Objectives This study aimed to determine if CD31 is a novel marker of a circulating angio-vasculogenic cell population and to establish their therapeutic effects on experimental ischemia. Background Emerging evidence suggested that therapeutic mechanisms underlying various bone marrow (BM)-derived cells are due to paracrine effects. Furthermore, the vasculogenic potential of these cells is under debate. CD31 is a well known marker for endothelial cells (ECs) but is also expressed in a fraction of peripheral blood (PB) mononuclear cells. Methods CD31+ cells were isolated from human PB by magnetic-activated cell sorting (MACS). The gene expression profile was examined by DNA microarray and real-time RT-PCR (qRT-PCR). Various in vitro endothelial differentiation or vasculogenic assays were conducted. Finally, cells were directly implanted into a mouse hindlimb ischemia (HLI) model to test angiogenic-vasculogenic and therapeutic effects. Results Fluorescent-activated cell sorter (FACS) analysis revealed that PB-CD31+ cells exhibited endothelial and hematopoietic stem/progenitor markers. CD31+ cells had higher levels of expression of pro-angiogenic genes on microarray and qRT-PCR and generated higher numbers of endothelial progenitor cells (EPCs) compared to CD31− cells. CD31+ cells spontaneously formed vascular tube-like structures and exhibited an endothelial cell phenotype in vitro. In a HLI model, CD31+ cell transplantation augmented blood perfusion and prevented limb loss. Both angiogenic cytokines and capillary density were increased, suggesting CD31+ cells augmented neovascularization. Conclusions CD31 is a novel marker that designates circulating angiogenic and vasculogenic cells. These cells are easily isolated from human PB and thus are a novel candidate for treatment of ischemic cardiovascular disease.

Rationale: Bone marrow (BM) cells play an important role in physiological and therapeutic neovascularization.However, it remains unclear whether any specific uncultured BM cell populations have higher angiogenic and vasculogenic activities. Moreover, there has been controversy regarding the vasculogenic ability of BM cells.Objective: Preliminary flow cytometric analysis showed that CD31, traditionally a marker for endothelial cells, is expressed in certain nonendothelial BM mononuclear cells in both human and mouse. Based on the conserved CD31 expression in the axis of hematopoietic stem/progenitor cells (HSC/HPCs) to endothelial cells, we further sought to determine the comprehensive vasculogenic and angiogenic characteristics of human and mouse BM-derived CD31 ؉ cells. Methods and Results: Flow cytometric analysis demonstrated that all CD31؉ cells derived from BM were CD45 ؉ and expressed markers for both HSC/HPCs and endothelial cells. Comprehensive gene expression analyses revealed that BM-CD31؉ cells expressed higher levels of angiogenic genes than CD31 ؊ cells. Endothelial progenitor cells, as well as HSC/HPCs, were almost exclusively confined to the CD31 ؉ cell fraction, and culture of CD31؉ cells under defined conditions gave rise to endothelial cells. Finally, injection of CD31 ؉ cells into ischemic hindlimb repaired ischemia, increased expression of angiogenic and chemoattractive factors, and, in part, directly contributed to vasculogenesis, as demonstrated by both 3D confocal microscopy and flow cytometry. Conclusions: These data indicate that BM-CD31؉ cells represent highly angiogenic and vasculogenic cells and can be a novel and highly promising source of cells for cell therapy to treat ischemic cardiovascular diseases. (Circ Res. 2010;107:602-614.) Key Words: bone marrow Ⅲ CD31 (PECAM-1) angiogenesis Ⅲ vasculogenesis Ⅲ peripheral vascular disease F ormation of new blood vessels (neovascularization) consists of 2 processes, vasculogenesis and angiogenesis. Vasculogenesis refers to the de novo development of blood vessels from endothelial progenitor cells (EPCs) or angioblasts which differentiate into endothelial cells (ECs). In contrast, angiogenesis is the formation of new vasculature from preexisting blood vessels through proliferation, migration, and remodeling of differentiated ECs. The identification of circulating EPCs in adult vertebrates suggested a role for BM cells in postnatal vasculogenesis, 1-3 and led to trials of BM cells for therapy for ischemic cardiovascular diseases.However, conflicting results from recent clinical trials 4,5 suggests a need for the discovery of new cell types 6 and more thorough investigation of the therapeutic mechanisms.Two of the most important questions in current EPC biology are whether the reported cultured EPCs or similar BM cells have true vasculogenic potential and whether a specific marker can prospectively identify true EPCs or vasculogenic cells. The endothelial differentiation or vasculogenic potential of early EPCs has been questioned. 7,3,8 -11 ...

Background-Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. Methods and Results-We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow-derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves.A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. Conclusions-We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy. Key Words: angiogenesis Ⅲ diabetes mellitus Ⅲ progenitor cells Ⅲ diabetic neuropathy P eripheral neuropathy is the most common complication of diabetes mellitus, affecting up to 60% of diabetic patients. 1 Loss of sensation in the feet, the most frequent manifestation of diabetic neuropathy (DN), frequently leads to foot ulcers and may progress into amputation of the limb. 2,3 Despite a continuous increase in the incidence of diabetes mellitus and DN, current treatments have yet to effectively treat DN. Our group reported that experimental DN is characterized by reduced microcirculation in peripheral nerves caused by the destruction of the vasa nervorum and thus administration of angiogenic factors such as vascular endothelial growth factors (VEGFs), sonic hedgehog (SHh), and statin could restore neural function by augmenting angiogenesis. 4 -6 In addition, deficiency of neurotrophic factors is regarded as one of the most plausible mechanisms underlying DN. 7 Alterations of nerve growth factor, ciliary neurotrophic factor, glial-derived neurotrophic factor, and brain-derived neurotrophic factor have been reported. 8 -12 However, in clinical trials, single neurotrophic cytokines turned out to be ineffective for treating DN. 13 Recently, many classic angiogenic factors were shown to possess neurotrophic activities and vice versa. VEGF, [14][15][16]17,18 Received April 29, 2008; accepted November 28, 2008. Cli...