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

肺动脉高压(PAH)的特征是平均肺动脉压升高，这导致 血管重塑和右心衰竭。虽然内皮细胞(EC)功能障碍会导致血管重构， 引发EC损伤的事件尚不清楚。内皮细胞和实验性血小板近端的血小板循环 在PAH模型中，耗竭已被证明可以减轻发病机制。然而，负责 这种效果仍然难以捉摸。值得注意的是，血小板是高度新陈代谢活跃的，并介导血管炎症， 但这些功能在PAH中还没有得到充分的考虑。我们显示了PAH患者的血小板显示 脂肪酸氧化(FAO)和线粒体氧化剂(MtROS)增加的代谢功能障碍。初步 数据表明，这种功能障碍是由线粒体GTP酶和丝裂原蛋白-1(MFN-1)上调引起的， 刺激依赖NLRP3的炎症体的激活。必需成分caspase-1的裂解 在炎症体中，导致白介素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)。新的数据表明，这些依赖于血小板的 事件触发EC氧化剂的产生(通过NADPH氧化酶-1；NOX1)和EC的增殖。假设：血小板 依赖Mfn1的线粒体功能障碍触发炎性小体介导的血栓炎症，这 导致PAH中的EC功能障碍。目的1：确定Mfn1依赖的血小板线粒体 功能障碍会触发炎症小体激活。通过对PAH患者的血小板进行生化检测，我们 会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，以 确定FAO和mtROS触发炎症小体激活的机制。多环芳烃将在 一种新的分泌组小鼠，用于确定PAH中血小板脱颗粒所释放的血管活性分子。目标2： 确定血小板驱动的血栓炎症信号引起EC的机制(S) PAH的功能障碍。利用人/鼠血小板-EC共培养，我们将表征血小板驱动的EC NOX1 激活和增殖。拮抗剂在IL-1β和血小板颗粒因子介导的信号转导中的应用 在目标1中，我们将确定导致EC功能障碍的血小板依赖信号轴。这个 P-选择素依赖的血小板-中性粒细胞聚集体的贡献也将被测试。我们将使用特定于EC的 NOX1基因敲除小鼠检测血小板介导的NOX1激活在PAH中的作用。目标3：确定VX-765， 其抑制caspase-1阻断炎性小体激活，减轻PAH的发病。我们会 测试VX-765(在非PAH病理的临床试验中)在PAH啮齿动物模型中的疗效。这些研究 将揭示一个新的血小板内到EC信号轴，它定义了血小板在PAH发病机制中的作用 揭示了潜在的治疗靶点。

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.