Simvastatin ameliorates established pulmonary hypertension through a heme oxygenase-1 dependent pathway in rats

Abstract

Background: Simvastatin has been shown to ameliorate pulmonary hypertension by several mechanisms in experimental animal models. In this study, we hypothesized that the major benefits of simvastatin in pulmonary hypertension occur via the heme oxygenase-1 pathway.

Methods: Simvastatin (10 mg/kgw/day) was tested in two rat models of pulmonary hypertension (PH): monocrotaline administration and chronic hypoxia. The hemodynamic changes, right heart hypertrophy, HO-1 protein expression, and heme oxygenase (HO) activity in lungs were measured in both models with and without simvastatin treatment. Tin-protoporphyrin (SnPP, 20 micromol/kg w/day), a potent inhibitor of HO activity, was used to confirm the role of HO-1.

Results: Simvastatin significantly ameliorated pulmonary arterial hypertension from 38.0 +/- 2.2 mm Hg to 22.1 +/- 1.9 mm Hg in monocrotaline-induced PH (MCT-PH) and from 33.3 +/- 0.8 mm Hg to 17.5 +/- 2.9 mm Hg in chronic hypoxia-induced PH (CH-PH) rats. The severity of right ventricular hypertrophy was significantly reduced by simvastatin in MCT-PH and CH-PH rats. Co-administration with SnPP abolished the benefits of simvastatin. Simvastatin significantly increased HO-1 protein expression and HO activity in the lungs of rats with PH; however co-administration of SnPP reduced HO-1 activity only. These observations indicate that the simvastatin-induced amelioration of pulmonary hypertension was directly related to the activity of HO-1, rather than its expression.

Conclusion: This study demonstrated that simvastatin treatment ameliorates established pulmonary hypertension primarily through an HO-1-dependent pathway.

Figure 1

Schematic outline of the experimental protocol. Monocrotaline-induced pulmonary hypertension (MCT-PH) was established by subcutaneous administration on day 1 (60 mg/kgw), and chronic hypoxia-induced pulmonary hypertension (CH-PH) was established by treatment in a hypobaric chamber (380 Torr, intermittent exposure) for 24 days. Simvastatin was given by intraperitoneal injection (10 mg/kgw/day) on day 21~23, and SnPP (20 μmol/kgw/day) was injected alone or with simvastatin simultaneously on days 21~23. All animals underwent experimental surgery on day 24.

Figure 2

Effects of simvastatin and SnPP on pulmonary arterial pressure in MCT-PH (A) and CH-PH (B) rats. Rats that received simvastatin (MS and HS groups) had significantly decreased pulmonary arterial pressure compared with non-simvastatin treated PH rats (M and H groups). Co-treatment with simvastatin and SnPP (MSP and HSP groups) failed to relieve the high pulmonary arterial pressure. Rats that received SnPP alone (MP and HP groups) maintained high pulmonary arterial pressure. Horizontal bars indicate groups that differed significantly from each other, * p < 0.05. (N = normal control, M = monocrotaline treatment, MS = monocrotaline + simvastatin treatment, MSP = monocrotaline + simvastatin + SnPP treatment, MP = monocrotaline + SnPP treatment, H = chronic hypoxia, HS = chronic hypoxia + simvastatin treatment, HSP = chronic hypoxia + simvastatin + SnPP treatment, HP = chronic hypoxia + SnPP treatment).

Figure 3

The effects of simvastatin and SnPP on right ventricular hypertrophy in MCT-PH (A) and CH-PH (B) rats. Simvastatin treatment (MS and HS groups) prevented progression and improved established right ventricular hypertrophy compared with the non-simvastatin treated PH rats (M and H groups). Rats that received SnPP alone (MP and HP groups) or received both simvastatin and SnPP (MSP and HSP groups) developed severe right ventricular hypertrophy. The index of right ventricular hypertrophy is given as the ratio of right ventricle to (left ventricle plus septum) weight [RV/(LV+S)]. Horizontal bars indicate groups that differed significantly from each other, * p < 0.05. (N = normal control, M = monocrotaline treatment, MS = monocrotaline + simvastatin treatment, MSP = monocrotaline + simvastatin + SnPP treatment, MP = monocrotaline + SnPP treatment, H = chronic hypoxia, HS = chronic hypoxia + simvastatin treatment, HSP = chronic hypoxia + simvastatin + SnPP treatment, HP = chronic hypoxia + SnPP treatment).

Figure 4

Western blot analysis of HO-1 protein expression in MCT-PH (A) and CH-PH (B) rat lung. Densitometric quantification revealed a significant increase in expression of HO-1 protein in the lungs of rats that received simvastatin and SnPP treatment. Densitometric units were normalized to β-actin and then divided by N group results. (N = normal control, NS = normal + simvastatin treatment, M = monocrotaline treatment, MS = monocrotaline + simvastatin treatment, MSP = monocrotaline + simvastatin + SnPP treatment, MP = monocrotaline + SnPP treatment, H = chronic hypoxia, HS = chronic hypoxia + simvastatin treatment, HSP = chronic hypoxia + simvastatin + SnPP treatment, HP = chronic hypoxia + SnPP treatment).

Figure 5

Immunohistochemical staining of the distribution of HO-1 in rat lung. Simvastatin treatment led to marked HO-1 immunoreactivity around pulmonary vascular sites. (A) normal control, (B) MCT-PH rats, (C) CH-PH rats, (D) normal rats treated with simvastatin, (E) simvastatin treated, MCT-PH rats, (F) simvastatin treated, CH-PH rats. Magnification: 400×. Bars represent 20 μm.

Figure 6

Changes in HO activity in MCT-PH (A) and CH-PH (B) rat lungs. Simvastatin treatment (MS and HS groups) significantly increased HO activity compared with non-simvastatin treatment in PH rats (M and H groups). Co-administration of simvastatin and SnPP (MSP and HSP groups) led to significantly less HO activity than in simvastatin-treated rats (MS and HS groups). The bilirubin concentration in each group was normalized to that of normal controls (Group N, value = 1.0). Horizontal bars indicate groups that differed significantly from each other, * p < 0.05.