Abstract：
Corneal wound healing requires epithelial reorganization and stromal extracellular matrix (ECM) remodeling, with ECM proteins such as Tenascin C (TnC) regulating and maintaining corneal homeostasis. The N-terminal globular domain and C-terminal fibrinogen-related domains of TnC are separated by epidermal growth factor (EGF)-like repeats, and upto fifteen fibronectin type III domains (Tn fn). Overexpression of Tn fn 1-5 and its splice variants occurs in varied pathologies. We have previously used Tn64 (a single chain variable fragment antibody cognate to Tn fn 1-5) to establish roles of Tn fn 1-5 in fibrotic pathologies such as rheumatoid arthritis and posterior capsular opacification. Here, we show that Tn64 binds to Tn fn repeats 3-5 (which constitute the major site for binding of soluble fibronectin within TnC). Unlike other Tn fn domains, Tn fn 3-5 displays no inhibition of fibronectin matrix assembly. Rather, the Tn fn 3-5 construct is pro-fibrotic and elicits increased expression of fibronectin. We examined corneal epithelial as well as stromal wound healing through Tn64 binding to Tn fn 3-5, using a human corneal epithelial cell (HCEC) line, primary cultures of human corneal fibroblasts (HCFs), and an ex-vivo corneal organ culture model. Tn64 enhanced proliferation and adhesion of corneal epithelial cells, while inhibiting the migration of corneal fibroblasts and myofibroblasts. Tn64 appears to attenuate inflammation through downregulation of TNF-α, prevent corneal fibrosis by limiting fibronectin polymerization, and promote regeneration of corneal epithelia and stroma, suggesting that it could be developed as a therapeutic agent for effective anti-fibrotic corneal wound healing.
摘要：
角膜伤口愈合需要上皮重组和基质细胞外基质（ECM）重塑，与ECM蛋白如肌腱蛋白C（TnC）调节和维持角膜稳态。TnC的N端球形结构域和C端纤维蛋白原相关结构域被表皮生长因子（EGF）样重复序列分开，和多达15个纤连蛋白III型结构域（Tnfn）。Tnfn1-5及其剪接变体的过表达发生在各种病理中。我们以前已经使用Tn64（与Tnfn1-5同源的单链可变片段抗体）来建立Tnfn1-5在纤维化病理如类风湿性关节炎和后囊混浊中的作用。这里,我们显示Tn64结合Tnfn重复3-5(其构成TnC内可溶性纤连蛋白结合的主要位点)。与其他Tnfn域不同，Tnfn3-5显示对纤连蛋白基质组装没有抑制。相反，Tnfn3-5构建体是促纤维化的并引起纤连蛋白表达增加。我们使用人角膜上皮细胞（HCEC）线检查了通过Tn64与Tnfn3-5结合的角膜上皮和基质伤口愈合，人角膜成纤维细胞（HCFs）的原代培养，和离体角膜器官培养模型。Tn64增强角膜上皮细胞的增殖和粘附，同时抑制角膜成纤维细胞和肌成纤维细胞的迁移。Tn64似乎通过下调TNF-α来减轻炎症，通过限制纤连蛋白聚合来预防角膜纤维化，促进角膜上皮和基质的再生，这表明它可以作为有效的抗纤维化角膜伤口愈合的治疗剂。
