Inhibition of phosphodiesterase 2 augments cGMP and cAMP signaling to ameliorate pulmonary hypertension

Abstract

Background: Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin contributes to PH pathogenesis, and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP, which underpin the bioactivity of NO and prostacyclin. PDE5 inhibitors (eg, sildenafil) are licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH.

Methods and results: The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide- and treprostinil-evoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), trepostinil, inorganic nitrate (NO donor), or a PDE5 inhibitor. Proliferation of pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension was reduced by BAY 60-7550, an effect further enhanced in the presence of atrial natriuretic peptide, NO, and treprostinil.

Conclusions: PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity and is additive with prostacyclin analogues, PDE5 inhibitor, and NO. PDE2 inhibition represents a viable, orally active therapy for PH.

Keywords: cyclic nucleotide; natriuretic peptide; nitric oxide; phosphodiesterase inhibitor; pulmonary hypertension.

Figure 1

Concentration-response curves to atrial natriuretic peptide (ANP), spermine-NONOate (S-NO), and treprostinil in pulmonary arteries in the absence or presence of BAY 60-7550 (BAY, 0.1 μmol/L) isolated from normoxic (A, C, E; n=4-6) and hypoxic (2 weeks 10% O₂; B, D, F; n=3-8) rats. Data are presented as mean ± SEM. Curves are compared using two-way analysis of variance with repeated measures. *P<0.05, ***P<0.001 BAY vs. Control.

Figure 2

Concentration-response curves to atrial natriuretic peptide (ANP), spermine-NONOate (S-NO), and treprostinil in aorta in the absence or presence of BAY 60-7550 (BAY, 0.1 μmol/L) isolated from normoxic (A, C, E; n=5-13) and hypoxic (2 weeks 10% O₂; B, D, F; n=5-8) rats. Data are presented as mean ± SEM. Curves are compared using two-way analysis of variance with repeated measures. ***P<0.001 BAY vs. Control.

Figure 3

Right ventricular systolic pressure (RVSP; A & B), right ventricle/left ventricle+septum ratio (RV/(LV+S); C & D), and mean arterial blood pressure (MABP; E & F) in normoxic (Nx, n=6-18), vehicle-treated hypoxic (3 weeks 10% O₂; n=10-11) or BAY-60-7550-treated (n=11-14) hypoxic mice (A, C, E) and saline- (n=15), vehicle- (Veh; n=21) or BAY-60-7550-treated (n=15) mice exposed to bleomycin (1mg/kg; B, D, F). Muscularisation of pulmonary small arteries (G) and representative images showing pulmonary vessel muscularisation using anti-alpha smooth muscle actin staining (H; scale bar = 20 μm, red stain). All mice were treated with either veh (0.5% carboxymethylcellulose + 10% polyethylene glycol) or BAY 60-7550 (BAY, 10 mg/kg/day) by oral gavage for 3 weeks. Data are presented as mean ± SEM. Statistical analysis by one-way analysis of variance. *P<0.05, **P<0.01 vs. Veh, ^#P<0.05, ^{# #}P<0.01, ^{# # #}P<0.001 vs. Nx or Saline as determined by Bonferroni post-hoc comparisons (2 in total: Nx/Saline v Veh and Veh v BAY).

Figure 4

Right ventricular systolic pressure (RVSP; A & B), right ventricle/left ventricle+septum ratio (RV/(LV+S); C & D), and mean arterial blood pressure (MABP; E & F) in wildtype (WT) and natriuretic peptide receptor A (NPR-A) knockout (KO) mice, and WT animals receiving N^ω-nitro-l-arginine methyl ester (l-NAME; 100 mg/kg/day in the drinking water) in hypoxia (3 weeks 10% O₂) -induced PH. Mice were administrated vehicle (Veh; n=10 WT, n=8 KO, n=15 WT + L-NAME) or BAY 60-7550 (BAY; 10 mg/kg/day, n=10 WT, n=7 KO, n=10 WT + L-NAME). Data are presented as mean ± SEM. Statistical analysis by one-way analysis of variance. ^#P<0.05 vehicle-treated KO/l-NAME vs. vehicle-treated WT, *P<0.05, **P<0.01, ***P<0.001 vs. Veh, as determined by Bonferroni post-hoc comparisons (3 in total: WT Veh v KO Veh, and Veh v BAY in both WT & KO groups).

Figure 5

Right ventricular systolic pressure (RVSP; A,B, H), right ventricle/left ventricle+septum ratio (RV/(LV+S); C & D), and mean arterial blood pressure (MABP; E & F) in hypoxia-induced PH. In A, C, E mice were exposed to normoxia (Nx; n=6) or 3 weeks hypoxia (10% O₂) with drug treatment from day 1. In B, D, F, G & H animals were exposed to normoxia (Nx; n=15) or 5 weeks hypoxia (10% O₂) with drug treatment at day 14. Hypoxic mice received one of vehicle (Veh; n=6-15), BAY 60-7550 (BAY, 10mg/kg/day, n=8-12; 30 mg/kg/day, n=10; or 100 mg/kg/day, n=9), ecadotril (E; 60mg/kg/day; n=6-10) or a combination of both (at the same doses; n=8-9). Plasma cGMP concentrations from animals exposed to 5 weeks hypoxia (10% O₂; G). Data are presented as mean ± SEM. Statistical analysis by one-way analysis of variance. *P<0.05, **P<0.01 vs. Veh, ^{# #}P<0.01, ^{# # #}P<0.001 vs. Nx as determined by Bonferroni post-hoc comparisons (4 in total: Nx v Veh, and Veh v each treatment group).

Figure 6

Right ventricular systolic pressure (RVSP; A & B), right ventricle/left ventricle+septum ratio (RV/(LV+S); C & D), mean arterial blood pressure (MABP; E & F), and plasma cAMP concentration (G) in mice exposed to normoxia (Nx) or 5 weeks hypoxia (10% O₂) with drug treatment from day 14. Animals received vehicle (Veh; n=15), sham-surgery plus vehicle (sham, n=4), BAY-60-7550 (BAY; 10 mg/kg/day; n=8), inorganic nitrate (N; 150mg/kg/day; n=7), treprostinil (T; 20mg/kg/day; n=8) or a combination of BAY plus nitrate or BAY plus treprostinil (at the same doses). Data are presented as mean ± SEM. Statistical analysis by one-way analysis of variance. *P<0.05, vs. Veh, ^{# # #}P<0.001 vs. Nx as determined by Bonferroni post-hoc comparisons (4 in total: Nx v Veh/Sham, and Veh/Sham v each treatment group).

Figure 7

PDE2A mRNA (A, D) and protein (B) expression, and activity (defined as 5′-GMP formation inhibitable by BAY 60-7550 [1μmol/L]; C, E), in isolated pulmonary arteries from normoxic (Nx) and hypoxic (Hx) rats (2 weeks 10% O₂; n=3-8; A, B, C) and pulmonary vascular smooth muscle cells from normal individuals and PAH patients (n=5; D, E). Proliferation of human pulmonary artery smooth muscle cells from PAH patients in the absence (Control; n=9) or presence of BAY 60-7550 (BAY; 1μmol/L; n=9), atrial natriuretic peptide (ANP; 1μmol/L; n=4), DETA-NONOate (DETA-NO; 10μmol/L; n=5), treprostinil (T; 3μmol/L; n=4) or combinations thereof (at the same concentrations; F, G, H). Data are shown as mean ± SEM. Statistical analysis by unpaired Students T-test (A-E) or two-way analysis of variance (F, G, H). *P<0.05, **P<0.01, ***P<0.001 versus control/normoxia/normal, ^#P<0.05, ^{# #}P<0.01 vs. BAY, ^≠P<0.05 versus treprostinil.

Figure 8

Right ventricular systolic pressure (RVSP; A), right ventricle/left ventricle+septum ratio (RV/(LV+S); B), and mean arterial blood pressure (MABP; C) in normoxic mice (Nx; n=6) or hypoxic (5 weeks 10% O₂) animals treated with vehicle (Veh; n=13), BAY 60-7550 (BAY, 10 mg/kg/day; n=9), sildenafil (S; 30 mg/kg/day; n=9) or a combination of sildenafil plus BAY (at the same doses; S + BAY, n=10). Concentration-responses curves to BAY 60-7550 (D) and atrial natriuretic peptide (ANP; E) in isolated pulmonary arteries from hypoxic (2 weeks 10% O₂) rats. Proliferation of human pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension in the absence (Control; n=9) and presence of BAY 60-7550 (1μmol/L; n=9), sildenafil (3umol/L; n=3) or a combination thereof (at the same concentrations; n=3) (F). Data are shown as mean ± SEM. Statistical analysis by one-way analysis of variance with Bonferroni post-hoc comparisons (4 in total: Nx v Veh, and Veh v each treatment group; A, B, C) or two-way analysis of variance (D, E, F). *P<0.05, ***P<0.001 versus vehicle control, ^≠P<0.05 vs. BAY, ^#P<0.05, ^###P<0.001 versus sildenafil + BAY.