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尾细胞/血管祖细胞。我们最近发现，在一种已建立的动脉粥样硬化小鼠模型中，即使ApoE基因表达没有恢复，血浆胆固醇水平（>1200 mg/dl）没有正常化，长期给药全骨髓（BM）细胞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老化是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这些细胞功能受损。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：确定从年轻小鼠和老年小鼠（野生型和ApoE -/-）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linSP细胞和从年轻小鼠和老年小鼠（野生型和ApoE -/-）中获得的未分离的linSP细胞和被剥夺了lin-SP部分的骨髓细胞在抑制ApoE -/-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损伤形成方面的作用，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；特异性目标3：确定在年轻apoE -/-和野生型BM的lin-SP部分中，a)使用Aim 1中鉴定的标记物分离的候选VPCs （CVPC，衰老小鼠中耗尽或功能受损的细胞），b)未分离的lin-SP细胞- CVPCs (USP-CVPC)，以及c)未分离的linSP细胞（USP）在抑制血浆IL-6， VEGF和其他炎症标志物水平升高以及预防apoE -/-小鼠动脉粥样硬化中的相对效力；特异性目的4：确定4-整合素、VCAM-1和e -选择素在介导apoE-/-小鼠血管祖细胞向慢性损伤血管壁移植中的作用。因此，细胞植入与非细胞自主机制对血管修复的相对贡献将被表征。

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 stern 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. This data underscores the importance of stem cells/vascular progenitor cells in vascular healing and atherogenesis and provide support for the use of stern/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 these cells are 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, and ApoE -/-) mice; Specific Aim 2: To establish that competent VPCs are encompassed in the lin-SP fraction in the marrow, which convey the anti-atherosclerotic efficacy, by testing the effects of unfractionated bone marrow cells, enriched linSP 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 -/- mice; Specific Aim 3: To determine within the lin-SP fraction of young apoE -/- and wild-type BM the relative efficacy of a) candidate VPCs (CVPC, cells that are either depleted or functionally impaired in aging mice) isolated using markers identified in Aim 1, b) unfractionated lin-SP cells minus CVPCs (USP-CVPC), and c) unfractionated linSP cells (USP) in suppressing elevated plasma levels of IL-6, VEGF and other inflammatory markers and in preventing atherosclerosis in ApoE -/- mice; and Specific Aim 4: To determine the role of a4-integrin, VCAM-1, and E-selectin in mediating the engraftment of vascular progenitor cells to chronically injured vessel wall in apoE-/- mice. As a corollary, the relative contribution of cell engraftment versus a non-cell autonomous mechanism to vascular repair will be characterized.