BACKGROUND & AIMS Activation of TGFB (transforming growth factor β) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers. METHODS We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1Δhep). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization. RESULTS ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl4-induced liver fibrosis, we found an inverse correlation between level of ECM1 and severity of fibrosis. CCl4-induced liver fibrosis was accelerated in ECM1Δhep mice compared with control mice. Hepatocytes produced the highest levels of ECM1 in livers of mice. Ectopic expression of ECM1 or soluble TGFBR2 in liver prevented fibrogenesis in ECM1-KO mice and prolonged their survival. Ectopic expression of ECM1 in liver also reduced the severity of CCl4-induced fibrosis in mice. CONCLUSIONS ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis.

中文翻译：

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ECM1防止小鼠中转化生长因子β，肝星状细胞和纤维化的激活。

背景与目的TGFB（转化生长因子β）的激活通过激活肝星状细胞（HSC）促进肝纤维化，但TGFB激活的机制尚不清楚。我们调查了与细胞外和结构蛋白相互作用的ECM1（细胞外基质蛋白1）在小鼠肝脏TGFB激活中的作用。方法我们对C57BL / 6J小鼠（对照），ECM1基因敲除（ECM1-KO）小鼠和肝细胞特异性基因敲除EMC1（ECM1Δhep）的小鼠进行了研究。注射腺相关病毒载体后，在小鼠肝脏中表达了ECM1或可溶性TGFBR2（TGFB受体2）。肝纤维化是通过四氯化碳（CCl4）给药诱导的。从小鼠收集肝脏，并通​​过组织学，免疫组织化学，原位杂交和免疫荧光分析进行分析。从小鼠肝脏中分离出肝细胞和HSC，并与ECM1一起孵育。定量细胞因子的产生和报道基因的激活。通过免疫组织化学和原位杂交对来自病毒性或酒精性肝炎（具有不同纤维化分期）的患者和健康肝脏个体的肝组织进行了分析。结果ECM1-KO小鼠自发发展为肝纤维化，并在2个月大时死亡，而没有明显的肝细胞损伤或炎症。在小鼠的肝组织中，我们发现ECM1通过与αv整合素相互作用来阻止HSC的活化，从而以非活性形式稳定了细胞外基质沉积的TGFB。在患者和CCl4诱导的肝纤维化小鼠的肝组织中，我们发现ECM1的水平与纤维化严重程度之间呈负相关。与对照小鼠相比，在ECM1Δhep小鼠中CCl4诱导的肝纤维化被加速。肝细胞在小鼠肝脏中产生最高水平的ECM1。ECM1或可溶性TGFBR2在肝脏中异位表达可阻止ECM1-KO小鼠的纤维化并延长其存活时间。肝脏中ECM1的异位表达也降低了CCl4诱导的小鼠纤维化的严重性。结论肝细胞产生的ECM1抑制TGFB的活化及其对HSC的活化，从而阻止小鼠肝脏中的纤维发生。可以制定提高肝脏中ECM1水平的策略来治疗纤维化。ECM1或可溶性TGFBR2在肝脏中异位表达可阻止ECM1-KO小鼠的纤维化并延长其存活时间。肝脏中ECM1的异位表达也降低了CCl4诱导的小鼠纤维化的严重性。结论肝细胞产生的ECM1抑制TGFB的活化及其对HSC的活化，从而阻止小鼠肝脏中的纤维形成。可以制定提高肝脏中ECM1水平的策略来治疗纤维化。ECM1或可溶性TGFBR2在肝脏中异位表达可阻止ECM1-KO小鼠的纤维化并延长其存活时间。肝脏中ECM1的异位表达也降低了CCl4诱导的小鼠纤维化的严重性。结论肝细胞产生的ECM1抑制TGFB的活化及其对HSC的活化，从而阻止小鼠肝脏中的纤维发生。可以制定提高肝脏中ECM1水平的策略来治疗纤维化。