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

描述（由申请人提供）：角膜瘢痕形成是全球视觉质量下降和视力丧失的主要原因。瘢痕形成是正常角膜结构和功能破坏的结果，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。尽管有超过25年的时间试图表征伤口愈合过程中的细胞因子信号传导，但没有合适的方法来控制角膜瘢痕形成。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方法来治疗或预防角膜瘢痕。我们的总体假设是，过氧化物酶体增殖物激活受体γ（PPAR 3）配体可预防角膜纤维化，并可作为治疗这种疾病的靶向药物，其疗效显著高于目前的临床治疗或阻断单一细胞因子的活性。目的1：检验PPAR 3配体抑制培养的角膜基质细胞的关键促纤维化活性的假设。我们使用免疫组化，增殖试验，创伤试验，Western印迹，狭缝印迹和Q-PCR来评估PPAR 3配体在调节最佳剂量的TGF 2刺激的培养角膜细胞中的增殖，迁移，CTGF，SMA，Thy-1，胶原I，胶原III，纤连蛋白及其mRNA的表达方面的相对有效性。目标二：检验PPAR 3配体通过PPAR 3依赖性和非依赖性途径抑制培养的角膜基质细胞中关键的促纤维化活性的假设。我们将使用药理学和遗传学的方法来测试我们的预测，即PPAR 3配体通过激活PPAR 3和抑制TGF 2调节的途径发挥作用。如果我们要开发PPAR 3配体作为角膜纤维化的最佳靶向疗法，了解这些机制在角膜基质细胞中的相对优势是至关重要的。目标3：检验PPAR 3配体在PRK诱导的角膜伤口愈合中是比抗TGF 2抗体、类固醇或丝裂霉素C更有效的纤维化抑制剂的假设。我们将在猫中进行双眼双盲，然后局部施用选择的PPAR 3配体、抗TGF 2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TGF 2抗体、类固醇或丝裂霉素C相比，PPAR 3配体将与较低的细胞死亡、较快的伤口愈合、较少的混浊和较低的高阶光学像差诱导相关。 公共卫生相关性：目前减少角膜纤维化和在眼损伤或手术后保持视觉质量的方法仅部分有效并具有显著的副作用。所提出的实验测试过氧化物酶体增殖物激活受体γ（PPAR 3）配体通过激活多个促纤维化细胞内途径来预防角膜纤维化的假设。这些结果将为控制角膜纤维化活性的分子机制提供重要的理论见解，并使我们能够测试PPAR 3配体作为角膜纤维化的新型原位疗法，其临床有效性显着高于目前的治疗方法或阻断单一细胞因子的活性，副作用更少。

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. PUBLIC HEALTH RELEVANCE: Current approaches to reduce corneal fibrosis and preserve visual quality following eye injury or surgery are only partially effective and carry significant side effects. The proposed experiments test the hypothesis that peroxisome proliferator activated receptor gamma (PPAR3) ligands prevent corneal fibrosis by activating multiple pro-fibrotic intracellular pathways. The results will provide significant theoretical insights into the molecular mechanisms that control corneal fibrotic activity and allow us to test PPAR3 ligands as a novel in situ therapy for corneal fibrosis, with significantly greater clinical effectiveness and fewer side-effects than current treatments or than blocking the activity of single cytokines.