Abstract
Purpose: :
Previous microarray data showed Caveolin-1 among the genes up-regulated after chronic oxidative stress in porcine trabecular meshwork TM (pTM) cells. We further investigated the role of caveolin-1 in stress induced senescence parameters.
Methods: :
Porcine TM cells were subjected to chronic oxidative stress by treatment with 200 µM H2O2 two times a day for 4 days. Senescence markers were analyzed by FacScan (sa-beta-galactosidase, SaBgal, using C12 FDG; autofluorescence; intracellular Reactive Oxygen Species, ROS, using H2DCFDA; and proliferation, using BrDu). Overexpression of caveolin-1 was achieved using adenovirus; caveolin-1 gene was cloned in the pAd/CMV-DEST vector. Overexpression was confirmed by Western blot. Null adenovirus was used as a control.
Results: :
pTM cells subjected to chronic oxidative stress exhibited markers of senescence, as an increase in ROS (182%), SaBgal (173%), autofluorescence (124%) and decrease in proliferation (48%), compared to non-stressed cells. Overexpression of caveolin-1 induced a significant increase in SaBgal (127%) and ROS (114%) and a decrease in proliferation (24%). These changes were not associated with an increase in phosphorylated p38 or total P53 proteins.
Conclusions: :
Caveolin-1 partially mediated some of the parameters observed in stress induced senescence including the increase in ROS generation, SaBgal activity , and decreased proliferation in pTM cells. Therefore, the observed upregulation of caveolin-1 after chronic oxidative stress may contribute to induced phenotypic changes characteristic of senescent cells that could, in turn, lead to functional alterations of the TM in aging and glaucoma.
Keywords: trabecular meshwork • oxidation/oxidative or free radical damage • adenovirus