Regulation of Stromal Wound Healing by Growth Factors

描述（由申请人提供）：由于没有fda批准的药物声称可以减少光散射角膜雾霭，外伤、手术或感染引起的视力受损角膜瘢痕仍然是一个主要的临床问题。抗炎类固醇的临床试验没有显示出统计学上显著的益处，丝裂霉素C治疗有严重副作用的风险。细胞培养研究、动物实验和近期的临床试验均有力地表明，由转化生长因子β (TGFb)诱导的结缔组织生长因子（CTGF）是角膜及其他组织（包括皮肤和肾脏）中主要的瘢痕诱导生长因子。CTGF通过上调不规则细胞外疤痕基质的合成，诱导静止的角质细胞转化为活化的成纤维细胞和肌成纤维细胞，从而刺激角膜疤痕的形成，这两种细胞共同产生了角膜疤痕的大部分光散射。我们的总体目标是了解角膜创面愈合的基本细胞和分子调控，并应用这些知识开发有效消除角膜瘢痕的治疗方法。在这项资助中，我们提出了四个具体的目标，进一步扩大我们对角膜伤口愈合的细胞和分子调节的基本知识，并将这些信息转化为防止角膜瘢痕形成的新疗法。Specific Aim #1将进一步评估CTGF在调节上皮愈合、基质瘢痕形成和角膜雾霭消退中的作用，使用我们在上次拨款期间开发的系统性和角膜上皮细胞特异性条件CTGF敲除（KO）转基因小鼠菌株。角膜创面愈合的表观遗传调控是一个重要但尚未开发的领域。使用PCR阵列，我们鉴定了44个microRNAs (mir)，在准分子消融后30分钟和24小时显著改变小鼠角膜的表达水平，包括miR-133b和miR-22，它们靶向TGFb1、CTGF、TGFb1、TGFbRI和胶原等关键疤痕基因的mrna。特异性目标#2将进一步表征角膜瘢痕形成和消退过程中关键时期miR表达模式的变化（第3,6和9天），并将利用小鼠角膜细胞培养物评估靶向关键瘢痕基因的其他pre-miR和anti-miR的效果。从细胞培养研究过渡到动物研究，特异性目标#3将比较准分子切除兔角膜治疗与特异性目标#2中确定的最有效的miR药物（例如，pre- miR-133b或anti-miR-22）的抗疤痕作用。此外，我们将测试一种有效的sirna三重组合，该组合是我们在上一次拨款中开发的，专门针对TGFb， TGFbRII和CTGF mrna。最后，我们假设最大限度地减少角膜疤痕将通过基因靶向药物和广泛的基因靶向表观遗传药物的组合来实现。为了验证这一假设，Specific Aim #4将使用基因特异性药物（sirna或ASOs）和广泛基因靶向表观遗传药物（miRs或SAHA，一种组蛋白去乙酰化酶抑制剂）的最佳组合来评估准分子消融兔角膜的抗疤痕效果。

DESCRIPTION (provided by applicant): Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, since there are no FDA-approved drugs with the claim of reducing light-scattering corneal haze. Clinical trials of anti-inflammatory steroids have not shown statistically significant benefits, and mitomycin C treatment carries the risk serious side effects. Cell culture studies, animal experiments, and recent clinical trials strongly indicate that connective tissue growth factor (CTGF), which is induced by transforming growth factor beta (TGFb), is the dominant scar-inducing growth factor in the cornea and in other tissues, including skin and kidney. CTGF stimulates corneal scar formation 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 cellular and molecular regulation of corneal wound healing and apply that knowledge to develop therapies that effectively eliminate corneal scarring. In this grant, we propose four specific aims that further expand our basic knowledge of cell and molecular regulation of corneal wound healing and translate that information into new treatments that prevent corneal scarring. Specific Aim #1 will further assess the roles of CTGF in regulating epithelial healing and stromal scarring and regression of corneal haze using systemic and corneal epithelial cell specific conditional CTGF knockout (KO) transgenic mice strains that we developed during the last grant. Epigenetic regulation of corneal wound healing is an important, but unexplored area. Using PCR arrays, we identified 44 microRNAs (miRs) that significantly change expression levels in mouse corneas at 30 minutes and at 24 hours after excimer ablation, including miR-133b and miR-22, which target mRNAs of key scarring genes including TGFb1, CTGF, TGFb1, TGFbRI, and collagen. Specific Aim #2 will further characterize changes in patterns of miR expression at key times during corneal scar formation and resolution (days 3, 6, and 9 days) and will assess the effects of additional pre-miRs and anti-miRs that target key scarring genes using cultures of mouse corneal cells. Transitioning from cell culture studies to animal studies, Specific Aim #3 will compare the anti-scarring effects in excimer ablated rabbit corneas of treatment with the most effective miR drugs identified in Specific Aim #2 (e.g., pre- miR-133b or anti-miR-22). In addition, we will test a potent, triple combination of siRNAs we developed during the last grant that specifically targets TGFb, TGFbRII and CTGF mRNAs. Finally, we hypothesize that maximum reduction of corneal scarring will be achieved by using a combination of gene targeted drugs and broad gene targeting epigenetic drugs. To test this hypothesis, Specific Aim #4 will evaluate antiscarring effects in excimer ablated rabbit corneas using the optimal combination of a gene specific drug (siRNAs or ASOs) and a broad gene targeting epigenetic drug (miRs or SAHA, a histone deacetylase inhibitor).