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。

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.