Tetrahydrobiopterin oral therapy recouples eNOS and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs

Abstract

We previously showed that newborn piglets who develop pulmonary hypertension during exposure to chronic hypoxia have diminished pulmonary vascular nitric oxide (NO) production and evidence of endothelial NO synthase (eNOS) uncoupling (Fike CD, Dikalova A, Kaplowitz MR, Cunningham G, Summar M, Aschner JL. Am J Respir Cell Mol Biol 53: 255-264, 2015). Tetrahydrobiopterin (BH₄) is a cofactor that promotes eNOS coupling. Current clinical strategies typically invoke initiating treatment after the diagnosis of pulmonary hypertension, rather than prophylactically. The major purpose of this study was to determine whether starting treatment with an oral BH₄ compound, sapropterin dihydrochloride (sapropterin), after the onset of pulmonary hypertension would recouple eNOS in the pulmonary vasculature and ameliorate disease progression in chronically hypoxic piglets. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received oral sapropterin starting on day 3 of hypoxia and continued throughout an additional 7 days of hypoxic exposure. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios (a measure of eNOS coupling), NO production, and superoxide (O₂^·-) generation. Although higher than in normoxic controls, pulmonary vascular resistance was lower in sapropterin-treated hypoxic piglets than in untreated hypoxic piglets. Consistent with eNOS recoupling, eNOS dimer-to-monomer ratios and NO production were greater and O₂^·- generation was less in pulmonary arteries from sapropterin-treated than untreated hypoxic animals. When started after disease onset, oral sapropterin treatment inhibits chronic hypoxia-induced pulmonary hypertension at least in part by recoupling eNOS in the pulmonary vasculature of newborn piglets. Rescue treatment with sapropterin may be an effective strategy to inhibit further development of pulmonary hypertension in newborn infants suffering from chronic cardiopulmonary conditions associated with episodes of prolonged hypoxia.

Keywords: nitric oxide signaling; pulmonary resistance arteries; sapropterin dihydrochloride; superoxide.

Fig. 1.

A: pulmonary artery pressure in control (normoxic) and chronically hypoxic piglets. High-dose (n = 12), but not low-dose (n = 4), treatment with oral BH₄ (sapropterin dihydrochloride) reduced pulmonary artery pressure in hypoxic piglets to values less than those in untreated (n = 8) hypoxic piglets. Pulmonary artery pressure was higher in all groups of hypoxic piglets than values in normoxic controls (n = 10). Moreover, pulmonary artery pressure was less in hypoxic piglets treated with the high- vs. low-dose oral BH₄. B: pulmonary vascular resistance (PVR) in control (normoxic) and chronically hypoxic piglets. High-dose (n = 12), but not low-dose (n = 4), treatment with oral BH₄ reduced PVR in hypoxic piglets to values less than those in untreated (n = 8) hypoxic piglets. PVR was higher in all groups of hypoxic piglets than values in normoxic controls (n = 10). Moreover, PVR was less in hypoxic piglets treated with the high- vs. low-dose oral BH₄. C: Fulton index (RV/LV+S) measurements of right ventricular mass in control (normoxic) and chronically hypoxic piglets. All three groups of chronically hypoxic piglets developed increased right heart mass compared with normoxia controls (normoxic controls, n = 10; untreated chronic hypoxia, n = 8; low-dose BH₄-treated chronic hypoxia, n = 4; high-dose BH₄-treated chronic hypoxia, n = 12). The high-dose, but not the low-dose, oral BH₄-treatment reduced right ventricular mass in chronically hypoxic piglets to values below those measured in untreated chronically hypoxic piglets. Values are means ± SE. Different from *control (normoxic), ⁺untreated chronic hypoxic, and !low-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.

Fig. 2.

Plasma NOx levels in control (normoxic) and chronically hypoxic piglets. Plasma NOx levels were greater in chronically hypoxic piglets receiving high-dose oral BH₄ (sapropterin dihydrochloride) treatment (n = 7) than in normoxic control piglets (n = 9), untreated chronically hypoxic piglets (n = 9), or in piglets receiving low-dose oral BH₄ treatment (n = 4). Values are means ± SE. Different from *control (normoxic), +untreated chronic hypoxic, and !low-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.

Fig. 3.

A: responses to the endothelium-dependent vasodilator, ACh, in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. Small pulmonary arteries from normoxic control piglets dilated to ACh (n = 8); ACh elicited a similar degree of constriction in small pulmonary arteries from the untreated group of hypoxic piglets (n = 4) and the low-dose oral BH₄ (sapropterin dihydrochloride)-treated group of hypoxic piglets (n = 4). ACh elicited less constriction in small pulmonary arteries from the high-dose oral BH₄-treated group of hypoxic piglets (n = 11) than measured in small pulmonary arteries from the other two groups of hypoxic piglets. B: responses to the NO donor, SNAP, in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. Responses to SNAP were similar for small pulmonary arteries from normoxic control piglets (n = 10) and high-dose oral BH₄-treated chronically hypoxic piglets (n = 11). Responses to SNAP were less for small pulmonary arteries from both the untreated group of hypoxic piglets (n = 6) and the low-dose oral BH₄-treated group of hypoxic piglets (n = 4) than for the other two groups of piglets. Values are means ± SE. Different from *control (normoxic), ⁺untreated chronic hypoxic, and !low-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.

Fig. 4.

A: BH₄ levels in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. BH₄ levels were greater in small pulmonary arteries from normoxic control piglets (n = 6) than in all groups of hypoxic piglets. BH₄ levels were greater in small pulmonary arteries from chronically hypoxic piglets receiving either low-dose (n = 4) or high-dose (n = 6) oral BH₄ treatment than in small pulmonary arteries from untreated chronically hypoxic piglets (n = 6). BH₄ levels were greater for pulmonary arteries from high-dose compared with low-dose oral BH₄-treated chronically hypoxic piglets. B: BH₂ levels in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. BH₂ levels were less in small pulmonary arteries from normoxic control piglets (n = 6) than in all groups of hypoxic piglets. BH₂ levels were less in small pulmonary arteries from chronically hypoxic piglets receiving either low-dose (n = 4) or high-dose (n = 6) oral BH₄ treatment than in small pulmonary arteries from untreated chronically hypoxic piglets (n = 6). BH₂ levels were less for pulmonary arteries from high-dose compared with low-dose oral BH₄-treated chronically hypoxic piglets. C: BH₄-to-BH₂ ratios (BH₄/BH₂) in small pulmonary arteries from control (normoxia) and chronically hypoxic piglets. BH₄/BH₂ were greater in small pulmonary arteries from normoxic control piglets (n = 6) than in all groups of hypoxic piglets. BH₄/BH₂ were greater in small pulmonary arteries from the high-dose oral BH₄-treated group of chronic hypoxic piglets (n = 6) than in the untreated group of chronic hypoxic piglets (n = 6). D: BH₄+BH₂ levels in small pulmonary arteries from control (normoxia) and chronically hypoxic piglets. BH₄+BH₂ ratios were greater in small pulmonary arteries from normoxic control piglets (n = 6) than in the untreated (n = 6) or low-dose oral BH₄-treated (n = 4) groups of chronic hypoxic piglets. Values are means ± SE. Different from *control (normoxic), ⁺untreated chronic hypoxic, and !low-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.

Fig. 5.

A: nitric oxide (NO) production by small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. NO production was greater for small pulmonary arteries from normoxic control piglets (n = 7) than all groups of hypoxic piglets. Small pulmonary arteries from both low- (n = 4) and high-dose (n = 6) oral BH₄ (sapropterin dihydrochloride)-treated groups of piglets had greater NO production than untreated (n = 7) hypoxic animals. B: superoxide production by small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. Superoxide production was less for small pulmonary arteries from normoxic control piglets (n = 6) than all groups of hypoxic piglets. Small pulmonary arteries from both low- (n = 4) and high-dose (n = 6) oral BH₄-treated groups of piglets had less superoxide production than untreated (n = 6) hypoxic animals. Superoxide production was less for pulmonary arteries from high-dose compared with low-dose oral BH₄-treated chronically hypoxic piglets. Superoxide production was unchanged by ex vivo l-NAME (10⁻⁴ M) treatment in small pulmonary arteries from control (normoxic) piglets (n = 6). Ex vivo treatment with l-NAME reduced superoxide generation in small pulmonary arteries from all three groups of hypoxic piglets. Values are means ± SE. Different from *control (normoxic), ⁺untreated chronic hypoxic, !low-dose BH₄-treated chronic hypoxic, and ĥigh-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.

Fig. 6.

A: representative Western blot for eNOS dimer-to-monomer ratios with corresponding densitometry shows that small pulmonary arteries from both low- and high-dose oral BH₄ (sapropterin dihydrochloride)-treated groups of chronic hypoxic piglets had greater eNOS dimer-to-monomer ratios than untreated hypoxic animals. In addition, eNOS dimer-to-monomer ratios were greater for pulmonary arteries from high-dose compared with low-dose oral BH₄-treated chronically hypoxic piglets. B: representative Western blot for eNOS expression with corresponding densitometry shows that eNOS expression was similar in small pulmonary arteries from all groups of piglets. Values are means ± SE. Different from *control (normoxic), ⁺untreated chronic hypoxic, and !high-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.

Fig. 7.

Representative Western blot for NOX1 with corresponding densitometry shows that small pulmonary arteries from both low- and high-dose oral BH₄ (sapropterin dihydrochloride)-treated groups of chronic hypoxic piglets had lower NOX1 expression than untreated hypoxic animals. In addition, NOX1 expression was less for pulmonary arteries from high-dose compared with low-dose oral BH₄-treated chronically hypoxic piglets. Different from *control (normoxic), ⁺untreated chronic hypoxic, and !high-dose BH₄-treated chronic hypoxic: P < 0.05; ANOVA with post hoc comparison test.