Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor β2

Background Transforming growth factor β （TGFβ） is one of the most important growth factors in the development of fibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFβ signal transduction. In recent years, effects of lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about the effects on cornea. This study aimed to determine the effects of lentiviral-mediated SmadTgene expression on keratocyte proliferation and fibrosis induced by TGF β2 in vitro. Methods Keratocytes were cultured from corneal tissue isolated from Sprague-Dawley （SD） rats and transfected with Smad7 expressing lentiviral vector （Lv-Smad7） or non-functioning control vector （Lv-blank）. Following the exposure to TGFβ2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream event of TGFβ/Smad signaling. Expression of fibrotic markers a-smooth muscle actin （a-SMA）, type III collagen （collagen III） were measured by Western blotting and quantitative real time polymerase chain reaction （PCR）. Overall cell proliferation was determined by 3-（4,5-dimethyl-thiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and the expression of cell cycle-related marker Ki67 at both mRNA and protein levels. Results The Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulating phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, a-SMA, collagen III were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated. Conclusion Smad7gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.

Inhibition of corneal fibrosis by Smad7 in rats after photorefractive keratectomy

Background Haze or corneal subepithelial fibrosis is one of the common complications after refractive surgery procedures, such as photorefractive keratectomy （PRK）, laser epithelial keratomileusis, and epipolis laser in situ keratomileusis, which would result in refractive regression, decreased visual quality, and corneal opacification. Haze directly resulted from corneal fibrosis mediated by transforming growth factor β （TGFβ）. SmadT, an inhibitory Smad, can inhibit TGFβ signal transduction. Recently, the effects of Smad7 on the inhibition of fibrosis in several organs have been studied, while little is known about the effects on cornea after PRK. This study was aimed to determine the effects of lentiviral-mediated Smad7 gene expression on corneal fibrosis in rats after PRK. Methods Four different experimental groups were established using right eyes of Sprague-Dawley rats. Thirty-two eyes underwent de-epithelialization only and served as a sham operation group （group 1）. Ninety-six eyes underwent PRK operation and were further divided into group 2 （the PRK group） without lentivector administration, group 3 （the Lv-blank group） with control lentiviral vector without Smad7 administration, and group 4 （the Lv-Smad7 group） with Smad7 expressing lentiviral vector Smad7 administration. At 1 day, 1 week, 1 month, and 3 months after PRK, the transfection efficiency was determined by measuring the fluorescence signal as well as Smad7 protein and mRNA levels. Corneas were further processed for immunoblotting to assess the phosphorylation of Smad2 as a downstream event of TGFβ/Smad signaling. The expression of fibrotic markers, such as c（-smooth muscle actin （c（-SMA）, Type III collagen （collagen III）, and cell cycle-related marker Ki67, was measured by quantitative real-time reverse transcription polymerase chain reaction （RT-PCR）. Results Lentivirus-mediated exogenous Smad7 gene expression in rat corneal tissue resulted in reduced activation of TGFβ/Smad signaling caused by downregulation of phosphorylation of Smad2. Smad7 also downregulated the expression of TGFβ2. Markers of cell proliferation and fibrosis, including Ki67, c（-SMA, and collagen III, were inhibited by Smad7 up to 3 months after PRK operation. Conclusion Smad7 gene transfer inhibits fibrogenic responses of cornea in rats after PRK.