Enalapril and nifedipine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Specific pathogen free 8–9-week-old male SHR and WKY rats were obtained from the Samtaco Animal Breeding Company (Osan, South Korea). All in vivo studies were approved by the Institutional Animal Care and Use Committee, Chonnam National University. Enalapril (40 mg/kg/day) or nifedipine (30 mg/kg/day) were administered orally every day for 3 weeks. The dosage and duration of administration were calculated considering the maximum therapeutic dose in humans and based on previous studies (8,9).

Systolic blood pressure (SBP) was measured using the CODA system (Kent Scientific Corporation, Torrington, CT, USA) as previously described (10). SBP and body weight were evaluated every week. After 3 weeks of administration, the rats were sacrificed and tissue weights of the heart, brain and liver were determined.

Samples of heart, brain and liver were homogenized, and total RNA was extracted using TRIzol^® reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). The concentration and purity of the total RNA were determined spectrophotometrically at 260 and 280 nm using an Agilent™ Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

Differential gene expression was measured using CodeLink Rat Whole Genome Bioarrays (<34,000 genes; Amersham, Piscataway, NJ, USA) following the manufacturer's protocol, and the data were analyzed as previously described (11). Briefly, for first-strand cDNA synthesis, 5 μg of total RNA was heated at 70°C for 10 min with control bacterial mRNA and a T7 oligo(dT) primer, followed by incubation with first-strand reaction components (10X buffer, 5 mM dNTP, RNase inhibitor and reverse transcriptase) for 1 h at 42°C. The second strand was produced by incubation of first-strand cDNA products with second-strand reaction components (10X buffer, 5 mM dNTP, RNase H, DNA polymerase) for 2 h at 16°C. Double-stranded cDNA was purified using a QIAquick purification kit (Qiagen, Valencia, CA, USA). Purified double-stranded cDNA was incubated at 37°C for 14 h with IVT mix (10X reaction buffer, biotinylated UTP, UTP, ATP, CTP, GTP and T7 enzyme) to enable in vitro transcription of cRNA. The biotin-labeled cRNA was purified using the Qiagen RNeasy mini kit (Qiagen). A mixture of 10 μg of cRNA and 5X fragmentation buffer was incubated at 94°C for 20 min, and then hybridized at 90°C for 5 min. After chilling on ice for 10 min, the hybridization mixtures were slowly injected into sealed microarray slides. The slides were shaken at 37°C and 300 rpm for 18–24 h prior to incubation at 46°C for 1 h with preheated 0.75X TNT buffer [1X TNT: 0.1 M Tris-HCl (pH 7.6), 0.15 M NaCl, and 0.05% Tween-20], followed by incubation at room temperature for 30 min with 3.4 ml of Cy5-streptavidin working solution. The slides were washed four times with 1X TNT buffer for 5 min and then rinsed with 0.1X SSC/0.05% Tween™ for 30 sec. Hybridization to the arrays was detected using a ScanArray Express scanner (Packard BioScience, Meriden, CT, USA) with the laser set at 635 nm. After scanning, the array images were analyzed by ImaGene 6.0 and GeneSight 4.1 software.

MOVAS cells (1x10⁵/ml), an immortalized mouse vascular aorta smooth muscle cells, were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco-BRL, Rockville, NY, USA) at 37°C in a humidified atmosphere containing 5% CO₂. When the cells were 70% confluent, the medium was replaced with serum-free DMEM. After serum starvation for 12 h, the cells were treated with 0.3 mM H₂O₂ (Merck, Darmstadt, Germany) for 6 h in the presence or absence of recombinant human (rh) IL-24 (50 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA).

Total RNA was isolated using TRIzol^® reagent (Molecular Research Center, Inc.). First-strand cDNA was synthesized from 5.0 μg of total RNA using an M-MLV reverse transcriptase kit (Promega, Madison, WI, USA), according to the manufacturer's instructions. For RT-PCR, the synthesized cDNAs were added to a PCR mixture consisting of 10X PCR buffer, 0.25 mM dNTP, 0.5 unit TaqDNA polymerase and 10 pmol of gene-specific primers. Primers used for PCR were: angiotensinogen, 5′-gtacagacagcaccctactt-3′ and 5′-cacgtcacggagaagttgtt-3′; endothelin-1, 5′-agctggtggaaggaaggaaactacg-3′ and 5′-gacagtgcagaaaggtgaggtagac-3′; angiotensin II type 1 receptor-associated protein (ATRAP), 5′-tgcttggggcaacttcactatc-3′ and 5′-acggtgcatgtggtagacgag-3′; platelet-derived growth factor (PDGF), 5′-tccagcgacaaggaacagaacg-3′ and 5′-ggagattcagattcaccactttgc-3′; β-actin, 5′-gagatggccactgccgcatcctct-3′ and 5′-atggtgctaggagccagagcagta-3′. PCR amplification was performed using 26 cycles (β-actin) or 28 cycles (all others) at 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, followed by a final extension for 10 min at 72°C. Equal volumes of reaction mixture from each sample were loaded onto 1% agarose gels and visualized by ethidium bromide staining.

Real-time PCR amplifications were performed in 48-well plates in a MiniOpticon™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., USA) in a total volume of 10 μl, including 1 μl of template cDNA, 10 pmol of gene-specific primers and iQ SYBR-Green Supermix (Bio-Rad Laboratories, Inc.). Each sample was analyzed in triplicate. Primers used for PCR were: myocyte enhancer factor 2D (Mef2d), 5′-cagcagccagcactacagag-3′ and 5′-ggcagggatgaccttgttta-3′; IL-24, 5′-ggcctgagcctaatccttct-3′ and 5′-ctgcagaacctgtggtttca-3′; occludin, 5′-gagggtacacagaccccaga-3′ and 5′-caggattgcgctgactatga-3′; peroxisome proliferator-activated receptor δ (Pparδ), 5′-aacatccccaacttcagcag-3′ and 5′-tactgcgcaagaactcatgg-3′; triadin, 5′-gcttccagacctgctttgtc-3′ and 5′-ggctcttttcctttcccatc-3′; moesin, 5′-cccaaagagtcttggagcag-3′ and 5′-atgttgagacccaaggcatc-3′; guanosine monophosphate reductase (Gmpr), 5′-gatgtggccaatgggtattc-3′ and 5′-ccgactcccactttgatgat-3′; β-actin, 5′-attgttaccaactgggacgacatg-3′ and 5′-cttcatgaggtagtctgtcaggtc-3′. PCR amplification was performed as follows: 95°C for 15 min, followed by 48 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. All data were normalized to β-actin expression levels.

For statistical analysis of data, P-values were analyzed using a two-way ANOVA and post-hoc test. Results are expressed as the means ± SEM, and differences are considered statistically significant when P<0.05.

SBP levels of SHR were higher than those of WKY rats (128±11 mmHg in WKY vs. 186±10 mmHg in SHR, P<0.05), while administration with enalapril or nifedipine for 3 weeks significantly decreased SBP levels of SHR (186±10 mmHg in SHR vs. 144±9 mmHg in enalapril-treated SHR, P<0.05 or 142±8 mmHg in nifedipine-treated SHR, P<0.05) (Table I). SHR had a lower body weight than WKY (233±6 g in WKY vs. 228±6 g in SHR, Day 0; 254±5 g in WKY vs. 246±7 g in SHR, Day 21) (Table I). The heart weights of SHR were greater than those of the WKY rats (1.00±0.05 g in WKY vs. 1.22±0.05 g in SHR, Day 0; 1.09±0.004 g in WKY vs. 1.30±0.05 g in SHR, Day 21, P<0.05), but weights of the brain and liver did not show apparent differences between SHR and WKY rats. Administration with enalapril or nifedipine did not significantly affect the body and tissue weights in SHR (Table I).

We used 34K gene chip microarray analysis to identify differentially expressed genes in the heart, brain, and liver from WKY and SHR as well as SHR treated with antihypertensive drugs. In total, 4,020 genes were found to be differentially expressed, with expression profiles clustering into three different groups using the K-means algorithm (Fig. 1). Heart, brain and liver samples showed 1,146, 265 and 172 upregulated genes, respectively, in SHR compared with the WKY controls. Administration of enalapril in SHR decreased the expression of 948, 104 and 45 of these same genes in the heart, brain and liver, respectively, compared with the untreated SHR. Similarly, nifedipine administration decreased the expression of 921, 111 and 69 of these genes in the heart, brain and liver, respectively.

In the converse analysis, 1,887, 283 and 267 genes showed decreased expression in the heart, brain and liver, respectively, from SHR compared with the WKY controls. Among these downregulated genes, expression of 1,490, 36 and 50 genes in the heart, brain and liver, respectively, was increased in SHR treated with enalapril compared with untreated SHR. Similarly, the expression of 1,564, 102 and 47 of these genes in the heart, brain and liver, respectively, was increased by nifedipine administration.

Several of the differentially expressed genes are known to be involved in the pathogenesis of cardiovascular diseases, as shown in Table II. Expression of 23 genes was markedly elevated in these three tissues from SHR compared with the WKY controls, whereas 10 genes showed decreased expression. Among these differentially regulated genes, analysis of heart samples from SHR showed that the expression of 11 genes was increased, while the expression of 7 genes was decreased. The expression of 9 genes was increased in brain samples from SHR, while the expression of 2 genes was decreased. Analysis of liver samples showed that the expression of 3 genes was elevated, while 1 was decreased. Markedly, these gene expression patterns were reversed by administration of either enalapril or nifedipine. We also identified 16 differentially expressed genes that had not previously been reported to be involved in the pathogenesis of cardiovascular diseases (Table III). In heart samples from SHR, 5 genes were upregulated compared with the WKY controls, and 3 genes were downregulated. In brain samples from SHR, 2 genes were upregulated, while 2 genes were downregulated. In liver samples from SHR, 3 genes were upregulated, while 1 gene was downregulated. Among these three tissues, the most prominent changes in gene expression were observed in the heart. These data show that administration of SHR with antihypertensive drugs led to changes in gene expression in a drug- and tissue-specific manner.

To validate the microarray data, the expression levels of 7 genes were measured using real-time PCR (Table IV). Expression of IL-24 in heart samples from SHR was increased by enalapril compared with SHR controls, but the expression of occludin and Pparδ did not change. Expression of triadin and moesin was elevated in brain samples of SHR compared with WKY controls, but it was decreased after administration of enalapril. Gmpr and Mef2d showed no change in expression in the heart or liver from SHR compared with WKY controls. In addition, the elevated expression of triadin, moesin, Gmpr and Mef2d in the heart, brain, and liver of SHR was lowered by nifedipine, while decreased expression of IL-24, occludin and Pparδ in the heart of SHR was raised by nifedipine. These results correlate with those from our microarray data.

The overproduction of intracellular ROS, such as superoxide anions (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH^-), has been implicated in the pathogenesis of cardiovascular diseases, including hypertension, heart failure, atherosclerosis and diabetes (12). ROS can induce the expression of genes associated with cardiovascular diseases in vascular smooth muscle cells (VSMCs) (13,14). IL-24 is among the differentially expressed genes described above, but no role of IL-24 in cardiovascular disease has been reported. To investigate the effect of IL-24 on ROS-mediated gene expression, RT-PCR was performed in MOVAS cells treated with H₂O₂ in the presence or absence of rhIL-24. The expression of angiotensinogen, endothelin-1, ATRAP and PDGF was increased by treatment with H₂O₂, but reduced by rhIL-24 (Fig. 2). There was no effect of IL-24 alone on the expression of the above genes. These data suggest that IL-24 might play a role in the suppression of ROS-induced gene expression in cardiovascular disease.