TGF-beta, matrix, and myofibroblasts in hepatic fibrosis

描述(由申请人提供)：此申请是对NOT-OD-09-058-NOT-NOT-OD-09-058-NOT-NIH宣布恢复法案资金可用于竞争性修订申请的回应。R01-DK058123的这一竞争性修订申请的目标是增加一个额外的特定目标--与现有的三个目标直接相关，并从已经产生的数据中得出--以研究桥接纤维化中的机械因素。母体基金的目标是开发一种融合了可溶性、细胞和机械因素的纤维化模型。最初的建议集中在早期纤维化，特别是肝脏的早期机械变化，导致肌成纤维细胞分化的有利环境。最初的三个具体目标是：1)确定整合素在肝星状细胞(HSC)肌成纤维细胞分化过程中的机械传感作用；2)建立包括HSC和门静脉成纤维细胞(PF)分化的纤维化力学模型，重点描述局部力学差异；以及3)确定纤维化中基质僵硬的介质，特别是赖氨酰氧化酶(LOX)胶原交联酶家族。我们最初的模型扩展到了HSC和PF分化为纤维化的肌成纤维细胞的程度。我们建议在此扩展该模型，以涵盖桥接纤维化中发生的架构变化。我们认为，桥接纤维化是由肝脏的局部机制与HSC和PF在僵硬环境中的收缩特性相结合的机械结果。我们提出以下假设：早期纤维化的机械异质性与肌成纤维细胞的收缩能力相结合可以解释桥接性纤维化的发展。新的具体目标是确定机械因素(肌成纤维细胞的收缩能力和肝脏硬度的局部增加)对桥接性纤维化发展的贡献。该研究计划包括1)对纤维化大鼠肝组织进行详细的形态分析，以寻找胶原纤维移位和排列的证据；2)使用二次谐波分析胶原纤维的取向，并使用微压痕技术比较早期纤维化中门脉和中间区域的硬度；以及3)使用ROCK1小鼠建立体内纤维化模型，以确定是否需要细胞介导的整合素黏附和牵引才能发生桥接纤维化。如果这一提议背后的假设被证明是正确的，它将对理解和潜在地治疗纤维化具有重大意义。 公共卫生相关性：晚期肝纤维化和疾病的终末期，即肝硬变，是慢性肝损伤的破坏性并发症。纤维化进展并最终演变为肝硬变的机制尚不清楚。这项申请提出了一个新的模型来解释纤维化进展的组成部分，如果得到这里提出的实验的支持，将对理解和治疗纤维化具有重要意义。

DESCRIPTION (provided by applicant): This application is in response to NOT-OD-09-058 - Notice entitled NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The goal of this competitive revision application for R01-DK058123 is to add an additional specific aim - directly related to the three existing aims and derived from data already generated - to investigate mechanical factors in bridging fibrosis. The goal of the parent grant is to develop a model of fibrosis that incorporates soluble, cellular, and mechanical factors. The original proposal concentrated on early fibrosis, in particular on early mechanical changes in the liver that resulted in a favorable environment for myofibroblastic differentiation. The three original specific aims were: 1) To determine the role of integrins in hepatic stellate cell (HSC) mechanosensing during myofibroblastic differentiation; 2) To develop a mechanical model of fibrosis that incorporated both HSC and portal fibroblast (PF) differentiation, with an emphasis on characterizing regional mechanical differences; and 3) To identify mediators of matrix stiffness in fibrosis, with a particular focus on the lysyl oxidase (LOX) family of collagen cross-linking enzymes. Our original model extended to the point where HSC and PF differentiated into fibrogenic myofibroblasts. We propose here to extend the model to encompass the architectural changes that occur in bridging fibrosis. We suggest that bridging fibrosis results mechanistically from the regional mechanics of the liver combined with the contractile properties of HSC and PF in a stiff environment. We propose the following hypothesis: that mechanical heterogeneity in early fibrosis combined with myofibroblast contractility can explain the development of bridging fibrosis. The new specific aim is to determine the contribution of mechanical factors (myofibroblast contractility and regional increases in liver stiffness) to the development of bridging fibrosis. The research plan includes 1) a detailed morphological analysis of fibrotic rat liver tissue for evidence of collagen fibril translocation and alignment; 2) an analysis of collagen fibril orientation using second harmonics and a comparison, using microindentation techniques, of the stiffness in portal and intervening regions in early fibrosis; and 3) use of ROCK1 mice in an in vivo fibrosis model to determine whether cell-mediated integrin adhesion and traction is required for bridging fibrosis to occur. If the hypothesis underlying this proposal is proven correct, it would have major implications for understanding and, potentially, treating fibrosis. PUBLIC HEALTH RELEVANCE: Late stage liver fibrosis and the end-stage of the disease, cirrhosis, are devastating complications of chronic liver injury. The mechanisms whereby fibrosis progresses, eventually becoming cirrhosis, are not well understood. This application proposes a new model to explain components of fibrosis progression, that, if supported by the experiments proposed here, would have significant implications for the understanding and treatment of fibrosis.