Dual role of HMGB1 in pathogenic platelet biology in pulmonary hypertension

项目摘要 肺动脉高压（PH）是一种以肺动脉压升高和异常为特征的致死性疾病 在血管生长中。虽然目前的疗法适度改善功能能力，但它们对长期的影响微乎其微。 长期生存，也许是因为他们忽视了发病机制。尽管有令人信服的证据表明血栓形成和炎症 在PH的机制，没有药物已经开发出针对这些机制。我们最近报道了高流动性 人和鼠PH中的HMGB 1升高，通过Toll样受体4促进发病 （TLR 4）依赖性机制。我们还报道了补体系统在PH中的作用。 补体级联或TLR 4诱导促炎、促粘附、促凝血表型，研究表明， TLR和补体系统之间的串扰。初步数据表明：（1）HMGB 1刺激TLR 4， 血小板从而导致PH，（2）在血小板TLR 4和补体系统之间存在串扰，和（3） 血小板是PH中HMGB 1的来源。这些数据构成了我们总体假设的基础，即HMGB 1依赖性 血小板TLR 4的活化引起血小板活化、分泌、血小板依赖性补体活化和HMGB 1 在目的1中，我们将描述HMGB 1对人和 小鼠血小板活化、分泌和聚集以及负责该作用的HMGB 1同种型。TLR4- 将探索HMGB 1刺激的血小板活化的依赖性。线粒体活性氧作为一个下游的作用， 研究HMGB 1/TLR 4依赖性血小板活化的信号转导机制。将WT血小板输注至 TLR 4-/-小鼠将证实血小板TLR 4在缺氧诱导的PH中的作用。 HMGB 1/TLR 4相互作用将进一步确定HMGB 1/TLR 4相互作用在PH中的作用 血小板TLR 4减弱缺氧诱导的PH中的补体激活。我们将测试血小板TLR 4激活的能力。 TLR 4激动剂活化补体，以及这些激动剂是否可通过补体成分增强血小板活化。 最后，我们将探讨TLR 4介导C3 a和C5 a依赖性血小板活化的机制。在目标3 我们将使用血小板特异性免疫荧光技术，探讨血小板释放的HMGB 1是否有助于PH的发病机制。 HMGB 1敲除小鼠。将WT血小板输回血小板特异性HMGB 1-/-小鼠将证实 血小板HMGB 1在缺氧诱导的PH中的表达。 患者以确定血小板是否在人类PAH中释放HMGB 1。我们的目标是确定血小板的病理生理学 和免疫系统异常的PH，并在这样做，应用知识，以改善PH患者的护理。

Project Abstract Pulmonary hypertension (PH) is a fatal disease characterized by increased pulmonary arterial pressure and abnormalities in blood vessel growth. While current therapies modestly improve functional capacity, they have marginal impact on long- term survival, perhaps because they neglect pathogenesis. Despite compelling evidence for thrombotic and inflammatory mechanisms in PH, no drugs have been developed to target these mechanisms. We recently reported that high mobility group box 1 (HMGB1) is elevated in human and murine PH contributing to the pathogenesis via toll like receptor 4 (TLR4)-dependent mechanisms. We also reported on the role of the complement system in PH. Activation of the complement cascade or TLR4 induces a pro-inflammatory, pro-adhesive, pro-coagulant phenotype and studies point to a cross talk between TLRs and the complement system. Preliminary data suggest that (1) HMGB1 stimulates TLR4 on platelets thus contributing to PH, (2) there is cross talk between platelet TLR4 and the complement system and (3) that platelets are a source of HMGB1 in PH. These data form the basis of our overarching hypothesis that HMGB1-dependent activation of platelet TLR4 causes platelet activation, secretion, platelet-dependent complement activation, and HMGB1 release thus contributing to the pathogenesis of PH. In Aim 1we will characterize the effect of HMGB1 on human and mouse platelet activation, secretion and aggregation as well as the HMGB1 isoform responsible for this effect. The TLR4- dependence of HMGB1-stimulated platelet activation will be explored. A role for mitochondrial ROS as a downstream signaling mechanism in HMGB1/TLR4-dependent platelet activation will be investigated. Transfusion of WT platelets into TLR4-/- mice will confirm a role for platelet TLR4 in hypoxia-induced PH. A novel HMGB1 inhibitor that specifically blocks HMGB1/TLR4 interaction will further identify the role of HMGB1/TLR4 interactions in PH. In aim 2 we will examine if loss of platelet TLR4 attenuates complement activation in hypoxia-induced PH. We will test the ability of platelets activated by TLR4 agonists to activate complement and if these agonists can enhance platelet activation by complement components. Finally, we will explore the mechanism(s) by which TLR4 mediates C3a and C5a-dependent platelet activation. In Aim 3 we will explore whether HMGB1 released from platelets contributes to the pathogenesis of PH using platelet specific HMGB1 knockout mice. Transfusion of WT platelets back into platelet specific HMGB1-/- mice will confirm a role for platelet HMGB1 in hypoxia-induced PH. Intracellular and platelet surface HMGB1 levels will be measured in platelets from patients to determine if platelets release HMGB1 in human PAH. It is our goal to define the pathophysiology of the platelet and immune system abnormalities in PH, and in so doing, apply the knowledge to improve the care of PH patients.