Molecular Mechanisms of Portal Hypertension

描述（由申请人提供）：门脉高压在肝硬化患者中引起显著的发病率和死亡率，尽管介导这种综合征的确切事件尚不完全清楚。该资助的总体目标是确定导致门静脉高压的肝窦内的分子机制，最终开发出可在人体中测试的新治疗方法。肝内皮细胞（LEC）和肝星状细胞（HSC）之间的旁分泌相互作用是窦状血管重构和收缩所必需的，这是门静脉高压的关键步骤。在这方面，我们的初步数据表明：1)LEC产生脂质信号分子鞘氨醇-1磷酸（S1P），促进HSC的迁移和收缩；2)FGF2和FGF21刺激LEC中的血管生成生长因子受体、成纤维细胞生长因子受体1 （FGFR）导致S1P的产生；3)LEC生长因子途径的药物抑制可减轻体内门静脉高压症的发展。我们将这些新发现运用到当前提案的中心假设中；LEC中FGFR的激活会增加S1P的产生，从而刺激HSC的募集和肝窦的收缩，从而导致门静脉高压。目的是验证以下子假设：1)LEC通过增加S1P的产生和细胞释放来招募和收缩HSC。目的1a将研究LEC的外泌体和旁分泌释放S1P分别如何刺激HSC的募集和收缩。Aim 1b将揭示FGFR信号如何通过染色质调控的表观遗传机制转录激活S1P生成酶鞘氨酸激酶1 （SK1）。2) LEC通过FGF21磷酸化FGFR和随后的核内体信号传导激活S1P的产生。Aim 2a将探索内分泌FGF家族成员FGF21调节特定FGFR残基磷酸化以激活FGFR信号传导和S1P产生的新机制。Aim 2b将关注FGF21如何促进FGFR内化到细胞内小体，从而控制FGFR信号传导和S1P的产生。3)抑制LEC中FGFR-S1P信号通过阻断HSC的募集和收缩，在体内减弱门静脉高压症的发展。Aim 3a将在LEC中FGFR基因缺失的小鼠中使用独特的FGF传递系统，剖析血管生成FGFR通路如何促进门静脉高压症。Aim 3b将利用一种创新的外泌体转移技术，在S1P产生基因缺陷的小鼠中揭示S1P在门静脉高压症中的作用。Aim 3c将使用我们最近获得的一种新的小鼠系，在体内测试FGFR内化在门静脉高压症发展中的作用。因此，本提案将利用各种创新但可行的细胞和

DESCRIPTION (provided by applicant): Portal hypertension accounts for significant morbidity and mortality in patients with liver cirrhosis although the precise events that mediate this syndrome remain incompletely understood. The Overall Objective of this grant is to define the molecular mechanisms within the hepatic sinusoids that are responsible for portal hypertension with the goal of eventually developing new therapeutic approaches that can be tested in humans. Paracrine interactions between liver endothelial cells (LEC) and hepatic stellate cells (HSC) are required for sinusoidal vascular remodeling and contraction, which are critical steps in portal hypertension. In this regard our Preliminary Data shows that 1) LEC generate the lipid signaling molecule sphingosine-1 phosphate (S1P) which promotes HSC migration and contraction, 2) stimulation of the angiogenic growth factor receptor, fibroblast growth factor receptor 1 (FGFR) in LEC by FGF2 and FGF21 leads to S1P production, and 3) pharmacologic inhibition of LEC growth factor pathways attenuates portal hypertension development in vivo. We have parlayed these novel findings into the Central Hypothesis of the current proposal; that FGFR activation in LEC increases S1P production which stimulates HSC recruitment and contraction of hepatic sinusoids leading to portal hypertension. The Aims are to test the sub-hypotheses that: 1) LEC recruit and contract HSC through increased production and cellular release of S1P. Aim 1a will examine how both exosome and paracrine release of S1P from LEC stimulates HSC recruitment and contraction, respectively. Aim 1b will discern how FGFR signaling transcriptionally activates the S1P generating enzyme, sphingosine kinase 1 (SK1) through an epigenetic mechanism of chromatin regulation. 2) Activation of S1P production from LEC occurs through FGFR phosphorylation by FGF21 and ensuing endosome based signaling. Aim 2a will explore new mechanisms by which the endocrine FGF family member, FGF21 regulates phosphorylation of specific FGFR residues to activate FGFR signaling and S1P production. Aim 2b will focus on how FGF21 promotes FGFR internalization into intracellular endosomes to govern FGFR signaling and S1P production. 3) Inhibition of FGFR-S1P signaling in LEC attenuates development of portal hypertension in vivo by blocking recruitment and contraction of HSC. Aim 3a will dissect how the angiogenic FGFR pathway contributes to portal hypertension using a unique FGF delivery system in mice with genetic deletion of FGFR from LEC. Aim 3b will uncover the role of S1P in portal hypertension using an innovative exosome-transfer technique in mice harboring a genetic defect in S1P production. Aim 3c will test the role of FGFR internalization on portal hypertension development in vivo using a new mouse line that we recently derived. Thus this proposal will utilize a variety of innovative yet feasible, cell and molecular approaches, both in vitro and in vivo to elucidate novel pathways that contribute to portal hypertension. The results, by advancing our understanding of sinusoidal structure and function, will set a trajectory towards new and significant advances to treat portal hypertension in humans.