Background— The delivery of autologous cells to increase angiogenesis is emerging as a treatment option for patients with cardiovascular disease but may be limited by the accessibility of sufficient cell numbers. The beneficial effects of delivered cells appear to be related to their pluripotency and ability to secrete growth factors. We examined nonadipocyte stromal cells from human subcutaneous fat as a novel source of therapeutic cells. Methods and Results— Adipose stromal cells (ASCs) were isolated from human subcutaneous adipose tissue and characterized by flow cytometry. ASCs secreted 1203±254 pg of vascular endothelial growth factor (VEGF) per 10 6 cells, 12 280±2944 pg of hepatocyte growth factor per 10 6 cells, and 1247±346 pg of transforming growth factor-β per 10 6 cells. When ASCs were cultured in hypoxic conditions, VEGF secretion increased 5-fold to 5980±1066 pg/10 6 cells ( P =0.0016). The secretion of VEGF could also be augmented 200-fold by transfection of ASCs with a plasmid encoding VEGF ( P <0.05). Conditioned media obtained from hypoxic ASCs significantly increased endothelial cell growth ( P <0.001) and reduced endothelial cell apoptosis ( P <0.05). Nude mice with ischemic hindlimbs demonstrated marked perfusion improvement when treated with human ASCs ( P <0.05). Conclusions— Our experiments delineate the angiogenic and antiapoptotic potential of easily accessible subcutaneous adipose stromal cells by demonstrating the secretion of multiple potentially synergistic proangiogenic growth factors. These findings suggest that autologous delivery of either native or transduced subcutaneous ASCs, which are regulated by hypoxia, may be a novel therapeutic option to enhance angiogenesis or achieve cardiovascular protection.

Abstract-It has been shown that stromal-vascular fraction isolated from adipose tissues contains an abundance of CD34 ϩ cells. Histological analysis of adipose tissue revealed that CD34 ϩ cells are widely distributed among adipocytes and are predominantly associated with vascular structures. The majority of CD34 ϩ cells from freshly isolated stromal-vascular fraction were CD31 Ϫ /CD144 Ϫ and could be separated from a distinct population of CD34 ϩ /CD31 ϩ /CD144 ϩ (endothelial) cells by differential attachment on uncoated plastic. The localization of CD34 ϩ cells within adipose tissue suggested that the nonendothelial population of these cells occupied a pericytic position. Analysis of surface and intracellular markers of the freshly isolated CD34 ϩ /CD31 Ϫ /CD144 Ϫ adipose-derived stromal cells (ASCs) showed that Ͼ90% coexpress mesenchymal (CD10, CD13, and CD90), pericytic (chondroitin sulfate proteoglycan, CD140a, and CD140b), and smooth muscle (␣-actin, caldesmon, and calponin) markers. ASCs demonstrated polygonal self-assembly on Matrigel, as did human microvascular endothelial cells. Coculture of ASCs with human microvascular endothelial cells on Matrigel led to cooperative network assembly, with enhanced stability of endothelial networks and preferential localization of ASCs on the abluminal side of cords. Bidirectional paracrine interaction between these cells was supported by identification of angiogenic factors (vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor), inflammatory factors (interleukin-6 and -8 and monocyte chemoattractant protein-1 and -2), and mobilization factors (macrophage colony-stimulating factor and granulocyte/macrophage colony-stimulating factor) in media conditioned by CD34 ϩ ASCs, as well a robust mitogenic response of ASCs to basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor-BB, factors produced by endothelial cells. These results demonstrate for the first time that the majority of adipose-derived adherent CD34

Abstract-Rapid induction and maintenance of blood flow through new vascular networks is essential for successfully treating ischemic tissues and maintaining function of engineered neo-organs. We have previously shown that human endothelial progenitor cells (EPCs) form functioning vessels in mice, but these are limited in number and persistence; and also that human adipose stromal cells (ASCs) are multipotent cells with pericytic properties which can stabilize vascular assembly in vitro.In this study, we tested whether ASCs would cooperate with EPCs to coassemble vessels in in vivo implants. Collagen implants containing EPCs, ASCs, or a 4:1 mixture of both were placed subcutaneously into NOD/SCID mice. After a range of time periods, constructs were explanted and evaluated with regard to vascular network assembly and cell fate; and heterotypic cell interactions were explored by targeted molecular perturbations. The density and complexity of vascular networks formed by the synergistic dual-cell system was many-fold higher than found in implants containing either ASCs or EPCs alone. Coimplantation of ASCs and EPCs with either pancreatic islets or adipocytes produced neoorgans populated by these parenchymal cells, as well as by chimeric human vessels conducting flow. This study is the first to demonstrate prompt and consistent assembly of a vascular network by human ASCs and endothelial cells and vascularization by these cells of parenchymal cells in implants. Mixture of these 2 readily available, nontransformed human cell types provides a practical approach to tissue engineering, therapeutic revascularization, and in vivo studies of human vasculogenesis. A ssembly of new vascular networks for therapeutic purposes has been critical but elusive for tissue engineering and angiogenesis. A general requirement for preserving viability within a regenerating region is that a vascular bed is assembled or expanded sufficiently to ensure adequate tissue perfusion. Also important to success of such applications is the ability of any network to anastomose or inosculate promptly with the vessels of immediately adjacent tissues. Cell-based revascularization therapies were tested in patients with various ischemic diseases. These studies used progenitor cells from bone marrow 1,2 and skeletal muscle 3,4 to treat patients with myocardial infarction, 1,2 heart failure, 3-6 and peripheral vascular disease. 7,8 Despite accumulating data and recent metaanalyses 9,10 that support the hypothesis that progenitor cells have high potential for promoting tissue revascularization and functional recovery, invasive methods of cells harvest and their low abundance may limit adoption some of progenitor cells for therapies. Recent approaches were focused on evaluating cells derived from more available tissues including adipose tissue 11,12 and umbilical cord blood. 13,14 Endothelial progenitor cells (EPCs) isolated from adult peripheral blood, 15 bone marrow, umbilical cord blood, 13 and vessel wall 16 were intensively studied over the past decade. ...

Adipose stromal cells (ASCs) express markers and functional properties of pericytes in vitro and, in combination with endothelial cells (ECs), are able to establish multilayer functional vessels in vivo. However, the factors that coordinate EC-ASC communications to promote migration of these cells toward one another, and their heterotypic assembly into vascular structures are not well defined. To understand the mechanisms of EC-ASC interaction, we developed an in vitro model of coculturing ECs with ASCs in a system containing serum but no additional exogenous cytokines or extracellular matrix (ECM) proteins. We demonstrated that ASCs have a profound potential to stimulate morphogenesis of ECs into branching networks of cord structures. The vascular networks developed in 6 days and were stable for at least 3 weeks. This process was associated with an increase in ECM protein production by ASCs and ECs, alpha-smooth muscle actin expression by ASCs, and increased CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1) surface presentation by ECs. The vascular network formation (VNF) was dependent on matrix metalloproteinase activity and cell communications through vascular endothelial growth factor, hepatocyte growth factor, and platelet-derived growth factor-BB pathways. ASCs exhibited significantly higher potential to stimulate VNF than smooth muscle cells and fibroblasts. Media conditioned by ASCs promoted VNF by ECs cultured on smooth muscle cells and fibroblasts, but could not replace the presence of ASCs in coculture. The presence of ASCs in EC-fibroblast cocultures in a low fraction efficiently stimulated VNF. These findings demonstrate that the vasculogenesis-promoting potential of ASCs depends on interaction with ECs involving contact and likely bi-directional interaction, resulting in modulated secretion of cytokines and ECM proteins.

Adipose stromal cells (ASC) are multipotential mesenchymal progenitor cells that are readily induced to undergo adipogenic differentiation, and we have recently demonstrated them to have functional and phenotypic overlap with pericytes lining microvessels in adipose tissues. In this study we addressed the hypothesis that modulation of ASC fate within this perivascular niche can occur via interaction with endothelial cells (EC), which serve to modulate the adipogenic potential of ASC. To this end, we investigated contact as well as paracrine effects of EC on ASC adipogenesis, in two-dimensional coculture and via conditioned medium and analyzed mutual gene expression changes by real-time reverse transcription polymerase chain reaction (PCR). A significant decrease in adipogenic differentiation was observed in ASC when they were cocultured with EC but not control fibroblasts. This endothelial cell-specific effect was accompanied by increased expression of factors involved in Wnt signaling, most prominently Wnt1, Wnt4, and Wnt10a, which are well-known inhibitors of adipogenesis. Suppression of Wnt1 but not Wnt 10a or scrambled control short interfering RNA in cocultures partially reversed the endothelial cell effect, thus increasing adipogenic differentiation, suggesting a plausible role of Wnt1 ligand in modulation of adipogenesis by the vasculature. Furthermore, addition of recombinant Wnt ligand or the Wnt signaling agonist inhibited adipogenic differentiation of ASC in the absence of EC. In conclusion, these data define the relationship in adipose tissue between ASC and EC in the perivascular niche, in which the latter act to repress adipogenesis, thereby stabilizing vasculature. It is tempting to speculate that abnormal endothelial function may be associated with pathologic derepression of adipogenesis.

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.

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