Simvastatin Exerts Both Anti-inflammatory and Cardioprotective Effects in Apolipoprotein E–Deficient Mice
Background—Simvastatin attenuates ischemia and reperfusion in normocholesterolemic animals by stabilizing endothelial nitric oxide synthase activity and inhibiting neutrophil-mediated injury. Because endothelial dysfunction is a detrimental effect of hypercholesterolemia, we examined whether short-term treatment with simvastatin could inhibit leukocyte-endothelium interaction and attenuate myocardial ischemia-reperfusion injury in apoE-deficient (apoE–/–) mice fed a high-cholesterol diet.
Methods and Results—We studied leukocyte-endothelium interactions in apoE–/– mice fed a normal or a high-cholesterol diet after short-term (ie, 18 hours) simvastatin treatment. We also studied simvastatin treatment in myocardial ischemia-reperfusion injury by subjecting apoE–/– mice to 30 minutes of ischemia and 24 hours of reperfusion. ApoE–/– mice fed a high-cholesterol diet exhibited higher blood cholesterol levels, which were not affected by short-term simvastatin treatment. However, the increased leukocyte rolling and adherence that occurred in cholesterol-fed apoE–/– mice (P<0.001 versus control diet) were significantly attenuated by simvastatin treatment (P<0.01 versus vehicle). Cholesterol-fed apoE–/– mice subjected to myocardial ischemia-reperfusion also experienced increased myocardial necrosis (P<0.01 versus control diet), which was significantly attenuated by simvastatin (P<0.01 versus vehicle). Simvastatin therapy also significantly increased vascular nitric oxide production in apoE–/– mice.
Conclusions—Simvastatin attenuates leukocyte-endothelial cell interactions and ameliorates ischemic injury in hypercholesterolemic mice independently of lipid-lowering actions.
The major action of the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (simvastatin, pravastatin, etc), which are collectively known as “statins,” is the inhibition of cholesterol synthesis in the liver.1 Recently, Vaughan et al2 suggested that several statins may exert effects that are distinct from their cholesterol-lowering actions. In this regard, it has been shown that simvastatin can extend the half-life of nitric oxide synthase (NOS) mRNA in endothelial cells in vitro under cholesterol-clamped conditions.3 Other investigators have shown that by increasing the bioavailability of NO in the vascular endothelium of normocholesterolemic animals, statins inhibit leukocyte-endothelium interactions in the microcirculation.4 As a result of these endothelial-preserving effects, statins can ameliorate neural injury during cerebral ischemia5 and protect the ischemic-reperfused myocardium from polymorphonuclear neutrophil (PMN)-mediated injury.6 These findings provide convincing evidence that statins can improve endothelial function and protect the cardiovascular system in normocholesterolemic subjects, independent of their cholesterol-lowering effects.
However, the highest incidence of coronary artery disease is observed in humans who have a long-standing history of hypercholesterolemia, including those who are affected by severe forms of familial hypercholesterolemia.7 8 To our knowledge, no data are available demonstrating that short-term statin therapy can exert anti-inflammatory effects or protect tissue in the setting of severe hypercholesterolemia.
Therefore, we tested the hypothesis that a widely used HMG-CoA reductase inhibitor, simvastatin, can inhibit leukocyte-endothelium interactions in the microcirculation of a genetic mouse model of hypercholesterolemia and can protect the myocardium of hypercholesterolemic mice against ischemia-reperfusion injury. In the present study, we used the apoE–/– mouse, a well-established genetic mouse model of atherogenic hypercholesterolemia, which is similar to hyperlipoproteinemia type III in humans.9 The study was designed so that blood cholesterol levels were not affected by statin treatment. We also used NOS gene-targeted mice to help determine the mechanism of action of short-term simvastatin therapy.
This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All animal protocols were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and the Animal Care and Use Committee of Louisiana State University Health Sciences Center.
Animal Protocols and Preparation of Simvastatin
ApoE–/– mice, 4 to 6 months of age (Jackson Laboratory, Bar Harbor, Maine), either were fed a high-cholesterol diet for 2 consecutive weeks (15.8% fat and 1.25% cholesterol; from Harlan Teklad) or were maintained on noncholesterol-fortified chow for an equal duration of time. Mice were then randomly divided into 1 of the following 3 groups: (1) apoE–/– mice fed a normal diet and given vehicle, (2) apoE–/– mice fed a high-cholesterol diet and given vehicle, and (3) apoE–/– mice fed a high-cholesterol diet and given simvastatin. Simvastatin was obtained from Merck Inc and was chemically activated by alkaline treatment before use, as previously described.10 Simvastatin (1 mg/kg SC) was administered to the apoE–/– mice 18 hours before study. At the end of the 2-week high-cholesterol feeding, mice were either used for intravital microscopy studies or subjected to in vivo myocardial ischemia and reperfusion.
In additional studies, wild-type mice (C57BL/6, Jackson Labs), endothelial NOS (eNOS)-deficient mice (Paul Huang, Harvard University, Boston, Mass), and inducible NOS (iNOS)-deficient mice (Jackson Labs) were treated with simvastatin (1 mg/kg) or an equal volume of vehicle 18 hours before myocardial ischemia.
Intravital microscopy of mouse peri-intestinal venules was performed according to previously described methods11 in pentobarbital-anesthetized mice. The ileum and mesentery of anesthetized animals were placed in a temperature-controlled Plexiglas chamber, and a modified Krebs-Henseleit solution was used to superfuse the mouse intestine. Red blood cell velocity was determined online using an optical Doppler velocimeter.12
The surgical protocol and infarct size determination were performed according to methods described previously.13 The mice were anesthetized with sodium pentobarbital (50 mg/kg IP) and ketamine (50 mg/kg IP). After the surgical procedure, mice were given butorphanol tartrate (≈0.08 mg/kg SC) for analgesia. The animals were given supplemental oxygen (100%) via a nasal cone, and they were allowed to recover in a temperature-controlled area.
Determination of Area at Risk and Infarct Size
After 24 hours of reperfusion, the mice were connected to a ventilator, the left anterior descending coronary artery was re-ligated, and Evans blue (1.2 mL of 1.0% solution) was retrogradely infused into the carotid artery catheter to delineate the ischemic zone from the nonischemic zone. The heart was serially sectioned along its long axis and incubated in 1.0% of 2,3,5-triphenyltetrazolium chloride for 5 minutes at 37°C to allow differentiation between the viable and necrotic areas of the myocardium previously rendered ischemic. Each of the five 1-mm-thick slices was weighed, and the areas of infarction, risk, and nonischemic left ventricle were assessed by a blinded observer using computer-assisted planimetry (NIH Image 1.57).
Assessment of Myocardial Neutrophil Infiltration
Routine histological staining was performed on multiple sections of midventricular cardiac sections to determine the extent of PMN infiltration. For each heart, the number of PMNs was counted in 12 fields of 3 independent tissue sections by a blinded observer.
Quantification of NO Released From Isolated Aortic Tissue
Freshly isolated thoracic aortas were isolated from wild-type and apoE–/– mice. Freshly dissected aortic rings measuring 5 to 6 mm in length were cut, opened from random segments of the aorta, and fixed by small pins with the endothelial surface up in 24-well culture dishes filled with 1 mL of Krebs-Henseleit solution. After equilibration at 37°C, NO released into the Krebs-Henseleit solution was measured by means of a shielded polarographic NO electrode connected to a NO meter (Iso-NO Mark II, World Precision Instruments), according to a previously described method.14
All data are presented as mean±SEM. Data on mean arterial blood pressure, venular shear rates, leukocyte rolling, and adherence were compared by ANOVA using Fisher’s post hoc analysis. Myocardial necrosis and PMN counts (between groups) were analyzed with a 2-tailed unpaired t test. All statistics were calculated with StatView 4.5 (Abacus Concepts). Probabilities of 0.05 or less were considered significant.
Effect of Simvastatin on Blood Cholesterol Levels
Wild-type mice exhibited total cholesterol levels <100 mg/dL and were unaffected by 18 hours of simvastatin treatment (Figure 1⇓). ApoE–/– mice that were maintained on a high-cholesterol diet exhibited elevated total blood cholesterol levels (2769±210 mg/dL) compared with apoE–/– mice fed normal chow (343±21 mg/dL; P<0.001). Administration of a single dose of simvastatin to cholesterol-fed apoE–/– mice did not significantly lower their elevated blood cholesterol levels.
Leukocyte-Endothelium Interaction in the Microcirculation
We examined mouse peri-intestinal venules ranging from 33 to 39 μm in diameter; there was no significant difference in venular diameter among any of the groups studied. The mean venular shear rates were also similar in all groups, ranging from 685±81 to 710±64 s–1. Mean arterial blood pressures ranged between 99±16 and 105±10 mm Hg over the 60-minute observation period, and they did not differ significantly among groups. Thus, the adhesive interactions observed between circulating leukocytes and the microvascular endothelium were not due to changes in hemodynamics brought about by high cholesterol feeding or by the subcutaneous administration of simvastatin 18 hours earlier.
Wild-type mice, whether given simvastatin or not, exhibited low levels of leukocyte rolling (Figure 2⇓) and a low number of adherent leukocytes (Figure 3⇓). Investigation of peri-intestinal venules in apoE–/– mice revealed that leukocyte rolling doubled and adherence increased nearly 4-fold in apoE–/– mice (Figure 3⇓) compared with wild-type controls. Moreover, at the end of the 2 weeks of high cholesterol feeding, both leukocyte rolling and firm adhesion increased nearly 2-fold compared with apoE–/– mice fed a control diet.
In contrast, simvastatin markedly attenuated both leukocyte rolling (Figure 2⇑) and adherence (Figure 3⇑) in cholesterol-fed apoE–/– mice (P<0.05 versus cholesterol-fed apoE–/– mice given vehicle). Therefore, systemic administration of simvastatin to apoE–/– mice exposed to a high-cholesterol diet for 2 weeks inhibited leukocyte-endothelium interaction, even in the absence of significant cholesterol lowering.
Effect of Simvastatin on Myocardial Ischemia-Reperfusion
Myocardial Area at Risk and Necrosis
Experiments in which wild-type controls were treated with simvastatin and then subjected to the myocardial ischemia-reperfusion protocol are shown in Figure 4A⇓. The areas of left ventricle placed at risk for infarction by coronary artery occlusion were similar in both groups of wild-type controls. Pretreatment with simvastatin significantly (P<0.01) reduced the extent of myocardial necrosis in wild-type mice.
Despite similar areas at risk, hearts from cholesterol-fed apoE–/– mice given vehicle suffered a significantly larger area of infarction after myocardial ischemia-reperfusion compared with both control diet–fed apoE–/– mice and cholesterol-fed apoE–/– mice given simvastatin. Summary data for areas at risk and amount of myocardial necrosis in apoE–/– mice are shown in Figure 4B⇑. All groups of animals experienced similar-sized areas at risk per left ventricular area. The areas at risk were 54% to 57% of the left ventricle for all groups of mice subjected to myocardial ischemia/reperfusion. Thus, the exposure of apoE–/– mice to high cholesterol feeding for 2 weeks resulted in increased myocardial injury after ischemia and reperfusion, and this was attenuated by a noncholesterol-lowering treatment with simvastatin.
The cardioprotection provided by simvastatin was influenced by the bioavailability of different isoforms of NOS, as depicted in the Table⇓. Thus, mice deficient in eNOS (eNOS–/– mice) exhibited markedly exacerbated myocardial necrosis (P<0.05 versus wild-type controls). Moreover, this cardiac reperfusion injury was unaffected by simvastatin treatment (Table⇓). In iNOS–/– mice, the degree of cardiac necrosis was comparable to that in the wild-type mice, but simvastatin did not influence this degree of injury (Table⇓). Thus, NO seems to play a key role in the cardioprotective effects of simvastatin. Both eNOS and iNOS seem to contribute to the enhanced NO released by simvastatin, although the contribution by eNOS seems to be somewhat greater.
Myocardial PMN Accumulation
The degree of infiltrated PMNs into the ischemic-reperfused myocardium after 30 minutes of myocardial ischemia and 24 hours of reperfusion is presented in Figure 5⇓. Twenty-four hours after reperfusion, PMN accumulation in ischemic-reperfused myocardium was 52±2 PMNs/field in wild-type mice (Figure 5A⇓). Accumulation was only 21±1 PMNs/mm2 in hearts taken from wild-type mice given simvastatin (P<0.01). The number of PMNs/mm2 in hearts from apoE–/– mice fed the control diet was 54±3 PMNs/mm2. This value increased by 40% in apoE–/– mice fed a high-cholesterol diet (Figure 5B⇓; P<0.01). In contrast, systemic administration of simvastatin to cholesterol-fed apoE–/– mice attenuated the number of neutrophils infiltrating into the ischemic/reperfused myocardium by 45% (P<0.01 versus cholesterol-fed apoE–/– mice given vehicle). These data closely correlate with data on leukocyte adherence obtained by intravital microscopy, and they also demonstrate a marked antiadhesive effect of simvastatin in the microcirculation of cholesterol-fed apoE–/– mice.
Effect of Simvastatin on Endothelial NO Release
We also measured NO release from aortic segments isolated from vehicle- and simvastatin-treated apoE–/– mice fed either a control or a high-cholesterol diet. Wild-type mice exhibited a NO release of 22±4 nmol/g tissue, and this increased by 53% after simvastatin treatment (Figure 6⇓). We detected a slightly reduced basal level of NO release in aortic rings isolated from apoE–/– mice fed a control diet (Figure 6⇓). However, exposure of apoE–/– mice to high-cholesterol diet for 2 weeks significantly reduced the basal release of NO to half of control values (P<0.02 versus control diet mice). However, 18 hours after the injection of 1 mg/kg simvastatin into cholesterol-fed apoE–/– mice, the basal release of NO measured in aortic rings increased to double the values observed in apoE–/– mice fed a normal diet (Figure 6⇓). Although not shown in Figure 6⇓, the addition of 100 μmol/L of N-nitro-l-arginine methyl ester, a NOS inhibitor, reduced NO values in all groups to approximately zero. Therefore, systemic administration of a single dose of simvastatin to either wild-type or apoE–/– mice significantly increased endothelium-derived NO over a period of 18 hours.
Endothelial cell dysfunction is one of the earliest events occurring during hypercholesterolemia,15 16 and it ultimately contributes to the development of overt atherosclerosis.17 18 This endothelial dysfunction is characterized by a marked reduction in biologically active NO released from the vascular endothelium, which can predispose the circulation to coronary artery disease. Increased hypercholesterolemia in rabbits in the absence of atherosclerotic plaque formation has been shown to exacerbate reperfusion injury after myocardial ischemia and reperfusion.19 20 Moreover, several animal and human studies17 21 indicate an improvement in endothelial function associated with the lowering of serum cholesterol.
One widely used means of controlling hypercholesterolemia is HMG-CoA reductase inhibitors (statins). Statins lower blood cholesterol levels by inhibiting the hepatic biosynthesis of cholesterol at the mevalonate step.1 This reduced cholesterol synthesis normalizes endothelial function in animals17 and humans22 23 and results in reduced coronary artery disease21 23 and stroke.24 These effects are apparently due to reduced cholesterol accumulation in endothelial cells and, consequently, to preservation of endothelial function, including maintenance of NO release.
More recently, statins reportedly directly increased NOS activity in cultured endothelial cells under cholesterol-clamped conditions.3 In normocholesterolemic animals, this action of statins is associated with the inhibition of leukocyte-endothelium interaction in the microcirculation,4 attenuation of myocardial ischemia-reperfusion injury,6 and stroke protection.5
These findings clearly point toward an action other than cholesterol lowering by statins. However, it is unknown whether the noncholesterol-lowering effects of statins have therapeutic significance in chronic hypercholesterolemic conditions. In this study, simvastatin administered to cholesterol-fed apoE–/– mice exerted a marked cardioprotective effect without lowering blood cholesterol levels. Our data also show that short-term treatment with statins exerts a distinct anti-inflammatory effect within the cardiovascular system, as demonstrated by attenuated neutrophil-endothelium interactions in the microvasculature and the reduced infiltration of neutrophils into the ischemic-reperfused myocardium of cholesterol-fed apoE–/– mice. This early anti-inflammatory effect of simvastatin was associated with enhanced NO release from the vasculature of cholesterol-fed apoE–/– mice. These findings are consistent with previous observations showing that simvastatin upregulates eNOS activity in endothelial cells by inhibiting the degradation of eNOS mRNA.3
In the present study, we extended our knowledge of the source of enhanced NO production by simvastatin. We presented data in this study showing that simvastatin-stimulated NO production occurs, to a large extent, from eNOS, but iNOS can also contribute to this process. Because iNOS is present in vascular smooth muscle cells, this may represent an augmentation of NO production by vascular components other than endothelium.
Several possibilities may explain the cardiovascular protective effects of statins, even in the absence of reduced blood cholesterol levels. These mechanisms all relate to the preservation of endothelial function, the inhibition of neutrophil-mediated tissue injury, or a combination of these 2 effects. Statins have been shown to stabilize eNOS mRNA, thus increasing the bioavailability of NO in cultured endothelial cells3 and in normocholesterolemic animals.4 6 In the present study, we showed for the first time that simvastatin significantly and acutely increases basal NO release from the vascular endothelium of genetically hypercholesterolemic animals by a specific mechanism involving NOS. NO has been shown to act as a physiological immunomodulator that can inhibit leukocyte-endothelium interaction25 by suppressing the upregulation of several endothelial cell adhesion molecules.26 27 Therefore, the maintenance of endothelium-derived NO may be a very important aspect of the cardioprotective effects of simvastatin. Although one cannot directly visualize the coronary microcirculation in vivo, all indications are that comparable events occur in the more readily visualized splanchnic microcirculation.
Salutary effects have been observed with simvastatin and lovastatin in mice subjected to cerebral ischemia-reperfusion.5 These effects were specifically dependent on enhanced NO formation because they did not occur in eNOS–/– mice28 and they were unrelated to the inhibitory effect of simvastatin on cholesterol biosynthesis. This endothelial preservation, with its subsequent attenuation of neutrophil activation, effectively prevents neutrophil-induced cardiac dysfunction.6 In previous work, we provided evidence that simvastatin significantly increases basal NO release from the normocholesterolemic vascular endothelium by a specific mechanism involving NOS.6 This increased bioavailability of NO induced by statins attenuates the upregulation of cell adhesion molecules both in the coronary microvascular endothelium6 and in the mesenteric microvasculature,4 thus limiting neutrophil adherence to the endothelium during inflammatory conditions.
In conclusion, therapeutic administration of statins during severe hypercholesterolemia may promote endothelial preservation and normalize vascular homeostasis before lowering cholesterol. These salutary effects were obtained with clinically used doses (ie, equivalent to 75 mg, single dose in a human subject), and these effects might represent a new strategy for the primary prevention of short and long-term inflammatory states in coronary artery disease, as was previously suggested in a large multicenter clinical study.29
Supported by research grants from Merck to A.M.L. and D.J.L., research grant #1-2000-68 from the Juvenile Diabetic Foundation to R.S., and research grants #RO1-HL-60849 and P01-DK-43785 from the National Institutes of Health to D.J.L..
- Received October 23, 2000.
- Revision received January 12, 2001.
- Accepted January 23, 2001.
- Copyright © 2001 by American Heart Association
Laufs U, La Fata V, Plutzky J, et al. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 1998;97:1129–1135.
Pruefer D, Scalia R, Lefer AM. Simvastatin inhibits leukocyte-endothelial cell interactions and protects against inflammatory processes in normocholesterolemic rats. Arterioscler Thromb Vasc Biol. 1999;19:2894–2900.
Endres M, Laufs U, Huang Z, et al. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998;95:8880–8885.
Lefer AM, Campbell B, Shin YK, et al. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation. 1999;100:178–184.
Simon A, Megnien JL, Levenson J. Coronary risk estimation and treatment of hypercholesterolemia. Circulation. 1997;96:2449–2452.
Zhang HS, Reddick RL, Piedrahita JA, et al. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258:468–471.
Scalia R, Armstead VE, Minchenko AG, et al. Essential role of P-selection in the initiation of the inflammatory response induced by hemorrhage and reinfusion. J Exp Med. 1999;189:931–938.
Hoffmeyer MR, Jones SP, Ross CR, et al. Myocardial ischemia/reperfusion injury in NADPH oxidase-deficient mice. Circ Res. 2000;87:812–817.
Guo JP, Murohara T, Buerke M, et al. Direct measurement of nitric oxide release from vascular endothelial cells. J Appl Physiol. 1996;81:774–779.
Osborne JA, Siegman MJ, Sedar AW, et al. Lack of endothelium-dependent relaxation in coronary resistance arteries of cholesterol-fed rabbits. Am J Physiol. 1989;256:C591–C597.
Scalia R, Appel JZ III, Lefer AM. Leukocyte-endothelium interaction during the early stages of hypercholesterolemia in the rabbit: role of P-selectin, ICAM-1, and VCAM-1. Arterioscler Thromb Vasc Biol. 1998;18:1093–1100.
Osborne JA, Lento PH, Siegfried MR, et al. Cardiovascular effects of acute hypercholesterolemia in rabbits: reversal with lovastatin treatment. J Clin Invest. 1989;83:465–473.
Verbeuren TJ, Jordaens FH, Zonnekeyn LL, et al. Effect of hypercholesterolemia on vascular reactivity in the rabbit, I: endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits. Circ Res. 1986;58:552–564.
Tilton RG, Cole PA, Zions JD, et al. Increased ischemia-reperfusion injury to the heart associated with short-term, diet-induced hypercholesterolemia in rabbits. Circ Res. 1987;60:551–559.
Golino P, Maroko PR, Carew TE. The effect of acute hypercholesterolemia on myocardial infarct size and the no-reflow phenomenon during coronary occlusion-reperfusion. Circulation. 1987;75:292–298.
Delanty N, Vaughan CJ. Vascular effects of statins in stroke. Stroke. 1997;28:2315–2320.
Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991;88:4651–4655.
De Caterina R, Libby P, Peng HB, et al. Nitric oxide decreases cytokine-induced endothelial activation: nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest. 1995;96:60–68.
Laufs U, Fata VL, Liao JK. Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated down-regulation of endothelial nitric oxide synthase. J Biol Chem. 1997;272:31725–31729.