Platelet Metabolic Stress Induces Thrombo-Inflammation to Drive Endothelial Dysfunction in PH

PH 中血小板代谢应激诱导血栓炎症导致内皮功能障碍

基本信息

批准号：
10736724
负责人：
Sruti Shiva
金额：
$ 72.14万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10736724
关键词：
Alpha Granule Animal Model Anticoagulants Attenuated Biochemical Blood Platelets Blood Vessels CASP1 gene Cell Proliferation Cells Chimeric Proteins Clinical Trials Coculture Techniques Complex Data Development Disease Endothelial Cells Endothelium Event Fatty Acids Functional disorder Genetic Guanosine Triphosphate Phosphohydrolases Human Hypoxia Inflammasome Inflammation Inflammation Mediators Inflammatory Injury Interleukin-1 beta Knockout Mice Label Leukocytes Link Lung Lung diseases Measures Mediating Mediator Metabolic Metabolic dysfunction Metabolic stress Metabolism Mitochondria Modeling Mus NADPH Oxidase 1 Natural Immunity Oxidants Oxidation-Reduction P-Selectin Pathogenesis Pathology Pathway interactions Patients Plasma Platelet Activation Production Proliferating Pulmonary Vascular Resistance Rodent Model Role Scheme Signal Transduction Surface Testing Thrombocytopenia Transgenic Organisms Up-Regulation Vascular remodeling Wild Type Mouse antagonist cell injury efficacy testing endothelial dysfunction experimental study fatty acid oxidation hypertension treatment in vivo interleukin-1beta-converting enzyme inhibitor lipidomics lung imaging mitochondrial dysfunction mouse model multiphoton imaging neutrophil novel pulmonary arterial hypertension pulmonary arterial pressure pulmonary artery endothelial cell pulmonary vascular remodeling receptor right ventricular failure targeted treatment therapeutic target therapy development thromboinflammation thrombotic vascular inflammation

项目摘要

Pulmonary arterial hypertension (PAH) is characterized by increased mean pulmonary artery pressure, that leads to vascular remodeling and right heart failure. While endothelial cell (EC) dysfunction drives vascular remodeling, the events that instigate EC injury are unclear. Platelets circulate proximal to ECs and experimental platelet depletion has been shown to attenuate pathogenesis in PAH models. However, the mechanisms responsible for this effect remain elusive. Notably, platelets are highly metabolically active and mediate vascular inflammation, but these functions have not been fully considered in PAH. We showed that platelets from PAH patients display metabolic dysfunction with increased fatty acid oxidation (FAO) and mitochondrial oxidants (mtROS). Preliminary data indicate this dysfunction is caused by upregulation of the mitochondrial GTPase and mitofusin-1 (MFN-1), stimulating activation of the NLRP3-dependent inflammasome. Cleavage of caspase-1, an essential component of the inflammasome, leads to secretion of interleukin-1β (IL-1 β), platelet degranulation, and surface expression of p-selectin (collectively labeled thrombo-inflammation). New data demonstrate that these platelet-dependent events trigger EC oxidant production (via NADPH oxidase-1; NOX1) and EC proliferation. Hypothesis: Platelet MFN1-dependent mitochondrial dysfunction triggers inflammasome mediated thrombo-inflammation, which leads to EC dysfunction in PAH. Aim 1: Determine how MFN1-dependent platelet mitochondrial dysfunction triggers inflammasome activation. Using biochemical measures in PAH patients’ platelets, we will link mitochondrial dysfunction to inflammasome activation. We will induce PAH in transgenic murine models of platelet-specific MFN1 and caspase-1 silencing, supplemented with lipidomics and redox modulation, to determine the mechanism by which FAO and mtROS triggers inflammasome activation. PAH will be induced in a novel secretome mouse to define vasoactive molecules released by platelet degranulation in PAH. Aim 2: Determine the mechanism(s) by which platelet-driven thrombo-inflammatory signaling causes EC dysfunction in PAH. Using human/murine platelet-EC co-cultures, we will characterize platelet-driven EC NOX1 activation and proliferation. Using antagonists to signaling mediated by IL-1β and platelet granule factors identified in Aim 1, we will determine the platelet-dependent signaling axis that causes EC dysfunction. The contribution of p-selectin dependent platelet-neutrophil aggregates will also be tested. We will utilize EC-specific NOX1 knockout mice to test the role of platelet-mediated NOX1 activation in PAH. Aim 3: Determine if VX-765, which inhibits caspase-1 to block inflammasome activation, and attenuates PAH pathogenesis. We will test the efficacy of VX-765 (in clinical trials for non-PAH pathologies) in rodent models of PAH. These studies will uncover a novel intra-platelet to EC signaling axis that defines the role of platelets in PAH pathogenesis and reveals potential therapeutic targets.

肺动脉高压（PAH）的特点是平均肺动脉压升高，导致血管重塑和右心衰竭虽然内皮细胞（EC）功能障碍驱动血管重塑，引起 EC 损伤的事件尚不清楚。研究表明，PAH 模型中的耗竭可以减轻发病机制，但其机制仍不清楚。这种效应仍然难以捉摸，值得注意的是，血小板具有高度代谢活性并介导血管炎症，但这些功能在 PAH 中尚未得到充分考虑。我们发现 PAH 患者的血小板显示出这些功能。脂肪酸氧化（FAO）和线粒体氧化剂（mtROS）增加导致代谢功能障碍。数据表明这种功能障碍是由线粒体 GTP 酶和线粒体融合蛋白-1 (MFN-1) 上调引起的，刺激 NLRP3 依赖性炎症小体的激活，裂解重要成分 caspase-1。炎症小体的作用，导致白细胞介素 1β (IL-1β) 的分泌、血小板脱颗粒和表面表达 p-选择素（统称为血栓炎症）的新数据表明，这些血小板依赖性。事件触发 EC 氧化剂产生（通过 NADPH 氧化酶-1；NOX1）和 EC 增殖。 MFN1 依赖性线粒体功能障碍会引发炎症小体介导的血栓炎症，目标 1：确定 MFN1 依赖性血小板线粒体如何导致 EC 功能障碍。我们通过对 PAH 患者血小板进行生化检测，发现功能障碍会引发炎症小体激活。将线粒体功能障碍与炎症小体激活联系起来我们将在转基因小鼠模型中诱导 PAH。血小板特异性 MFN1 和 caspase-1 沉默，辅以脂质组学和氧化还原调节，确定FAO和mtROS触发PAH炎症体激活的机制。一种新型分泌组小鼠，用于定义 PAH 中血小板脱颗粒释放的血管活性分子。确定血小板驱动的血栓炎症信号传导引起 EC 的机制使用人/鼠血小板-EC 共培养，我们将表征血小板驱动的 EC NOX1。使用 IL-1β 和血小板颗粒因子介导的信号传导拮抗剂在目标 1 中确定后，我们将确定导致 EC 功能障碍的血小板依赖性信号轴。还将测试 p-选择素依赖性血小板中性粒细胞聚集体的贡献，我们将利用 EC 特异性。 NOX1 敲除小鼠测试血小板介导的 NOX1 激活在 PAH 中的作用目标 3：确定 VX-765 是否有效。它抑制 caspase-1 以阻止炎症小体激活，并减弱 PAH 发病机制。这些研究测试了 VX-765（在非 PAH 病理学临床试验中）在 PAH 啮齿动物模型中的功效。将揭示一种新的血小板内 EC 信号轴，该轴定义了血小板在 PAH 发病机制中的作用，揭示潜在的治疗靶点。