expression of total eNOS in the mesenteric arteries was significantly

reduced as was the proportion of eNOS expressed as the dimer and the expression of Nox2 was

significantly increased (Figure 6.6). Treatment with linagliptin did not affect the expression or

dimerization of eNOS or the expression of Nox2 in normal rats. By contrast, in diabetic

mesenteric arteries after linagliptin treatment there was a significant increase in eNOS, both in

total and as a dimer, and a decrease in Nox2 expression

176

Figure 6.6 Western blot analysis of protein expression of eNOS (a, 130 kDa), eNOS dimers and

monomers (b, 260 kDa) and Nox2 (c, 58 kDa) in normal and diabetic mesenteric arteries with or without

linagliptin treatment. In diabetic mesenteric arteries, the expression of eNOS significantly reduced and the

proportion of eNOS expressed as the dimer reduced, and the expression of Nox2 increased. Treatment

with linagliptin increased the expression of eNOS significantly and reduced Nox2 expression and

increased the proportion of eNOS expressed as the dimer. Representative blots are shown on each of the

corresponding graphs. n=6 experiments. Results are shown as mean±s.e.m. *p<0.05, **p<0.01,

***p<0.001, ****p<0.0001.

177

6.4 Discussion

This study showed that treatment of type1 STZ-induced diabetic rats with the DPP-4

inhibitor linagliptin (2 mg/Kg, daily oral gavage) for 4 weeks decreases the levels of vascular

oxidative stress and improves endothelium-dependent relaxation in mesenteric arteries.

Importantly in this type 1 diabetes model the beneficial vascular effects of linagliptin were

achieved without affecting plasma concentrations of glucose or HbA1c demonstrating that

linagliptin has actions in addition to the capacity to lower plasma glucose. A similar dose of

linagliptin (1 mg/kg po) to that used in this study (2 mg/kg po) has been demonstrated to

effectively inhibit DPP-4 activity and to increase GLP-1 in Zucker diabetic fatty rats (Jones et

al., 2014) but in this type 1 model of diabetes where the beta pancreatic cells are destroyed by

necrosis it is not possible to elevate insulin secretion to decrease glucose. The dysfunction of

endothelial cells in the mesenteric arteries of diabetic rats is accompanied with an impairment of

the contribution of both NO and EDH-mediated relaxation to endothelium-dependent relaxation.

Treatment of diabetic rats with linagliptin improves the contribution of NO-mediated relaxation

to endothelium-dependent relaxation which associated with an increased expression of total

eNOS, decreased eNOS uncoupling and downregulation of Nox2 expression. In addition, the

contribution of EDH to endothelium-dependent relaxation in diabetic mesenteric arteries was

improved by the treatment with linagliptin.

6.4.1. Effect of linagliptin on vascular endothelial function

In the present study, Type 1 STZ-induced diabetes increased the level of vascular ROS

generation concomitant with a selective impairment of endothelium-dependent relaxation and an

increase in the expression of Nox2 in the mesenteric arteries consistent with previous reports (De

178

Vriese et al., 2000, Leo et al., 2011b, Ding et al., 2005). In diabetic rats, the in vivo treatment

with the DPP-4 inhibitor linagliptin (2 mg/kg, per day, oral gavage) for 4 weeks improved

endothelium-dependent relaxation in mesenteric arteries and decreased the generation levels of

oxidative stress associated with decreased Nox2 and improved coupling of eNOS in the

mesenteric arteries of diabetic rats. Our observations are similar to that of other observations that

have shown that treatment with the DPP-4 inhibitor linagliptin protects against cardiovascular

injury induced by salt-sensitive hypertension via a reduction of oxidative stress (Koibuchi et al.,

2014) or improves endothelial function and reduces vascular stress in experimental sepsis in

large conduit aortic artery (Kroller-Schon et al., 2012). Whilst there are various reports that the

DPP-4 inhibitor, linagliptin could ameliorate endothelial dysfunction induced by diabetes,

however, the mechanism of the vasoprotective effect of linagliptin remains ambiguous

particularly on the contribution of NO and EDH to endothelium-dependent relaxation in

diabetes.

6.4.2. Effects of linagliptin on NO-mediated relaxation in rats mesenteric arteries

In order to investigate NO-mediated relaxation, the EDH-mediated relaxation was

blocked by endothelial KCa channel blockers. This clearly showed impaired release of NO. Under

those conditions of blocking NO-mediate relaxation, the response to ACh and the maximum

relaxation was significantly reduced in the mesenteric arteries from diabetic rats in comparison

to mesenteric arteries from normal rats. Chronic treatment with linagliptin in vivo for 4 weeks

significantly increased the maximum relaxation to ACh in diabetic mesenteric arteries in

comparison to untreated diabetic mesenteric arteries. Furthermore, beside stimulated NO release,

basal NO release was also reduced in mesenteric arteries of diabetic rats, in response to NOS

179

inhibition the contraction was impaired and this was reversed in arteries from linagliptin treated

diabetic rats. Therefore, treatment with linagliptin was able to preserve the contribution of NO-

mediated relaxation to the endothelium-dependent relaxation in diabetic mesenteric arteries. In

our results, consistent with the impairment of NO mediated-relaxation we found that in the

diabetic mesenteric arteries the expression of eNOS was reduced and the proportion of eNOS

expressed as a dimer was decreased which indicated uncoupling of the total eNOS in the diabetic

mesenteric arteries. In addition, in linagliptin treated diabetic rats, the expression of eNOS was

comparable to the normal rats. Moreover, treatment with linagliptin stimulated the re-coupling of

eNOS, which was expressed as a dimer more than monomer compared to untreated mesenteric

arteries in diabetic rats and this normalization of eNOS expression and re-coupling of eNOS by

chronic treatment with linagliptin would elucidate the improvement of the endothelium-

dependent relaxation in mesenteric arteries of diabetic rats. A further factor to the repair of the

activity of NO by chronic treatment with liangliptin could be the decrease of vascular superoxide

generation in diabetic mesenteric arteries, and this could then increase the bioactivity of NO

through avoiding the forming of peroxynitrite (ONOO-) via the degradation of NO by superoxide

action. The reduction of ROS activity and prevention of peroxynitrite formation in diabetic

vasculature could be either due to the rapid free radical scavenging effect of linagliptin in order

to preserve the bioavailability of NO linagliptin through the inhibition of xanthine oxidase

(Yamagishi et al., 2014) or by reducing the expression and/or activity of the enzymatic source of

superoxide generation in the diabetic mesenteric arteries such as NAPDH oxidase subunit Nox2.

Taken together, chronic treatment with liangliptin reserved the beneficial activity of NO via

improving of eNOS expression, re-coupling of eNOS and reducing the expression of Nox2

which mediate superoxide generation in the mesenteric arteries of diabetic rats.

180

6.4.3 Effect of linagliptin on EDH-mediated relaxation in rat mesenteric arteries

The endothelium-dependent relaxation in rat mesenteric arteries is mediated by NO,

classical EDH and non-classical EDH pathways (Edwards et al., 2010). In order to evaluate the

contribution of EDH to endothelium-dependent relaxation in diabetes, we investigated the

endothelium-dependent relaxation in the presence of L-NNA and ODQ inhibitions to block NO

formation and sGC activity respectively. In addition to inhibit NOS, the guanylate cyclase

inhibitor was also used to confirm the inhibition of the action of NO derived from nitrosothiols, a

non-NOS sources of NO, which was earlier described to act as a source of NO in diabetic

conditions (Leo et al., 2010). The sensitivity to ACh in diabetic mesenteric arteries was

decreased significantly in comparison to normal mesenteric arteries in the presence of L-

NNA+ODQ, indicating that diabetes impaired the contribution of EDH to endothelium-

dependent relaxation, which is consistent with other reports (Leo et al., 2011b, Fukao et al.,

1997, Matsumoto et al., 2003, Matsumoto et al., 2005). Chronic in vivo treatment with

linagliptin improved EDH-mediated relaxation in mesenteric arteries of diabetic rats, indicating

that treatment with linagliptin preserved the contribution of EDH to endothelium-dependent

relaxation in diabetes. Although the main cause of the impairment of EDH in diabetic conditions

remains uncertain (Leo et al., 2011b, Matsumoto et al., 2003, Matsumoto et al., 2006, Makino et

al., 2000, Burnham et al., 2006a, Weston et al., 2008), however, is partly associated with over

generation of superoxide and oxidative stress. It is reported that auto-oxidation of pyrogallol

causes an increase in superoxide anions which could impair EDH-mediated relaxation in rat

mesenteric arteries (Ma et al., 2008). Our results in this study demonstrated that the impairment

of EDH-mediated relaxation in diabetes may be in part due to the overproduction of superoxide

181

and treatment of diabetic rats with lingaliptin improved the contribution of EDH to endothelium-

dependent relaxation in mesenteric arteries which could be as a result of the reduction of

superoxide levels by linagliptin treatment.

6.4.4 Conclusion

In this study we have demonstrated that the DPP-4 inhibitor linagliptin improves

endothelial function in mesenteric arteries in diabetic rats by preserving the activities of both NO

and EDH-mediated relaxation. In order to reduce inactivation of NO, linagliptin has protective

actions via at least two mechanisms. The DPP-4 inhibitor linagliptin was able to reduce the

generation of superoxide which may be due to direct radical scavenging action and/or attenuating

the enzymatic source for superoxide generation where the chronic treatment of diabetic rats with

liangliptin in vivo for 4 weeks prevents the uncoupling of eNOS, increases the expression of total

eNOS, and decreases Nox2, hence facilitates maintenance of endothelium-dependent relaxation.

Interestingly, the beneficial effects of linagliptin to preserve NO activity and improve EDH role

in relaxation demonstrated in this study displays that linagliptin has the ability to act as a

therapeutic compound independent of its glucose lowering action for use in the prevention of the

microvasculature complications of diabetes.

183

Chapter 7

General Discussion

7.1 Effects of diabetes and WD on vascular function

Diabetes mellitus is one of the fast growing diseases all over the world both in developed

and developing countries (Dunstan et al., 2002, Wild et al., 2004). Diabetes affects all the organs

in the body, however cardiovascular complications are a major cause of high prevalence of

morbidity and mortality in individuals with diabetes (Boyle et al., 2010). Vascular endothelial

function is identified as a vital independent risk factor for cardiovascular disease. In diabetes, the

endothelial dysfunction plays a critical role in the initiation and development of cardiovascular

complications (De Vriese et al., 2000, Halcox et al., 2002, Al Suwaidi et al., 2000). In this

thesis, we investigated the mechanisms underlying endothelial dysfunction over the course of

expose to high glucose concentration, STZ-induced type-1 diabetes, and western diet feeding

regime.

It is widely acknowledged that hyperglycaemia causes an increase in oxidative stress

which is known to cause endothelial dysfunction and is unfavourably related to an imbalance

between NO and superoxide production in vascular endothelial cells. In investigations of small

blood vessels (mesenteric arteries) exposed to high glucose, our findings (Chapter 3) indicate

that there was an endothelial dysfunction related to an increase in ROS production and

impairment in NO and EDH-mediated relaxation. Further investigation showed that the vascular

endothelial function improved by increased bioavailability of NO derived from eNOS pathway

184

and improved EDH-type response which leads to increasing the contribution of these mediated

relaxation factors to the endothelium-dependent relaxation in microvasculature. The results also

suggested that both NO and EDH-mediated relaxation are vital modulators of vascular health and

pharmacological compounds targeted to increase the bioavailability of NO and improve EDH-

type response could be used to maintain vascular endothelial function under the pathological

condition of diabetes.

In diabetes, prolonged hyperglycaemia causes overgeneration of ROS causing oxidative

stress which leads to an impairment of the action of NO and EDH-mediated relaxation and

subsequent endothelial dysfunction (De Vriese et al., 2000, Fatehi-Hassanabad et al., 2010)

(Chapters 4 and 6). During diabetes, hyperglycaemia results in oxidative stress via the increase in

NADPH oxidase expression/activity and uncoupling of eNOS that causes impairment of vascular

endothelial function. This impairment in endothelial function was concomitant with a reduction

in the contribution of both NO and EDH-mediated relaxation to the endothelium-dependent

relaxation. This indicates that the increase in the expression/activity of NADPH oxidase and

eNOS uncoupling are early contributors to hyperglycaemia-induced endothelial dysfunction. The

increase in expression/activity of endothelial NADPH-oxidase, suggests that the observed

impairment of EDH-type response may also be due to oxidative stress as a result of

overgeneration of superoxide in the microvasculature which cause KCa-channels (IKCa and SKCa)

alteration and interruption of downstream reactions of these channels (Leo et al., 2011b,

Burnham et al., 2006a) (Chapters 4 and 6). Thus, therapies targeting ROS derived from NADPH-

oxidase and eNOS uncoupling could have the potential to prevent microvascular complications

and endothelial dysfunction concomitant with diabetes.

185

Metabolic syndrome is generally defined as a combination of risk factors or abnormalities

strongly associated with obesity and insulin resistance which are noticeably associated with the

increase in the risk of development of cardiovascular disease and diabetes mellitus (Stein and

Colditz, 2004). ROS production is proposed to have an important role in the development of

insulin resistance (Furukawa et al., 2004, Lin et al., 2005, Urakawa et al., 2003, Diniz et al.,

2006). This study (Chapter 5) showed that the consumption of high fat diet (western diet) caused

endothelial dysfunction in the mesenteric microvasculature. The findings showed that the ROS

production in small blood vessels was significantly increased due to the consumption of western

diet. This increase in oxidative stress was through the increase in the expression/activity of

NADPH-oxidase and eNOS uncoupling that leading to vascular endothelial dysfunction. These

findings suggest that ROS production may be the early event promoting western diet-induced

pathologies and therefore may be an attractive therapeutic targeted for inhibiting insulin

resistance and obesity caused by consumption of high fat diet or western diet.

7.2 Protection of diabetes or WD-induced endothelial dysfunction by the DPP-4

inhibitors

Exposure of blood vessels to high glucose induced oxidative stress, that has been

demonstrated as a primary cause of premature vascular disease (Cosentino et al., 1997,

Cosentino et al., 2003, Tesfamariam and Cohen, 1992). A recent study demonstrated that the

DPP-4 inhibitor linagliptin, currently used as an antihyperglycaemic agent, was markedly more

potent than other DPP-4 inhibitors in its anitioxidant capacity (Kroller-Schon et al., 2012). In

this study (Chapter 3), the acute effects of the DPP-4 inhibitors linaglitin, sitagliptin and

vildagliptin and the GLP-1 receptor agonist exendin-4 were examined on the endothelial function

186

of blood vessels exposed to high glucose. The findings demonstrated that high glucose impaired

endothelium-dependent, but not endothelium-independent, relaxation in rat mesenteric arteries,

due to an increased production of superoxide and the acute treatment with linagliptin and

exendin-4 reduced vascular superoxide and improved endothelium-dependent relaxation and the

beneficial effect on endothelium-dependent relaxation of exendin-4, but not linagliptin, was

mediated by the GLP-1 receptor. Also, the NO and EDH-mediated relaxation were both impaired

in the presence of a high glucose concentration and the actions of both mediators were improved

by linagliptin. Taken together, the findings suggested that linagliptin and exendin-4 have

antioxidant effects that may not be shared with other DPP-4 inhibitors. This antioxidant action

may have additional benefit to the glucose lowering effects of linagliptin in the treatment of

diabetes.

In Chapter 4, the acute effect of the DPP-4 inhibitor linagliptin in vitro on microvascular

function from STZ-induced type 1 diabetic rats was investigated, demonstrating that acute

treatment with linagliptin markedly reduce the high level of superoxide and improved endothelial

function in diabetes. Endothelial dysfunction in the diabetic rats was associated with impairment

in the modulatory role of NO and EDH-mediated relaxation. Acute treatment with linagliptin in

vitro improved the contribution of both NO and EDH to the endothelium-dependent relaxation in

small blood vessels from the diabetic rats, indicating that the DPP-4 inhibitor has potential as

therapeutic agent for use in the reduction and/or prevention of microvascular complications

associated with diabetes beyond its current use in type 2 diabetes. In Chapter 5, the acute

treatment with DPP-4 inhibitor linagliptin in vitro on the endothelial function of small blood

vessels from western diet fed rats was investigated. The observed findings indicated that

superoxide production is increased in mesenteric arteries from western diet fed rats and in the

187

acute presence of linagliptin, the vascular superoxide production was significantly reduced.

Western diet caused endothelial dysfunction and both the NO and the EDH-mediated relaxation

was impaired in the microvasculature. The results showed that the acute presence of linagliptin

improved endothelium-dependent relaxation in the mesenteric arteries from western high fat diet

fed rats by selectively preserving the contribution of both NO and EDH-mediate relaxation. This

indicates that linagliptin could be in favour of the prevention of the pathological events caused

by western diet including the development of endothelial dysfunction in small arteries.

In Chapter 6, treatment with linagliptin in vivo in STZ-induced type 1 diabetic rats had no

effect on the plasma glucose level, indicating that the DPP-4 inhibitor improves the function in

blood vessels independently of a glucose lowering pathway. Furthermore, in normal animals,

treatment with linagliptin had no effect on vascular function. The beneficial action of linaglipin,

when administered in vivo may have at least two mechanisms of action. The linagliptin may

exhibiting its protective action via either as an antioxidant by rapidly directly scavenging ROS

and/or inhibit the enzymatic source for superoxide production (i.e. NADPH oxidase and eNOS

uncoupling) to preserve NO bioavailability or through a GLP-1 receptor dependent pathway

through increased activation of GLP-1R secondary to DPP-4 inhibition. The beneficial action of

linagliptin via its ability to reduce the oxidative stress in diabetic vasculature by inhibiting

superoxide-derived from NADPH oxidase and eNOS uncoupling makes the DPP-4 inhibitor

linagliptin a potential adjunctive treatment for diabetic vascular complications.

188

7.3 Future directions

Whilst the acute relaxant effects of the GLP-1 agonist exendin-4 are of interest, of great

significance is the investigation of the capacity of long-term chronic treatment with GLP-1

agonist exendin-4 to improve endothelial function in conditions of cardiovascular risk including

diabetes and western, high fat diet. This would help in better understanding of the effects of their

effect on endothelial function.

Acute treatment of small blood vessels from western high fat diet fed rats with the DPP-4

inhibitor linagliptin markedly improved vascular function may have been by inhibiting the

activity of NADPH oxidase derived ROS. Hence, future experiments would be useful to

investigate the effects of long-term chronic treatment with linagliptin on endothelial function in

conditions of western high fat diet.

The observations of preclinical studies have indicated a possible beneficial cardiovascular

action of DPP-4 inhibitors through both glucagon-like peptide 1 (GLP-1)-dependent and GLP-1-

independent pathways. Clinically, DPP-4 inhibitors improve several risk factors such as

improvement of blood glucose homeostasis, reduced blood pressure, improve postprandial

lipaemia, reduced inflammatory markers, reduced oxidative stress, and improved endothelial

function in individuals with type 2 diabetes. Some beneficial effect was also observed in patients

with ischaemic heart disease or congestive heart failure (Bose et al., 2005, Anagnostis et al.,

2011, Jose and Inzucchi, 2012). However, the actual relationship between the beneficial effects

of DPP-4 inhibitors such as linagliptin and cardiovascular events remains to be demonstrated,

thus investigation of the effect of DPP-4 inhibitor linagliptin on the ischemic/reperfusion cardiac

injury would help insight into the effects of gliptins such as linagliptin on cardiovascular

outcomes.

189

7.4 Conclusion

Taken together, the set of studies conducted in the present thesis have contributed to

better understanding of the beneficial action of the DPP-4 inhibitor linagliptin on endothelial

function independent of decreasing blood glucose levels. The data presented in the thesis

provides an insight into the effects of diabetic conditions and western high fat diet on the

mechanism(s) of endothelium-dependent relaxation. Also, data obtained in this thesis would help

in understanding the mechanism of action of the DPP-4 inhibitor linagliptin used in the treatment

of endothelial dysfunction in disorders such as diabetes and metabolic syndrome. Endothelial

dysfunction is the initial step of the commencement of cardiovascular complication such as

atherosclerosis. Conservation of endothelial function would delay and/or avoid complications of

cardiovascular risk factors and reduce the prevalence of the mortality and morbidity among

diabetic patients. As the DPP-4 inhibitor linagliptin treatment has demonstrated valuable effects

in improving endothelial function, it may be a useful adjunct therapy in the treatment of

endothelial dysfunction in diabetes independent of lowering blood glucose. This would make the

range of patients able to benefit from the use of this drug to be greatly expanded.

191

Chapter 8

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