Abstract
Purpose :
To investigate the effect of exogenous nitric oxide (NO) on primarily cultured human keratocytes.
Methods :
The primary culture of human keratocytes was isolated from descemet's membrane and epithelium. The corneal stroma was sliced into quarters and digested overnight with 2.0 mg/mL collagenase and 0.5 mg/mL hyaluronidase in DMEM at 37 °C. Isolated cells were washed in DMEM and cultured in DMEM/F12 supplemented with 10% fetal bovine serum. Keratocytes were treated with different concentrations of NaNO2 for 24 h and 48h and following incubation, cells were assayed for the measurement of intracellular ROS using a Fluorometric Intracellular ROS Kit. Cell viability assay was performed as previously described using a cell counting kit reagent according to the manufacturer’s protocol. Different concentrations of NaNO2 were added to the culture media for 24 h, keratocytes were lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer. 20 μg of total cell protein were separated by SDS-PAGE and transferred to PVDF membrane.
Results :
NO had no deleterious effect on keratocyte viability. The decrease of viability in extreme high concentration (100mM) of sodium nitrite is due to osmolar stress. In addition, NO had no effect of mTOR and autophagy pathway of keratocytes. Stimulation with TGFβ1 increased SMA expression from the keratocytes which was more prominent after 72 h exposure. However, vimentin expression was not affected by TGFβ1 until 72 h. Sodium nitrite, one of the NO donors, attenuated the expression level of αSMA in TGFβ1-stimulated keratocytes, however, both proteins, αSMA and vimentin were not affected in resting keratocytes, without TFG β1.
Conclusions :
We found NO can weakly attenuate the expression level of αSMA protein following stimulation using TGF-β1 in keratocytes. These results suggest that myofibroblast differentiation of human keratocytes can be controlled by NO treatment.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.