在角膜损伤后纤维化形成的期间,III型中间丝(IF)蛋白波形蛋白和结蛋白在基质肌成纤维细胞中依次过表达。先前的研究发现,波形蛋白缺陷小鼠在碱损伤后可显著防止角膜纤维化,这表明这种IF蛋白是角膜纤维化的重要调节因子。尚未证明结蛋白是否以任何重要方式导致角膜纤维化。在这里,我们在角膜碱损伤模型中使用了结蛋白缺陷(DesKO)小鼠,并显示受伤的DesKO小鼠在损伤后14天发展为纤维化,并显示出与野生型(WT)小鼠相似的角膜混浊水平,并且即使在损伤后30d仍保留该表型。损伤小鼠的DesKO角膜显示波形蛋白和α-平滑肌肌动蛋白表达上调至与WT角膜相当的水平,阐明结蛋白缺乏不会干扰肌纤维分化。使用含AfferinA(WFA)的小分子,波形蛋白的抑制剂,我们表明,WFA治疗导致波形蛋白和丝氨酸38磷酸化波形蛋白的稳态水平降低,后者是与角膜纤维化相关的生物标志物,并通过阻断肌成纤维细胞分化改善角膜透明度。为了进一步探讨结蛋白缺乏的纤维化机制,我们检查了上皮中角蛋白8的表达,并发现与WT角膜相比,受伤的DesKO角膜中这种细胞角蛋白的水平降低。该发现还证实了与WT角膜相比,受损的DesKO角膜中细胞增殖的减少。DesKO角膜的纤维化表型还具有丰富的血管形成,进一步举例说明角膜病理的程度。一起,这些发现表明,在这种损伤模型中,结蛋白对角膜纤维化没有显著贡献。
The type III intermediate filament (IF) proteins vimentin and desmin are sequentially overexpressed in stromal myofibroblasts over the period when fibrosis sets in after corneal injury. Prior findings have revealed vimentin-deficient mice are significantly protected from corneal fibrosis after alkali injury, which has implicated this IF protein as an important regulator of corneal fibrosis. It has remained as yet unproven whether desmin contributes in any significant manner to corneal fibrosis. Here we have employed desmin-deficient (Des KO) mice in the corneal alkali injury model and show that injured Des KO mice develop fibrosis and show similar levels of corneal opacity at 14 days post-injury as wild type (WT) mice and retain this phenotype even at 30d post injury. Des KO corneas from injured mice show upregulation of vimentin and alpha-smooth muscle actin expression to equivalent levels as WT corneas, illuminating that desmin deficiency does not interfere with myofibrobast differentiation. Employing the small molecule withaferin A (WFA), an inhibitor of vimentin, we show that WFA treatment causes the decrease in steady state levels of vimentin and serine 38 phosphorylated vimentin, the latter a biomarker associated with corneal fibrosis, and improved corneal clarity through blockade of myofibroblast differentiation. To investigate further the mechanism of fibrosis in desmin deficiency, we examined keratin 8 expression in the epithelium, and found reduced levels of this cytokeratin in injured Des KO corneas compared to WT corneas. This finding also corroborates the decrease of cell proliferation in injured Des KO corneas compared to that in WT corneas. The fibrotic phenotype of Des KO corneas also features abundant vascularization, further exemplifying the magnitude of corneal pathology. Together, these findings illuminate that desmin does not contribute significantly to corneal fibrosis in this injury model.