Regulation of Stromal Wound Healing by Growth Factors

描述（由申请人提供）：角膜感染后瘢痕形成；

DESCRIPTION (provided by applicant): Corneal scarring following infection, trauma, or surgery can lead to impaired vision, yet relatively little is known about which genes contribute to corneal scarring or what molecules regulate changes in expression of these fibrotic genes. Our-overall objective is to more fully define the molecular regulation of corneal wound healing, and to develop agents that will control scaring. In our previous grant, we hypothesized that the TGFbeta system played a dominant role in regulating corneal scarring, and we measured changes in the TGFbeta system during healing of a standardized excimer ablation wound model in rat corneas that strongly support this hypothesis. We also developed agents (antisense oligonucleotides and ribozymes) that selectively inhibited the TGFbeta system in vitro, and we established basic parameters for in vivo transfection of corneal cells with oligonucleotides and plasmids. During our investigations of the TGFbeta system, we identified a new downstream growth factor connective tissue growth factor (CTGF) that mediated the effects of TGFbeta on synthesis of collagen in corneal fibroblasts. In this grant, we enlarge our previous hypothesis to include investigating the actions of CTGF on corneal scarring. Specifically, we hypothesize that CTGF increases corneal scar formation. We further hypothesize that: TGFbeta will upregulate CTGF expression; that CTGF is the down stream mediator of many of the actions of TGFbeta on corneal fibroblasts; levels of CTGF and CTGF-R mRNA and protein will increase during corneal scarring; the type II IGF receptor is the CTGF receptor; CTGF will regulate multiple genes involved in scarring; selective reduction of CTGF and CTGF-R by antisense oligonucleotides or ribozymes will reduce corneal scarring; expression of genes during healing of corneal wounds will change in clustered patterns that are determined by the function and by the molecular regulators of the genes. We will test these hypotheses in 5 specific aims. Specific Aim #1 will measure the changes in CTGF and CTGF-R levels in rat corneas during healing at 9 time points up to 90 days after excimer ablation. Specific Aim #2 will determine CTGF-induced changes in gene expression in cultures of human corneal fibroblasts using Affymetrix microarrays. Specific Aim #3 will identify the CTGF receptor and develop and test antisense oligonucleotides and ribozymes targeting CTGF and CTGF-R genes in-vitro. Specific Aim #4 will test the antisense oligonucleotides and ribozymes targeting TGFbeta, CTGF and CTGF-R for in vivo reduction of scarring in excimer ablated corneas. Specific Aim #5 will determine the patterns of changes in gene expression at 12 times during healing of rat corneal wounds using Affymetrix microarrays then use the clustered data to test previous hypotheses about the roles of genes implicated in corneal scarring, generate new hypotheses about the regulation of corneal scar formation, and identify new target genes to reduce scar formation. These integrated, hypothesis-based experiments will increase our general understanding of how corneal scarring is regulated at the molecular level, will test the new hypotheses that CTGF regulates important aspects of corneal scarring and will evaluate new selective anti-scarring agents.