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CARDIOVASCULAR
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California
Received for publication
November 2, 2004
Accepted
January 20, 2005.
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Abstract |
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). This event is mediated by the surface expression of endothelial adhesion molecules and chemotactic proteins induced by risk factors such as hypercholesterolemia. The exact mechanisms by which hypercholesterolemia accelerates monocyte adhesion (and, therefore, atherosclerotic disease) have not been fully delineated; however, recent evidence indicates that increased local oxidative stress may play a major role. In addition to inducing vascular smooth muscle cell proliferation (Griendling and Fitzgerald, 2003a), excess oxygen-derived free radicals can increase the expression of proinflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1) (Marui et al., 1993) and monocyte chemotactic protein-1 (MCP-1) (Tsao et al., 1997), both of which are involved with the recruitment of monocytes.
One of the most important innovations in the treatment of atherosclerotic disease has been the development and use of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). Several major clinical trials have demonstrated a clear benefit of statins upon cardiovascular endpoints as well as surrogate markers of coronary artery disease (Anonymous, 1994, 1998a,b; Heart Protection Study Collaborative Group, 2002). Yet, the mechanisms by which LDL cholesterol initiates atherogenesis are not completely delineated. Significant research has focused on how the cells within atherosclerotic lesions accumulate lipid. As such, simple lowering of cholesterol levels would reduce the rate of lipid accumulation and inhibit atherogenesis. However, closer examination of clinical trial data has led to the concept that the benefit of statins may go beyond their ability to lower circulating LDL levels (Anonymous, 1998a). In addition, recent preclinical studies have shown that statins can, indeed, directly affect intracellular processes. These actions are as diverse as enhancing endothelial expression of endothelial nitric-oxide synthase (eNOS) (Laufs et al., 1998; Laufs and Liao, 1998), increasing the number of circulating endothelial progenitor cells (Vasa et al., 2001), and modulating inflammatory cell activation (Kallen et al., 1999; Weitz-Schmidt et al., 2001). Rosuvastatin is a new statin that has potent inhibitory effects on hepatic HMG-CoA reductase. Comparative dose-ranging studies indicate that rosuvastatin is highly effective in reducing LDL cholesterol in hypercholesterolemic patients with large decreases at starting doses (Olsson et al., 2002). We hypothesized that rosuvastatin would not only lower circulating cholesterol levels, but would also attenuate early inflammatory processes involved with atherogenesis. Therefore, the influence of rosuvastatin upon endothelial adhesiveness as well as cellular signaling mechanisms essential for monocyte binding were investigated in the hypercholesterolemic apoE-deficient mouse.
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Materials and Methods |
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Animal Studies. All animals were housed in a room with a 12-h light/dark cycle and an ambient temperature of 22°C. The experimental protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.
Eight-week-old female mice (both apoE knockout and their genetic controls, C57BL/6) were fed a normal chow diet (0.022% cholesterol, 10% fat by weight; Purina Mills, Richmond, IN) for a period of 12 weeks. ApoE-deficient mice were administered vehicle or rosuvastatin at a dose of 0, 1, 5, or 20 mg/kg by subcutaneous injection at the same time daily for a period of 2 or 6 weeks prior to sacrifice. At 20 weeks of age, mice received their final dose of drug and were sacrificed by an overdose of pentobarbital. Whole blood was collected in EDTA by intracardiac puncture and isolated plasma stored at –80°C for measurement of total cholesterol, HDL cholesterol, triglycerides by enzymatic method (Sigma-Aldrich, St. Louis, MO), and 8-epiprostaglandin (PG) F2
using a commercially available enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI).
Monocyte binding studies were performed as previously described (Tsao et al., 1994). Briefly, animals were sacrificed by cervical dislocation and thoracic aortae were removed, placed into ice-cold, oxygenated phosphate-buffered saline, and cleaned of adventitial tissue. Aortic segments were then carefully opened longitudinally and placed into 35-mm culture dishes containing 2 ml of HBSS. Aortic strips were fixed to the culture dish and placed on a rocking platform at room temperature. After 10 min, HBSS was replaced by 2 ml of HBSS containing fluorescently labeled WEHI 78/24 cells (2 x 106/ml) for 30 min, rotating the dishes 120° every 10 min. Nonadherent monocytes were then washed away and adherent cells counted by epifluorescent microscopy from at least 25 different sites. Data are expressed as a percentage of adherent cells on thoracic aorta compared with a control animal studied in parallel.
mRNA Expression. To investigate potential underlying mechanisms for altered inflammatory processes, aortic segments were harvested, quickly cleaned of adventitia, and flash frozen in liquid nitrogen. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. After DNase treatment, cDNA was synthesized from 5 mg of total RNA using Moloney murine leukemia virus reverse transcriptase (SuperScript II kit; Invitrogen). Relative expression levels of mRNA was determined by quantitative real-time polymerase chain reaction (q-RT-PCR) using the following primers and fluorescently labeled TaqMan probes: VCAM-1 (forward, CCGTCGCGAGGTTGTTTAGA; reverse, TCAGTCCAAGCAACACTCTCTGAT; probe, TCAGTCCAAGCAACACTCTCTGAT), MCP-1 (forward, CTGCTCAACTTGGCCATCTCT; reverse, GTGAGCCCAGAATGGTAATGTG; probe, ACCTGCTCTTCCTGC), metalloproteinase (MMP)-9 (forward, CAGCTGGCAGAGGCATACTTG; reverse, GCTTCTCTCCCATCATCTGGG; probe, ACCGCTATGGTTACACCCGGGCC), and p22phox (forward, 5'-ACCTGACCGCTGTGGTGAA-3'; reverse, 5'-GTG GAG GAC AGC CCG GA-3'; probe, 5'-CCCGCTGCCCACACCTCTTGAACT-3'). TaqMan 18S RNA probes and primers were supplied in a control reagent kit and used according to manufacturer's instructions (Applied Biosystems, Foster City, CA).
The sensitivity and specificity of the assays were assessed from serial dilutions of reference and target templates in separate PCR reactions. Optimal primer concentrations were determined as the minimum primer concentration to yield maximum change in emission intensity and minimum cycle number of fluorescence signal liberated upon dissolution of the specific probe that crosses an arbitrary threshold set within the exponential phase of the PCR (Ct). Amplification was carried out in triplicate at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Threshold cycle was calculated for each target and normalized to the Ct for 18S RNA in the same sample. 
Ct-Ct method was then used to determine differences between experimental groups and control (normocholesterolemic) samples.
Evaluation of Vascular ROS Production. ROS production was measured as previously described (Uemura et al., 2001). Briefly, freshly isolated segments of aorta were cleaned of adventitia and incubated in Dulbecco's modified Eagle's medium supplemented with a spin-trapping agent 4-amino-2,2,6,6,-tetramethylpiperidino-1-oxyl [Tempamine (TA)] for 2 h in standard cell culture conditions. Electron paramagnetic resonance (EPR) spectra of conditioned medium were recorded at room temperature with a spectrometer (model 8400, Resonance Instruments, Skokie, IL) operating at X-band with a transverse magnetic 110 cavity and flat cell. The spectrometer settings are: modulation frequency, 100 kHz; modulation amplitude, 0.5 G; scan time, 30 s; microwave power, 10 to 20 mW; and microwave frequency, 9.5 GHz. Spectral simulations are performed on a personal computer and matched directly with experimental data to extract the spectral paramagnetic parameters. Quantification of the EPR signal intensity was determined by comparing the double integration of the recorded first derivative EPR peak of each sample with a standard TA spin solution.
Data Analysis. All values in the text are expressed as mean ± S.E.M. of n independent experiments. Differences between specific means were tested by analysis of variance followed by Bonferroni post hoc analysis. A value of p < 0.05 was accepted as being statistically significant.
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Results |
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Monocyte Binding. To monitor the effects of rosuvastatin on monocyte-endothelial cell interactions, thoracic aortae were carefully harvested and functional binding assays performed using fluorescently labeled monocytoid cells (Fig. 1). Elevated cholesterol levels at 20 weeks of age were consistently associated with increased endothelial adhesiveness for monocytes compared with normocholesterolemic control animals (Fig. 2). Two-week treatment with rosuvastatin diminished monocyte binding at the highest dose tested (20 mg/kg). More robust effects were observed with 6 weeks of treatment in that both 5 and 20 mg/kg doses of rosuvastatin significantly reduced endothelial adhesiveness by 33 and 55%, respectively.
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Vascular Gene Expression and Oxidative Signaling. To investigate potential underlying mechanisms that regulate endothelial adhesiveness, we determined the expression levels of known inflammatory mediators. Quantitative RT-PCR indicated that vascular mRNA levels of VCAM-1, MCP-1, and MMP-9 were higher in hypercholesterolemic animals (Fig. 3); rosuvastatin (20 mg/kg) potently inhibited this expression. Hypercholesterolemia is also known to induce an oxidative stress in several different cell types including vascular endothelial and smooth muscle cells. Indeed, it has been demonstrated that reactive oxygen species play an important role in the activation of several atherogenic genes as well as in the process of lipid peroxidation. Analysis of the mRNA levels of p22phox, an important regulatory component of the vascular NAD(P)H oxidase, indicated elevated expression in the setting of hypercholesterolemia (Fig. 4A). To determine whether this was associated with enhanced oxidative stress, oxygen-derived free radical production from vascular segments was analyzed by EPR using TA. As shown in Fig. 4B, hypercholesterolemia induced superoxide production from the vessel wall. In addition, the plasma concentrations of the oxidative marker 8-epi-PGF2 were also elevated (Fig. 5). Consistent with its role as an anti-inflammatory compound, rosuvastatin reduced both vascular oxidative stress as well as levels of 8-epi-PGF2. This response to rosuvastatin was observed after 2 weeks but was more evident with 6 weeks of treatment.
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Discussion |
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The beneficial effects of statins on cardiovascular morbidity and mortality have been demonstrated in several primary and secondary prevention trials. These have included patients with a wide range of plasma cholesterol levels as well as concomitant risk factors. However, post hoc analysis of the West of Scotland Coronary Prevention Study (WOSCOPS) (Anonymous, 1998a), a study in which pravastatin was investigated in patients with moderate risk, indicated that some of the beneficial effects of statin treatment may go beyond its effects on cholesterol reduction. Specifically, patients in the treatment arm experienced greater benefit than would be predicted by the Framingham risk model. Furthermore, when pravastatin-treated patients were compared with placebo patients with similar LDL levels at the end of the study, the pravastatin cohort had a much lower risk of cardiovascular events.
These epidemiological findings are bolstered by preclinical animal studies with the same conclusion. One of the most convincing studies was one performed by Williams and colleagues (Williams et al., 1998) in cynomologous monkeys placed on high-fat diets. After 2 years, the animals received either low-fat or high-fat diets with pravastatin for an additional 2 years. Animals were closely monitored and diets were modified to produce the same level of total cholesterol, LDL, and HDL in the plasma of both groups. At the end of the study, isolated vessels from the statin-treated animals displayed greater endothelium-dependent vasodilatation. Moreover, histological analysis of vessels indicated reduced atherosclerotic burden as well as less macrophage content in the lesions, suggesting a potential anti-inflammatory effect of statins.
Other animal models also displayed evidence that statins may modulate inflammatory processes. Pretreatment with simvastatin produced a beneficial effect similar to indomethacin in the carrageen-induced footpad model of local inflammation (Sparrow et al., 2001). Maggard et al. (1998) showed that pravastatin prevented the aggressive coronary vasculopathy in a rat model of heart transplantation. This effect was also associated with reduced inflammatory infiltrate into the lesional areas. In addition, statins can alter the expression and activity of immunomodulatory molecules such as nitric oxide (NO). Peng et al. (1995) demonstrated that statins enhance the expression and activity of eNOS, whereas Lefer and colleagues (Lefer et al., 1999) found that statins increased coronary flow and reduced neutrophil infiltrate in the isolated perfused rat heart due to enhanced bioactivity of NO. Kline and Scalia (2003) also showed that rosuvastatin effectively prevented microcirculation leukocyte-endothelium interactions in the diabetic (db/db) mouse independent of cholesterol or glucose lowering activity. As a radical species, NO is quickly neutralized by other free radicals such as superoxide anion. Thus, the effect of simvastatin upon NO bioavailability could be due to increased NO production or reduced inactivation by reactive oxygen species. Since we detected reduced free radical production from the vessel wall, bioactivity of NO should be increased. Coupled with the ability of rosuvastatin to up-regulate endothelial NOS levels (Laufs et al., 1998) the resultant increase in NO bioavailability could help explain the observed effects on endothelial adhesiveness.
Many of the direct effects of statins on inflammatory processes are dependent on their ability to inhibit intracellular HMG-CoA reductase and, thereby, reduce intracellular concentrations of isoprenoid compounds such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate (Laufs and Liao, 1998). These isoprenoids are essential for post-translational modification of proteins that allow anchoring to lipid membranes. Signaling molecules whose functions are dependent upon isoprenoid modification include the Rho family of GTPases. Indeed, the effect of statins on eNOS expression and activity is dependent upon inhibition of Rho GTPase geranylgeranylation and is reversible by mevalonate (Laufs and Liao, 1998).
Several markers of inflammation have also been used to investigate the potential anti-inflammatory effects of statins in humans. The best known inflammatory marker used in clinical trials is C-reactive protein (CRP). High circulating levels of CRP are indicative of acute as well as chronic inflammatory situations. Indeed, significantly elevated CRP levels have been associated with cardiovascular disease as well as most risk factors for coronary heart disease (Willerson and Ridker, 2004). A substudy of the Cholesterol and Recurrent Events (CARE) trial determined that baseline CRP, as well as another marker of inflammation serum amyloid A, were both significantly higher in patients who later developed recurrent nonfatal myocardial infarction or fatal coronary event compared with matched controls who remained event free throughout the study (Ridker et al., 1999). Moreover, patients with elevated inflammatory markers had a 2-fold greater benefit from statin therapy than those without chronic inflammation, indicating an added effect of pravastatin.
Longer clinical studies have shown a small but consistent effect of statins to elevate HDL levels (Kjekshus and Pedersen, 1995; Downs et al., 1998). HDL is thought to have positive effects on atherogenesis by both reducing vascular cholesterol levels (i.e., reverse cholesterol trafficking) as well as by direct anti-inflammatory actions, presumably proteins found in the HDL moiety such as paraoxonase (Durrington et al., 2001). This indirect mechanism may not explain the effects of rosuvastatin in the current study since there were not any significant alterations in HDL levels observed. However, we cannot discount a direct effect of rosuvastatin on paraoxonase activity.
Our EPR results indicate for the first time that rosuvastatin can attenuate the enhanced superoxide anion production from vascular segments derived from apoE-deficient mice. Although several enzymatic sources have been implicated in vascular superoxide production, various lines of evidence indicate that the vascular NAD(P)H oxidase plays a significant role (Griendling and Fitzgerald, 2003a,b; Spiekermann et al., 2003). Overexpression of the p22phox subunit of the NAD(P)H oxidase increases vascular smooth muscle cell production of reactive oxygen species (Fukui et al., 1997; Griendling and Fitzgerald, 2003a). Activation of NAD(P)H oxidase can then stimulate local inflammatory processes leading to leukocyte-endothelial cell interactions (Stokes et al., 2001). In the current study, rosuvastatin reduced expression of p22phox and attenuated superoxide production, further implying a role of NAD(P)H oxidase. In support of this idea, Wassman et al. (2002) demonstrated that atorvastatin could inhibit angiotensin II-induced superoxide production as well as NAD(P)H oxidase subunit expression in cultured endothelial cells. Membrane translocation of the Rac1 GTPase, another NAD(P)H oxidase subunit required for enzyme activation, was also inhibited by atorvastatin. Reversal of these effects by the addition of mevalonate argues that atorvastatin decreases geranylgeranylation-dependent translocation of Rac1. These results, together with the findings of the current study, indicate that statins can simultaneously modulate several aspects of ROS production in vivo.
Inhibition of cellular oxidative stress is likely to play a pivotal role in rosuvastatin-mediated modulation of local inflammatory processes. Reactive oxygen species can activate nuclear factor-
B (NF-B), a transcription factor that is essential for the transcription of several inflammatory molecules including VCAM-1, MCP-1, macrophage colony stimulating factor, MMP-9, and CRP (Marui et al., 1993; Tsao et al., 1997; Jovanovic et al., 2000; Yao et al., 2000; Agrawal et al., 2003). Thus, inactivation of ROS production and NF-B by rosuvastatin would modulate the expression of several molecules involved with leukocyte trafficking. Moreover, studies by Cammarano and Minden (2001) suggest that NF-B is activated Rho GTPase, offering another explanation for the anti-inflammatory actions of rosuvastatin.
Statins may also reduce inflammatory gene expression by up-regulating members of the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors (Inoue et al., 2000; Zelvyte et al., 2002). Zelvyte and colleagues (Zelvyte et al., 2002) demonstrated an increase in PPAR-
expression after incubation of cultured monocytes with pravastatin. Intriguingly, the promoters of several inflammatory molecules, including VCAM-1 and MMP-9, contain PPAR- binding sites. Likewise, Inoue et al. (2000) showed that reduced expression of p22phox by statin was dependent upon enhanced expression of PPAR-. These findings may have particular importance when statins are used in combination with PPAR agonists for the treatment of diabetes. Fibric acid derivatives (PPAR- agonists) and thiazolidinediones (PPAR- agonists) are commonly used to reduce triglyceride levels and enhance insulin sensitivity, respectively, in patients with type 2 diabetes. Thus, the combinatorial effects of statins with PPAR agonists upon inflammatory gene transcription may help explain the added benefit of statins in diabetics observed in several large prevention trials (Pyorala et al., 1997; Goldberg et al., 1998).
In conclusion, we have demonstrated a potent anti-inflammatory effect of rosuvastatin in a murine model of hypercholesterolemia. This effect is associated with reduced vascular oxidative stress and local expression of inflammatory genes. Since many of these effects are directly related to the inhibition of HMG-CoA reductase and since rosuvastatin is considered one of the most effective statin compounds to date, it is presumed that these findings will translate into the clinical realm and have important implications for the role of rosuvastatin in the development of cardiovascular disease.
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Footnotes |
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ABBREVIATIONS: VCAM-1, vascular cell adhesion molecule-1; MCP-1, monocyte chemotactic protein-1; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LDL, low-density lipoprotein; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; apoE, apolipoprotein E; HBSS, Hanks' balanced salt solution; HDL, high-density lipoprotein; PG, prostaglandin; RT-PCR, real-time polymerase chain reaction; MMP, metalloproteinase; ROS, reactive oxygen species; TA, 4-amino-2,2,6,6,-tetramethylpiperidino-1-oxyl; CRP, C-reactive protein; NF-B, nuclear factor of the -enhancer in B cells; PPAR, peroxisome proliferator-activated receptor.
Address correspondence to: Dr. Philip S. Tsao, Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5246. E-mail: ptsao{at}stanford.edu
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, p < 0.05 from 0 mg/kg; 
