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共同终末途径， 在肝病患者中占显著的发病率和死亡率。然而， 治疗方法来改善PHTN。本提案的总体目标是阐明 中性粒细胞和血小板对PHTN发病机制的贡献，其治疗目标是促进 设计降低门静脉压力的疗法。PHTN的病理生理学是复杂的， 在多个水平，包括窦状隙内的旁分泌信号传导，微血管血栓形成， 内皮功能障碍我们以前已经确定了一个新的，但关键的作用，中性粒细胞在 PHTN的发病机制。我们发现，充血性肝病（CH）引起的周期性牵张诱导 激活肝窦内皮细胞（LSEC）内的机械敏感性压电通道。压电 通道激活机械分泌信号通路，其最终分泌中性粒细胞趋化因子 细胞因子CXCL 1。CXCL 1诱导中性粒细胞浸润到肝窦中。中性粒细胞与 血小板，导致中性粒细胞胞外陷阱或NET的形成。我们发现基因和 在CH小鼠模型中，NET形成的药物抑制显著降低门静脉压力， PHTN。尽管血小板在某些情况下与NET的形成有关，但 募集血小板并调节其与中性粒细胞的相互作用以产生正弦NET需要进一步的研究。 调查韦贝尔-帕拉德小体（Webel-Palade bodies，WPB）是一种产生细胞外囊泡的内皮细胞器 (EVs)含有炎症和止血因子。WPB衍生EV对血小板募集的影响 对肝窦的影响还没有研究过。我们已经提出了一个中心假设，即血小板被 WPB衍生的EV与中性粒细胞上的CD 11b/CD 18整合素受体相互作用以刺激NET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， 在技术上和概念上都是创新的。首先，我们将测试压电通道 作为主机械传感器，其激活调节中性粒细胞和血小板的生成， 调节门静脉压力的趋化因子。我们认为压电激活产生血小板 LSEC中源自WPB的EV内的趋化因子。最后，使用小鼠模型和临床- 相关形式的血小板抑制，我们将测试的假设，中性粒细胞整合素受体的相互作用， CD 11b/CD 18与血小板糖蛋白受体GPIbα驱动NET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，这是一种毁灭性的， 目前只能通过肝移植治愈的流行病。

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.