Introduction Directory UMM :Data Elmu:jurnal:A:Atherosclerosis:Vol153.Issue1.Nov2000:

Atherosclerosis 153 2000 23 – 35 7b-Hydroxycholesterol induces Ca 2 + oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells Mikko P.S. Ares a, , M. Isabella Po¨rn-Ares b , Sara Moses c , Johan Thyberg d , Lisa Juntti-Berggren e , Per-Olof Berggren e , Anna Hultga˚rdh-Nilsson c , Bengt Kallin f , Jan Nilsson a a Wallenberg Laboratory, Uni6ersity Hospital MAS, Lund Uni6ersity, S- 20502 Malmo¨, Sweden b Di6ision of Experimental Pathology, Uni6ersity Hospital MAS, Lund Uni6ersity, S- 20502 Malmo¨, Sweden c Department of Cell and Molecular Biology, Section for Connecti6e Tissue Biology, Lund Uni6ersity, P.O. Box 94 , S- 221 00 Lund, Sweden d Department of Cell and Molecular Biology, Karolinska Institutet, S- 17177 Stockholm, Sweden e Rolf Luft Center for Diabetes Research, Department of Endocrinology, Karolinska Hospital, Karolinska Institutet, S- 17176 Stockholm, Sweden f Department of Medicine, Atherosclerosis Research Unit, King Gustaf Vth Research Institute, Karolinska Hospital, Karolinska Institutet, S- 17176 Stockholm, Sweden Received 23 June 1999; received in revised form 25 November 1999; accepted 7 January 2000 Abstract In the present study, we characterize the early cytotoxic effects of 7b-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7b-hydroxycholesterol induced Ca 2 + oscillations with a frequency of : 0.3 – 0.4 min − 1 . A few hours later, thapsigargin-sensitive Ca 2 + pools were depleted, indicating that 7b-hydrox- ycholesterol perturbs intracellular Ca 2 + homeostasis. The mitogen-activated protein kinases MAPKs ERK1 and ERK2 but not JNK were activated within 5 min after addition of 7b-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxyc- holesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7b-hydroxycholesterol and cholesterol-5a,6a- epoxide, as determined by TUNEL staining. Addition of TNFa 10 ngml and IFNg 20 ngml enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7b-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4 – 8 h as determined by [ 35 S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca 2 + and ERK signals preceded the ultrastructural changes induced by 7b-hydroxycholesterol. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : 7b-Hydroxycholesterol; Ca 2 + oscillations; MAP kinase activation www.elsevier.comlocateatherosclerosis

1. Introduction

Death of smooth muscle cells SMCs in the fibrous cap region of the atherosclerotic plaque is believed to play an important role in plaque rupture [1]. A high incidence of apoptotic cell death among SMCs in this region has been demonstrated [2,3]. Similarly, human vascular SMCs derived from coronary plaques are characterized by increased frequency of apoptosis [4]. Moreover, electron microscopic studies suggest that additional cell death by necrosis occurs in parallel [5]. Atherosclerotic plaques have been shown to contain higher amounts of oxysterols oxidized cholesterol derivatives than normal arteries [6 – 10]. As oxysterols are generally cytotoxic [11 – 13], they may contribute to cell death in atherosclerotic lesions. We have previously shown that 25-hydroxycholes- terol induces activation of the protease caspase-3 [14], a key mediator of apoptotic cell death in many systems [15,16]. In the present study, we have compared the ability of different oxysterols to induce cell death and characterize several early events during exposure of cells to 7b-hydroxycholesterol, reportedly a major cyto- Corresponding author. Tel.: + 46-40-337674; fax: + 46-40- 332550. E-mail address : mikko.aresmedforsk.mas.lu.se M.P.S. Ares. 0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 0 0 0 0 3 8 0 - 4 toxin in oxidized LDL [17,18]. 7b-Hydroxycholesterol can also be produced endogenously from cholesterol [19]. Ca 2 + mobilization can induce apoptosis [20 – 23] by activating Ca 2 + -dependent proteases and endonucle- ases and by depleting intracellular Ca 2 + stores [24,25], the maintenance of which is required for cell survival. In the present study we show that 7b-hydroxy- cholesterol induces intracellular Ca 2 + oscillations which lead to depletion of thapsigargin-releasable Ca 2 + stores within a few hours. Mitogen-activated protein kinases MAPKs play im- portant roles in cell proliferation. Activation of the extracellular signal-regulated kinases ERKs generally inhibits cell death [26], but pro-apoptotic effects have been reported as well [27,28]. ERKs have been impli- cated in CD95-mediated apoptosis [29]. The results of the present study show that several different oxysterols induce apoptosis, which is potentiated by the inflamma- tory cytokines TNFa and IFNg, which are known to be expressed in atherosclerotic vessels [30,31].

2. Materials and methods