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，缺乏基因匹配的供者，以及与扩增肝细胞相关的技术障碍，限制了原本可以通过肝或肝细胞移植有效治疗的患者数量。因此，识别允许肝细胞扩增和植入并增强肝再生的分子和细胞通路将具有重要的治疗意义。我们发现，70%肝部分切除(PH)后，肝窦内皮细胞(LSECs)通过产生旁分泌因子(称为血管分泌因子)而激活，从而诱导肝再生。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，该网络与肝细胞直接细胞接触，支持肝脏再生(Ding等人，《自然》2010)。PH后，LSECs的激活启动并维持剩余肝叶的再生。血管内皮生长因子受体(VEGFR2)和Id1通路的激活上调了WNT2和肝细胞生长因子(HGF)促肝细胞增殖的血管分泌表达。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，造血细胞的亚群(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。根据这些数据，我们推测，PH后，造血细胞，特别是激活的血小板，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。我们将使用我们实验室开发的肝再生和血管生成模型来检验这些假说，通过执行以下实验：目的1.确定在VEGFR2+LSECs中激活CXCR7支持血管分泌介导的肝细胞再生的机制。目的2.明确招募的造血细胞，特别是血小板在介导LSEC激活驱动肝再生中的作用。目的3.确定血小板与Akt激活的LSECs之间的相互作用在血管分泌因子诱导和促进肝细胞增殖中的作用。我们通过揭示LSECs支持肝再生的机制来促进肝细胞的扩张，这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，我们建议的实验将允许开发策略，其中通过适当地激活LSECs，将为在临床环境下加速肝脏再生奠定基础。 公共卫生相关性：肝移植是因肝炎、遗传疾病和肿瘤转移而导致的终末期肝病患者的主要治疗方法。然而，基因匹配供者的缺乏导致许多本来可以通过肝或肝细胞移植有效治疗的患者的发病率和死亡率增加。事实上，每年有超过5万人死于肝脏疾病的并发症，有超过1.5万名患者等待肝脏移植。肝细胞移植提供了一种临床上可行的改善肝功能的方法。因此，识别允许肝细胞扩增和长期植入或增强肝再生的分子和细胞通路将具有重要的治疗作用。我们已经证明，由专门的内皮细胞排列的肝血管会产生一组特定的生长因子，支持肝细胞的扩张和增强肝脏的再生。在这项建议中，我们提出了一些实验，以确定刺激肝血管以支持肝细胞长期植入肝脏以及增强肝脏再生的因素。我们希望我们的研究将为设计临床前和临床试验，以开发肝血管内皮细胞的潜力，以改善肝细胞和肝移植的结果奠定基础。

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.