Targeting myofibroblast activation in chronic fibrotic disease

描述(由申请人提供)：愈合伤口中肌成纤维细胞的慢性积聚与过度收缩、间质胶原过度沉积和破坏性组织重塑有关。血管平滑肌α-肌动蛋白(SMalphaA)是一种由分化的肌成纤维细胞瞬时表达的收缩蛋白，用于产生张力 闭合开放伤口所需的力量。在慢性纤维性疾病中，肌成纤维细胞分化功能障碍 我们发现SmaA和I型胶原基因的激活都需要分子信号 在这些细胞中也提供了负面反馈，这可能潜在地限制多动的招募 伤口愈合和破坏性重塑过程中的肌成纤维细胞。这项提案中概述的研究包括 有望揭示DNA和mRNA结合蛋白YB-1，Purα， 和Pur beta与SMalphaA和I型胶原启动子结合，并阐明这些蛋白是如何受促纤维化影响的 如TGFbeta1和凝血酶等药物，如果不加检查，可能会导致肌成纤维细胞进展为 肥厚性疤痕。实验旨在启动、放大或减弱肌成纤维细胞分化，以 更好地了解在转录和转录水平控制SMalphaA和I型胶原基因输出的策略 翻译水平以及揭示可能有助于最大限度地减少异常的新的干预策略 伤口愈合的结果。Aim 1将研究TGFbeta1调控的YB-1和PUR蛋白抑制物之间的相互作用 利用SMalphaA和胶原启动子DNA以及转录激活剂Sp1、SRF和Smads 2，3，描绘 这种功能相互作用所需的阻遏多肽链的区域，并试图破坏复合体 利用多肽诱饵和小分子形成和抑制肌成纤维细胞的病理分化 药理上的抑制剂。目标2将确定凝血酶是否在水平上促进肌成纤维细胞分化 翻译调控的作用因此作为TGFbeta1的补充，或者相反地通过以下方式拮抗这种生长因子 通过诱导抗纤维化转录调控阻断转录和肌成纤维细胞募集 蛋白质，Egr-1。目的3研究将探索肌成纤维细胞的替代、Smad非依赖性机制 分化和纤维化。基于药物抑制TGFbeta1/Smad的功能丧失途径 将使用激酶或磷脂酰肌醇-3-激酶(PI3K)/Akt激酶信号转导来评估其可能性 体外对小鼠心肌成纤维细胞活化和缺血再灌注后心肌纤维化的抑制作用 受伤。TGFbeta1和凝血酶开发血管内皮细胞DNA、RNA和蛋白质结合特性的能力 Yb-1和PUR蛋白为肌成纤维细胞基因表达调控提供了新的动态视角 这可能揭示了慢性纤维性疾病治疗的最佳策略。

DESCRIPTION (provided by applicant): Chronic accumulation of myofibroblasts in healing wounds is associated with hypercontractility, excessive deposition of interstitial collagens, and destructive tissue remodeling. Vascular smooth muscle alpha-actin (SMalphaA) is a contractile protein transiently expressed by differentiated myofibroblasts for generating tensile force required to close open wounds. In chronic fibrotic disease, myofibroblast differentiation is dysfunctional and we discovered that molecular signaling required for activation of both the SMaA and type I collagen genes in these cells also provides negative feedback that could potentially limit the recruitment of hyperactive myofibroblasts during wound healing and destructive remodeling. Studies outlined in this proposal are expected to reveal novel forms of functional interplay of the DNA- and mRNA-binding proteins YB-1, Pur alpha, and Pur beta with the SMalphaA and type I collagen promoters and clarify how these proteins are affected by pro-fibrotic agents such as TGFbeta1 and thrombin that, if unchecked, may cause myofibroblast progression to hypertrophic scarring. Experiments are designed to initiate, amplify, or attenuate myofibroblast differentiation to better understand strategies for controlling SMalphaA and type I collagen gene output at the transcriptional and translation levels as well as reveal novel interventional strategies that might be useful for minimizing aberrant wound healing outcomes. Aim 1 will examine TGFbeta1-regulated interaction of YB-1 and Pur protein repressors with SMalphaA and collagen promoter DNA and the transcriptional activators Sp1, SRF, and Smads 2,3, delineate regions of repressor polypeptide chains required for this functional interplay, and attempt to disrupt complex formation and disable pathobiologic myofibroblast differentiation using peptide decoys and small molecule pharmacologic inhibitors. Aim 2 will determine if thrombin potentiates myofibroblast differentiation at the level of translational control thus functioning as a TGFbeta1 supplement or instead antagonizes this growth factor by blocking transcription and myofibroblast recruitment by inducing the anti-fibrotic transcriptional regulatory protein, Egr-1. Aim 3 studies will explore alternative, Smad-independent mechanisms of myofibroblast differentiation and fibrosis. Loss-of-function approaches based on pharmacologic inhibition of TGFbeta1/Smad kinase- or phosphatidylinositol-3-kinase (PI3K)/Akt kinase signaling will be used to evaluate their possible suppressive effect on myofibroblast activation in vitro and cardiac fibrosis in mice after ischemia/reperfusion injury. The ability of TGFbeta1 and thrombin to exploit the unique DNA-, RNA-, and protein-binding properties of YB-1 and Pur proteins adds a new dynamic perspective to control of gene expression during myofibroblast differentiation that may reveal optimum strategies for therapeutic management of chronic fibrotic diseases.