Analysis of Smad3 in the modulation of stromal extracellular matrix proteins in corneal scarring after alkali injury.

Purpose: During ocular trauma, excessive proliferation and transdifferentiation of corneal stromal fibroblasts cause haze/fibrosis in the cornea. Transforming growth factor β (TGFβ) plays a key role in corneal fibrosis through the Smad signaling pathway. The aberrant activity of TGFβ signaling during ocular trauma (viz. mechanical, infectious, chemical, or surgically altered TGFβ/Smad signaling) leads to regulating the predominant expression of myogenic proteins and the extracellular matrix (ECM). We sought to investigate the functional role of Smad3 in corneal wound repair and stromal ECM assembly using Smad3^+/+ wild-type and Smad3^-/- deficient mice.

Methods: Corneal injury was introduced with the topical application of an alkali-soaked 2-mm filter disc on the central cornea in the Smad3^+/+ (C57BL/6J) and Smad3^-/- (129-Smad3^tm1Par/J) mouse strains. Slit-lamp and stereo microscopy were used for clinical assessment and corneal haze grading in live animals. Hematoxylin and eosin and Masson's trichrome staining were used to study comparative morphology and collagen level alterations between the groups. Real-time qRT-PCR, western blot, and immunohistochemistry were used to measure changes in profibrotic genes at the mRNA and protein levels.

Results: Slit-lamp clinical exams and stereo microscopy detected notably less opaque cornea in the eyes of Smad3^-/- compared with Smad3^+/+ mice at 3 weeks (p<0.01) in live animals. Corneal tissue sections of Smad3^-/- mice showed significantly fewer α-smooth muscle actin-positive cells compared with those of the Smad3^+/+ animals (p<0.05). The corneas of the Smad3^-/- mice showed significantly lower mRNA levels of pro-fibrotic genes, α-smooth muscle actin, fibronectin, and collagen I (p<0.05, p<0.01, and p<0.001). In addition, the matrix metalloproteinase and tissue inhibitors of metalloproteinase levels were significantly increased (p<0.001) in the corneal tissue during alkali injury in both Smad3^+/+ wild-type and Smad3^-/- deficient mice.

Conclusions: The significant changes in profibrotic genes and stromal ECM proteins revealed a direct role of Smad3 in stromal ECM proteins and TGFβ/Smad-driven wound healing. Smad3 appears to be an attractive molecular target for limiting abnormal stroma wound healing to treat corneal fibrosis in vivo.