Investigating the Role of Shear Stress in Coronary Artery Development

项目摘要/摘要 冠状动脉疾病(CAD)是美国主要的死亡原因，是由冠状动脉狭窄引起的 动脉，其结果是心脏灌注减少，潜在的心肌梗死和/或心力衰竭。 治疗冠心病的一个有希望的方法是再生动脉并恢复缺血心脏组织的血液流动。 然而，为了使动脉再生成为CAD治疗的现实，我们需要更详细的 了解动脉是如何形成的。先前的研究表明，内皮细胞(ECs)暴露于 血流对冠状动脉的发育至关重要。冠状动脉的形成是一个循序渐进的过程 1)内皮细胞符合动脉表型；2)毛细血管内皮细胞向发育中的动脉迁移。这个 血流刺激这些形态发生过程的机制尚不清楚。以前的研究 已经表明，将培养的内皮细胞暴露在剪切力下会导致动脉特异基因的上调，这是一个关键 执行动脉EC规范。此外，我们实验室和其他实验室的研究结果表明， 趋化因子CXCL12在动脉内皮细胞中的表达丰富，这是一种高剪应力环境。 我们还发现，CXCL12-CXCR4(CXCL12受体)信号通路促进内皮细胞逆方向迁移 在体外的流动。这些观察结果使我假设剪切力既使祖细胞完全动脉化 并刺激它们释放趋化因子，将附近的内皮细胞吸引到发育中的动脉。我要测试一下 这一假设解决了以下具体目标。在具体目标1中，我将确定剪切的影响 强调血管内皮细胞的动脉规范。为了实现这一目标，我将利用一种新的人类体外系统 多能干细胞可分化成纯净的动脉内皮细胞群 用动脉化的生化信号进行治疗。测量动脉EC参数以响应不同的 剪切力和动脉化生化信号的组合将揭示 切应力将内皮细胞推向动脉的命运。在特定的目标2中，我将确定趋化因子在 在体内协调流动诱导的EC迁移。也就是说，我将使用以下命令干扰EC CXCL12-CXCR4信令 从动脉内皮细胞中删除CXCL12或从毛细血管内皮细胞中删除CXCR4的小鼠。正在评估 这些小鼠的冠状动脉形成将使我能够确定动脉CXCL12-毛细血管CXCR4 信号转导内皮细胞从低(毛细血管)切应力环境向高(动脉)切应力环境迁移。结果 这些研究将对切应力促进动脉的机制产生实质性的洞察。 规范和冠状动脉重塑。这项工作的发现可能会在治疗上被利用 开发在体内再生动脉或在体外生成组织工程动脉的策略。

PROJECT SUMMARY/ABSTRACT Coronary artery disease (CAD), the leading cause of death in the U.S., is caused by a narrowing of coronary arteries, the result of which is reduced cardiac perfusion and potentially myocardial infarction and/or heart failure. One promising approach to treating CAD is to regenerate arteries and restore blood flow to ischemic heart tissue. In order to make arterial regeneration a reality for CAD treatment, however, we need a more detailed understanding of how arteries are formed. Previous studies indicate that exposure of endothelial cells (ECs) to blood flow is critical for coronary artery development. Coronary artery formation is a stepwise process involving 1) specification of ECs to an arterial phenotype and 2) migration of capillary ECs into developing arteries. The mechanisms by which blood flow stimulates each of these morphogenic processes are unclear. Previous studies have shown that exposing cultured ECs to shear stress leads to upregulation of artery-specific genes, one key step in arterial EC specification. Furthermore, findings from our laboratory and others have shown that expression of the chemokine Cxcl12 is enriched in the arterial endothelium, a high shear stress environment. We also found that Cxcl12-Cxcr4 (Cxcl12 receptor) signaling promotes the migration of ECs against the direction of flow in vitro. These observations have led me to hypothesize that shear stress both fully arterializes progenitor ECs and stimulates them to release chemokines which attract nearby ECs to the developing artery. I will test this hypothesis by addressing the following Specific Aims. In Specific Aim 1, I will determine the effects of shear stress on arterial specification of ECs. To accomplish this goal, I will utilize a novel in vitro system of human arterial differentiation in which pure populations of arterial ECs can be generated from pluripotent stem cells treated with arterializing biochemical signals. Measuring arterial EC specification in response to different combinations of shear stress and arterializing biochemical signals will reveal molecular mechanisms by which shear stress drives ECs towards an arterial fate. In Specific Aim 2, I will determine the role of chemokines in orchestrating flow-induced EC migration in vivo. Namely, I will perturb EC Cxcl12 – Cxcr4 signaling by using mice in which either Cxcl12 is deleted from arterial ECs or Cxcr4 is deleted from capillary ECs. Assessing coronary artery formation in these mice will allow me to determine whether arterial Cxcl12 – capillary Cxcr4 signaling directs the migration of ECs from environments of low (capillary) to high (artery) shear stress. Results from these studies will generate substantial insight into the mechanisms by which shear stress promotes arterial specification and coronary artery remodeling. Findings from this work may be leveraged therapeutically to develop strategies for regenerating arteries in vivo or generating tissue-engineered arteries in vitro.