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: 10816153
• 负责人: Joann Trejo
• 金额: $ 4.98万
• 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: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10816153
• 关键词: Anti-Inflammatory Agents; Apoptosis; Apoptotic; Arr2; Arrestins; Binding; CRISPR/Cas technology; Caveolae; Cell membrane; Cells; Cytoprotection; Endothelial Cells; Endothelium; Exhibits; Future; G-Protein-Coupled Receptors; GRK5 gene; Goals; Human; Inflammatory Response; Injury; Knock-out; Mediating; Mediator; Membrane Microdomains; Morbidity - disease rate; PAR-1 Receptor; Pathway interactions; Phosphorylation; Physiological; Research; SPHK1 enzyme; Sepsis; Signal Induction; Signal Pathway; Signal Transduction; System; Testing; Therapeutic; Thrombin; Transducers; Vascular Diseases; activated Protein C; arrestin 2; caveolin 1; desensitization; endothelial dysfunction; improved; innovation; mortality; new therapeutic target; novel; receptor; 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 -arrestin-2 (-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 -arr2 transducer bias to promote endothelial cytoprotection. We hypothesize that distinct GRK5 determinants and co-receptors facilitate APC/PAR1-induced -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 are 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 -arrestin transducer bias. It is not known how -arrestin transducer bias (signaling vs. desensitization) is induced by APC- vs. Th-activated PAR1 nor how APC/PAR1 promotes two distinct -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 -arrestin and GPCR co-receptors control different -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, -arr2 and Cav1 drive APC/PAR1 endothelial cytoprotective responses in a physiologically relevant context and will help advance the status of new targets as future therapeutics for the treatment of endothelial dysfunction.

摘要/摘要 目前改善血管疾病内皮功能障碍的治疗选择有限，例如 脓毒症，导致高发病率和死亡率。内皮功能障碍导致内皮细胞活化， 该提案的长期目标是破坏内皮屏障功能和细胞凋亡敏感性。 描绘内皮细胞抵抗损伤和破坏以促进进展的途径 活性蛋白 C (APC) 是一种很有前途的治疗方法，并且具有一定的治疗开发新靶点。 多种有益作用，包括稳定内皮屏障和抗凋亡活性。 激活受体-1 (PAR1) 是一种 G 蛋白偶联受体 (GPCR)，是 APC 细胞的中心介质 信号传导，需要 Caveolin-1 (Cav1) 和细胞膜穴区室化，我们发现 APC-。 主要通过 -arrestin-2 (-arr2) 激活 PAR1 信号以促进内皮屏障保护，而不是 该提案的总体目标是开发异三聚体 G 蛋白，例如凝血酶 (Th) 激活的 PAR1。 对 APC/PAR1 如何产生 -arr2 传感器偏压以促进内皮细胞的机制的理解 我们追求不同的 GRK5 决定簇和共受体促进 APC/PAR1 诱导。 -arr2 传感器偏向通过 Cav1 启用的途径促进内皮细胞保护 我们提出了三个具体目标 目标 1：描述使 GRK5 能够磷酸化的机制。 明显调节 APC 与 Th 诱导的偏向信号传导，GRK5 是 APC 刺激信号传导所必需的。 然而，实现不同 GRK5 功能的机制却并非如此。 我们将确定不同的 GRK5 功能是否通过定位到离散的质膜来调节。 使用人类培养的内皮细胞（一种允许进行研究的天然系统）进行微结构域（例如小窝） 目的 2：确定内源性 PAR1 和 GRK5 以及 HEK293 CRISPR/Cas9 敲除细胞。 APC 与凝血酶控制 -arrestin 传感器偏差的机制尚不清楚 -arrestin 是如何实现的。 传感器偏差（信号转导与脱敏）是由 APC 与 Th 激活的 PAR1 以及 APC/PAR1 如何引起的 促进两种不同的 -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 调节抗凋亡活性。 将确定 -arrestin 和 GPCR 共受体的不同决定簇是否控制不同的 -arr2 结合模式 目标 3：确定 APC/PAR1 的调节机制。 Cav1 的功能是促进细胞保护，APC/PAR1 会刺激 Cav1 磷酸化，但它是如何调节的。 Cav1 的功能以及是否整合到细胞保护途径中尚不清楚，并将确定。 拟议的研究具有创新性，因为它将测试新的假设来解释 GRK5、-arr2 和 Cav1 如何 在生理相关环境中驱动 APC/PAR1 内皮细胞保护反应，并将有助于推进 作为治疗内皮功能障碍的未来疗法的新靶点的地位。