Regulation of Stromal Wound Healing by Growth Factors

描述（由申请人提供）：由创伤、手术或感染引起的损伤后视力受损的角膜瘢痕形成仍然是一个主要的临床问题，但没有FDA批准的药物声称可以减少光散射角膜混浊。抗炎类固醇的临床试验没有显示出统计学显著的益处，丝裂霉素C治疗有严重副作用的风险。细胞培养研究、动物实验和最近的临床试验强烈表明，结缔组织生长因子（CTGF）是角膜和其他组织（包括皮肤和肾脏）中的主要瘢痕诱导生长因子。CTGF通过上调不规则细胞外瘢痕基质的合成并诱导静止的角膜细胞转化为活化的成纤维细胞和肌成纤维细胞来产生角膜瘢痕，所述活化的成纤维细胞和肌成纤维细胞联合收割机产生角膜瘢痕中的大部分光散射。我们的总体目标是了解角膜伤口愈合的基本分子调控，并将这些知识应用于开发更有效的治疗方法以减少角膜瘢痕。在这项新提案中，我们提出了三个具体目标，进一步扩大我们对CTGF系统在角膜伤口愈合中的知识基础，并将这些信息转化为消除/减少角膜瘢痕形成的新临床疗法。具体目标#1将使用三种方法进一步定义CTGF在调节角膜瘢痕形成中的关键作用。我们将使用CTGF启动子-GFP转基因小鼠和上皮细胞、基质细胞和内皮细胞样品的Q-RT-PCR分析来鉴定角膜中在伤口愈合期间合成CTGF的细胞。我们将确定通过使用我们将开发的新的CTGF条件性敲除小鼠品系（CTGFfloxed/floxed CreCAG-cre/Esr/+）消除受伤角膜中的CTGF合成可以实现的角膜瘢痕形成的最大减少，该小鼠品系在暴露于他莫昔芬后停止CTGF合成。我们还将使用这种独特的CTGF条件性敲除小鼠品系评估其他生长因子系统是否可以部分补偿CTGF合成的消除并有助于瘢痕形成。角膜伤口愈合的表观遗传调控是一个相对未探索的领域。具体目标#2将使用miRNA微阵列研究miRNA模式的变化。我们还将评估两类表观遗传药物microRNA（miRNA）和组蛋白去乙酰化酶抑制剂（HDI）对角膜上皮细胞、成纤维细胞CTGF合成的影响。 和内皮细胞。最后，我们假设，最大限度地减少角膜瘢痕形成将实现基因靶向和广泛基础的表观遗传敲除的组合。因此，具体目标#3将评价CTGF-ASO和表观遗传治疗的组合的抗瘢痕形成作用，所述表观遗传治疗使用HDI、SAHA和微RNA、mir26 a和mir18 a，所述微RNA和mir26 a和mir18 a已被报道在其他细胞类型中减少CTGF的合成。 公共卫生相关性：由创伤、手术或感染引起的损伤后视力受损的角膜瘢痕形成仍然是一个主要的临床问题，但没有药物能有效地减少角膜瘢痕形成而没有严重副作用的风险。(1)我们的研究确定了两种生长因子是角膜瘢痕形成的主要调节因子，转化生长因子β（TGF β）及其下游介质结缔组织生长因子（CTGF）。(4)这笔赠款将继续我们的基础研究，以了解CTGF系统如何在角膜伤口愈合过程中使用CTGF条件性敲除小鼠和细胞培养系统发挥作用，并将这些知识转化为新型药物疗法，减少角膜瘢痕形成，而不会产生严重的长期副作用使用特定的基因靶向疗法和广泛的表观遗传疗法。

DESCRIPTION (provided by applicant): Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, yet no drugs are FDA-approved with the claim of reducing light-scattering corneal haze. Clinical trials of anti-inflammatory steroids have not shown statistically significant benefit, and mitomycin C treatment carries the risk serious side effects. Cell culture studies, animal experiments, and recent clinica trials strongly indicate that connective tissue growth factor (CTGF) is the dominant scar-inducing growth factor in the cornea and in other tissues, including skin and kidney. CTGF generates corneal scar by up-regulating synthesis of irregular extracellular scar matrix and inducing transformation of quiescent keratocytes into activated fibroblasts and myofibroblasts, which combine to produce the majority of light scattering in corneal scars. Our overall goal is to understand the basic molecular regulation of corneal wound healing and apply that knowledge in the development of even more effective therapies to reduce corneal scarring. In this new proposal, we propose three specific aims that further expand our knowledge base of the CTGF system in corneal wound healing and translate that information into new clinical therapies that eliminate/reduce corneal scarring. Specific Aim #1 will further define the key roles of CTGF in regulating corneal scarring using three approaches. We will identify the cells in the cornea that synthesize CTGF during wound healing using CTGF promoter-GFP transgenic mice and Q-RT-PCR analysis of samples of epithelial cells, stromal cells and endothelial cells. We will determine the maximum reduction in corneal scarring that can be achieved by eliminating CTGF synthesis in wounded corneas using a new CTGF conditional knockout mouse strain that we will develop that stops synthesis of CTGF after exposure to tamoxifen (CTGFfloxed/floxed CreCAG-cre/Esr/+). We will also use this unique CTGF conditional knockout mouse strain assess if other growth factor systems may partially compensate for elimination of CTGF synthesis and contribute to scarring. Epigenetic regulation of corneal wound healing is a relatively unexplored area. Specific Aim #2 will investigate changes in miRNAs patterns using miRNA microarrays. We also will assess the effects two classes of epigenetic drugs, microRNAs (miRNA) and histone deacetylase inhibitors (HDI), on synthesis of CTGF in corneal epithelial cells, fibroblasts and endothelial cells. Finally, we hypothesize that maximum reduction of corneal scarring will be achieved using a combination of gene targeted and broad based epigenetic knockdown. Therefore, Specific Aim #3 will evaluate antiscarring effects of a combination of CTGF-ASOs and epigenetic treatments using the HDI, SAHA, and microRNAs, mir26a, and mir18a that have been reported to reduce synthesis of CTGF in other cell types. PUBLIC HEALTH RELEVANCE: Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, yet no drugs effectively reduce corneal scarring without the risk of serious side effects.(1),(2),(3) Our research identified two growth factors that are the major regulators of scar formation in the cornea, transforming growth factor beta (TGFb), and its downstream mediator, connective tissue growth factor (CTGF).(4) This grant will continue our basic research to understand how the CTGF system functions during corneal wound healing using CTGF conditional knockout mice and cell culture systems and translate that knowledge into novel drug therapies that reduce corneal scarring without producing serious, long-term side effects using both specific gene-targeted therapies and broad-based epigenetic therapies.