Role of NO-sensitive guanylyl cyclase during liver fibrosis in mice

本实验室主要研究NO受体--NO敏感的鸟苷酸环化酶(NO-GC)的生理和病理生理作用。我们已经产生了对NO-GC缺乏全局或细胞特异性缺陷的小鼠。最近的研究主要集中在NO对肠蠕动、血压调节和血管生成的影响。本课题旨在探讨NO/cGMP信号转导通路在肝纤维化中的作用。初步资料显示，NO-GC表达于肝星状细胞(HSC)，即肝脏的周细胞。已有研究表明，HSC参与了肝损伤后创面愈合和纤维化反应。我们的初步数据表明，肝小叶中存在肝星状细胞和“真”周细胞。这两种类型的细胞都表达NO-GC，但它们的小叶分布不同。在第一个子项目中，我们将详细描述NO-GC在肝脏不同细胞中的表达(包括NO-GC亚单位)。在第二部分中，我们将使用原代培养的HSC和周细胞来研究NO-GC在体外的作用机制。我们将继续描述NO-GC在体内参与纤维化反应的情况。我们将在小鼠中采用四氯化碳肝纤维化模型，该模型模拟了人类疾病的几个方面。为了评估HSC和周细胞的作用，我们将使用我们不同的CRE/KO株(Lrat-CRE；SMMHC-CreERT2)。使用的方法包括使用共聚焦显微镜的免疫组织化学、报告菌株的谱系追踪、定量聚合酶链式反应、胶原蛋白分析或原位杂交。此外，我们计划研究一种非GC刺激物的抗纤维化作用。通过这种翻译方法，我们将深入了解NO-GC刺激的潜在治疗用途。综上所述，我们期望对HSC和周细胞中NO-GC如何参与肝纤维化有一个详细的认识。通过比较体内和体外两种不同的肝纤维化模型，我们不仅期望得到机制细节，而且还将收集关于NO/cGMP级联通路治疗纤维化疾病的有价值的信息。

Our lab focuses on the physiological and pathophysiological role of the NO receptor, NO-sensitive guanylyl cyclase (NO-GC). We have generated mice with global or cell-specific deficiency for NO-GC. Recent work has been focusing on the effect of NO on gut peristalsis, blood pressure regulation and angiogenesis. With this project we would like to investigate the influence of NO/cGMP signaling on liver fibrosis.Preliminary data show NO-GC to be expressed in hepatic stellate cells (HSC), the pericytes of the liver. HSC have been shown to participate in wound healing and fibrotic response after liver injury. Our preliminary data indicate the existence of HSC and 'true' pericytes in the liver lobule. Both cell types express NO-GC but they differ in their lobular distribution. In the first subproject, we will carefully describe NO-GC expression (including NO-GC subunits) in different cells of the liver. In the second part, we will use primary cultures of HSC and pericytes to investigate the mechanistic contribution of NO-GC in vitro. We will proceed in describing the participation of NO-GC in the fibrotic response in vivo. We will employ the carbon tetrachloride model for liver fibrosis in the mouse that mimics several aspects of the human disease. In order to evaluate the role of HSC and pericytes, we will use our different Cre/KO strains (Lrat-Cre; SMMHC-CreERT2). Methods used include immunohistochemistry using a confocal microscope, lineage tracing with reporter strains, qPCR, collagen assays or in situ hybridization. In addition, we plan to investigate the antifibrotic effects of a NO-GC stimulator. With this translational approach, we will fathom the potential therapeutic use of NO-GC stimulation.In summary, we expect to obtain a detailed view on how NO-GC in HSC and pericytes participates in liver fibrosis. By comparing two different models of liver fibrosis in vitro and in vivo we not only expect mechanistic details but we also will gather valuable information on the therapeutic use of the NO/cGMP cascade for fibrotic diseases.