CXCR4/SDF-1 Axis in Proliferation Diabetic Retinopathy

描述(由申请人提供)： 增殖性糖尿病视网膜病变(PDR)以异常新生血管为特征，是美国成人失明的主要原因。PDR失明的破坏性后果是视网膜表面异常新生血管的结果，而不是视网膜本身的生理性血管生成。现在已经认识到，内皮细胞前体在血流中循环，被招募到新血管生成的部位，并参与新血管的形成。这些前体细胞表达趋化因子受体CXCR4，并在基质衍生因子1(SDF-1)作用下迁移。SDF-1是一种强有力的血管内皮生长因子刺激因子，目前被认为是糖尿病视网膜病变中视网膜新生血管的主要效应因子。我们的假设是，PDR是SDF-1介导的内皮前体细胞迁移的一个或多个缺陷的结果，导致新生血管在视网膜表面而不是在视网膜内生成。这一建议将解决以下假设：1)糖尿病患者视网膜中SDF-1的模式改变或内皮祖细胞上CXCR4的表达水平在PDR中明显；2)PDR患者的祖细胞对SDF-1的反应发生改变；以及3)SDF-/CXCR4相互作用的拮抗将阻止体内视网膜血管生成。这些假说将通过下列特定目标进行检验：目的1)通过细胞为基础的酶联免疫吸附试验，检测糖尿病患者循环内皮细胞和循环内皮祖细胞的数量，并将这些细胞上CXCR4的表达与PDR的存在与否相关联；B)通过原位杂交检测SDF-1在糖尿病合并和不合并PDR患者和正常对照组身体视网膜中的RNA表达模式；C)采用ELISA法测定糖尿病合并和不合并PDR患者和非糖尿病对照组玻璃体液中SDF-1的水平。目的：用改良的Boyden小室迁移实验、Matrigel管形成实验和ELISA法检测有无PDR的糖尿病患者和正常对照组的祖细胞内皮细胞对SDF-1的依赖反应。目的：A)检测SDF-1和CXCR4在新生小鼠氧诱导视网膜病变模型中的时空表达；B)确定CXCR4拮抗剂对成年小鼠视网膜血管生成的影响。这些研究通过引入趋化因子和祖细胞内皮细胞生物学领域的专业知识，将为增殖性糖尿病视网膜病变的发病机制提供重要信息。

DESCRIPTION (provided by applicant): Proliferative diabetic retinopathy (PDR), characterized by aberrant neovascularization, is the leading cause of adult blindness in the United States. The devastating consequence of blindness in PDR is the result of aberrant neovascularization on the surface of the retina rather than physiological vascularization within the retina itself. It is now recognized that endothelial cell precursors circulate in the bloodstream, are recruited to sites of neoangiogenesis, and participate in new blood vessel formation. These precursors have been shown to express the chemokine receptor CXCR4 and migrate in response to stromal derived factor 1 (SDF-1). SDF-1 is a potent stimulator of vascular endothelial growth factor (VEGF), which is currently viewed as the major effector for retinal neovascularization in diabetic retinopathy. It is our hypothesis that PDR is the result of one or more defects in SDF- 1-mediated migration of endothelial progenitor cells leading to neoangiogenesis on the surface of the retina rather than within the retina. This proposal will address the following hypotheses: 1) Alterations in the pattern of SDF-1 in the retinas of diabetic patients or level of expression of CXCR4 on endothelial progenitor cells are evident in PDR; 2) Progenitor cells from patients with PDR have altered responses to SDF-1; and 3) Antagonism of SDF-/CXCR4 interactions will prevent retinal angiogenesis in vivo. These hypotheses will tested through the following Specific Aims: Aim 1: A) In diabetic patients, correlate the numbers of circulating endothelial cells and circulating endothelial progenitor cells by cell-based ELISA and correlate the expression of CXCR4 on these cells with the presence or absence of PDR; B) Examine the pattern of RNA expression of SDF-1 by in situ hybridization in cadaveric retinas of diabetic patients with and without PDR and normal controls; C) Determine the levels of SDF-1 in the vitreous fluid of diabetic patients with and without PDR and non-diabetic controls by ELISA. Aim 2: Examine SDF-1-dependent response of progenitor endothelial cells from diabetic patients with and without PDR and normal controls in a migration assay using a modified Boyden chamber, a tube formation assay on Matrigel, and an ELISA for VEGF expression. Aim 3: A) Examine the spatial and temporal expression of SDF-1 and CXCR4 in a neonatal mouse model of oxygen-induced retinopathy; B) Determine the effect of a CXCR4 antagonist on the development of retinal angiogenesis in an adult mouse model of induced retinopathy. These studies by bringing in expertise from the areas of chemokine and progenitor endothelial cell biology will provide important information on the pathogenesis of proliferative diabetic retinopathy.