University of Rochester School of Medicine and Dentistry, University of Rochester, Rochester, NY, United States.
Exp Eye Res. 2012 Jan;94(1):136-45. doi: 10.1016/j.exer.2011.11.018. Epub 2011 Dec 8.
A critical component of corneal scarring is the TGFβ-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-methyl ester (CDDO-Me) and 15-deoxy-Δ(-12,14)-prostaglandin J(2) (15d-PGJ(2)) for inhibiting TGFβ-induced myofibroblast differentiation in vitro. TGFβ was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ(2) were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFβ-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ(2)) potently inhibited TGFβ-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ(2) are strong inhibitors of TGFβ-induced corneal fibroblast to myofibroblast differentiation in vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.
角膜瘢痕形成的一个关键组成部分是 TGFβ诱导角膜成纤维细胞向肌成纤维细胞分化。这种分化的抑制剂可能对角膜瘢痕形成具有治疗作用。在这项研究中，我们测试了两种亲电过氧化物酶体增殖物激活受体γ (PPARγ)配体的相对有效性和作用机制：氰基-3,12-二氧代-1,9-二烯-28-酸甲酯 (CDDO-Me)和 15-脱氧-Δ(-12,14)-前列腺素 J2 (15d-PGJ2)，以抑制 TGFβ诱导的体外培养原代人角膜成纤维细胞向肌成纤维细胞分化。用 TGFβ诱导培养的原代人角膜成纤维细胞向肌成纤维细胞分化。将 CDDO-Me 和 15d-PGJ2 添加到培养物中，以测试它们抑制该过程的能力。通过测量肌成纤维细胞特异性蛋白 (αSMA、I 型胶原和纤维连接蛋白) 和 mRNA (αSMA 和胶原 III) 的表达来评估肌成纤维细胞分化。通过基因和药理学操作的细胞测试了这些药物抑制肌成纤维细胞分化中 PPARγ 的作用。最后，我们通过 Western blot 和免疫荧光法检测了这些药物在 TGFβ诱导的 αSMA 表达中的亲电性在作用中的重要性。两种亲电 PPARγ 配体 (CDDO-Me 和 15d-PGJ2) 均能强烈抑制 TGFβ诱导的肌成纤维细胞分化，但 PPARγ 仅部分参与两种药物对肌成纤维细胞分化的抑制。亲电 PPARγ 配体抑制肌成纤维细胞分化的能力强于非亲电 PPARγ 配体，这表明亲电性在该过程中起重要作用。CDDO-Me 和 15d-PGJ2 是体外 TGFβ诱导的角膜成纤维细胞向肌成纤维细胞分化的强抑制剂，表明这类药物可能是治疗角膜瘢痕的新方法，值得在临床前动物模型中进一步研究。
