Combination of sildenafil and simvastatin ameliorates monocrotaline-induced pulmonary hypertension in rats

Abstract

Sildenafil, a phosphodiesterase-5 inhibitor, and simvastatin, a cholesterol lowering drug, both have therapeutic effects on PAH; however, the combination of these drugs has not been tested in the treatment of PAH. The purpose of this study was to determine whether the combination of sildenafil and simvastatin is superior to each drug alone in the prevention of MCT-induced PAH. Phosphorylated Smad levels were decreased in lung tissue in MCT-injected rats, whereas ERK protein levels were increased. This indicates a possible role for an increase in mitogenic ERK activity in addition to decreased proapoptotic Smad signaling in the MCT model of PAH. Combination sildenafil and simvastatin treatment prevented the MCT-induced increases in right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH), exerted an anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC). Our results indicate that combination therapy with sildenafil and simvastatin attenuated the development of pulmonary hypertension more than either treatment alone.

Figure 1. MCT induces pulmonary hypertension and vascular remodeling 3 weeks after injection

mPAP, RVSP and RVH (RV/LV+S wet weight ratio) were measured in rats sacrificed at the times indicated after MCT injection (A). The week 0 group was injected with saline and measurements taken the subsequent day. Representative hematoxylin and eosin (H&E) staining of cross sections of resistance pulmonary arterioles is shown in (B). Scale bas = 10 μm. Based on digital analysis of these images, medial thickness (MT), MT percent (MT/2%) and percent wall area were calculated; the summarized data for the different groups are presented in (C). Data are presented as mean ± SD; n=8/group; * P<0.05 vs. 0 week control group; ** P<0.01 vs. 0 week control group.

Figure 2. MCT-induced PH is characterized by increased pulmonary artery smooth muscle cell proliferation

Lung sections from each rat in the different groups were fixed in formalin and embedded in paraffin. PCNA staining was used to indicate pulmonary artery smooth muscle cell proliferation over the time course indicated. Representative sections are shown in (A) and the summarized data in (B). Data are presented as mean ± SD; n=8/group; * P<0.05 vs. 0 week group.

Figure 3. MCT induces an increase in ERK protein levels and a decrease in activated (or phosphorylated) Smad1 protein in whole rat lung

mRNA and protein were collected from whole lung from rats injected with MCT at the indicated timepoints after injection. The week 0 group was injected with saline, and protein and mRNA was collected the next day. mRNA was used in RT-PCR to quantify ERK1 mRNA levels (A) using the primer listed in Table 1. This signal was normalized against GAPDH mRNA levels. Protein lysate resolved electrophoretically was transferred to a nitrocellulose membrane which was immunoblotted for phosphorylated Smad1 (p-Smad1) and ERK. Representative gels are shown in (Ba) and summarized data in (Bb and Bc). Protein signals were normalized to a-tubulin protein levels. Data are presented in arbitrary units (a.u.) as mean ± SD; n=5/group; ** P<0.01 vs. 0 week control group.

Figure 4. Time course of hemodynamic and molecular changes induced by MCT injection

Summarized data from Figures 1–3, including changes in pulmonary artery smooth muscle cell proliferation, ERK and p-Smad1 protein, right ventricular systolic pressure and remodeling, are presented over the 4 week time course after MCT injection. Dashed grey line represents control levels in normotensive saline-injected rats.

Figure 5. Combination Sildenafil and Simvastatin treatment is more effective at preventing MCT-induced pulmonary hypertension than either Sildenafil or Simvastatin alone

MCT induces pulmonary hypertension 4 weeks after injection as indicated by the increases in right ventricular systolic pressure (RVSP), RV/LV+S weight ratio, medial thickness (MT), MT/2% and wall area parameters in MCT-injected animals compared to sham (saline)-injected animals (A). MCT-injected rats were treated with sildenafil, simvastatin or combination sildenafil + simvastatin therapy and sacrificed after 4 weeks. Summarized data of RVSP and RV/LV+S weight ratio for the MCT-injected controls and the three treatment groups are shown in (B). Representative hematoxylin and eosin (H&E) staining of cross sections of resistance pulmonary arterioles are shown in (C). Scale bars = 10 μm. Based on digital analysis of these images, MT, MT/2% and percent wall area were calculated; the summarized data for the different groups are presented in (D). Data are presented as mean ± SD; n=8/group; ** P<0.01 vs. sham injected group (A) or vs. MCT-injected control group (B, D); ⁺P<0.05 vs. combination group.

Figure 6

Combination Sildenafil and Simvastatin treatment is more effective than single therapy at attenuating the MCT-induced increase in PASMC proliferation in pulmonary arterioles. Lung sections taken from each rat in each treatment group 4 weeks after MCT injection were fixed in formalin and embedded in paraffin. PCNA staining was used to indicate pulmonary artery smooth muscle cell proliferation in resistance pulmonary arterioles [external diameter: 50–150 μm] from sham-treated saline-injected, MCT-injected control, MCT+sildenafil, MCT+simvastatin and MCT+sildenafil+simvastatin groups. Representative histological sections are shown in (A) and the summarized data in (B). Treatment group data are presented as a percent of the MCT-injected control animals which was set at 100%. Scale bars = 10 μm. Data are presented as mean ± SD; n=8/group; ** P<0.01 vs. sham-injected (Ba) or vs. MCT-injected control (Bb); ⁺⁺ P<0.01 vs. combination group (Bb).