The Effect of Hypoxia and Elevated cAMP on Corneal Keratocyte Myofibroblast Transformation

Abstract

Purpose:: Our previous study showed that hypoxia preconditioning protects cornea stromal cells from induced apoptosis. However hypoxia treatment has been shown to increase pSmad levels which are associated with TGFß dependent myofibroblast formation. On the other hand, elevated cAMP is known to reduce myofibroblast formation in cardiac and pulmonary fibroblasts. Therefore we asked whether hypoxia or elevated cAMP could influence keratocyte transformation to myofibroblasts.

Methods:: Rabbit keratocytes were cultured in DMEM without serum. Cells were analyzed 80h after treatment with TGFß1 or TGFß2. In the hypoxic preconditioning group, cells were treated with periodic hypoxia (4 h per day at 1% O₂) for three days then incubated with TGFß. In hypoxia and Forskolin treatment groups, cells were treated with hypoxia or Forskolin together with TGFß. Cells were stained for α-SMA, F-actin and counterstained with DAPI for immunofluorescence analysis of myofibroblast phenotype. Whole cell lysates were collected for westernblot of α-SMA.

Results:: Immunofluorescence analysis showed that 5ng/mL TGFß1 induced 3.1%±0.2% myofibroblast phenotype whereas 5ng/mL TGFß2 induced 3.6%±1.6%. Forskolin 2uM reduced TGFß induced myofibroblast phenotype to 0.3% ± 0.2%. Hypoxia treatment reduced TGFß induced myofibroblast phenotype to 2.1% ± 1% whereas hypoxia preconditioning (3.0%±1.3%) had no significant effect. Controls for all conditions showed very few myofibroblast phenotype with 0.1%±0.1% in primary cultured cells, 0.2%±0.1% in Forskolin control cells and 0.2%±0.1% in hypoxic control cells. Westernblots showed that TGFß 5ng/mL could induce a 3.27±1.37 fold increase in a-SMA level compared to control. Our preliminary data showed that hypoxia preconditioning had no effect on TGFß induced α-SMA whereas hypoxia treatment reduced it by 23.7%. Periodic hypoxia alone increased α-SMA level by 67% compared to control even though we did not observe any increase in myofibroblast phenotype by periodic hypoxic treatment. Forskolin completely blocked TGFß induced α-SMA.

Conclusions:: Our data shows that periodic hypoxia during TGFß treatment (but not hypoxia preconditioning) could reduce the myofibroblast phenotype and α-SMA level. Forskolin blocked TGFß induced myofibroblast phenotype and α-SMA expression. Interestingly, hypoxia treatment alone increased α-SMA level without an increase in the myofibroblast phenotype.

Keywords: cornea: stroma and keratocytes • wound healing • differentiation