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，这种蛋白分解活性导致眼内抗血管生成因子和促血管生成因子之间失去平衡，导致眼部新生血管。在患有病理性血管生成疾病的眼睛中，PEDF蛋白水平降低，而在这些疾病组织中发现基质金属蛋白酶活性上调。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，在N-末端附近的抗血管生成区域和C-末端附近的丝氨酸反应中心环内，蛋白酶MMP-9裂解PEDF。被基质金属蛋白酶-9切割的PEDF消除了在小鼠主动脉环实验中测得的PEDF的抗血管生成活性，并将PEDF转化为内皮趋化因子。最后，用玻璃体内注射一种基质金属蛋白酶-2和-9抑制剂对小鼠眼睛进行预注射，可以使小鼠玻璃体内注射的PEDF的量增加三倍。在此，我们建议1)确定基质金属蛋白酶-2和-9靶向的PEDF内的特定序列；2)确定经基质金属蛋白酶处理的PEDF的分离片段的活性；以及3)通过在小鼠脉络膜新生血管模型中评估基质金属蛋白酶耐药突变的PEDF来检验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，在发达国家是导致失明的主要原因。这些疾病威胁着迅速增长的高危人口的前景，尽管手头有有限的治疗干预措施，但到目前为止还没有治愈的方法。这项研究将促进对病理性眼血管生成机制的基本认识，并可能导致此类疾病的治疗进展。在发达国家，眼部新生血管疾病是导致失明的主要原因，治疗措施有限。我们假设基质金属蛋白酶调节色素上皮源性因子的抗血管生成活性，这种蛋白分解活性导致眼内抗血管生成因子和促血管生成因子之间失去平衡，从而导致眼部新生血管。我们的研究将促进对病理性眼血管生成机制的基本认识，并可能导致此类疾病的治疗进展。

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.