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Audrey M. Bernstein, Rajiv R. Mohan, Jonathan C. Tovey, Nadia Pelaez, Lingyan Wang; Tgfβ Concentration And Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2011.
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We have investigated the role of TGFβ concentration in fibroblast migration and corneal wound healing as a means to enhance stromal repopulation and improve corneal stromal wound healing after LASIK.
Human corneal fibroblasts (HCFs) were cultured on collagen in supplemented serum-free media (SSFM). Cell migration was assessed by a scratch-wound assay and results were quantified using T-Scratch software. Immunocytochemistry was used to evaluate SMAD 2/3 localization and α-SMA expression. Active TGFβ was quantified by co-culturing HCFs with TMLC cells containing the PAI-1 promoter linked to the luciferase gene. Wounding in vivo was performed by creating a corneal flap of 8mm in diameter and 200µm in depth with a FlapMaker Disposable Microkeratome in New Zealand White rabbits (2.5-3.0kg). At 0 and 24 hours, corneas were excised and embedded for histological examination to measure wound closure and α-SMA expression. The contralateral eyes served as unoperated controls.
We demonstrated that endogenous levels of active TGFβ1 (0.01ng/ml) stimulated cell migration in vitro since neutralizing antibody to TGFβ1 inhibited cell migration by 3.6-fold compared to IgG control, which had no effect. Addition of 1.0 ng/ml TGFβ1 (100-fold higher than endogenous levels) also reduced cell migration by 3.2-fold, and promoted fibrotic markers: SMAD 2/3 nuclear translocalization and myofibroblast differentiation. Similarly after corneal wounding in rabbits in vivo, application of 0.01ng/ml TGFβ1 significantly increased the rate of wound closure with reduced α-SMA expression compared to 1.0 ng/ml TGFβ1.
These in vitro and in vivo data suggest that low levels of TGFβ1 (0.01ng/ml) promote cell migration and wound closure with a reduced fibrotic response. Thus controlling but not eliminating TGFβ may be a key to promoting regenerative healing in the cornea.
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