- Email: [email protected]
− mice aortas
Biochemical and Biophysical Research Communications 356 (2007) 681–686 www.elsevier.com/locate/ybbrc
OxLDL-induced gene expression patterns in CASMC are mimicked in apoE / mice aortas Janice L.V. Reeve a, Catherine Stenson-Cox a, Aideen O’Doherty b, Isabella Po¨rn-Ares d, Mikko Ares e, Timothy O’Brien b,c, Afshin Samali a,c,* a
d
Department of Biochemistry, National University of Ireland, Galway, Ireland b Department of Medicine, National University of Ireland, Galway, Ireland c National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Department of Biomedicine/Biochemistry, Helsinki University, Haartmaninkatu 8, 00014 Helsinki, Finland e Wallenberg-Lab, University Hospital MAS, Malmo¨, Sweden Received 27 February 2007 Available online 12 March 2007
Abstract Oxidized low density lipoprotein (oxLDL) contributes to the pathophysiology of atherosclerosis, partly by altering gene expression in vascular cells. Here, we show 221 genes differentially regulated by oxLDL in coronary artery smooth muscle cells (CASMC), using oligonucleotide microarrays. These genes were classified into 14 functional groups. A comparable gene expression pattern was detected in apoE / mice. OxLDL induced an oxidative stress response in CASMC, but not the unfolded protein response. OxLDL also caused CASMC death which was accompanied by increased expression of FasL, Bax, and p53 but was caspase-independent. This approach provides further insight into disease pathology and prognosis. 2007 Elsevier Inc. All rights reserved. Keywords: Apolipoprotein E; Cell death; Gene expression; Oxidative stress; OxLDL; Smooth muscle cell
Atherosclerosis, leading to coronary heart disease (CHD) and cerebral stroke, is the most common cause of death and morbidity in industrialized societies. Low density lipoprotein (LDL) and its oxidized derivative, oxLDL, initiate and promote atherosclerosis [1]. In vivo LDL may be oxidized by vascular cells and later by infiltrating immune cells [2]. OxLDL is detected in atherosclerotic plaques and in the plasma of atherosclerosis patients, where it contributes to disease evolution [1]. Depending on the extent of its oxidation, oxLDL can induce proliferation, monocyte chemotaxis, apoptosis or necrosis of vascular endothelial (EC) and smooth muscle cells (VSMC) [1,3]. * Corresponding author. Address: National Centre for Biomedical Engineering Science, National University of Ireland, University Road, Galway, Ireland. Fax: +353 91 494596. E-mail address: [email protected] (A. Samali).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.027
The vascular cell response to oxLDL is regulated, in part, at the level of transcription and oxLDL-induced gene expression profiles have been investigated in EC, macrophage and VSMC [4–7]. The overall effect may initially be cytoprotective allowing the cell to adapt to the stress, however, when prolonged; such cellular stress responses result in cellular demise and disease manifestations. Uptake of oxLDL by human macrophages increased the expression of the antioxidant genes, glutathione and thioredoxin, to protect the cell from oxLDL and the resultant oxidative stress [4]. OxLDL also increased the expression of genes involved in cell–cell interaction, transport, transcription and apoptosis, and decreased expression of genes involved in protein biosynthesis and lipid metabolism in human aortic SMC [6]. The proteomics study which followed revealed similar patterns suggesting that gene expression profiling can provide insight into the cellular response [5,6]. This
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prompted us to investigate the global gene expression pattern modulated by oxLDL in primary human CASMC and to compare them to that found in vivo in the aortas of apoE / mice, the well-established atherosclerotic model [8]. Additionally, we investigated the effect of oxLDLinduced alterations in gene expression on overall biological function. Materials and methods Cell culture. Human coronary artery smooth muscle cells (CASMC) (Clonetics) were cultured in SMC basal medium (Cambrex), supplemented with Clonetics SMC bullet kit containing 5% fetal bovine serum, at 37 C with 5% CO2 CASMC, when not stated otherwise, were treated with 60 lg/ml oxLDL or sterile PBS as vehicle control. Preparation and oxidation of low density lipoproteins. LDL was isolated as previously reported [9]. For oxidation, 300 lg/ml LDL was incubated with 5 lM CuSO4 at 37 C until the desired oxidation state was obtained. The oxidation state was determined by measuring the thiobarbituric acidreactive substances (TBARS) using malondialdehyde (MDA) as a standard. OxLDL used contained 15–30 nmol of TBARS as MDA equivalents/mg of LDL proteins and was used within 6 h of preparation. Preparation of RNA and hybridization to oligonucleotide microarrays. The total RNA was isolated from oxLDL-treated and untreated CASMC using Tri reagent (MRC Inc.) according to the manufacturer’s instructions. Complementary DNA was synthesized using the Microarray cDNA Synthesis Kit (Roche Diagnostics) according to the manufacturer’s instructions. After purification with High Pure RNA Tissue Kit (Roche Diagnostics), cRNA was transcribed and labeled with cyanine 3-UTP or cyanine 5-UTP (100 nmol; Amersham Biosciences) dyes using the MEGAscript T7 High Yield Transcription Kit (Ambion). Labeled cRNA (10 lg) was combined and fragmented with buffer containing 20 mM tris acetate, pH 8.1, 50 mM potassium acetate, and 15 mM magnesium acetate for 15 min at 94 C. Fragmented cRNA was applied to human 10 K oligonucleotide microarrays (microarray ‘‘A’’, MWG Biotech) and incubated overnight agitating at 42 C. Microarrays were scanned and analyzed by MWG Biotech with genes differentially expressed by 2-fold analyzed for proposed function using gene ontology programs, GeneCards (www.genecards.org) and Bioinformatic Harvester (www.harvester.embl.de). Reverse transcriptase polymerase chain reaction (RT-PCR). Reverse transcription was carried out with 2 lg total RNA and oligo(dT) 12–18 (Invitrogen) using AMV Superscript (Invitrogen) according to the manufacturer’s instructions. The cDNA product was subjected to PCR using primers from Supplementary material Table 1. GAPDH primers were used for normalization. Extraction of mouse aortas and sample preparation. ApoE / mice, strain B6.129P2-Apoetm1Unc/J; Jackson Laboratory, were fed a Western diet of 0.15% cholesterol (Harlan Teklad) for 1 month and were sacrificed at 4–8 months of age. The abdominal cavity was opened. Visceral organs were moved to the side and an angled incision was performed above the heart. The aorta was excised along with the heart as far as the aortic bifurcation in the groin area. Frozen sections were cut from OCTembedded aortas on a rotary freezing microtome at 20 lm and lifted onto polylysine-coated slides. Sections were incubated at 37 C overnight and stained for lipid-rich lesions with sequential incubations as follows: dH2O for 10 s, 60% isopropyl alcohol for 10 s, Sudan IV stain (0.5% Sudan IV, 35% ethanol, 50% acetone) for 4 min, 60% isopropyl alcohol for 10 s, dH2O for 30 s, Harris hematoxylin for 1 min, dH2O for 1 min 3·, and mounted using aqueous mounting solution (Dako). Sections were examined by light microscopy (Olympus BX51). The total RNA was extracted from homogenized aortas as described earlier. All animal experiments were performed in accordance with the ethical regulations of NUI Galway. Western blot analysis. Protein samples were prepared as previously reported [10]. Equal amounts of protein (30 lg) were denatured in Laemmli’s buffer and resolved on 10–12% SDS–PAGE and transferred onto nitrocellulose membrane. After blocking in 5% (w/v) non-fat milk
in PBS containing 0.05% Tween 20 the membranes were incubated with rabbit polyclonal antibodies to Bax, caspase-3, PARP (1:1000; Cell Signaling Technologies), caspase-8, CHOP (1:1000; Santa Cruz), HSF-1 ˚ bo Akademi University, (1:500, a kind gift from Prof. Lea Sistonen, A Finland), actin (1:500; Sigma–Aldrich), and mouse monoclonal antibodies to Hsp70 and KDEL (recognizes Grp78 and Grp94; 1:1000; Stressgen). Horseradish peroxidase-conjugated secondary antibodies (1:10,000) were obtained from Pierce. Protein bands were visualized with Supersignal Ultra Chemiluminescent Substrate (Pierce) on X-ray film (AGFA). Cell morphology. Cells were fixed in methanol for 5 min followed by staining in Harris hematoxylin solution for 15 s and Eosin Y for 15 s. Subsequently, cells were observed by phase contrast microscopy (Olympus IX71). Caspase activity assay. Cell lysates (25 ll) and 50 lM DEVD-AMC (Peptide Institute Inc.) in reaction buffer (100 mM Hepes, 10% sucrose, 5 mM DTT, 0.0001% Igepal-630, and 0.1% CHAPS, pH 7.25) were added in duplicate to a microtiter plate. Liberated AMC was measured using 355 nm excitation and 460 nm emission wavelengths, over time and the data analyzed by linear regression to determine enzyme activity. Enzyme activity was expressed as fold change in DEVDase activity compared to untreated control cells. Lactate dehydrogenase assay. Relative lactate dehydrogenase (LDH) levels in the supernatant were measured using a Cytotoxicity Detection LDH assay kit (Roche Diagnostics Ltd.) as per manufacturer’s instructions. Results are expressed as fold change in absorbance at 490 nm compared to untreated CASMC controls.
Results and discussion Global gene expression analysis of oxLDL-treated CASMC OxLDL is a pro-atherogenic risk factor responsible for many of vascular cell alterations observed during atherosclerosis. To determine the oxLDL-induced alterations in gene expression, CASMC were incubated with 60 lg/ml (15–30 nmol of MDA/mg LDL) of Cu2+-oxLDL for 24 h, RNA was isolated, fluorescently labeled and hybridized to oligonucleotide microarrays. The experiment was performed in duplicate and included a dye-swap experiment to compensate for potential dye bias. Two hundred and twenty-one genes exhibited P2-fold change in expression, 174 were putatively upregulated while 47 were downregulated, following oxLDL treatment (Supplementary material Tables 2 and 3). The differentially expressed genes were classified into 14 functional groups encompassing redox homeostasis, Ca2+ signaling, immune response and apoptosis (Supplementary material Table 4). All of which have been implicated in the pathology of atherosclerosis. A random selection of genes identified as putatively regulated by the microarray study was chosen for RT-PCR analysis. As shown in Fig. 1A, oxLDL induced the expression of glutathione reductase, NAD(P)H dehydrogenase-1, adenosine A2A receptor, calcium/calmodulin-dependent protein kinase kinase 2b (CAMKK2b), heat shock factor-1 (HSF-1), and interleukin 12A (IL-12A); whilst it reduced the mRNA levels of vascular endothelial growth factor A (VEGFA) precursor, hydroxysteroid 17b dehydrogenase 4 (HSD17B4) and apoptosis inhibitor 5 (API5), compared to vehicle treated controls.
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Fig. 1. The gene expression pattern in oxLDL treated CASMC and atherosclerotic aortas. (A) CASMC were incubated with 60 lg/ml oxLDL for 24 h at 37 C. The total RNA from treated and untreated cells was subjected to RT-PCR for the mRNA expression levels of glutathione reductase, NAD(P)H dehydrogenase-1, adenosine A2A receptor, CAMKK2b, HSF-1, IL-12A, VEGFA precursor, HSD17B4, and API5. (B) Transverse sections of apoE / mouse aorta were prepared and stained with Sudan IV and hematoxylin. Depicted are representative sections from apoE / mouse aorta containing an established lesion (upper panel) and a section free from lesions (lower panel). (C) RT-PCR analysis of mRNA levels of HSF-1, IL-12A, VEGFA precursor, HSD17B4, and API5 in apoE / and WT mice. GAPDH was used as an internal control.
Differential gene expression in apoE
/
mice
To investigate the pathophysiological relevance of this altered gene expression, the profile of selected genes were examined in apoE / mice. The presence of plaques in the aortas of apoE / mice was confirmed by Sudan IV staining (Fig. 1B). The total RNA from the aortas of apoE / and WT mice was examined by RT-PCR for the expression of HSF-1, IL-12A, VEGFA precursor, HSD17B4, and API5 (Fig. 1C). Significantly, oxLDLinduced alterations in CASMC gene expression were comparable in aortas of apoE / mice. As anticipated, the expression of IL-12A and HSF-1 was upregulated, an indication of the pro-inflammatory response, as previously reported in animal models of atherosclerosis [11,12]. We observed a decrease in VEGFA precursor and HSD17B4 expression, associated with cell proliferation and cholesterol metabolism, respectively. Interestingly, HSD17B4 is associated with oxidative stress in humans [13,14]. Taken together these in vitro and in vivo findings bear relevance to the development of atherosclerosis. Despite the decrease in API5 observed in the microarray study, its expression remained unchanged in apoE / mice. Overall, despite the increased complexity of the atherosclerotic mouse model, the changes in gene expression observed in vitro are immediately comparable to the in vivo situation. OxLDL-induced CASMC injury is therefore highly signif-
icant in the context of the pathology and the progression of atherosclerosis. OxLDL induces an oxidative stress response, but not the unfolded protein response Having established that the alterations in gene expression induced by oxLDL are comparable to the more complex in vivo setting, we wished to extrapolate the potential cellular consequences. Of the genes regulated in oxLDLtreated CASMC, a number were associated with the cellular stress response. The expression of redox-sensitive genes can be induced as part of an adaptive response to oxidative stress [15–17]. Since redox-sensitive genes, present on the microarray were induced in response to oxLDL we set out to determine whether oxLDL induced an oxidative stress response in CASMC. To that end, the expression of additional antioxidant genes, not present on the array but positively associated with the oxidative stress response, was assessed [18–20]. As demonstrated in Fig. 2A oxLDL induced the expression of manganese superoxide dismutase (MnSOD), hemoxygenase-1 (HO-1) and ferritin. In addition, protein levels of Hsp70 and HSF-1, whose gene expression was increased in the microarray study, was also found to be increased (Fig. 2B). Taken together these data suggest the initiation of an adaptive oxidative stress response in CASMC in response to oxLDL. We hypothesis
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Fig. 2. OxLDL induces an oxidative stress response, but not the unfolded protein response. CASMC were incubated with 60 lg/ml oxLDL for 24 h at 37 C. (A) RT-PCR analysis of mRNA expression levels of MnSOD, HO-1, ferritin, ATF3, ATF4, XBP-1, Egr-1, and Grp78. GAPDH was used as internal control. (B) Western blot analysis of Hsp70, HSF-1, CHOP, and KDEL. Thapsigargin (2 lM; 36 h) treated HeLa cells were used as a positive control (+ve). Anti-actin antibody was used to determine equal sample loading.
that this response is an attempt to alleviate cellular oxidative stress. As oxidative stress can induce secondary effects such as increasing cytosolic Ca2+ concentrations [21] and oxLDL can decrease the expression of ER resident Ca2+-pumps [22,23] we next investigated the possible effect of oxLDL on ER stress and the unfolded protein response (UPR). ER stress is sensed by three ER transmembrane receptors; ATF6, Ire1, and PERK [24]. Activation of these sensors launches the UPR which attempts to restore ER homeosta-
sis. However, failure of the UPR to restore normal ER function, can result in apoptosis [24]. The activation of Ire1 and PERK was monitored by detecting the induction of ATF3, ATF4, Egr-1, XBP-1, CHOP, Grp94, and Grp78. As demonstrated in Fig. 2A mRNA levels of ATF3, ATF4, Egr-1, XBP-1 or Grp78 were not altered by oxLDL nor did protein levels of Grp78, Grp94 or CHOP change (Fig. 2B). This would indicate that oxLDL does induce an oxidative stress response in CASMC but this is not accompanied by activation of the UPR.
Fig. 3. OxLDL induces CASMC death and induces the expression of pro-apoptotic genes. CASMC were incubated with either 60 or 150 lg/ml oxLDL for 24 h at 37 C. (A) Cellular morphology was examined by phase contrast and light microscopy of hematoxylin and eosin stained cells. (B) RT-PCR analysis for FasL and p53 mRNA expression. GAPDH mRNA was utilized as an internal control. Western blot analysis of Bax expression. Anti-actin antibody was used to determine equal sample loading.
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Fig. 4. OxLDL-induced CASMC death is independent of caspases. CASMC were incubated with 60 lg/ml oxLDL for 24 h at 37 C. (A) Western blot analysis using antibodies to caspase-8, caspase-3 and PARP. Apoptotic HeLa cells were used as a positive control. (B) Caspase-3-like activity was detected by monitoring the cleavage of synthetic substrate DEVD-AMC. Staurosporine-treated (0.25 lM; 4 h, sts) apoptotic cells were used as a positive control. (C) Loss of outer plasma membrane integrity was determined by LDH release assay. CASMC exposed to 60 lg/ml oxLDL for 24 h were compared to untreated CASMC.
OxLDL-induced CASMC death is caspase-independent Since excessive or prolonged oxidative stress can induce cell death, we investigated the effect of oxLDL on CASMC. In terms of cell morphology, CASMC exhibited a loss in cell volume and appeared condensed following treatment with 60 lg/ml oxLDL for 24 h, while administration of 150 lg/ml oxLDL induced cytolysis (Fig. 3A). As demonstrated in Fig. 3B, administration of 60 lg/ml oxLDL also induced the expression of FasL, p53, and Bax, which are associated with apoptosis. However, despite the induction of such early apoptotic molecules, processing of pro-caspase-8, pro-caspase-3 or its downstream substrate PARP was not detected (Fig. 4A). In addition DEVDase activity was not evident (Fig. 4B) and a loss in outer plasma membrane integrity, as detected by LDH release, was observed (Fig. 4C). Therefore, oxLDL-induced CASMC death, under these experimental conditions appeared to be independent of caspase activation. This is complimentary to the previous findings by Porn-Ares et al., in oxLDL-treated endothelial cells [9]. As previously demonstrated by Samali et al., oxidative stress can inactivate caspases thus converting the mode of cell death from apoptosis to necrosis [25]. OxLDL can contribute to the induction of either apoptosis or necrosis depending on its oxidation state [26]. Therefore, despite the presence of the apoptotic machinery and the altered expression of pro-apoptotic genes, the high levels of oxidative stress induced by oxLDL may compel the CASMC to die in a caspase-independent manner. Acknowledgments ˚ bo The authors are grateful to Prof. Lea Sistonen, A Akademi University, Finland for providing anti-HSF1 antibody. This work was supported in part by grants from the Millennium Research Fund of NUI Galway and Higher Education Authority. J.R. was supported by an
EMBARK Fellowship from IRCSET. A.S. is an SFI Principal Investigator. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2007.03.027. References [1] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Witztum, Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity, N. Engl. J. Med. 320 (1989) 915–924. [2] M. Navab, G.M. Ananthramaiah, S.T. Reddy, B.J. Van Lenten, B.J. Ansell, G.C. Fonarow, K. Vahabzadeh, S. Hama, G. Hough, N. Kamranpour, J.A. Berliner, A.J. Lusis, A.M. Fogelman, The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL, J. Lipid Res. 45 (2004) 993–1007. [3] S.A. Thorne, S.E. Abbot, P.G. Winyard, D.R. Blake, P.G. Mills, Extent of oxidative modification of low density lipoprotein determines the degree of cytotoxicity to human coronary artery cells, Heart 75 (1996) 11–16. [4] D. Hagg, M.C. Englund, M. Jernas, C. Schmidt, O. Wiklund, L.M. Hulten, B.G. Ohlsson, L.M. Carlsson, B. Carlsson, P.A. Svensson, Oxidized LDL induces a coordinated up-regulation of the glutathione and thioredoxin systems in human macrophages, Atherosclerosis 185 (2006) 282–289. [5] S. Sukhanov, P. Delafontaine, Protein chip-based microarray profiling of oxidized low density lipoprotein-treated cells, Proteomics 5 (2005) 1274–1280. [6] S. Sukhanov, Y. Hua Song, P. Delafontaine, Global analysis of differentially expressed genes in oxidized LDL-treated human aortic smooth muscle cells, Biochem. Biophys. Res. Commun. 306 (2003) 443–449. [7] W. Takabe, Y. Kanai, A. Chairoungdua, N. Shibata, S. Toi, M. Kobayashi, T. Kodama, N. Noguchi, Lysophosphatidylcholine enhances cytokine production of endothelial cells via induction of L-type amino acid transporter 1 and cell surface antigen 4F2, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 1640–1645. [8] S.H. Zhang, R.L. Reddick, J.A. Piedrahita, N. Maeda, Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E, Science 258 (1992) 468–471.
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[9] M.I. Porn-Ares, T.C. Saido, T. Andersson, M.P. Ares, Oxidized lowdensity lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells, Biochem. J. 374 (2003) 403–411. [10] T. Ni Chonghaile, C.G. Concannon, E. Szegezdi, A.M. Gorman, A. Samali, Dexamethasone inhibits apoptosis in C6 glioma cells through increased expression of Bcl-XL, Apoptosis 11 (2006) 1247–1255. [11] K. Ali, M. Middleton, E. Pure, D.J. Rader, Apolipoprotein E suppresses the type I inflammatory response in vivo, Circ. Res. 97 (2005) 922–927. [12] B. Metzler, R. Abia, M. Ahmad, F. Wernig, O. Pachinger, Y. Hu, Q. Xu, Activation of heat shock transcription factor 1 in atherosclerosis, Am. J. Pathol. 162 (2003) 1669–1676. [13] S. Ferdinandusse, B. Finckh, Y.C. de Hingh, L.E. Stroomer, S. Denis, A. Kohlschutter, R.J. Wanders, Evidence for increased oxidative stress in peroxisomal D-bifunctional protein deficiency, Mol. Genet. Metab. 79 (2003) 281–287. [14] E.G. van Grunsven, E. van Berkel, L. Ijlst, P. Vreken, J.B. de Klerk, J. Adamski, H. Lemonde, P.T. Clayton, D.A. Cuebas, R.J. Wanders, Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency, Proc. Natl. Acad. Sci. USA 95 (1998) 2128–2133. [15] F. Bea, F.N. Hudson, A. Chait, T.J. Kavanagh, M.E. Rosenfeld, Induction of glutathione synthesis in macrophages by oxidized lowdensity lipoproteins is mediated by consensus antioxidant response elements, Circ. Res. 92 (2003) 386–393. [16] N. Gotoh, A. Graham, E. Nikl, V.M. Darley-Usmar, Inhibition of glutathione synthesis increases the toxicity of oxidized low-density lipoprotein to human monocytes and macrophages, Biochem. J. 296 (Pt. 1) (1993) 151–154. [17] V. Radjendirane, P. Joseph, Y.H. Lee, S. Kimura, A.J. Klein-Szanto, F.J. Gonzalez, A.K. Jaiswal, Disruption of the DT diaphorase
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OxLDL-induced gene expression patterns in CASMC are mimicked in apoE / mice aortas Janice L.V. Reeve a, Catherine Stenson-Cox a, Aideen O’Doherty b, Isabella Po¨rn-Ares d, Mikko Ares e, Timothy O’Brien b,c, Afshin Samali a,c,* a
d
Department of Biochemistry, National University of Ireland, Galway, Ireland b Department of Medicine, National University of Ireland, Galway, Ireland c National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Department of Biomedicine/Biochemistry, Helsinki University, Haartmaninkatu 8, 00014 Helsinki, Finland e Wallenberg-Lab, University Hospital MAS, Malmo¨, Sweden Received 27 February 2007 Available online 12 March 2007
Abstract Oxidized low density lipoprotein (oxLDL) contributes to the pathophysiology of atherosclerosis, partly by altering gene expression in vascular cells. Here, we show 221 genes differentially regulated by oxLDL in coronary artery smooth muscle cells (CASMC), using oligonucleotide microarrays. These genes were classified into 14 functional groups. A comparable gene expression pattern was detected in apoE / mice. OxLDL induced an oxidative stress response in CASMC, but not the unfolded protein response. OxLDL also caused CASMC death which was accompanied by increased expression of FasL, Bax, and p53 but was caspase-independent. This approach provides further insight into disease pathology and prognosis. 2007 Elsevier Inc. All rights reserved. Keywords: Apolipoprotein E; Cell death; Gene expression; Oxidative stress; OxLDL; Smooth muscle cell
Atherosclerosis, leading to coronary heart disease (CHD) and cerebral stroke, is the most common cause of death and morbidity in industrialized societies. Low density lipoprotein (LDL) and its oxidized derivative, oxLDL, initiate and promote atherosclerosis [1]. In vivo LDL may be oxidized by vascular cells and later by infiltrating immune cells [2]. OxLDL is detected in atherosclerotic plaques and in the plasma of atherosclerosis patients, where it contributes to disease evolution [1]. Depending on the extent of its oxidation, oxLDL can induce proliferation, monocyte chemotaxis, apoptosis or necrosis of vascular endothelial (EC) and smooth muscle cells (VSMC) [1,3]. * Corresponding author. Address: National Centre for Biomedical Engineering Science, National University of Ireland, University Road, Galway, Ireland. Fax: +353 91 494596. E-mail address: [email protected] (A. Samali).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.027
The vascular cell response to oxLDL is regulated, in part, at the level of transcription and oxLDL-induced gene expression profiles have been investigated in EC, macrophage and VSMC [4–7]. The overall effect may initially be cytoprotective allowing the cell to adapt to the stress, however, when prolonged; such cellular stress responses result in cellular demise and disease manifestations. Uptake of oxLDL by human macrophages increased the expression of the antioxidant genes, glutathione and thioredoxin, to protect the cell from oxLDL and the resultant oxidative stress [4]. OxLDL also increased the expression of genes involved in cell–cell interaction, transport, transcription and apoptosis, and decreased expression of genes involved in protein biosynthesis and lipid metabolism in human aortic SMC [6]. The proteomics study which followed revealed similar patterns suggesting that gene expression profiling can provide insight into the cellular response [5,6]. This
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prompted us to investigate the global gene expression pattern modulated by oxLDL in primary human CASMC and to compare them to that found in vivo in the aortas of apoE / mice, the well-established atherosclerotic model [8]. Additionally, we investigated the effect of oxLDLinduced alterations in gene expression on overall biological function. Materials and methods Cell culture. Human coronary artery smooth muscle cells (CASMC) (Clonetics) were cultured in SMC basal medium (Cambrex), supplemented with Clonetics SMC bullet kit containing 5% fetal bovine serum, at 37 C with 5% CO2 CASMC, when not stated otherwise, were treated with 60 lg/ml oxLDL or sterile PBS as vehicle control. Preparation and oxidation of low density lipoproteins. LDL was isolated as previously reported [9]. For oxidation, 300 lg/ml LDL was incubated with 5 lM CuSO4 at 37 C until the desired oxidation state was obtained. The oxidation state was determined by measuring the thiobarbituric acidreactive substances (TBARS) using malondialdehyde (MDA) as a standard. OxLDL used contained 15–30 nmol of TBARS as MDA equivalents/mg of LDL proteins and was used within 6 h of preparation. Preparation of RNA and hybridization to oligonucleotide microarrays. The total RNA was isolated from oxLDL-treated and untreated CASMC using Tri reagent (MRC Inc.) according to the manufacturer’s instructions. Complementary DNA was synthesized using the Microarray cDNA Synthesis Kit (Roche Diagnostics) according to the manufacturer’s instructions. After purification with High Pure RNA Tissue Kit (Roche Diagnostics), cRNA was transcribed and labeled with cyanine 3-UTP or cyanine 5-UTP (100 nmol; Amersham Biosciences) dyes using the MEGAscript T7 High Yield Transcription Kit (Ambion). Labeled cRNA (10 lg) was combined and fragmented with buffer containing 20 mM tris acetate, pH 8.1, 50 mM potassium acetate, and 15 mM magnesium acetate for 15 min at 94 C. Fragmented cRNA was applied to human 10 K oligonucleotide microarrays (microarray ‘‘A’’, MWG Biotech) and incubated overnight agitating at 42 C. Microarrays were scanned and analyzed by MWG Biotech with genes differentially expressed by 2-fold analyzed for proposed function using gene ontology programs, GeneCards (www.genecards.org) and Bioinformatic Harvester (www.harvester.embl.de). Reverse transcriptase polymerase chain reaction (RT-PCR). Reverse transcription was carried out with 2 lg total RNA and oligo(dT) 12–18 (Invitrogen) using AMV Superscript (Invitrogen) according to the manufacturer’s instructions. The cDNA product was subjected to PCR using primers from Supplementary material Table 1. GAPDH primers were used for normalization. Extraction of mouse aortas and sample preparation. ApoE / mice, strain B6.129P2-Apoetm1Unc/J; Jackson Laboratory, were fed a Western diet of 0.15% cholesterol (Harlan Teklad) for 1 month and were sacrificed at 4–8 months of age. The abdominal cavity was opened. Visceral organs were moved to the side and an angled incision was performed above the heart. The aorta was excised along with the heart as far as the aortic bifurcation in the groin area. Frozen sections were cut from OCTembedded aortas on a rotary freezing microtome at 20 lm and lifted onto polylysine-coated slides. Sections were incubated at 37 C overnight and stained for lipid-rich lesions with sequential incubations as follows: dH2O for 10 s, 60% isopropyl alcohol for 10 s, Sudan IV stain (0.5% Sudan IV, 35% ethanol, 50% acetone) for 4 min, 60% isopropyl alcohol for 10 s, dH2O for 30 s, Harris hematoxylin for 1 min, dH2O for 1 min 3·, and mounted using aqueous mounting solution (Dako). Sections were examined by light microscopy (Olympus BX51). The total RNA was extracted from homogenized aortas as described earlier. All animal experiments were performed in accordance with the ethical regulations of NUI Galway. Western blot analysis. Protein samples were prepared as previously reported [10]. Equal amounts of protein (30 lg) were denatured in Laemmli’s buffer and resolved on 10–12% SDS–PAGE and transferred onto nitrocellulose membrane. After blocking in 5% (w/v) non-fat milk
in PBS containing 0.05% Tween 20 the membranes were incubated with rabbit polyclonal antibodies to Bax, caspase-3, PARP (1:1000; Cell Signaling Technologies), caspase-8, CHOP (1:1000; Santa Cruz), HSF-1 ˚ bo Akademi University, (1:500, a kind gift from Prof. Lea Sistonen, A Finland), actin (1:500; Sigma–Aldrich), and mouse monoclonal antibodies to Hsp70 and KDEL (recognizes Grp78 and Grp94; 1:1000; Stressgen). Horseradish peroxidase-conjugated secondary antibodies (1:10,000) were obtained from Pierce. Protein bands were visualized with Supersignal Ultra Chemiluminescent Substrate (Pierce) on X-ray film (AGFA). Cell morphology. Cells were fixed in methanol for 5 min followed by staining in Harris hematoxylin solution for 15 s and Eosin Y for 15 s. Subsequently, cells were observed by phase contrast microscopy (Olympus IX71). Caspase activity assay. Cell lysates (25 ll) and 50 lM DEVD-AMC (Peptide Institute Inc.) in reaction buffer (100 mM Hepes, 10% sucrose, 5 mM DTT, 0.0001% Igepal-630, and 0.1% CHAPS, pH 7.25) were added in duplicate to a microtiter plate. Liberated AMC was measured using 355 nm excitation and 460 nm emission wavelengths, over time and the data analyzed by linear regression to determine enzyme activity. Enzyme activity was expressed as fold change in DEVDase activity compared to untreated control cells. Lactate dehydrogenase assay. Relative lactate dehydrogenase (LDH) levels in the supernatant were measured using a Cytotoxicity Detection LDH assay kit (Roche Diagnostics Ltd.) as per manufacturer’s instructions. Results are expressed as fold change in absorbance at 490 nm compared to untreated CASMC controls.
Results and discussion Global gene expression analysis of oxLDL-treated CASMC OxLDL is a pro-atherogenic risk factor responsible for many of vascular cell alterations observed during atherosclerosis. To determine the oxLDL-induced alterations in gene expression, CASMC were incubated with 60 lg/ml (15–30 nmol of MDA/mg LDL) of Cu2+-oxLDL for 24 h, RNA was isolated, fluorescently labeled and hybridized to oligonucleotide microarrays. The experiment was performed in duplicate and included a dye-swap experiment to compensate for potential dye bias. Two hundred and twenty-one genes exhibited P2-fold change in expression, 174 were putatively upregulated while 47 were downregulated, following oxLDL treatment (Supplementary material Tables 2 and 3). The differentially expressed genes were classified into 14 functional groups encompassing redox homeostasis, Ca2+ signaling, immune response and apoptosis (Supplementary material Table 4). All of which have been implicated in the pathology of atherosclerosis. A random selection of genes identified as putatively regulated by the microarray study was chosen for RT-PCR analysis. As shown in Fig. 1A, oxLDL induced the expression of glutathione reductase, NAD(P)H dehydrogenase-1, adenosine A2A receptor, calcium/calmodulin-dependent protein kinase kinase 2b (CAMKK2b), heat shock factor-1 (HSF-1), and interleukin 12A (IL-12A); whilst it reduced the mRNA levels of vascular endothelial growth factor A (VEGFA) precursor, hydroxysteroid 17b dehydrogenase 4 (HSD17B4) and apoptosis inhibitor 5 (API5), compared to vehicle treated controls.
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Fig. 1. The gene expression pattern in oxLDL treated CASMC and atherosclerotic aortas. (A) CASMC were incubated with 60 lg/ml oxLDL for 24 h at 37 C. The total RNA from treated and untreated cells was subjected to RT-PCR for the mRNA expression levels of glutathione reductase, NAD(P)H dehydrogenase-1, adenosine A2A receptor, CAMKK2b, HSF-1, IL-12A, VEGFA precursor, HSD17B4, and API5. (B) Transverse sections of apoE / mouse aorta were prepared and stained with Sudan IV and hematoxylin. Depicted are representative sections from apoE / mouse aorta containing an established lesion (upper panel) and a section free from lesions (lower panel). (C) RT-PCR analysis of mRNA levels of HSF-1, IL-12A, VEGFA precursor, HSD17B4, and API5 in apoE / and WT mice. GAPDH was used as an internal control.
Differential gene expression in apoE
/
mice
To investigate the pathophysiological relevance of this altered gene expression, the profile of selected genes were examined in apoE / mice. The presence of plaques in the aortas of apoE / mice was confirmed by Sudan IV staining (Fig. 1B). The total RNA from the aortas of apoE / and WT mice was examined by RT-PCR for the expression of HSF-1, IL-12A, VEGFA precursor, HSD17B4, and API5 (Fig. 1C). Significantly, oxLDLinduced alterations in CASMC gene expression were comparable in aortas of apoE / mice. As anticipated, the expression of IL-12A and HSF-1 was upregulated, an indication of the pro-inflammatory response, as previously reported in animal models of atherosclerosis [11,12]. We observed a decrease in VEGFA precursor and HSD17B4 expression, associated with cell proliferation and cholesterol metabolism, respectively. Interestingly, HSD17B4 is associated with oxidative stress in humans [13,14]. Taken together these in vitro and in vivo findings bear relevance to the development of atherosclerosis. Despite the decrease in API5 observed in the microarray study, its expression remained unchanged in apoE / mice. Overall, despite the increased complexity of the atherosclerotic mouse model, the changes in gene expression observed in vitro are immediately comparable to the in vivo situation. OxLDL-induced CASMC injury is therefore highly signif-
icant in the context of the pathology and the progression of atherosclerosis. OxLDL induces an oxidative stress response, but not the unfolded protein response Having established that the alterations in gene expression induced by oxLDL are comparable to the more complex in vivo setting, we wished to extrapolate the potential cellular consequences. Of the genes regulated in oxLDLtreated CASMC, a number were associated with the cellular stress response. The expression of redox-sensitive genes can be induced as part of an adaptive response to oxidative stress [15–17]. Since redox-sensitive genes, present on the microarray were induced in response to oxLDL we set out to determine whether oxLDL induced an oxidative stress response in CASMC. To that end, the expression of additional antioxidant genes, not present on the array but positively associated with the oxidative stress response, was assessed [18–20]. As demonstrated in Fig. 2A oxLDL induced the expression of manganese superoxide dismutase (MnSOD), hemoxygenase-1 (HO-1) and ferritin. In addition, protein levels of Hsp70 and HSF-1, whose gene expression was increased in the microarray study, was also found to be increased (Fig. 2B). Taken together these data suggest the initiation of an adaptive oxidative stress response in CASMC in response to oxLDL. We hypothesis
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Fig. 2. OxLDL induces an oxidative stress response, but not the unfolded protein response. CASMC were incubated with 60 lg/ml oxLDL for 24 h at 37 C. (A) RT-PCR analysis of mRNA expression levels of MnSOD, HO-1, ferritin, ATF3, ATF4, XBP-1, Egr-1, and Grp78. GAPDH was used as internal control. (B) Western blot analysis of Hsp70, HSF-1, CHOP, and KDEL. Thapsigargin (2 lM; 36 h) treated HeLa cells were used as a positive control (+ve). Anti-actin antibody was used to determine equal sample loading.
that this response is an attempt to alleviate cellular oxidative stress. As oxidative stress can induce secondary effects such as increasing cytosolic Ca2+ concentrations [21] and oxLDL can decrease the expression of ER resident Ca2+-pumps [22,23] we next investigated the possible effect of oxLDL on ER stress and the unfolded protein response (UPR). ER stress is sensed by three ER transmembrane receptors; ATF6, Ire1, and PERK [24]. Activation of these sensors launches the UPR which attempts to restore ER homeosta-
sis. However, failure of the UPR to restore normal ER function, can result in apoptosis [24]. The activation of Ire1 and PERK was monitored by detecting the induction of ATF3, ATF4, Egr-1, XBP-1, CHOP, Grp94, and Grp78. As demonstrated in Fig. 2A mRNA levels of ATF3, ATF4, Egr-1, XBP-1 or Grp78 were not altered by oxLDL nor did protein levels of Grp78, Grp94 or CHOP change (Fig. 2B). This would indicate that oxLDL does induce an oxidative stress response in CASMC but this is not accompanied by activation of the UPR.
Fig. 3. OxLDL induces CASMC death and induces the expression of pro-apoptotic genes. CASMC were incubated with either 60 or 150 lg/ml oxLDL for 24 h at 37 C. (A) Cellular morphology was examined by phase contrast and light microscopy of hematoxylin and eosin stained cells. (B) RT-PCR analysis for FasL and p53 mRNA expression. GAPDH mRNA was utilized as an internal control. Western blot analysis of Bax expression. Anti-actin antibody was used to determine equal sample loading.
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Fig. 4. OxLDL-induced CASMC death is independent of caspases. CASMC were incubated with 60 lg/ml oxLDL for 24 h at 37 C. (A) Western blot analysis using antibodies to caspase-8, caspase-3 and PARP. Apoptotic HeLa cells were used as a positive control. (B) Caspase-3-like activity was detected by monitoring the cleavage of synthetic substrate DEVD-AMC. Staurosporine-treated (0.25 lM; 4 h, sts) apoptotic cells were used as a positive control. (C) Loss of outer plasma membrane integrity was determined by LDH release assay. CASMC exposed to 60 lg/ml oxLDL for 24 h were compared to untreated CASMC.
OxLDL-induced CASMC death is caspase-independent Since excessive or prolonged oxidative stress can induce cell death, we investigated the effect of oxLDL on CASMC. In terms of cell morphology, CASMC exhibited a loss in cell volume and appeared condensed following treatment with 60 lg/ml oxLDL for 24 h, while administration of 150 lg/ml oxLDL induced cytolysis (Fig. 3A). As demonstrated in Fig. 3B, administration of 60 lg/ml oxLDL also induced the expression of FasL, p53, and Bax, which are associated with apoptosis. However, despite the induction of such early apoptotic molecules, processing of pro-caspase-8, pro-caspase-3 or its downstream substrate PARP was not detected (Fig. 4A). In addition DEVDase activity was not evident (Fig. 4B) and a loss in outer plasma membrane integrity, as detected by LDH release, was observed (Fig. 4C). Therefore, oxLDL-induced CASMC death, under these experimental conditions appeared to be independent of caspase activation. This is complimentary to the previous findings by Porn-Ares et al., in oxLDL-treated endothelial cells [9]. As previously demonstrated by Samali et al., oxidative stress can inactivate caspases thus converting the mode of cell death from apoptosis to necrosis [25]. OxLDL can contribute to the induction of either apoptosis or necrosis depending on its oxidation state [26]. Therefore, despite the presence of the apoptotic machinery and the altered expression of pro-apoptotic genes, the high levels of oxidative stress induced by oxLDL may compel the CASMC to die in a caspase-independent manner. Acknowledgments ˚ bo The authors are grateful to Prof. Lea Sistonen, A Akademi University, Finland for providing anti-HSF1 antibody. This work was supported in part by grants from the Millennium Research Fund of NUI Galway and Higher Education Authority. J.R. was supported by an
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