Angiotensin-Converting Enzyme 2 Activator Ameliorates Severe Pulmonary Hypertension in a Rat Model of Left Pneumonectomy Combined With VEGF Inhibition

Figures

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  Figure 1. The design of animal study. In the early treatment protocol, there were sham-operated rats, SU5416 combined with left pneumonectomy rats (SuPNx42), and SuPNx42 rats given with 15 mg DIZE/kg/day from day 1 to day 42 (SuPNx42/DIZE1−42). In the late treatment protocol, there were sham-operated rats (sham42), SuPNx42, and a SuPNx42 rats given with 15 mg DIZE/kg/day from day 29 to day 42 (SuPNx42/DIZE29−42). SuPNx, Su5416 with left pneumonectomy; DIZE, diminazene aceturate.

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  Figure 2. Comparison of (A) right ventricle systolic pressure (RVSP), (B) Fulton index, the weight ratio of right ventricle to left ventricle plus septum (RV/LV+S), (C) the lung weight and (D) the body weight among the sham42, SuPNx42, SuPNx42/DIZE1−42 and SuPNx42/DIZE29−42 groups. RVSP levels were significantly higher in the SuPNx42 group (45.17 ± 4.62 mmHg) than in sham-operated rats (18.37 ± 1.36 mmHg, p < 0.001). Both DIZE treatment groups (SuPNx42/DIZE1−42 and SuPNx42/DIZE29−42) showed significant decreases in RVSP when compared with that of the SuPNx42 rats (early, 25.10 ± 1.38 mmHg, p < 0.01; late 31.23 ± 4.06 mmHg, p < 0.05). The Fulton index was significantly increased in the SuPNx42 group (0.55 ± 0.06) than in sham-operated group (0.35 ± 0.04, p < 0.05), but no significant changes were observed in the SuPNx42/DIZE1−42 and SuPNx42/DIZE29−42 groups when compared with the SuPNx42 group. There was no significant change in either the lung or body weight in all groups. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (n = 6–7).

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  Figure 3. (A) The plasma brain natriuretic peptide (BNP) and (B) RV BNP levels. The plasma BNP level was significantly higher in the SuPNx42 rats than in sham-operated rats (130.2 ± 19.76 vs. 59.50 ± 7.74 pg/ml; P < 0.001). Treatment with DIZE for 42 days significantly lowered the plasma BNP levels when compared with that of the vehicle-treated group (31.14 ± 2.83 vs. 130.2 ± 19.76 pg/ml; P < 0.05). There was a trend that the SuPNx42/DIZE29−42 rats had a decreased plasma BNP level when compared with the SuPNx42 rats, but the difference was not statistically significant. The RV BNP was significantly higher in SuPNx42 rats than that of the sham42 rats (103.4 ± 10.01 vs. 31.05 ± 2.57 pg/ml; P < 0.0001). Both DIZE treatment groups had significantly lowered RV BNP than in the SuPNx42 rats (early: 46.82 ± 6.62 pg/ml vs. 103.4 ± 10.01 pg/ml, P < 0.0001; late: 61.83 ± 5.10 pg/ml vs. 103.4 ± 10.01 pg/ml, P < 0.001). Values represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (n = 6–8).

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  Figure 4. The representative results of Hematoxylin-eosin stating of lung tissue (A), Verhoeff-van Gieson staining (B), and double immunohistochemical staining of smooth muscle actin (green) and PCNA (brown) (C). (D) The ratio of medial wall thickness of pulmonary arterioles. The value for the sham42 group was set at 1.0. The muscular layer of pulmonary arterioles (50–100 μm in diameter) was significantly thicker in the SuPNx42 rats than in sham42, SuPNx42/DIZE1−42, but not SuPNx42/DIZE29−42 rats (n = 6). (E) the vascular occlusion score (VOS). The VOS of early treatment group (SuPNx42/DIZE1−42) was decreased compared to SuPNx42 (1.27 ± 0.74 vs. 1.07 ± 0.56, n = 6) (magnification, 200x. Values represent the mean ± SEM. *P < 0.05, **P < 0.01.

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  Figure 5. (A) A representative Western blot of eNOS, ACE, ACE2, and the MAS receptor in sham42, SuPNx42, SuPNx42/DIZE1−42, and SuPNx42/DIZE29−42 rats. Representative results from one of three separate Western blotting experiments are shown. (B) The normalized eNOS/actin ratio revealed the expression of eNOS was higher in the SuPNx42/DIZE1−42 rats than in SuPNx42 rats. (C) The normalized ratio of ACE/actin showed that the expression of ACE was higher in SuPNx42 rats than in sham42 rats. DIZE administration decreased ACE expression in the early treatment (SuPNx42/DIZE1−42) and the late treatment (SuPNX42/DIZE29−42) groups. (D) The normalized ratio of ACE2/actin revealed that an increase in the early treatment group (SuPNX42/DIZE1−42) compared to the SuPNx42 rats. No significant differences were found among the sham42, SuPNx42, and SuPNX42/DIZE29−42 groups. (E) The normalized MAS/actin ratios were not changed in the all groups. The value for the sham42 group was set at 1.0 in (B–E). Values represent the mean ± SEM *P < 0.05, **P < 0.01 (n = 6–7).

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  Figure 6. (A) The levels of RV Ang-(1-7). The level of RV Ang-(1-7) was significantly higher in the SuPNx42 /DIZE1−42 and SuPNx42 /DIZE29−42 rats than in SuPNx42 rats. (B) The levels of pulmonary Ang-(1-7). The expression of pulmonary Ang-(1-7) was significantly higher in SuPNx42 /DIZE1−42 rats than in SuPNx42 rats. Values represent the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 (n = 6–7).