Therapeutic Vasculogenesis Using Human Cord Blood-Derived Endothelial Progenitors

Peripheral blood of adult species contains endothelial progenitor cells (EPCs) that participate in neovascularization, consistent with postnatal vasculogenesis. Abundant EPCs can be isolated from a relatively small volume of human umbilical cord blood, and that culture-expanded EPCs participate in endothelial network formation in vitro. Transplanted EPCs incorporated into sites of active neovascularization and formed capillaries among preserved skeletal myocytes in the ischemic hindlimb of athymic nude rats in vivo. Furthermore, transplantation of EPC quantitatively and effectively augmented neovascularization in response to hindlimb ischemia. Thus, human umbilical cord blood seems to be a novel source for EPCs, and the transplantation of cord blood-derived EPCs may become a useful strategy to modulate postnatal neovascularization.     

人和大鼠内皮祖细胞培养及其生长特性的比较

目的建立人内皮祖细胞（hEPCs）离体培养的方法,探讨培养条件,观察细胞生长状态、形态。xd同时行大鼠EPCs（rEPCs）培养。建立hEPCs和rEPCs培养体系,比较两者不同的生长条件和生长状态。方法：取人脐带血80-120ml/袋,先分离单个核细胞,后使用磁珠细胞分选法（MACS）,分选出CD133＋/VEGFR2＋的细胞,进行流式细胞检测,发现双阳性细胞占48.79%。用差速贴壁法培养5-9天,倒置相差显微镜观察细胞形态并照相、免疫荧光染色后荧光显微镜下观察照相。取大鼠骨髓,冲洗骨髓腔,离心沉淀出细胞,差速贴壁培养法培养、观察。结果：（1）hEPCs在普通光镜下呈索条状、卵圆形。（2）细胞吞噬DiI-acLDL、UEA染料后可在荧光显微镜下特异显色,证明细胞有吞噬功能,推断为hEPCs。（3）CD133＋/VEGFR2＋的hEPCs细胞,占使用MACS筛选后细胞比例为48.79%。（4）从骨髓中分离出的rEPCs生长活力明显优于hEPCs。结论：（1）该方法培养的hEPCs和rEPCs生长活性好,在普通光镜下呈索条状、卵圆形或铺路石样;（2）hEPCs在细胞数量上可不少于rEPCs,从骨髓中培养出的rEPCs增殖力优于脐血来源的hEPCs。此实验比较并完善了两种不同来源内皮祖细胞培养的方法学及其生长特征,有利于根据不同的细胞培养特性来选择应用于内皮祖细胞的实验研究。     

Critical roles of muscle-secreted angiogenic factors in therapeutic neovascularization

The discovery of bone marrow-derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 "no-option" patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1beta (IL-1beta). We then examined an experimental model of limb ischemia using IL-1beta-deficient mice. Implantation of IL-1beta-deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1beta-deficient mononuclear cells increased expression of IL-1beta and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1beta markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.     

Mechanisms underlying the impairment of ischemia-induced neovascularization in matrix metalloproteinase 2-deficient mice

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2(+/+) and MMP-2(-/-) mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2(-/-) young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti-l-lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2(-/-) mice compared with the age-matched MMP-2(+/+) mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2(-/-) mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow-derived endothelial progenitor cell (EPC)-like c-Kit(+) cells from MMP-2(-/-) showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2(-/-). Flow cytometry showed that the numbers of EPC-like CD31(+)c-Kit(+) cells in peripheral blood markedly decreased in MMP-2-deficient mice. Transplantation of bone marrow-derived mononuclear cells from MMP-2(+/+) mice restored neovascularization in MMP-2(-/-) young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.     

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.     

Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia

Circulating progenitor cells home to sites of postnatal neovascularization and differentiate into endothelial cells but questions remain regarding the source of these cells. Indeed, a recent study suggests that nonbone marrow-derived cells may be even more important than bone marrow-derived cells in the setting of transplant arteriosclerosis. Thus, we aimed to thoroughly investigate the contribution of nonbone marrow-derived progenitor cells for neovascularization. We exclusively identified nonbone marrow-derived progenitor cells by combining a parabiosis model with reverse bone marrow transplantation followed by hindlimb ischemia. In this model, nonbone marrow-derived circulating progenitor cells attributed for 74+/-13% of the circulating progenitor cells that incorporated into the ischemic hindlimb. Increasing evidence suggests that organs such as small intestine and liver contain a considerable number of tissue resident progenitor cells and, thus, represent putative sources for nonbone marrow-derived progenitors. To track organ-derived progenitors, we transplanted sex-mismatched small intestine or liver, respectively, into rats followed by induction of hindlimb ischemia. These experiments show that organ-derived progenitor cells are contributing to postnatal vasculogenesis (intestine: 4.7+/-3.7%; liver: 6.3+/-2.2%). Based on the subsequent observation that liver-derived nonhematopoietic c-kit(+)CD45(-) progenitors are mobilized on induction of hindlimb ischemia, we prospectively isolated and intravenously infused these progenitors from murine livers. The isolated cells demonstrated a marked capacity for enhancing neovascularization and restoring blood flow to the ischemic hindlimb (no cells: 26.4+/-4.8% of normal blood flow; c-kit(+)CD45(-) cells: 67.0+/-8.0% of normal flow; P<0.01). In conclusion, we find that nonbone marrow-derived c-kit(+)CD45(-) progenitors contribute to postnatal neovascularization to an extent that is similar to that of bone marrow-derived progenitor cells. Intestine and liver represent a rich source for mobilized tissue-residing progenitor cells.     

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.     

Endothelial cell growth factors and the vessel wall

The role of endothelial cell growth factors in the maintenance of the blood vessel wall is, as we have described here, much more complex than merely stimulating the mitogenesis of endothelial cells. The FGFs are capable of eliciting an array of responses in endothelial cells, some, or all, of which are important for neovascularization and the control of clot dissolution. These endothelial cell responses include protease elaboration, chemotaxis, and mitogenesis. That these growth factors seem neither to be constitutively released into the medium of cultured cells that synthesize bFGF, nor released into the bloodstream in vivo suggests that the temporal and local control of neovascularization may involve the regulation of growth factor release from cells such as endothelial cells, fibroblasts, and macrophages. Although there is no known example of this for bFGF, it is well known that both thrombin and Factor Xa stimulate the release of a mitogenic protein from endothelial cells and that low oxygen tension stimulates the release of macrophage-derived angiogenesis factor. In addition, both TGF beta and heparin alone appear to play a role in wound healing and vessel wall maintenance. The work of Roberts et al suggests that TGF beta is not only angiogenic, but also stimulates the growth of fibrotic tissue as well. Studies on mast cells demonstrated that released heparin is chemotactic for endothelial cells and can potentiate tumor angiogenesis. An attractive hypothesis is that these molecules not only act as FGF potentiators or inhibitors but that they also may exert their angiogenic effects by inducing FGF release from cells. Perhaps angiogenin, an angiogenic molecule with no mitogenic activity, works in this way. However, no evidence as yet exists concerning this point. A second level of control of neovascularization may involve the interaction of FGF with other molecules released into the same microenvironment. For example, thrombin and TGF beta released from platelets, as well as heparin released from mast cells, have all been demonstrated to affect bFGF activity in vitro and may act as modifiers of FGF activity in vivo. Since bFGF can modulate fibrinolytic activity, one could imagine that its release into a wound region of the vasculature could have detrimental effects on clot formation and subsequent wound healing. Thus, the transient inhibition of bFGF activity by TGF beta would allow clot formation before the induction of neovascularization by bFGF, TGF beta thereby playing a role in the regulation of the sequence in which events occur.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical role of endothelial Notch1 signaling in postnatal angiogenesis

Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1(+/-) mice, but not Notch4(-/-) mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of gamma-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of gamma-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates gamma-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs.     

Inhibition of neovascularization by environmental agents

The formation of new blood vessels, neovascularization, occurs by two unique processes: vasculogenesis, the de novo assembly of blood vessels from angioblast precursors, and angiogenesis, the formation of new capillary sprouts from pre-existing vessels. There are many potential targets by which environmental pollutants may inhibit neovascularization and thus there are many possible phenotypic outcomes. Two examples of environmental pollutants that have been demonstrated to inhibit neovascularization include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical halogenated aromatic hydrocarbon, and constituents found in environmental tobacco smoke. Studies have shown that TCDD disrupts neoangiogenesis by inhibiting the expression of angiogenic stimuli as well as by reducing the responsiveness of endothelial cells to those stimuli. Additionally, studies have shown that constituents of environmental tobacco smoke, including pyradine and pyrazine derivatives, can potently inhibit the angiogenic process of branching as well as the vasculogenic process involved in capillary plexus formation. Further, the inhibition of neovascularization by either TCDD or environmental tobacco smoke constituents is associated with reduced endothelial cell proliferation and altered expression of extracellular matrix proteins. Future research that identifies the specific angiogenic signaling pathways that are disrupted by these pollutants will improve our ability to assess their risk to human health. Finally, it is likely that many other environmental pollutants impact neovascularization; however, very few have been studied in sufficient detail. Thus, additional research also is needed to identify those environmental agents that mediate their toxicity by disrupting neovascularization.