Matrix Regulation of Cell Function During Wound Healing

致病性瘢痕形成和纤维化是重要的临床问题， 对患者造成的后果，包括正常组织再生和邻近组织受损 功能我们的长期目标是确定控制成纤维细胞的关键调节机制， 分化成肌成纤维细胞，从而开发控制瘢痕形成和纤维化的新策略。转化生长因子 ~是这种机制的核心成分，我们最近确定了一种粘着斑蛋白， Hic-5在正常人皮肤成纤维细胞中由TGF-β 1诱导表达，Hic-5在正常人皮肤成纤维细胞中也持续表达。 增生性瘢痕肌成纤维细胞（HTSF）。重要的是，Hic-5是HTSF中TGF-α产生所必需的 从而通过TGF-β自分泌环调节其自身的表达。此外，当Hic-5被击倒时， 利用RNAi，HTSF表型回复到正常真皮成纤维细胞的表型，支持治疗性的 通过靶向肌成纤维细胞中Hic-5表达阻断纤维化的策略。我们提议一个为期两年， ARRA赞助的研究项目中，我们测试了TGF-β诱导Hic-5表达的假设。 通过Rho GTP酶依赖性途径，从而建立自分泌环和持久的 肌成纤维细胞表型在目的1中，我们将定义调节TGF-β依赖性的细胞内途径， 肌成纤维细胞中的Hic-5表达。正常人Hic-5的诱导机制 致病性成纤维细胞（HTSF） 将被确定。实验将使用药理学抑制剂，遗传沉默 （RNAi）和cDNA重建。Hic-5和候选信号分子的水平将是 使用磷酸化特异性抗体测定活性变化。在目标2中，我们将 增生性瘢痕中Hic-，5调控基因的鉴定与分析。将使用表达（cDNA）阵列 鉴定HTSF中受Hic-5调控的基因产物。这些将被交叉引用到基因 在增生性瘢痕中有明显的调节。Hic-5依赖性基因产物 从这些cDNA阵列中鉴定的基因也将作为候选基因进行分析，这些候选基因也将调节TGF-β 1的表达。 自分泌环或介导特定的肌成纤维细胞功能，然后将纳入关键基因产物 定量PCR（qPCR）阵列，并用从新鲜切除的肥大细胞中获得的RNA进行探测。 伤疤将采用免疫组织学来确认这些Hic-5的表达和定位。 瘢痕中的依赖性基因产物。从这项工作中获得的结果将填补以下方面的重要空白： 了解致病性肌成纤维细胞调节，并提出新的治疗策略， 严重烧伤或创伤后纤维化。

Pathogenic scarring and fibrosis represent important clinical problems with potentially serious consequences for patients, including impairment of normal tissue regeneration and neighboring tissue function. Our long-term goal is to identify the critical regulatory mechanisms that govern fibroblast differentiation Into myofibroblasts thereby developing novel strategies to control scarring and fibrosis. TGF- ~ is a central component of this mechanism and we recently determined that a focal adhesion protein, termed Hic-5, is induced by TGF-~ in normal human dermal fibroblasts; Hic-5 is also persistently expressed in hypertrophic scar myofibroblasts (HTSF). Importantly, Hic-5 is required for TGF-~ production in HTSF thereby regulating its own expression via the TGF-~ autocrine loop. Moreover, when Hic-5 is knocked down with RNAi the HTSF phenotype reverts to that of a normal dermal fibroblast supporting a therapeutic strategy of blocking fibrosis by targeting Hic-5 expression in myofibroblasts. We propose a two-year, ARRA sponsored, research program in which we test the hypothesis that TGF-~ induces Hic-5 expression through Rho GTPase-dependent pathway(s) thereby establishing an autocrine loop and a persistent myofibroblast phenotype. In Aim 1, we will define the intracellular pathways that regulate TGF-~-dependent, Hic-5 expression in myofibroblasts. The mechanisms through which Hic-5 is induced in normal human fibroblasts and perpetuated in pathogenic fibroblasts (HTSFs) will be determined. Experiments will be performed using pharmacological inhibitors, genetic silencing (RNAi) and cDNA reconstitution, in vitro. The levels of Hic-5 and candidate signaling molecules will be determined and changes in activity measured using phosphorylation-specific antibodies. In Aim 2, we will identify and analyze Hic-,5 regulated genes in hypertrophic scars. Expression (cDNA) arrays will be used to identify the genes products in HTSF that are regulated by Hic-5. These will be cross-referenced to genes that others have reported are markedly regulated in hypertrophic scars. Hic-5-dependent gene products identified from these cDNA arrays will also be ,analyzed as candidate genes that eitlier reguiate the TGF-~ autocrine loop or mediate specific myofibroblast functions, Key gene products will then be incorporated into quantitative PCR (qPCR) arrays and probed with RNA obtained from freshly resected hypertrophic scars. Immunohistology will be employed to confirm the expression and localization of these Hic-5- dependent gene products in scars. Findings obtained from this work will fill important gaps in understanding pathogenic myofibroblast regulation and suggest new therapeutic strategies to modulate fibrosis following serious burn or traumatic injury.