study of vascular endothelial growth factor (vegf) and bcl 2 protein levels in serum and vitreous...

STUDY OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)

AND BCL-2 PROTEIN LEVELS IN SERUM AND VITREOUS HUMOR

Of PATIENTS WITH PROLIFERATIVE DIABETIC

RETINOPATHY

by

Ola Hussein Aly Elgaddar

ADVISORS

Prof. Dr. Ahmad Mohamad Zaki Professor of Chemical Pathology

Medical Research Institute

Alexandria University

Prof. Dr. Ahmad Magdy Bedda Professor of Ophthalmology

Faculty of Medicine

Alexandria University

Dr. Amel Abd El-Fattah Kamel Assistant Professor of Chemical Pathology

Medical Research Institute

Alexandria University

Dr. Hoda Ali El-Attar Assistant Professor of Chemical Pathology

Medical Research Institute

Alexandria University

INTRODUCTION INTRODUCTION

DIABETIC RETINOPATHY (DR)

Normal vision DR vision

DR is a devastating microvascular complication

of diabetes mellitus.

It is considered the leading cause for adult

blindness.

Its prevalence among diabetic patients in Egypt

is 42%.

Risk factors includes: poor glycemic control,

duration of diabetes, hypertension,

hyperlipidemia and proteinuria.

DR can be classified into:

Early non-proliferative diabetic

retinopathy

Advanced non-proliferative diabetic

retinopathy

Proliferative diabetic retinopathy

Non-proliferative Diabetic Retinopathy

Proliferative Diabetic Retinopathy

Hypoxia occurring early in the course of DR

triggers the release of several growth

factors that promote retinal

neovascularization.

Among these growth factors are:

Insulin-like growth factor-I (IGF-I)

Basic fibroblast growth factor (bFGF)

Hepatocyte growth factor (HGF)

Vascular endothelial growth factor (VEGF)

VASCULAR ENDOTHELIAL

GROWTH FACTOR (VEGF)

VEGF-A is a 34- to 42-kDa dimeric

glycoprotein.

It is a member of the VEGF family that

currently comprises seven members;

VEGF-A (hereafter referred to as VEGF),

VEGF-B, VEGF-C, VEGF-D, VEGF-E,

VEGF-F, and placental growth factor (PlGF)

Monomer

Monomer Dimer

VEGF STRUCTURE

ACTIVITIES OF VEGF

A. Mitogenesis, angiogenesis, and endothelial

cell survival.

B. Stimulatory effect on bone marrow cells and

hematopoiesis.

C. Enhancement of vascular permeability.

REGULATION OF

VEGF PRODUCTION

I) Hypoxia: The main stimulus

II) Growth factors & hormones: PlGF, TNF- α, bFGF, TGF-ß, PDGF,Ang-1, Ang-2,

IGF-1, IL-1 and IL-6

TSH, ACTH, estrogens & progestins

III) Glucose: Hyperglycemia or hypoglycemia???

VEGF RECEPTORS

Receptor tyrosine kinases family (RTKs):

VEGFR-1 (Flt-1)

VEGFR-2 (KDR)

VEGFR-3 (Flt-4)

Co receptors:

Neuropilin-1&-2

VEGF IN PATHOLOGICAL CONDITIONS

A) Solid & hematological tumors:

As in Lung, breast, renal, ovarian and

intracranial tumors as well as in some

lymphomas and leukemias.

In these tumors, VEGF Induces Bcl-2 production

thus increasing tumor cells survival.

B) Intraocular neovascular syndromes:

Like Diabetes mellitus, occlusion of central retinal vein and neonatal prematurity.

Retinal ischemia is the main stimulus for VEGF production in such conditions.

In the eye, VEGF is produced by retinal pericytes, endothelial cells and glial cells.

It leads to ocular neovascularization, hemorrhages and vascular permeability, which results in visual impairment/blindness.

B-Cell lymphocyte/leukemia-2

(Bcl-2)

Bcl-2 family members function as

regulators of apoptosis.

All family members share the presence of

at least one Bcl-2 homology (BH)

domain.

BCL-2 PROTEIN

Bcl-2 is the founding antiapoptotic member of this family of proteins.

It is a 25 KDa protein.

It is a membrane protein that is localized to the outer mitochondrial membrane, endoplasmic reticulum membrane, and nuclear envelope.

Lymphoid germinal center.

Proliferative precursor cells in the bone marrow.

Glandular epithelium of the breast, thyroid & prostate.

Normally, it is expressed in cells and

tissues characterized by apoptotic turn

over, like:

Pathologically, Bcl-2 is over expressed in

many malignant & non-malignant conditions:

Malignant

• B- Cell lymphoma

• Ovarian cancer

• HCC

• Malignant melanoma

• Breast cancer

• Colorectal cancer

• Lung cancer

• Kaposi sarcoma

Non-malignant

• Epilepsy

• Endometriosis

• Liver cirrhosis

• Multiple sclerosis

1) Blocking the release of cytochrome- c from the

mitochondria to the cytosol.

2) Inhibition of the proapoptotic effects of the Bcl-2

family proteins (e.g., Bax and Bak).

3) Maintenance of sufficient Ca++ in the

sarcoplasmic/endoplasmic reticulum and

mitochondria.

4) Direct antioxidant activity.

The mechanisms by which Bcl-2 suppresses apoptosis

So……

Is there a role for VEGF and Bcl-2

in the pathogenesis of PDR?

Is it possible that the angiogenic effect of VEGF in PDR is via Bcl-2 up regulation?

AIM OF THE WORK

This study aimed at evaluating the

levels of VEGF and Bcl-2 protein

in the serum and vitreous humor

of patients with proliferative

diabetic retinopathy

SUBJECTS

SUBJECTS

40 subjects were included in the present study divided as follow:

Patients group

25 subjects with PDR

Undergoing vitrectomy

Control group

15 non-diabetic subjects

Undergoing vitrectomy

METHODS

To all studied subjects the following was done:

I) Full clinical examination.

II) Laboratory investigations:

Preliminary tests in serum.

(F.B.G, RFTs, lipid profile & aminotransferases activities)

Measurement of glycated hemoglobin (Hb A1C

) by resin chromatography technique.

Estimation of serum and vitreous humor levels of both VEGF & Bcl-2 by ELISA technique.

VITREOUS HUMOR SAMPLING

VITREOUS HUMOR

A clear avascular gel which occupies the posterior compartment of the eye.

It has the following composition:

• Water (99%)

• Network of collagen fibrils

• Large molecules of hyaluronic acid

• Peripheral cells (hyalocytes)

• Inorganic salts, sugar and ascorbic acid

VITRECTOMY

RESULTS

0

20

40

60

80

100

120

140

160

180

mg/ dl

1 2

Controls Patients

F.B.G in the studied groups

VEGF IN THE VITREOUS HUMOR

PatientsControls

Vitre

ou

s V

EG

F (p

g/m

l)

500

400

300

200

100

0

p= 0.001

VEGF IN THE SERUM

PatientsControls

Se

ru

m V

EG

F (p

g/m

l)

500

400

300

200

100

0

BCL-2 IN THE VITREOUS HUMOR

p= 0.003

PatientsControls

Vitre

ou

s B

cl-2

(n

g/m

l)

35

30

25

20

15

10

5

0

-5

p= 0.001

BCL-2 IN THE SERUM

PatientsControls

Se

ru

m B

cl-2

(n

g/m

l)

35

30

25

20

15

10

5

0

CORRELATION BETWEEN SERUM

VEGF AND BCL-2 IN THE PATIENTS

Serum VEGF (pg/ml)

7006005004003002001000

Se

ru

m B

cl-2

(n

g/m

l)

50

40

30

20

10

0

CONCLUSIONS

From the present study the following could be concluded:

1) Levels of VEGF and Bcl-2 were

significantly higher in the vitreous

humor of patients with PDR when

compared to their corresponding levels

in the control group suggesting that both

factors are incriminated in the pathogenesis

of this disease.

2) VEGF and Bcl-2 did not show any

statistical difference in the serum of the

studied groups. This finding may support

the hypothesis that their increased levels

in the vitreous is probably attributed to

intraocular synthesis, in response to local

retinal hypoxia, rather than to serum filtration.

3) A significant positive correlation was

found between serum Bcl-2 and serum

VEGF in the proliferative diabetic

retinopathy patients. This might suggest

that the role of VEGF in inducing

pathological angiogenesis in PDR might be

in part due to up regulation of the anti-

apoptotic protein Bcl-2.

RECOMMENDATIONS

VEGF together with Bcl-2 will hopefully lead

to the discovery of new targets for future

therapy for proliferative diabetic

retinopathy as well as other diseases with a

neovascular component.

THANK YOU