Mesenchymal stem cells in the treatment of hindlimb ischemia in diabetic mice

描述（由申请人提供）：该项目的目标是增强使用间充质干细胞（MSC）治疗外周动脉疾病的手段，特别是糖尿病患者。骨髓间充质干细胞移植基于其多能性，以及其在旁分泌输送促血管生成因子方面的功效，作为PAD的治疗干预手段具有很大的前景。然而，我们已经确定糖尿病MSC比健康（WT） MSC表现出更大的氧化应激。糖尿病间充质干细胞表现出有限的多能性，倾向于脂肪细胞分化而不是内皮细胞分化；当移植到WT宿主时，糖尿病骨髓间充质干细胞会损害缺血后的新生血管，并在缺血后肢产生脂肪浸润。项目假设是，糖尿病MSC的氧化应激限制了它们的多能性，从而限制了它们的新生血管能力。为了验证这一假设，提出了三个目标。特异性目的1将证明氧化剂的产生是糖尿病间充质干细胞有限多能性的基础，在培养的间充质干细胞中使用还原方法。实验[1]将确定nox4来源的H2O2是否驱动糖尿病MSC中的脂肪细胞分化。[2]将确定ppar3过表达在糖尿病MSC中产生脂肪细胞分化的作用。Exp[3]将确定未偶联的eNOS在糖尿病间充质干细胞中产生氧化应激的作用，并评估eNOS重新偶联的策略。[4]实验将确定VEGF-Akt-eNOS通路的缺陷是否是糖尿病间充质干细胞内皮分化受损的基础。特异性目标2将使用体内MSC移植范例来证明糖尿病MSC或糖尿病宿主的抗氧化治疗

DESCRIPTION (provided by applicant): The goal of this project is to enhance the means to use mesenchymal stem cells (MSC) for the treatment of peripheral artery disease, particularly in the diabetic patient. MSC transplantation hold great promise as a therapeutic intervention for PAD based on their pluripotency, as well as their efficacy in paracrine delivery of proangiogenic factors. However, we have determined that diabetic MSC manifest greater oxidant stress than healthy (WT) MSC. Diabetic MSC display restricted pluripotency, favoring adipocytic over endothelial differentiation; when transplanted into a WT host, diabetic MSC impair post-ischemic neovascularization and generate fatty infiltration in the ischemic hindlimb. The project hypothesis is that oxidant stress in diabetic MSC restricts their pluripotency and hence their neovascularization capacity. Three aims are proposed to test this hypothesis. Specific Aim 1 will demonstrate that oxidant production is the basis for restricted pluripotency in diabetic MSC, using a reductionist approach in cultured MSC. Exp [1] will determine if Nox4-derived H2O2 drives adipocyte differentiation in diabetic MSC. Exp [2] will determine the role of PPAR3-overexpression in diabetic MSC in generating adipocyte differentiation. Exp [3] will determine the role of uncoupled eNOS in generating oxidant stress in diabetic MSC and evaluate strategies for eNOS recoupling. Exp [4] will determine if deficiencies of the VEGF-Akt-eNOS pathway are the basis for impaired endothelial differentiation in diabetic MSC. Specific Aim 2 will use an in vivo MSC transplant paradigm to demonstrate that antioxidant treatment of diabetic MSCs or the diabetic host improves the efficacy of MSC transplant vis-a-vis post-ischemic neovascularization. Exp [1] will determine if ex vivo treatment of diabetic MSC with N-acetylcysteine (NAC) or other agents with direct or indirect antioxidant properties (resveratrol, rosiglitazone, rosuvastatin) improve their function upon subsequent transplant into a WT host in the setting of hindlimb ischemia. Exp [2] will determine if treatment of the db/db recipient mouse with NAC, or the other agents just noted, improve the outcome of MSC transplant in the setting of hindlimb ischemia. Specific Aim 3 will demonstrate that genetic engineering of MSC to enhance their expression of proangiogenic factors improves their participation in post-ischemic neovascularization. Exp [1] will determine if overexpression of wild type eNOS or constitutively active Akt in MSCs prior to transplant increases eNOS activation and eNOS-derived NO production, and in turn improves their efficacy in the treatment post-ischemic neovascularization. Exp [2] will determine if selection MSC expressing the chemokine receptor CXCR4 increases the homing of these cells to the ischemic hindlimb. If homing is improved, then this CXCR4+ MSC will undergo genetic engineering to maximize their in vivo functional capacity. PUBLIC HEALTH RELEVANCE: Current treatments for peripheral artery disease (PAD) are either palliative and treatment options are particularly limited in patients with the common co-morbidity of diabetes mellitus; discovery of novel therapeutic options are an urgent matter of national health care priority. Autologous transplantation with mesenchymal stem cells (MSC), a pluripotent cell that par- ticipates in post-ischemic neovascularization, holds great promise as a means to effect symptomatic relief by reversing disease pathology. The proposed work will elucidate the mechanisms responsible for the loss of pluripotency and therapeutic efficacy of diabetic MSC, as well as greatly increase knowledge regarding the basic and applied vascular biology of MSC in the treatment of lower extremity ischemia.