Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase

Abstract

Background: Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO(3)(-)) and nitrite (NO(2)(-)) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity.

Methods and results: Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol.

Conclusions: These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH.

Figure 1

(A) Right ventricular systolic pressure (RVSP) and (B) right ventricle:left ventricle plus septum ratio (RV/LV+S) in normoxic (control) WT mice and WT animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrite (NO₂⁻; 0.6mM) or inorganic nitrate (NO₃⁻; 15mM or 45mM). ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=17-25 for each group.

Figure 2

(A) % Muscularized vessels in normoxic (control) WT mice and WT animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrite (NO₂⁻; 0.6mM) or inorganic nitrate (NO₃⁻; 15mM or 45mM). ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=17-25 for each group. (B) Representative light-microscopic images (80× magnification) of pulmonary arteries from normoxic, hypoxic and nitrate (45mM)-treated animals; the hypoxic vessels exhibit a marked muscularization that is reduced in the presence of nitrate (45mM).

Figure 3

Vessel wall thickness in (A) subpopulations and B) all arteries in the pulmonary circulation of normoxic (control) WT mice and WT animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrite (NO₂⁻; 0.6mM) or inorganic nitrate (NO₃⁻; 15mM or 45mM). ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=120 for each group.

Figure 4

Plasma (A) nitrite (NO₂⁻), (B) nitrate (NO₃⁻) and (C) cGMP concentrations in normoxic (control) WT mice and WT animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrite (NO₂⁻; 0.6mM) or inorganic nitrate (NO₃⁻; 15mM or 45mM). ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=12-16 for each group.

Figure 5

Total lung and urinary nitrite (NO₂⁻) (A & C) and nitrate (NO₃⁻) (B & D) concentrations in normoxic (control) WT mice and WT animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrite (NO₂⁻; 0.6mM) or inorganic nitrate (NO₃⁻; 15mM or 45mM). *P<0.05 versus hypoxia. n=12-16 for each group.

Figure 6

Right ventricular systolic pressure (RVSP) and right ventricle:left ventricle plus septum ratio (RV/LV+S) in normoxic (control) WT mice and WT animals exposed to 5 weeks hypoxia (10% O₂) (A & B) or bleomycin (1mg/kg) (C & D) in the absence and presence of inorganic nitrate (NO₃⁻; 45mM). Inorganic nitrate was administered at weeks 3-5 in the hypoxic studies and weeks 0-3 in the bleomycin model. ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=6-10 for each group.

Figure 7

Right ventricular systolic pressure (RVSP) (A), right ventricle:left ventricle plus septum ratio (RV/LV+S) (B), and plasma nitrite (NO₂⁻) (C) and nitrate (NO₃⁻) (D) concentrations in eNOS KO normoxic mice and eNOS KO animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrite (NO₂⁻; 0.6mM) or inorganic nitrate (NO₃⁻; 45mM). ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=6-12 for each group.

Figure 8

Right ventricular systolic pressure (RVSP) (A), right ventricle:left ventricle plus septum ratio (RV/LV+S) (B) in normoxic mice and animals exposed to 3 weeks hypoxia (10% O₂) in the absence and presence of inorganic nitrate (NO₃⁻; 45mM) and allopurinol (1mM). ^#P<0.05 v normoxia; *P<0.05 versus hypoxia. n=6-12 for each group. Lung nitrite reductase activity in response to sodium nitrite (10-300μM) in the absence and presence of L-NMA (300μM) or allopurinol (100μM) (C). *P<0.05 versus control across entire curve. n=3-5 for each group. Correlation between lung nitrite (NO₂⁻) and plasma cGMP concentrations [D]. n=34.