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

Abstract

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, NFκB 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.

Introduction

Apart from the induction of adhesion molecules in endothelial cells, proliferation of smooth muscle cells (SMC) in the subendothelial layer is one of the early events in atherogenesis [1]. Proliferation of SMC finally leads to intima thickening and the development of fibrous plaques. Proliferation is well documented to be stimulated by several compounds, including oxidized LDL (oxLDL), in aortic SMC [2], [3], [4], in rabbit femoral SMC [5] and in SMC from aortic segments from human kidneys [6]. SMC are known to take up oxLDL by a particular receptor referred to as the scavenger receptor [7]. However, which of the various oxidized compounds in the LDL particle are triggering the proliferation has not yet been addressed systematically. Oxidized fatty acids belong to the putative stimulators of atherogenesis. They have been found in atherosclerotic lesions together with oxLDL [8]. They induce ICAM-1 in HUVEC [9] and obviously enhance TNF-induced CAM expression and PMA-induced T cell adhesion in 15-lipoxygenase-transfected endothelial cells [10]. Their exact involvement in SMC proliferation, however, remains unknown. Hydroxy fatty acids, the reduction products of the corresponding hydroperoxy fatty acids, proved to be even stronger inducers of the endothelial cell adhesion molecule ICAM-1 [9]. They also induce the scavenger receptor CD36 responsible for oxLDL uptake in monocytes/macrophages [11]. In chemically oxidized LDL, oxidized fatty acids were primarily found esterified to cholesterol [12]. They can also be produced by the action of 15-lipoxygenases in cultured endothelial cells [13], macrophages [14], 15-lipoxygenase overexpressing endothelial cells [10] and fibroblasts [15], and after treatment of LDL with purified rabbit reticulocyte 15-lipoxygenase [16]. However, these findings are not undebated [17].

The selenium-containing glutathione peroxidases [18] inhibit the activation of lipoxygenases in vitro [19], [20], [21] and efficiently reduce products thereof [18]. Their role in the prevention of atherogenesis in vivo is widely discussed, since epidemiological studies revealed a correlation of cardiovascular diseases and selenium deficiency [22], [23]. Therefore, the manipulation of the peroxide metabolism was considered in SMC as a promising approach to analyze the relevance of lipid hydroperoxides present in oxLDL to SMC biology.

While all glutathione peroxidases reduce H₂O₂ or soluble alkyl hydroperoxides [24], only the phospholipid hydroperoxide glutathione peroxidase (PHGPx) efficiently reduces hydroperoxy groups of complex lipids including those of phospholipids [18], [25] and cholesterolesters [26] even when present in lipoproteins [27]. Overexpression of PHGPx was therefore considered the optimum tool to define the effects of hydroperoxy and hydroxy lipids of oxLDL on cellular targets. With this in mind PHGPx was overexpressed in SMC, verified its function in situ, and tested the proliferative response to oxLDL in rabbit aortic SMC.