Evidence that simvastatin prevents induction of nitric oxide synthase by LPS in the porcine isolated coronary artery

Introduction

Several retrospective studies suggest that prior use of statins can reduce hospital mortality in patients diagnosed with either bacteraemia or sepsis. In an accompanying abstract we demonstrated that pre-treatment with simvastatin prevents LPS-induced hyporesponsiveness of the porcine isolated coronary artery (PCA); an observation consistent with the clinical data. Although this effect of simvastatin is qualitatively similar to that of a known inhibitor of inducible nitric oxide synthase (1400 W), it unlikely that direct inhibition of the enzyme is implicated. We have investigated whether the beneficial effect of simvastatin on LPS-induced changes in the PCA involves alteration in the induction of nitric oxide synthase (iNOS).

Methods

Segments of the PCA were dissected from hearts and incubated (2 × 5 mm) in DMEM at 37°C in the presence of an antibiotic mixture (60 μg/ml benzylpenicillin, and 20 μg/ml streptomycin sulphate), with or without 1 μg/ml LPS, 3 μM simvastatin or a combination of the two (simvastatin added 60 minutes before LPS). The medium also contained 1 mM L-arginine. After 24 hours, segments were removed and weighed (mg wet weight). The nitrite/nitrate content (nmol) of the bathing medium was determined by spectrophotometry using the Griess reaction. In a separate experiment, segments were prepared for immunohisto-chemical determination of the presence of iNOS and CD31. Differences between mean values were assessed by ANOVA (post-hoc Dunnett test).

Results

Under control conditions the coronary artery segments produced 25.9 ± 3.7 nmol/mg wet weight nitrite/nitrate (n = 8) over 24 hours. Exposure to 1 μg/ml LPS caused a sevenfold increase in nitrite/nitrate production (199.0 ± 40.6 nmol/mg wet weight, n = 8). Although 3 μM simvastatin did not affect basal nitrite/nitrate production, it inhibited the response to LPS (35.6 ± 7.4 nmol/mg wet weight nitrite/nitrate) by 94.7 ± 5.7% (n = 8). Immunohistochemical assessment of four arteries revealed the presence of CD31 on endothelial cells under control conditions. Exposure to 1 μg/ml LPS was associated with an increase in endothelial CD31 and the appearance of iNOS in the adventitia. Co-incubation of segments with 3 μM simvastatin and LPS produced a profile similar to that of control segments (CD31-positive, iNOS-negative, n = 4).

Conclusion

Simvastatin suppressed the induction of nitric oxide caused by LPS and the associated increase in nitrite/nitrate production. This finding helps to explain our observation that simvastatin prevented LPS-induced hyporesponsiveness of the coronary artery, and is also consistent with clinical studies suggesting that prior use of statins may afford protection against bacterial sepsis.