Abstract
Purpose::
To assess the role of SMAD pathway in TGF-ß1-induced human tenon’s fibroblasts (HTFs) transdifferentiation into myofibroblasts and to identify the potential pharmacologic target for the inhibition of scar formation after glaucoma surgery.
Methods::
HTFs were obtained from patients with cataract during surgery. They were induced by 10µg/L TGF-ß1. TGF-ß1-induced expression of p-Smad2 was determined by Western blot analysis. TGF-ß-induced mRNA expressions of alpha smooth muscle actin (α-SMA, a marker of myofibroblast) and connective tissue growth factor (CTGF, the potent mediator of transdifferentiation) were analyzed by RT-PCR. The protein expressions of α-SMA and CTGF were determined by Western blot analysis and immunocytochemistry. A specific pharmacologic kinase inhibitor was used to confirm the involvement of SMAD-dependent pathways.
Results::
The increased expressions of p-Smad2 were first observed when the cells were incubated with TGF-ß for 5 min, which lasted up to 24 hours. There were two peaks of the elevated expression, at 1 hour and 12 hours, respectively. The relative mRNA expression of CTGF was up-regulated, with a peak at 12 hours after TGF-ß simulation. The relative mRNA expression of α-SMA was also increased, with the peak at 24 hours after TGF-ß simulation. The protein changes of CTGF and α-SMA demonstrated the same pattern of the changes in mRNA expression. The peaks for CTGF and α-SMA were at 24 and 48 hours respectively. SMAD inhibitor prevented the up-regulations of CTGF and α-SMA, at both protein and RNA levels.
Conclusions::
SMAD pathway plays an important role in TGF-ß-induced HTFs transdifferentiation into myofibroblasts. SMAD inhibitor could abrogate TGF-ß1-induced fibroblast transdifferentiation, and thus, seems to be able to inhibit the scar formation after glaucoma surgery.
Keywords: signal transduction • wound healing