Functional requirement of CMG2 for endothelial cell chemotaxis and resulting angiogenesis

CMG2 内皮细胞趋化性和由此产生的血管生成的功能要求

• 批准号: 10522258
• 负责人: MICHAEL SEAN ROGERS
• 金额: $ 41万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522258
• 关键词: ANTXR2 gene; Affect; Angiogenesis Inhibitors; Arthritis; Behavior; Binding; Biological Assay; Blindness; Blood capillaries; Bypass; Cardiovascular Diseases; Cell Polarity; Cell Surface Receptors; Cell surface; Cells; Cellular biology; Chemotaxis; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Cornea; Corneal Neovascularization; Dangerousness; Data; Defect; Development; Diabetic Retinopathy; Disease; Drug Targeting; Endophthalmitis; Endothelial Cells; Exhibits; Extracellular Domain; Extracellular Matrix; Eye; FDA approved; FGF2 gene; Genes; Genetic; Graft Rejection; Growth Factor; Health; Heart Diseases; Human; Individual; Integral Membrane Protein; Integrins; Keratoplasty; Knock-out; Life; Ligands; Macular degeneration; Malignant Neoplasms; Mediating; Mediator of activation protein; Meningitis; Methods; Microfluidics; Migration Assay; Modeling; Morphogenesis; Movement; Mus; Mutagenesis; Mutation; Neovascular Glaucoma; Ocular Pathology; Pain; Pathologic Neovascularization; Pathology; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phenocopy; Physiologic Neovascularization; Platelet-Derived Growth Factor; Positioning Attribute; Process; Proteins; Proteomics; Retinopathy of Prematurity; Risk; Role; Signal Pathway; Signal Transduction; Solid; Source; Sphingosine-1-Phosphate Receptor; Tail; Testing; Therapeutic; Time; Travel; United States; Vascular Endothelial Growth Factors; Vision; Western Blotting; Work; angiogenesis; anthrax protective factor; base; bevacizumab; cell motility; cellular imaging; confocal imaging; corneal epithelium; design; experimental study; extracellular; in vivo; infection risk; inhibitor; member; migration; mutant; novel; ocular neovascularization; off-label use; receptor; response; rho; side effect; small molecule; targeted treatment; therapeutic target; vessel regression; wound

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.

角膜新生血管大大增加了角膜移植排斥反应的风险，因此有助于 严重的视力丧失、眼内炎的风险和危及生命的脑膜炎。它折磨着多达140万新 患者每年，并与其他病理性血管生成是致盲的主要原因， 美国的血管生成也有助于从癌症到关节炎的疾病。广泛的 蛋白质生长因子（例如VEGF、bFGF、PDGF）刺激血管生成，但目前仅VEGF是靶向的 用于眼部的抗血管生成治疗。CMG 2是一种整合素样跨膜蛋白，具有细胞外膜结构。 结合ECM蛋白的结构域和与已知功能结构域不同源的细胞内尾部。我们 发现通过蛋白质抑制剂PASSSR、CMG 2结合小分子或CMG 2敲除靶向CMG 2 显著抑制角膜新生血管形成，但其作用机制尚不清楚。 血管生成需要内皮细胞向生长因子迁移。这种迁移既有 运动成分（运动性=化学运动）和方向成分（趋化性）。但这些 使用传统的（伤口划痕或transwell）分析不能区分组分。使用微流体 随着时间的推移跟踪单个细胞的迁移试验，我们最近发现CMG 2完全靶向 破坏趋化性但不破坏趋化作用。多种生长因子（bFGF、VEGF， PDGF）和迄今为止尝试的所有靶向方法（CRISPR敲除、PASSSR、阻断肽）。因此我们 假设CMG 2是生长因子诱导趋化性所需途径中的关键中间体， 有效的血管生成。我们将通过以下方式来检验这一假设：1）确定细胞内相互作用所需的 CMG 2介导的趋化性; 2）鉴定CMG 2介导的趋化性所需的细胞外相互作用 3）评估RhoA对CMG 2定向趋化性的贡献。 我们的CMG 2信号传导的工作模型是基于我们实验室和其他人的初步数据，这些数据表明 CMG 2定位于RhoA和几个Rho通路成员附近。因此，CMG 2被定位为直接 调节定向迁移（趋化性）所需的细胞极性。我们观察到， RhoA表型模仿CMG 2抑制。最后，我们可以通过S1 P激活RhoA来绕过CMG 2信号传导。 受体，使得趋化性不再对CMG 2靶向敏感。因此，RhoA位于CMG 2的下游。 成功完成拟议的工作将确定强抗血管生成的潜在机制， 在体内靶向CMG 2时观察到的作用，并加速了对这种潜在靶点的广泛利用。 光谱抗血管生成疗法。此外，这项工作将使药效学的发展 用于快速评估药物先导物的分析。最后，本提案的关键方面旨在产生可能的 治疗引导。从这些研究中产生的药物可以补充抗VEGF疗法来治疗失明 由眼部新生血管形成以及许多其它血管生成依赖性疾病引起。