Atherosclerosis 152 2000 307 – 316
Overexpression of PHGPx inhibits hydroperoxide-induced oxidation, NFkB activation and apoptosis and affects
oxLDL-mediated proliferation of rabbit aortic smooth muscle cells
Regina Brigelius-Flohe´
a,b,
, Stefanie Maurer
a,b
, Katharina Lo¨tzer
a
, Gaby-Fleur Bo¨l
a
, Hanna Kallionpa¨a¨
c
, Paulina Lehtolainen
c
, Helena Viita
c
, Seppo Yla¨-Herttuala
c
a
German Institute of Human Nutrition, Uni6ersity of Potsdam, Potsdam-Rehbru¨cke, Arthur-Scheunert-Allee
114
-
116
,
14558
Bergholz-Rehbru¨cke, Germany
b
Institute for Nutritional Science, Uni6ersity of Potsdam, Potsdam-Rehbru¨cke, Arthur-Scheunert-Allee
114
-
116
,
14558
Bergholz-Rehbru¨cke, Germany
c
A.I. Virtanen Institute and Department of Medicine, Uni6ersity of Kuopio, Kuopio, Finland Received 19 July 1999; received in revised form 1 November 1999; accepted 6 December 1999
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 SMCPHGPx 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 hydroperox- ide-induced toxicity, dihydrorhodamine oxidation, NFkB activation and apoptosis. SMC grown in 1 FCS responded to oxidized
LDL oxLDL with a marked proliferation, as measured by [
3
H]thymidine incorporation, irrespective of selenium supplementa- tion. In SMCPHGPx 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 SMCPHGPx compared to untransfected SMC up to 24 h of incubation. After 40 h, however, selenite supplementation restored maximum proliferation response to oxLDL
in SMCPHGPx. 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. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Glutathione peroxidases; Selenium; Smooth muscle cells; Proliferation; Overexpression; Atherosclerosis; Apoptosis www.elsevier.comlocateatherosclerosis
1. 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 – 4], in rabbit femoral
SMC [5] and in SMC from aortic segments from hu- man kidneys [6]. SMC are known to take up oxLDL by
a particular receptor referred to as the scavenger recep- tor [7]. However, which of the various oxidized com-
pounds
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 atheroscle- rotic 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 prolifera-
tion, however, remains unknown. Hydroxy fatty acids,
Corresponding author. Tel.: + 49-33200 88353; fax: + 49-33200 88407.
E-mail address
:
flohewww.dife.de R. Brigelius-Flohe´. 0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 1 5 0 9 9 0 0 4 8 6 - 4
the reduction products of the corresponding hydroper- oxy fatty acids, proved to be even stronger inducers of
the endothelial cell adhesion molecule ICAM-1 [9]. They also induce the scavenger receptor CD36 respon-
sible for oxLDL uptake in monocytesmacrophages [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]. How-
ever, these findings are not undebated [17].
The selenium-containing glutathione peroxidases [18] inhibit the activation of lipoxygenases in vitro [19 – 21]
and efficiently reduce products thereof [18]. Their role in the prevention of atherogenesis in vivo is widely
discussed, since epidemiological studies revealed a cor- relation of cardiovascular diseases and selenium defi-
ciency [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 H
2
O
2
or soluble alkyl hydroperoxides [24], only the phospho-
lipid 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 consid- ered the optimum tool to define the effects of hydroper-
oxy 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.
2. Methods
