Origin and Fate of Myofibroblasts in Alcoholic Liver Disease

酒精性肝病中肌成纤维细胞的起源和命运

• 批准号: 8597097
• 负责人: DAVID A. BRENNER
• 金额: $ 18.11万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8597097
• 关键词: Acetylation; Alcohol abuse; Alcohol withdrawal syndrome; Alcoholic Liver Diseases; Alcohols; Animals; Binding Sites; Biochemical; CYP2E1 gene; California; Cell Culture Techniques; Cell model; Cells; ChIP-seq; Chronic; Cirrhosis; Clinical; Collaborations; Collagen; Consumption; DNA Binding; Data; Defect; Deposition; Development; Diet; Epigenetic Process; Ethanol; Event; Exhibits; Extracellular Matrix Proteins; Fatty Liver; Fatty acid glycerol esters; Fibrosis; Flow Cytometry; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glial Fibrillary Acidic Protein; Goals; Healthcare; Hepatic Stellate Cell; Human; Hybrids; In Vitro; Inflammation; Infusion procedures; Injury to Liver; Investigation; Knockout Mice; Link; Liquid substance; Liver; Liver Cirrhosis; Liver Fibrosis; Location; Maintenance; Mediating; Messenger RNA; Modeling; Molecular; Mus; Myofibroblast; Nuclear Receptors; Pathway interactions; Patients; Phenotype; Population; Production; Proteins; Recovery; Regulation; Regulatory Element; Repression; Research; Signal Transduction; Simulate; Site; Small Interfering RNA; Source; Staging; Testing; Transgenic Mice; Transplantation; Up-Regulation; Vitamin A; base; blocking factor; care burden; feeding; genome wide methylation; genome-wide; histone modification; improved; in vivo; insight; interest; intrahepatic; knock-down; knockout gene; mRNA Expression; mortality; mouse model; novel; overexpression; prevent; protein activation; response; therapeutic target; transcription factor

Alcohol-induced liver injury activates hepatic stellate cells (HSCs) resulting in hepatic fibrosis. Withdrawal from alcohol leads to regression of liver fibrosis and disappearance of HSCs/myofibroblasts. We have recently demonstrated that some activated HSCs apoptose, while other HSC inactivate and revert to a quiescent-like phenotype. The overall goals of this Project are to identify the molecular factors that may prevent HSC activation into myofibroblasts, or revert activated HSCs (aHSCs) into a quiescent-like state. Our central hypothesis is that genome wide epigenetic changes regulate HSC phenotype by activation (or suppression) of transcriptional activity in HSCs. We also hypothesize that activation of PPARg-target genes regulates the quiescent (gHSC) and inactivated (iHSC) phenotypes. AIM1A: We undertake two approaches to improve the model of intragastric ethanol infusion: optimization of the regiment of ethanol and high fat diet (HFD) administration, and utilization of "humanized" transgenic mice expressing human CYP2E1 protein in Cyp2e1-null mice that make them more susceptible to alcohol-induced liver injury. We hypothesize that these mice also exhibit a defect in HSC inactivation during recovery from alcohol-induced liver fibrosis. "AIM1B: We will use a translafional approach to explore whether our findings in mice apply to patients. Using flow cytometry, we will examine if HSC inactivation also occurs in patients. Inactivation of HSCs will be simulated in vitro by TGF-pi cessation, or in vivo by intrahepatic transplantation of human HSCs into Rag2-/- yc-/- mice. iHSCs will be identified by Vitamin A*aSMAGFAPlowPPARYhi phenotype. AIM2A: We will use Chip- Seq analysis to access the genome wide methylation and acetylation sites in qHSCs, aHSCs and iHSCs to identify motifs and transcription factors critical for HSC inactivation. Using primary HSC cultures, we will determine if siRNA knockdown or over-expression of these factors blocks HSC activation, or trigger HSC inactivation. AIM2B: We have demonstrated that PPARy is re-expressed in HSCs during inactivafion. To gain a greater insight into mechanisms of HSC inactivation, we will conduct a broad investigation of the epigenetic changes that regulate PPARy-target genes in distinct HSC phenotypes (qHSCs, aHSCs and IHSCs). We will test if functional inhibition of these genes compromises the qHSC and iHSC phenotypes. We anticipate that the results obtained in this study will identify specific factors that can revert aHSC into iHSCs and will give greater insight into mechanisms underlying HSC inactivation, validating potential therapeutic targets that can induce inactivation of already existing HSCs/myofibroblasts in fibrotic liver.

酒精性肝损伤激活肝星状细胞(HSCs)，导致肝纤维化。戒酒会导致肝纤维化的消退和肝干细胞/肌成纤维细胞的消失。我们最近已经证明，一些激活的HSC凋亡，而另一些HSC失活，并恢复到静止的样表型。该项目的总体目标是确定可能阻止HSC激活为肌成纤维细胞或将激活的HSCs恢复到静止状态的分子因素。我们的中心假设是全基因组的表观遗传变化通过激活(或抑制)HSC的转录活性来调节HSC的表型。我们还假设PPARg靶基因的激活调节静止(GHSC)和失活(IHSC)表型。AIM1A：我们采取了两种方法来改进乙醇灌胃模型：优化乙醇和高脂饮食(HFD)的给药方案，以及在CYP2E1基因缺失的小鼠中使用表达人CYP2E1蛋白的人源化转基因小鼠，使它们更容易受到酒精性肝损伤的影响。我们假设这些小鼠在酒精性肝纤维化恢复过程中也表现出肝星状细胞失活缺陷。“AIM1B：我们将使用一种跨性别的方法来探索我们在小鼠身上的发现是否适用于患者。使用流式细胞术，我们将检查HSC失活是否也发生在患者身上。HSCs的失活将在体外通过停止转化生长因子-pi来模拟，或通过将人HSCs肝内移植到Rag2-/-YC-/-小鼠体内来模拟。IHSCs将通过维生素A*aSMAGFAPlowPPARYHI表型进行鉴定。AIM2A：我们将使用芯片序列分析来访问qHSCs、AHSCs和iHSCs中全基因组的甲基化和乙酰化位点，以确定HSC失活的关键基序和转录因子。利用原代HSC培养，我们将确定siRNA敲除或过度表达这些因子是否阻止HSC激活或触发HSC失活。AIM2B：我们已经证明了PPARy在HSCs失活过程中重新表达。为了更深入地了解HSC失活的机制，我们将对不同HSC表型(qHSCs、AHSCs和iHSCs)中调控PPARy靶基因的表观遗传学变化进行广泛的研究。我们将测试这些基因的功能抑制是否会影响qHSC和IHSC的表型。我们预计，本研究的结果将确定可以将AHSC转化为iHSC的特定因素，并将更深入地了解HSC失活的机制，验证潜在的治疗靶点，这些靶点可以诱导纤维化肝脏中已有的HSCs/肌成纤维细胞失活。