Metalloproteinase Expression in Corneal Wounds

描述(申请人提供)：角膜新生血管(NV)是世界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1(MT1-MMPs)活性及其蛋白分解功能在角膜新生血管形成中的作用。我们的实验室发现，在角膜基质成纤维细胞中，每个细胞的MT1-MMP2激活原MMP2，降解胶原，并导致血管内皮生长因子(VEGFs)的上调。我们还证实了成纤维细胞生长因子-2上调基质成纤维细胞中MT1-MMPs的表达，并且MT1-MMPs和FGF2对间质成纤维细胞中的VEGF表达有协同作用。我们还进行了其他实验，显示从培养的基质成纤维细胞衍生的外体富含活性的MT1-基质金属蛋白酶。我们推测，间质成纤维细胞中成纤维细胞生长因子-2上调MT1-MMP会导致膜相关活性定位于迁移的间质成纤维细胞的前沿。我们进一步假设基质成纤维细胞相关的活性MT1-MMP通过两种可能的机制促进角膜新生血管的形成：(I)通过与成纤维细胞/FGFR1的协同作用上调血管内皮生长因子，以及(Ii)基质成纤维细胞系留或外周体系留的MT1-MMP分解细胞外基质。这项研究将验证我们的假设的各个方面：目的A)确定MT1-MMP酶活性在体内的时空定位以及对FGF-2/FGFR1激活的响应；目的B)确定MT1-MMP酶活性在MT1-MMPs诱导的间质成纤维细胞中上调VEGF的必要性；以及目的C)检测间质成纤维细胞膜和外膜相关的MT1-MMP酶活性对胶原降解和血管生成的影响。研究MT1-基质金属蛋白酶活性在角膜新生血管形成中的时空关系，评价角膜新生血管形成的两种可能机制，将有助于确定角膜新生血管生成治疗干预的潜在靶点。 公共卫生相关性：MT1-MMP酶活性在角膜血运重建中起着重要作用。转录上调的血管内皮生长因子和细胞周围/跨细胞的ECM降解可能是酶活性的MT1-MMP促血管生成的两种不同的机制。

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.