Objective-We examined whether plasma levels of angiogenic factors are altered in plasma of patients with peripheral arterial disease (PAD) and whether these factors affect endothelial progenitor cell-induced angiogenesis. Methods and Results-Plasma was collected from 184 patients with PAD and 330 age-matched healthy controls. Vascular endothelial growth factor and placental growth factor concentrations did not differ between the groups, whereas we found a linear correlation between PAD disease and thrombospondin (TSP)-1 plasma level. TSP-1 was expressed in newly formed vessels in PAD patients having received local injections of bone marrow mononuclear cells. To analyze the functional role of TSP-1 during neoangiogenesis, we used a Matrigel-plug assay and showed that vascularization of implanted Matrigel-plugs was increased in TSP-1 Ϫ/Ϫ mice. Moreover, injections of TSP-1 in C57Bl6/J mice after hindlimb ischemia induced a significant decrease of blood flow recovery. To investigate the effects of TSP-1 on human endothelial colony-forming cell (ECFC) angiogenic potential, recombinant human TSP-1 and a small interfering RNA were used. In vitro, TSP-1 N-terminal part significantly enhanced ECFC adhesion, whereas recombinant human TSP-1 had a negative effect on ECFC angiogenic potential. This effect, mediated by CD47 binding, modulated stromal cell-derived factor 1/CXC chemokine receptor 4 pathway. Key Words: angiogenesis Ⅲ arterial thrombosis Ⅲ endothelial progenitor cells P eripheral arterial disease (PAD), characterized by atherosclerosis of the lower extremities, affects up to 15% of people older than 55 years. 1 The main clinical manifestations of PAD are intermittent claudication and critical limb ischemia (CLI). Intermittent claudication is characterized by reproducible pain on exertion that is relieved by rest. CLI is the most severe form of PAD and is characterized by the inability of arterial blood flow to meet the metabolic demands of resting muscle or tissue, resulting in rest pain and/or tissue necrosis and frequently necessitating amputation. Currently, PAD diagnosis is based on the ankle-brachial systolic pressure index (ABI), but the ABI is a poor marker of PAD severity. There are no other reliable diagnostic tests for PAD, and new biomarkers would therefore be useful. Conclusion-TSP-1 is a potential biomarker of PAD and ECFC-induced angiogenesisAtherosclerosis induces occlusion of the arterial tree and tissue hypoxia, which is a strong stimulus for angiogenesis. Collateral vessels develop physiologically in patients with CLI, mainly driven by an enhanced angiogenic response. 2 However, the capacity of this compensatory mechanism is rapidly exceeded, and normal flow is not restored. Autologous endothelial progenitor cells (EPCs) are candidates for angiogenic therapy. Because of their scarcity in human samples, EPCs have been characterized by culture methods. At least 2 populations of EPCs have been described. 3 "Early" EPCs appear within 4 to 7 days of culture, whereas "late" EPCs, also called endothe...

Objective-To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. Methods and Results-Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization.Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. W ingless related proteins (Wnts) are powerful regulators of cell proliferation and differentiation, and their signaling pathway involves proteins that participate directly in both gene transcription and cell adhesion. 1 The major signaling pathway of Wnt is the canonical pathway that results from Wnt binding to the frizzled and low-densitylipoprotein-receptor-related protein (LRP) families on the cell surface. Complex formation induces ␤-catenin nuclear entry and forms a complex with transcription factor T cell factor (TCF) and/or lymphoid enhancer factor (LEF) to activate transcription of Wnt target genes. The Wnt pathway is modulated by several Wnt antagonists, including Dickkopfs (DKKs). 2 The DKK family encodes secreted proteins of 255 to 350 amino acids and comprises 4 main members in vertebrates (DKK1 to DKK4). DKK1, the most widely studied member of this family, has been implicated in various physiological and pathological processes in human adults. DKK1 mediates inflammation in atherosclerotic lesions 3 and mobilizes progenitor cells by activating the bone marrow endosteal stem cell niche. 4 Recently, serum levels of DKK1 correlated with the extent of bone disease in patients with multiple malignant neoplasms, such as breast cancer. 5 In tumoral angiogenesis, endothelial cells have distinct gene expression profiles when compared with normal endothelial cells. Notably, they express high levels of DKK3. 6,7 However, the role of DKK1 in tumoral angiogenesis and postnatal vasculogenesis by endothelial progenitor cells (EPCs) has not yet been studied. Conclusion-DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFCBone marrow-derived cells have been shown to contribute to tumor neovasculature. 8,9 The active cell population in bone marrow-derived cells has...

BackgroundPulmonary vasodilators in general and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary arterial hypertension (PAH). As endothelial dysfunction is a key feature of PAH, and as endothelial progenitor cells (EPC) may contribute to vascular repair in PAH, we suspected that prostacyclin therapy might enhance EPC numbers and functions. In the present study, objectives were to determine whether EPC may contribute to vasodilator treatment efficacy in PAH.MethodsWe quantified CD34+ cells, CFU-Hill and ECFC (endothelial colony forming cells) in peripheral blood from children with idiopathic PAH (n = 27) or PAH secondary to congenital heart disease (n = 52). CD34+ were enumerated by flow cytometry, CFU-Hill and ECFC by a culture assay. ECFC grown ex vivo were tested for their angiogenic capacities before and after prostacyclin analog therapy (subcutaneous treprostinil).ResultsECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors. CD34+ cell and CFU-Hill counts were unaffected. ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia.ConclusionsECFC may partly mediate the clinical benefits of prostanoids in pulmonary arterial hypertension.

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IPF is basically associated with both a vascular injury and a repair defect. This study highlights an adaptative process of EPC mobilization in the most severe forms of IPF, that could reflect enhanced homing to the pulmonary vasculature, which clinical consequences remain to be determined.

Upregulation of hypoxia-inducible transcription factor-1a (HIF-1a), through prolyl-hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell-based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF-1a were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow-derived mesenchymal stem cells (hBM-MSC)-based therapy. PHD2 silencing enhanced hBM-MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF-1a and its target gene, VEGF-A. In addition, PHD2-transfected hBM-MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF-1a or VEGF-A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM-MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD-2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF-1a. In conclusion, silencing of PHD2, by the transient upregulation of HIF-1a and its target gene VEGF-A, might improve the efficiency of hBM-MSC-based therapies. STEM CELLS 2014;32:231-243

The profibrotic cytokine transforming growth factor-b1 increases endothelial progenitor cell angiogenic properties. J Thromb Haemost 2012; 10:Summary. Background: Transforming growth factor-b1 (TGF-b1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF-b1 modulation of EPC angiogenic properties. Methods: TGF-b1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF-b1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony-forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGFb1 in ECFCs by using short interfering RNA. Results:Total TGF-b1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF-b1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF-b1 receptors. Conclusions:TGF-b1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF-b1 may play a role during vascular remodeling in fibrotic disease states via EPCs.

The proinflammatory chemokine interleukin 8 exerts potent angiogenic effects on endothelial cells by interacting with its receptors CXCR1 and CXCR2. As thrombin is also a potent inflammatory factor, and as endothelial progenitor cells (EPC) express functional PAR-1 thrombin receptor, we examined whether PAR-1 stimulation interferes with the IL-8 pathway in EPC. EPC were obtained from adult blood (AB) and cord blood (CB). The effect of PAR-1 stimulation by the peptide SFLLRN on IL-8, CXCR1 and CXCR2 expression was examined by RTQ-PCR and at the protein level in AB and