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

基本信息

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
项目类别：
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

项目摘要

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.

角膜新血管形成大大增加了角膜移植排斥的风险，因此有助于严重的视力丧失，内嗜性的风险和威胁生命的脑膜炎。它折磨了140万个新的患者每年及其其他病理血管生成是失明的主要原因美国。血管生成还导致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，但目前仅针对VEGF 眼睛中的抗血管生成疗法。 CMG2是一种整联蛋白样的跨膜蛋白，细胞外结合ECM蛋白的结构域和无同源性的细胞内尾巴与已知功能的域。我们找到通过蛋白抑制剂通过，CMG2结合小分子或CMG2敲除靶向CMG2 深刻抑制角膜新血管形成，但是这种作用的基础机制尚不清楚。血管生成需要内皮细胞向生长因子迁移。这个迁移都有一个运动成分（运动=趋化因子）和方向成分（趋化性）。但是，这些无法使用传统（伤口刮擦或transwell）测定法进行区分组件。使用微流体随着时间的推移，我们最近发现CMG2完全靶向了单个单元的迁移测定法破坏趋化性，但不会破坏趋化因子。有多种生长因子观察到这种效果（BFGF，VEGF，迄今为止，PDGF）和所有靶向方法（CRISPR敲除，PASSSR，阻止肽）。因此，我们假设CMG2是生长因素诱导趋化性和有效的血管生成。我们将通过：1）确定所需的细胞内相互作用来检验此假设 CMG2介导的趋化性； 2）确定CMG2介导的趋化性所需的细胞外相互作用响应生长因子和3）评估RhoA对CMG2定向趋化性的贡献。我们的CMG2信号的工作模型基于我们实验室的初步数据，其他指示该CMG2位于Rhoa和几个Rho Pathway成员附近。因此，CMG2定位为直接调节定向迁移所需的细胞极性（趋化性）。确实，我们观察到抑制 RhoA表型CMG2抑制。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是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基础机制观察到的CMG2靶向体内的效果，并加速对这种潜在目标的剥削谱抗血管生成疗法。此外，这项工作将使药效学的发展迅速评估药物铅的测定。最后，该提案的关键方面旨在产生可能治疗铅。这些研究引起的药物可以补充抗VEGF疗法以治疗失明由眼部新血管形成以及许多其他血管生成依赖性疾病引起。