European Journal of Pharmacology 669 (2011) 1–6

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European Journal of Pharmacology

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Review

The promise of EPC-based therapies on vascular dysfunction in diabetes

Adriana Georgescu a,b,⁎, Nicoleta Alexandru a,b, Andrei Constantinescu b, Irina Titorencu a,b, Doina Popov b

a ‘Petru Poni’ Institute of Macromolecular Chemistry, Iasi, Romaniab Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of Romanian Academy, Bucharest, Romania

⁎ Corresponding author at: Cellular Physiology anInstitute of Cellular Biology and Pathology ‘Nicolae Simio8, BP Hasdeu Street, PO Box 35-14, 050568-Bucharest, Rofax: +40 21 319 4519.

E-mail address: adriana.georgescu@icbp.ro (A. GeorgURL: http://www.icbp.ro (A. Georgescu).

0014-2999/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.ejphar.2011.07.035

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 February 2011Received in revised form 29 June 2011Accepted 21 July 2011Available online 5 August 2011

Keywords:DiabetesVascular dysfunctionCirculating endothelial progenitor cell

Diabetes mellitus is one of the most common metabolic diseases in the world and the vascular dysfunctionrepresents a challenging clinical problem. In diabetes, endothelial cells (ECs), lining the inner wall of bloodvessels, do not function properly and contribute to impaired vascular function. Circulating endothelialprogenitor cells (EPCs), the precursor of mature EC, actively participate in endothelial repair, by moving to thevascular injury site to formmature EC and new blood vessels. Knowing that the therapeutic interventions canimprove only a part of EC dysfunction in diabetes, this review addresses recent findings on the use of EPCs forcell therapy. The strategies proposed in review are based on in vivo and in vitro studies and, thus, theirphysiological relevance is confirmed. EPC therapy shows great promise for the prevention and cure ofdiabetes-induced vascular dysfunction.

d Pharmacology Department,nescu’ of Romanian Academy,mania. Tel.: +40 21 319 4518;

escu).

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Endothelial physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Endothelial cell dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24. Endothelial progenitor cells (EPCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25. The role of EPCs in vascular dysfunction in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26. The mechanisms underlying EPC reduction in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37. EPC as biomarkers and therapeutic strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1. Introduction

Diabetes mellitus is a clinical condition characterized by early andwidespread endothelial dysfunction (McVeigh et al., 1992; Simionescuet al., 2005). By means of soluble factors, which can alternativelymediate vasoconstriction or vasodilation, the endothelium is cruciallyinvolved in the maintenance of adequate vascular tone and function.The term endothelial dysfunction refers to a condition in whichendothelium loses its physiological ability to promote vasodilation,

fibrinolysis and anti-aggregation. The responsible factors for theendothelial dysfunction are vasodilators: nitric oxide (NO), prostacyclin,endothelium-derived hyperpolarizing factor (EDHF), and vasoconstric-tors and growth-promoting substances such as: superoxide anions,endoperoxides, thromboxane A2, endothelin-1, and angiotensin II. Thecontribution of each of these signals varies from a type of blood vessel toanother.

In addition to well known mechanisms by which diabetes inducesendothelial dysfunction, some evidences indicate that alterations innumber or function of bone marrow-derived endothelial progenitorcells (EPCs) are involved in the pathogenesis of vascular complica-tions in diabetes. EPCs are a heterogeneous subpopulation of bonemarrow mononuclear cells with an enhanced potential for differen-tiation within the endothelial cell lineage. In response to vascularinjury, EPCs are mobilized from the bone marrow to the peripheralcirculation, and home to the sites of new vessel growth, where they

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become incorporated into the growing vasculature (Roncalli et al.,2008).

2. Endothelial physiology

The endothelial cells (ECs) line the internal lumen of blood vesselsand serve as a biological barrier between the blood and vascularsmooth muscle cell (VSMC) layer of the wall. The physiologicalfunction of ECs includes the modulation of vascular tone (vasocon-striction and vasodilation), hemostasis, regulation of growth anddifferentiation of VSMC, andmodulation of inflammation (Singh et al.,2010). ECs modulate vascular tone by regulating the release ofvasodilators such as NO and EDHF and vasoconstrictors such asendothelin-1, prostaglandin H2 (Fleming and Busse, 1999), reactiveoxygen species (ROS), angiotensin II, and thromboxane A2 (Schiffrin,2001). ECs express also a range of adhesionmolecules such as ICAM-1,VCAM-1, and selectins (E, P and L-selectin) (Hwang et al., 1997).These molecules are modulated by ECs to regulate the dissociation ofleukocytes (Miyamoto et al., 1997) and platelets from the vascularbed by release of NO (Colwell and Nesto, 2003). As a major regulatorof local vascular homeostasis, the endothelium maintains the balancebetween vasodilatation and vasoconstriction, the inhibition andpromotion of proliferation and migration of VSMCs, the preventionand stimulation of adhesion and aggregation of platelets, as well asthrombogenesis and fibrinolysis. Upsetting this tightly regulatedbalance leads to endothelial dysfunction (Davignon and Ganz, 2004).

3. Endothelial cell dysfunction

A lot of reports directly link the diabetic vascular complications toendothelial dysfunction. These studies are based on data from animalmodels, as well as from clinical trials. EC dysfunction occurs early indiabetes and insulin resistance condition (Madonna and De Caterina,2011). Thedefinitionof endothelial dysfunctionvariesdependingon theorgan studied; however, in general, endothelial dysfunction is charac-terized by impaired endothelium dependent dilatation to agonists, toshear stress, or to local ischemia. In general, diabetic microvascularcomplications are typically associated with dysregulation of vascularremodeling and vascular growth, with decreased responsiveness toischemic/hypoxic stimuli, and impaired or abnormal neovasculariza-tion. Lack of endothelial regeneration and impaired angiogenesiscontribute to the progression of diabetic micro- and macrovascularcomplications. The presence of endothelial dysfunction was assessedalso in hypertensive streptozotocin injected mice. The results demon-strated a diminished reactivity of the renal arteries in response to10−4 M noradrenaline, 10−4 M acetylcholine, and 10−4 M sodiumnitroprusside (Georgescu et al., 2007). Also, on the experimental modelof simultaneously hyperlipemic–hyperglycemic hamster (Simionescuet al., 1996), the vascular reactivity ofmesenteric resistance arterieswasfound tobemodified, essentially in termsof an enhanced contractility toPGF2α (Georgescu and Popov, 2003) and a diminished endothelium-dependent vasodilation (Georgescu et al., 2001).

When testing the effects of depolarizing K+ (64.1 mM) both phasicand tonic components of K+ stimulated contractionwere diminished inthe resistance arteries of hyperlipemic hamster, and were particularlyreduced in hyperlipemic–hyperglycemic hamster (Georgescu andPopov, 2001). It was reported that both normal biological aging anddiabetes induced in aged hamsters conduct to the dysfunction ofresistance arteries (Georgescu et al., 2003). The endothelial dysfunctionmay generate various pathophysiological complications such asenhanced expression of adhesion molecules resulting in increasedleukocyte–endothelial cell adhesions (Goldberg, 2009), promotion of aprocoagulant state as a result of increased activation of platelets andclotting factors (Ding and Triggle, 2005), and impaired NO release(Georgescu et al., 2011). These conditions may conduct to defective

modulation of vascular growth and remodeling in the vesselwall (Rudicand Sessa, 1999; Spinetti et al., 2008).

Currently, the clinical management of diabetic complications reliesexclusively on pharmacological therapeutic that minimally affects theendothelial repair or regeneration. These treatments have modestinfluence on end organ dysfunction. Hence, there is a need fortherapeutic interventions aimed to accelerate the repair of dysfunc-tional ECs and to restore the blood flow, resulting in the functionaltissue generation. A rapid progression of EPCs from the “bench to thebedside” occurred via translational studies even in the absence of aconsensus about the true identity of EPCs (Jarajapu and Grant, 2010).

4. Endothelial progenitor cells (EPCs)

The definition and biology of EPCs are complex and under a heavydebate. Today, the term EPCs is used for a heterogeneous group ofcells including circulating and culture-differentiated cells. Protocolsfor enumeration and cultivation are as heterogeneous as the cellsthemselves (Steinmetz et al., 2010). Bone marrow derived EPCs play acritical role in vascular maintenance and repair. There is still greatdispute about the most appropriate markers that define an EPC. EPCscan be isolated using cell sorting by surface phenotype selection or invitro cell culture (Li Calzi et al., 2010).

Asahara et al. (1997) described first early EPCs that mainlyconsisted of CD34-derived cells. Today, it is known that EPCs could bereleased from bone marrow, fat tissue, vessel wall (especiallyadventitia) and possibly spleen, liver and intestine. EPCs enter theblood as circulating EPCs, where they express CD133 (at the earlystage), then CD34/Flk-1, and also VEGFR2 (Xu, 2007). EPCs as definedby the depicted markers can be further mobilized to contribute toendothelial repair, but can also promote plaque growth, neovascular-ization and instability (Hristov and Weber, 2009). Clinically, thenumber and function of EPCs may reflect the balance betweenendothelial integrity and repair; both measures have been suggestedas surrogate markers of endothelial function and cardiovasculardiseases (Hamed et al., 2010).

5. The role of EPCs in vascular dysfunction in diabetes

The possible role for EPCs in diabetic vascular disease was firstinvestigated in mice. Infusion of human CD34-positive leukocytes, asan EPC-enriched population, accelerated the blood flow restoration indiabetic nude mice with experimental hindlimb ischemia (Harraz etal., 2001). Decreased angiogenic potential of EPCs has been demon-strated in diabetic animals (Tamarat et al., 2004). Also, reduction incirculating EPCs and functional impairment of cultured EPCs havebeen reported both in type 1 and type 2 diabetic patients. It wasshowed that peripheral blood mononuclear cell (PBMC)-derived EPCsisolated from type 2 diabetic (Tepper et al., 2002) and type 1 diabeticpatients (Loomans et al., 2004) displayed a reduced proliferation ratein culture, compared to control subjects, a weaker adherence toactivated human umbilical vein endothelial cells (HUVECs) and areduced incorporation into vascular structures in vitro. The rate of EPCproliferation from plated PBMCs in diabetic patients was inverselycorrelated with the levels of glycated hemoglobin, suggesting apossible relation between glucose control and EPC function. Reducedadhesion of EPCs to HUVECs demonstrated altered cell-to-cellinteractions which could indicate that EPCs are recruited less avidlyin vivo at sites of ischemia, as well that the reendothelization bymeans of bone-marrow derived cells is less likely to take place in thepresence of EPC dysfunction. Lambiase et al. (2004) have shown that apoor coronary collateral development (typical for diabetes), may berelated to low levels of circulating EPCs. Also, patients with diabetesmellitus and high high-sensitivity C-reactive protein (hs-CRP) levelsshowed a marked decrease in the number of EPCs compared withnon-diabetic patients with low hs-CRP levels (Koshikawa et al., 2010).

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Recently, it was demonstrated that insulin signaling and EPC survivalare impaired in Zucker fatty insulin resistant rats and that this defectcan be significantly ameliorated by a knockdown of NF-κB (Desouzaet al., 2011).

6. The mechanisms underlying EPC reduction in diabetes

In diabetes, mechanisms underlying EPC reduction are largelyunknown. To date, weak bone marrow mobilization, impairedperipheral differentiation, and short survival in peripheral blood arethe candidates. Several factors, such as granulocyte colony-stimulat-ing factor (G-CSF), stromal cell derived factor-1 (SDF-1), VEGF,erythropoietin (Epo, via AKT protein kinase pathway activation andeNOS), and receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), α4 integrin, CXC chemokine receptor-2 and -4 (CXCR2,4), and β1-and β2-integrins, were demonstrated to mediate EPC mobilization,proliferation, migration and their differentiation and homing to sites

Fig. 1. Contribution of EPCs in vascular repair in diabetes. In a diabetic environment proteifactors are increased above non-diabetic levels while NO is reduced. This results in a blunted rsecreted soluble factors, like hypoxia-inducible factor-1α (HIF-1α) and stromal cell-deriprogenitor cells from bone marrow (or fat tissue, vessel wall, especially adventitia and pmobilized from bone marrow by cytokines, growth factors, hormones, chemokines and diabetissue, EPCs adhere to the vessel wall and migrate along gradients of chemotactic factors (blood vessels mainly via an angiogenesis mechanism. (EPCs—endothelial progenitor cells;oxide; ROS—reactive oxygen species; VEGF—vascular endothelial growth factor; SDF-1—str

of vascular injury (Abou-Saleh et al., 2009) (Fig. 1). In particular, itwas found that the expression of angiogenic factors VEGF andhypoxia-inducible factor-1 (HIF-1) is reduced in the hearts of diabeticpatients during acute coronary syndromes, and that expression ofHIF-1 gene is decreased in hyperglycemic conditions and is associatedwith myocardial infarct size in rat (Marfella et al., 2002, 2004). SDF-1,also known as CXCL12, and its receptor CXCR4 play a critical role inregulating hematopoietic cell trafficking (Wieczorek et al., 2005). Innon-obese diabetic mice, the onset of diabetes is significantly delayedby reducing the level of SDF-1, either by antibody-mediatedneutralization or G-CSF-induced suppression of SDF-1 transcription(Kared et al., 2005; Semerad et al., 2005). Despite these initialobservations, however, how chemokine SDF-1 affects the develop-ment of type 1 diabetes has not been fully investigated. Also,numerous data underline that EPCs might decrease because ofincreased apoptosis. Bruhl et al. (2004) revealed a dose-dependentrelation between levels of p21Cip1, that control cell cycle progression

n kinase C (PKC), reactive oxygen species (ROS), proinflammatory and antiangiogenicesponse to vascular injury andmarginal repair at the site of injury. In response to injury,ved factor-1 (SDF-1), induce proliferation, differentiation, and liberation of vascularossibly spleen, liver, intestine) into blood. EPCs circulate in peripheral blood and aretic conditions. EPCs then migrate to the site of injury and initiate blood vessel repair. Ine.g. VEGF, SDF-1, P-selectin, integrins). Circulating EPCs contribute to repair of injuredECs—endothelial cells; SMCs—smooth muscle cells; PKC—protein kinase C; NO—nitricomal cell derived factor 1).

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and apoptosis in mature ECs, and the level of circulating EPCs indouble and single p12Cip1 knockout mice. In rats with streptozotocininduced diabetes, the reduced circulating EPC levels were associatedwith uncoupled eNOS in bone marrow (Thum et al., 2007). Also, thereare data which show that EPCs are more protected against oxidativestress compared to mature ECs, and therefore it seems unlikely thatdecrease and dysfunction of EPCs are mediated by an increasedoxidative stress (Dernbach et al., 2004). Furthermore, EPC dysfunctionin type 2 diabetes patients was associated with oxidative stress due toexcessive generation of reactive oxygen species (ROS). It was showedthat prolonged exposure of EPCs to high glucose concentrations invitro increased superoxide anion production, and reduced NObioavailability (Hamed et al., 2009). Generation of superoxide anionsappears by several processes that include glucose auto-oxidation, andincreased protein kinase C (PKC) and NAD(P)H oxidase activity(Galasso et al., 2006). Moreover, in diabetic patients, the presence ofAGE adducts on the basement membrane compromises the EPC repairfunction, a process with implications for vasodegeneration during themicrovasculopathy (Bhatwadekar et al., 2008). In vitro studies onthrombosis model, indicated that platelets influence EPC recruitmentto sites of vascular injury, adhesion and activation and promote theirdifferentiation to an endothelial phenotype. The platelet adhesionconstitutes an essential step for the targeting of EPCs to sites ofendothelial dysfunction (Stellos et al., 2008). Recently, it wasdemonstrated that EPCs bind platelets via P-Selectin and inhibitplatelet activation, aggregation, adhesion to collagen in vitro andthrombus formation in vivo, predominantly by upregulation of COX-2and secretion of PGI2 (Abou-Saleh et al., 2009). It was showed thathealthy volunteer platelets provide a source of soluble factors toimprove the number and function of EPCs from patients withcardiovascular risk factors, particularly diabetes mellitus (Dernbachet al., 2008).

7. EPC as biomarkers and therapeutic strategies

EPCs have recently generated great attention as potential noveldiagnostic/prognostic biomarkers for vascular integrity and thera-peutic clinical approaches, and the use of these cells is ongoing(Hristov and Weber, 2007). Although still not well assessed, EPCnumber and function could be regarded as a surrogate marker forvascular endothelial function. In diabetic patients, the use ofantioxidants and/or other medications, such as prostacyclin or statins,can enhance EPC number and function at least throughNO-dependentmechanisms (Hamed et al., 2010). Notably, ACE inhibitors such as,ramipril (Min et al., 2004), enalapril (Wang et al., 2005) and AT IIinhibitors, like valsartan (Bahlmann et al., 2005) were shown toincrease EPC levels both in the diabetic experimental model and inpatients, probably interfering with the D26/dipeptidylpeptidase IVsystem. Thus, it appears that the vasculoprotective effect of thesecompounds may be partly due to their action on EPCs.

Other studies suggested that either PI3-K/Akt/eNOS/NO signalingpathway or the interaction between hyperglycemia and hyperlipid-emia in diabetes patients, is a potential therapeutic target forabolishing the impaired function of EPCs and for restoring theirneovascularization capacity (Hamed et al., 2010). Ceradini et al.(2008) demonstrated that prevention of hyperglycemia-induced ROSgeneration significantly improved EPC-induced revascularization inischemic tissues in genetically-engineered diabetic mice that over-expressed superoxide dismutases (SOD), or after treating diabeticmicewith SOD. Neutralization of the p66ShcAgene, which regulates theapoptotic response to oxidative stress, prevented high glucose-induced EPC impairment in vitro (Di Stefano et al., 2009). Ohshimaet al. (2009) reported that the antioxidant therapy with SOD indiabetic mice reduced oxidative stress, and increased EPC count andtheir potential to differentiate into ECs. It was reported that treatingglucose-stressed EPCs with SOD restored their proliferative ability

through a NO-dependent mechanism and it was suggested that theextent of interaction between NO and superoxide anion is importantto the development of EPC dysfunction; the resultant product,peroxynitrite, can reduce the EPC count and impair their proliferation(Hamed et al., 2009).

In addition, a new inhibitor of CXCR4, AMD3100, was found toaccelerate blood flow restoration to ischemic tissue in diabetic mice(Jiao et al., 2006). Also, the treatment with AMD3100 in diabeticpatients improved wound healing by correcting EPC mobilization andhoming (Marchac et al., 2010). AMD3100 is now approved for use as amobilization agent of EPCs in United States; new data provide enticingevidence regarding its therapeutic effect in human myocardialinfarction (Jujoa et al., 2010).

Another approach to improve the vascular dysfunction could bethe therapy developed by using EPC transplantation. The possibility ofcellular therapy with EPCs for treatment of diabetic macular ischemiaand the vasodegenerative phase of diabetic retinopathywas discussedby Li Calzi et al. (2010). In a very recent study it was demonstratedthat intravenous administration of circulating human EPCs hasbeneficial effects on ischemic brain injury in a mouse model oftransient middle cerebral artery occlusion (Fan et al., 2010).Transplantation of human cord blood-derived EPCs was reported tocontribute to neovascularization in various ischemic diseases. Also, itwas showed that EPC transplantation on diabetic wounds has abeneficial effect mainly achieved by their direct paracrine action onkeratinocytes, fibroblasts, and ECs, rather than through their physicalengraftment into host tissues (vasculogenesis). In addition, EPC-conditioned medium was shown to be therapeutically equivalent toEPCs, at least for the treatment of diabetic dermal wounds (Kim et al.,2010). Also, it was found that EPCs given to Zucker fatty rats decreaseneointimal hyperplasia post-carotid angioplasty (Desouza et al.,2011).

8. Concluding remarks

Diabetes-associated vascular complications are a growing concernworldwide and a major clinical problem. There is thus an urgent needto characterize the appropriate diagnostic markers that can providean early prognostic indicator of developing vascular disease. Endo-thelial dysfunction is an early indicator of vascular disease and maydirectly or indirectly be associated with EC senescence, and decreasesin the number and viability of EPCs. Assays of the status of EPCs arelikely to prove to be critical for assessing the health of vascular system,and interventions that enhance EPC number and restore angiogenicactivity in diabetes may prove to be particularly beneficial.

Acknowledgments

Our studies are supported by CNCSIS–UEFISCSU, project number1159, PNII—IDEI code 1043/2008, by CNMP project number 42138,PNII—Parteneriat code 3334/2008, by European Social Fund—“Cristo-for I. Simionescu” Postdoctoral Fellowship Programme (ID POS-DRU/89/1.5/S/55216), Sectoral Operational Programme HumanResources Development 2007–2013, by CNCSIS–UEFISCSU, Pro-gramme Human Resources, Postdoctoral Research Project; projectnumber 124/6.08.2010, code PD134 and by Romanian Academy.

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