Inhibition of p38 MAPK reverses hypoxia-induced pulmonary artery endothelial dysfunction

Abstract

Hypoxia-induced endothelial dysfunction plays a crucial role in the pathogenesis of hypoxic pulmonary hypertension. p38 MAPK expression is increased in the pulmonary artery following hypoxic exposure. Recent evidence suggests that increased p38 MAPK activity is associated with endothelial dysfunction. However, the role of p38 MAPK activation in pulmonary artery endothelial dysfunction is not known. Sprague-Dawley rats were exposed to 2 wk hypobaric hypoxia, which resulted in the development of pulmonary hypertension and vascular remodeling. Endothelium-dependent relaxation of intrapulmonary vessels from hypoxic animals was impaired due to a reduced nitric oxide (NO) generation. This was despite increased endothelial NO synthase immunostaining and protein expression. Hypoxia exposure increased superoxide generation and p38 MAPK expression. The inhibition of p38 MAPK restored endothelium-dependent relaxation, increased bioavailable NO, and reduced superoxide production. In conclusion, the pharmacological inhibition of p38 MAPK was effective in increasing NO generation, reducing superoxide burden, and restoring hypoxia-induced endothelial dysfunction in rats with hypoxia-induced pulmonary hypertension. p38 MAPK may be a novel target for the treatment of pulmonary hypertension.

Fig. 1.

The measurement of hypoxia-induced pulmonary hypertension in rats. Immunostaining for α-smooth muscle actin demonstrated muscularization of pulmonary arteries (PAs) in sections from chronically hypoxic rats compared with normotensive rats (A). Quantification of thickening of the PA wall showed muscularization in chronically hypoxic lung sections immunostained for α-smooth muscle actin compared with normotensive lungs (B). Chronically hypoxic rats had increased right ventricular weight (RV)/left ventricular weight (LV) + septum weight (S) ratio (C) and hematocrit (D) compared with control animals. Results are expressed as means ± SE; n = 6 experiments. Scale bar = 50 μm. *P < 0.001 vs. control.

Fig. 2.

The effect of acute hypoxia on endothelium-dependent and -independent relaxation in rat PA. Cumulative concentration-response curves to relaxations induced by carbachol (A) and the nitric oxide (NO) donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (NOC-22; B) in isolated PAs are shown. The artery rings were precontracted with U-46619, and relaxation is expressed as percentage of the maximum U-46619-induced contraction. PA rings were examined in parallel under the following conditions: oxygenated control untreated (⧫), oxygenated and pretreated with 10 μM SB-203580 (◊), during hypoxia but with no other treatment (▴), during hypoxia and pretreated with 10 μM SB-203580 (▵), and oxygenated and pretreated with 10 μM anisomycin (▪). For carbachol (A), the significant differences were as follows: #P < 0.05, anisomycin vs. oxygenated control; *P < 0.01, hypoxia and pretreated with SB-203580 vs. hypoxia with no other treatment; and **P < 0.001, hypoxia vs. oxygenated control. Results are expressed as means ± SE; n = 6 experiments.

Fig. 3.

The effect of pulmonary hypertension induced by chronic hypoxia on endothelium-dependent and -independent relaxation in rat PA. Cumulative concentration-response curves to relaxations induced by carbachol (A) and the NO donor NOC-22 (B) in isolated PAs from either normoxic rats or chronically hypoxic rats are shown. The artery rings were precontracted with U-46619, and relaxation is expressed as percentage of the maximum U-46619-induced contraction. PA rings were examined in parallel under the following conditions: normoxic rat control untreated (⧫), chronic hypoxic rat with no other treatment (○), normoxic rat with the PA being pretreated with 10 μM SB-203580 (◊), and chronic hypoxic rat with the PA being pretreated with 10 μM SB-203580 (•). The significant differences for carbachol (A) were as follows: *P < 0.01, chronic hypoxic rat vs. chronic hypoxic rat with the artery ring pretreated with SB-203580; and **P < 0.001 chronic hypoxic rat vs. normoxic rat. The significant difference for NOC-22 (B) was as follows: #P < 0.05, chronic hypoxic rat vs. chronic hypoxic rat with the artery ring pretreated with SB-203580. Results are expressed as means ± SE; n = 6 experiments.

Fig. 4.

The effect of chronic hypoxia on endothelial NO synthase (eNOS) expression in small (<100 μm) and large (400–700 μm) PAs. A: sections of small and large PAs from normoxic (a and b) and chronically hypoxic (c and d) rats immunostained for eNOS. B: quantitative measurement of eNOS immunostaining in small and large PAs from normoxic and chronically hypoxic rats. C and D: representative Western blot (C) and corresponding densitometric analysis (D) showing of eNOS protein expression in control and chronically hypoxic PAs (400–700 μm). β-Actin was probed to confirm equal protein loading. Results are expressed as means ± SE; n = 6 experiments. Scale bar = 50 μm. *P < 0.001 vs. small PA control, #P < 0.01 vs. large PA control.

Fig. 5.

The effect of chronic hypoxia on endothelium-derived NO production in rat PA. Carbachol-stimulated NO production was measured with NO electrode in control and chronically hypoxic PA and following pretreatment with 10 μM SB-203580. Results are expressed as means ± SE; n = 6 experiments. #P < 0.001 vs. control; *P < 0.01 vs. chronic hypoxia.

Fig. 6.

A: representative confocal microscopic images of superoxide production in isolated PA sections stained with dihydroethidium (DHE, 10⁻⁵ M). B: the production of superoxide was quantified in arbitrary units of DHE fluorescence in control, chronically hypoxic, SB-203580-pretreated chronically hypoxic, and anisomycin-treated control PAs. Results are expressed as means ± SE; n = 4 to 6 experiments. *P < 0.001 vs. control; **P < 0.01 vs. chronic hypoxia.

Fig. 7.

The effects of hypoxia (acute and chronic) and anisomycin stimulation on p38 MAPK phosphorylation in the PA. Representative Western blot (A) and corresponding densitometric analysis (B) of phosphorylation of p38 MAPK in PA: 1) control, 2) 10 μM anisomycin, 3) acute hypoxia (35 mmHg O₂), 4) chronic hypoxia, 5) control + 10 μM SB-203580, 6) acute hypoxia + 10 μM SB-230580, and 7) chronic hypoxia + 10 μM SB-230580. Total p38 MAPK was probed to confirm equal protein loading. Results are expressed as means ± SE; n = 4 experiments. *P < 0.01 vs. control; **P < 0.001 vs. control; #P < 0.001 vs. acute hypoxia; †P < 0.001 vs. chronic hypoxia.