Regulation of portal hypertension through neutrophil-platelet interactions in liver sinusoids

项目摘要 门静脉高压症(PHTN)是多种形式慢性肝病的常见终末通路。 在肝病患者中造成显著的发病率和死亡率。然而，有一种稀缺的 可用于改善PHTN的治疗方法。这项建议的总体目标是澄清 中性粒细胞和血小板在PHTN发病机制中的作用，其治疗目的是促进 降低门脉压力的疗法设计。PHTN的病理生理机制是复杂的，并且是受调节的 在多个水平上，包括窦内旁分泌信号，微血管血栓的形成，以及 内皮功能障碍。我们之前已经确定了中性粒细胞在 PHTN的发病机制。我们发现，充血性肝病(CH)造成的周期性拉伸 肝窦内皮细胞(LSECs)机械敏感压电通道的激活。压电体 通道激活机械分泌信号通路，最终导致中性粒细胞趋化作用的分泌 细胞因子CXCL1。CXCL1诱导中性粒细胞渗入肝窦。中性粒细胞与 导致中性粒细胞胞外陷阱或Net形成的血小板。我们发现基因和 药物抑制Net形成显著降低CH和CHD模型小鼠的门脉压力 PHTN.尽管在某些情况下，血小板参与了网络的形成，但其机制 招募血小板并调节其与中性粒细胞的相互作用以产生正弦网络需要进一步 调查。Webel-Palade小体(WPBs)是产生细胞外泡的内皮细胞器 (EVS)含有炎症和止血因子。WPB来源的EVS对血小板募集的影响 对肝窦的影响尚未见研究。我们已经提出了一个中心假设，即血小板被 WPB来源的EV与中性粒细胞表面CD11b/CD18整合素受体相互作用刺激网络形成 和PHTN。我们将通过以下独立但综合的具体目标来检验这一假设 在技术和概念上都是创新的。首先，我们将检验如下假设：Piezo通道 作为主要的机械传感器，其激活调节中性粒细胞和血小板的产生 调节门脉压力的趋化因子。我们认为，Piezo激活会产生血小板 白血球来源的EV中的趋化因子。最后，使用小鼠模型和临床- 相关形式的血小板抑制，我们将检验中性粒细胞整合素受体相互作用的假设 CD11b/CD18与血小板膜糖蛋白受体GPIBα共同驱动网状结构的形成。我们的建议意义重大 因为它有可能阐明新的治疗靶点，以更好地管理PHTN，这是一种毁灭性和 目前只有通过肝移植才能治愈的流行疾病。

PROJECT ABSTRACT Portal hypertension (PHTN) is a common final pathway of multiple forms of chronic liver disease which accounts for significant morbidity and mortality among patients with liver disease. However, there is a paucity of therapies available to ameliorate PHTN. The overall objective of this proposal is to elucidate the contribution of neutrophils and platelets to the pathogenesis of PHTN, with the therapeutic goal of facilitating the design of therapies to decrease portal pressure. The pathophysiology of PHTN is complex and is regulated at multiple levels, including paracrine signaling within sinusoids, formation of microvascular thrombosis, and endothelial dysfunction. We have previously identified a novel but critical role of neutrophils in the pathogenesis of PHTN. We found that cyclic stretch imposed by congestive hepatopathy (CH) induces activation of mechanosensitive Piezo channels within liver sinusoidal endothelial cells (LSECs). Piezo channels activate mechanocrine signaling pathways which culminate in secretion of the neutrophil chemotactic cytokine CXCL1. CXCL1 induces infiltration of neutrophils into liver sinusoids. Neutrophils form complexes with platelets which lead to the formation of neutrophil extracellular traps, or NETs. We found that genetic and pharmacologic inhibition of NET formation significantly decreases portal pressures in murine models of CH and PHTN. Although platelets have been implicated in NET formation in certain settings, the mechanisms which recruit platelets and regulate their interactions with neutrophils to generate sinusoidal NETs require further investigation. Weibel-Palade bodies (WPBs) are endothelial organelles which generate extracellular vesicles (EVs) containing inflammatory and hemostatic factors. The impact of WPB-derived EVs on platelet recruitment to liver sinusoids has not been studied. We have formulated the central hypothesis that platelets activated by WPB-derived EVs interact with the CD11b/CD18 integrin receptor on neutrophils to stimulate NET formation and PHTN. We will test this hypothesis through the following independent but integrated specific aims which are both technically and conceptually innovative. First, we will test the hypothesis that Piezo channels serve as master mechanosensors whose activation regulates the generation of both neutrophil- and platelet- chemotactic factors which modulate portal pressures. We propose that Piezo activation generates platelet chemotactic factors within EVs derived from WPBs in LSECs. Finally, using murine models and clinically- relevant forms of platelet inhibition, we will test the hypothesis that interaction of the neutrophil integrin receptor CD11b/CD18 with the platelet glycoprotein receptor GPIbα drives NET formation. Our proposal is significant because it has the potential to elucidate novel therapeutic targets to better manage PHTN, a devastating and prevalent disease which is currently curable only with liver transplantation.