Abstract-We recently demonstrated that marrow stromal cells (MSCs) augment collateral remodeling through release of several cytokines such as VEGF and bFGF rather than via cell incorporation into new or remodeling vessels. The present study was designed to characterize the full spectrum of cytokine genes expressed by MSCs and to further examine the role of paracrine mechanisms that underpin their therapeutic potential. Normal human MSCs were cultured under normoxic or hypoxic conditions for 72 hours. The gene expression profile of the cells was determined using Affymetrix GeneChips representing 12 000 genes. A wide array of arteriogenic cytokine genes were expressed at baseline, and several were induced Ͼ1.5-fold by hypoxic stress. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC conditioned media (MSC CM ). MSC CM promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partially attenuated these effects. Similarly, MSC CM promoted smooth muscle cell proliferation and migration in a dose-dependent manner. Using a murine hindlimb ischemia model, murine MSC CM enhanced collateral flow recovery and remodeling, improved limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy compared with control media. These data indicate that paracrine signaling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for their effects.

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Background-T lymphocytes, components of the immune and inflammatory systems, are involved in such normal processes as wound healing and host defense against infection and in such pathological processes as tumor growth and atherosclerotic plaque development. Angiogenesis is a mechanism common to each. Because CD4ϩ T lymphocytes are active in regulating humoral and cellular responses of the immune system, we determined whether CD4ϩ cells contribute to collateral vessel development by using the mouse ischemic hindlimb model. Methods and Results-One week after ischemia, CD4Ϫ/Ϫ mice showed reduced collateral flow induction, macrophage number, and vascular endothelial growth factor levels in the ischemic muscle compared with wild-type mice. There was also delayed recovery of hindlimb function and increased muscle atrophy/fibrosis. Spleen-derived purified CD4ϩ T cells infused into CD4Ϫ/Ϫ mice selectively localized to the ischemic limb and significantly increased collateral flow as well as macrophage number and vascular endothelial growth factor levels in the ischemic muscle. Muscle function and damage also improved. Conclusions-These results indicate an important role of CD4ϩ cells in collateral development, as demonstrated by a 25%decrease in blood flow recovery after femoral artery ligation. Our data also suggest that CD4ϩ T cells control the arteriogenic response to acute hindlimb ischemia, at least in part, by recruiting macrophages to the site of active collateral artery formation, which in turn triggers the development of collaterals through the synthesis of arteriogenic cytokines.

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