Nifedipine improves endothelial function: role of endothelial progenitor cells

Nifedipine has been shown to improve endothelial function. Recent studies have indicated that endothelial function is correlated with the number of circulating endothelial progenitor cells (EPCs), but it is unclear whether nifedipine affects the number and function of EPCs. The aims of this study were to determine the effects of nifedipine on the number and function of EPCs and to investigate the relationship between improvement of endothelial function and EPC numbers in patients with hypertension. Stage 1 hypertensive men (n=37) were randomly divided into the nifedipine group and the control untreated group. The nifedipine group was administered slow-release nifedipine (20 mg) once daily. At baseline and after 4 weeks, flow-mediated dilation, blood pressure, biochemical data, and number of circulating CD34+CD133+ progenitor cells and EPCs were measured. The direct effects of nifedipine on EPC number and function were assessed in vitro. In the nifedipine group, flow-mediated dilation and the numbers of circulating CD34+CD133+ progenitor cells and EPCs were increased, along with a decrease of serum malondialdehyde low-density lipoprotein. The improvement of flow-mediated dilation by nifedipine was correlated with the increase of circulating CD34+CD133+ progenitor cells. Nifedipine also improved angiogenesis-related functions of EPCs (differentiation, migration, and resistance to oxidative stress) in vitro. Thus, nifedipine improved endothelial function and EPC function in stage 1 hypertensive subjects. The latter action may be mediated by reduction of oxidative stress and suppression of EPC apoptosis. These results demonstrate that nifedipine preserves endothelial integrity in patients with hypertension, at least partly, by enhancing EPC numbers and activity.     

Human CD34+AC133+VEGFR-2+ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors

OBJECTIVE: Endothelial progenitor cells (EPCs) are used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk. However, there is no uniform definition of an EPC, which confounds EPC studies. EPCs are widely described as cells that coexpress the cell-surface antigens CD34, AC133, and vascular endothelial growth factor receptor-2 (VEGFR-2). These antigens are also expressed on primitive hematopoietic progenitor cells (HPCs). Remarkably, despite their original identification, CD34+AC133+VEGFR-2+ cells have never been isolated and simultaneously plated in hematopoietic and endothelial cell (EC) clonogenic assays to assess the identity of their clonal progeny, which are presumably the cellular participants in vascular regeneration. METHODS: CD34+AC133+VEGFR-2+ cells were isolated from human umbilical cord blood (CB) or granulocyte colony-stimulating factor-mobilized peripheral blood and assayed for either EPCs or HPCs. RESULTS: CD34+AC133+VEGFR-2+ cells did not form EPCs and were devoid of vessel forming activity. However, CD34+AC133+VEGFR-2+ cells formed HPCs and expressed the hematopoietic lineage-specific antigen, CD45. We next tested whether EPCs could be separated from HPCs by immunoselection for CD34 and CD45. CD34+CD45+ cells formed HPCs but not EPCs, while CD34+CD45- cells formed EPCs but not HPCs. CONCLUSIONS: Therefore, CD34+AC133+VEGFR-2+ cells are HPCs that do not yield EC progeny, and the biological mechanism for their correlation with cardiovascular disease needs to be reexamined.     

Increased circulating hematopoietic and endothelial progenitor cells in the early phase of acute myocardial infarction

Endothelial progenitor cell (EPC) mobilization has been reported following tissue damage, whereas no data are available regarding the mobilization of hematopoietic progenitor cells (HPCs). We performed the phenotypic and functional analysis of circulating CD34+ progenitor cells in patients with acute myocardial infarction (AMI), assessed from admission up to 60 days, in patients with stable angina pectoris (SA), and in healthy controls (CTRLs). In patients with AMI at admission (T0), the number of circulating CD34+ cells was higher (P < .001) than in CTRLs and became comparable with CTRLs within 60 days. Both the number of CD34+ cells coexpressing CD33, CD38, or CD117 and the number of HPCs was higher (P < .02 for all) in patients with AMI at T0 than in CTRLs, as was the number of hematopoietic colonies (P < .03). Patients with AMI (T0) had a significantly increased number of CD34+ vascular endothelial growth factor receptor 2-positive (VEGFR-2+) cells (P < .002) with respect to CTRLs, including CD34(+) CD133(+)VEGFR-2+ and CD34+ CD117(+)VEGFR-2+ EPCs. The number of endothelial colonies was higher in patients with AMI (T0) than in CTRLs (P < .05). No significant difference was documented between patients with SA and CTRLs. Spontaneous mobilization of both HPCs and EPCs occurs within a few hours from the onset of AMI and is detectable until 2 months.     

Role of progenitor endothelial cells in cardiovascular disease and upcoming therapies.

The field of cell-based transplantation has expanded considerably and is poised to become an established cardiovascular therapy in the near future. In this review, we will focus on endothelial progenitor cells (EPCs), which are immature cells capable of differentiating into mature endothelial cells. EPCs share many surface marker antigens such as CD34, AC133, Flk-1, etc. with hematopoietic stem cells (HSCs) and the major source of EPCs as well as HSCs is the bone marrow (BM). BM-derived EPCs are mobilized into peripheral blood and recruited to the foci of pathophysiological neovascularization and reendothelialization, thereby contributing to vascular regeneration. Severe EPC dysfunction is an indicator of poor prognosis and severe endothelial dysfunction. Indeed, number of circulating EPCs and their migratory activity are reduced in patients with diabetes, coronary artery disease (CAD), or subjects with multiple coronary risk factors. Effective neovascularization induced by EPC transplantation for hindlimb, myocardial, and cerebral ischemia has been demonstrated in many preclinical studies, and early clinical trials of EPC transplantation in chronic and acute CAD indicate safety and feasibility of myocardial cell-based therapies. For therapeutic reendothelialization in patients undergoing percutaneous coronary intervention, CD34 antibody-coated stents have been used clinically to capture circulating EPCs at the injury sites and enhance reendothelialization and safety of stents. Further development in cell processing technology for efficient isolation, expansion, mobilization, recruitment, and transplantation of EPCs into target tissues are underway and expected to be tested in clinical trials in the near future.     

Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors

Emerging data suggest that a subset of circulating human CD34(+) cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 +/- 0.3% of CD34(+) cells isolated from fetal liver (FL), 2 +/- 0.5% from mobilized peripheral blood, and 1.4 +/- 0.5% from cord blood were VEGFR-2(+). In addition, most CD34(+)VEGFR-2(+) cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34(+) cells phenotypically identifies a unique population of CEPs. CD34(+)VEGFR-2(+) cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34(+)VEGFR-2(+) cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34(+) cells that give rise to endothelial colonies, CD34(+) cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2(+) cells with VEGF and FGF-2 resulted in differentiation of AC133(+ )VEGFR-2(+) cells into adherent AC133(-)VEGFR-2(+)Ac-LDL(+ )(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133(+)VEGFR-2(+) cells. These data suggest that circulating CD34(+) cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.     

Differentiation of endothelial cells from human umbilical cord blood AC133−CD14+ cells

Endothelial progenitor cells (EPCs) participate in neovascularization and are consistent with postnatal vasculogenesis. In vitro, they differentiate into endothelial cells (ECs). Prior reports have suggested that circulating human AC133(+) cells have the capacity to differentiate into ECs as progenitor cells. However, recent studies have demonstrated that circulating CD34(-)CD14(+) cells also have EPC-like properties in vitro and in vivo. We tested whether AC133(-)CD14(+) cells from human umbilical cord blood (HUCB) have the potential to differentiate into ECs. The AC133(-)CD14(+) cells were isolated from HUCB by magnetic bead selection and cultured on fibronectin-coated six-well trays in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin growth factor (IGF-1). The AC133(-)CD14(+) cells adhered slightly within 1 day of culture and subsequently underwent a distinct process of morphological transformation to spindle-shaped cells that sprouted from the edge of the cell clusters. After 14 days, the cells formed cord- and tubular-like structures. The AC133(-)CD14(+) cells showed a strong increase in the endothelial marker P1H12 over time, whereas CD14 decreased, and CD45 did not change, respectively. In addition, the cells expressed endothelial markers von Willebrand's factor (vWF), platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1, VEGFR-2/Flk-1, eNOS, and VE-cadherin, but did not express Tie-2 after 7 days of culture. The present data indicate that AC133(-)CD14(+) cells from HUCB are able to develop endothelial phenotype with expression of endothelial-specific surface markers and even form cord- and tubular-like structures in vitro as progenitor cells.     

冠心病患者胰岛素水平与内皮祖细胞及冠状动脉病变的相关性

目的 探讨冠心病患者不同胰岛素水平与循环内皮祖细胞(EPC)数量、功能及冠状动脉病变程度的关系并探讨相关临床意义.方法 69例经选择性冠状动脉造影证实的冠心病患者,按胰岛素水平高低分为胰岛素抵抗(IR)组和胰岛素敏感(IS)组,另设25例健康对照者.采集研究对象外周血以激酶插入区域受体(KDR)和CD133双阳性为循环EPC标记行流式细胞分析,同时采血进行EPC的分离培养,7 d后鉴定并检测增殖及迁移能力,将各组的一般临床资料,循环EPC数量、迁移、增殖能力指标、稳态模型胰岛素抵抗指数(HOMA-IR)及冠状动脉病变Gensini评分进行统计学分析.结果 IR组循环EPC数量明显少于IS组[(0.34±0.08)‰比(0.47±0.09)‰,P<0.01],HOMA-IR自然对数与循环EPC数量呈负相关(r=-0.291,P=0.01),循环EPC数量与Gensini评分呈负相关(r=-0.3984,P=0.006).IR组的增殖能力和迁移能力均低于IS组减弱(P<0.05).结论 冠心病患者血清胰岛素水平与循环EPC数量呈负相关.循环EPC数量及功能与冠状动脉病变程度呈负相关;IR或高胰岛素血症可能部分通过损害循环EPC的数量及功能,从而影响冠状动脉病变程度.     

VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells

Human CD133 (AC133)(+)CD34(+) stem and progenitor cells derived from fetal liver and from bone marrow and blood incorporate a functional population of circulating endothelial precursor cells. Vascular endothelial growth factor receptor 3 (VEGFR-3) regulates cardiovascular development and physiological and pathological lymphangiogenesis and angiogenesis. However, the origin of VEGFR-3(+) endothelial cells (ECs) and the mechanisms by which these cells contribute to postnatal physiological processes are not known, and the possible existence of VEGFR-3(+) lymphatic or vascular EC progenitors has not been studied. Using monoclonal antibodies to the extracellular domain of VEGFR-3, we show that 11% +/- 1% of CD34(+) cells isolated from human fetal liver, 1.9% +/- 0.8% CD34(+) cells from human cord blood, and 0.2% +/- 0.1% of CD34(+) cells from healthy adult blood donors are positive for VEGFR-3. CD34(+)VEGFR-3(+) cells from fetal liver coexpress the stem/precursor cell marker CD133 (AC133). Because mature ECs do not express CD133, coexpression of VEGFR-3 and CD133 on CD34(+) cells identifies a unique population of stem and progenitor cells. Incubation of isolated CD34(+)VEGFR-3(+) cells in EC growth medium resulted in a strong proliferation (40-fold in 2 weeks) of nonadherent VEGFR-3(+) cells. Plating of these cells resulted in the formation of adherent VEGFR-3(+)Ac-LDL(+) (Ac-LDL = acetylated low-density lipoprotein) EC monolayers expressing various vascular and lymphatic endothelial-specific surface markers, including CD34, VE-cadherin, CD51/61, CD105, LYVE-1, and podoplanin. These data demonstrate that human CD34(+)CD133(+) cells expressing VEGFR-3 constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal lymphangiogenesis and/or angiogenesis.     

Endurance training increases the number of endothelial progenitor cells in patients with cardiovascular risk and coronary artery disease

OBJECTIVE: As regular physical exercise improves endothelial dysfunction and promotes cardiovascular health, we investigated the effect of training on angiogenesis by measuring the number of circulating endothelial progenitor cells (EPC), the level of EPC-mobilizing growth factors and tested vascular function by flow-mediated dilation (FMD) in patients with coronary artery disease (CAD) and cardiovascular risk factors (CVRF). In addition, degradation products of the NO pathway (NOx) were determined. METHODS AND RESULTS: Twenty patients with documented CAD and/or CVRF joined a 12-week supervised running training. Circulating EPCs--defined by the surface markers CD34, KDR and CD133--were measured at baseline and after exercise training by flow cytometry. We found a significant increase in circulating EPCs (2.9+/-0.4-fold increase; P < .0001), which was positively correlated with both, the change of FMD (r = .81, P < .001) and the increase of NOx synthesis (r = .83, P < .001). Plasma VEGF and erythropoietin did not change in response to exercise. However, we observed a positive correlation between the number of EPCs and erythropoietin at baseline (r = .70, P < .01) and after training (r = .73, P < .01). CONCLUSIONS: Regular exercise training augments the number of circulating EPCs in patients with CVRF and CAD and is associated with improved vascular function and NO synthesis.     

Impaired progenitor cell activity in age-related endothelial dysfunction

OBJECTIVES: We investigated whether human age-related endothelial dysfunction is accompanied by quantitative and qualitative alterations of the endothelial progenitor cell (EPC) pool. BACKGROUND: Circulating progenitor cells with an endothelial phenotype contribute to the regeneration and repair of the vessel wall. An association between the loss of endothelial integrity and EPC modification may provide a background to study the mechanistic nature of such age-related vascular changes. METHODS: In 20 old and young healthy individuals (61 +/- 2 years and 25 +/- 1 year, respectively) without major cardiovascular risk factors, endothelial function, defined by flow-mediated dilation of the brachial artery via ultrasound, as well as the number and function of EPCs isolated from peripheral blood, were determined. RESULTS: Older subjects had significantly impaired endothelium-dependent dilation of brachial artery (flow-mediated dilation [FMD] 5.2 +/- 0.5% vs. 7.1 +/- 0.6%; p < 0.05). Endothelium-independent dilation after glycerol trinitrate (GTN) was not different, but the FMD/GTN ratio was significantly lower in old subjects (49 +/- 4% vs. 37 +/- 3%; p < 0.05), suggesting endothelial dysfunction. There were no differences in the numbers of circulating EPCs, defined as CD34/KDR or CD133/KDR double-positive cells in peripheral blood. In contrast, lower survival (39 +/- 6 cells/mm(2) vs. 65 +/- 11 cells/mm(2); p < 0.05), migration (80 +/- 12 vs. 157 +/- 16 cells/mm(2); p < 0.01), and proliferation (0.20 +/- 0.04 cpm vs. 0.44 +/- 0.07 cpm; p < 0.05) implicate functional impairment of EPCs from old subjects. The FMD correlated univariately with EPC migration (r = 0.52, p < 0.05) and EPC proliferation (r = 0.49, p < 0.05). Multivariate analysis showed that both functional features represent independent predictors of endothelial function. CONCLUSIONS: Maintenance of vascular homeostasis by EPCs may be attenuated with age based on functional deficits rather than depletion of CD34/KDR or CD133/KDR cells.