肝脏星状细胞表观遗传调控及肝硬化的机理

1. Significance：Tissue fibrosis is a patho-physiological response and abnormal wound healing for tissue injury. Two fundamental questions remain inadequately addressed. First, how soft ECM in the space of Disse configures hepatic stellate cells into a quiescent state (qHSCs), exhibiting as accumulation of vitamin A droplets and high capability to express MMPs upon injury signals. Second, how, in chronic infection/injury, the qHSCs are trans-differentiated into myofibroblasts (myoHSCs), by which many MMP genes are silenced and vitamin A droplets are vanished. .. 2. Hypothesis：Soft 3D extracellular matrix in the space of Disse, through up regulation of cysteine cathepsins (Cts) to degrade class-II HDAC and YAP1, configuring HSCs into an epigeneticaly permissive/quiescent state in normal liver. Conversely, through sensing the stiffed/fibrotic interface, the cysteine cathepsins are down regulated, leading to accumulation of class-II HDACs and YAP1, which consequently confers the chromatin and MMP loci into epigenetic non-permissive/closed states, favoring ECM accumulation and persistency of liver fibrosis. .. 3. Supporting evidence：We reasoned that soft 3D ECM in the space of Disse, nested by hepatic stellate cells themselves, is critical for the quiescence/permissiveness of HSCs. To such regard we cultured the primary rat HSCs in soft 3D ECM, by which the cells exhbit vitamin A droplets and are able to express MMPs upon injury signal challenge. Conversely, on stiffed interface to simulate fibrosis, the stellate cells undergo transformation to form myofibroblasts, showing lose of vitamin A droplets, and silencing of MMP genes (JBC. 2004, JBC. 2007, Plos One, 2011). During the cellular transformation, class-II HDACs are progressively accumulated in the myofibroblasts, while their mRNA levels are without change, indicating a potential proteolytic regulation (Am J Pathology, 2010). In continuation of our effort, we recently discovered that cathepsin H (CtsH) can directly digest HDAC4 (Am J of Pathology, 2017). Importantly, in human cirrhotic tissues, we uncovered that up regulation of class-II HDACs is related to down regulation of CtsH in the fibrotic septa, suggesting a potential new mechanism for liver fibrosis. .. 4. Specific aims：Aim #1 is to depict changing epigenetic landscapes, at genome-wide, during trans-differentiation of rat hepatic stellate cells. We anticipate that the mmp loci and many other genes related to fibrosis may undergone epigenetic re-programing during HSC trans-differentiation. Using chromatin-immunoprecipitation-genomic sequencing (ChIP-seq) and through monitoring the changing epigenetic landscapes and bioinformatics based data mining we will uncover new loci or genes that are critical for fibrosis. Aim #2 is to study the roles of CtsH and class-II HDACs in (1) phenotype dermination, including mmp responsiveness, and LRAT expression, and cell cycle regulation, and (2) epigenetic re-programing, including histone acetylation/methylation in mmp loci and LRAT gene in the hepatic stellate cells. Aim #3 is to generate transgenic mice to determine the roles of CtsH in liver function and fibrosis. Two transgeneic mice will be generated: (1) hepatic stellate cell specifically expressed CtsH mice (CMV-lox-STOP-lox-CtsH), and (2) hepatic stellate cell specific knockout CtsH mice (lox-CtsH-lox). Through crossing with LRAT-Cre or desmin-Cre mice, we will examine our hypothesized cascade: “soft ECM>CtsH up>class-II-HDAC down” in physiology and "fibrotic stiffed ECM > CtsH down >class-II HDAC up in liver fibrosis.

在损伤信号的刺激下，肝星状细胞发生表型转分化，形成肌成纤维母细胞。我们致力于回答两个问题: (1)软化的胞外基质，以怎样的机理赋予肝星状细胞以正常生理表型？(2)在纤维硬化的环境中，肝星状细胞是怎样被转分化的？在此我们提出新假设：柔软的3D胞外基质，通过上调半胱氨酸组织蛋白酶(Cts)以降解II型组蛋白去乙酰基酶(class-II HDAC)以及原癌基因YAP1，产生大规模的表观遗传重塑，赋予星状细胞以允许状态(permissiveness), 表现为储存维生素A，以及在损伤信号的刺激下高表达基质金属蛋白酶(MMPs)。反之，在纤维硬化环境中，Cts表达下调，导致class-II HDAC以及YAP1累积，发生大规模的表观遗传改变，MMP基因被抑制，维生素A丢失，形成肌成纤维母细胞。以此，纤维化得以持续。我们将通过3D-ECM细胞，表观分子生物学，遗传动物模型来验证该理论。

继续我本人在美国南加州大学开创的工作，探索在机体损伤条件下，细胞转分化的机理。我们致力于比较细胞在不同柔软组织环境的生理代谢。为此我们开展了多个工作。第一，我们提问，组织纤维化/硬化是怎样促进肿瘤细胞的激活；反之，我们继续提问，癌症细胞在柔软组织环境中为什么能够长期处于休眠状态？为此，我们发现在柔软组织环境中细胞的Pten被激活，mTOR被抑制，导致auotphagy-lysosome的生物发生加强·，从而YAP被溶酶体降解。相反，在坚硬的纤维化状态中，细胞Pten失活，而mTOR被激活，抑制细胞自噬-溶酶体，导致YAP累积，促进细胞进一步癌变。一篇论文在修改状态。第二，我们发现缺乏阳光照射可能促进肝硬化的发生。具体来讲，我们发现肝脏损伤导致维生素D-25-羟基化降低，而缺乏维生素D的激活会损伤小肠潘氏细胞，防御肽降低，细菌上升，内毒素入血，进一步加重肝炎症以及组织硬化。论文发表在Am J Pathology， 2021. 此外，我们开展了一个临床试验，测定补充维生素D2是否能够改善脂肪肝/肝硬化。第三，类似与肝脏，胰腺组织也会发生纤维化，后者可以进一步促进胰腺癌的发展。肠道细菌紊乱导致的内毒素入血是导致多种慢性疾病的重要原因。我们发现肠道细菌内毒素能够激活mTOR，抑制细胞autophagy-lysosme flux，导致YAP累积，促进细胞迁移。采用胰腺癌转基因动物模型，我们发现通过口服阳离子树脂（考来烯胺）能够有效地清除肠道微生物产生的LPS，从而能够抑制胰腺癌的恶性转化。论文正在评审阶段。第四，我们做了一些关于饮食成分对肿瘤，对慢性肠炎（IBD）的影响。我们发现服用绿色素chlorophyllin能够抑制AKT，从而缓解肝硬化，缓解脂肪肝，缓解IBD；我们发表了两篇论文，Frontiers in Physiology。此外，我们发现穿心莲内脂也能够抑制mTOR，抑制肿瘤细胞。在该基金的资助下，4年来我们培养了10位研究生，包括博士研究生以及硕士研究生。在该基金的资助下，我们制备了两种转基因小鼠，flox-Vdr，以及flox-Tfeb，我们正在以这两种转基因小鼠开展工作，探索维生素D以及溶酶体发生在肝硬化，肠炎，胰腺癌中发关键作用。