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HUVEC were isolated as described (1) and cultured in Medium-199 (BioWhittaker) supplemented with 50 m g/ml endothelial cell growth supplement (Collaborative Research), 100 m g/ml heparin (Sigma), 100 units/ml penicillin plus 100 m g/ml streptomycin (BioWhittaker), 2 mM L-Glutamine (GIBCO), and 20% FBS (BioWhittaker). EC (passage 1) were plated at an initial density of 70,000 cells per cm² on 0.1% gelatin (Difco)-coated polystyrene plastic (Plaskolite, Columbus, OH). After 24 h, the confluence of each EC monolayer was verified by phase contrast examination, and the cell culture plate was assembled in the dynamic flow system. EC monolayers were then exposed to the desired shear stress waveforms for 24 h. Culture medium in the shear apparatus was replenished during the experiment at an exchange rate of 0.07 ml/min, and the enclosed environment was maintained at 37°C in a humidified 5% CO₂/95% air atmosphere. EC from the same passage were plated on an identical plastic plate assembly and incubated at 37°C in a 5% CO₂/95% air atmosphere under static (no flow) conditions.

Total RNA was isolated by using TRIzol reagent (Invitrogen) and the RNeasy kit (Qiagen, Valencia, CA). Total RNA was DNase-treated with the RNase-free DNase kit (Qiagen) according to the manufacturer’s protocol and purified on RNeasy mini columns (Qiagen). RNA quality was verified by Agilent’s 2100 Bioanalyzer with RNA 6000 Nano LabChip Kit. The concentration of RNA was measured by spectrophotometric analysis at 260 nm. To choose a consistent control for standard data set comparisons, we prepared the reference RNA by pooling RNA from various sources to achieve broad gene coverage on human microarrays. The reference RNA used in our experiments was comprised of the universal human reference RNA (Stratagene) plus HUVEC exposed to laminar shear stress (10 dyn/cm², 24 h) and HUVEC exposed to an inflammatory stimulus (IL-1, 10 units/ml, 4 h), and angiogenic stimulus (vascular endothelial growth factor,, 50 ng/ml, 24 h).

For cDNA microarray analysis, three independent sets of experiments were performed. Each experiment consisted of cells cultured under athero-prone, athero-protective, and static (no flow) conditions for 24 h. Twenty micrograms of total RNA isolated from each experimental condition and 20 m g of reference RNA were reverse-transcribed into cDNA in the presence of Cy5- and Cy3-labeled dNTPs (fluorescent direct label kit, Agilent Technologies, Palo Alto, CA). The resulting labeled cDNA samples were purified by using QIAquick spin columns (Qiagen). Cy3- and Cy5-labeled cDNA were then combined and hybridized to one array set (human 1 cDNA microarray, Agilent Technologies). The Agilent human 1 cDNA array contains 14,836 array spots representing 10,456 genes. The clones spotted on the array are sourced from Incyte’s sequence-verified UniGene 1 and human drug target cDNA clone sets. The microarray slides were hybridized at 65°C for 17 h followed by washing with 0.5x SSC, 0.01% SDS for 5 min, and 0.06x SSC for 2 min at room temperature. The hybridized array was then scanned by an Agilent G2565AA microarray scanner at 5-m m resolution. Array images were analyzed and quantified by using Agilent FEATURE EXTRACTION software (version A.6.1.1) to obtain the spot intensity and background level of the two channels (Cy3 and Cy5). Data were then transferred to a custom-designed database (Argus) previously developed in our laboratory (2) to facilitate data analysis.

Array data were analyzed by using statistical techniques developed in our laboratory especially adapted to analyze data sets with large number of genes and comparatively small number of array replicates. The method is an intensity-based variance estimation algorithm based on the Z-statistics, which outperforms two other methods for ranking differentially regulated genes (t test, penalized t test) in both accuracy and precision. Detailed statistical algorithm and implementation can be found in ref. 3. For comparison between any two given experimental conditions, the arrays were processed in pairs (e.g., athero-prone vs. athero-protective, athero-protective vs. static, or athero-prone vs. static). Data from three independent experiments were analyzed, and the averaged ratio and P value were obtained for each paired comparison. For any given gene, the regulation was considered as statistically significant when P < 0.05.

Purified, DNase-treated RNA (1.5 m g) was reversed-transcribed by using a MultiScribe based reverse transcription reaction (Applied Biosystems). The cDNA were then subjected to a real-time TaqMan PCR in a GeneAmp 5700 sequence detection system (Applied Biosystems). The relative gene expression was normalized to b ₂-microglobulin. The primer pairs and TaqMan probes used in the reactions are shown in Table 1.

EC were cultured under static (no flow), athero-prone, and athero-protective waveforms for 24 h, and in certain experiments subsequently treated with IL-1b (1 unit/ml) for 4 h (for E-selectin) or 6 h (for VCAM-1) under static conditions. EC were then rinsed with 4°C PBS, detached with cell dissociation buffer (Invitrogen) containing EDTA, and centrifuged. Cells were washed with PBS and resuspended in PBS containing 1% BSA and an anti-VCAM-1 antibody (1:500, mouse monoclonal IgG, Dako Cytomation) or anti-E-selectin (1:400, H18/7 mouse monoclonal IgG anti-human E-selectin). The cell suspension was incubation at 4°C for 30 min, then centrifuged at 1,000 rpm for 5 min, and washed twice with PBS. The EC were then incubated with Alexa Fluor goat anti-mouse IgG (1:500, Molecular Probes) for 30 min at 4°C. After two final washes in PBS, the cells were fixed in PBS containing 4% paraformaldehyde and analyzed in a Becton Dickinson FACScan Flow Cytometer.

At the conclusion of each experiment, EC were rinsed in cold PBS and fixed in 4% paraformaldehyde for 10 min and then permeabilized in 0.1% Triton X-100 for 2 min at room temperature. To reduce nonspecific Ig binding, cells were preincubated in 1% BSA for 1 h. For F-actin staining, cells were incubated with Alexa Fluo 488 phalloidin (1:50 (Molecular Probes) for 30 min at room temperature. Cells were rinsed with PBS and incubated with SYTOX orange (Molecular Probes) at 0.01 m M for 2 min to stain the nuclei. For NF-k B, Cx37, and Cx43 staining, cells were incubated with anti-p65 antibody (1:500, rabbit polyclonal IgG, Santa Cruz Biotechnology), anti-Cx37 antibody (1:200, rabbit polyclonal IgG, Alpha Diagnostic International, San Antonio, TX), or anti-Cx43 antibody (1:500, mouse monoclonal IgG, BD Biosciences) for 1 h at room temperature, rinsed with 0.1% BSA in PBS, and incubated for 30 min with 2% goat serum in PBS. Cells were incubated with Alexa Fluor 488 goat anti-rabbit or goat anti-mouse (1:500, Molecular Probes) for 1 h at room temperature, rinsed in PBS, and mounted by using Gel-Mount (Biomeda, Foster City, CA).

Cells were gently harvested in the presence of enzyme-free cell dissociation buffer (Invitrogen) by using a rubber policeman. Nuclear and cytoplasmic proteins were then extracted by using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology). The protein concentration was measured by BCA protein assay (Pierce Biotechnology). Ten micrograms of nuclear or cytoplasmic extracts from each condition was then subjected to Western blot analysis of p65 protein (anti-p65 antibody 1:500, rabbit polyclonal IgG, Santa Cruz Biotechnology). To verify the purity of the nuclear and cytoplasmic protein fraction, the same membrane was incubated in Western blot stripping buffer (Pierce Biotechnology) for 10 min at room temperature and reprobed with anti-histone H3 antibody (1:200, rabbit polyclonal, Cell Signaling Technology) or anti-a -tubulin (1:500, mouse monoclonal, Zymed).

1. Garcia-Cardena, G., Comander, J., Anderson, K. R., Blackman, B. R. & Gimbrone, M. A., Jr. (2001) Proc. Natl. Acad. Sci. USA 98, 4478-4485.

2. Comander, J., Weber, G. M., Gimbrone, M. A., Jr., & Garcia-Cardena, G. (2001) Genome Res. 11, 1603-1610.

3. Comander, J., Natarajan, S., Gimbrone, M. A., Jr., & Garcia-Cardena, G. (2004) BMC Genomics 5, 17.