REGULATION OF VASCULAR SMOOTH MUSCLE CELL CHEMOTAXIS THROUGH CALCIUM SIGNALING

通过钙信号传导调节血管平滑肌细胞趋化性

• 批准号: 6097891
• 负责人: Steven J Sollott
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6097891
• 关键词: atherosclerosis; biological signal transduction; calcium; calcium channel; calcium flux; calmodulin dependent protein kinase; cell migration; chemotaxis; laboratory rat; platelet derived growth factor; restenosis; vascular smooth muscle

The chemotaxis, or directed migration, of vascular smooth muscle cells (VSMCs) is critical in the development of atherosclerosis and post-angioplasty restenosis, yet the cytosolic signaling pathways regulating this event are still largely undetermined. Our laboratory has demonstrated that: 1) specifically-timed Cai elevations, which in turn activate Ca2+/ calmodulin-dependent protein kinase II (CaMK), are required for chemotaxis, and 2) that the responses of individual cells need be examined due to the asynchrony and rarity of migration (only 5-10% of cells undergo chemotaxis by 4 hours). The precise role of CaMK, however, has yet to be resolved. Therefore, the specific aim was to determine whether compartmentalization of CaMK, and its activity, occurs within cells, since discrete localization of a kinase (or its activity) could implicate its function and target. The intracellular distribution of CaMK was determined in both fixed and live individual VSMCs respectively using immunocytochemistry (with three antibodies specific for different epitopes on CaMK) and live-cell imaging (with AS2, a novel fluorescent probe for CaMK activity). Subcellular domains of CaMK exist within VSMCs with a certain pool of CaMK decorating the polymerized actin cytoskeleton. Dynamic shifts in the state of actin polymerization (which accompany chemotaxis) result in an apparent translocation between a "soluble" pool and an "immobilized" pool of CaMK. The CaMK that is localized to polymerized actin stress fibers is activated (above the basal state) in quiescent, non-migrating VSMCs. Reactive oxygen species may provide a mechanism for "local" maintenance of this cytoskeletal-associated CaMK activity during quiescence. In migrating VSMCs, activated CaMK is localized to the cortical shell of actin that develops during chemotaxis, which suggests that CaMK may play a critical role in modulating force generation associated with directed migration. Thus, discrete localization of CaMK may enable specific functional targeting and regulation of the cytoskeleton during both quiescence and chemotaxis.

血管的趋化性或定向迁移 平滑肌细胞（VSMCs）在发展中至关重要， 动脉粥样硬化和血管成形术后再狭窄，但细胞溶质 调控这一事件的信号通路在很大程度上仍然是 不确定。我们的实验室已经证明：1） 特定时间的Cai升高，这反过来又激活了Ca 2 +/ 钙调蛋白依赖性蛋白激酶II（CaMK），是必需的， 趋化性，和2）单个细胞的反应需要 由于迁移的复杂性和罕见性（仅 5-10%的细胞在4小时内经历趋化性）。的确切作用 然而，CaMK还没有得到解决。因此，具体目标 是为了确定CaMK的区室化及其 由于激酶的离散定位，活性发生在细胞内 (or其活动）可能涉及其功能和目标。的 在固定的和未固定的细胞中测定CaMK的细胞内分布。 应用免疫细胞化学方法， (with对CaMK上的不同表位具有特异性的三种抗体）和 活细胞成像（用AS 2，一种新型的CaMK荧光探针 活动）。CaMK的亚细胞结构域存在于VSMCs内， 一定量的CaMK修饰聚合的肌动蛋白 细胞骨架肌动蛋白聚合状态的动态变化 （伴随趋化性）导致明显的易位 在CaMK的“可溶性”池和“固定化”池之间。的 CaMK定位于聚合的肌动蛋白应力纤维， 在静止、非迁移状态下激活（高于基础状态） 血管平滑肌细胞。活性氧物质可能提供了一种机制， 这种细胞内相关的CaMK活性的“局部”维持 在静止期。在迁移的VSMCs中，激活的CaMK是 定位于肌动蛋白的皮质壳， 趋化性，这表明CaMK可能在 调节与定向迁移相关的力产生。 因此，CaMK的离散定位可能使特定的功能性 在静止期和静止期， 和趋化性。