Endothelial Cell Migration and Retinal Angiogenesis

描述(申请人提供)：内皮细胞迁移对正常新生血管至关重要，而新生血管又对视网膜发育和功能至关重要。相反，血管生成异常是绝大多数导致视力丧失的疾病的主要原因，包括老年性黄斑变性和糖尿病视网膜病变。虽然在理解促进视网膜血管生成的因素方面已经取得了一些进展，但驱动潜在的内皮细胞迁移过程的机制还不是很清楚。本文概述的研究将开发一种新的直接相关方法，从机械上剖析内皮细胞在体外和体内迁移所需的细胞运动功能。作为一个测试案例，这项提案将研究Cofilin的作用，Cofilin是一种肌动蛋白结合蛋白，已知对其他类型细胞的运动性很重要。在目标1中，将通过Cofilin突变体的表达和RNA干扰来检测Cofilin在体外对内皮细胞迁移的作用。定量荧光图像分析和活细胞延时显微镜将被用来确定在Cofilin功能受扰的细胞中肌动蛋白动力学是如何改变的，以及这如何影响细胞迁移行为。在目标2中，Cofilin的功能将在内皮前体干细胞中受到类似的干扰，然后将其注射到新生小鼠的玻璃体内。这些干细胞在视网膜新生血管形成过程中的迁移能力，以及整合到视网膜血管中的能力，将通过视网膜的定量荧光显微镜分析进行监测。 不同时间间隔的血管形成。这种新颖的综合方法将导致一个根本的 了解视网膜所特需的内皮细胞迁移过程 新生血管的形成，并将为未来全面研究监管 控制视网膜血管生成的网络。此外，这项研究还将推动智能网的发展 具有更强预测能力的体外试验用于识别新的靶点和化合物 抗血管生成治疗。

DESCRIPTION (provided by applicant): Endothelial cell migration is critical to normal neovascularization, which is in turn essential for retinal development and function. Conversely, abnormal angiogenesis is the primary cause in the vast majority of diseases that result in vision loss, including age-related macular degeneration and diabetic retinopathy. While there has been some progress in understanding the factors that promote retinal angiogenesis, the mechanisms that drive the underlying endothelial cell migration process are not well understood. The research outlined here will develop a novel direct correlation approach to mechanistically dissect the cell locomotion functions required for endothelial migration both in vitro and in vivo. As a test case, this proposal will investigate the role of cofilin, an actin binding protein known to be important for motility in other cell types. In Aim 1, the function of cofilin in endothelial cell migration in vitro will be examined by expression of cofilin mutants and RNA interference. Quantitative fluorescence image analysis and live-cell, time-lapse microscopy will be used to determine how the actin dynamics are altered in cells with perturbed cofilin function, and how this affects cell migration behavior. In Aim 2, the functions of cofilin will be similarly perturbed in endothelial-precursor stem cells, which will then be intravitreally injected into neonatal mice. The ability of these stem cells to migrate during retinal neovascularization, and to integrate into the retinal vasculature will be monitored by quantitative fluorescence microscopy analyses of the retinal vasculature at various time intervals. This novel integrated approach will lead to a fundamental understanding of the endothelial cell migration processes that are specifically required for retinal neovascularization, and will lay the groundwork for future comprehensive studies of regulatory networks controlling retinal angiogenesis. Furthermore, this research will initiate development of in vitro assays with greater predictive power for identification of novel targets and compounds for anti-angiogenesis treatments.