The role of PPAR{gamma} ligands in corneal wound healing and optics

描述（申请人提供）：角膜瘢痕形成是世界范围内视力下降和视力丧失的主要原因。无论是感染、激光屈光手术、角膜移植、眼外伤（化学或物理）还是角膜营养不良，都会对正常的角膜结构和功能造成破坏。没有合适的方法来控制角膜瘢痕，尽管超过25年试图表征伤口愈合过程中的细胞因子信号。我们的长期目标是了解角膜伤口愈合的机制，我们的目标是设计有效的治疗或预防角膜瘢痕形成的疗法。我们的总体假设是，过氧化物酶体增殖物激活受体γ (PPAR3)配体可以预防角膜纤维化，并且可以作为这种情况的靶向治疗，其疗效明显高于目前的临床治疗或阻断单一细胞因子的活性。目的1：验证PPAR3配体抑制培养角膜角质细胞关键促纤维化活性的假设。我们使用免疫组织化学、增殖试验、损伤试验、western blots、slot blots和Q-PCR来评估PPAR3配体在调节最佳剂量TGF2刺激下培养的角化细胞增殖、迁移、CTGF、1SMA、Thy-1、胶原I、胶原III、纤维连接蛋白及其mrna表达方面的相对有效性。目的2：验证PPAR3配体通过PPAR3依赖性和非依赖性途径抑制培养角膜角质细胞关键促纤维化活性的假设。我们将使用药理学和遗传学方法来验证我们的预测，即PPAR3配体通过激活PPAR3和抑制tgf2调节的途径来起作用。如果我们要开发PPAR3配体作为角膜纤维化的最佳靶向治疗方法，了解这些机制在角膜角质细胞中的相对优势是至关重要的。目的3：验证PPAR3配体在PRK诱导的角膜伤口愈合中比抗tgf2抗体、类固醇或丝裂霉素c更有效地抑制纤维化的假设。我们将在猫身上进行双目PRK，然后局部给药选择PPAR3配体、抗tgf2抗体、类固醇或丝裂霉素c。我们将使用免疫组织化学来对比伤口愈合反应的关键细胞方面。我们预测，与术后使用抗tgf2抗体、类固醇或丝裂霉素C相比，PPAR3配体将与更低的细胞死亡、更快的伤口愈合、更少的阴霾和更低的高阶光学像差的诱导相关。

DESCRIPTION (provided by applicant): Corneal scarring is a major cause of decreased visual quality and vision loss worldwide. Scarring follows disruption to normal corneal structure and function, whether from infection, laser refractive surgery, corneal transplantation, ocular trauma (chemical or physical) or corneal dystrophies. There is no suitable means of controlling corneal scaring despite more than 25 years trying to characterize cytokine signaling during wound healing. Our long-term objective is to understand mechanisms of corneal wound healing and our goal is to design effective therapies to treat or prevent corneal scarring. Our overall hypothesis is that peroxisome proliferator activated receptor gamma (PPAR3) ligands prevent corneal fibrosis and can be targeted as a therapy for this condition, with significantly greater efficacy than current clinical treatments or than blocking the activity of single cytokines. Aim 1: Test the hypothesis that PPAR3 ligands inhibit key pro-fibrotic activities of cultured corneal keratocytes. We use immunohistochemistry, proliferation assays, wounding assays, western blots, slot blots and Q-PCR to assess the relative effectiveness of PPAR3 ligands at modulating proliferation, migration, expression of CTGF, 1SMA, Thy-1, collagen I, collagen III, fibronectin and their mRNAs in cultured keratocytes stimulated by optimal doses of TGF2. Aim 2: Test the hypothesis that PPAR3 ligands inhibit key pro-fibrotic activities in cultured corneal keratocytes through both PPAR3-dependent and -independent pathways. We will use both pharmacological and genetic approaches to test our prediction that PPAR3 ligands act both by activating PPAR3 and by inhibiting TGF2-regulated pathways. Knowing the relative strengths of these mechanisms in corneal keratocytes is critical if we are to develop PPAR3 ligands as optimally-targeted therapies for corneal fibrosis. Aim 3: Test the hypothesis that PPAR3 ligands are more efficient inhibitors of fibrosis in PRK-induced corneal wound healing than anti-TGF2 antibodies, steroids or Mitomycin C. We will perform binocular PRK in cats followed by the topical administration of select PPAR3 ligands, anti-TGF2 antibodies, steroids or Mitomycin C. We will use immunohistochemistry to contrast key cellular aspects of the wound healing reaction. We predict that PPAR3 ligands will be associated with lower cell death, faster wound healing, less haze and lower induction of higher-order optical aberrations than use of anti-TGF2 antibodies, steroids or Mitomycin C post-operatively.