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Gene transfer of Smad7 modulates injury-induced conjunctival wound healing in mice
Osamu Yamanaka1, Kazuo Ikeda, Shizuya Saika
1Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. yamanaka@wakayama-med.ac.jp
Purpose:
Smad7 is a molecule that blocks the Smad2/3 signal. Herein, we examined the effects of Smad7 gene introduction on post-injury conjunctival wound healing in mice. Its effects on the cultured human subconjunctival fibroblasts (SCFs) were also investigated.
Methods:
A circumferential incision was made in the equatorial conjunctiva by using scissors in the right eye of fully anesthetized adult C57BL/6 mice (n=72). Smad7 cDNA-expressing adenoviral vector was topically applied. The control eye received nonfunctioning adenoviral vector. After 2, 5, 7, and 30 days the eyes were processed for histological or immunohistochemical examination to evaluate wound healing of conjunctiva. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of Smad7 gene introduction on the cultured human SCFs were also studied.
Results:
Marked Smad7 protein expression was detected in the vector-treated conjunctival epithelium and fibroblasts that coincided with green fluorescein protein expression, whereas faint endogenous Smad7 expression was observed in the control tissue. In vivo Smad7 gene introduction blocked Smad2/3 nuclear translocation with suppression of alpha-smooth muscle actin (alphaSMA) and vascular endothelial growth factor (VEGF) in fibroblasts and invasion of macrophages. Smad7 gene transfer suppressed mRNA expressions of connective tissue growth factor (CTGF), VEGF, and monocyte chemoattractant protein-1 in vivo and those of type-I collagen, alphaSMA, and CTGF in vitro.
Conclusions:
Smad7 gene transfer modulates injury-induced wound healing of conjunctival tissue in mice, suggesting that this strategy may be effective in preventing excessive scarring following filtration surgery. The mechanism might include suppression of activation of fibroblasts and reduction of macrophage invasion.
Insights
Smad7 gene transfer effectively modulates conjunctival wound healing in mice by blocking key signaling pathways. This approach shows promise for preventing excessive scarring after eye surgery.
Area of Science:
- Ophthalmology
- Molecular Biology
- Wound Healing Research
Background:
- Conjunctival wound healing is a complex process involving cellular signaling pathways.
- Excessive scarring after ocular surgery can impair vision.
- Smad7 is an endogenous inhibitor of the TGF-beta/Smad signaling pathway.
Purpose of the Study:
- To investigate the therapeutic potential of Smad7 gene transfer in modulating conjunctival wound healing post-injury.
- To examine the effects of Smad7 on human subconjunctival fibroblasts (SCFs) in vitro.
Main Methods:
- Adenoviral vector-mediated Smad7 gene transfer into mouse conjunctival wounds.
- Histological and immunohistochemical analysis of conjunctival tissues.
- Real-time RT-PCR to assess gene expression of collagen and growth factors.
- In vitro studies on cultured human SCFs.
Main Results:
- Smad7 gene transfer successfully upregulated Smad7 expression in conjunctival tissues.
- In vivo, Smad7 suppressed Smad2/3 nuclear translocation, alphaSMA, VEGF, and macrophage invasion.
- Smad7 inhibited mRNA expression of CTGF, VEGF, type-I collagen, and alphaSMA both in vivo and in vitro.
Conclusions:
- Smad7 gene transfer effectively modulates conjunctival wound healing in mice.
- This strategy may prevent excessive scarring in ocular filtration surgery.
- Mechanisms involve suppressing fibroblast activation and reducing macrophage infiltration.
