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Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor β2
Ti Wang1, Xing-tao Zhou, Yan Yu
1Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
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 Smad7 gene 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 α-smooth muscle actin (α-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, α-SMA, collagen III were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated.
Conclusion:
Smad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.
Insights
Smad7 gene transfer inhibited corneal fibrosis and keratocyte proliferation induced by TGFβ2. This study shows Smad7 can suppress fibrogenic responses in corneal cells.
Area of Science:
- Ophthalmology
- Molecular Biology
- Regenerative Medicine
Background:
- Transforming growth factor beta (TGFβ) drives corneal fibrosis and scarring.
- Smad7 inhibits TGFβ signaling, but its role in corneal fibrosis is unclear.
- Lentiviral vectors offer a method for gene delivery to corneal cells.
Purpose of the Study:
- To investigate the effect of lentiviral-mediated Smad7 gene expression.
- To determine Smad7's impact on keratocyte proliferation and fibrosis.
- To assess Smad7's efficacy in inhibiting TGFβ2-induced fibrotic responses in vitro.
Main Methods:
- Cultured rat corneal keratocytes and transfected with Smad7 lentiviral vector (Lv-Smad7).
- Induced fibrosis using TGFβ2 and analyzed Smad2 phosphorylation, fibrotic markers (α-SMA, collagen III), and cell proliferation (MTT, Ki67).
Main Results:
- Smad7 gene transfer effectively suppressed TGFβ/Smad signaling by reducing Smad2 phosphorylation.
- Introduction of Smad7 inhibited key markers of proliferation (Ki67) and fibrosis (α-SMA, collagen III).
- Keratocyte proliferation rates were significantly attenuated following Smad7 gene transfer.
Conclusions:
- Smad7 gene transfer is effective in inhibiting TGFβ2-induced fibrogenic responses in corneal keratocytes.
- Smad7 demonstrates potential as a therapeutic agent to mitigate corneal fibrosis and scarring.
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