Overexpression of PHGPx inhibits hydroperoxide-induced oxidation, NFkappaB activation and apoptosis and affects oxLDL-mediated proliferation of rabbit aortic smooth muscle cells.

Rabbit abdominal aortic smooth muscle cells (SMC) were stably transfected with the cDNA of porcine phospholipid hydroperoxide glutathione peroxidase (PHGPx) by means of a retroviral gene transfer technique, to create a model for studying cellular processes relevant to atherogenesis. The transfected cells (SMC/PHGPx) had approximately 4-fold higher PHGPx activity when cultured in the presence of selenite whereas the parental cells did not show any significant increase in PHGPx or total GPx activity upon selenium supplementation. In situ functionality of PHGPx was validated by inhibition of linoleic acid hydroperoxide-induced toxicity, dihydrorhodamine oxidation, NFkappaB activation and apoptosis. SMC grown in 1% FCS responded to oxidized LDL (oxLDL) with a marked proliferation, as measured by [3H]thymidine incorporation, irrespective of selenium supplementation. In SMC/PHGPx grown with or without selenite under control conditions or exposed to native LDL, thymidine incorporation was generally depressed. Also, oxLDL-induced proliferation was lower in SMC/PHGPx compared to untransfected SMC up to 24 h of incubation. After 40 h, however, selenite supplementation restored maximum proliferation response to oxLDL in SMC/PHGPx. The results suggest a proliferative effect of endogenous hydroperoxides in SMC. They further reveal that hydroperoxy lipids of oxLDL contribute to the induction of proliferation, but also suggest involvement of hydroxy lipids in the response to oxLDL.

Utilization of selenium from different chemical entities for selenoprotein biosynthesis by mammalian cell lines.

Four different cell lines (Hep G2, THP-1, EL 4 6.1, and ECV 304) were grown in a selenium-deficient standard medium (5% fetal calf serum in RPMI 1640 resulting in 5.5 nM selenium of unknown bioavailability) and supplemented with increasing concentration of selenium in the form of sodium selenite, selenomethionine and serum-bound selenium. The activities of two types of glutathione peroxidases (cGPx and PHGPx) were measured to estimate the availability of selenium for selenoprotein synthesis. Only sodium selenite between 1 and 100 nM was found to consistently induce GPx activity in all cell lines, whereas selenomethionine in equal concentrations was practically ineffective. Only THP-1 cells were able to utilize selenium from serum as efficiently as sodium selenite. PHGPx activity similarly responded to selenium supplementation, but was not increased in EL 4 6.1 cells. Our data demonstrate that conventional tissue culture media require selenium supplementation to guarantee adequate selenoprotein biosynthesis in cultured cells. The chemical nature of the selenium compound used for such supplement is as critical for in vitro cultivated cells as for dietary intake.