Simvastatin reduces interleukin-1β secretion by peripheral blood mononuclear cells in patients with essential hypertension

Clinica Chimica Acta 344 (2004) 195 – 200 www.elsevier.com/locate/clinchim

Simvastatin reduces interleukin-1h secretion by peripheral blood mononuclear cells in patients with essential hypertension Shuiping Zhao *, Quanzhong Li, Ling Liu, Zhumei Xu, Jiezhi Xiao Department of Cardiology, The Second Xiangya Hospital, Central South University, 86# Renmin Middle Road, Changsha, Hunan 410011, PR China Received 29 September 2003; received in revised form 27 February 2004; accepted 3 March 2004

Abstract Background: Chronic low-grade inflammation may contribute to the increased risk of atherosclerosis in essential hypertension. Statins have been reported to have anti-inflammatory effects. We studied whether individuals with essential hypertension have increased interleukin-1h (IL-1h) secretion in peripheral blood mononuclear cells (PBMCs) and whether treatment with simvastatin lowered IL-1h secretion by PBMCs. Methods: PBMCs were isolated by gradient centrifugation from 24 individuals with essential hypertension (EH) and 12 normotensive subjects. The IL-1h concentrations in the supernatant from PBMCs were measured by enzyme-linked immunosorbent assay (ELISA). The patients with EH were then randomized to treatment with valsartan 80 mg/day or matching group who took the same drug valsartan 80 mg/day plus simvastatin 40 mg/day for 1 week. IL-1h secretion by PBMCs was also measured. Results: Compared with controls, patients with EH had increased IL-1h [992 F 151 pg/ml, 912 F 102 pg/ml vs. 599 F 93 pg/ml; P < 0.05] secretion by PBMCs after stimulated by angiotensin II. Simvastatin treatment had a significant effect of decreasing IL-1h [668 F 98 vs. 923 F 67 pg/ml; P < 0.05] secretion in PBMCs. The reductions were not correlated to changes in plasma lipids. Conclusions: This study shows that EH is associated with increased PBMCs activation and that treatment with simvastatin may partly attenuate this abnormality. D 2004 Elsevier B.V. All rights reserved. Keywords: Essential hypertension; Inflammation; Interleukin-1h; Simvastatin

1. Introduction Statins, a structurally related group of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are widely used to treat hyperlipidemia. Clinical trials with statins have demonstrated a significant reduction in adverse coronary events [1– 7].

* Corresponding author. Tel.: +86-731-4895989; fax: +86-7314895989. E-mail address: [email protected] (S. Zhao). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.03.003

These reductions may be related not only to the effects on plasma lipids, but also to other non-lipid-lowering benefits of these agents, including anti-inflammation effect [8]. Hypertension follows closely behind lipids as one of the major risk factors for atherosclerosis and is commonly associated with the development of atherosclerosis. Increasing evidence suggests that inflammation may participate in hypertension [9 –11]. In this study, we investigated whether individuals with essential hypertension (EH) have increased proinflammatory cytokine interleukin-1h (IL-1h) secretion in peripheral blood mononuclear cells (PBMCs) and

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whether treatment with statin can lower IL-1h secretion from PBMCs.

2. Materials and methods 2.1. Subjects We recruited 24 patients with essential hypertension without severe dyslipidemia at screening and 12 normotensive healthy control of similar age for the study. None of the participants had diabetes mellitus, macroproteinuria, creatinemia, hypothyroidism, abnormal liver and muscle enzymes, acute and/or chronic infections, autoimmune diseases or neoplastic diseases. Hypertension was defined as a phase V Korotkoff diastolic blood pressure (DBP) with a minimum of 90 mm Hg and/or a systolic blood pressure (SBP) of 140 mm Hg, the average of the readings obtained on at least 3 different occasions. The blood pressure was determined using a mercury sphygmomanometer in a supine position. None reported a history of cardiovascular disease or were taking medication or other agents known to affect inflammatory response. None of the patients were smokers. Patients had not received antihypertensive drugs before. Patients with secondary hypertension were excluded (n = 1). 2.2. Cross-sectional study and intervention At baseline, PBMCs was collected from all participants after an overnight fast (14 h). Patients then were randomized to one of the two groups for 1 week: one group only took valsartan 80 mg/day alone (group A), and the other group took the same drug plus simvastatin 40 mg/day (group B). After treatment, PBMCs was also collected from all patients after an overnight fast. All patients were advised to continue their habitual isocaloric diet and to keep physical exercise constant.

vested from the Ficoll-plasma interface and washed three times in RPMI1640 medium (Gibco-BRL) containing 2 mmol/l glutamine (Sigma) and 10% heatinactivated fetal calf serum (Gibco-BRL). Cells were suspended at 1  106/ml in RPMI1640 supplemented as above. Cell viability was always >95%, as estimated by trypan blue exclusion. The cell suspension was plated at 1 ml per well in 24-well flat-bottomed tissue culture plates (Costar). After 12 h of incubation with or without a physiologically relevant concentration of angiotensin II (Ang II, 10 10 mol/l) (Sigma) [11] at 37 jC in 95% humidified air and 5% CO2, cell supernatants were harvested and stored at 70 jC for cytokine analysis. 2.4. Cytokine assays Supernatant concentrations of IL-1h were assessed using a specific sandwich enzyme-linked immunosorbent assay (ELISA) kit manufactured by JinMei. All samples were analyzed in duplicate. The sensitivity was 3.0 pg/ml and the intra-assay and inter-assay precision variability was < 9.6%. 2.5. Statistical analysis All analyses were carried out using SPSS 10.0 (SPSS Software). Because the distributions of IL-1h were skewed, they were natural-log-transformed for all analyses. Differences between controls and patients (groups A and B) at baseline were determined by one-way ANOVA and Newman –Keuls method. Within-group comparisons were performed with paired t-tests. Two-group comparisons (groups A vs. B) were performed with independent t-tests. Values were stated as mean F S.D. Statistical significance was defined as P < 0.05.

3. Results 3.1. Characteristics of the subjects

2.3. Isolation of peripheral blood mononuclear cells Five milliliters of peripheral blood were drawn into sterile 15 ml tubes containing 30 Al of sodium heparin, layered on to an equal volume of Ficoll and centrifuged at 1500  g for 20 min. Cells were har-

The clinical and biochemical characteristics of the hypertension and healthy individuals are shown in Table 1. Age and plasma cholesterol, triglycerides, LDL-C and HDL-C ( P < 0.05) were not significantly different among the groups. Both groups A and B had

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Table 1 Characteristics of participants at baseline

Men/women Age (years) Weight (kg) SBP (mm Hg) DBP (mm Hg) TG (mmol/l) TC (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l)

Control (n = 12)

Group A (n = 12)

Group B (n = 12)

P

6/6 63.3 F 11 64.3 F 6.9 124.5 F 10.9 75.0 F 7.5 1.37 F 0.57 4.46 F 0.95 1.34 F 0.41 2.59 F 0.81

7/5 66.1 F 14 62.9 F 8.4 165.8 F 25.2* 100.4 F 15.8* 1.45 F 0.59 4.91 F 1.34 1.52 F 0.41 2.85 F 1.08

7/5 67.3 F 8.0 63.2 F 7.2 158 F 22.8* 93.7 F 12.0* 1.38 F 0.43 4.63 F 1.09 1.43 F 0.35 2.78 F 0.76

NS NS NS < 0.05 < 0.05 NS NS NS NS

SBP: systolic blood pressure; DBP: diastolic blood pressure; TG: triglyceride; TC: total cholesterol; HDL-c: high density lipoprotein cholesterol; LDL: low density lipoprotein cholesterol. NS: not significant. * P < 0.05 vs. control.

a significantly higher blood pressure compared with the healthy controls, but neither systolic nor diastolic blood pressure between groups A and B were significantly different ( P < 0.05).

#

Fig. 1. The pre- and post-treatment secretion of IL-1h (pg/ml) by PBMCs after stimulated by Ang II (10 10 mol/l) in patients. PBMCs were isolated by gradient centrifugation. After 12 h of incubation, IL-1h concentrations in supernatant from PBMCs were measured by ELISA. *P < 0.05 vs. Group A; #P < 0.05 vs. pretreatment.

reduced the IL-1h secretion stimulated by Ang II significantly.

4. Discussion 3.2. Secretion of IL-1b into culture supernatants by PBMCs At baseline, spontaneous secretion of IL-1h into culture supernatants by PBMCs did not differ significantly between hypertensive patients and normotensive controls. But IL-1h secretion stimulated by Ang II was significantly higher in patients with essential hypertension (both groups A and B) vs. healthy individuals (Table 2). Spontaneous secretion of IL-1h by PBMCs after treatment did not change significantly (186 F 54 vs. 183 F 66 pg/ml; 150 F 63 vs. 148 F 45 pg/ml).The changes of IL-1h concentrations stimulated by Ang II after treatment with simvastatin in group A and B are shown in Fig. 1. It shows that simvastatin Table 2 Secretion of IL-1h (pg/ml) by PBMCs at baseline

Spontaneous + Ang II

Control (n = 12)

Group A (n = 12)

Group B (n = 12)

146 F 45 599 F 93*

183 F 66 992 F 151*,#

150 F 63 912 F 102*,#

PBMCs: peripheral blood mononuclear cells. + Ang II: stimulated by angiotensin II (10 10 mol/l). * P < 0.05 vs. spontaneous secretion. # P < 0.05 vs. control.

There is an accumulating body of evidence that atherosclerosis involves an ongoing inflammatory response [9]. These findings provide important links between risk factors and the mechanisms of atherosclerosis. Hypertension is a classical risk factor for atherosclerosis. Increasing evidence suggests that inflammation may participate in hypertension, which may provide a pathophysiological link between hypertension and atherosclerosis [12]. Under our experiment condition, we confirmed that individual with essential hypertension have a significantly higher PBMCs secretion of IL-1h than healthy controls after stimulation with Ang II, and the treatment with statin was associated with significant reductions in PBMCs secretion of IL-1h. The changes in IL-1h with statin treatment were not related to changes in plasma lipids and blood pressure because circulating cholesterol levels were not altered after therapy and there were not any differences in the reduction of blood pressure between the two groups (data not shown). We studied PBMCs rather than monocytes or macrophages in this study to avoid possible artifacts related to cell isolation techniques, which involve an adherence step that may itself activate these cells and maintain the integrity of the cellular interactions.

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In our study, though spontaneous secretion of IL1h by PBMCs did not differ significantly between hypertensive patients and normotensive controls, IL1h secretion stimulated by Ang II was significantly higher in patients with essential hypertension. It is well documented that AngII plays a critical role in the blood pressure regulation and in the pathophysiology of cardiovascular disorders [13]. Our results suggest that Ang II may be directly involved in the process of PBMCs activation. IL-1h, a proinflammatory cytokine with a variety of activities, has been shown to have important effects on the cell types that constitute atherosclerotic lesions [14 – 16]. Using a IL-1h gene knock-out mice model, Kirii et al. [17] demonstrated that IL-1h deficiency induced an approximately 33% reduction in atherosclerotic lesions in apoE ( / ) mice which suggests that IL-1h exerts an atherogenetic action. Studies in spontaneous hypertension rats have shown that the expression and release of IL-1h and tumor necrosis factor-a (TNF-a) is increased due to monocyte and endothelial cell activation [18]. Moreover, infusion of IL-1h in rats induced dosedependent vasopressor responses with increased blood pressure and enhanced abdominal symphathetic discharge [19]. In a study with 537 aged subjects, after adjusting for age, sex, insulin resistance syndrome score, and severity of carotid atherosclerosis, serum IL-1h, IL-1ra concentration and the insulin resistance syndrome score were the only predictors of diastolic blood pressure [20]. In another study, Dalekos et al. [21] reported that plasma concentrations of IL-1h in patients with EH were increased. Our results suggest that IL-1h secretion by PBMCs may partly account for the increased secretion of IL-1h in circulation. Recent studies, both in vitro and in vivo, have suggested that the beneficial effects of statins may extend to mechanisms beyond cholesterol reduction [22 – 25]. It has been suggested that statins possess anti-inflammatory properties partly by their ability to reduce the production of pro-inflammatory cytokines. In one study, statins were found to significantly inhibit Ang II-induced secretion of IL-6 in cultured human vascular smooth muscle cells [26], whereas in another study, fluvastatin reduced production of IL-1h in human endothelial cells [27]. In nonhuman primates (adult male cynomolgus monkeys), statins were found to reduce inflammatory cytokine IL-1h expression in intimal lesions independent of their effect on plasma

cholesterol level. A recent study with 107 hypercholesterolemic patients demonstrated that simvastatin treatment for 6 weeks led to a decreased expression of mRNA of peptide cytokines in circulating PBMCs and of serum monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-6 levels in hypercholesterolemic patients [28]. Though it had been reported that valsartan had anti-inflammatory effects [29], we failed to observe any significant changes in our study. It may partly be due to the short period of treatment and higher reactivity of PBMCs to Ang II after valsartan treatment. In this study, we found that statin (plus valsartan) treatment for 1 week resulted in a significant decrease of proinflammatory cytokines IL-1h secretion by PBMCs in EH normocholesterolemic patients. Little is known about the mechanism(s) that underlies the anti-inflammatory effects of statins. Statins may act through multiple receptors and pathways in their target cells. It has been suggested that statins inhibit the activation of nuclear factor-[kappa]B, a major nuclear factor that regulates the expression of diverse cytokines,including MCP-1, IL-1h, and TNF-a [30]. In macrophages, lovastatin has been shown to downregulate expression of genes regulated by nuclear factor-[kappa]B such as inducible nitric oxide synthase and the pro-inflammatory cytokines TNF-a, IL-1h, and IL-6 in macrophages [31]. On the basis of the above findings, it is tempting to speculate that the statin-induced downregulation of cytokine production in PBMCs may involve, in part, a loss of functional nuclear factor-[kappa]B. In a patient study [32], Nickenig et al. showed that statin treatment effectively reduced Ang II type 1 (AT1) receptor density and decreased the blood pressure-elevating effects of Ang II. This effect might explain some of the cholesterol-independent statin effects. Conceivably, the effects could also be related to a cholesterol-independent decreased AT1 receptor expression in response to statin treatment. It suggests that simvastatin and valsartan may have synergetic effect but further investigation is needed. In summary, our study suggests that PBMCs in EH are preactivated and inflammatory mechanisms may be important participants in the pathophysiology of hypertension. This study supports the view that hypertension is a low-grade chronic inflammatory state and that treatment with statin may be beneficial to attenuate this abnormality. The evalu-

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ation of the efficacy of anti-inflammatory treatments with statins in blood pressure regulation and in the pathophysiology of cardiovascular disorders will allow progress in our ability to combat the complications of hypertension and other cardiovascular diseases.

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