Endothelial Cytoprotective Signaling by Activated Protein C/Protease-activated Receptor-1

激活蛋白 C/蛋白酶激活受体 1 的内皮细胞保护信号传导

• 批准号: 10594367
• 负责人: Joann Trejo
• 金额: $ 60.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594367
• 关键词: ARRB1 gene; ARRB2; Agonist; Anti-Inflammatory Agents; Anticoagulants; Apoptosis; Apoptotic; Arginine; Binding; CRISPR/Cas technology; Caveolae; Cell membrane; Cells; Cytoprotection; Endothelial Cells; Endothelium; Exhibits; Future; G-Protein-Coupled Receptors; GRK5 gene; GTP-Binding Proteins; Goals; Heterotrimeric GTP-Binding Proteins; Human; In Vitro; Inflammation; Inflammatory Response; Injury; Knock-out; Ligands; Mediating; Mediator; Membrane; Membrane Microdomains; Morbidity - disease rate; N-terminal; PAR-1 Receptor; Pathogenesis; Pathologic; Pathway interactions; Peptide Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Research; SPHK1 enzyme; Sepsis; Signal Induction; Signal Pathway; Signal Transduction; Site; Specificity; Sphingosine-1-Phosphate Receptor; System; Testing; Therapeutic; Thrombin; Transducers; Vascular Diseases; activated Protein C; beta-arrestin; caveolin 1; desensitization; endothelial dysfunction; improved; in vivo; innovation; insight; mortality; new therapeutic target; novel; pharmacologic; preclinical study; receptor; resilience; response; therapeutic development

Summary/Abstract There are currently limited treatment options for improving endothelial dysfunction in vascular diseases such as sepsis, resulting in high morbidity and mortality. Endothelial dysfunction results in endothelial cell activation, disruption of endothelial barrier function and sensitivity to apoptosis. The long-term goal of this proposal is to delineate the pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated protein C (APC) is a promising therapeutic and exhibits multiple beneficial effects including stabilization of endothelial barriers and anti-apoptotic activities. Protease- activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the central mediator of APC cellular signaling, which requires caveolin-1 (Cav1) and compartmentalization in caveolae. We discovered that APC- activated PAR1 signals primarily through b-arrestin-2 (b-arr2) to promote endothelial barrier protection, and not heterotrimeric G proteins like thrombin (Th)-activated PAR1. The overall objective of this proposal is to develop a mechanistic understanding of how APC/PAR1 generates b-arr2 transducer bias to promote endothelial cytoprotection. We hypothesize that distinct GRK5 determinants and co-receptors facilitate APC/PAR1-induced b-arr2 transducer bias to promote endothelial cytoprotection through pathways enabled by Cav1 phosphorylation. We propose three specific aims. Aim 1: To delineate the mechanisms that enable GRK5 to distinctly regulate APC- vs. Th-induced biased signaling. GRK5 is required for APC-stimulated signaling and desensitization of Th-induced signaling. However, the mechanisms that enable distinct GRK5 functions is not known. We will determine if distinct GRK5 functions are regulated by localization to discrete plasma membrane microdomains such as caveolae using human cultured endothelial cells, a native system that permits the study of endogenous PAR1 and GRK5 and HEK293 CRISPR/Cas9 knockout cells. Aim 2: To determine the mechanisms by which APC vs. thrombin control b-arrestin transducer bias. It is not known how b-arrestin transducer bias (signaling vs. desensitization) is induced by APC- vs. Th-activated PAR1 nor how APC/PAR1 promotes two distinct b-arr2-mediated cytoprotective signaling pathways: dishevelled2 (Dvl2)-Rac1 controls endothelial barrier protection whereas sphingosine kinase 1 (SphK1)-Akt regulates anti-apoptotic activities. We will determine if distinct determinants of b-arrestin and GPCR co-receptors control different b-arr2 binding modes and functions induced by APC vs. thrombin. Aim 3: To define the mechanisms by which APC/PAR1 regulates Cav1 function to promote cytoprotection. APC/PAR1 stimulates Cav1 phosphorylation but how this modulates Cav1 function and is integrated into the cytoprotective pathway is not known and will be determined. The proposed research is innovative because it will test novel hypotheses to explain how GRK5, b-arr2 and Cav1 drive APC/PAR1 endothelial cytoprotective responses in a physiologically relevant context and will help advance the status of new targets as a future therapeutics for the treatment of endothelial dysfunction.

摘要/摘要 目前有有限的治疗方法可改善血管疾病中的内皮功能障碍，例如 败血症，导致高发病率和死亡率。内皮功能障碍导致内皮细胞激活， 内皮屏障功能的破坏和对凋亡的敏感性。该提议的长期目标是 描述内皮可以抵抗伤害和破坏以促进进步的途径 用于治疗发展的新目标。活化的蛋白C（APC）是一种有前途的治疗性和展示 多种有益的效果，包括稳定内皮屏障和抗凋亡活性。蛋白酶 - 活化的受体1（PAR1），G蛋白偶联受体（GPCR），是APC细胞的中心介体 信号传导，需要小窝蛋白-1（Cav1）和小窝中的隔室化。我们发现APC- 激活的PAR1信号主要通过B-arrestin-2（B-ARR2）来促进内皮屏障保护，而不是 异三聚合物G蛋白如凝血酶（Th）激活的PAR1。该提议的总体目的是发展 对APC/PAR1如何产生B-arr2传感器偏置以促进内皮的机械理解 细胞保护。我们假设不同的GRK5决定因素和共受体促进了APC/PAR1诱导的 B-arr2传感器偏置通过CAV1启用的途径促进内皮细胞保护 磷酸化。我们提出了三个具体目标。目标1：描述使GRK5的机制 明显调节APC与TH诱导的偏置信号传导。 APC刺激的信号传导需要GRK5和 TH诱导的信号传导的脱敏。但是，启用不同GRK5功能的机制不是 已知。我们将确定是否通过定位来调节不同的GRK5函数到离散的质膜 使用人培养的内皮细胞的微域，例如小窝，这是一种允许研究的天然系统 内源性PAR1和GRK5和HEK293 CRISPR/CAS9基因敲除细胞。目标2：确定 APC与凝血酶控制B- arterstin传感器偏置的机制。尚不知道B-arrestin 传感器偏置（信号传导与脱敏）由APC与TH激活的PAR1诱导，也不是APC/PAR1如何 促进两个不同的B-arr2介导的细胞保护信号通路：DISHEVELLED2（DVL2）-RAC1控制 内皮屏障保护，而鞘氨醇激酶1（SPHK1）-AKT调节抗凋亡活动。我们 将确定B-arrestin和GPCR共受体的不同决定因素是否控制不同的B-arr2结合模式 和APC与凝血酶引起的功能。目标3：定义APC/PAR1调节的机制 CAV1功能以促进细胞保护作用。 APC/PAR1刺激CAV1磷酸化，但如何调节 CAV1功能并集成到细胞保护途径中，并将确定。这 拟议的研究具有创新性，因为它将检验新的假设，以解释GRK5，B-ARR2和CAV1如何 在生理上相关的环境中驱动APC/PAR1内皮细胞保护反应，并将有助于进步 新靶标作为治疗内皮功能障碍的未来治疗剂的地位。