The renin-angiotensin system (RAS) is an important factor in the structural and functional modulation of the cardiovascular system.1-3 In particular, RAS activation is associated with hypertension and with the occurrence of acute vascular events in patients with dyslipidemia.4 In the pathogenesis of atherosclerosis (ATH), increased angiotensin (Ang) II activity may act at various levels, such as modification of lipid metabolism, endothelial dysfunction, and plaque instability.3 Ang II exerts its effects through multiple receptors; however, pro-ATH effects depend mainly on activation of the Ang II type 1 receptors (AT1Rs).2 Hypercholesterolemia and, in particular, elevated plasma levels of low-density lipoprotein (LDL) increase AT1R expression on vascular smooth cells (VSMC) in animal models5 and in humans,6 an effect likely contributing to the accelerated progression of vascular lesions2; in humans hypercholesterolemia is directly and strongly correlated with AT1R expression on VSMCs5 and endothelial cells (ECs).7 Available experimental evidences thus suggest that downregulation of AT1R-operated pathways could significantly contribute to the overall reduction of the cardiovascular risk, interfering with the processes leading to ATH.8
Several reports show that statin treatment may directly counteract the effects of Ang II.9-11 Moreover, in patients with hypercholesterolemia statin (atorvastatin or simvastatin) treatment reduced Ang II (infusion)-induced increase of blood pressure.6 Evidence from clinical studies thus supports the general conclusion that inhibition of RAS results in a significant reduction of risk in subjects at high risk for vascular events and reduces the risk for further events in patients with cardiovascular disease.12,13
Increasing experimental evidence points to polymorphonuclear leukocytes (PMNs) as key players in the ATH process.14 We have recently reported that PMNs from subjects of high risk of occurrence of cardiovascular events (at least 20% within 10 years according to the Framingham algorithm15) show altered functional properties, resulting in increased inflammatory responses. In particular, we have shown that in PMNs obtained from high-risk patients, the production of the proinflammatory cytokine interleukin (IL)-8 was higher with respect to PMNs obtained from healthy donors and that simvastatin treatment reduced IL-8 production to levels of healthy controls.16
The present longitudinal study was therefore performed to assess whether statin treatment in high-risk subjects may result in downregulation of AT1Rs on PMNs. To this end, by means of real-time polymerase chain reaction (PCR) and flow cytometry, we measured the expression of AT1Rs in PMNs from high-risk subjects before and during treatment with simvastatin, in comparison to cells from age- and sex-matched healthy controls. Moreover, in in vitro experiments we investigated in human PMNs the ability of simvastatin to interfere with Ang II-dependent activation of Rac 1, which is a guanosine triphosphate (GTP)-binding protein playing a key role in Ang II-operated signaling pathways.17 Ang II is increasingly regarded as a proinflammatory agent,3 and the results of this study shed more light on its immune effects and on the mechanisms possibly involved in the antiinflammatory effects of statins.
We enrolled 28 subjects [8 females, 20 males; age (mean ± standard deviation, or SD): 57 ± 11 years] at high risk for vascular events according to the Adult Treatment Panel (ATP) III.15 High-risk factors were evaluated “counting major risk factors and estimating the 10 year coronary heart disease (CHD) risk.”15 Subjects were selected to start a lipid-lowering pharmacological treatment with an HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A)-reductase inhibitor (simvastatin, 20 mg per day assumed at 10:00 pm). The patients were visited for the first time in our Lipid Clinic, University Department of Clinical Medicine (Ospedale di Circolo, Varese, Italy) and were included in the study if no disease (apart from dyslipidemia and/or diabetes and/or undiagnosed hypertension) was found after a clinical examination and routine laboratory tests. Thus we enrolled patients with a 10 year cardiovascular risk ≥20% when calculated according to the Framingham algorithm and/or with diabetes, whereas patients with documented ischemic heart disease or “equivalent ischemic heart disease”15 were excluded from the study. Clinical indication for a lipid-lowering pharmacological treatment with statins was also required for inclusion. Exclusion criteria included smoking, involvement in competitive sporting activities, need for any other pharmacological treatment (except for antidiabetic medications, see later), serum creatinine levels >150 μmol/L, aspartate aminotransferase and/or alanine aminotransferase levels >1.5 times the upper normal limits and creatine kinase > upper normal limit, and intake of any drug (with the exclusion of the previously mentioned antidiabetic treatments) in the 4 weeks before starting the study.
Evaluation included familiar and personal history, calculation of body mass index, measurement of waist circumference, and measurement of standard sphygmomanometric blood pressure. The presence of a metabolic syndrome was diagnosed according to ATP III criteria.15 Seven patients (6 males, 1 female) with type-2 diabetes (a risk factor included in the ATP III) were included in the study on the basis of good glycemic control observed at the first visit that required no change of antidiabetic medications; thus, patients with diabetes did not change treatment throughout the study. Drug treatments for these patients included metformin (5 patients) and glimepiride (2 patients). The 5 females enrolled in the study were all postmenopausal.
After enrollment, all the patients started statin treatment only after 6 weeks of lifestyle modification, including dietary treatment and recommendations for mild physical activity.
The subjects were then studied 3 times: before starting the treatment with simvastatin (visit 1), after 3 days (visit 2), and after 30 days of treatment (visit 3). At baseline clinical examination (visit 1), their blood pressure was 135 ± 15/85 ± 7 mm Hg and their body mass index was 27.2 ± 3.7 kg/m2.
For each patient, an age- and sex-matched healthy control was enrolled. The healthy subjects (8 females, 20 males; age; mean ± SD: 54 ± 14 years) were selected from a population evaluated for a general clinical checkup at our department. Exclusion criteria were presence of any of the risk factors included in the ATP III, intake of any drug in the 4 weeks before starting the study, cigarette smoking, and participation in competitive sporting activities. The study protocol was approved by the local Ethics Committee, and all the participants gave informed consent before inclusion in the study.
At the 3 evaluations, venous blood samples were obtained after a fasting night, between 8:00 and 9:00 am by use of heparinized tubes, and circulating leukocytes were isolated for subsequent studies. An aliquot of blood was used to monitor lipid profile-including total cholesterol, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), triglycerides, ApolipoproteinA, ApolipoproteinB, high-sensitivity (hs) C-reactive protein (CRP), creatine kinase, lipoprotein (a)-and to perform routine laboratory examinations (haemocromocytometric examination, plasma creatinine, urea, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transpeptidase, alkaline phosphatase, and HbA1c). A urine sample was also taken and assayed for microalbuminuria.
Isolation of Polymorphonuclear Leukocytes
Whole blood was allowed to sediment on dextran at 37°C for 30 minutes. Supernatant was recovered and PMNs were separated from peripheral blood mononuclear cells (PBMCs) by Ficoll-Paque Plus density-gradient centrifugation. Contaminating erythrocytes were eliminated by 10 minute hypotonic lysis in distilled water with added (g/L) NH4Cl 8.25, KHCO3 1.00, and ethylenediamine tetraacetic acid (EDTA) 0.04. Cells were then washed 3 times in NaCl 0.15 M and resuspended in 1 mL phosphate buffered saline with added bovine serum albumin 0.1%. A counting chamber was used to enumerate cell number. No platelets or erythrocytes could be detected by either light microscopic examination or flow cytometric analysis. The purity of the preparations was assessed by flow cytometry and was usually 98%-99%, whereas the viability was 95%-98% by the trypan blue exclusion test.
RNA Isolation and Real-Time PCR Analysis of AT1R mRNA
Total mRNA was extracted from 1 × 106 cells by Perfect RNA Eukaryotic Mini kit (Eppendorf, Hamburg, Germany), and the amount of extracted RNA was estimated by spectrophotometry at 260 nm. Total RNA was reverse transcribed using the high-capacity cDNA Archive Kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer's instructions.
Real-time PCR was performed by means of an ABI prism 7000 apparatus (Applied Biosystems) using the assay on demand kit for human AT1Rs (Applied Biosystems). Cycles included one 2 minute hold (50°C) and one 10 minute and 45 15 second cycles of denaturation (95°C). Raw data were analyzed by the ABI prism SDS software (Applied Biosystems).
Threshold cycle values (Ct) for human AT1Rs were (mean ± SD) 25.73 ± 0.82. Ct value was used to calculate a linear regression line generated by performing serial dilutions (1:10; 1:50; 1:500; 1:1,000; 1:10,000) of the total mRNA obtained from human PMN. The values were then normalized for Ct values of 18S ribosomal RNA.
Whole Blood Collection and Flow Cytometric Analysis of AT1R Expression
In a subgroup of 6 high-risk subjects (6 males; age: 55 ± 16 years) and 6 healthy subjects (6 males; age: 56 ± 14 years) the expression of AT1Rs on the cell membrane of the various leukocyte subsets was also evaluated. To this end, 1 mL of whole blood was used and the analysis was performed by using conventional immunofluorescence techniques together with a multiparametric flow cytometric analysis as previously described.18 A minimum of 50,000 cells were analyzed from each sample, and results were expressed as both percent of AT1R-positive cells (%) and AT1R density on positive cells (mean fluorescence intensity, or MFI).
Western Blotting of Rac 1
PMNs were obtained starting from buffy coat preparations derived from the whole blood of healthy donors. Cells were washed with cold PBS (composition: 130 mM NaCl, 4 mM Na2HPO4, 1.5 mM NaH2PO4, pH 7.4) and disrupted by incubation (30 minutes, 4°C) in hypotonic buffer (composition: 5 mM Tris-HCl, 5 mM KCl, 0.1 mM EGTA, 1.5 mM MgCl2, pH 7.4), containing a mixture of protease inhibitors (Complete, Roche Diagnostic GmbH, Penzberg Germany) and sonicated for 5 seconds. Cells were then centrifuged (21,000 rpm, 1.5 hours at 4°C) and membrane and cytosolic fractions were obtained. Fractions were resuspended in Triton X 100 buffer (0.1%) in PBS, containing the mixture of protease inhibitors (Complete), and the protein concentration was determined by the BCA assay (Pierce Biotechnology, Illinois, USA). Twenty-five micrograms of protein/fraction/lane were loaded on 11% polyacrylamide gels and separated under denaturing conditions. Protein samples were then transferred on polyvinylidene fluoride membranes (Amersham Biosciences, Buckinghamshire, UK) and after incubation in blocking solution (5% nonfat milk, Bio-Rad Laboratories, California, USA), Western blot analysis was performed by standard techniques using a rabbit anti-Rac 1 polyclonal antibody (C-11 Santa Cruz Biotechnology, California, USA; 1:100 overnight at 4°C). Proteins were visualized using a peroxidase-conjugated antirabbit immunoglobulin secondary antibody (Amersham Biosciences, 1:1000 for 2 hours at room temperature) and the ECL Plus Western Blotting Detection Reagents (Amersham Biosciences). The densities of protein bands were analyzed by Scion Image software (Scion Corporation, Maryland, USA) and expressed as pixels area.
Data are presented as means ± SD, with n indicating the number of observations. Statistical significance of the differences between groups was assessed by 2-tailed Student's t test or 1-way analysis of variance followed by Bonferroni or Dunnet posttest for paired or unpaired data, as appropriate. Analysis of the correlation between selected variables was performed by linear regression analysis. Calculations were performed using commercial software (GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com).
Total cholesterol, LDL-c, triglycerides, ApolipoproteinB, and hs-CRP in high-risk subjects at visit 1 were significantly higher than in controls. Treatment with simvastatin significantly reduced total cholesterol, LDL-c, and ApolipoproteinB (Table 1). All the other parameters were always within the normal range in both controls and high-risk subjects, before starting statin treatment as well as throughout the study (data not shown).
AT1R mRNA Levels
AT1R mRNA levels were higher in PMNs of high-risk subjects compared with controls (Fig. 1). In high-risk patients, simvastatin treatment resulted in a generalized reduction of the expression of AT1R mRNA (Fig. 2). The largest reduction occurred between visit 1 and visit 2 (ie, after 3 days of statin treatment). It is interesting that in individual patients we observed an early reduction of AT1R mRNA levels; mRNA expression at visit 2 always reduced with respect to visit 1. At visit 3 (ie, after 30 days of statin treatment) values did not decrease further (% variation with respect to visit 2; P > 0.05), and in a few patients with respect to visit 2 an increase was even observed, thus leading to final values that were less than 10% different from pretreatment levels (2 cases). Therefore, in comparison to basal values (visit 1) at visit 3 the mean decrease of AT1R mRNA levels was −23.0 ± 23.0% (P < 0.01 vs visit 1).
As shown in Figure 3, an inverse correlation was found between AT1R mRNA levels in PMNs from high-risk subjects before simvastatin treatment (visit 1) and after 30 days of treatment [ie, the difference (Δ) between AT1R mRNA levels at visit 3 and at visit 1 was directly related to pretreatment levels].
AT1R Expression on Cell Membranes
AT1Rs were identified on isolated PMNs in agreement with our previous observations.18 No significant differences, however, were found between high-risk subjects and healthy controls and no changes were observed in high-risk subjects throughout the study (Table 2).
Rac 1 Expression
Western blot analysis showed that Rac 1 is present in human PMNs in both the membrane and the cytosol fraction of the cells (Fig. 4). Twenty-four-hour treatment with Ang II (10 μM) increased membrane-associated Rac 1 without affecting Rac 1 levels into the cytosolic fraction. This effect was prevented by coincubation with simvastatin (10 μM), which per se had no effect on Rac 1 in either the membrane or the cytosolic fraction (Fig. 4).
The main findings of this study are as follows: (1) PMNs obtained from venous blood of subjects at high risk for cardiovascular events express higher levels of AT1R mRNA in comparison to cells obtained from healthy controls; (2) treatment with simvastatin reduces AT1R mRNA expression to an extent that is directly proportional to AT1R mRNA pretreatment levels; and (3) in isolated PMNs, simvastatin prevents Ang II-dependent intracellular signaling.
Until now, information about the expression of AT1Rs on human leukocytes was fragmentary. Binding sites for Ang II in human mononuclear leukocytes had been described nearly 3 decades ago,19 and more recently the occurrence of AT1Rs has been further characterized on human monocytes/macrophages20 and lymphocytes.21 We have recently shown that in leukocytes from healthy subjects AT1Rs are expressed not only in lymphocyte subsets and monocytes but also in PMNs.18
According to our data, AT1R mRNA is higher in patients with respect to control subjects, whereas membrane receptor expression does not differ in the 2 groups. The functional implications of these differences cannot be inferred by the present data; however, it may be of interest that in previous studies in human immune cells we found that the activity of expressed proteins was directly related to the levels of their mRNA and not to the levels of protein expression (eg, tyrosine hydroxylase in lymphocytes).22 Moreover, mRNA levels of G protein-coupled receptors such as AT1Rs usually correlate with receptor responsiveness,23 suggesting that receptor function may be better predicted by mRNA levels rather than by protein expression.
Thus the observation that AT1R mRNA levels are higher in patients is of particular interest, in view of the ability of Ang II to stimulate their function; indeed, treatment of human PMNs with Ang II induces NADPH oxidase and nuclear factor (NF)-κB activation, as well as increased ROS production, all of which in vivo are likely to result in vascular damage and subsequent pathology.24 The role of PMNs in cardiovascular diseases is now well established (for a review see Brown et al25). In our previous report16 we showed that PMNs from high-risk subjects have increased oxidative metabolism and produce more IL-8 than cells from healthy controls and that simvastatin treatment results in reduction of PMN function. In agreement with such results, it has been recently shown that PMNs from patients with coronary heart disease treated with atorvastatin show an impairment of superoxide anion (O2−) generation.26 Indeed, increased circulating PMNs in ATH is a well-known phenomenon and a defined risk factor for disease progression.27 Thus the present findings that AT1R mRNA expression is increased in PMNs from high-risk subjects and that it is reduced after simvastatin treatment deserves careful consideration. The key role of AT1Rs in the proinflammatory effects of Ang II has been documented by several experimental studies. Overexpression of AT1Rs in the vascular wall is involved in the induction of oxidative stress and in turn of endothelial dysfunction and plaque instability.2
It is well known that statins, beyond their lipid-lowering properties, exert pleiotropic effects on different cell types.28 Clinical studies point to the beneficial effects of statin treatment in improving endothelial dysfunction, a key step of ATH, an effect that depends largely on the reduction of oxidized LDL, in turn are the main responsible of the reduction of endothelial NO.29,30 Statin-induced inhibition of isoprenoid intermediates formation however, besides leading to reduction of circulating cholesterol levels, also prevents the synthesis of mediators that have an important role in the intracellular signaling cascade.
In line with this notion, the results of our in vitro experiments show that simvastatin directly interferes with Ang II-dependent intracellular signaling in isolated PMNs, as indicated by its ability to inhibit Ang II-induced activation of Rac-1. The membrane translocation of Rac 1 represents a key step in initiating the proinflammatory events induced by increased cholesterol levels,17 and inhibition of this step may thus contribute to the beneficial effects of statins.9
Altogether, our results suggest that simvastatin interferes with Ang II-dependent pathways at both receptor and intracellular signal transduction levels. It can be easily hypothesized that downregulation of Ang II-operated receptor pathways in PMNs is likely to result in beneficial consequences (also in view of the established role of these cells in ATH). In our patients, statin-induced reduction of AT1R mRNA expression in PMNs was already maximal after 3 days of treatment, an observation that, together with in vitro results showing the ability of simvastatin to acutely interfere with the activity of Ang II, strengthens the hypothesis that statins may exert early effects likely independent from their lipid-lowering activity and independently of the reduction of C-reactive protein and with any significant change in blood pressure. It has been reported that statin treatment (24 weeks) reduces blood pressure in men with mild hypertension.31 We can hypothesize that 4 weeks of treatment is short in order to obtain a reduction of blood pressure, but the effects observed in our and in other studies confirm that the effects of statins are more complex and include different mechanisms. These observations may be relevant particularly in view of the results of recent studies in cardiac ischemia and in acute stroke, pointing to the importance of early statin treatment after an acute vascular event to reduce or limit the damage.13,32 AT1R mRNA expression is increased in lymphocytes from cardiac donors with spontaneous intracerebral hemorrhage, and AT1R mRNA levels in these cells is a strong independent predictor of transplant vasculopathy in transplant recipients.21 These observations support the involvement of AT1R-operated mechanisms in vascular damage and indirectly point to the potential therapeutic relevance of strategies aimed at downregulating these receptor pathways.
In conclusion, our findings extend current knowledge about the expression of AT1Rs in immune cells and about their involvement in cardiovascular risk. The ability of simvastatin to affect AT1R mRNA expression in PMNs from high-risk subjects, as well as the in vitro interference of the drug with Ang II activity in these cells, provides further insights into the mechanisms contributing to the effects of statins on the cellular events associated with ATH, on the significance of these cells in the progression of disease, and on the relevance of new therapeutic strategies pointing to reduce inflammatory markers in immune cells.
The skillful assistance of Dr Massimiliano Legnaro in performing polymorphonuclear leukocyte AT1R expression assays is gratefully acknowledged.
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