Novel regulation of the anti-angiogenic activity of PEDF by pro-angiogenic MMPs

描述（申请人提供）：我们假设基质金属蛋白酶（MMPs）调节色素上皮衍生因子（PEDF）的抗血管生成活性，并且这种蛋白水解活性导致眼睛中抗血管生成因子和促血管生成因子之间的平衡丧失，从而导致眼部新生血管形成。在患有病理性血管生成疾病的眼睛中，PEDF蛋白水平降低，而在这些病变组织中，发现MMP活性上调。我们发现PEDF是促血管生成基质金属蛋白酶-2和-9的底物。我们还发现蛋白酶MMP-9在n端附近的抗血管生成区域和c端附近的蛇形蛋白反应中心环内切割PEDF。在小鼠主动脉环实验中，MMP-9对PEDF的切割消除了PEDF的抗血管生成活性，并将PEDF转化为内皮化因子。最后，通过玻璃体内注射一种MMP-2和-9抑制剂对小鼠眼睛进行预处理，用一种设计用于表达PEDF的腺载体在玻璃体内转导的小鼠眼睛中观察到的PEDF量增加了两倍。我们在此提出1)确定MMP-2和-9靶向PEDF内的特定序列；2)确定mmp处理的PEDF分离片段的活性，3)通过在小鼠脉络膜新生血管模型中评估mmp抗性突变PEDF，验证MMPs在体内调节PEDF活性的假设。眼部新生血管疾病，最常见的是糖尿病视网膜病变和渗出性老年性黄斑变性，是发达国家致盲的主要原因。这些疾病威胁着快速增长的高危人口的前景，尽管现有的治疗干预措施有限，但迄今为止还没有治愈方法。这一研究将促进对病理性眼血管生成机制的基本认识，并有可能导致此类疾病的治疗取得进展。眼部新生血管疾病是发达国家致盲的主要原因，治疗干预措施有限。我们假设基质金属蛋白酶调节色素上皮衍生因子的抗血管生成活性，这种蛋白水解活性导致眼睛中抗血管生成因子和促血管生成因子之间的平衡丧失，从而导致眼部新生血管形成。我们的研究将推进病理性眼部血管生成机制的基础知识，并可能导致此类疾病的治疗取得进展。

DESCRIPTION (provided by applicant): We hypothesize that matrix metalloproteinases (MMPs) modulate the anti-angiogenic activity of pigment-epithelium derived factor (PEDF), and that this proteolytic activity contributes to a loss of balance between anti-angiogenic and pro-angiogenic factors in the eye leading to ocular neovascularization. The level of PEDF protein is decreased in eyes afflicted with pathological angiogenic conditions while MMP activity is found to be upregulated in these diseased tissues. We have found that PEDF is a substrate for the pro-angiogenic matrix-metalloproteinases-2, and -9. We also found that the proteinase MMP-9 cleaves PEDF within the anti-angiogenic region near the N-terminus and within the serpin reactive center loop near the C-terminus. Cleavage of PEDF by MMP-9 eliminates the anti-angiogenic activity of PEDF as measured in the mouse aortic ring assay, and converts PEDF into an endothelial chemoattractant factor. Finally, pretreatment of mouse eyes with intravitreal injection of an MMP-2 and -9 inhibitor tripled the observed amount of PEDF in mouse eyes intravitreally transduced with an adenovector designed to express PEDF. We propose herein 1) to determine the specific sequences within PEDF targeted by MMP-2 and -9; 2) determine the activities of the isolated fragments of MMP-treated PEDF, and 3) test the hypothesis that MMPs modulate PEDF activity in vivo by evaluating MMP-resistant mutant PEDF in a mouse model of choroidal neovascularization. Diseases of ocular neovascularization, the most prevalent being diabetic retinopathy and exudative age-related macular degeneration, are the leading cause of blindness in developed countries. These diseases threaten the sight of a quickly growing population-at-risk, and although there are limited therapeutic interventions at hand, there is, as yet, no cure. This research will advance the basic knowledge of the mechanism of pathological ocular angiogenesis, and will likely lead to advances in treatment of such diseases. Diseases of ocular neovascularization are the leading cause of blindness in developed countries and there are limited therapeutic interventions. We hypothesize that matrix metalloproteinases modulate the anti-angiogenic activity of pigment-epithelium derived factor, and that this proteolytic activity contributes to a loss of balance between anti-angiogenic and pro-angiogenic factors in the eye leading to ocular neovascularization. Our research will advance the basic knowledge of the mechanism of pathological ocular angiogenesis, and will likely lead to advances in treatment of such diseases.