Rosuvastatin ameliorates the development of pulmonary arterial hypertension in the transgenic (mRen2)27 rat

Abstract

We have recently reported that transgenic (mRen2)27 rats (Ren2 rats) exhibit pulmonary arterial hypertension (PAH), which is, in part, mediated by oxidative stress. Since 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exhibit beneficial vascular effects independent of cholesterol synthesis, we hypothesized that rosuvastatin (RSV) treatment ameliorates PAH and pulmonary vascular remodeling in Ren2 rats, in part, by reducing oxidative stress. Six-week-old male Ren2 and Sprague-Dawley rats received RSV (10 mg x kg(-1) x day(-)1 ip) or vehicle for 3 wk. After treatment, right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. To evaluate treatment effects on pulmonary arteriole remodeling, morphometric analyses were performed to quantitate medial thickening and cell proliferation, whereas whole lung samples were used to quantitate the levels of 3-nitrotyrosine, superoxide, stable nitric oxide (NO) metabolites [nitrates and nitrites (NO(x))], and expression of NO synthase isoforms. In the Ren2 rat, RVSP is normal at 5 wk of age, PAH develops between 5 and 7 wk of age, and the elevated pressure is maintained with little variation through 13 wk. At 8 wk of age, left ventricular function and blood gases were normal in the Ren2 rat. Ren2 rats exhibited elevations in medial hypertrophy due to smooth muscle cell proliferation, 3-nitrotyrosine, NO(x), NADPH oxidase activity, and endothelial NO synthase expression compared with Sprague-Dawley rats. RSV significantly blunted the increase in RVSP but did not reduce MAP in the Ren2 rat; additionally, RSV significantly attenuated the elevated parameters examined in the Ren2 rat. These data suggest that statins may be a clinically viable adjunct treatment of PAH through reducing peroxynitrite formation.

Fig. 1.

Time course for the development of pulmonary hypertension in male transgenic (Ren2)27 rats (Ren2 rats). Right ventricular systolic pressures (RVSPs) of Sprague-Dawley (SD) and Ren2 rats are plotted versus age (n = 2–8 rats/age group). Values are means ± SE. Solid lines indicate best-fit regressions for 7- to 13-wk-old rats; dotted lines are estimates of RVSPs in Ren2 rats.

Fig. 2.

In vivo cine MRI analysis of left ventricular (LV) function in SD and Ren2 rats. Nine-week-old male Ren2 and SD rats were imaged by in vivo cine MRI. A and B: representative images of end systole (A) and end diastole (B) of a representative SD rat are shown. Movies for a representative SD and Ren2 rat are shown in Supplemental Fig. 1. C: no differences or similarities were found in the diastolic filling rate/end-diastolic volume (EDV). D: the ejection fraction between SD and Ren2 rats was significantly similar (power = 0.842) via a noninferiority test.

Fig. 3.

Lung expression of RNA transcripts of renin-angiotensin system components in SD and Ren2 rats. Angiotensin type 1 receptor (AT₁R; A), angiotensinogen (B), and angiotensin-converting enzyme (ACE; C) mRNA were examined via RT-PCR in 9-wk-old male SD and Ren2 rats. Bar graphs show band densities (means ± SE) for each transcript normalized to GAPDH (n = 5). Bands displayed above each bar graph are representative of each transcript and corresponding GAPDH. *Statistically significant difference between the two groups.

Fig. 4.

Effects of rosuvastatin (RSV) on pulmonary and systemic blood pressure in SD and Ren2 rats. RSV reduced RVSP (A) but not mean arterial pressure (MAP; B) in Ren2 rats. Con, control. Rats were divided into the following groups: SD control (SDC), RSV-treated SD (SDR), Ren2 control (R2C), and RSV-treated Ren2 (R2R). Significant differences were determined via ANOVA with a Tukey-Kramer post hoc test. *P < 0.05, SDC vs. R2C; §P < 0.05, SDC vs. R2R; †P < 0.05, SDR vs. R2R; ^ΩP < 0.05, SDR vs. R2C; ‡P < 0.05, R2C vs. R2R.

Fig. 5.

Microscopic analysis of small pulmonary arterioles from control and RSV-treated SD and Ren2 rats. A–E: Verhoeff vanGeison-stained SDC (A), SDR (B), R2C (C), and R2R (D) small pulmonary arterioles were quantified for medial (E) and lumen thickness; the medial area was plotted as a percentage of the total arteriole area (media + lumen, n ≥ 4). F–I: representative small pulmonary arterioles from SDC (F), SDR (G), R2C (H), and R2R (I) were imaged by confocal microscopy for α-smooth muscle actin (green) and endothelial cell von Willibrand factor (red). Scale bars = 50 μm. J–M: confocal imaging was also used to count 4′,6-diamidino-2-phenylindole-stained nuclei; representative images of small pulmonary arterioles from SDC (J), SDR (K), R2C (L), and R2R (M) are shown. The white lines in J–M trace the outer and inner medial boundaries. N: average total number of nuclei in a cross section of the medial layer, that is, between the white lines. O: average number of nuclei divided by the cross-sectional area of the medial layer (n = 5). Significant differences were determined via ANOVA with a Tukey-Kramer post hoc test. *P < 0.05, SDC vs. R2C; ^ΩP < 0.05, SDR vs. R2C; ‡P < 0.05, R2C vs. R2R.

Fig. 6.

Effects of RSV on pulmonary 3-nitrotyrosine (3-NTY) in SD and Ren2 rats. A–D: representative micrographs of lung sections from SDC (A), SDR (B), R2C (C), and R2R (D) rats stained for 3-NTY. E: average grayscale intensity (±SE) of the 3-NTY immunostaining (n = 4). F: representative lanes from a slot blot of whole lung homogenates probed for 3-NTY and normalized to total protein by amido black staining are shown over a histogram quantifying 3-NTY (n ≥ 4). Significant differences were determined via ANOVA with a Tukey-Kramer post hoc test. *P < 0.05, SDC vs. R2C; ^ΩP < 0.05, SDR vs. R2C; ‡P < 0.05, R2C vs. R2R.

Fig. 7.

Effects of RSV on ROS and nitric oxide (NO) production in the lungs of SD and Ren2 rats. A: lucigenin was used to measure superoxide (O₂⁻) from fresh lung samples. B: NADPH oxidase-specific assays were also preformed (n ≥ 7). C: the sum of stable NO metabolites [nitrate and nitrite (NO_x)] was measured in lung homogenates via the Griess reaction (n ≥ 5). Significant differences were determined via ANOVA with a Tukey-Kramer post hoc test. *P < 0.05, SDC vs. R2C; ^ΩP < 0.05, SDR vs. R2C; ‡P < 0.05, R2C vs. R2R.

Fig. 8.

Effects of RSV on pulmonary NO synthase (NOS) isoform expression in SD and Ren2 rats. A–E: pulmonary mRNA and protein were examined via RT-PCR (A, C, and E) and Westen blot analysis (B and D), respectively, for endothelial NOS (eNOS; A and B), inducible NOS (iNOS; C and D), and neuronal NOS (nNOS; E). All analyses were conducted on the same gel/blot; representative blots are shown above each histogram except for nNOS, which was barely detectable, and aligned to match the labels for the histogram (n ≥ 3). Significant differences were determined via ANOVA with a Tukey-Kramer post hoc test. *P < 0.05, SDC vs. R2C; #P < 0.05, SDC vs. SDR; §P < 0.05, SDC vs. R2R; ^ΩP < 0.05, SDR vs. R2C; ‡P < 0.05, R2C vs. R2R.