Monocyte chemoattractant protein-1 (MCP-1) and peroxisome proliferator-activated receptor-γ (PPAR-γ) play a significant role in monocyte activation, vascular inflammation, and atherogenesis. Angiotensin receptor blockers and calcium channel blockers are antihypertensive drugs with established efficacy and a favorable safety profile. We investigated the effect of telmisartan—an angiotensin receptor blocker with PPAR-γ agonist activity—and amlodipine on the activation state of peripheral blood monocytes with respect to MCP-1 and PPAR-γ gene expression in hypertensives. We recruited 31 previously untreated patients with essential hypertension who were randomly assigned to receive treatment with telmisartan (n = 16) or amlodipine (n = 15). Blood samples were taken before and 3 months after therapy initiation. Mononuclear cells were isolated and mRNAs of MCP-1 and PPAR-γ were estimated by real-time quantitative reverse transcription–polymerase chain reaction each time. The 2 treatments decreased all blood pressure components significantly (p <0.001). In contrast, in the amlodipine group, MCP-1 gene expression was significantly downregulated after treatment with telmisartan (from 21.4 ± 20.5 to 8.1 ± 6.5, p = 0.009), whereas the amlodipine group did not show any significant change (12.5 ± 8.5 vs 17.6 ± 16.4, p = NS). In addition, PPAR-γ mRNA levels showed a significant increase in telmisartan-treated patients (from 20 ± 18.5 to 42.6 ± 36, p = 0.006) and no significant alterations in the amlodipine group (from 29.6 ± 42.5 to 24.2 ± 27.7, p = NS). In conclusion, treatment with telmisartan results in a significant attenuation of MCP-1 gene expression and an increase of PPAR-γ gene expression in peripheral monocytes in patients with essential hypertension. Our findings may provide new insights into the cardiovascular protection of telmisartan in hypertensives.
Circulating peripheral monocytes and their activation play an important role in the early and late stages of atherosclerotic lesion formation. Arterial hypertension is accompanied by functional changes in monocytes, promoting expression of adhesion molecules and cytokines, leads to increased monocyte adhesion to the vascular wall, and induces the migration of monocytes/macrophages into the vascular wall in target organs, contributing to the development of atherosclerosis. Antihypertensive medication has been shown to exert beneficial effects in hypertensives often independently of blood pressure (BP) decrease, by inducing several pleiotropic effects. Angiotensin receptor blockers (ARBs) and calcium channel blockers are antihypertensive drugs with established efficacy and a favorable safety profile. We compared the effect telmisartan—an ARB that can partly activate the peroxisome proliferator activated receptor-γ (PPAR-γ)—on monocyte chemoattractant protein-1 (MCP-1) gene expression to amlodipine in peripheral monocytes of patients with essential hypertension. We also examined their effects with respect to expression of the PPAR-γ gene, a nuclear receptor that controls several metabolism-related genes and is involved in various cellular and molecular events such as those related to inflammatory processes.
Methods
Thirty-one patients with mild to moderate hypertension participated in this study. The study population was recruited from the cardiology outpatient department and consisted of subjects who had untreated grade 1 or 2 essential hypertension, with no indications of other organic heart disease. Diagnosis of hypertension was based on 3 outpatient measurements of BP >140/90 mm Hg at intervals ≤2 weeks according to recommendations of the European Society of Hypertension/European Society of Cardiology. Patients had not previously taken any hypertensive medication and did not take any other drugs for 3 weeks before the studies. The following were criteria for exclusion: smokers, diabetics, pregnant or lactating women or women potentially childbearing, previous or medication for hypertension, patients with grade 3 hypertension or secondary hypertension, tachyarrhythmias or bradyarrhythmias, coronary artery disease, heart failure, cerebrovascular, liver or renal disease, albumin excretion rate >200 μg/min, history of drug or alcohol abuse, any long-term inflammatory or other infectious disease during the previous 6 months, thyroid gland disease, body mass index >30 kg/m 2 , and vascular, metabolic, or neoplastic disease. Patients were randomly assigned to telmisartan (group 1, n = 16) or amlodipine (group 2, n = 15). Medications administered were open-label and initially consisted of telmisartan 40 mg/day or amlodipine 5 mg/day. If systolic BP was >140 mm Hg or diastolic BP was >90 mm Hg after 1 month, the dose was doubled to telmisartan 80 mg or amlodipine 10 mg. BP was measured before and 4, 8, and 12 weeks after treatment. At least 2 measurements were made and mean values of these measurements were used. After enrollment and randomization, a standard echocardiographic study was performed and blood samples were taken for analysis of full clinical chemistry and hematology markers. The same procedure was repeated at the end of the treatment period (12 weeks). In addition, homeostasis model assessment (HOMA) for insulin resistance was calculated each time as fasting blood sugar multiplied by insulin/405. Blood samples were collected into collection tubes containing ethylenediaminetetra-acetic acid. Peripheral blood mononuclear cells were isolated by Histopaque-Ficoll (Sigma, St. Louis, Missouri) centrifugation and CD14 + monocytes were purified from peripheral blood mononuclear cells by positive selection using high-gradient magnetic separation columns (MACS), type MS, and negative magnetic bead selection. Purity assessed by fluorescence-activated cell sorting (FACSCalibur; Becton Dickinson, San Jose, California) analysis was >95%. Total RNA was isolated from monocytes using the TRI Reagent (Ambion, Austin, Texas) and RNA 1 μg was reverse-transcribed with oligo-(dT) using the Reverse Transcription System (Promega, Madison, Wisconsin) in 20-μL reactions. Measurements of mRNA levels were performed by real-time reverse transcription–polymerase chain-reaction using the Stratagene Mx3000P Detection System (Santa Clara, California). Polymerase chain reaction assays were performed in cDNA template 1 μL using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, California). The standard-curve method was used for absolute quantification of amplification products and specificity was determined by performing melting-curve analysis. The housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase ( GAPDH ) was used as an endogenous reference gene. Relative quantification values were normalized to the endogenous reference gene. For each subject, normalized values at follow-up were expressed as fold differences from values before treatment. Primers used were 5′-ATTCCCCAAGGGCTCGCTCA-3′ (sense) and 5′-GCACAGATCTCCTTGGCCACAA-3′ (antisense) for MCP1 , 5′-AGATGACAGCGAC TTGGCAAT-3′ (sense) and 5′-GGAGCAGCTTGGCAAACAG-3′ (antisense) for PPAR -γ, 5′-CCATCTT CCAGGAGCGAG-3′ (sense) and 5′-GCAGGAGGCATTGCTGAT-3′ (antisense) for GAPDH . All patients gave written informed consent. The institutional ethics committee approved the study.
Summary descriptive statistics are presented as mean ± SD or count (percentage), as appropriate. Continuous variables were compared between the 2 groups by 2-tailed t tests or Mann-Whitney tests, as appropriate. Pearson correlation and linear regression was employed to assess the association between continuous parameters. All tests were performed at the 5% level of significance. SPSS 17 (SPSS, Inc., Chicago, Illinois) was used for all analyses.
Results
There was no statistical difference in baseline data between the 2 groups ( Table 1 ). No adverse effects of antihypertensive drugs were noticed. The 2 treatments significantly decreased systolic and diastolic BPs, but there was no significant difference in decrease in BP between the 2 groups. More specifically, amlodipine decreased systolic BP to 139 ± 4 mm Hg and diastolic BP to 87 ± 7 mm Hg (difference in systolic BP 16 ± 3 mm Hg, difference in diastolic BP 6 ± 3 mm Hg, p <0.001 for the 2 comparisons); telmisartan decreased systolic BP to 141 ± 5 mm Hg and diastolic BP to 88 ± 4 mm Hg (difference in systolic BP 15 ± 6 mm Hg, difference in diastolic BP 4 ± 2 mm Hg, p <0.001 for the 2 comparisons). No changes in patients’ physical characteristics—body weight or body mass index—were observed at the end of the treatment period (data not shown). However, insulin levels and HOMA were significantly improved in the telmisartan group (insulin after treatment 10.8 ± 4.1 IU/ml, p = 0.041, and HOMA after treatment 2.4 ± 1.1, p = 0.028), whereas the amlodipine group did not show any significant change (insulin after treatment 10.6 ± 3.7 IU/mL and HOMA after treatment 2.3 ± 0.9, p = NS for the 2 comparison). In addition, our data showed that treatment with telmisartan significantly downregulated MCP-1 gene expression in peripheral monocytes. MCP-1 mRNA levels decreased significantly to 8.1 ± 6.5 in the telmisartan group (p = 0.009), whereas in the amlodipine group they did not show any significant change during the study period (MCP-1 mRNA levels after treatment 17.6 ± 16.4, p = NS). In addition, PPAR-γ gene expression showed a significant increase in telmisartan-treated patients to 42.6 ± 36 (p = 0.006), but there was no significant change in the amlodipine group (PPAR-γ gene expression after treatment 24.2 ± 27.7, p = NS). Nevertheless, our analysis did not reveal any correlation between changes in systolic and diastolic BPs and fold induction of MCP-1 and PPAR-γ gene expression in the groups of patients we examined ( Figure 1 ).
| Variable | Amlodipine | Telmisartan |
|---|---|---|
| (n = 15) | (n = 16) | |
| Age (years) | 55 ± 8 | 56 ± 6 |
| Men/women | 9/6 | 10/6 |
| Smokers | 9 | 9 |
| Body mass index (kg/m 2 ) | 27 ± 3 | 26 ± 3 |
| Fasting glucose (mg/dl) | 87 ± 19 | 97 ± 10 |
| Insulin (IU/ml) | 10.6 ± 4.3 | 11.6 ± 5.1 |
| Homeostasis model assessment for insulin resistance (units) | 2.5 ± 1.1 | 2.8 ± 1.5 |
| Hemoglobin (g/dl) | 13.6 ± 1.1 | 14 ± 0.96 |
| Total cholesterol level (mg/dl) | 224 ± 42 | 227 ± 32 |
| Triglyceride level (mg/dl) | 189 ± 87 | 221 ± 102 |
| Low-density lipoprotein cholesterol level (mg/dl) | 143 ± 52 | 164 ± 63 |
| High-density lipoprotein cholesterol level (mg/dl) | 34 ± 40 | 37 ± 15 |
| Uric acid (mg/dl) | 6.7 ± 1.4 | 5.9 ± 1.8 |
| Treatment with a statin | 11 | 10 |
| Systolic blood pressure (mm Hg) | 155 ± 4 | 157 ± 7 |
| Diastolic blood pressure (mm Hg) | 92 ± 4 | 95 ± 8 |
| Heart rate (beats/min) | 68 ± 7 | 70 ± 4 |
| Ejection fraction (%) | 62 ± 5 | 61 ± 5 |
| Monocyte chemoattractant protein-1 mRNA levels (arbitrary units) | 21.4 ± 20.5 | 12.5 ± 8.5 |
| Peroxisome proliferator-activated receptor-γ mRNA levels (arbitrary units) | 29.6 ± 42.5 | 20 ± 18.5 |
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