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. ...

The connection between obesity and accelerated cancer progression has been established, but the mediating mechanisms are not well understood. We have shown that stromal cells from white adipose tissue (WAT) cooperate with the endothelium to promote blood vessel formation through the secretion of soluble trophic factors. Here, we hypothesize that WAT directly mediates cancer progression by serving as a source of cells that migrate to tumors and promote neovascularization. To test this hypothesis, we have evaluated the recruitment of WAT-derived cells by tumors and the effect of their engraftment on tumor growth by integrating a transgenic mouse strain engineered for expansion of traceable cells with established allograft and xenograft cancer models. Our studies show that entry of adipose stromal and endothelial cells into systemic circulation leads to their homing to and engraftment into tumor stroma and vasculature, respectively. We show that recruitment of adipose stromal cells by tumors is sufficient to promote tumor growth. Finally, we show that migration of stromal and vascular progenitor cells from WAT grafts to tumors is also associated with acceleration of cancer progression. These results provide a biological insight for the clinical association between obesity and cancer, thus outlining potential avenues for preventive and therapeutic strategies.

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.

Rationale: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. Objectives: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. Methods: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. Measurements and Main Results: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. Conclusions: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.

The use of adipose-derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual-cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC-shHGF and ASCshCtrl, respectively). Transduced ASC-shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC-shHGF were also significantly impaired, compared with ASC-shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP ؉ cells were detected at 3 weeks in ischemic limbs of mice treated with ASCshHGF compared with those treated with ASC-shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell-based therapies. STEM CELLS 2007;25:3234 -3243 Disclosure of potential conflicts of interest is found at the end of this article.

ASCs may ultimately represent a valuable therapeutic option in tissue rescue and repair based on their ready availability, proangiogenesis and antiapoptotic factor secretion, immunomodulatory effects, and capacity for multilineage differentiation and ready expansion.