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DOI: 10.1161/01.HYP.0000168923.92885.f7
2005;46;7-18; originally published online Jun 13, 2005;
Hypertension Melo
Victor J. Dzau, Massimiliano Gnecchi, Alok S. Pachori, Fulvio Morello and Luis G.
Therapeutic Potential of Endothelial Progenitor Cells in Cardiovascular Diseases
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Therapeutic Potential of Endothelial Progenitor Cells in
Cardiovascular Diseases
Victor J.Dzau,Massimiliano Gnecchi,Alok S.Pachori,Fulvio Morello,Luis G.Melo
Abstract—Endothelial dysfunction and cell loss are prominent features in cardiovascular disease.Endothelial progenitor cells(EPCs)originating from the bone marrow play a significant role in neovascularization of ischemic tissues and in re-endothelialization of injured blood vessels.Several studies have shown the therapeutic potential of EPC transplan-tation in rescue of tissue ischemia and in repair of blood vessels and bioengineering of prosthetic grafts.Recent small-scale trials have provided preliminary evidence of feasibility,safety,and efficacy in patients with myocardial and critical limb ischemia.However,several studies have shown that age and cardiovascular disease risk factors reduce the availability of circulating EPCs(CEPCs)and impair their function to varying degrees.In addition,the relative scarcity of CEPCs limits the ability to expand these cells in sufficient numbers for some therapeutic applications.Priority must be given to the development of strategies to enhance the number and improve the function of CEPCs.Furthermore, alternative sources of EPC such as chord blood need to be explored.Strategies for improvement of cell adhesion, survival,and prevention of cell senescence are also essential to ensure therapeutic viability.Genetic engineering of EPCs may be a useful approach to developing these cells into efficient therapeutic tools.In the clinical arena there is pressing need to standardize the protocols for isolation,culture,and therapeutic application of https://www.doczj.com/doc/437438187.html,rge-scale multi-center randomized trials are required to evaluate the long-term safety and efficacy of EPC therapy.Despite these hurdles,the outlook for EPC-based therapy for cardiovascular disease is promising.(Hypertension.2005;46:7-18.)
Key Words:coronary artery diseaseⅢendothelial progenitor cellsⅢgenetic engineeringⅢmyocardial infarction
ⅢneovascularizationⅢvascular repair
I t is now well-established that endothelial dysfunction underlies all of the major cardiovascular diseases.1In normal conditions,the vascular endothelium produces and secretes substances that modulate vascular tone and protect the vessel wall from inflammatory cell infiltration,thrombus formation,and vascular smooth muscle cell proliferation.2 Pathologic conditions such as hyperlipidemia,hyperglyce-mia,and hypertension impair the ability of the vascular endothelium to produce vasodilatory and anti-adhesion moi-eties and increase the production of vasoconstrictor,pro-adhesion,and pro-thrombotic molecules,leading to elevated vascular tone,enhanced cell adhesion,proliferation of media smooth muscle cells,and propensity toward thrombosis.1,3 Endothelial cell loss and turnover are accelerated in the presence of hemodynamic and biochemical alterations and are a prominent feature of vascular injury resulting from percutaneous coronary intervention.4The loss of endothelial function and integrity sets in motion the cascade of events that lead to atherosclerosis and restenosis after percutaneous revascularization.5,6Given the role of endothelial cell loss in the pathogenesis of vascular diseases,the attention has recently turned to the development of strategies to enhance rapid endothelial recovery.7,8Several studies have suggested that circulating endothelial progenitor cells(CEPCs)originat-ing in the bone marrow play a significant role in endogenous neovascularization of ischemic tissues9–12and in the re-endothelization of injured vessels.13–15In addition,EPC mobilization and proliferation was reported to contribute to the salutary effects of3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors16,17and estrogen.18EPC transplantation has been shown to induce new vessel forma-tion in ischemic myocardium and hind limb19–21and to accelerate re-endothelialization of injured vessels and pros-thetic vascular grafts in humans and in various animal models,22,23demonstrating their therapeutic potential as a cell-based strategy for rescue and repair of ischemic tissues and injured blood vessels.Furthermore,EPCs are amenable to genetic manipulation,underscoring their usefulness as vectors for local delivery of therapeutic genes.23–25However, despite the excitement regarding the possible clinical use of EPC,recent studies have shown that age and other risk factors for cardiovascular disease reduce the availability of EPC and
Received February16,2005;first decision March4,2005;revision accepted April21,2005.
From the Department of Medicine(V.J.D.,M.G.,A.S.P.,F.M.),Duke University Medical Center,Durham,NC;and the Department of Physiology (L.G.M.),Queen’s University,Kingston,Ontario,Canada.
Correspondence to Victor J.Dzau,MD,Department of Medicine,Duke University Medical Center,DUMC3701,Durham,NC27710.E-mail victor.dzau@https://www.doczj.com/doc/437438187.html, or Luis G.Melo,MD,Department of Physiology,18Stuart St,Queen’s University,Kingston,Ontario K7L3N6,Canada.E-mail melol@post.queensu.ca
?2005American Heart Association,Inc.
Hypertension is available at https://www.doczj.com/doc/437438187.html, DOI:10.1161/01.HYP.0000168923.92885.f7
Brief Review
impair their function to varying degrees,26–28thus limiting their therapeutic usefulness in these patient populations. Furthermore,the relative scarcity of CEPCs and their finite proliferative potential limits the ability to expand these cells in sufficient numbers for some therapeutic applications.29
In this article,we discuss the biology of EPCs,their therapeutic potential in the treatment of cardiovascular dis-eases,and the limitations facing their use in the clinic.We end with a discussion of the outstanding issues and a perspective on future developments in the field.
Isolation,Characterization,and Genetic
Modification of EPCs
The isolation and characterization of endothelial progenitor cells from peripheral blood was first reported by Asahara et al in1997.30Since the original report several other groups, including ours,have confirmed the original finding and have reported various modifications of the methods for the isola-tion,characterization,and culture of EPCs.9,23,31–33The cells are thought to originate from a common hemangioblast precursor in the bone marrow;9,33,34however,nonhematopoi-etic mesenchymal precursors in the bone marrow(multipo-tent adult progenitor cells)have also been reported to trans-differentiate into EPCs in culture.35Other studies have reported that myeloid/monocyte lineage cells(CD14?)can differentiate into cells with EPC characteristics.36,37In addi-tion,some tissues harbor stem cells that may differentiate into various lineages including endothelial cells.38–40Thus, CEPCs appear to be a heterogenous group of cells originating from multiple precursors within the bone marrow and present in different stages of endothelial differentiation in peripheral blood.For this reason,the accurate characterization of EPCs is difficult because many of the cell surface markers used in phenotyping are shared by hematopoietic stem cells and by adult endothelial cells.In culture,EPCs emerge as late(?2 weeks)outgrowth colonies after plating in endothelium spec-ified medium.Typically,the cells are defined on the basis of expression of cell surface markers such as CD34,Flk-1,and CD-133.30,31The cells have high proliferative potential,albeit for a finite number of cell divisions.10,19–21,30,34As the cells differentiate,they acquire endothelial lineage markers,vas-cular endothelium-cadherin,PECAM-1(CD31),von Wille-brand factor,endothelial nitric oxide synthase(eNOS),and E-selectin,and incorporate acetylated low-density lipoprotein cholesterol.23,30,31,33,34,41The loss of hematopoietic stem cell marker CD133expression coincides with EPC differentiation into cells with functional characteristics of adult endothelial cells.42
The relative abundance of CEPC is low in basal condi-tions.30However,the number of circulating cells increases several fold after exogenous stimulation with cytokines and hormones9–13,18,21,43–46(Table1).In addition to endogenous agonists,statins and physical exercise have also been reported to stimulate EPC mobilization.16,47–49The mechanisms gov-erning the mobilization,homing,and differentiation of the EPC in vivo remain largely unknown.
We have reported a streamlined method for isolation, cultivation,and expansion of EPCs from peripheral blood based on density centrifugation and selective adherence to fibronectin-coated plastic dishes(Figure1).23The unfraction-
TABLE1.Factors Influencing EPC Mobilization Growth and Differentiation
Factors Effect on EPC References
Physiological
Chemokines
SCF-1,G-CSF,GM-CSF Recruitment,mobilization10,13,21,32,81,94,101,102
SDF-1Recruitment,mobilization,homing44,82–84,58–60
Cytokines/growth factors
FGF,VEGF,PIGF Mobilization,differentiation11,30,43
Angiopoietin,PDGF Differentiation
Hormones
Erythropoietin Mobilization,replication45
Estrogen Mobilization18
Signaling molecules
NO,Akt Mobilization,differentiation72
Phamacological
3-HMG-COA inhibitors(statins)Mobilization,migration,homing15–17,47,48,104
PPAR-?agonists Mobilization,differentiation
Physical
Exercise,hypoxia Mobilization49
Pathological
Coronary artery disease Mobilization,homing11,27,55
Acute myocardial infarction Mobilization,homing12,56,60
Peripheral limb ischemia Mobilization,homing10,25,30,43
Vascular injury and inflammation
8Hypertension July2005
ated mononuclear cells (MNCs)are cultivated in medium enriched with endothelial specific growth factors such as vascular endothelial growth factor (VEGF).Within days of plating,colonies of adherent cells proliferate rapidly to form a monolayer with the cobblestone morphology typical of endothelium.After 2weeks,the cells adopt endothelial-like characteristics such as expression of von Willebrand factor,uptake of acetylated low-density lipoprotein cholesterol,and the ability to assemble into vascular tube-like structures (Figure 2).Using this approach,we are able to expand the circulating cells in culture to yield sufficient number for autologous transplantation onto injured blood vessels and prosthetic grafts in rabbits 23.
EPCs are highly amenable to genetic modification with viral vectors,rendering them useful as vehicles for delivery of therapeutic genes.23–25We use a pseudotyped murine stem cell retroviral vector 50for ex vivo genetic modification of
EPCs.23,24The pseudotyped murine stem cell retroviral vector vector transduces EPC with nearly 100%efficiency,without any noticeable effects on cell phenotype or engraftment in vivo.23Furthermore,because the retroviral genome integrates into the host genome,it can lead to long-lasting transgene expression.We have documented transgene expression in vivo up to 1e month after transplantation of the genetically modified cells.23,24One potential shortcoming of retroviral vectors is their proneness to transcriptional silencing,which may shorten the duration of transgene expression.51In addi-tion the retroviral DNA randomly integrates into the host genome,posing a potential risk of oncogenesis.52Other viral vectors such as adenovirus,25lentivirus,53and herpes virus 54have also been reported to transduce EPCs,but they have been used far less extensively than retroviral vectors.
Endothelial Progenitor Cells in Cardiovascular Disease and Aging
EPC and Cardiovascular Diseases
Differences in EPC number and function have been observed in a number of pathologies 27,28(Table 2).An inverse corre-lation was recently reported between the number and migra-tory activity of CEPCs and risk factors for coronary artery disease.27In a group of 45men with various degrees of cardiovascular risk,as defined by the combined Framingham risk factor score,the number of CEPCs correlated with endothelial function.28Interestingly,these investigators found that the number of CEPC was a better predictor of endothelial function than the presence or absence of traditional risk factors,suggesting that the abundance of CEPC may be a useful marker of vascular function and overall cardiovascular risk.In patients with severe coronary artery disease,the colony-forming capacity and migratory activity of bone marrow-derived CD34?/CD133?MNCs was markedly re-duced and associated with reduced neovascularization after transplantation in ischemic hind limb of nude rats,despite no difference in the total number of hematopoietic progenitor cells between patients and healthy control subjects.55EPC (CD133?/Flk-1?)was also reported to be inversely related to the severity of congestive heart failure.55,56Reduced number of EPCs was also seen in cardiac transplantation patients
with
Figure 1.Isolation,cultivation and genetic engineering of endothelial pro-genitor cells (EPCs)for therapeutic appli-cation.EPCs are isolated from the
mononuclear cell fraction of bone mar-row,peripheral blood,or umbilical chord blood.The mononuclear cells are expanded ex vivo under endothelial-speci?c growth conditions and may be genetically modi?ed to overexpress one or several therapeutic genes.The differ-entiated cells are then used in transplan-tation protocols for rescue and repair of damaged tissues such as infarcted myo-cardium,ischemic limb,or injured mus-cle.The cells may also be used for en-dothelialization of damaged blood vessels and for the bioengineering of vascular prosthetic grafts and arti?cial blood
vessels.
Figure 2.Immunohistochemical characterization of culture-expanded EPCs.A,EPCs at 2weeks after plating shows the cobblestone morphology typical of endothelial cells (100?).B,EPC stains positive for cytoplasmic factor VIII (von Willebrand factor)(200?).C,EPCs take up acetylated low-density lipopro-tein in culture medium (200?).D,EPCs form vascular structures when plated in Matrigel (40?).
Dzau et al Endothelial Progenitor Cells 9
vasculopathy57and in patients with in-stent restenosis.58In contrast,elevated EPC levels were reported in patients with unstable angina59and in patients with acute myocardial infarction60in association with elevation in plasma C-reactive protein levels.Interestingly,C-reactive protein reduces EPC proliferation,survival,differentiation,and function,61sug-gesting that the ability of C-reactive protein to inhibit EPC differentiation and function may play a role in the develop-ment of cardiovascular disease.
Diabetes,a major condition associated with cardiovascular diseases,also adversely affects endothelial function and number.62,63EPC are markedly reduced in patients with either type I62or type II63diabetes.Furthermore,the EPCs from diabetic patients showed reduced capacity to induce angio-genesis in vitro.62,63These defects in EPC function may underlie some of the vascular complications associated with diabetes,such as endothelial dysfunction,that predisposes to diffuse atherosclerosis and impaired neovascularization after ischemic events.In this regard,Schatteman et al64showed that transplantation of CD34?-derived angioblasts from non-diabetic mice markedly accelerates blood flow restoration in ischemic hind limb of diabetic mice in association with enhanced neovascularization.Chronic renal failure,another disease well known to predispose to coronary artery disease and heart failure,is characterized by enhanced coronary atherosclerosis and impaired angiogenesis.In patients with renal failure on hemodialysis,the number and colony-forming capacity of EPCs recovered from venous blood was decreased by?40%.65In addition,the EPCs from these patients showed reduced migratory activity and impaired ability to assemble into vascular tubes,suggesting that EPC deficiency may play a role in the progression of the disease.EPC and Aging
Age appears to affect EPC availability and function as well.26,66,67It has been reported that in young patients with stable coronary artery disease after coronary artery bypass grafting,the number of CEPCs increases,whereas the oppo-site occurs in older patients.66The age-related deficiency in the number of CEPCs was not related to differences in cardiovascular risk factor or cardiac function and it may be caused,at least in part,by reduced levels of angiogenic and mobilizing cytokines.This deficiency may be at the root of impaired neovascularization of ischemic tissues and attenu-ated re-endothelialization of injured tissues commonly ob-served in older patients.68,69Evidence for this hypothesis was provided by an elegant study by Eldeberg et al,67who showed that neovascularization of cardiac allografts in aged mice occurred only after transplantation of bone marrow-derived EPCs from young animals.Cardiovascular disease further compounds the effects of aging on EPC number and function. The presence of cardiovascular risk factors increases the rate of EPC senescence,even in the absence of overt disease.28 Chronic treatment of apolipoprotein E?/?mice with bone marrow-derived progenitor cells form young mice without atherosclerosis attenuates the progression of atherosclerosis of animals maintained in an atherogenic diet,26despite underlying hypercholesterolemia,suggesting that progressive depletion of EPCs with aging may precipitate the develop-ment of atherosclerosis,particularly in the presence of risk factors such as hypercholesterolemia.Reduction in progenitor cell mobilization with age may be caused by defects in the bone marrow stem cell niche and in the production of angiogenic cytokines and chemokines.VEGF and nitric oxide production have been reported to decrease with age,66,70–74 and it is known that these2factors play synergistic roles in the mobilization,migration,proliferation,and survival of endothelial cells.72,73
The alterations in EPC number and properties seen in aging and cardiovascular disease may be caused by a combination of factors.The chronic exposure to risk factors and presence of underlying cardiovascular disease accentuates endothelial injury,which may require continuous replacement of dam-aged endothelial cells.This may lead to exhaustion of the pool of progenitor cells available in the bone marrow,which may be exacerbated by accelerated senescence and apoptosis of the remaining cells.28,72,75In addition,the reduced avail-ability of mobilizing,homing,and differentiation/survival signals may limit the ability of EPC to repair injured tissues. Paradoxically,the functional impairment of EPC by cardio-vascular disease and aging may limit their therapeutic use-fulness in the patients who need it most.
Endothelial Progenitor Cells,Endogenous Repair,and Rescue in Cardiovascular Disease Multiple lines of evidence suggest that EPC are recruited to sites of injury where they participate in the repair of damaged tissues.9,10,21,30,33,41,76,77For example,the bone marrow-derived mononuclear cells(BM-MNCs),containing also the EPC population,home to ischemic myocardium,12,21,60 brain,78and hind limb,10,25,30,34,43where they participate in neovascularization.Marked increase in mobilization and
TABLE2.Diseases Characterized by Alterations in EPC Levels and Function
Disease Type Effect on
EPC N
Effect on
EPC Function References
Myocardial
Coronary artery disease2211,12,27,28,55 Congestive heart failure2256,60 Unstable angina2ND59 Myocardial infarction1260 Vascular
Atherosclerosis2227,55
Acute vascular injury and inflammation 1ND14,15,17,18,32,
80,81
Peripheral limb ischemia1ND10,25,30,34,43 Transplant arteriopathy2ND57
In-stent restenosis2ND58 Hypertension ND ND
Hyperlipidemia2226 Diabetes2262,63 Renal failure
Hemodialysis2265
ND indicates not determined.
10Hypertension July2005
homing of CD34?cells was seen in patients with myocardial infarction12,60or hind limb43ischemia.Furthermore,in pa-tients with inoperable coronary artery disease,mobilization and homing of EPCs to the ischemic myocardium increased up to9weeks after VEGF gene transfer,in parallel with increase in circulating levels of VEGF,11and in patients with critical limb ischemia VEGF gene transfer led to marked increase in CEPCs.43
Regeneration of endothelium is a fundamental process in vascular repair.1,7,8Mature endothelial cells have limited ability to regenerate damaged endothelium because these cells are terminally differentiated.79Accessory mechanisms such as EPCs may play a significant role in vascular repair and healing,8,32,76,80and strategies aimed at rapid endothelial recovery should reduce cardiovascular events associated with endothelial cell loss,including thrombosis,restenosis,and hypertension.8EPCs were reported to repopulate implanted vascular grafts and damaged blood vessels as part of endog-enous repair mechanism.13,14,15,17,18,32,80,81
Therapeutic Potential of the Endothelial
Progenitor Cells
Studies in animal models of tissue ischemia and vascular injury have confirmed the therapeutic efficacy of EPCs in rescue of ischemia,10,11,19–21,25,30,31,34,35,82–84in repair of blood vessels,and in bioengineering of prosthetic grafts.13,15,17,22–24,32Several small-scale trials have provided preliminary evidence of feasibility and safety of EPC transplan-tation in patients in myocardial and limb ischemia(Table3). Cell Therapy for Myocardial and Peripheral Ischemic Disease Using EPCs
Cell-based neovascularization has been achieved either by directly injecting purified EPC into the ischemic region19–21,30 or by mobilizing the cells using cytokines or chemokines such as VEGF,granulocyte colony-stimulating factor(G-CSF),or stromal derived factor(SDF).10,11,43,44,83–85Various studies have confirmed the efficacy of EPC transplantation in inducing neovascularization of ischemic myocardium and hind limb(Table3).19–21,25,30,34,43Kocher et al21 showed that intravenous delivery of human CD34?cells into athymic nude rats with myocardial infarction leads to marked angiogenesis in the peri-infarct region,resulting in decreased myocyte apoptosis,reduced interstitial fibrosis, and recovery of left ventricular function.Transplantation of CD31?cells from peripheral blood improved left ventricular perfusion and function in a porcine amaroid model of myocardial ischemia.86Similarly,implantation of whole20or CD34?-selected human peripheral blood-mononuclear cells(PB-MNCs)into nude rats immediately after myocardial infarction led to significant neovascular-ization and improved function in the infarcted myocardi-um.86Others showed that transendocardial delivery of unfractionated autologous BM-MNCs induced collateral formation and rescued ventricular function in hibernating porcine myocardium.87–90Orlic et al reported that implan-tation of bone marrow-derived Lin?/c-kit?cells,a sub-population of BM-MNCs,into the infarct border enhanced new vessel formation.91In rats with limb ischemia,local intramuscular delivery of autologous BM-MNCs restored blood flow and exercise in association with enhanced neovascularization of the ischemic muscle.92Interestingly, in one study93the transplanted peripheral blood MNCs did not incorporate into the new capillaries,but contributed to new vessel formation by secreting pro-angiogenic cyto-kines.This observation suggests that a paracrine effect is probably an important mechanism contributing to the increased neovascularization observed after EPC transplantation.
Mobilization of EPC with cytokines or conventional phar-macological agents used in treatment of cardiovascular dis-ease such as statins has been reported to enhance angiogen-esis of ischemic tissues.Orlic et al94showed that mobilization of bone marrow cells by G-CSF and SCF led to decreased postinfarction mortality and functional recovery in mice with myocardial infarction in association with significant regener-ation and angiogenesis of the infarcted myocardium.In athymic nude mice with hind limb ischemia,local injection of SDF-1stimulated homing of systemically delivered human PB-MNCs to the ischemic muscle and induced vasculogen-esis.44Other groups reported that statin therapy increases the number of CEPC in animal models and in patients with stable coronary artery disease,27,47,48suggesting that the therapeutic effect of these drugs may be mediated,at least in part,via mobilization of EPCs.
EPC Cell Therapy for Pulmonary Hypertension EPC-based cell therapy might be beneficial as a treatment of pulmonary hypertension.Chord blood-derived human EPCs overexpressing adrenomedullin markedly reduced pulmonary vascular resistance and improved survival rates after intrave-nous administration into nude rats with monocrotaline-induced pulmonary hypertension compared with control an-imals treated with either saline or EPCs alone.95Similarly, intraparenchymal injection of autologous EPCs in dogs with pulmonary hypertension brought about significant improve-ments in mean pulmonary artery pressure,cardiac output,and pulmonary vascular resistance96
Endothelial Cell Therapy for Vascular Repair and Bioengineering of Grafts
An emerging area in which endothelial progenitor cell trans-plantation and genetic manipulation may have therapeutic potential is in repair of damaged vessels and in the bioengi-neering of prostheses and artificial organs.13–17,22–24,81,97–100 Autologous EPC transplantation may be used to promote rapid re-endothelialization and restoration of homeostasis in blood vessels injured during revascularization proce-dures22–24,97–100or for seeding of prosthetic grafts,stents, or engineered blood vessels to create a bioactive endothe-lial layer.22,23,97,98We showed recently that transplantation of autologous PB-EPCs leads to rapid re-endothelialization of balloon-denuded rabbit carotid arteries,resulting in significant reduction of neointimal hyperplasia.23Using a similar approach,Gulati et al98reported that transplanta-tion of cultured autologous MNCs at the time of injury markedly reduced neointima hyperplasia in association with rapid re-endothelization of the damaged vessel.Oth-Dzau et al Endothelial Progenitor Cells11
TABLE3.Preclinical and Clinical Cell-Based Therapy for Therapeutic Angiogenesis for Myocardial and Hind Limb Ischemia
Target Donor Recipient Type and Source of Cells Method of Delivery Therapeutic Effects References Preclinical
Myocardial ischemia Swine Autologous CD31?,peripheral blood Transendocardial with NOGA
mapping 1Rentrop score,1EF,1
capillary density
86
Myocardial ischemia Swine Autologous MNC,bone marrow Transendocardial1capillary density,1collateral
flow,1myocardial contractility
90
Hibernating myocardium Swine Autologous MNC,peripheral blood Transendocardial1EF,1capillary density,1
flow
87
MNC,bone marrow1EF,1collateral flow88 Myocardial ischemia Rat Autologous MNC,bone marrow Intramyocardial1capillary density89 Myocardial infarction Human Nude rat CD34?,peripheral blood Intramyocardial1EF,1capillary density,2
fibrosis
20,
MNC,peripheral blood Intravenous1EF,1capillary density,2
fibrosis
86
Myocardial infarction Human Nude rat CD34?,bone marrow Tail vein injection1EF,1capillary density,2
fibrosis,
21
2apoptosis,2infarct size Myocardial infarction GFP mouse Syngenic mouse Lin?c-kit?,bone marrow Peri-infarct region1LVDP,1capillary density,2
infarct,
91
Myocardial infarction Mouse Autologous bone marrow mobilization Homing1EF,1capillary density,2
remodelling,
94
Hind limb ischemia Rat Autologous MNC,bone marrow Intramuscular injection in1capillary density,1blood
flow,
92
Gastrocnemius2AVDO2,1exercise capacity
Hind limb ischemia Rabbit Autologous MNC,bone marrow Intramuscular injection in thigh1capillary density,1blood
flow
Hindlimb ischemia Human Athymic mice MNC,peripheral blood Intracardiac injection1capillary density,1blood
flow
19
Hind limb ischemia Human Athymic mice MNC,peripheral blood
overexpressing VEGF Tail vein injection2autoamputation,1capillary
density,
25
1blood
Hind limb ischemia Human Nude rat MNC,peripheral blood Intramuscular injection in thigh1capillary density,1blood
flow
93
MNC,chord blood
Clinical
Myocardial infarction Human Autologous CD133?,bone marrow Infarct border1EF,1collateral flow(SPECT)107 Myocardial infarction Human Autologous MNC,bone marrow Intracoronary balloon catheter2infarct size,1wall motion,108
Myocardial infarction (TOPCARE-AMI trial)Human Autologous MNC,bone marrow,
peripheral blood
Intracoronary balloon catheter1contractility,1myocardial
perfusion2remodeling1EF
109–112
Myocardial infarction(BOOST
trial)
Human Autologous CD34?,bone marrow Intracoronary during PCA
Myocardial infarction Human Autologous CD34?/CD117?/AC133?Intracoronary with PCA1EF,1LV wall thickness,2
ESV
113
Myocardial ischemia(Unstable angina)Human Autologous MNC,bone marrow Transendocardial with NOGA
mapping
2anginal episodes,1wall
thickening,1wall motion,1
EF
114,115
Myocardial infarction Human Autologous CD34?1:intracoronary,2:G-CSF1EF,1exercise time,
1myocardial
120 (MAGIC trial)mobilization perfusion,1angiogenesis
Hind limb ischemia Human Autologous MNC,bone marrow Intramuscular injection in
Gastrocnemius 1ankle-brachial index,1
pain-free walking,1
transcutaneous PO2
116
AVDO2indicates arteriovenous oxygen difference;EF,ejection fraction;G-CSF,granulocyte colony-stimulating factor;MNC,mononuclear cells;PO2,partial pressure of oxygen.
12Hypertension July2005
ers have shown the ability of transplanted EPCs to restore endothelial function in damaged vessels.99,100We have also demonstrated the suitability of EPC to seed prosthetic grafts.Seeding of EPCs led to rapid endothelialization of expanded polytetrafluoroethylene(ePTFE)grafts after ca-rotid interpositional grafting.23Using a similar strategy, Kaushal et al22showed that implantation of EPCs into decellularized porcine iliac vessels implanted as coronary interposition grafts reconstituted a functional endothelial layer that conferred improved vasodilatory function and prolonged patency of the grafts.EPCs may also be useful for genetic engineering of stents.Shirota et al97reported that EPCs are capable of efficiently seeding photo-cured gelatin-coated metallic and microporous thin segmented polyurethane stents,suggesting that seeding of stents before implantation may provide a strategy for prevention of in-stent restenosis and thrombosis.
Exogenous mobilization of EPC from the bone marrow may provide a less cumbersome and potentially more effec-tive strategy to enhance re-endothelialization of damaged vessels.Bhattacharya et al101and Shi et al102reported that mobilization of bone marrow by exogenous G-CSF enhanced endothelialization and patency of small caliber prosthetic grafts.We showed that treatment with G-CSF before balloon injury of rat carotid arteries led to accelerated re-endotheli-alization and concomitant reduction in neointima of the injured vessels,in association with an increase in the number of CEPCs.13Others have reported that statin therapy15,17and estrogen18increases the number of CEPCs and reduces neointima hyperplasia in animal models of arterial injury, presumably by stimulating eNOS activity.103Interestingly, statins also reduce senescence and stimulate proliferation of PB-EPCs by regulating the activity of telomerase and cell cycle genes.89,104Thus the therapeutic potential of endothelial progenitor cells could potentially be enhanced by noninvasive pharmacological manipulation and used to accelerate re-en-dothelialization of injured vessels and inhibit neointimal hyperplasia after revascularization procedures.
Potential of Genetic Engineering of
Endothelial Progenitor Cells
The ability to genetically modify EPCs with expression vectors provides the opportunity to use the cells as vehicles for local delivery of therapeutic genes and allows the design of strategies to enhance the biological properties of EPCs.For example,the EPCs could be genetically modified to overex-press antithrombotic,vasodilatory,or antiproliferative genes to improve the function of the implanted cells in injured vessels or prostheses and to prevent thrombosis and resteno-sis,53,105or to increase the capability of the cells to synthesize angiogenic,vasodilatory,or cytoprotective factors for main-tenance and survival of the grafts.24We reported recently that
the therapeutic effect of EPCs could be enhanced by overex-pressing eNOS.24Our results showed that the denuded carotid vessels treated with the eNOS-expressing EPCs had enhanced inhibition of neointima and reduced incidence of thrombosis compared with vessels treated with control vector(Figure3). We postulated from these observations that the transplanta-tion of autologous EPCs expressing vasculoprotective genes may be a useful strategy to prevent postintervention compli-cations such as thrombosis and restenosis after revasculariza-tion procedures.
EPCs have also been used as vectors for delivery of pro-angiogenic factors.The genetically modified EPC appear to contribute to new vessel growth by proliferating and differentiating at the site of implantation,and by
secreting Figure3.Inhibition of neointimal proliferation by EPCs in balloon-injured carotid arteries.All sections were stained with Accustain elastic stain.Arrows indicate neointima.A and B, Saline-injected(untreated)arteries2weeks after injury.C and D, Arteries treated with genetically modi?ed EPCs expressing GFP.
E and F,Arteries treated with genetically modi?ed EPCs expressing endothelial nitric oxide synthase(eNOS).G,Morpho-metric analysis of neointima hyperplasia in unseeded and seeded injured arteries.EPC transplantation signi?cantly reduced neointima/media ratios and genetic modi?cation with eNOS further reduced neointima.*Saline vs GFP-EPC;#saline vs NOS-EPC;?GFP-EPC vs NOS-EPC;P?0.05.Data modi?ed from Kong D,Melo LG,Mangi AA,Zhang L,Lopez-Ilasaca M, Perrella MA,Liew CC,Pratt RE,Dzau VJ.Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells.Circulation.2004;109:1769–1775.
Dzau et al Endothelial Progenitor Cells13
growth factors that stimulate growth of pre-existing ves-sels.Overexpression of mobilizing cytokines and chemo-kines such as G-CSF and SDF-1may be used to further potentiate the angiogenic effect of locally implanted EPCs by potentiating the recruitment and homing of other progenitor cells to the sites of ischemic injury.Genetic engineering may also be useful in the design of strategies to improve cell adhesion and survival and prevention of senescence.For example,overexpression of human telom-erase reverse-transcriptase was reported to enhance the proliferative and migratory capacity of EPCs in response to VEGF stimulation,leading to improved neovasculariza-tion of ischemic limb.29The combination of genetic modification and endogenous mobilization of EPCs may have synergistic effects,106but the development of a strategy that will target mobilization of EPCs exclusively presents a difficult challenge.
Clinical Application of Endothelial
Progenitor Cells
Several small-scale feasibility and safety clinical trials have been performed recently to evaluate the use of bone marrow cell transplantation in treatment of myocardial infarction and peripheral limb ischemia(Table3).107–113All the results published so far indicate that such therapeutic approach is feasible and safe.Moreover,in the case of myocardial ischemia,an improvement in ventricular function in associ-ation with enhanced coronary perfusion has been reported after cell therapy(Table2).Stamm et al107injected autolo-gous AC133?bone marrow cells into the infarct border during coronary artery bypass grafting in6patients that had experienced earlier myocardial infarction.The authors re-ported improved perfusion of the infarcted area and signifi-cant enhancement of global left ventricular function3to9 months after surgery and no incidence of adverse events in5 of the6patients.Similarly,intracoronary delivery of unfrac-tionated autologous mononuclear bone marrow cells in pa-tients undergoing PCA5to9days after myocardial infarction led to a reduction in infarct size and improvement in ventricular function and chamber geometry for up to3 months after transplantation compared with patients treated with standard therapy for myocardial infarction alone.108 Intracoronary infusion of ex vivo-expanded BM-MNCs or PB-MNCs to a randomized group of20patients with reper-fused acute myocardial infarction4days after infarction (Transplantation of Progenitor Cells and Regeneration En-hancement in Acute Myocardial Infarction[TOPCARE-AMI]trial)led equally to significant improvements in global left ventricle ejection fraction and wall motion in the infarct zone and reduced end systolic dimensions at4months.109The improved echocardiographic parameters were accompanied by increases in coronary flow reserve in the infarct artery and in myocardial viability in the infarct zone.109Subsequently, the authors reported increase in global ejection fraction and decreases in end-systolic volume and infarct size at the 4-month follow-up period in the cell-treated patients,suggest-ing the infusion of progenitor cells attenuates postinfarct remodeling.110Interestingly,the beneficial effects of progen-itor cell infusion in post infarction functional recovery and prevention of remodeling was highly correlated with the migratory capacity of the progenitor cells,110suggesting that the ability of the cells to migrate to the infarct site is a major determinant of infarct remodeling and regeneration.The recently published final1-year follow-up results for this phase I trial showed sustained improvement in left ventricular function,reduction in end-systolic volumes,and smaller infarct sizes in the2groups of patients treated with cell therapy.111Wollert et al reported recently the results of the BOOST randomized controlled trial evaluating the therapeu-tic effect of autologous bone marrow cell transfer in patients with myocardial infarction.112In this trial,60patients with myocardial infarction were randomized into2groups of30 patients after PCA and treated with medical therapy for myocardial infarction alone or with medical therapy and intracoronary infusion of autologous CD34?bone marrow cells.The authors reported improvement in global left ven-tricular ejection fraction in the patients treated with the bone marrow cells6months after cell transfer.112Furthermore,cell administration did not lead to adverse cardiac events or in-stent restenosis,thus indicating that the method of cell delivery is safe.However,the total number of cells adminis-tered and the mechanisms responsible for the functional improvement after cell delivery were not identified in this https://www.doczj.com/doc/437438187.html,parable results were also reported by Fernandez-Aviles et al113using intracoronary delivery of CD34?/ CD117?/CD133?cells in patients with reperfused myocardial infarction.However,it is not clear whether the cells used by Wollert et al112are the same as those used in the later study.113Two other groups have reported that transendocar-dial delivery of autologous BM-MNCs using NOGA map-ping led to significant improvements in left ventricular perfusion and performance and reduced incidence of ische-mic episodes in patients with end-stage ischemic heart dis-ease114or stable angina.115
Recently,autologous BM-MNCs were injected into the gastrocnemius muscle of patients with unilateral or bilateral leg ischemia caused by severe peripheral artery disease.116 Four weeks after cell transplantation,ankle–brachial indexes were significantly improved in the legs of patients treated with BM-MNCs but not in patients treated with saline.Rest pain and pain-free walking were also reported to be signifi-cantly improved during the24-week duration of the study. The authors suggest that BM-MNC transplantation may be a safe and effective strategy for treatment of peripheral ische-mic disease.
It must be pointed out that all the trials mentioned had several https://www.doczj.com/doc/437438187.html,ly,they consisted of a limited number of patients,were single-center,not blinded,and almost all of them were uncontrolled.Moreover,in most of these studies the cell population was not preselected;rather, the whole bone marrow MNC fraction was injected without further purification.Thus the results to date,although encour-aging,should be considered preliminary.There is pressing need for large multicenter,controlled trials to test the efficacy of preselected pure EPC transplantation in the treatment of cardiovascular diseases.
14Hypertension July2005
Potential Problems With Therapeutic Use
of EPCs
Although the preclinical and clinical studies reviewed here generally lend support to the therapeutic potential of autolo-gous EPCs in the treatment of tissue ischemia and repair of injured blood vessels,the clinical application of EPCs is limited by several factors.First,the scarcity of CEPCs makes it difficulty to expand sufficient number of cells for thera-peutic application without incurring the risk of cell senes-cence and change in phenotype.30,34Furthermore,EPCs from patients with cardiovascular diseases display varying degrees of functional impairment.16,27,28,55,58,62,63Aging and diabetes markedly reduce the availability and impair the function of EPCs.28,62–64,66,67Because older and diabetic patients are the most vulnerable populations for cardiovascular diseases,this severely restricts the ability to treat with autologous EPCs the patients who theoretically need them most.
The purity and developmental stage of the cells used for transplantation are important factors.Yoon et al reported recently that injection of total bone marrow cells into the heart of infarcted rats could potentially lead to severe in-tramyocardial calcifications.117In contrast,animals receiving the same number of clonally expanded bone marrow cells did not show myocardial calcification.Thus,this finding brings attention to the potential risks of transplanting unselected bone marrow cells and cautions against their premature use in the clinical setting.
Exogenous mobilization of bone marrow with hematopoi-etic growth factors and other endothelial cell growth factors may recruit progenitor cells to sites of occult neoplasia, leading to vascularization of dormant tumors.In addition, mobilization could potentially accelerate progression of ath-erosclerotic plaque by recruiting inflammatory and vascular smooth muscle cell progenitor cells into the plaque,contrib-uting to neointima hyperplasia and transplant arteriopa-thy.118,119Increased rate of in-stent restenosis led recently to the cancellation of the MAGIC clinical trial using G-CSF for endogenous mobilization of progenitor cells in patients with myocardial infarction.120Finally,there has been one study that has shown evidence that EPC may themselves contribute to allograft vasculopathy by promoting neovascularization of the plaque.121However,another study failed to show evi-dence that EPCs contribute significantly to transplant arteriosclerosis.122
Outstanding Issues and Perspective for
Future Direction
Despite the encouraging results regarding the therapeutic potential of EPCs,several issues currently stand in the way of their wide clinical application.Strategies need to be devel-oped to enhance the number of EPCs to allow the harvesting of adequate number for therapeutic application.The limited ability to expand PB-MNC–derived EPCs in culture to yield sufficient number for clinical application indicates that alter-native sources of cells(ie,chord blood)or strategies to increase their number endogenously need to be explored.We believe that further characterization of the biology of EPCs, the nature of the mobilizing,migratory and homing signals, and the mechanisms of differentiation and incorporation into the target tissues need to be identified and further character-ized.Strategies to improve retention and survival of the transplanted cells need to be developed as well.The issues of the timing of cell administration,the appropriate clinical condition,the optimal cell number,and,most importantly,the safety of cell transplantation must be defined.There is urgent need to standardize the protocols for isolation,cultivation, and therapeutic application for cell-based therapy.Finally, large-scale randomized,controlled,multi-centric trials will be essential to evaluate the long-term safety and efficacy of EPC therapy for treatment of tissue ischemia and vessel repair amid concerns of potential side effects such as neovascular-ization of occult neoplasias and the development of age-and diabetes-related vasculopathies.Despite these hurdles,the outlook for EPC-based therapies for tissue ischemia and blood vessel repair appears promising.Genetic engineering of EPC may provide an important strategy to enhance EPC mobilization,survival,engraftment,and function,thereby rendering these cells efficient therapeutic modalities for cardiovascular diseases.
Acknowledgments
Dr Dzau is supported by grants from the National Institutes of Health (HL72010,HL73219,HL58576,and HL356107)and donations from the Edna Mandel Foundation.Dr Melo is Canada Research Chair in Molecular Cardiology and a New Investigator of the Heart and Stroke Foundation of Canada and is supported by grants from the Canadian Institutes of Health Research,the Canadian Foundation for Innovation,Ontario Innovation Fund,and the Heart and Stroke Foundation of Canada.
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