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The article presents a study which examines the potential of human fetal aorta-derived CD133+ progenitor cell to promote the healing of diabetic ischemic ulcers. The study utilizes a preclinical model of diabetic ischemic foot ulcer using streptozocin-induced diabetic mice. The study reveals that CD133+ progenitor cells promote wound healing through paracrine mechanisms and activation of Wnt signaling pathway, and opens new perspectives on the treatment of diabetic ulcers.

The article presents a study on the role of kinin B2 receptor in the recruitment of circulating progenitor cells (CPCs) to sites of ischemia. B2 receptor expression and bradykinin (BK) sensitivity in CPCs from cardiovascular disease (CVD) patients is discussed. It notes that CPCs from CVD patients exhibit low B2 receptor levels with a decreased migratory capacity of BK. It contends that the reduction of sensitivity in progenitor cells from patients might contribute to impaired neovascularizaton.

Objective: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process., Approach and Results: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation., Conclusions: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Objective: This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis., Approach and Results: A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1-positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1-positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1-positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1-positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E-deficient mice, which contributed to ≈ 30% of neointimal SMCs., Conclusions: We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.

Background: Clinical trials in ischemic patients showed the safety and benefit of autologous bone marrow progenitor cell transplantation. Non-bone marrow progenitor cells with proangiogenic capacities have been described, yet they remain clinically unexploited owing to their scarcity, difficulty of access, and low ex vivo expansibility. We investigated the presence, antigenic profile, expansion capacity, and proangiogenic potential of progenitor cells from the saphenous vein of patients undergoing coronary artery bypass surgery., Methods and Results: CD34-positive cells, negative for the endothelial marker von Willebrand factor, were localized around adventitial vasa vasorum. After dissection of the vein from surrounding tissues and enzymatic digestion, CD34-positive/CD31-negative cells were isolated by selective culture, immunomagnetic beads, or fluorescence-assisted cell sorting. In the presence of serum, CD34-positive/CD31-negative cells gave rise to a highly proliferative population that expressed pericyte/mesenchymal antigens together with the stem cell marker Sox2 and showed clonogenic and multilineage differentiation capacities. We called this population "saphenous vein-derived progenitor cells" (SVPs). In culture, SVPs integrated into networks formed by endothelial cells and supported angiogenesis through paracrine mechanisms. Reciprocally, endothelial cell-released factors facilitated SVP migration. These interactive responses were inhibited by Tie-2 or platelet-derived growth factor-BB blockade. Intramuscular injection of SVPs in ischemic limbs of immunodeficient mice improved neovascularization and blood flow recovery. At 14 days after transplantation, proliferating SVPs were still detectable in the recipient muscles, where they established N-cadherin-mediated physical contact with the capillary endothelium., Conclusions: SVPs generated from human vein CD34-positive/CD31-negative progenitor cells might represent a new therapeutic tool for angiogenic therapy in ischemic patients.

We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

Objective: Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity., Methods and Results: Here, we demonstrate that tissue kallikrein knockout mice (KLK1-/-) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9-/- mice, whereas its proarteriogenic action was preserved in ApoE-/- mice, an atherosclerotic model of impaired angiogenesis., Conclusions: These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression.

Objective: We evaluated whether phosphatidylinositol 3-kinase gamma (PI3Kgamma) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function., Methods and Results: Unilateral limb ischemia was induced in mice lacking the PI3Kgamma gene (PI3Kgamma-/-) or expressing a catalytically inactive mutant (PI3Kgamma(KD/KD)) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3Kgamma-/- ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3Kgamma(KD/KD). In PI3Kgamma-/- muscles, endothelial cell proliferation was reduced, apoptosis was increased, and interstitial space was infiltrated with leukocytes but lacked cKit+ progenitor cells that in WT muscles typically surrounded arterioles. PI3Kgamma is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3Kgamma-/- EPCs showed a defect in proliferation, survival, integration into endothelial networks, and migration toward SDF-1. The dysfunctional phenotype was associated with nuclear constraining of FOXO1, reduced Akt and eNOS phosphorylation, and decreased nitric oxide (NO) production. Pretreatment with an NO donor corrected the migratory defect of PI3Kgamma-/- EPCs. PI3Kgamma(KD/KD) EPCs showed reduced Akt phosphorylation, but constitutive activation of eNOS and preserved proliferation, survival, and migration., Conclusions: We newly demonstrated that PI3Kgamma modulates angiogenesis, arteriogenesis, and vasculogenesis by mechanisms independent from its kinase activity.