Smooth muscle Rac1 contributes to pulmonary hypertension

Abstract

Background and purpose: Pulmonary hypertension (PH) is a multifactorial chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signalling in PA plays a major role in PH. We recently described the involvement of the Rho protein Rac1 in the control of systemic blood pressure through its involvement in NO-mediated relaxation of arterial smooth muscle cell (SMC). The aim of this study was to analyse the role of SMC Rac1 in PH.

Experimental approach: PH is induced by exposure of control and SMC Rac1-deficient (SM-Rac1-KO) mice to chronic hypoxia (10% O₂ , 4 weeks). PH is assessed by the measurement of RV systolic pressure and hypertrophy. PA reactivity is analysed by isometric tension measurements. PA remodelling is quantified by immunofluorescence in lung sections and ROS are detected using the dihydroethidium probe and electronic paramagnetic resonance analysis. Rac1 activity is determined by immunofluorescence.

Key results: Rac1 activation in PA of hypoxic mice and patients with idiopathic PH. Hypoxia-induced rise in RV systolic pressure, RV hypertrophy and loss of endothelium-dependent relaxation were significantly decreased in SM-Rac1-KO mice compared to control mice. SMC Rac1 deletion also limited hypoxia-induced PA remodelling and ROS production in pulmonary artery smooth muscle cells (PASMCs).

Conclusion and implications: Our results provide evidence for a protective effect of SM Rac1 deletion against hypoxic PH. Rac1 activity in PASMCs plays a causal role in PH by favouring ROS-dependent PA remodelling and endothelial dysfunction induced by chronic hypoxia.

Keywords: Rac1; pulmonary hypertension; smooth muscle.

FIGURE 1

Smooth muscle (SM) Rac1 deletion prevents chronic hypoxia‐induced increase in right ventricular systolic pressure and right ventricular remodelling. (a) Representative images of Rac‐GTP immunofluorescence (red) in cryosections of lung from SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks to normoxia or hypoxia. Nuclei are detected by DAPI staining (blue) and smooth muscle by SM22α immunofluorescence labelling (green). Scale bar = 80 μm. (b) Quantification of Rac‐GTP labelling fluorescence intensity in SMCs. (c) Right ventricular systolic pressure (RVSP, left panel), left ventricular systolic pressure (LVSP, middle panel) and Fulton index (RV/(LV + S); right panel) in SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks to normoxia or hypoxia. Data are expressed as mean ±SEM. *P < 0.05

FIGURE 2

Smooth muscle Rac1 deletion prevents hypoxia‐induced reduction of endothelium‐dependent relaxation. Contractile responses to potassium chloride (KCl), 5‐HT and endothelin‐1 (ET‐1), and relaxation of phenylephrine (PhE)‐induced tension (1 μM) by acetylcholine (ACh) and S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP) in pulmonary artery (PA) from SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks in normoxia or hypoxia (Normoxia SM‐Rac1^Lox/Lox n = 9 mice; Normoxia SM‐Rac1‐KO n = 7 mice; hypoxia SM‐Rac1^Lox/Lox n = 11 mice; hypoxia SM‐Rac1‐KO n = 5 mice). Data are expressed as mean ± SEM. *P < 0.05

FIGURE 3

Smooth muscle Rac1 deletion prevents pulmonary artery (PA) remodelling induced by hypoxia. (a) Representative images of SM22alpha labelling on haematoxylin and eosin‐stained sections of lung from SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks to normoxia or hypoxia. Scale bar = 80 μm. (b and c) quantification of the number of muscularized PA (b) and wall thickness (c). Data are expressed as mean ± SEM. *P < 0.05

FIGURE 4

Smooth muscle Rac1 deletion prevents hypoxia‐induced ROS production in pulmonary artery (PA) and pulmonary artery smooth muscle cell (PASMC) proliferation. (a) Representative images of dihydroethidium (DHE) staining of lung sections from SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks to normoxia or hypoxia. Scale bar = 20 μm. L, lumen; P, parenchyma; * indicates DHE positive cell. (b) Quantification of DHE positive cells in PA (left panel) and in lung parenchyma (right panel). Data are expressed as mean ± SEM. *P < 0.05. (c) Detection of O₂ ⁻ by electronic paramagnetic resonance (right panel) and xanthine oxidase activity in lungs from SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks to normoxia or hypoxia (left panel). Data are expressed as mean ± SEM. *P < 0.05. (d) Analysis by real‐time PCR of the relative expression of HIF1a, eNOS (NOS3), NOX1 and NOX2 in pulmonary arteries from SM‐Rac1^lox/lox and SM‐Rac1‐KO mice exposed for 4 weeks to hypoxia compared to SM‐Rac1^lox/lox exposed for 4 weeks to normoxia. Data are expressed as mean ± SEM. (e) Representative images of DHE (red) and DAPI (blue) staining of PASMCs cultured in normoxic or hypoxic condition, and quantification of DHE positive cells and PASMCs proliferation. When indicated, cells were treated with EHT1864 (10⁻⁵ M) or 1.5‐mM tempol. Scale bar = 10 μm. Data are expressed as mean ± SEM. *P < 0.05

FIGURE 5

Rac1 is overactivated in pulmonary artery (PA) of idiopathic pulmonary arterial hypertension (iPAH) patients. (a) Representative confocal images of Rac‐GTP immunofluorescence (blue) in cryosections of lung from control and iPAH patients. Arteries was detected by elastin autofluorescence (red), smooth muscle by SM22α immunofluorescence (green), and Rac1 activity by Rac‐GTP immunofluorescence (blue). Scale bar = 80 μm. (b) Quantification of smooth muscle (SM) layer thickness and Rac1 activity (Rac‐GTP labelling fluorescence intensity) in lung sections from control and iPAH patients. (c) Magnification corresponding to the white square in (a) and spatial profile of fluorescence intensity for indicated fluorescence channels for the white line positioned on the image. Data are expressed as mean ± SEM