Endothelial Cell Migration and Retinal Angiogenesis

描述（申请人提供）：内皮细胞迁移是正常新生血管形成的关键，这反过来对视网膜发育和功能至关重要。相反，血管生成异常是导致视力丧失的绝大多数疾病的主要原因，包括年龄相关性黄斑变性和糖尿病性视网膜病变。虽然在了解促进视网膜血管生成的因素方面取得了一些进展，但驱动潜在内皮细胞迁移过程的机制尚不清楚。本文概述的研究将发展一种新的直接相关方法，以机械地解剖体外和体内内皮细胞迁移所需的细胞运动功能。作为一个测试案例，本提案将研究cofilin的作用，cofilin是一种已知对其他细胞类型的运动性很重要的肌动蛋白结合蛋白。在Aim 1中，将通过cofilin突变体的表达和RNA干扰来检测cofilin在体外内皮细胞迁移中的功能。定量荧光图像分析和活细胞延时显微镜将用于确定肌动蛋白动力学是如何改变的细胞与受干扰的cofilin功能，以及这如何影响细胞迁移行为。在Aim 2中，cofilin的功能将在内皮前体干细胞中受到类似的干扰，然后将其静脉注射到新生小鼠中。这些干细胞在视网膜新生血管形成过程中的迁移能力和融入视网膜血管系统的能力将通过视网膜的定量荧光显微镜分析来监测

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.