Caveolin plays a central role in endothelial progenitor cell mobilization and homing in SDF-1-driven postischemic vasculogenesis

When neovascularization is triggered in ischemic tissues, angiogenesis but also (postnatal) vasculogenesis is induced, the latter requiring the mobilization of endothelial progenitor cells (EPC) from the bone marrow. Caveolin, the structural protein of caveolae, was recently reported to directly influence the angiogenic process through the regulation of the vascular endothelial growth factor (VEGF)/nitric oxide pathway. In this study, using caveolin-1 null mice (Cav(-/-)), we examined whether caveolin was also involved in the EPC recruitment in a model of ischemic hindlimb. Intravenous infusion of Sca-1(+) Lin(-) progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization in Cav(-/-) mice, suggesting a defect in progenitor mobilization. The adhesion of Cav(-/-) EPC to bone marrow stromal cells indeed appeared to be resistant to the otherwise mobilizing SDF-1 (Stromal cell-Derived Factor-1) exposure because of a defect in the internalization of the SDF-1 cognate receptor CXCR4. Symmetrically, the attachment of Cav(-/-) EPC to SDF-1-presenting endothelial cells was significantly increased. Finally, EPC transduction with caveolin small interfering RNA reproduced this advantage in vitro and, importantly, led to a more extensive rescue of the ischemic hindlimb after intravenous infusion (versus sham-transfected EPC). These results underline the critical role of caveolin in ensuring the caveolae-mediated endocytosis of CXCR4, regulating both the SDF-1-mediated mobilization and peripheral homing of progenitor cells in response to ischemia. In particular, a transient reduction in caveolin expression was shown to therapeutically increase the engraftment of progenitor cells.     

Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization.

AIMS: Erythropoietin (EPO) improves cardiac function and induces neovascularization in chronic heart failure (CHF), although the exact mechanism has not been elucidated. We studied the effects of EPO on homing and incorporation of endothelial progenitor cells (EPC) into the myocardial microvasculature and myocardial expression of angiogenic factors. METHODS AND RESULTS: CHF was induced in rats by coronary artery ligation resulting in myocardial infarction (MI) after bone marrow had been replaced by human placental alkaline phosphatase (hPAP) transgenic cells. We studied the effects of darbepoetin alfa treatment (EPO, 40 microg/kg, every 3 weeks, starting 3 weeks after MI) on longitudinal changes in left ventricular (LV) function, circulating EPC, myocardial histology, and expression of vascular endothelial growth factor (VEGF) determined 9 weeks after MI. EPO prevented LV-dilatation and improved cardiac function (all P < 0.05), which was associated with 42% increased capillary growth (P < 0.01). EPO-induced mobilization of EPC from the bone marrow (P < 0.01), which resulted in a three-fold increased homing of EPC into the cardiac microvasculature. The percentage of the endothelium that consisted of bone marrow derived cells was significantly increased (3.9 +/- 0.5 vs. 11.4 +/- 1%, P < 0.001) comprising 30% of the newly formed capillaries. In addition, EPO treatment resulted in a 4.5-fold increased myocardial expression of VEGF, which correlated strongly with neovascularization (r = 0.67; P < 0.001). VEGF was equally expressed by endothelial cells of myocardial and bone marrow origin. CONCLUSION: EPO-induced neovascularization in post-MI heart failure is mediated through a combination of EPC recruitment from the bone marrow and increased myocardial expression of VEGF.     

Reduction of connexin43 in human endothelial progenitor cells impairs the angiogenic potential

Our previous work showed that arsenic trioxide down-regulated Cx43 and attenuated the angiogenic potential of human late endothelial progenitor cells (EPC). However, the relation between Cx43 and angiogenic activity of the EPC remained unclear. In the study, human late EPC were treated with siRNA specific to Cx43 (Cx43siRNA). The expression profiles as well as activity of the treated cells were examined. In parallel, the angiogenic potential of human EPC treated with Cx43siRNA was evaluated using murine hind limb ischemic model. The results showed that, in the EPC treated with Cx43siRNA, the activity of migration, proliferation, and angiogenic potential were attenuated, accompanied by reduction in vascular endothelial growth factor (VEGF) expression. In hind limb ischemia mice, EPC treated with Cx43siRNA lost the therapeutic angiogenic potential. VEGF supplementation partially recovered the activity impaired by Cx43 down-regulation. In conclusion, reduced Cx43 expression per se in the EPC causes decreased expression of VEGF and impaired angiogenic potential of the cells. Prevention of Cx43 reduction is a potential target to maintain the angiogenic potential of the EPC.     

SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit(+) stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting alpha(2), alpha(4), and alpha(5) integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit(+) cells, while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4) restored such differentiation, suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection, plasma SDF-1 levels were up-regulated, while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally, using an in vivo assay such as injection of matrigel plugs, we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit(+) cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells.     

Protein kinase C alpha promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor

AIMS: Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined. METHODS AND RESULTS: mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms alpha, delta and epsilon in HUVEC. Knockdown of PKC alpha diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC alpha activation and PKC-dependent VEGF upregulation. CONCLUSIONS: PKC alpha activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC alpha-dependent positive feedback mechanism.     

Effects of exercise training on endothelial progenitor cells in patients with chronic heart failure

BackgroundThe enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34^pos/KDR^pos EPCs, as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines, were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT).Methods and ResultsThe levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay, respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT, the levels of both EPC and VEGF/SDF-1 markedly increased (P < .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential, but on DSAT the baseline level was incompletely attained.ConclusionsIn response to SAT, patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential.     

Arterio-venous gradients of endothelial progenitor cells reveal a complex kinetics in human limb ischemia

Ischemic recruitment of endothelial progenitor cells (EPCs) in involved in compensatory angiogenic in animal models, but this still needs to be substantiated in humans. We enrolled 12 patients, who underwent surgical correction of abdominal aortic aneurysm without atherosclerosis of leg arteries (n = 4) or lower limb atherosclerosis obliterans (AO; n = 8). We measured VEGF, SDF-1, lactate and CD34+ KDR+ EPCs in the arterial and venous circulation of lower limbs. We found that, irrespectively of AO stage and lactate production, there was no consistent arterio-venous gradient of EPC, VEGF and SDF-1. Notably, in 4/8 patients, EPCs were more abundant in the vein than in the artery. EPC gradient was directly correlated with VEGF gradient and inversely correlated with SDF-1 gradient. In conclusion, we failed to show any consistent gradient of EPCs across ischemic limbs in relation to severity of atherosclerosis obliterans, but we speculatively suggest that a bidirectional traffic of EPCs in and out the ischemic tissue might be regulated by VEGF and SDF-1.     

Bone marrow subpopulations contain distinct types of endothelial progenitor cells and angiogenic cytokine-producing cells

Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). However, the mechanism of BMC-mediated neovascularization remains to be clarified. We investigated the differential activities of bone marrow subpopulations in angiogenesis and cytokine production. BMCs were separated into positive and negative fractions by surface expression of Mac-1, Gr-1, CD19, and c-kit, respectively. After 7 days of culture in the presence of vascular endothelial growth factor (VEGF), the cells produced adherent cells which incorporate acetylated low-density lipoprotein (acLDL). Mac-1(+) and Mac-1(-) cells produced almost equal numbers of acLDL(+) cells, but only Mac-1(-) cells expressed endothelial markers, including Flk-1, vWF, and CD31. Similarly, the expression of endothelial markers was detected in Gr-1(-), CD19(-), and c-kit(+) BMC fractions at 7-day cultures, but not in Gr-1(+), CD19(+), or c-kit(-) cells. In contrast, freshly isolated Mac-1(+) and Gr-1(+) BMCs expressed higher levels of mRNAs for angiogenic cytokines (including VEGF-A, FGF-2, and HGF) than Mac-1(-) and Gr-1(-) cells, respectively. Moreover, Mac-1(+)/c-kit(+) BMC subpopulation expressed higher levels of VEGF-A and SDF-1 mRNAs than other subpopulations. These data demonstrate that a relatively small proportion of VEGF-cultured adherent cells are true endothelial cells with a Flk-1(+)/vWF(+)/CD31(+) phenotype. Moreover, endothelial stem/progenitor cells (EPCs) are limited primarily to Mac-1(-), Gr-1(-), and c-kit(+) BMC populations. In contrast, angiogenic cytokine mRNAs were also produced by Mac-1(+), Gr-1(+), and c-kit(-) BMCs, suggesting the heterogeneity of effector cell types for neovasculatization therapy.     

Age-dependent impairment of endothelial progenitor cells is corrected by growth-hormone-mediated increase of insulin-like growth-factor-1

Aging is associated with an increased risk for atherosclerosis. A possible cause is low numbers and dysfunction of endothelial progenitor cells (EPC) which insufficiently repair damaged vascular walls. We hypothesized that decreased levels of insulin-like growth factor-1 (IGF-1) during age contribute to dysfunctional EPC. We measured the effect of growth hormone (GH), which increases endogenous IGF-1 levels, on EPC in mice and human subjects. We compared EPC number and function in healthy middle-aged male volunteers (57.4+/-1.4 years) before and after a 10 day treatment with recombinant GH (0.4 mg/d) with that of younger and elderly male subjects (27.5+/-0.9 and 74.1+/-0.9 years). Middle-aged and elderly subjects had lower circulating CD133(+)/VEGFR-2(+) EPC with impaired function and increased senescence. GH treatment in middle-aged subjects elevated IGF-1 levels (126.0+/-7.2 ng/mL versus 241.1+/-13.8 ng/mL; P<0.0001), increased circulating EPC with improved colony forming and migratory capacity, enhanced incorporation into tube-like structures, and augmented endothelial nitric oxide synthase expression in EPC comparable to that of the younger group. EPC senescence was attenuated, whereas telomerase activity was increased after GH treatment. Treatment of aged mice with GH (7 days) or IGF-1 increased IGF-1 and EPC levels and improved EPC function, whereas a two day GH treatment did not alter IGF-1 or EPC levels. Ex vivo treatment of EPC from elderly individuals with IGF-1 improved function and attenuated cellular senescence. IGF-1 stimulated EPC differentiation, migratory capacity and the ability to incorporate into forming vascular networks in vitro via the IGF-1 receptor. IGF-1 increased telomerase activity, endothelial nitric oxide synthase expression, phosphorylation and activity in EPC in a phosphoinositide-3-kinase/Akt dependent manner. Small interference RNA-mediated knockdown of endothelial nitric oxide synthase in EPC abolished the IGF-1 effects. Growth hormone-mediated increase in IGF-1 reverses age-related EPC dysfunction and may be a novel therapeutic strategy against vascular disorders with impairment of EPC.     

Transplantation of endothelial progenitor cells improves neovascularization and left ventricular function after myocardial infarction in a rat model

Cell transplantation has recently emerged as a novel therapy for ischemic heart disease. The presented study investigated the effect of intramyocardial transfer of human endothelial progenitor cells (EPCs) and stromal-cell derived factor-1α (SDF-1α) on left ventricular function in a chronic setting after myocardial infarction in cyclosporine treated rats. BrdU-labeled EPCs (10⁶), 10 μg SDF-1α, EPCs+SDF-1α or placebo medium were injected directly into the border infarct zone 4 weeks after acute myocardial infarction. Eight weeks after transplantation, echocardiography identified significantly improved fractional shortening after EPC or EPCs+SDF-1α injection as compared with injection of placebo medium. Investigating isolated hearts revealed a significant increase in left ventricular developing pressure after transplantation of SDF-1α or EPCs+SDF-1α. Furthermore, coronary flow rates were significantly elevated, especially after transplantation of EPCs+SDF-1α (under catecholamine stress 24.2 ± 1.55 ml/min vs. 13.1 ± 1 ml/min in the control) correlating with increased density of CD31+ vessel structures in the EPC as well as EPCs+SDF-1α groups, thus defining a higher rate of neovascularization. Notably, SDF-1α injected hearts showed only a trend towards improvement in coronary flow. BrdU+ signals were detected in infarct areas, partially integrating into vascular networks. The rate of apoptotic cells as well as the amount of inflammatory cells was significantly elevated in the placebo control group. In conclusion, transplantation of EPCs as well as EPCs+SDF-1α associated with improvement in cardiac function after infarction, which was attributable to enhanced neovascularization and decreased inflammation. These results imply a combined benefit of EPCs+SDF-1α in the treatment of myocardial infarction. A. Schuh and E.A. Liehn contributed equally to this article. Returned for 1. revision: 6 June 2007 1. Revision received: 27 August 2007 Returned for 2. revision: 24 September 2007 2. Revision received: 1 October 2007     

Nifedipine improves the migratory ability of circulating endothelial progenitor cells depending on manganese superoxide dismutase upregulation

BACKGROUND: Migratory ability of resident endothelial cells and their circulating progenitors, that is endothelial progenitor cells (EPCs), represent a crucial event in vascular repairing processes. Although oxidants are known to counteract the migratory ability of resident endothelial cells, their possible role in modulating EPC motility is unknown. EPCs exhibit stronger resistance to oxidants than mature endothelial cells mainly because of higher expression of manganese (Mn) superoxide dismutase (SOD). As nifedipine is a dihydropyridine calcium antagonist known to enhance MnSOD expression in mature endothelial cells, we investigated the effects of nifedipine on MnSOD expression and motility in EPCs. METHODS AND RESULTS: EPCs were isolated from peripheral blood of healthy donors and cultured in fibronectin-coated flasks. Nifedipine improved both motility of cultured EPCs (+55% vs. control, P = 0.007) and their adhesion to tumor necrosis factor-alpha-activated mature endothelial cells (+33% vs. control, P = 0.03). Reduction of EPC dichlorofluorescein content (-32% vs. control, P = 0.009) and a parallel increase in nitrite plus nitrate concentration in EPC supernatants (+25% vs. control, P = 0.009) were also observed. The study of SOD showed a nifedipine-dependent upregulation of MnSOD in a time-dependent and dose-dependent manner. MnSOD expression blockade by RNA interference abolished nifedipine effect on EPC motility. Although nifedipine also increased vascular endothelial growth factor (VEGF) release from EPCs, its effect on EPC motility was unaffected by an anti-VEGF antibody. CONCLUSION: Nifedipine improves EPC motility due to MnSOD upregulation. The capability of this dihydropyridine calcium antagonist to reduce cardiovascular events might also be related to improved EPC function.     

内皮祖细胞分泌SDF-1及其抗内皮祖细胞凋亡作用

目的探讨内皮祖细胞（EPC）能否分泌基质细胞衍生因子-1（SDF-1）,及SDF-1能否抑制紫杉醇、AMD3100（特异性阻断SDF-1与其受体CXCR4结合）等诱导的EPC凋亡。方法取不同时间段的EPC培养液上清,采用ELISA检测上清中SDF-1a浓度。用不同浓度的紫杉醇、AMD3100及无血清培养诱导EPC凋亡;或在上述基础上加不同浓度的SDF-1a处理;48 h后采用TUNEL法和流式细胞仪检测各组EPC凋亡率。结果EPC培养液上清中SDF-1a浓度显著高于对照组（P<0.01）。紫杉醇、AMD3100和无血清培养一样,均可显著增高EPC凋亡率（P<0.01）;加入外源性SDF-1a可显著降低紫杉醇组、无血清培养组EPC凋亡率（P<0.01）,却不能降低AMD3100组EPC凋亡率。结论体外培养的EPC可分泌SDF-1a,采用AMD3100阻断其作用可诱导EPC凋亡;SDF-1a可对抗紫杉醇和无血清培养诱导的EPC凋亡,但对AMD3100诱导的凋亡无效。     

SDF-1/CXCR4轴在内皮祖细胞治疗脑缺血中的作用

近年来的研究表明,SDF-1/CXCR4轴在血管形成过程中起着重要作用.脑缺血时,缺血组织内的基质细胞衍生因子(stromal cell-derived factor,SDF)-1表达增加,其与内皮祖细胞(endothelial progenitor cell,EPC)表面的CXCR4受体结合,进而促进骨髓EPC的动员、增殖并向缺血区迁移和归巢.EPC能在缺血区分化为成熟的血管内皮细胞,促进新血管形成,从而改善缺血组织的血液供应.文章就SDF-1/CXCR4轴在EPC治疗脑缺血中作用进行了综述.     

Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization

Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced. Ex vivo expanded hEPCs may thus have utility as a "supply-side" strategy for therapeutic neovascularization.     

Insulin modulates ischemia-induced endothelial progenitor cell mobilization and neovascularization in diabetic mice

Decreased levels of circulating endothelial progenitor cells (EPCs) predict increased risk of cardiovascular events in diabetic patients. Insulin treatment exerts important cardiovascular protection. Whether and how insulin participates in the EPC regulation of postnatal neovascularization are currently unclear. We employed a mouse hindlimb ischemia model to study EPC mobilization in non-diabetic and streptozotocin-induced diabetic mice. Insulin was administered to diabetic animals postoperatively. To determine the role of EPCs contributing to postnatal vasculogenesis, we used bone marrow-transplanted mice whose bone marrow cells selectively expressed enhanced green fluorescent protein (EGFP). Insulin treatment improved EPC mobilization into peripheral blood, accelerated transcutaneous oxygen pressure restoration and increased capillary density in the ischemic limb associated with partial incorporation of EGFP-positive cells into the capillaries. Insulin treatment restored ischemia-induced release of stromal-derived growth factor 1α and vascular endothelial growth factor (VEGF), and consequently enhanced the activity of Akt and endothelial nitric oxide synthase (eNOS) as well as matrix metalloproteinase-9 in bone marrow. Insulin also augmented tissue-level activation of VEGF/Akt/eNOS pathway. However, all such effects of insulin were completely blocked by combined treatment with a NOS inhibitor. Our data suggested that insulin treatment improved ischemia-induced EPC mobilization and contributed to compensatory neovascularization in diabetic mice through a VEGF/eNOS-related pathway.     

Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects

Preclinical studies in animal models and early results of clinical trials in patients suggest that intramuscular injection of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) can promote neovascularization of ischemic tissues. Such neovascularization has been attributed exclusively to sprout formation of endothelial cells derived from preexisting vessels. We investigated the hypothesis that VEGF gene transfer may also augment the population of circulating endothelial progenitor cells (EPCs). In patients with critical limb ischemia receiving VEGF gene transfer, gene expression was documented by a transient increase in plasma levels of VEGF. A culture assay documented a significant increase in EPCs (219%, P<0.001), whereas patients who received an empty vector had no change in circulating EPCs, as was the case for volunteers who received saline injections (VEGF versus empty vector, P<0.001; VEGF versus saline, P<0.005). Fluorescence-activated cell sorter analysis disclosed an overall increase of up to 30-fold in endothelial lineage markers KDR (VEGF receptor-2), VE-cadherin, CD34, alpha(v)beta(3), and E-selectin after VEGF gene transfer. Constitutive overexpression of VEGF in patients with limb ischemia augments the population of circulating EPCs. These findings support the notion that neovascularization of human ischemic tissues after angiogenic growth factor therapy is not limited to angiogenesis but involves circulating endothelial precursors that may home to ischemic foci and differentiate in situ through a process of vasculogenesis.     

Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease

Transplantation of bone marrow cells as well as circulating endothelial progenitor cells (EPC) enhances neovascularization after ischemia. The chemokine receptor CXCR4 is essential for migration and homing of hematopoietic stem cells. Therefore, we investigated the role of CXCR4 and its downstream signaling cascade for the angiogenic capacity of cultured human EPC. Ex vivo, differentiated EPC derived from peripheral blood abundantly expressed CXCR4. Incubation of EPC from healthy volunteers with neutralizing antibodies against CXCR4 profoundly inhibited vascular endothelial growth factor- and stromal-derived factor-1-induced migration as well as EPC-induced angiogenesis in an ex vivo assay. Preincubation of transplanted EPC with CXCR4 antibody reduced EPC incorporation and impaired blood-flow recovery in ischemic hindlimbs of nude mice (57+/-4% of normal perfusion versus untreated EPC: 80+/-11%, P<0.001). Bone marrow mononuclear cells (BM-MNC) or EPC of heterozygous CXCR4(+/-) mice displayed reduced CXCR4 expression and disclosed impaired in vivo capacity to enhance recovery of ischemic blood flow in nude mice (blood flow 27+/-11% versus 66+/-25% using wild-type cells, P<0.01). Importantly, impaired blood flow in ischemic CXCR4(+/-) mice was rescued by injection of wild-type BM-MNC. Next, we investigated the role of CXCR4 for functional capacities of EPC from patients with coronary artery disease (CAD). Surface expression of CXCR4 was similar in EPC from patients with CAD compared with healthy controls. However, basal Janus kinase (JAK)-2 phosphorylation was significantly reduced and less responsive to stromal-derived factor-1 in EPC from patients with CAD compared with healthy volunteers, indicating that CXCR4-mediated JAK-2 signaling is dysregulated in EPC from patients with CAD. The CXCR4 receptor signaling profoundly modulates the angiogenic activity and homing capacity of cultured human EPC. Disturbance of CXCR4 signaling, as demonstrated by reduced JAK-2 phosphorylation, may contribute to functional impairment of EPC from patients with CAD. Stimulating CXCR4 signaling might improve functional properties of EPC and may rescue impaired neovascularization capacity of EPC derived from patients with CAD.     

Circulating human CD34+ progenitor cells modulate neovascularization and inflammation in a nude mouse model

CD34+ progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34+ cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34+ cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34+ implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34+ cells did not incorporate in neovessels, but formed vWF+/CD31+/VEGF+ cell clusters that were present up to day 14. However, CD34+ cells induced host neovascularization, as demonstrated by increased presence of murine CD31+ and vWF+ vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34+ implants at all time points. Gene expression of chemotactic cytokines MCP-1 and IL-8 was detected on CD34+ cells in vitro and confirmed immunohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34+ cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages.     

Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow

To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.