Regulation of Stromal Wound Healing

描述（由申请人提供）：由创伤、传染病或屈光手术引起的角膜伤口瘢痕过多导致视力受损。试图用抗炎类固醇或抗代谢物（5 FU，丝裂霉素-C）减少瘢痕形成可能会导致副作用，并长期降低角膜基质细胞密度。我们的长期目标是了解角膜瘢痕形成的分子调控，然后设计基因靶向方法来减少瘢痕形成。在我们之前的资助中，我们专注于生物化学定义两个关键的纤维化生长因子TGF β和CTGF在角膜瘢痕形成中的作用。我们发现，在人角膜成纤维细胞（HCF）培养物中，TGF β上调CTGF的表达，并且CTGF介导TGF β刺激胶原合成和基质收缩。我们鉴定了CTGF受体（M6 P/IGF-II-R），发现在大鼠角膜准分子激光损伤愈合过程中，CTGF mRNA、蛋白和受体水平均显著增加。我们发现，MEK 1/2，ERK 1/2，STATS级联反应是HCF中CTGF的主要信号通路。准分子消融的大鼠角膜的Affyellow微阵列分析显示基因表达模式的主要变化，包括角膜晶体蛋白，TGF β和CTGF。我们开发了核酶和反义寡核苷酸，其选择性地降低HCF培养物中TGF β和CTGF mRNA和蛋白水平，并建立了兔角膜中AAV载体转导的条件。在这项新的资助中，我们将扩展这些结果，以进一步了解TGF β和CTGF在角膜瘢痕形成中的机制和生理作用，包括最近发现的CTGF蛋白水解裂解成刺激细胞增殖的N端片段和刺激胶原蛋白合成的C端片段。我们将（1）鉴定将CTGF切割成N/C末端片段的细胞蛋白酶;（2）表征在准分子消融伤口愈合过程中N/C末端蛋白的比例;（3）鉴定N/C末端片段的受体并建立它们在HCF中的信号转导通路;（4）充分表征HCF和CTGF敲除成纤维细胞中N/C末端蛋白的基因表达模式（5）使用核酶和shRNA组合优化TGF β和CTGF mRNA和蛋白质的敲低;（6）评估来自自身互补AAV（scAAV）的组合的核酶和shRNA表达载体，允许在角膜准分子激光伤口愈合的兔模型中快速开启高表达的转基因。这些综合实验将扩大对TGF β和CTGF系统对角膜瘢痕形成的分子调节的理解，并评估基因靶向治疗的翻译以控制角膜瘢痕形成。

DESCRIPTION (provided by applicant): Excessive scarring of corneal wounds caused by trauma, infectious diseases or refractive surgery leads to impaired vision. Attempts to reduce scarring with anti-inflammatory steroids or anti-metabolites (5FU, mitomycin-C) can cause side effects and chronically reduce corneal keratocyte density. Our long term goals are to understand the molecular regulation of corneal scarring and then design gene-targeted approaches to reduce scarring. In our previous grant, we focused on biochemically defining the roles of two key fibrogenic growth factors, TGFp and CTGF, in corneal scarring. We showed that TGFp up-regulated expression of CTGF and that CTGF mediated TGFp-stimulated collagen synthesis and matrix contraction in cultures of human corneal fibroblasts (HCF). We identified the CTGF receptor (M6P/IGF-II-R) and found that CTGF mRNA, protein and receptor levels all dramatically increased during healing of excimer laser injuries of rat corneas. We showed that the MEK1/2, ERK1/2, STATS cascade is a major signaling pathway for CTGF in HCF. Affymetrix microarray analyses of excimer ablated rat corneas showed major changes in patterns of gene expression including corneal crystallins, TGFp and CTGF. We developed ribozymes and antisense oligonucleotides that selectively reduced TGFp and CTGF mRNA and protein levels in HCF cultures and established conditions for AAV vector transduction in rabbit corneas. In this new grant, we will extend these results to further understand the mechanism and physiological roles of TGFp and CTGF in corneal scarring, including the recently discovered proteolytic cleavage of CTGF into a N-terminal fragment that stimulates cell proliferation and a C-terminal fragment that stimulates collagen synthesis. We will (1) identify the cellular protease that cleaves CTGF into N/C terminal fragments; (2) characterize the ratio of N/C terminal proteins during healing of excimer ablation wounds; (3) identify receptors for the N/C-terminal fragments and establish their signal transduction pathways in HCF; (4) fully characterize gene expression patterns for N/C- terminal proteins in HCF and CTGF knockout fibroblasts (Affymetrix microarray); (5) optimize knock down of TGFp and CTGF mRNAs and proteins using a ribozyme and shRNA combination; (6) evaluate combined ribozyme and shRNA expression from a self complementary AAV (scAAV) vector that allows rapid turn on with high expression of transgenes in a rabbit model of corneal excimer laser wound healing. These integrated experiments will expand the understanding of molecular regulation of corneal scarring by the TGFp and CTGF systems and evaluate translation of gene-targeted therapy to control corneal scarring.