Targeting myofibroblast activation in chronic fibrotic disease

描述（由申请方提供）：愈合伤口中肌成纤维细胞的慢性蓄积与过度收缩、间质胶原过度沉积和破坏性组织重塑相关。血管平滑肌α-肌动蛋白（-SMA）是一种由分化的肌成纤维细胞瞬时表达的收缩蛋白，用于产生闭合开放性伤口所需的张力。在慢性纤维化疾病中，肌成纤维细胞分化功能障碍，我们发现这些细胞中β-SMA和I型胶原蛋白基因激活所需的分子信号传导也提供了负反馈，这可能会限制伤口愈合和破坏性重塑期间过度活跃的肌成纤维细胞的募集。本提案中概述的研究有望揭示DNA和mRNA结合蛋白YB-1、Pur β和Pur β与β-SMA和I型胶原启动子的功能性相互作用的新形式，并阐明这些蛋白如何受到促纤维化剂如TGF β 1和凝血酶的影响，如果不加抑制，可能导致肌成纤维细胞进展为肥大性瘢痕。实验旨在启动、扩增或减弱肌成纤维细胞分化，以更好地理解在转录和翻译水平控制β-SMA和I型胶原基因输出的策略，以及揭示可能有助于最大限度减少异常伤口愈合结果的新型干预策略。目的1将研究TGF β 1调节的YB-1和Pur蛋白阻遏物与β-SMA和胶原启动子DNA以及转录激活因子Sp1、SRF和Smads 2，3的相互作用，描绘这种功能相互作用所需的阻遏物多肽链区域，并尝试使用肽诱饵和小分子药理学抑制剂破坏复合物的形成并使病理生物学肌成纤维细胞分化失效。目的2将确定凝血酶是否在翻译控制水平上增强肌成纤维细胞分化，从而作为TGF β 1补充剂起作用，或者通过诱导抗纤维化转录调节蛋白Egr-1阻断转录和肌成纤维细胞募集来拮抗这种生长因子。目的3研究将探索肌成纤维细胞分化和纤维化的替代性、非Smad依赖性机制。基于TGF β 1/Smad激酶-或磷脂酰肌醇-3-激酶（PI 3 K）/Akt激酶信号传导的药理学抑制的功能丧失方法将用于评价它们对体外肌成纤维细胞活化和缺血/再灌注损伤后小鼠心脏纤维化的可能抑制作用。TGF β 1和凝血酶利用YB-1和Pur蛋白独特的DNA、RNA和蛋白质结合特性的能力为肌成纤维细胞分化过程中基因表达的控制提供了新的动态视角，这可能揭示慢性纤维化疾病治疗管理的最佳策略。项目叙述：拟议的研究调查了心脏和肺组织中结缔组织细胞的细胞和分子变化，这些变化是手术后正常伤口愈合所需的。在移植患者以及被诊断患有各种形式的慢性心肺疾病的个体中，这些过程被加速，导致永久性疤痕形成、心脏和肺的结构畸形、组织功能丧失和早期死亡。本申请中概述的新研究将检查驱动正常伤口愈合的个体生物化学途径，并尝试确定药物治疗的新靶点，这将有助于控制破坏性伤口愈合活动，并改善患有慢性疾病的个体的生活质量，这些慢性疾病导致心脏和肺部异常瘢痕组织形成。

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 ((-SMA) 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 (-SMA 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 (, and Pur ( with the (-SMA and type I collagen promoters and clarify how these proteins are affected by pro- fibrotic agents such as TGF(1 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 (-SMA 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 TGF(1-regulated interaction of YB-1 and Pur protein repressors with (-SMA 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 TGF(1 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 TGF(1/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 TGF(1 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. Project Narrative: The proposed research investigates cellular and molecular changes in connective tissue cells in heart and lung tissues that are required for normal wound healing after surgery. In transplant patients as well as individuals diagnosed with various forms of chronic cardiopulmonary diseases, these processes are accelerated leading to permanent scar formation, structural deformities in the heart and lung, loss of tissue function, and early death. New research studies outlined in this application will examine the individual biochemical pathways that drive normal wound healing and attempt to identify new targets for drug therapy that will help control destructive wound healing activity and improve the quality of life for individuals suffering from chronic diseases that cause abnormal scar tissue formation in the heart and lung.