Impact of Aging on Stem Cell Repair in Atherosclerosis

描述（由申请人提供）：动脉粥样硬化是一种异常的、持续的修复性炎症过程，是对血管壁反复损伤的反应。的确，心血管系统不断受到各种各样的损害，这些损害的影响随着时间的推移而累积。参与修复过程的细胞有两种来源：(1)从邻近血管段迁移过来的局部分化血管内皮细胞和平滑肌细胞；(2)通过外周循环从骨髓中募集干细胞/血管祖细胞。我们最近发现，在已建立的动脉粥样硬化小鼠模型中，即使ApoE / C57/B6小鼠喂养高脂肪/高胆固醇，西式饮食，即使没有恢复ApoE基因表达和血浆胆固醇水平正常化（>1200 mg/dl），全骨髓（BM）细胞的慢性给药也能显著减少动脉粥样硬化病变的形成。此外，来自年轻而不是年老ApoE-/-小鼠的BM细胞能够恢复血管活力和预防动脉粥样硬化。这些数据强调了干细胞/血管祖细胞在血管愈合和动脉粥样硬化中的重要性，并为使用干细胞/祖细胞治疗作为动脉粥样硬化的一种新的预防和/或治疗策略提供了支持，特别是对于那些骨髓及其陈旧成为心血管系统长期完整性瓶颈的个体。在这个项目中，我们提出验证这样一个假设，即在修复动脉粥样硬化后，维持心血管系统稳态所需的特定类型的血管祖细胞（VPCs）的供应以某种方式耗尽或功能受损。如果VPC“备件”可以外包，修复过程可以在适当的间隔内加快，动脉粥样硬化的后果可能会无限期地推迟。特异性目的1：确定从年轻小鼠与老年小鼠（野生型、ApoE /和LDLR -/-）获得的全骨髓细胞或谱系阴性群体（lin-SP）细胞的定量组成（FACS分析）、后代功能特征和基因表达表型（微阵列分析）；特异性目的2：通过测试未分离的骨髓细胞、富集的lin-SP细胞和从年轻小鼠和老年小鼠（野生型和ApoE -/）中获得的lin-SP部分的骨髓细胞在抑制ApoE 4小鼠血浆中升高的化学细胞因子和生长因子水平以及防止动脉粥样硬化病变形成方面的作用，确定在骨髓中包含有能力的VPCs，这传递了抗动脉粥样硬化的作用；特异性目的3：确定从年轻小鼠和老年小鼠（野生型和LDLR 4-）获得的未分离骨髓细胞、lin-SP细胞和骨髓细胞减lin-SP部分在抑制血浆化学细胞因子和生长因子水平升高以及预防LDLR t-小鼠动脉粥样硬化（类似家族性高胆固醇血症）方面的功效。我们将确定循环炎症因子，包括IL-6、M-CSF、TNFc_、VEGF和金属蛋白酶-9，是否在血管损伤反应中作为调动和募集血管祖细胞的信号。这些研究的结果将深入了解与动脉粥样硬化相关的骨髓干细胞生物学，并揭示可用于分离甚至富集骨髓中血管祖细胞的新标志物，特别是老年骨髓。这些发现也将有助于确定治疗动脉粥样硬化的最佳血管祖细胞表型。

DESCRIPTION (provided by applicant): Atherosclerosis represents an aberrant, continuous reparative inflammatory process in response to repeated injuries to the vessel wall. Indeed, the cardiovascular system is continuously exposed to a multitude of insults whose impact cumulates with the passage of time. Two sources of cells participating in the repair process exist: (1) local, differentiated, vascular endothelial and smooth muscle cells that migrate from adjacent vessel segments; and (2) recruited stem cells/vascular progenitor cells from the bone marrow, via peripheral circulation. We have recently discovered that chronic administration of whole bone marrow (BM) cells significantly reduced atherosclerotic lesion formation in an established mouse model of atherosclerosis--ApoE / C57/B6 mice fed high fat/cholesterol, Western-type diet--even in the absence of restoration of ApoE gene expression and normalization of plasma cholesterol levels (>1200 mg/dl). Furthermore, BM cells from young, but not old, ApoE-/- mice were capable of vascular rejuvenation and atherosclerosis prevention. These data underscore the importance of stem cells/vascular progenitor cells in vascular healing and atherogenesis and provide support for the use of stem/progenitor cell therapy as a novel preventative and/or treatment strategy for atherosclerosis, particularly for individuals whose bone marrow, and its obsolescence, represents the bottleneck for long-term integrity of the cardiovascular system. In this project, we propose to test the hypothesis that after a lifetime of repairing atherosclerotic arteries, the supply of the specific type(s) of vascular progenitor cells (VPCs) needed to maintain the homeostasis of the cardiovascular system is somehow exhausted or functionally impaired. If the VPC "spare parts" can be outsourced, the repair process can be boosted at appropriate intervals and atherosclerotic consequences delayed, perhaps indefinitely. Specific Aim 1: To determine the quantitative composition (FACS analysis), progeny functional characteristics, and gene expression phenotype (microarray analysis) of whole bone marrow cells or lineage negative side population (lin-SP) cells obtained from young versus old (wild-type, ApoE / and LDLR -/-) mice; Specific Aim 2: To establish that competent VPCs are encompassed in the linSP fraction in the marrow, which convey the anti-atherosclerotic efficacy, by testing the effects of unfractionated bone marrow cells, enriched lin-SP cells and bone marrow cells deprived of lin-SP fraction obtained from young versus old (wild-type and ApoE -/) mice in suppressing elevated plasma levels of chemo-cytokines and growth factors and in preventing atherosclerotic lesion formation in ApoE 4 mice; and Specific Aim 3: To determine the efficacy of unfractionated bone marrow cells, lin-SP cells and bone marrow cells minus lin-SP fraction obtained from young versus old (wild-type and LDLR 4-) mice in suppressing elevated plasma levels of chemo-cytokines and growth factors and in the prevention of atherosclerosis in LDLR t- mice--a trait resembling familial hypercholesterolemia. We will determine whether circulatory inflammatory factors, including IL-6, M-CSF, TNFc_, VEGF and metalloproteinase-9, serve as signals to mobilize and recruit vascular progenitor cells in response to vascular injury. Results from these studies will provide insight into BM stern cell biology in relation to atherosclerosis, and reveal new markers that could be used to isolate and even enrich vascular progenitor cells in the BM, especially old BM. The findings will also help determine the optimal vascular progenitor cell phenotypes for the treatment of atherosclerosis.