Expanded Methods

R2

Expanded Methods

Cell Culture

Human coronary artery smooth muscle cells (CASMCs) and human THP-1 cells were purchased from Cambrex and ECACC, respectively. Cell culture was performed as described previously.1 THP-1 cells differentiated into macrophages after culture in RPMI 1640 medium (MP Biomedicals) plus phorbol 12-myristate 13-acetate (100 ng/ml, Sigma) as a stimulant for 3 days and became attached to the plates as a subconfluent monolayer before use in the following experiments.

Quantitative RT–PCR

Quantitative RT–PCR was performed as described previously.2 In brief, CASMCs or THP-1 cells were incubated for 12 hours with 7-KC (80 mM) in the absence or presence of 3.3 mM N-acetylcysteine (NAC) or 10 mM glutathione (GSH), which are both potent antioxidants (Sigma). RNA was extracted using Trizol reagent (Invitrogen) from cultured cells or DCA specimens (SAP; n=4, UAP; n=4). After extraction, 1 mg of total RNA was reverse-transcribed using Omniscript RT (Qiagen). Quantitative RT–PCR was performed with TaqMan technology using the ABI Prism 7700 detection system (Applied Biosystems) according to the manufacturer’s instructions. Data were normalized for the level of GAPDH expression. Each sample was analyzed in duplicate and the experiments were replicated twice for the full set of genes. The primers and probes for GAPDH, GRP78, and CHOP were obtained as TaqMan Assays-on-Demand products (Applied Biosystems).

Measurement of ROS generation

Intracellular production of ROS was measured by microscopy with 2’,7’-dichlorofluorescin diacetate (DCFH-DA, Invitrogen), as described previously.3 In brief, SMCs plated on Lab-Tek chambered cover-glass slides were preincubated with or without 7-KC (80 mM) for 12 h in the absence or presence of 10 mM glutathione (GSH) and were then loaded with 10 mM DCFH-DA for 10 min. Detection of Apoptosis in Cultured CASMCs and THP-1 Cells

CASMCs or THP-1 cells were incubated with 0, 10, 40, or 100 mM 7-KC (Sigma) for 24 hours to assess the dose-response effect, and with 40 mM 7-KC for 0, 12, 24, or 36 hours to assess the influence of the incubation period. At the end of incubation, the cells were assayed for early/middle-stage apoptosis by staining with FITC-labelled annexin V (BD PharMingen) and for late apoptosis by staining with PI, as described previously.4

Immunofluorescent staining

CASMCs or THP-1 cells were incubated for 24 hours with 80 mM 7-KC. In brief, CASMCs or THP-1 cells were incubated under the indicated conditions, were fixed with 3% PFA for 20 minutes, and then permeabilized in 0.2% Triton X-100 for 10 minutes. Double immunofluorescence staining was performed as previously described5 with anti-KDEL antibody (1:200) and anti-CHOP antibody (1:100). Terminal dUTP nick-end labeling (TUNEL) reaction was performed as described previously5. Cells were incubated with tunicamycin (1.0 mg/ml, Sigma) as a positive control. DAPI (Vector Laboratories) was used for nuclear staining. Either Alexa 488 or 546 antibody or an ApoTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon) was employed for immunofluorescence. Numbers of either KDEL-, or CHOP-, or TUNEL-positive cells counted in DAPI-positive cells (n=300) are expressed as percentages.

Inhibition of GADD153 with Short Interfering RNA (siRNA)

Transfection of CASMCs with siRNA was performed as described previously.2 CASMCs at 80-90% confluence were transfected with Lipofectamine RNAi Max (Invitrogen) plus siRNA at 50 nM for 4 hr in a dish containing serum free OPTI-MEM medium (Invitrogen) at 4-6 hr after plating. The transfection efficiency was measured with Block-it Alexa Fluor Red Fluorescense Oligo (Invitrogen) (Supplementary Figure 4). THP-1 cells were transfected in the same way after PMA stimulation for 3 days at 80-90% confluence. siRNA targeting CHOP and the nonsilencing control (S5C-0600) were synthesized by B-Bridge International (Mountain View, CA), and their sequences were as follows: CHOP (1) sense gcaugaaggagaaagaacaTT, CHOP (2): sense gcaccaagcaugaacaauuTT.

Supplementary analyses of autopsy specimens

For the pairwise analysis of 17 pairs of unruptured and ruptured plaques obtained from 17 patients with ACS (Supplementary Figure 1), as representative unruptured plaques, we chose the unruptured lesion with the most numerous positive cells in each patient enrolled in this analysis. Thus, one pair of plaques was compared per patient, and Wilcoxon’s signed-rank test was used for evaluating the differences.

For the subgroup analysis of risk factors (Supplementary Table 2 and Supplementary Figure 3), we chose the plaque (n=71) with the most numerous positives cells in each patient (n=71). Mann-Whitney U test was used for evaluating the difference between the presence or absence of risk factors (Supplementary Table 2). Spearman’s rank correlation analysis was used to evaluate the correlation between the number of traditional cardiovascular risk factors and the number of CHOP-positive cells/mm2 (Supplementary Figure 3). In all analyses, P<0.05 was accepted as indicating statistical significance.

References:

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2. Shintani Y, Takashima S, Asano Y, Kato H, Liao Y, Yamazaki S, Tsukamoto O, Osamu Seguchi O, Yamamoto H, Fukushima T, Sugahara K, Kitakaze M, Hori M. Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling. EMBO Journal. 2006;25:3045–3055

3. Pedruzzi E, Guichard C, Ollivier V, Driss F, Fay M, Prunet C, Marie JC, Pouzet C, Samadi M, Elbim C, O’Dowd Y, Bens M, Vandewalle A, Gougerot-Pocidalo MA, Lizard G, and Ogier-Denis E. NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol. 2004;24:10703–10717.

4. Feng B, Yao PM, Li Y, Devlin CM, Zhang D, Harding HP, Sweeney M, Rong JX, Kuriakose G, Fisher EA, Marks AR, Ron D, Tabas I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol. 2003;5:781–792.

5. Okada K, Minamino T, Tsukamoto Y, Liao Y, Tsukamoto O, Takashima S, Hirata A, Fujita M, Nagamachi Y, Nakatani T, Yutani C, Ozawa K, Ogawa S, Tomoike H, Hori M, Kitakaze M. Prolonged endoplasmic reticulum stress in hypertrophic and failing heart after aortic constriction. Circulation. 2004;110:705-712