Functional requirement of CMG2 for endothelial cell chemotaxis and resulting angiogenesis

角膜新生血管极大地增加了角膜移植排斥反应的风险，从而导致 严重的视力丧失、眼内炎和危及生命的脑膜炎。它困扰着多达140万名新患者 每年的患者和其他病理性血管生成是导致失明的主要原因 美国。血管生成也会导致从癌症到关节炎的各种疾病。范围广泛的 蛋白质生长因子(如血管内皮生长因子、碱性成纤维细胞生长因子、血小板衍生生长因子)刺激血管生成，但目前仅以血管内皮生长因子为靶点 用于眼部抗血管生成治疗。CMG2是一种整合素样跨膜蛋白，具有细胞外 与ECM蛋白和细胞内尾巴结合的结构域，与已知功能的结构域没有同源性。我们 发现通过蛋白抑制剂PASSSR、CMG2结合的小分子或CMG2基因敲除来靶向CMG2 深刻地抑制角膜新生血管，但其机制尚不清楚。 血管生成需要内皮细胞向生长因子迁移。此迁移既有 运动成分(运动性=趋化性)和方向性成分(趋化性)。然而，这些 使用传统的(创伤、划痕或井间)分析不能区分成分。使用微流控芯片 随着时间的推移跟踪单个细胞的迁移试验，我们最近发现CMG2完全靶向 会破坏趋化作用，但不会影响趋化作用。多种生长因子(碱性成纤维细胞生长因子、血管内皮生长因子、 PDGF)和迄今为止尝试过的所有靶向方法(CRISPR基因敲除、PASSSR、封闭肽)。因此，我们 假设CMG2是生长因子诱导趋化所需的途径中的关键中介 高效的血管生成。我们将通过以下方式检验这一假设：1)确定细胞内的相互作用 CMG2介导的趋化作用；2)鉴定CMG2介导的趋化作用所需的细胞外相互作用 3)评价RhoA对CMG2定向趋化的贡献。 我们的CMG2信令工作模型基于我们实验室和其他机构的初步数据，这些数据表明 CMG2定位于RhoA和几个Rho通路成员附近。因此，CMG2定位为直接 调节定向迁移(趋化)所需的细胞极性。事实上，我们观察到，抑制 RhoA对CMG2有抑制作用。最后，我们可以通过S1P激活RhoA来绕过CMG2信号 受体，因此趋化性不再对CMG2靶向敏感。因此，RhoA位于CMG2的下游。 成功完成拟议的工作将确定强大的抗血管生成的机制 观察CMG2在体内的靶向作用，并加速这一潜在靶点的开发 光谱抗血管生成治疗。此外，这项工作将使药效学的发展成为可能。 快速评估药物先导的化验。最后，这项提案的关键方面旨在产生可能的 治疗线索。这些研究产生的药物可以补充抗血管内皮生长因子疗法来治疗失明 由眼部新生血管以及许多其他依赖血管生成的疾病引起。

Corneal neovascularization greatly increases the risk for corneal graft rejection, and thus contributes to severe vision loss, risk of endophthalmitis, and life-threatening meningitis. It afflicts up to 1.4 million new patients annually and together with other pathological angiogenesis is the leading cause of blindness in the United States. Angiogenesis also contributes to diseases that range from cancer to arthritis. A wide range of protein growth factors (e.g. VEGF, bFGF, PDGF) stimulate angiogenesis, but only VEGF is currently targeted for antiangiogenic therapy in the eye. CMG2 is an integrin-like transmembrane protein with an extracellular domain that binds ECM proteins and an intracellular tail without homology to domains of known function. We find that targeting CMG2 via the protein inhibitor PASSSR, CMG2-binding small molecules, or CMG2 knockout profoundly inhibits corneal neovascularization, but the mechanism underlying this effect is unknown. Angiogenesis requires endothelial cells to migrate towards growth factors. This migration has both a movement component (motility = chemokinesis) and a directional component (chemotaxis). However, these components cannot be distinguished using traditional (wound scratch or transwell) assays. Using a microfluidic migration assay that tracks individual cells over time, we recently discovered that CMG2 targeting completely disrupts chemotaxis, but not chemokinesis. This effect is observed with multiple growth factors (bFGF, VEGF, PDGF) and all targeting methods tried thus far (CRISPR knockout, PASSSR, blocking peptide). Thus, we hypothesize that CMG2 is a key intermediary in a pathway required for growth-factor induced chemotaxis and efficient angiogenesis. We will test this hypothesis by: 1) identifying intracellular interactions required for CMG2-mediated chemotaxis; 2) identifying extracellular interactions required for CMG2-mediated chemotaxis in response to growth factors, and 3) evaluating the contribution of RhoA to CMG2-directed chemotaxis. Our working model of CMG2 signaling is based on preliminary data from our lab and others that indicates that CMG2 localizes near RhoA and several Rho pathway members. Thus, CMG2 is positioned to directly regulate the cell polarity required for directional migration (chemotaxis). Indeed, we observe that inhibiting RhoA phenocopies CMG2 inhibition. Finally, we can bypass CMG2 signaling by activating RhoA via the S1P receptor, so that chemotaxis is no longer sensitive to CMG2 targeting. Thus, RhoA is downstream of CMG2. Successful completion of proposed work will identify the mechanism underlying the strong antiangiogenic effects observed upon CMG2 targeting in vivo and accelerate exploitation of this potential target for broad- spectrum antiangiogenic therapy. In addition, this work will enable the development of pharmacodynamic assays to rapidly evaluate drug leads. Finally, key aspects of this proposal are designed to produce possible therapeutic leads. Drugs arising from these studies could supplement anti-VEGF therapies to treat blindness caused by ocular neovascularization as well as many other angiogenesis-dependent diseases.