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Simvastatin Decreases Nitric Oxide Overproduction and Reverts the Impaired Vascular Responsiveness Induced by Endotoxic Shock in Rats

Giusti-Paiva, Alexandre; Martinez, Maria Regina; Cestari Felix, Jorge Vinicius; da Rocha, Maria Jose Alves; Carnio, Evelin Capellari; Elias, Lucila Leico Kagohara; Antunes-Rodrigues, Jose

doi: 10.1097/10.shk.0000115756.74059.ce
Article

Lipopolysaccharides (LPS) can be used to induce experimental endotoxic shock, which is characterized by a significant decrease in mean arterial pressure (MAP) and a decreased vasoconstrictor response that have been attributed to excessive nitric oxide production. Inhibitors of 3-hydroxi-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), in addition to lowering serum cholesterol levels, exert many pleiotropic effects, including anti-inflammatory action. In the present study, we investigated the effect of simvastatin, an HMG-CoA reductase inhibitor, on the production of nitric oxide and the cardiovascular response to LPS. Male Wistar rats were pretreated with different doses of simvastatin (10, 20, 40, and 80 mg/kg, i.p.) or saline 20 min before i.v. injection of LPS (1.5 mg/kg) or saline (control). MAP was continuously recorded and nitrate plasma concentration was determined during the 6-h experimental session at 1-h intervals. The pressor response to phenylephrine (1 μg/kg) was evaluated before and 6 h after LPS administration. In the LPS-treated group, there was a time-dependent increase in nitrate plasma concentration (P < 0.001), and this response was decreased in simvastatin pretreated rats (P < 0.001). We also observed that LPS decreased the pressor response to phenylephrine (P < 0.001), an effect that was reverted by simvastatin pretreatment (P < 0.05). However, simvastatin did not modify the decrease of MAP induced by LPS. We concluded that simvastatin decreases nitrate plasma concentration in response to LPS and recovers vascular responsiveness during an experimental endotoxic shock. These data suggest the potential use of HMG-CoA reductase inhibitors as a coadjuvant in the treatment of septic shock.

Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Brazil

Received 8 Oct 2003;

first review completed 23 Oct 2003; accepted in final form 16 Dec 2003

Address reprint requests to Dr. Jose Antunes-Rodrigues. Departamento de Fisiologia da Faculdade de Medicina de Ribeirao Preto, Av Bandeirantes 3900, Ribeirao Preto – SP, Brazil 14090-900. E-mail: antunes@fmrp.usp.br.

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INTRODUCTION

Endotoxic shock is thought to result from the release of proinflammatory cytokines such as tumor necrosis factor-α and interleukin 1β. These cytokines increase the inflammatory response, inducing target cells, to release additional and potentially harmful host mediators, such as cytokines, nitric oxide (NO), and eicosanoids. Severe infection with gram-negative bacteria results in an increased production of cytokines induced by the action of lipopolysaccharides (LPS), a constituent of the outer membrane of these bacteria. LPS is a potent activator of immune system cells (1). In fact, the administration of LPS to experimental animals results in an endotoxic shock characterized by hypotension, increased vascular permeability, and inadequate response to vasoconstrictors (2–4). NO has received attention as a potential mediator of the endotoxic shock, as it is one of the key elements in the modulation of inflammation, vascular reactivity, and cardiac function (2–5). It has been suggested that the production of NO generated from the conversion of l-arginine by NO synthase (NOS) can largely account for hypotension and hyporeactivity to vasoconstrictors. This is supported by in vivo animal models of endotoxic shock showing that NOS inhibitors revert much of the cardiovascular changes (5–8).

The overproduction of NO is central to the development of septic shock (2). NO synthesis is catalyzed either by the constitutive or the inducible isoform of NOS (iNOS). Constitutive NOS regulates the NO concentrations necessary for homeostatic processes such as regulation of vascular tone and neurotransmission. On the other hand, endotoxin and cytokines stimulate the iNOS expression in macrophages, vascular smooth muscle cells, and endothelium. The impaired pressor response to catecholamines in experimental endotoxic shock has been attributed to excessive NO formation by iNOS (2,5–7).

Endotoxic shock affects cholesterol metabolism. LPS has been reported to provoke an increase in hepatic cholesterol synthesis in rodents that is accompanied by an increase in the synthesis and activity of 3-hydroxi-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (9,10). HMG-CoA reductase is a rate-limiting enzyme in the biosynthesis of cholesterol and catalyzes the transformation of HMG-CoA to mevalonate. Inhibitors of HMG-CoA reductase have been extensively used in lipid-lowering therapy (11). In addition to lowering cholesterol, inhibitors of HMG-CoA reductase, known as statins, also reduce cellular isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylphyrophosphate (GGPP) (11,12), which are necessary for prenylation of critical membrane proteins that regulate cellular communication in the inflammatory response (11,13). Several studies have shown that HMG-CoA reductase inhibitors have various nonlipid effects, including anti-inflammatory effects (14,15). It has also been reported that an HMG-CoA reductase inhibitor could inhibit LPS-induced iNOS gene expression and production of NO by macrophages, which was reverted by mevalonate (16,17).

In the present study, we investigated the effects of simvastatin, an HMG-CoA reductase inhibitor, on the production of NO and the pressor response to phenylephrine in LPS-induced endotoxic shock in rats.

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MATERIALS AND METHODS

Animals

Adult male Wistar rats weighing 250 to 300 g were maintained under controlled temperature (23°C ± 1°C) and were exposed to a daily 12:12-h light-dark cycle (7:00am-7:00 pm), with free access to tap water and pelleted food. All experimental protocols were performed according to the guidelines of the Ethical Committee for Animal Use of the School of Medicine of Ribeirao Preto, University of Sao Paulo.

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Animal preparation

The rats were anesthetized with 2,2,2-tribromoethanol (25 mg/100 g body weight i.p., Aldrich, Milwaukee, WI) and a silastic catheter was inserted into the right external jugular vein for i.v. drug administration and blood sample collections. The animals were also fitted with a polyethylene catheter into the femoral artery for direct recording of blood pressure; the free end of the catheter was guided under the skin to exit on the back of the animal’s neck, and the catheter was filled with 0.3% heparin in sterile isotonic saline (0.15 M NaCl), locked, and sealed with a stylet. The rats were left 24 h to recover from surgery.

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Drugs

LPS (Escherichia coli serotype 0111:B4), N-ω-L-nitro-arginine methyl ester (L-NAME, a nonselective inhibitor of NOS), aminoguanidine (AG, a selective inhibitor of iNOS), and phenylephrine hydrochloride were purchased from Sigma Chemical (St Louis, MO). Simvastatin was obtained from Merck Sharp & Dohme Farmaceutica e Veterinaria (Campinas, Sao Paulo, Brazil). All drugs were dissolved in pyrogen-free isotonic saline.

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Experimental groups

Twelve hours and 20 min before the i.v. administration of LPS (1.5 mg/kg, n = 5 per group), rats received an i.p. injection of simvastatin (80 mg/kg) or isotonic saline (1.0 mL/kg). A dose-response curve of simvastatin on plasma nitrate concentration, determined 6 h after LPS administration, was performed using different doses of HMG-CoA inhibitor (0, 10, 20, 40, and 80 mg/kg) 12 h and 20 min before the LPS injection. This dose-response curve was used to determine the dose (80 mg/kg) for the maximum effective inhibitory effect on nitrate production induced by LPS. In a second set of experiments, animals were pretreated 20 min with an i.p. injection of L-NAME (30 mg/kg) or AG (100 mg/kg) before the LPS injection. The rats were observed every hour after LPS injection for 6 h, and septic shock symptoms such as fever, lethargy, diarrhea, and the number of rats surviving in each group were recorded. To assess liver function, 0.5 mL of heparinized blood was drawn at 6 h after LPS injection. Blood was centrifuged (3000 rpm for 10 min at 4°C) and plasma was frozen until use. Alanine aminotransferase (ALT) was determined by commercial kit (Celm, Sao Paulo, Brazil).

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Nitrate plasma determination

Blood samples (0.2 mL) for nitrate determination were collected just before LPS administration and thereafter at hourly intervals, during the subsequent 6 h with the volume being replaced with isotonic saline. Blood samples were centrifuged (3000 rpm for 10 min at 4°C) and plasma was kept in a freezer at −20°C. On the day of the assay, plasma samples were thawed and deproteinized with 95% ethanol (at 4°C) for 30 min, subsequently centrifuged, and the supernatant was used for measurement of nitrate according to the NO/ozone chemiluminescence technique as previously described by Archer (18), using a Sievers NO Analizer (Sievers 280 NOA; Sievers, Boulder, CO). Sodium nitrate (Sigma Chemical) was used as standard reference.

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Blood pressure measurements

The mean arterial pressure (MAP) of conscious, freely moving rats (n = 6 per group) was continuously recorded using a Grass polygraph (Grass model P122; Grass Instruments, Quincy, MA) connected to a pressure transducer (Grass model P23XL-1; Grass Instruments). The rats received the first injection of simvastatin 12 h before the MAP measurement. On the day of experiment, the MAP was allowed to stabilize for 30 min. After recording baseline hemodynamic parameters, the pressor response to phenylephrine (1 μg/kg, i.v.) was recorded, and 10 min later the animals were treated with the second injection of simvastatin (80 mg/kg) or vehicle (saline). Twenty minutes later, the animals received LPS (1.5 mg/kg, i.v.) and had their MAP monitored for 6 h. To evaluate the effect of NOS inhibition in the blood pressure during experimental septic shock, animals were pretreated for 20 min with an i.p. injection of L-NAME (30 mg/kg) or AG (100 mg/kg) before the LPS injection. The pressor response to phenylephrine was reassessed 6 h after LPS administration.

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Statistical analysis

Values are presented as mean ± SEM. Statistical differences were assessed by analysis of variance (ANOVA) followed by Tukey’s test or nonlinear regression for analysis of dose response. Comparisons of percentage of survival were performed by Fisher’s exact test. Differences were accepted as significant at P < 0.05.

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RESULTS

Plasma nitrate levels increased gradually from 1 to 6 h and were significantly higher than those observed in the control group throughout the experiment after the 2nd h of LPS treatment (P < 0.001). This effect was significantly reduced by pretreatment with simvastatin (P < 0.001;Fig. 1). The pretreatment with saline (control) or simvastatin (80 mg/kg), did not alter nitrate plasma concentration after intravenous injection of saline. The injection of different doses of simvastatin (10–80 mg/kg) reduced nitrate plasma concentration 6 h after LPS administration in a dose-dependent pattern (r2 = 0.97;Fig. 2).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Saline (control group) or simvastatin alone did not induce significant changes in the MAP during the experimental period. A single injection of LPS (1.5 mg/kg i.v.) produced a prolonged hypotension (P < 0.01;Fig. 3) compared with the control group. Pretreatment with simvastatin (80 mg/kg i.p.) did not significantly change the hypotension induced by LPS. Phenylephrine promoted an increase in MAP in all experimental groups under baseline conditions (Fig. 4). The pressor response to phenylephrine was not modified by simvastatin pretreatment in rats injected with saline. On the other hand, LPS treatment significantly attenuated the pressor response to phenylephrine at 6 h (P < 0.001) compared with the initial response. The pretreatment with simvastatin in LPS-treated rats reverted the impaired pressor response to phenylephrine when compared with rats treated with saline followed by LPS (P < 0.01;Fig. 4).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Table 1 shows the values of MAP, nitrate plasma, pressor response to phenylephrine, ALT, and survival rate in the LPS-treated group with or without NOS inhibitors or simvastatin. As expected, there was a reduction of nitrate plasma concentration (P < 0.05) and a recovery of MAP and the pressor response to phenylephrine (P < 0,05) in the L-NAME- or AG-pretreated animals that received LPS compared with saline plus LPS-treated animals. However, the nonselective inhibition of NOS by L-NAME resulted in a significant increase of ALT (P < 0.05) and reduction of survival rate to 45.3% compared with rats pretreated with saline or AG. The pretreatment with simvastatin did not alter the increase of ALT induced by LPS and also did not modify the survival rate.

Table 1

Table 1

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DISCUSSION

This study demonstrates that simvastatin, an HMG-CoA reductase inhibitor, attenuates the increase in NO production and reverts the impaired vascular responsiveness to phenylephrine elicited by prolonged period of endotoxemia in rats. However, this reduction of NO production was not accompanied by changes in the LPS-induced hypotension.

The predominant hypothesis for hypotension during sepsis is that there is an increase in NO production by iNOS (2–5), which leads to vasodilation and a decrease of vascular responsiveness to vasoconstrictors. We observed a clear, dose-dependent reduction in plasma nitrate concentration after LPS administration in animals pretreated with simvastatin. This result suggests that simvastatin inhibits the production of NO, and this effect might be due to a decrease of iNOS activity during endotoxic shock.

NO blockade results in a rise in arterial pressure and restored the effects of catecholamines. Overall, beneficial effects have been observed with selective iNOS inhibitors, whereas high doses of nonselective NOS inhibitors have been shown to be detrimental (5,7). Indeed, it has been demonstrated that nonselective NO inhibitors decrease blood flow, tissue oxygenation, and survival (5). Our results confirmed the effects of nonselective NOS inhibitors increasing the animal mortality. On the other hand, the beneficial effects of selective iNOS inhibitors were shown by the attenuation of circulatory failure, renal, hepatic, and pancreatic dysfunction, and improvement of survival in rats submitted to endotoxemia (5,19–21). The present data show that inhibition of HMG-CoA reductase with simvastatin is similar to treatment with aminoguanidine by restoring response to phenylephrine and reducing NO production without affecting the survival rate.

In endotoxic shock, multiple cellular processes are involved and many humoral cascades are triggered, so that the blocking a single component may be insufficient to arrest the inflammatory process. The statins have a strong protective effect against sepsis by the diverse anti-inflammatory properties that are independent of their lipid-lowering ability (14). Most of these effects are mediated by the inhibition of isoprenoid synthesis (14,22–25). HMG-CoA reductase inhibitor interrupts the isoprenylation of small G proteins (e.g., Ras and Rho) by decreasing FPP and GGPP level, leading to the accumulation of inactive small G proteins in the cytoplasm. The reduction of cellular isoprenoid, as well as the lowering membrane levels and activity of Ras/Rho proteins, may be important mechanisms mediating the direct cellular effects of statins on the vascular wall (22).

It is difficult to define in vivo the specific molecular mechanism underlying the effect of simvastatin on plasma nitrate concentration and the vascular response to LPS. Increasing evidence suggests that statins are able to down-regulate NOS gene transcription as a consequence of interference with the small G proteins/ NF-κB transduction pathway (26). In inflammatory conditions, NF-κB, activated by small G proteins, is an essential factor that promotes the transcription of iNOS gene (27).

Endotoxic shock is associated with a decrease in endothelial NOS (5,28) and a delayed increase in iNOS expression (5,27). NO produced by eNOS is an important regulator of vasomotor tone, blood flow, permeability, and is an inhibitor of leukocyte and platelet adhesion as well as a modulator of coagulation activation. During sepsis, the endothelial function is delayed by apoptosis induced by LPS, which has a crucial role in the pathogenesis of sepsis and multiple organ dysfunctions (5,29). Recently, it was demonstrated that statins upregulate the expression and increase eNOS activity (30), which has an antiapoptotic effect, and in native endothelial cells in situ decreased cytokine-stimulated iNOS expression (25), independent of cholesterol levels. Thus, statins could contribute to a favorable balance between eNOS and iNOS and restore the vascular responsivity in the endotoxic shock.

The present data show that simvastatin reestablished the pressor response to phenylephrine during endotoxic shock. The smooth muscle contraction elicited by most vasoconstrictor agents is regulated by an increase in cytosolic calcium levels from intracellular stores or by calcium influx through calcium channels located in the cell membrane. Phenylephrine, an α-adrenergic receptor agonist causes vasoconstriction through opening of receptor-operated membrane calcium channels with a resulting influx of extracellular calcium; activation of phospholipase C and hydrolysis of phosphatidyl inositol biphosphate to diacyl glycerol, which activates myosin light-chain kinase through protein kinase C; and production of inositol 1,4,5-triphosphate (IP3), which in turn causes calcium release from intracellular stores in the endoplasmic reticulum (31). A possible interference of the NO in these signal transduction mechanisms has previously been suggested by Ji and collaborators (32), who reported that NO selectively inhibits IP3-induced intracellular calcium release in aortic smooth muscle of rats. NO was also shown to inhibit calcium-permeable cation channels and suppress the increase in intracellular calcium levels in vascular smooth muscle (33). Endotoxemia is accompanied by a generalized contractile defect secondary to a decrease in the influx of calcium into the cell and reduction of release of calcium from intracellular stores (34). The dominant mechanism involved in this impairment might be the activation of iNOS by endotoxin and the subsequent overproduction of NO (3). In the present model, simvastatin reduces NO production, leading to the recovery to vasoconstrictor agent effect during the endotoxemia. In fact, a significant decrease in mortality rates was recently reported among patients with bacteremia who were receiving statins (35).

In conclusion, this work shows that simvastatin reduces the production of NO and reverts the impaired vascular responsiveness observed in endotoxin shock. These results suggest a possible therapeutic benefit and provide insight into putative mechanisms involved in the pathophysiology of endotoxic shock. A comprehensive understanding of the cellular mechanisms involved in the induction of iNOS and cytokines should help to identify novel targets for therapeutic intervention of NO-mediated changes in inflammatory diseases. The potential role of statins as adjuvant drugs in the treatment of septic shock and other inflammatory states remains to be defined.

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ACKNOWLEDGMENTS

The authors are grateful to Marina Holanda, Maria Valci Silva, and Wagner L. Reis for their excellent technical support. This research was supported by grants from FAPESP, CNPq, and PRONEX.

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Keywords:

Sepsis; lipopolysaccharide; blood pressure; nitric oxide synthase; HMG-CoA reductase

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