Regulation of Stromal Wound Healing

描述(申请人提供)：因外伤、传染病或屈光手术造成的角膜伤口结疤过多，导致视力受损。试图用抗炎类固醇或抗代谢产物(5FU，丝裂霉素-C)来减少瘢痕形成会引起副作用，并慢性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，然后设计基因靶向的方法来减少瘢痕形成。在我们之前的资助中，我们专注于从生化角度确定两种关键的纤维化生长因子TGFp和CTGF在角膜瘢痕形成中的作用。我们发现，在培养的人角膜成纤维细胞(HCF)中，TGFp上调CTGF的表达，并且CTGF介导的TGFp刺激胶原合成和基质收缩。我们鉴定了CTGF受体(M6P/IGF-II-R)，发现在大鼠准分子激光角膜损伤愈合过程中，CTGF的mRNA、蛋白和受体水平均显著升高。我们认为MEK1/2、ERK1/2、STATS级联信号通路是HCF中CTGF的主要信号转导途径。准分子激光切割大鼠角膜的Affymetrix微阵列分析显示，包括角膜晶体蛋白、TGFp和CTGF在内的基因表达模式发生了重大变化。我们开发了核酶和反义寡核苷酸，选择性地降低了HCF培养中TGFp和CTGF的mRNA和蛋白水平，并为AAV载体在兔角膜中的转导奠定了条件。在这项新的授权中，我们将扩展这些结果，以进一步了解TGFp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的蛋白水解性切割成刺激细胞增殖的N末端片段和刺激胶原合成的C末端片段。我们将(1)鉴定将CTGF裂解成N/C末端片段的细胞蛋白酶；(2)鉴定准分子消融伤口愈合过程中N/C末端蛋白质的比例；(3)鉴定N/C末端片段的受体并建立它们在HCF中的信号转导途径；(4)充分表征HCF和CTGF基因敲除成纤维细胞(Affymetrix微阵列)中N/C末端蛋白质的基因表达模式；(5)利用核酶和shRNA组合优化TGFp和CTGF mRNAs和蛋白质的下调；(6)评估自互补AAV(ScAAV)载体的核酶和shRNA的组合表达，该载体可以在兔准分子激光角膜创伤愈合模型中快速启动并高表达转基因。这些综合实验将扩大对TGFp和CTGF系统对角膜瘢痕形成分子调控的理解，并评估基因靶向治疗控制角膜瘢痕形成的翻译。

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.