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

描述(由申请人提供)：角膜疤痕是全球范围内视觉质量下降和视力丧失的主要原因。无论是由于感染、激光屈光手术、角膜移植、眼外伤(化学或物理)或角膜营养不良，瘢痕形成都会破坏正常的角膜结构和功能。尽管有超过25年的时间试图确定伤口愈合过程中的细胞因子信号，但目前还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创伤愈合的机制，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形成。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，可以作为治疗这种疾病的靶向，比目前的临床治疗方法或比阻断单一细胞因子的活性明显更有效。目的1：验证PPAR3配体抑制培养的角膜基质细胞主要促纤维化活性的假设。我们采用免疫组织化学、增殖分析、损伤分析、免疫印迹、缝隙印迹和Q-PCR等方法研究了PPAR3配体在最佳剂量的TGF2刺激下对培养的角质形成细胞的增殖、迁移、CTGF、1SMA、Thy-1、I型胶原、III型胶原、纤维连接蛋白及其mRNAs表达的影响。目的：验证PPAR3配体通过PPAR3依赖和非依赖途径抑制培养的角膜基质细胞主要促纤维化活性的假说。我们将使用药理学和遗传学方法来验证我们的预测，即PPAR3配体既通过激活PPAR3又通过抑制TGF2调节的途径发挥作用。如果我们要开发PPAR3配体作为治疗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强度是至关重要的。目的：验证PPAR3配体在PRK诱导的角膜创伤愈合中比抗TGF2抗体、类固醇或丝裂霉素C更有效地抑制纤维化的假设。我们将对猫进行双眼PRK，然后局部应用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方法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、更少的雾化和更低的高阶光学像差的诱导有关。 公共卫生相关性：目前减少眼外伤或手术后角膜纤维化和保持视觉质量的方法只有部分有效，且有显著的副作用。建议的实验验证了PPAR3配体通过激活多条促纤维化的细胞内通路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动的分子机制提供重要的理论见解，并使我们能够测试PPAR3配体作为一种新型的角膜纤维化原位治疗方法，与目前的治疗方法或阻断单一细胞因子的活性相比，其临床疗效显著更高，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.