Inactivation of Hepatic Stellate Cells During Reversal of Liver Fibrosis

描述(申请人提供)：慢性肝损伤导致肝纤维化，静止的肝星状细胞(QHSC)激活成肌成纤维细胞，沉积广泛的细胞外基质(ECM)蛋白。停止促纤维化刺激通常会导致退行性变 肝纤维化，与活化的肝星状细胞(AHSCs)/肌成纤维细胞的消失有关。我们最近已经证明，一些AHSCs凋零，而另一些AHSCs失活(IHSCs)进入静止的表型。该项目的总体目标是确定可能阻止HSC激活成肌成纤维细胞的分子因素，或使AHSC恢复到失活状态的分子因素。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，PPARHSC靶基因的激活调节静止和失活的γ表型。目的：我们将使用CHIP-SEQ技术评估qHSCs、AHSCs和iHSCs的全基因组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。使用体外系统，我们将确定f siRNA击倒或过表达这些因子阻止HSC激活，或触发HSC失活。目的：我们已经证明，在失活过程中，PPARγ在HSC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PPARγ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。目的：我们将在体内利用基因敲除小鼠评估PPARγ和PPARγ靶基因在肝星状细胞生物学中的作用，在HSC中，PPARγ被结构性或诱导性地特异性缺失。PPARγ在维持静止的HSC表型和失活AHSCs中的特定作用将在PPARγ缺陷的HSCs和野生型HSCs中进行评估。我们预计，在AIMS 1-3中获得的集体结果将确定特定的因素，可以使AHSCs恢复到失活的静止状态。AIM4：我们的 小鼠的研究结果必须转化为纤维性肝病患者。为了研究人的HSCs是如何灭活的，将静止的人HSCs移植到RAG2-/-γc-/-小鼠的肝脏中，并在体内用CCl_4诱导其激活，然后恢复。在每个时间点分离人qHSCs、AHSCs和iHSCs，并用RT-PCR、人类基因组芯片和流式细胞术进行分析。接下来，将对人的iHSCs进行芯片序列分析，并识别失活的特异性靶点(并与小鼠的iHSCs进行比较)。一种能力 在这些目标中，将使用siRNA敲除或将人HSCs移植到RAG2-/-γc-/-小鼠体内，在体外和体内测试将人AHSCs灭活到静止状态。我们的研究结果将使我们对小鼠和患者HSC失活的机制有新的认识，确定可以在纤维化肝脏中诱导激活的HSC/肌成纤维细胞失活的潜在治疗靶点。

DESCRIPTION (provided by applicant): Chronic liver injury results in hepatic fibrosis, in which quiescent hepatic stellate cells (qHSCs) activate into myofibroblasts, depositing extensive extracellular matrix (ECM) proteins. Cessation of fibrogenic stimuli often results in regression of liver fibrosis and is associated with the disappearance of activated HSCs (aHSCs)/myofibroblasts. We have recently demonstrated that some aHSCs apoptose, while other aHSCs inactivate (iHSCs) into a quiescent-like phenotype. The overall goals of this Project are to identify the molecular factors that may prevent HSC activation into myofibroblasts, or that revert aHSC into an inactivated 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 PPARγ-target genes regulates quiescent and inactivated HSC phenotypes. AIM1: We will assess the genome wide methylation and acetylation sites using ChIP-Seq in qHSCs, aHSCs and iHSCs in order to identify motifs and transcription factors critical for HSC inactivation. Using in vitro systems, we will determine f siRNA knock-down or overexpression of these factors blocks HSC activation, or triggers HSC inactivation. AIM2: We have demonstrated that PPARγ is re-expressed in HSCs during inactivation. To gain a greater insight into the mechanisms of HSC inactivation, we will conduct a broad investigation of the epigenetic changes that regulate PPARγ-target genes in distinct HSC phenotypes (qHSCs, aHSCs and iHSCs). We will test if functional inhibition of these genes affects the qHSC and iHSC phenotypes. AIM3: We will assess the role of PPARγ and PPARγ target genes in HSC biology in vivo using knockout mice, in which PPARγ is constitutively or inducibly deleted specifically in HSCs. A specific role of PPARγ in the maintenance of quiescent HSC phenotype and inactivation of aHSCs will be assessed in PPARγ-deficient HSCs versus wild type HSCs. We anticipate that the collective results obtained in AIMs 1-3 will identify specific factors that can revert aHSCs into an inactivated quiescent-like state. AIM4: Our findings in mice must be translated to patients with fibrotic liver disease. To examine how human HSCs inactivate, quiescent human HSCs will be engrafted into livers of Rag2-/-γc-/- mice, and their activation will be induced in vivo by CCl4 followed by recovery. Human qHSCs, aHSCs and iHSCs will be isolated at each time point and analyzed by RT-PCR, Human genome microarray and flow cytometry. Next, human iHSCs will be subjected to ChIP-Seq analysis, and inactivation-specific targets will be identified (and compared to that in mouse iHSCs). The ability of these targets to inactivate human aHSCs into a quiescent-like state will be tested in vitro and in vivo using siRNA knock-down or intrahepatic transplantation of human HSCs into Rag2-/-γc-/- mice. The results of our studies will give new insight into mechanisms underlying HSC inactivation in mice and patients, identifying potential therapeutic targets that can induce inactivation of activated HSCs/myofibroblasts in fibrotic liver.