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

描述(申请人提供)：该项目的目标是加强使用间充质干细胞(MSC)治疗外周动脉疾病，特别是糖尿病患者的手段。骨髓间充质干细胞移植基于其多能性以及在旁分泌递送促血管生成因子方面的有效性，被认为是一种治疗PAD的有效方法。然而，我们已经确定，糖尿病的MSC比健康的(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，有利于脂肪细胞的分化，而不是内皮分化；当移植到WT宿主体内时，糖尿病骨髓间充质干细胞会损害缺血后的新生血管，并在缺血的后肢产生脂肪渗透。项目假设是糖尿病间充质干细胞中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。为了检验这一假设，本文提出了三个目标。具体目标1将证明氧化剂的产生是糖尿病间充质干细胞限制多能性的基础，在培养的间充质干细胞中采用还原方法。EXP[1]将确定NOX4衍生的过氧化氢是否推动糖尿病间充质干细胞的脂肪细胞分化。实验[2]将确定糖尿病间充质干细胞中PPAR3过表达在脂肪细胞分化中的作用。实验[3]将确定非偶联eNOS在糖尿病MSC中产生氧化应激中的作用，并评估eNOS重新偶联的策略。EXP[4]将确定血管内皮生长因子-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移植范例来证明对糖尿病MSCs或糖尿病宿主的抗氧化治疗 与缺血后新生血管相比，提高了骨髓间充质干细胞移植的有效性。EXP[1]将确定用N-乙酰半胱氨酸(NAC)或其他具有直接或间接抗氧化特性的药物(白藜芦醇、罗格列酮、瑞舒伐他汀)体外治疗糖尿病MSC是否会在随后移植到WT宿主的后肢缺血环境中改善它们的功能。实验[2]将确定在后肢缺血的情况下，用NAC或其他药物治疗db/db受体小鼠是否能改善MSC移植的结果。具体目标3将证明，通过基因工程提高MSC的促血管生成因子的表达，可提高其在缺血后新生血管中的参与程度。EXP[1]将确定移植前在MSCs中过度表达野生型eNOS或结构性活性Akt是否会增加eNOS的激活和eNOS衍生的NO的产生，从而提高它们在治疗缺血后新生血管方面的疗效。EXP[2]将确定表达趋化因子受体CXCR4的选择MSC是否增加了这些细胞对缺血后肢的归巢。如果归巢功能得到改善，那么CXCR4+MSC将接受基因工程，以最大限度地发挥其在体内的功能。 公共卫生相关性：目前外周动脉疾病(PAD)的治疗要么是姑息治疗，治疗选择尤其有限，尤其是在糖尿病常见并存的患者中；发现新的治疗方案是国家卫生保健优先事项的紧急事项。自体移植间充质干细胞(MSC)是一种参与缺血后新生血管形成的多能细胞，作为一种通过逆转疾病病理来缓解症状的手段，具有很大的前景。这项拟议的工作将阐明糖尿病MSC丧失多能性和治疗效果的机制，并极大地增加对MSC在治疗下肢缺血中的基础和应用血管生物学的认识。

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.