Identification of vascular inductive signals in liver regeneration

描述（申请人提供）：肝移植是终末期肝病患者的主要治疗方法。然而，基因匹配供体的缺乏和与扩大肝细胞相关的技术障碍限制了本来可以通过肝或肝细胞移植有效治疗的患者数量。因此，鉴定允许肝细胞扩张和植入并增强肝再生的分子和细胞途径将具有重大的治疗影响。我们发现，在70%的肝部分切除术（PH）后，通过分泌旁分泌因子（定义为血管分泌因子）激活肝窦内皮细胞（LSECs），诱导肝脏再生。我们已经定义了LSECs的表型和操作定义，并表明LSECs由一个专门的血管网络组成，该血管网络与肝细胞直接接触，支持肝脏再生（Ding等人，Nature 2010）。PH后，LSECs的激活启动并维持肝脏剩余叶的再生。LSECs中VEGF-A酪氨酸激酶受体（VEGFR2）和Id1通路的激活上调了Wnt2和肝细胞生长因子（HGF）的血管分泌表达，刺激肝脏增殖。然而，PH诱导LSECs产生肝细胞活性血管分泌因子的机制尚不清楚。我们发现，在PH后，造血细胞亚群（即血小板）被招募到肝窦，并通过沉积VEGF-A和SDF-1激活LSECs。除VEGFR2外，SDF-1的趋化因子受体CXCR7在LSECs上特异性上调。基于这些数据，我们假设PH后，造血细胞，特异性活化的血小板，被招募到肝脏LSECs，并通过部署VEGF-A和SDF-1刺激VEGFR2+CXCR7+ LSECs产生肝细胞活性血管分泌因子，启动和维持肝脏再生。我们将采用我们实验室开发的肝脏再生和血管生成模型，通过执行以下实验来检验这些假设：目的1。确定VEGFR2+ LSECs中CXCR7激活支持血管分泌素介导的肝细胞再生的机制。目标2。明确募集的造血细胞，特别是血小板在介导LSEC激活驱动肝脏再生中的作用。目标3。确定血小板和akt激活的LSECs在血管分泌因子诱导和加速肝细胞增殖中的相互作用。我们通过揭示LSECs支持肝脏再生的机制来改善肝细胞扩张的方法，将为识别支持功能性肝细胞长期增殖和植入肝脏的血管分泌因子铺平道路。此外，我们提出的实验将允许制定策略，其中通过适当激活LSECs，将为在临床环境中加速肝脏再生奠定基础。

DESCRIPTION (provided by applicant): Liver transplantation is the mainstay of treatment for patients with end-stage liver disease. However, the paucity in genetically matched donors and technical hurdles associated with expanding hepatocytes has limited the number patients that could have otherwise been treated effectively with liver or hepatocyte transplantation. Therefore, identification of the molecular and cellular pathways that allow expansion and engraftment of hepatocytes and augment liver regeneration will have significant therapeutic impact. We have found that after 70% partial hepatectomy (PH), activation of liver sinusoidal endothelial cells (LSECs) by production of paracrine factors, defined as angiocrine factors, induce liver regeneration. We have defined phenotypic and operational definition of LSECs and have shown that LSECs compose of a specialized vascular network that are in direct cellular contact with hepatocytes, supporting liver regeneration (Ding et al, Nature 2010). After PH, activation of LSECs initiates and sustains the regeneration of remaining lobes of the liver. The activation of the VEGF-A tyrosine kinase receptor (VEGFR2) and Id1 pathway in LSECs upregulated the angiocrine expression of Wnt2 and hepatocyte growth factor (HGF) stimulating hepatic proliferation. However, the mechanism by which PH induces LSECs to produce hepatocyte-active angiocrine factors is unknown. We show that after PH subsets of the hematopoietic cells, (i.e. platelets), are recruited to liver sinusoids and activate LSECs by depositing VEGF-A and SDF-1. In addition to VEGFR2, chemokine receptor for SDF-1, CXCR7, is upregulated specifically on LSECs. Based on these data, we hypothesize that after PH, hematopoietic cells, specifically activated platelets, are recruited to liver LSECs and by deploying VEGF-A and SDF-1 stimulate VEGFR2+CXCR7+ LSECs to produce hepatocyte-active angiocrine factors that initiate and maintain liver regeneration. We will employ liver regeneration and angiogenic models developed in our laboratory to examine these hypotheses by executing the following experiments: Aim 1. Determine the mechanism by which activation of CXCR7 in VEGFR2+ LSECs support angiocrine-mediated hepatocyte regeneration. Aim 2. Define the role of recruited hematopoietic cells, specifically platelets in mediating LSEC activation driving liver regeneration. Aim 3. Determine the role of reciprocal interaction between platelets and Akt-activated LSECs in angiocrine factor induction and accelerating hepatocyte proliferation. Our approach to improve expansion of hepatocytes by unraveling the mechanism by which LSECs support liver regeneration will pave the way for identification of angiocrine factors that support long-term proliferation of functional hepatocytes and engraftment into the liver. In addition, our proposed experiments will allow for development of strategies in which by proper activation of LSECs, will set the stage for accelerating liver regeneration in the clinical setting. PUBLIC HEALTH RELEVANCE: Liver transplantation is the mainstay of treatment for patients with end-stage liver disease due to hepatitis, genetic disorders and tumor metastasis. However, the paucity in genetically matched donor results in increased morbidity and mortality of many patients who could have otherwise been treated effectively with liver or hepatocyte transplantation. Indeed, every year more than 50,000 individuals succumb to the complications of liver disease with more than 15,000 patients awaiting a liver transplant. Hepatocyte transplantation provides for a clinically plausible approach to improve liver function. Therefore, identification of the molecular and cellular pathways that allow expansion and long-term engraftment of hepatocyte or augment liver regeneration will have significant therapeutic impact. We have shown that liver blood vessels that are lined by specialized endothelial cells produce specific set of growth factors that support expansion of hepatocytes and augment liver regeneration. In this proposal, we have proposed experiments to identify the factors that stimulate liver blood vessels to support long-term hepatocyte engraftment into the liver as well as augmenting liver regeneration. We hope our studies will lay the foundation for designing pre-clinical and clinical trials to exploit the potential of liver blood vessel endothelial cells to imrove the outcome of hepatocyte and liver transplantation.