Review Pharmacological approaches to improve endothelial repair mechanisms Expert Rev. Cardiovasc. Ther. 6(8), xxx–xxx (2008) of ro Author for correspondence Cardiovascular Center, University Hospital Zurich, Raemistrassse 100, 8091 Zurich, Switzerland Tel.: +41 442 559 595 Fax: +41 442 554 251 [email protected] ho † Endothelial injury is thought to play a pivotal role in the development and progression of vascular diseases such as atherosclerosis, hypertension or restenosis, and their complications, including myocardial infarction or stroke. Accumulating evidence suggests that bone marrow-derived endothelial progenitor cells (EPCs) promote endothelial repair and contribute to ischemia-induced neovascularization. Coronary artery disease and its risk factors, such as diabetes, hypercholesterolemia, hypertension and smoking, are associated with a reduced number and impaired functional activity of circulating EPCs. Moreover, initial data suggest that reduced EPC levels are associated with endothelial dysfunction and an increased risk of cardiovascular events, compatible with the concept that impaired EPC-mediated vascular repair promotes progression of vascular disease. In this review we summarize recent data on the effects of pharmacological agents on mobilization and functional activity of EPCs. In particular, several experimental and clinical studies have suggested that statins, angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor blockers, PPAR-γ agonists and erythropoietin increase the number and functional activity of EPCs. The underlying mechanisms remain largely to be defined; however, they likely include activation of the PI3-kinase/Akt pathway and endothelial nitric oxide synthase, as well as inhibition of NAD(P)H oxidase activity of progenitor cells. rP Christian Besler, Carola Doerries, Giovanna Giannotti, Thomas F Lüscher and Ulf Landmesser† Keywords: endothelial regeneration • endothelium • EPC • nitric oxide • oxidant stress Role of the endothelium & endothelial function in cardiovascular disease A ut During the last 20 years, numerous experimental and clinical studies have demonstrated that the endothelium plays a crucial role in the regulation of vascular tone and structure [1–3] . Under physiological conditions, the endothelial monolayer does not only maintain the balance between vasodilation and vasoconstriction, but also inhibits leukocyte and platelet adhesion, and platelet aggregation as well as exerting anticoagulant and profibrinolytic effects (F igure 1) [1,4] . Endothelial dysfunction has been identified as a common link between the known cardiovascular risk factors, such as diabetes, hypercholesterolemia, smoking and hypertension and is characterized by a proinflammatory and prothrombotic phenotype of the endothelium [5,6] . Early on, atherosclerosis was considered to be an inflammatory–fibroproliferative response to various forms of insult to the endothelium [7] . Today it has become clear that atherosclerosis www.expert-reviews.com 10.1586/14779072.6.8.xxx is an inflammatory disease [8,9] and the degree of inflammation has prognostic significance [10] . Activation of endothelial cells therefore plays a crucial role in recruitment and adhesion of leukocytes, whose infiltration into the arterial wall is a critical step in development of atherosclerotic lesions [11,12] . Hence, disruption of endothelial integrity, both functionally and structurally, either in response to major cardiovascular risk factors or by direct mechanical injury (i.e., after percutaneous coronary intervention), induces a variety of proinflammatory and proliferative responses in the arterial wall, contributing to initiation and progression of atherosclerotic plaque formation, vascular remodeling and development of restenosis [13–15] . Therefore, there is increasing interest in the effect of pharmacological approaches, to maintain structural and functional integrity of the endothelium, in part by promoting endothelial repair and preventing endothelial cell apoptosis. © 2008 Expert Reviews Ltd ISSN 1477-9072 1 Review Besler, Doerries, Giannotti, Lüscher & Landmesser A B Neovascularization Circulating EPCs Functions of endothelial progenitor cells Anticoagulant and profibrinolytic effects Anti-inflammatory effects Endothelial cell functions Antihypertrophic effects on vascular smooth muscle cells rP Endothelium-dependent vasodilation Direct incorporation ro Paracrine effects of Antithrombotic effects Reendothelialization Expert Rev. Cardiovasc. Ther. © Future Science Group Ltd (2008) ho Figure 1. Vasoprotective functions of the healthy endothelium and potential role of EPCs in vascular repair processes. (A) The healthy endothelium not only mediates endothelium-dependent vasodilation, but also exerts anti-inflammatory, antithrombotic and anticoagulant effects and suppresses vascular smooth muscle cell hypertrophy. (B) EPCs have been shown to promote endothelial repair and augment ischemia-induced neovascularization, either by paracrine effects on adjacent mature endothelial cells or direct incorporation into the endothelial monolayer or a combination of both. EPC: Endothelial progenitor cell. EPCs & cardiovascular events Endothelial repair has long been thought to depend exclusively on the proliferation and migration of local adjacent endothelial cells [16] . In 1997, Asahara et al. described for the first time a population of putative endothelial progenitor cells (EPCs) in human peripheral blood [17] . In this study, selected circulating CD34 + cells were shown to differentiate into mature endothelial cells ex vivo and to contribute to neoangiogenesis in hindlimb ischemia (Figure 1) . Since then, numerous studies have suggested that blood-derived EPCs promote endothelial repair after vascular injury [18–20] , contribute to ischemia-induced myocardial and peripheral neovascularization [21] and improve endothelial function [22] . Moreover, a recent study by Foteinos et al. has suggested that bone-marrow derived endothelial progenitor cells contribute to endothelial repair in lesion-prone areas of experimental atherosclerosis [23] . Importantly, several cardiovascular risk factors reduce circulating numbers and impair functional activity of circulating EPCs, suggesting a loss of endogenous endothelial repair capacity in patients at high risk for cardiovascular events (Figure 2) [24,25] . Altered EPC levels were demonstrated in several clinical studies, for patients with stable coronary artery disease [26] , heart failure [27] , peripheral vascular disease [28] and cerebrovascular disease [29] . In a study of 519 patients with coronary disease, reduced circulating EPC levels were associated with an adverse cardiovascular outcome in a follow-up period of 12 months; that is to say, lower numbers of CD34 +/kinase insert domain receptor (KDR)+ double-positive blood-derived mononuclear cells were associated with a higher risk of cardiovascular events [30] . Moreover, in a study of 120 individuals by Schmidt-Lucke et al. including patients with stable coronary disease, acute coronary syndrome and control subjects, reduced numbers of peripheral blood CD34 +/KDR+ progenitor cells independently predicted cardiovascular events over a median follow-up period of 10 months [31] . Of note, in this study, the number of circulating EPCs remained an independent predictor of poor prognosis after adjustment for common cardiovascular risk factors, suggesting that impaired EPC-mediated vascular repair may be relevant for progression of cardiovascular disease [31] . A ut Endothelial repair mediated by circulating EPCs 2 EPC subpopulations A large number of bone marrow transplantation studies suggest that EPCs can be derived from the bone marrow [32] . More recently it has become clear that tissue-resident stem cells are an additional source of circulating progenitor cells [33] . However, there is currently no precise agreement on the exact definition Expert Rev. Cardiovasc. Ther. 6(8), (2008) Pharmacological approaches to improve endothelial repair mechanisms PPARγ agonists Benidipine Statins Review cells; however, the amount of cells obtained after FACS was rather low and this may be a limiting factor that should be considered in the interpretation of these data. Culture analysis PPARγ Culture analysis of EPCs is mostly performed by using blood-derived mononuclear cells. With respect to characterization of EPCs in culture, it has become clear that there are PPARγ Proliferation + several cell populations in peripheral blood + differentiation Akt mononuclear cells, which may promote migration – – endothelial repair or neovascularization Apoptosis in different ways [32] . In particular, recent data have suggested that at least two EPC + subpopulations can be grown from periph– NAD(P)H oxidase eral blood mononuclear cells, namely early eNOS EPCs and late-outgrowth endothelial cells (OECs), promoting angiogenesis in different ways [36] . Early EPCs, which have been NO suggested to arise primarily from a CD14 + O2subpopulation of peripheral blood mononuclear cells [37] , failed to form vascular netReendothelialization capacity works and to incorporate in endothelial-like Expert Rev. Cardiovasc. Ther. © Future Science Group Ltd (2008) structures in a newly developed angiogenesis assay, but contributed to tubulogenesis in a Figure 3. Postulated mechanisms mediating the effects of pharmacological agents on EPCs. Statins, EPO and benidipine have been suggested to exert their effects paracrine fashion. OECs, likely arising from on EPC functional activity, at least in part, via the phosphoinositide 3-kinase (PI3K)/Akt CD14- peripheral blood mononuclear cells pathway. Furthermore, endothelial nitric oxide synthase has been implicated for the [37] , augmented tubulogenesis by directly effects of statins, PPAR-γ agonist and EPO on EPCs. PPAR-γ agonists may exert their incorporating into newly formed vascular effect on EPCs, at least part, via antioxidant NAD(P)H oxiase-inhibiting effects. EPC: networks, but failed to stimulate tubulogenEndothelial progenitor cells; EPO: Erythropoietin. esis in a paracrine fashion when separated of endothelial progenitor cells. The examination of EPCs is at from mature endothelial cells in a transwell technique [36] . Of note, it has been observed that hematopoietic progenitor cells present largely performed by two ways: by FACS analysis of total blood cells or circulating mononuclear cells; and by analysis of can differentiate towards both endothelial and vascular smooth muscle-like cells [38,39] . Circulating progenitor cell-derived vascultured blood-derived mononuclear cells. cular smooth muscle-like cells have been suggested to contribute to vascular remodeling after wire-induced neointima formation FACS analysis As described previously, hematopoietic progenitor/stem cell mark- or restenosis [38,39] . Therefore, the differentiation of circulating ers, in particular CD34 and/or CD133, are used in combination progenitor cells towards either endothelial or vascular smooth with endothelial cell markers (particular KDR, also known as muscle like cells may also play an important role for the overall VEGFR-2) to quantify circulating EPCs in peripheral blood [34] vascular effects of circulating progenitor cells. Thus, additional studies are necessary, not only to define the and for these measurements, the initial data suggest a relation to cardiovascular prognosis, as described previously. The rationale for precise origin, phenotype and subtypes of circulating EPCs, but using these markers is, at least in part, based on the observation that also to characterize the functional role and differentiation potenboth isolated CD34+ and isolated KDR+ cells from peripheral blood tial of circulating progenitor cell-derived vascular smooth musclemay differentiate in vitro towards endothelial cells and contribute to like cells in more detail. ischemia-induced neovascularization [17] . However, this approach has not been supported by a precise phenotypic and functional Effect of pharmacological therapies in clinical use on characterization of these double-positive cells. Recently, Case et al. endothelial repair mechanisms have assessed the capacity of CD34 +/CD133 +/KDR+ cells (isolated Several pharmacological agents have been shown to impact on the by magnetic-activated cell sorting and FACS) to form endothelial- number and function of EPCs in experimental and small-scale like cell colonies [35] . In this study, the authors could not detect prospective clinical studies. Besides clinically used pharmacologiendothelial colony formation from CD34 +/CD133 +/KDR+ cells cal agents, a number of cytokines, growth factors (e.g., VEGF and questioned their capacity to differentiate towards endothelial and IGF) and hormones (e.g., estrogen) have been studied for PI3K A ut ho rP ro of EPO www.expert-reviews.com 3 Review Besler, Doerries, Giannotti, Lüscher & Landmesser their impact on EPC-mediated endothelial repair in experimental studies. In this review, however, we focus on recent data about the effects of pharmacological agents in clinical use for treatment of cardiovascular risk factors that affect mobilization and functional activity of EPCs (Table 1) . Statins & EPC homing Statins A ut ho rP The mechanisms by which statins increase EPC numbers and functional activity are not completely understood. Of note, the statin-induced increase in EPC numbers and myocardial neovascularization in the infarct border zone was dependent on endo thelial nitric oxide synthase (eNOS), since we did not observe these responses in eNOS -/- deficient mice [44] . A reduced bone marrow matrix metalloproteinase (MMP)-9 activity has been suggested to contribute to impaired stem-cell and progenitor cell mobilization in eNOS-deficient mice [45] ; however, recent studies did not observe an effect of statin therapy on bone marrow MMP-9 activity despite a significant mobilization of EPCs [46] . Therefore, the effects of statins on eNOS appear to be critical for EPC mobilization; however, the exact mechanisms whereby statins mobilize EPCs remain to be explored. Whereas several studies have shown that short-term statin treatment increases the number of circulating EPCs as detected by CD34 +/KDR+ cells or by culture assays of early EPCs, a recent retrospective study has suggested that long-term statin therapy (>8 weeks) may be associated with reduced EPC numbers, potentially as a result of increased EPC homing [47] . This observation, however, needs further confirmation. ro Statins & EPC mobilization of Several studies have suggested that the number and functional activity of circulating EPCs, as characterized by both, FACS and culture analysis, are increased by statins in mice and in patients with coronary disease or heart failure [19,20,40–43] . Moreover, in experimental studies, statin therapy accelerated re-endothelialization after balloon injury, that was associated with increased mobilization and incorporation of bone marrow–derived EPCs at the site of injury and a subsequently decreased neointima formation [19,20] Importantly, studies of bone marrow transplantation using Tie2/ lacZ mice [19] , or studies using retrovirally transfected bone marrow cells [20] , revealed that bone marrow-derived EPCs are directly recruited to endothelium-denudated areas of the arterial wall. Statin treatment of human early EPCs was found to upregulate the expression of endothelial integrin subunits α5 and β1, composing the fibronectin receptor, and αv and β5, which was associated with increased adhesiveness of EPCs towards endothelial cells in vitro [19] and may contribute to increased homing of EPCs to sites of vascular injury. Since changes in EPC number and function during statin treatment generally occured without significant correlations with LDL-cholesterol levels or statin-induced changes in LDL serum levels [42] , it has been argued that the enhancement of EPC’s functional activity by statins may represent a novel pleiotropic effect of statin therapy [52] . Of note, in a recent study, we have observed that a 4-week statin treatment, but not ezetimibe therapy, markedly increased functionally active EPCs in patients with chronic heart failure, despite a similar change in LDL cholesterol levels, suggesting that, at least short-term effects of statin treatment on EPCs are mediated by LDL cholesterol-independent mechanisms [43] . In conclusion, accumulating evidence suggests that statins mobilize EPCs, increase their functional activity and, probably their homing capacity to sites of vascular injury. These effects may contribute to the endothelial-protective effects of statins and are at least in part independent of their lipid-lowering properties. Statins & EPC proliferation, differentiation & senescence Several studies have suggested that statins increase proliferation, migration and survival of EPCs derived from peripheral blood [41,48–51] . Statins promote EPC differentiation and proliferation in human peripheral blood mononuclear cells via the PI3K/Akt pathway (Figure 3) [41] , whereas differentiation of blood-derived mononuclear cells towards vascular smooth muscle progenitor cells may be reduced by statins [48] . Ex vivo statin treatment of cultured early EPCs increased expression of cell cycle-promoting proteins [49] , protected telomeres by induction of telomere repeat-binding factor-2 [50] and inhibited TNF α-induced apoptosis [51] , suggesting that statins reduce EPC senescence and apoptosis, at least in vitro. 4 ACE inhibitors There is emerging evidence suggesting that modulation of the renin–angiotensin system by angiotensin-converting enzyme (ACE)-inhibitors or angiotensin II type 1 receptor blockers (ARBs) may have an impact on the number and functional activity of EPCs in different experimental and clinical settings. Treatment with enalapril, an ACE-inhibitor, increased the number of circulating EPCs in a murine hindlimb ischemia model and improved incorporation of EPCs into sites of active neovascularization, associated with enhanced blood flow recovery in ischemic hindlimbs [53] . These beneficial effects of enalapril were accompanied by reduced stromal cell-derived factor (SDF)-1 concentrations in bone marrow, but higher SDF-1 levels in peripheral blood of enalapril-treated mice, suggesting that reduced binding of EPCs to SDF-1 in bone-marrow may contribute to their release and mobilization after ACE-inhibition [53] . Of note, reduced SDF-1 levels in bone marrow after ACE-inhibition may, at least in part, result from increased bone marrow activation of dipeptidylpeptidase IV (DPP IV; CD26), a cell surface endopeptidase cleaving chemokines such as SDF-1α [53] . Notably, blockade of DPP IV by Diprotin-A, a DPP IV antagonist, prevented the effect of enalapril on ischemia-induced EPC mobilization [53] . Recently, treatment with another ACE-inhibitor, such as perindopril, in rats alone or in combination with the diuretic indapamide, restored both impaired levels and the angiogenic capacity of circulating EPCs in a hindlimb ischemia model in spontaneously hypertensive rats [54] . Moreover, in a small-scale clinical trial, treatment with ramipril for 4 weeks improved both number and functional activity of EPCs in patients with stable coronary artery disease, suggesting that stimulation of EPCs Expert Rev. Cardiovasc. Ther. 6(8), (2008) Pharmacological approaches to improve endothelial repair mechanisms Cardiovascular risk factors (hypertension, dyslipidemia, diabetes and smoking) – + EPC mobilization Review Statins, ACE inhibitors, ARBs, PPARγ agonists and EPO Physical exersice EPC repair capacity (e.g., homing, migration and paracrine effects) + of – Enhanced reendothelialization Improved endothelial function Direct incorporation rP Paracrine effects ro Diminished neointima formation Development and progression of atherosclerosis/restenosis ho Reendothelialization Expert Rev. Cardiovasc. Ther. © Future Science Group Ltd (2008) ut Figure 2. Proposed effects of cardiovascular risk factors and pharmacological treatment approaches on mobilization and functional repair capacity of EPCs. Cardiovascular risk factors have been shown to impair EPC mobilization from the bone marrow and EPC repair capacity in terms of homing (i.e., adhesion, migration, invasion or release of growth factors) and differentiation. On the other hand, several pharmacological therapies have been suggested to improve number and functional activity of EPCs in patients with cardiovascular disease or diabetes. Enhancement of endogenous vascular repair mechanisms may potentially contribute to the overall treatment effects of these drugs. A by ACE inhibitors may indeed contribute to beneficial vascular effects of ACE inhibitor therapy in patients with coronary artery disease [55] . Angiotensin II type 1 receptor antagonists Initial evidence that ARBs affect the number of circulating EPCs was obtained in patients with Type 2 diabetes treated with olmesartan or irbesartan for 12 weeks [56] . Treatment with either of these ARBs increased the number of EPCs in peripheral blood, with a significant effect in the irbesartan group already apparent after 4 weeks of therapy. Studies in spontaneously hypertensive rats have shown that candesartan and losartan augment the number and colony formation capacity of circulating EPCs and exert a favorable effect on EPC migration, at least in part by inhibiting oxidant stress in EPCs, as measured by the thiobarbituric reactive substances assay (TBARS) [57,58] . Of note, bone-marrow-derived CD34 + hematopoietic progenitor cells have been shown to express the angiotensin II type 1 receptor, suggesting that direct effects of angiotensin II on progenitor cells are www.expert-reviews.com possible [59,60] . In line with this concept, Imanishi et al. [61] observed that angiotensin II exerts detrimental effects on proliferation of EPCs from healthy human subjects in vitro, diminished telomerase activity and accelerated EPC senescence, possibly by induction of gp91phox-mediated peroxynitrite formation in EPCs. However, in a previous study from the same group, it was described that angiotensin II stimulated VEGFR-2 mRNA and protein expression in human EPCs, resulting in enhanced VEGF-induced proliferation of EPCs and vascular network formation in a matrigel assay [62] . Hence, these studies seem to be at least partly contradictory and provide evidence for both a stimulating and an inhibitory effect of angiotensin II on the proliferative capacity of EPCs. This question will therefore have to be clarified in further studies. Other antihypertensive agents Dihydropyridine calcium channel blockers Recent in vitro observations have suggested that another group of antihypertensive agents – calcium channel blockers – affect EPC number and functional activity. 5 Besler, Doerries, Giannotti, Lüscher & Landmesser β-blockers nebivolol & carvedilol ut of ho Several studies have suggested that carvedilol and nebivolol directly improve endothelial function, that is, they increase nitric oxide bioavailability and augment endothelium-dependent vasodilation in patients with coronary artery disease and essential hypertension [65,66] . In a recent study in C57BL/6 mice with extensive anterior myocardial infarction we have observed an improvement in endothelial function of aortic ring segments and an increase in the number of circulating EPCs after 4 weeks of treatment with nebivolol, whereas metoprolol succinate did not augment EPC levels during the treatment period, consistent with the notion that nebivolol may exert effects on EPCs independent of its β1-receptor blocking effect [C Dörries & U L andmesser ; Unpublished Data] . A Antidiabetic medication We and others have shown that EPC number and functional activity, such as migration, tube formation and re-endothelialization capacity, are substantially impaired in patients with diabetes mellitus [18,28,67–69] . Several recent studies have suggested that treatment with the PPAR-γ-agonists rosiglitazone and pioglitazone increases EPC number and functional activity, likely at least in part independent of their effects on glucose [18,70–73] . In addition, recent in vitro data suggest that insulin exerts an effect on EPC function via activation of the IGF-1 receptor [74] ; however, whether this occurs in diabetic patients treated with insulin remains to be determined. Thiazolidinediones: PPAR-γ agonists PPAR-γ agonists, such as rosiglitazone or pioglitazone, favorably influence glucose homeostasis by interfering with both adipogenesis and insulin resistance. In addition, PPAR-γ agonists have 6 been shown to reduce vascular inflammation, improve endothelial function and inhibit neointima formation [75,76] . A growing body of evidence suggests that vascular effects of PPAR-γ agonists are probably mediated by direct effects on vascular cells [75,76] and, as described later, on progenitor cells. Several studies have observed that both rosiglitazone and pioglitazone increase the number and functional activity of EPCs [18,70–73] . In an uncontrolled clinical study it was suggested that rosiglitazone therapy increased the number and in vitro migratory activity of circulating early EPCs [71] . We have observed in a prospective, randomized, placebo-controlled study that 2 weeks of therapy with rosiglitazone increased the number and in vivo re-endothelialization capacity of EPCs derived from patients with diabetes [18] . Pioglitazone therapy has been reported to increase the number, migratory and adhesion capacity of EPCs in patients with Type 2 diabetes after 8 weeks of therapy [72] . Moreover, incubation of diabetic EPCs with pioglitazone increased EPC proliferation and attenuated EPC apoptosis during ex vivo culture [72,73] . Notably, rosiglitazone therapy reduced superoxide production and increased nitric oxide production by EPCs derived from diabetic patients [18] . Furthermore, rosiglitazone treatment reduced NAD(P)H oxidase activity of EPCs from diabetic patients, representing a potential novel mechanism whereby PPAR-γ agonism promotes vascular repair [18] . In experiments using siRNA, we have observed that the effects of rosiglitazone on EPC nitric oxide availability and oxidative stress were mediated via the PPAR-γ receptor [18] . Moreover, ex vivo exposure of human peripheral blood mononuclear cells to rosiglitazone increased colony formation and likely promoted differentiation towards the endothelial lineage, whereas differentiation toward the smooth muscle cell lineage may be reduced [70] . In mice, treatment with pioglitazone for 10 days upregulated Sca1+/VEGFR-2 + EPCs in peripheral blood and bone marrow as well as the number of ex vivo cultured spleen-derived EPCs [73] . The increase in EPC number was accompanied by augmented vessel growth in a subcutaneously implanted polyvinyl sponge and improved migratory capacity of EPCs in vitro [73] . Furthermore, ex vivo pioglitazone treatment of EPCs increased expression of telomere repeat-binding factor 2 and prevented apoptosis induction in EPCs [73] . Several recent meta-analyses have suggested that pioglitazone therapy reduce cardiovascular risk more than rosiglitazone therapy [77,78] . Whereas both PPAR-γ agonists exert similar effects on endothelial progenitor cells, rosiglitazone therapy probably has a less favorable effect on the lipid profile [79] , which may be relevant for a potentially less favorable overall effect on clinical outcome. rP Ex vivo treatment of murine mononuclear cells with benidipine, a dihydropyridine-calcium channel blocker, increased the number of early EPCs after 7 days of culture [63] . Simultaneous treatment with wortmannin, a PI3K inhibitor, attenuated the effects of benedipine on cultured EPCs, suggesting that the PI3K pathway is involved. Moreover, incubation of EPCs with benidipine promoted Akt phosphorylation, further suggesting a role for the PI3K/Akt pathway in producing the observed effects of benidipine on early EPCs (Figure 3) [63] . Nifedipine – a dihydropyridine-calcium channel blocker capable of stimulating manganese superoxide dismutase (MnSOD) expression in mature endothelial cells – enhanced VEGF release from EPCs, improved migratory capacity of EPCs in a Boyden chamber system as well as adhesion capacity on TNF-α activated human umbilical vein endothelial cells [64] . Nifedipine increased MnSOD expression in EPCs and siRNAinduced knockdown of MnSOD abolished the beneficial effects of nifedipine on EPC migratory capacity, suggesting a role of this antioxidant enzyme system for the effects of nifedipine on EPCs. At present, however, no data on the effect of calcium channel antagonist therapy on in vivo EPC number and function have been published. ro Review Insulin An inital in vitro study has suggested that treatment of peripheral blood mononuclear cells with insulin dose-dependently increases the formation of EPC colony forming units and improves the tube formation capacity of EPCs, an effect that was in part mediated through extracellular signal-related kinase 1/2 and Expert Rev. Cardiovasc. Ther. 6(8), (2008) Pharmacological approaches to improve endothelial repair mechanisms EPO & its analogs Lipid-modifying therapies • Statins Inhibitors of the renin-angiotensin system • Angiotensin-converting enzyme inhibitors • Angiotensin II type 1 receptor blocker Other antihypertensive drugs • Dihydropyridine calcium channel blocker • b -blockers: carvedilol and nebivolol Antidiabetic medications • PPAR- g agonists: pioglitazone and rosiglitazone • Insulin Other • Erythropoietin eNOS phosphorylation and NO bioavailiability in EPO-treated cells (Figure 3) [86] . The effects of EPO treatment on vascular repair processes may depend, at least in part, on the vascular EPO receptor system, since VEGF expression, mobilization of EPCs, microvascular growth and blood flow recovery were impaired after femoral artery ligation in wild-type, bone-marrow transplanted EPO receptor knockout mice [87] . Moreover, EPO treatment for 14 days failed to increase CD34 + hematopoietic stem and progenitor cell numbers in eNOS-deficient mice, underlining the role of eNOS-derived NO for EPO-mediated mobilization of progenitor cells [88] . A ut ho rP ro Originally, erythropoietin (EPO) was described as a hematopoietic cytokine, regulating proliferation and differentiation of erythroid precursor cells. However, several studies have suggested that EPO confers several antiapoptotic and anti-inflammatory effects in cardiac, renal and neuronal cells, beyond regulation of hematopoiesis. Bahlmann et al. demonstrated, in patients with renal anemia and healthy subjects, that treatment with very low doses of recombinant human EPO and darbepoetin α, a recombinant EPO analog, increases the number of circulating CD34 + hematopoietic stem cells as well as the number and function of ex vivo cultured EPCs [81,82] . Heeschen et al. demonstrated that erythropoietin treatment increases the number and proliferation of bone-marrow derived EPCs, which was accompanied by improved neovascularization in a murine hindlimb ischemia model [83] . To test the relevance of their findings in humans, the authors isolated bone marrow-resident and circulating EPCs from patients with angiographically documented coronary artery disease and detected EPO serum levels. Of note, EPO serum levels correlated with the number and function of EPCs isolated from both bone marrow and peripheral blood, further suggesting that EPO may regulate EPCs in vivo [83] . Likewise, in patients with a first acute myocardial infarction, a single bolus injection of darbepoetin‑α before primary coronary intervention potently increased EPC number in peripheral blood [84] , whereas chronic EPO treatment of patients with congestive heart failure for a mean period of 28 months, failed to increase levels of hematopoietic progenitor cells in peripheral blood, but augmented proliferation of ex vivo cultured EPCs [85] . Initial results suggesting that EPO-induced increases of circulating EPCs exert beneficial effects on vascular repair processes, were obtained in a wire injury model of the femoral artery in mice [86] . Injection of recombinant human EPO for 3 days after induction of injury augmented levels of circulating EPCs, accelerated re-endothelialization of denudated areas of the femoral artery and inhibited neointima formation [86] . Mechanistically, EPO treatment affected the differentiation of circulating bone marrow-derived EPCs and proliferation of mature resident endothelial cells located next to the mechanical injury, which might in part be explained by an increase in Akt/ Box 1. Pharmaceutical agents that may increase number and functional activity of endothelial progenitor cells. of protein-kinase 38 [74] . Neutralizing antibodies and antisense oligonucleotides against the insulin receptor had no effect on EPC outgrowth, whereas inhibition of human IGF-1 receptor by neutralizing antibodies abrogated the insulin-induced effects on EPC number and function, suggesting that insulin exerts effects on EPCs via the IGF-1 receptor [74] . Whether this is also observed in diabetic patients treated with insulin remains to be determined. Initiation of insulin therapy in diabetic patients has been shown to increase the number of circulating CD34 +/ CD133 + hematopoietic progenitor cells in peripheral blood of patients with poorly controlled Type 2 diabetes mellitus after a mean time of 5.4 weeks [80] ; however, further information is required. Review www.expert-reviews.com HDL & EPCs HDL-targeted therapies are currently intensely studied as a potential novel therapeutic option in patients with cardiovascular disease. Besides promoting reverse cholesterol transport, HDL may exert direct vasoprotective effects [89] . In animal models, infusion of reconstituted HDL has been shown to increase the number of Sca-1+ hematopoietic stem cells, to promote endothelial repair in the thoracic aorta from apoE knockout mice and to augment angiogenesis in a murine hindlimb ischemia model [90,91] . Similarly, increased HDL levels induced by adenoviral transfer of human apolipoprotein (apo) A-I, the major structural HDL apo, improved both EPC number and function, and enhanced endothelial repair of transplanted carotid allografts in mice [92] . Pharmacological effects of apoA-I mimetics, such as D-4F, on reverse cholesterol transport and HDL-induced vasoprotective effects, are currently being evaluated. In a rat model of diabetes, long-term treatment with D-4F increased the number of EPC colonies in vitro and the expression of eNOS and heme oxygenase-1 in ex vivo cultured EPCs, suggesting a beneficial effect of D-4F on the proliferative and antioxidant capacity of EPCs in the diabetic state [93] . A first small clinical study in humans suggested that infusion of reconstituted HDL augments the number of CD34 +/VEGFR-2 + cells in peripheral blood of patients with Type 2 diabetes, 1 week after administration [94] . 7 Review Besler, Doerries, Giannotti, Lüscher & Landmesser Five-year view Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. A ut ho rP ro Expert commentary Since the initial description of putative bone-marrow-derived endothelial progenitor cells in human peripheral blood in 1997, numerous studies have suggested that circulating bone marrowderived EPCs promote re-endothelialization after vascular injury and ischemia-induced neovascularization. Importantly, patients with cardiovascular risk factors or coronary disease have, in most studies, a reduced number and functional capacity of EPCs as compared to healthy subjects, suggesting that cardiovascular risk factors do not only directly alter vascular endothelial function, but also impair the endothelial repair capacity mediated by circulating progenitor cells. Notably, several pharmacological treatment approaches, such as statin therapy, PPAR-γ, ACE-inhibitors and ARB therapy, likely have a direct impact on circulating progenitor cells, that may contribute to their overall effects on the vascular wall. Furthermore, several pharmacological agents may help to optimize cell-based treatment approaches in cardiovascular medicine, given the significant impairment of autologous, patientderived progenitor cells currently used in clinical studies. Endothelial progenitor cells are currently assessed by several methods, including FACS analysis of peripheral blood, using markers such as CD34 and KDR, culture and/or colony-forming assays of blood-derived mononuclear cells and by transplantation of culture-derived EPCs into nude mouse models. There is a growing consensus that there are likely several subsets of EPCs. ‘Early’ EPCs are most likely derived from CD14 + cells, are obtained after short-term culture of mononuclear cells and act largely by paracrine effects, whereas ‘late’ EPCs may transdifferentiate into endothelial cells, i.e., may incorporate into the endothelial layer, but are substantially lower in numbers. Within the next years our understanding of the definition and functional role of subsets of EPCs will hopefully improve and we will learn more about mechanisms leading to their ‘dysfunction’ in cardiovascular disease. EPCs may represent an interesting therapeutic target for pharmaceutical strategies, but also for cell-based therapies of vascular and cardiac disease. However, the mechanisms whereby drug therapy alters number and function of circulating EPCs will have to be determined in more detail. In particular, the impact of pharmacological agents on direct incorporation of EPCs into the endothelial monolayer and the release of paracrine mediators by EPCs will have to be characterized. In addition, studies comparing effects of different pharmacological agents from the same class of drugs on EPC number and function, are of interest to detect potential differences between pharmacophores. Research on EPCs will likely lead to novel developments in the field of bioengineering, such as bioengineered stents and autologous ‘living’ heart valves [97] . of Ex vivo treatment of human EPCs with either sphingosine-1-phosphate (S1P), an HDL-associated lysophospholipid, or its synthetic analog FTY720, improved blood flow after transplantation of EPCs in a murine hindlimb ischemia model, at least in part via S1P3 receptor-induced phosphorylation of the CXCR4 receptor [95] . Of note, CXCR4 receptor signaling is critical for the repair capacity of EPCs and is involved in homing, migration and functional integration of progenitor cells in ischemic tissues, suggesting a possible mechanism whereby HDL may exert its beneficial effects on functional activity of EPCs. In addition, HDL may increase the number of EPCs in peripheral blood by inhibiting EPC apoptosis, since incubation of isolated human EPCs with pooled HDL from healthy donors attenuated homocysteine-induced caspase-3 activity in vitro, whereas eNOS expression in EPCs increased in the presence of HDL derived from healthy donors [96] . Thus, raising HDL with beneficial vascular effects or apoA-I may increase the repair capacity of EPCs. Key issues • Functional and structural disruption of the endothelium is thought to play a critical role in development and complications of atherosclerotic vascular disease and restenosis after percutaneous coronary interventions. • Accumulating evidence suggests that endothelial progenitor cells (EPCs) promote re-endothelialization of injured arteries and ischemiainduced neovascularization. • Notably, recent studies have suggested that number and functional repair capacity of circulating EPCs are profoundly reduced in patients with cardiovascular risk factors or established cardiovascular disease. • Several pharmacological agents have been suggested to increase number and/or functional activity of EPCs that may play a role in their therapeutic effects, such as statins, PPAR-γ-agonists, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers and erythropoietin. • In addition, in vitro data have suggested that dihydropyridine calcium channel blockers, insulin and reconstituted HDL increase functional activity of EPCs. • The mechanisms whereby drug therapy alters EPC number and function will have to be determined in more detail, but likely include effects on the PI3-kinase-Akt-endothelial nitric oxidesynthase pathway. • Several pharmacological agents may play a role for optimization of cell-based treatment approaches in the cardiovascular field, for example by improving the functional and homing capacity of autologous patient-derived progenitor cells. 8 Expert Rev. 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Affiliations • Christian Besler, MD Cardiovascular Center, University Hospital Zurich; and, Cardiovascular Research, Institute of Physiology; and, Center for Integrative Human Physiology, University of Zurich, Switzerland Tel.: +41 446 355 097 Fax: +41 446 356 827 [email protected] 11 • Giovanna Giannotti, MD Cardiovascular Center, University Hospital Zurich; and, Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland Tel.: +41 446 355 081 Fax: +41 446 356 827 [email protected] • Thomas F Lüscher, MD Cardiovascular Center, University Hospital Zurich; and, Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland Tel.:+41 442 552 121 Fax: +41 442 554 251 [email protected] • Ulf Landmesser, MD Cardiovascular Center, University Hospital Zurich, Raemistrassse 100, 8091 Zurich, Switzerland Tel.: +41 442 559 595 Fax: + 41 442 554 251 [email protected] ro Carola Doerries, MD Cardiovascular Center, University Hospital Zurich; and, Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland Tel.: +41 446 355 097 Fax: +41 446 356 827 [email protected] A ut ho • of Besler, Doerries, Giannotti, Lüscher & Landmesser rP Review 12 Expert Rev. Cardiovasc. Ther. 6(8), (2008)
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