Thrombosis Research 130 (2012) S90–S94

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Thrombosis Research

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Pro-angiogenic cell-based therapy for the treatment of ischemiccardiovascular diseases☆

Jean-Sébastien Silvestre ⁎Paris Cardiovascular Research Center, INSERM U970, Université Paris Descartes, 56 rue Leblanc, 75015 Paris, France

☆ Manuscript will be presented at the 22nd Internati6–9 October 2012, Nice, France.⁎ Tel.: +33 1 53988060; fax: +33 1 53987951.

E-mail address: jean-sebastien.silvestre@inserm.fr.

0049-3848/$ – see front matter © 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.thromres.2012.08.287

a b s t r a c t

Pro-angiogenic cell therapy has emerged as a promising option to treat patients with acute myocardial infarc-tion or with critical limb ischemia. Exciting pre-clinical studies have prompted the initiation of numerousclinical trials based on administration of stem/progenitor cells with pro-angiogenic potential. Most of theclinical studies performed so far have used bone marrow-derived or peripheral blood-derived mononuclearcells and showed, overall, a modest but significant benefit on tissue remodeling and function in patients withischemic diseases. These mixed results pave the way for the development of strategies to overcome the lim-itation of autologous cell therapy and to propose more efficient approaches. Such strategies includepretreatment of cells with activators to augment cell recruitment and survival in the ischemic target areaand/or the improvement of cell functions such as their paracrine ability to release proangiogenic factorsand vasoactive molecules. In addition, efforts should be directed towards stimulation of both angiogenesisand vessel maturation, the development of a composite product consisting of stem/progenitor cells encapsu-lated in a biomaterial and the use of additional sources of regenerative cells.

© 2012 Elsevier Ltd. All rights reserved.

Introduction

Insufficient organ perfusion following thrombotic vessel obstructionof the feeding artery is a major determinant of post-ischemic remodel-ing, ultimately leading to atrophy of the affected territory, importantloss of function and serious health consequences. Although the promptre-establishment of a patent artery has significantly reduced subse-quent complications and mortality, deleterious remodeling still occurssince this therapy cannot be offered to a substantial proportion of pa-tients with acute disease. In addition, insufficient neovascularizationleading to tissue hypoperfusion is an integral component of tissue re-modeling and loss of organ function following ischemic injury. Hence,therapeutic angiogenesis is viewed as a highly promising strategy to en-sure revascularization of ischemic tissues by promoting the growth ofnew vessels or the maturation of pre-existing ones.

Advances in the field of vascular biology lead to the discovery of pu-tative circulating endothelial progenitor cells (EPCs) in adults [1] andhas triggered a massive amount of research regarding EPCs biologyand their therapeutic potential for ischemic diseases, mainly in patientswith acute myocardial (MI) or with critical limb ischemia (CLI), in thebeginning of the past decade [2,3]. EPCs mainly originate from thebone marrow, but extramedullary EPCs can also be recruited towardsischemic tissues [4]. Consequently, whole bone marrow derived

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mononuclear cells (BMCs) or medullar cell selected on differentmarkers (CD34+, CXCR4+, Lin-ckit+…) have often been used assource of EPCs for pre-clinical studies of cell therapy for therapeutic an-giogenesis. Nevertheless, the true identity of EPCs is still under debate.Indeed, although efforts have recently been made to standardize thecell surface markers, isolation procedure and phenotypic propertiesthat define bona-fide EPCs [5,2], a large number of different EPCs orEPCs-like populations have been used in experimental or clinical stud-ies, and hamper a comprehensive understanding of the existing litera-ture. Typically, these cells are defined on the basis of expression of cellsurface markers such as CD34, Flk-1 and CD-133 but EPCs appear tobe a heterogeneous group of cells originating from multiple precursorsand present in different stages of endothelial differentiation in periph-eral blood. At least two types of EPCs with divergent properties can beobtained in vitro [5]. “Early” EPCs possess a strong paracrine activitybut no paracrine potential, while “Late” EPCs have low paracrine activ-ity, but can incorporate into newly formed vessels [5]. Interestingly,both cell types can promote post-ischemic angiogenesis, and act syner-gistically when co-transplanted [6].

Mechanisms of EPCs-induced vascular regeneration

Although a substantial number of studies have demonstrated thepro-angiogenic and therapeutic effect of EPCs in experimental modelsof MI and CLI, the mechanisms of EPCs-induced neovascularization re-main undefined [3]. After the seminal discovery of Asahara, the firstmechanism of EPCs-induced angiogenesis to be proposed has been in-corporation of EPCs into newly formed vascular structure, a process

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referred to as post-natal vasculogenesis [1]. However, a first criticalpoint is the identification of cellular mechanisms governing progenitorcells ability to differentiate into EPC and subsequently, endothelial cells.Among these mechanisms, apoptotic or activated cells shed submicronmicroparticles (MPs) released after ischemia have been considered asendogenous signals leading to postischemic vasculogenesis. MPs frommice ischemic hind-limb muscle are detected by electron microscopy48 hours after unilateral femoral artery ligation as vesicles of 0.1- to1-microm diameter. After isolation by sequential centrifugation, flowcytometry analyses show that the annexin V(+) MPs concentration is3.5-fold higher in ischemic calves than control muscles (1392+/−406 versus 394+/−180 annexin V(+) MPs per 1 mg; Pb0.001) andcome mainly from endothelial cells (71% of MPs are CD(144+)). MPsisolated from ischemic muscles induce more potent in vitro BMCs dif-ferentiation into cells with endothelial phenotype than those isolatedfrom control muscles. MPs effects on postischemic revascularizationwere then examined in an ischemic hind-limb model. MPs isolatedfrom ischemic muscles were injected into ischemic legs in parallelwith venous injection of BMCs. MPs increase the proangiogenic effectof BMCs transplantation, and this effect is blunted by NOX2 deficiency.In parallel, BMCs proangiogenic potential also is reduced in ABCA1knockout mice with impaired vesiculation. Hence, MPs produced dur-ing tissue ischemia stimulate progenitor cell differentiation and subse-quently promote postnatal neovascularization [7].

A second critical point is that extensive studies have shown that EPCsincorporation into neovessels was generally low, and it appears nowa-days that EPCs mainly act through the paracrine release of multiple fac-tors and post-natal vasculogenesis cannot solely account for theirpro-angiogenic effects [3]. Nevertheless, differentiation of bonemarrowprogenitors into cells of the vascular lineage is required for theirlong-term beneficial effect after myocardial infarction, as eliminationof bone marrow progenitors expressing an inducible suicide geneunder the control of an endothelial or smooth muscle cells specificgene promoter late after transplantation reverses the original therapeu-tic benefit [8]. The pro-angiogenic effect of EPCs might at least partiallydepend on paracrine signals, as EPCs express various pro-angiogenic cy-tokines such as VEGF or Interleukin-8 [5], and injection of a culture me-dium conditioned by EPCs recapitulates the pro-angiogenic andtherapeutic effects of cell transplantation [9]. In addition, proangiogeniccells may also release vasoactive signals. BMCs and human EPCs derivedfrom cord blood interactwith ischemic femoral arteries through CXCL12and CXCR4 signaling and release nitric oxide (NO) via an endothelialnitric oxide synthase (eNOS)-dependent pathway. BMCs and EPCs-induced NO production promote a marked vasodilation and disruptedvascular endothelial-cadherin/beta-catenin complexes, leading to in-creased vascular permeability. Of note, NO-dependent vasodilationand hyperpermeability are critical for progenitor cells infiltration in is-chemic tissues and their proangiogenic potential in a model of hindlimbischemia in mice [10]. In addition, EPCs also release proteases, such ascathepsin L, and promote a concomitant increase in matrix degradationthat enables endothelial cell migration and vascular remodelling [11].Intramyocardial delivery of BMCs in infarcted mice has been shown toregulate the expression of cardiac MicroRNAs (miRNAs) and down-regulate the proapoptotic miR-34a. Insulin Growth Factor-1 (IGF-1) sig-nificantly inhibits H(2)O(2)-induced miR-34a expression, and miR-34aoverexpression abolishes the antiapoptotic effect of IGF-1 suggestingthat BMCs release IGF-1, which inhibits the processing of miR-34a,thereby blocking cardiomyocyte apoptosis [12]. Finally, EPCs can alsopromote tissue repair and post-MI angiogenesis by inducing recruit-ment of endogenousbonemarrowderived cells or progenitor cells local-ized in the ischemic tissue [13–15].

Clinical trials

After the discovery of EPCs and a tremendous amount of ex-perimental evidence pointing towards EPCs or BMCs therapeutic

potential for the treatment of MI [16], clinical trials evaluating thebenefit of intracoronary administration of BMCs for therapeutic an-giogenesis have been conducted in patients with ischemic diseases[17,18,18–23]. However, clinical trials showed mixed results. For ex-ample, although Schachinger et al demonstrated that intracoronaryBMCs delivery early after MI (3 to 7 days) improved cardiac functionat 4 months and reduced death, recurrence of MI and need for a re-vascularization procedure at 1 year [18], a simultaneously publishedtrial did not show any benefit of early BMCs transplantation at6 months of follow-up [19]. Moreover, the BOOST trial evidenced abenefit of BMCs transplantation after 6 months that was lost at18 months follow up [24]. Recently, intracoronary transplantationof BMCs 2 to 3 weeks after MI do not produce any functional bene-fits [25]. Nevertheless, a 2008 meta-analysis of 13 trials with a totalof more than 800 patients has shown that BMCs transplantation ledto a modest (+2.99%) but significant increase in Left Ventricle (LV)ejection fraction in patients receiving BMCs when compared to pla-cebo [26]. In this line, recently, a total of 50 studies (enrolling 2,625patients) identified by database searches through January 2012were analyzed. Transplantation of adult BMCs improves LV function,infarct size, and remodeling in patients with ischemic heart diseasecompared with standard therapy, and these benefits persist duringlong-term follow-up. BMCs transplantation also reduces the inci-dence of death, recurrent MI, and stent thrombosis in patientswith ischemic heart disease.

Activation of adult stem/progenitor cells regenerative potential

The use of autologous cells is fraught with several hurdles, particu-larly their often defective functionality in patients with atherosclerosisdiseases. Indeed,more than being just causes for coronary artery diseaseand the onset of MI or CLI, cardiovascular risk factors such as hyperten-sion [27], diabetes [28,29] or hyperlipidemia profoundly impair endog-enous and therapeutically induced post-ischemic angiogenesis, andrepresent a major difficulty to circumvent in pro-angiogenic therapy[30]. Along this line, several reports have highlighted bonemarrow pro-genitor cell deficiency in diabetic [31], dyslipidemic [32] patients or inhypertension [33,34]. Hence, in a model of hindlimb ischemia, basalpostischemic neovascularization is reduced in Spontaneously Hyperten-sive Rats (SHR) compared to normotensive animals (WKY). Treatmentswith Angiotensin Converting Enzyme (ACE) inhibitor (perindopril) orangiotensin type 1 receptor blocker (Losartan) or cotreatment withACE inhibitor and diuretic (indapamide) decrease blood pressure levelsand restore vessel growth in SHR to WKY levels. Interestingly, 14 daysafter BMCs transfusion, angiographic scores, capillary density, and footperfusion are decreased by 1.4-, 1.5-, and 1.2-fold, respectively in SHRtransfused with BMCs isolated from SHR compared to those receivingBMCs ofWKY. Alteration in BMCs proangiogenic potential is likely relat-ed to the reduction in their ability tomobilize into peripheral circulation,as revealed by the 2.9-fold decrease in number of circulating CD34+/CD117+ cells and to differentiate into cells with endothelial phenotype,as revealed by the 2.1-fold reduction in percentages of DilLDL/BS-1 lec-tin positive cells. In addition, reactive oxygen species (ROS) levels are in-creased by 2.2-fold in SHRBMCs compared toWKYBMCs, as assessed byL-012 luminescence. Cotreatmentwith ACE inhibitor, angiotensin type 1receptor blocker, or ACE inhibitor and diuretic or antioxidants (NAC3 mmol/L, Apocynin 200 micromol/L) reduced ROS levels, improvedthe number of DilLDL/BS-1 lectin-positive cells and restored BMCsproangiogenic effects in ischemic hindlimb [33,34].

The alternate use of banked allogeneic cells would provide a moreconsistent and readily available product but the expectedly rapid rejec-tion of these cells could only be avoided by an immunosuppressivetreatmentwhich carries its own safety risks. Therefore, our effort shouldbe moved towards the development of strategies that may circumventstem/progenitor cells dysfunction.

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Activation of paracrine potential

First, as previsouly mentioned, the benefits of cell therapy in MI andCLI are predominantlymediated by the paracrine effects of the cells andNOhas been shown to be critical for the proangiogenic function of BMCsand EPCs [10]. Of interest, transgenic eNOS overexpression in diabetic,atherosclerotic, and wild-type mice induced a 1.5- to 2.3-fold increasein postischemic neovascularization compared with control in micewith hindlimb ischemia. eNOS overexpression in diabetic or atheroscle-rotic BMCs restores their reduced proangiogenic potential in ischemichind limb. This effect is associated with a raise in BMCs ability to differ-entiate into cells with endothelial phenotype in vitro and in vivo and anincrease in BMCs paracrine function, including VEGF-A release andNO-dependent vasodilation. Hence, cell-based eNOS gene therapy hasboth proangiogenic and antiatherogenic effects and should be furtherinvestigated for the development of efficient therapeutic neovascu-larization designed to treat ischemic cardiovascular disease [35].

Increase survival

Second, the efficacy of cell therapy is limited by the pathological mi-croenvironment and the fact that few of the intravenously injected cellsaccumulate at sites of tissue damage. Hence, the control of stem/progenitor cells survival may also increase their therapeutic potential.C/EBP homologous protein-10 (CHOP-10), also known as growth arrestand DNA-damage-inducible gene 153 (GADD153), is a member of theCCAAT/enhancer binding proteins (C/EBPs) family of transcriptionalfactors. Of interest, CHOP-10 is a novel developmentally regulated nu-clear protein that emerges as critical transcriptional integrator amongpathways regulating differentiation, proliferation and survival. We ana-lyzed the role of CHOP-10 in postnatal neovascularization. Ischemiawasinduced by right femoral artery ligation in wild-type (WT) andCHOP-10−/− mice. In capillary structure of skeletal muscle, CHOP-10mRNA and protein levels are upregulated by ischemia and diabetes. An-giographic score, capillary density and foot perfusion were increased inCHOP-10−/−mice compared toWT. This effect is associatedwith a re-duction in apoptosis and anupregulation of eNOS levels in ischemic legsof CHOP-10−/−mice compared toWT. In line with these results, eNOSmRNA and protein levels are significantly upregulated in CHOP-10siRNA-transfected human endothelial cells whereas overexpression ofCHOP-10 inhibited basal transcriptional activation of the eNOS promot-er. Using chromatin immunoprecipitation assay, we also showed thatCHOP-10 bound to the eNOS promoter. Interestingly, enhanced post-ischemic neovascularization in CHOP-10−/−mice is fully blunted inCHOP-10/eNOS double knock out animals. Induction of diabetes is asso-ciated with a marked upregulation of CHOP-10 that substantially in-hibits post-ischemic neovascularization. Finally, CHOP-10 is as animportant transcription factor modulating BMCs therapeutic potential.BMCs isolated from control or diabetic mice lacking CHOP-10markedlyimprove post-ischemic vessel growth. These effects are blunted byco-treatment with NOS inhibitor or deficiency of both eNOS andCHOP-10 [36].

Increase recruitment

Alternatively, the control of stem/progenitor cells recruitment tothe ischemic tissue may also increase their therapeutic potential.Erythropoietin-producing humanhepatocellular carcinoma (Eph) recep-tors and their ephrin ligands are key regulators of vascular development.In a nude mouse model of hind limb ischemia, we unraveled that EphB4activation with an ephrin-B2-Fc chimeric protein increases the angio-genic potential of human EPCs. This effect is abolished by EphB4 siRNA,confirming that it is mediated by EphB4. EphB4 activation enhances Pselectin glycoprotein ligand-1 (PSGL-1) expression and EPCs adhesion.Inhibition of PSGL-1 by siRNA reverses the proangiogenic and adhesiveeffects of EphB4 activation. Moreover, neutralizing antibodies to E

selectin and P selectin block ephrin-B2-Fc-stimulated EPC adhesionproperties. Thus, activation of EphB4 enhances EPC proangiogenic capac-ity through induction of PSGL-1 expression and adhesion to E selectinand P selectin [37]. Recently, this approach has been validated in amore relevant clinical situation. Treatment of peripheral bloodmononu-clear cells (PB-MNCs) from diabetic patients with ephrin-B2/Fc has beenshown to improve their proangiogenic therapeutic potential in diabeticischemic experimental models. Indeed, paw skin blood flow, angio-graphic score, and capillary density are significantly increased in ische-mic leg of diabetic mice receiving ephrin-B2/Fc-activated diabeticPB-MNCs versus those receiving nontreated diabetic PB-MNCs. ephrin-B2/Fc binds to PB-MNCs and increases the adhesion and transmigrationof PB-MNCs. Finally, ephrin-B2/Fc-activated PB-MNCs raise the numberof circulating vascular progenitor cells in diabetic nudemice and increasethe ability of endogenous BMCs to differentiate into cells with endothe-lial phenotype and enhance their proangiogenic potential. Therefore,ephrin-B2/Fc treatment of PB-MNCs abrogates the diabetes-inducedstem/progenitor cell dysfunction and opens a new avenue for the clinicaldevelopment of an innovative and accessible strategy in diabetic patientswith critical ischemic diseases [38].

Use of alternative sources of stem cells for vasculartissue regeneration

Resident Cardiac Progenitor Cells

Although the myocardium is generally considered to possess poorregenerative ability, the description of multipotent resident cardiacprogenitor cells (CPCs) isolated from human and mammalian adulthearts [39] has offered a new alternative for regenerative therapeu-tics for MI. Similarly to EPCs, consensus is lacking regarding the trueidentity of CPCs as several markers (Lin-Ckit+, sca1+, Isl+) or isola-tion procedures (direct isolation followed by expansion or isolation ofCPCs arising from cardiospheres during in vitro culture of myocardialbiopsies) have been used so far [39]. Nevertheless, it is generally rec-ognized that CPCs have the ability to differentiate in several cardiaclineages including myocytes, vascular endothelial and smooth musclecells in vitro and in vivo, and might thus promote neovascularizationas well as cardiomyocyte repopulation of the injuredmyocardium. Al-though the therapeutic effect of CPCs in animal models of MI has beendocumented [40,41], the mechanisms mediating their therapeutic ef-fect, and mainly their ability to differentiate into cardiomyocytes orvascular cells in vivo is still controversial, as preclinical studies havereported inconsistent results [42]. Therapeutic benefit of CPCs trans-plantation might mostly rely on their paracrine activity, as intra-coronary infusion of CPCs in a rat model of MI resulted in very lowrates of exogenous cell incorporation [41]. Recently, the therapeuticuse of CPCs in MI patients has been evaluated in two phase 1 trials[43,44]. Although the primary endpoint of these studies was to assessthe safety of CPCs transplantation, both reported encouraging results.Indeed, the SCIPIO trial reported increased LV ejection fraction in theCPCs treated patients one year after cell infusion, while in theCADUCEUS study, CPCs delivery led to reduced scar mass, increasedviable heart mass, regional contractility and regional systolic wallthickening without significantly improving ejection fraction.

Induced pluripotent Stem Cells

A potential breakthrough in regenerative medicine has emergedfrom the discovery that adult somatic stem cells such as skin fibroblastscould be reprogrammed to a pluripotent stem cell state by as few as 4distinct factors [45]. These cells, termed induced Pluripotent Stemcells (iPS), were subsequently shown to possess the ability to differen-tiate in cardiac lineage cells in vitro, including cardiomyocytes, vascularendothelial and mural cells [46]. iPS were notably able to promote re-pair of ischemic myocardium after infarction, where they differentiated

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into cardiomyocytes, endothelial cells and smooth muscle cells [47].Endothelial cells differentiated in vitro from iPSwere also shown to pro-mote angiogenesis in a hindlimb ischemia model [48]. Although iPS ob-viously represent a potentially valuable tool for cell therapy aftermyocardial infarction, as they would provide an autologous source ofcells with multilineage differentiation capacity, further research iswarranted before therapeutic clinical use can be considered. One im-portant concern is the ability of iPS to form teratomas in vivo.Moreover,patient specific generation of iPS is a timely process which might notcompute with the requirement for acute therapy after an ischemicevent [49].

Alternative sources of stem cells

Other endogenous adult progenitor cell types, and in particularMes-enchymal Stem Cells and Adipose Tissue Derived Stem Cells [50] havebeen endowed with vasculogenic or at least pro-angiogenic potentialand have been successfully used to promote post-ischemic angiogenesisin experimental studies [50,51].

Futur directions

First, in addition to the dysfunction of regenerative cells observedin patients with MI or CLI, it is likely that methodological caveats suchas way of delivery or isolation/culture procedures for stem/progenitorcells might partially explain the failure of some of the clinical trials.Hence, effort should be directed towards the development of a com-posite product consisting of allogeneic cells encapsulated in a bioma-terial which would allow them to escape the immune system longenough for releasing the various cytokines and growth factors thatunderpin their paracrine effects.

Second, it appears that delivery of a single cell type does not supportangiogenesis sufficiently to promote tissue repair. Along this line, sever-al experimental reports have proposed alternatives to optimizepro-angiogenic therapies, and strategies aiming at simultaneously pro-moting angiogenesis and vessel maturation appear promising. Com-bined administration of an angiogenic and an arteriogenic growthfactor, namely FGF-2 and PDGF-BB, synergistically enhances angiogen-esis in the ischemic heart, but this concept is also relevant tocell-based therapy. Hence, co-administration of EPCs and smooth mus-cle progenitor cells is superior to EPCs alone for therapeutic angiogene-sis, and produces a mature vascular network in ischemic tissues [52].

Conclusion

Pro-angiogenic therapy appeared a promising strategy for thetreatment of patients with ischemic diseases. Exciting pre-clinicalstudies have prompted the initiation of numerous clinical trialsbased on administration of stem/progenitor cells from different ori-gins. Nonetheless, these clinical trials showed mixed results pavingthe way for the optimization of pro-angiogenic therapies includingdevelopment of innovative and potentially valuable strategies for im-proving recruitment, survival and function of stem/progenitor cells tosites of neovascularization. In addition, efforts should be directed to-wards stimulation of both angiogenesis and vessel maturation, thedevelopment of a composite product consisting of stem/progenitorcells encapsulated in a biomaterial and the use of additional sourcesof regenerative cells.

Conflict of interest statement

None.

Role of the funding source

J-S.S is supported by grants from Fondation de la RechercheMédicale, ANR Chemrepair (2010 BLAN 1127 02). J-S.S . is a recipientof a Contrat d'Interface from Assistance Publique-Hôpitaux de Paris.J-S.S, C.C, K.C are supported by fondation Leducq transatlantic net-work (09-CVD-01). The study sponsors had no role in the writing ofthe manuscript and in the decision to submit the manuscript forpublication.

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