ENDOTHELIUM IN RELATION TO ATHEROGENESIS

其目的是阐明分子和微观力学。 内皮细胞(EC)周转的基础已在 这项资助下的研究是引起焦点的一个关键因素 低密度脂蛋白通透性增加，因此区域易感性 动脉分叉和弯曲区域的动脉粥样硬化 树。我们的假设是，细胞水平的复杂流动模式 这些病变易感区域诱导机械力化学转导。 EC修改有丝分裂和细胞凋亡的调控，导致 这两个过程都在加速。最终结果是 保持EC单层的融合性，代价是 周转加快，低密度脂蛋白随之增加 渗透性。实验将通过使用两个新的 设计了流动装置，以产生复杂的流型，具有 强调剪应力梯度：阶跃流道产生 具有非定常再附着区的回流 在几个单元格的长度范围内来回振荡； T型狭缝流道产生较大的剪应力空间梯度 在单个细胞的长度尺度上。三个具体目标是 提出用来检验我们的假设。(一)确定影响 EC有丝分裂中分子事件的复杂流动模式 细胞凋亡，我们将使用阶梯状流动通道来研究EC 细胞周期蛋白A、B和D1的表达与流型的关系 以及过表达显性负性突变体的效果 细胞有丝分裂中的细胞周期蛋白依赖性激酶(CDC2、CDK4和CDK6) 和细胞凋亡。(2)破译信号转导途径 复杂的血流反应下潜在的EC有丝分裂和细胞凋亡 模式，我们将使用与特定目标1中类似的策略来测试 Raf-MEK-ERK和Mekk-JNKK-JNK的假设 信号转导通路在细胞有丝分裂和细胞凋亡中起着至关重要的作用。(3)至 在单个细胞水平上阐明微观机械机制， 血流动力导致EC有丝分裂和细胞凋亡， 我们将使用T形狭缝流道来研究 细胞基底膜上的整合素和粘着斑激酶 EC，除了腔上膜上的蛋白质外，在 涉及这些蛋白质的信号转导。这一交叉学科 研究将对分子机制产生新的见解 通过这种复杂的流动模式导致丝裂原加速和 单个内皮细胞的细胞凋亡，并加深我们对 脂质积聚局灶性的病理生理学基础 和动脉粥样硬化的形成。

The objective is to elucidate the molecular and micromechanical bases of endothelial cell (EC) turnover which has been found in studies under this grant to be a key factor in causing the focal increase of LDL permeability, and hence regional susceptibility to atherosclerosis, in bifurcations and curved areas of the arterial tree. Our hypothesis is that cellular-level complex flow patterns in these lesion-prone regions induce mechanochemical transduction in the EC to modify the regulation mitosis and apoptosis, leading to the acceleration of both processes. The end result is the preservation of confluency of the EC monolayer at the expense of an accelerated turnover and the consequent increase in LDL permeability. Experiments will be conducted by using two newly designed flow devices to generatecomplex flow patterns, with an emphasis on shear stress gradient: The step flow channel generates a recirculating flow with an unsteady reattachment region that oscillates back and forth over the lenght scale of a few cells; the T-slit flow channel generates large spatial gradients in shear stress on the length scale of an individual cell. Three specific aims are proposed to test our hypothesis. (1) To determine the influence of complex flow patterns on the molecular events in EC mitosis and apoptosis, we will use the step flow channel to study the EC expression of cyclins A,B, and D1 as a function of the flow regime and the effects of overexpressing the dominant negative mutants of cyclin dependent kinases (Cdc2, Cdk4, and Cdk6) on EC mitosis and apoptosis. (2) To decipher the signal transduction pathways underlying EC mitosis and apoptosis in response to complex flow patterns, we will use similar strategies as in specific aim 1 to test the hypothesis that the Raf-MEK-ERK and MEKK-JNKK-JNK pathways are crucial in mediating mitosis and apoptosis. (3) To elucidate the micromechanical mechanism, at individual cell level, by which hemodynamic forces lead to EC mitosis and apoptosis, we will use the T-slit flow channel t investigate the roles of integrins and focal adhesion kinase on the basal membrane of the EC, in addition to the proteins on the luminal membrane, in the signal transduction involving these proteins. This interdisciplinary research will generate new insights into the molecular mechanisms by which complex flow patterns lead to accelerated mitoxis and apoptosis of individual Ecs and enhance our understanding of the pathophysiological basis of the focal nature of lipid accumulation and atherogenesis.