Matrix Regulation of Cell Function During Wound Healing

病理性瘢痕和纤维化是重要的临床问题，具有潜在的严重性。 对患者的后果，包括损害正常组织再生和邻近组织 功能。我们的长期目标是确定控制成纤维细胞的关键调控机制 分化为肌成纤维细胞，从而开发控制瘢痕形成和纤维化的新策略。转化生长因子- ~是这一机制的中心成分，我们最近确定了一种焦点黏附蛋白， Hic-5是由转化生长因子-β在正常人真皮成纤维细胞中诱导的；Hic-5也持续表达 在增生性瘢痕肌成纤维细胞(HTSF)中。重要的是，Hic-5在HTSF中是产生转化生长因子-~的必需的 从而通过转化生长因子-β自分泌环路调节自身的表达。此外，当Hic-5被击落时 在RNAi作用下，HTSF表型恢复为支持治疗性治疗的正常真皮成纤维细胞的表型 通过靶向肌成纤维细胞Hic-5表达来阻断纤维化的策略。我们提议为期两年， ARRA赞助的研究计划，在该计划中，我们检验了转化生长因子-5诱导Hic-5表达的假设 通过Rho GTP酶依赖的途径(S)，从而建立自分泌环路和持久的 肌成纤维细胞表型。在目标1中，我们将定义调节转化生长因子依赖的细胞内通路， HIC-5在肌成纤维细胞中的表达。Hic-5在正常人体内的诱导机制 成纤维细胞和致病成纤维细胞(HTSFs)的永生化 将会被确定。实验将使用药物抑制剂，遗传沉默 (RNAi)和cDNA重组，在体外。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和测量活性的变化。在目标2中，我们将 鉴定和分析增生性瘢痕中的Hic-5调控基因。将使用表达(CDNA)阵列 鉴定hTSF中受Hic-5调控的基因产物。这些将与基因相互参照。 其他人报告的基因在增生性瘢痕中受到明显调控。HIC-5依赖基因产物 从这些cDNA阵列中鉴定出的候选基因也将被分析为更容易调节转化生长因子-1的基因。 自分泌循环或介导特定的肌成纤维细胞功能，关键基因产物将被整合 进入定量聚合酶链式反应(QPCR)阵列，并用从新鲜切除的肥大组织中获得的RNA进行探测 伤痕累累。将采用免疫组织化学的方法来确认这些Hic-5- 疤痕中的依赖基因产物。从这项工作中获得的发现将填补 了解病理性肌成纤维细胞的调节并提出新的治疗策略来调节 严重烧伤或创伤性损伤后的纤维化。

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.