Impact of Aging on Stem Cell Repair in Atherosclerosis

描述（由申请方提供）：动脉粥样硬化是一种异常的、持续的修复性炎症过程，对血管壁的反复损伤做出反应。事实上，心血管系统持续暴露于大量的损伤，其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）从相邻血管节段迁移的局部分化的血管内皮细胞和平滑肌细胞;和（2）通过外周循环从骨髓募集的干细胞/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，即使在ApoE基因表达没有恢复和血浆胆固醇水平没有正常化（>1200 mg/dl）的情况下，长期给予全骨髓（BM）细胞也能显著减少动脉粥样硬化病变的形成--ApoE -/- C57/B6小鼠，喂食高脂肪胆固醇，西式饮食。此外，来自年轻而非老年ApoE-/-小鼠的BM细胞能够血管再生和预防动脉粥样硬化。该数据强调了干细胞/血管祖细胞在血管愈合和动脉粥样硬化形成中的重要性，并为使用干细胞/祖细胞疗法作为动脉粥样硬化的新型预防和/或治疗策略提供了支持，特别是对于骨髓及其陈旧性代表了心血管系统长期完整性的瓶颈的个体。在这个项目中，我们提出了一个假设，即经过一生的动脉粥样硬化修复，维持心血管系统稳态所需的特定类型的血管祖细胞（VPC）的供应在某种程度上耗尽或这些细胞功能受损。如果VPC的“备件”可以外包，修复过程可以在适当的时间间隔和动脉粥样硬化的后果推迟，也许是无限期的。具体目标1：确定定量成分（FACS分析）、后代功能特征和基因表达表型（微阵列分析）从年轻人与老年人获得的全骨髓细胞或谱系阴性侧群（lin-SP）细胞（野生型和ApoE -/-）小鼠;特定目标2：为了证实骨髓中的lin-SP组分中包含有活性VPC，其传递抗动脉粥样硬化功效，通过测试未分级骨髓细胞的作用，富集的lin-SP细胞和从年轻人与老年人获得的去除lin-SP组分的骨髓细胞（野生型和ApoE -/-）小鼠中抑制升高的化学细胞因子和生长因子的血浆水平以及预防ApoE -/-小鼠中动脉粥样硬化病变形成的作用;具体目标3：在年轻apoE -/-和野生型BM的lin-SP部分内确定a）候选VPC的相对功效（CVPC，在衰老小鼠中耗尽或功能受损的细胞）分离，使用目的1中鉴定的标记物，B）未分级分离的lin-SP细胞减去CVPC c）未分级的linSP细胞（USP）在ApoE -/-小鼠中抑制升高的IL-6、VEGF和其它炎性标志物的血浆水平和预防动脉粥样硬化中的作用;和确定α 4-整合素、VCAM-1和E-选择素在介导血管祖细胞植入apoE-/-小鼠慢性损伤血管壁中的作用。作为推论，将表征细胞植入与非细胞自主机制对血管修复的相对贡献。

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.