Metalloproteinase Expression in Corneal Wounds

描述（由申请人提供）：角膜新生血管（NV）是全球范围内致盲的主要原因。我们的研究目的是确定膜型基质金属蛋白酶（MT 1-MMP）的活性及其蛋白水解功能在角膜NV中的作用。我们的实验室已经发现每细胞MT 1-MMP激活proMMP-2，降解胶原，并导致角膜基质成纤维细胞中血管内皮生长因子（VEGF）上调。我们还证明了成纤维细胞生长因子-2（FGF-2）上调MT 1-MMP，并且MT 1-MMP和FGF-2对基质成纤维细胞中VEGF的上调具有协同作用。我们进行了额外的实验，显示来源于培养的基质成纤维细胞的外泌体富含活性MT 1-MMP。我们推测FGF-2上调基质成纤维细胞中MT 1-MMP导致位于迁移基质成纤维细胞前缘的膜相关活性。我们进一步假设基质成纤维细胞相关的活性MT 1-MMP通过两种潜在的机制促进角膜新生血管形成：（i）通过与FGF-2/FGFR 1的协同活性上调VEGF和（ii）通过基质成纤维细胞系留或外泌体系留的MT 1-MMP分解细胞外基质。本研究将从多个方面验证我们的假设：目的A）确定MT 1-MMP酶活性在体内的时空定位以及对FGF-2/FGFR 1活化的响应：目的B）确定MT 1- MMP酶活性对MT 1-MMP诱导的基质成纤维细胞VEGF上调的必要性;目的C）检测基质成纤维细胞膜和外来体相关的MT 1-MMP对胶原降解和血管生成的酶活性。MT 1- MMP活性在角膜血管生成中的空间和时间关系的表征和两种提出的角膜NV机制的评价对于确定角膜NV治疗中的治疗干预的潜在靶点将是有价值的。 公共卫生相关性：MT 1-MMP酶活性在角膜血运重建中起重要作用。VEGF的转录上调和细胞周/跨细胞ECM降解可能是酶活性MT 1-MMP促进血管生成的两种不同机制。

DESCRIPTION (provided by applicant): Corneal neovascularization (NV) is a major cause of blindness worldwide. Our research objective is to identify the role of membrane type 1 matrix metalloproteinase (MT1-MMP) activity and its proteolytic functions in corneal NV. Our laboratory has found that per cellular MT1-MMP activates proMMP-2, degrades collagen, and results in vascular endothelial growth factor (VEGF) upregulation in corneal stromal fibroblasts. We also have demonstrated that fibroblast growth factor-2 (FGF-2) upregulates MT1-MMP and that MT1-MMP and FGF-2 have a synergistic effect on VEGF upregulation in stromal fibroblasts. We performed additional experiments showing that exosomes derived from cultured stromal fibroblasts are rich in active MT1-MMP. We hypothesize that FGF-2 upregulation of MT1-MMP in stromal fibroblasts results in membrane-associated activity localized at the leading edge of migrating stromal fibroblasts. We further hypothesize that stromal fibroblast-associated active MT1-MMP promotes corneal neovascularization through two potential mechanisms: (i) upregulation of VEGF by synergistic activity with FGF-2/FGFR1 and (ii) breakdown of the extracellular matrix by stromal fibroblast-tethered or exosome-tethered MT1-MMP. The proposed research will test various aspects of our hypotheses: Aim A) determine the spatio-temporal localization of MT1-MMP enzymatic activity in vivo and in response to FGF-2/FGFR1 activation; Aim B) determine the necessity of MT1- MMP enzymatic activity on MT1-MMP-induced upregulation of VEGF in stromal fibroblasts; and, Aim C) examine the enzymatic activity of stromal fibroblast membrane- and exosome-associated MT1-MMP on collagen degradation and angiogenesis. Characterization of the spatial and temporal relationships of MT1- MMP activity in corneal angiogenesis and evaluation of the two proposed mechanisms of corneal NV will be valuable for identifying potential targets for therapeutic intervention in the treatment of corneal NV. PUBLIC HEALTH RELEVANCE: MT1-MMP enzymatic activity plays an important role in corneal revascularization. Transcriptional up regulation of VEGF and pericellular/transcellular ECM degradation may be two distinct mechanisms by which enzymatically active MT1-MMP promotes angiogenesis.