Protective role of the antidiabetic drug metformin against chronic experimental pulmonary hypertension

Abstract

Background and purpose: Pulmonary arterial hypertension (PAH) is associated with increased contraction and proliferation of pulmonary vascular smooth muscle cells. The anti-diabetic drug metformin has been shown to have relaxant and anti-proliferation properties. We thus examined the effect of metformin in PAH.

Experimental approach: Metformin effects were analysed in hypoxia- and monocrotaline-induced PAH in rats. Ex vivo and in vitro analyses were performed in lungs, pulmonary artery rings and cells.

Key results: In hypoxia- and monocrotaline-induced PAH, the changes in mean pulmonary arterial pressure and right heart hypertrophy were nearly normalized by metformin treatment (100 mg.kg(-1).day(-1)). Pulmonary arterial remodelling occurring in both experimental models of PAH was also inhibited by metformin treatment. In rats with monocrotaline-induced PAH, treatment with metformin significantly increased survival. Metformin increased endothelial nitric oxide synthase phosphorylation and decreased Rho kinase activity in pulmonary artery from rats with PAH. These effects are associated with an improvement of carbachol-induced relaxation and reduction of phenylephrine-induced contraction of pulmonary artery. In addition, metformin inhibited mitogen-activated protein kinase activation and strongly reduced pulmonary arterial cell proliferation during PAH. In vitro, metformin directly inhibited pulmonary artery smooth muscle cell growth.

Conclusions and implications: Metformin protected against PAH, regardless of the initiating stimulus. This protective effect may be related to its anti-remodelling property involving improvement of endothelial function, vasodilatory and anti-proliferative actions. As metformin is currently prescribed to treat diabetic patients, assessment of its use as a therapy against PAH in humans should be easier.

Figure 1

Metformin prevents chronic hypoxia-induced PAH. (A) Mean PAP, (B) right ventricular wall thickness, (C) pulmonary artery flow acceleration and (D) [RV/(LV + S)] ratio determined in control rats (normoxia), rats chronically treated for 21 days with metformin (100 mg·kg⁻¹·day⁻¹), rats exposed to hypoxia for 21 days, and metformin-treated rats exposed to hypoxia. (E) Mean PAP and (F) [RV/(LV + S)] ratio determined in rats exposed to hypoxia for 21 days non-treated (0) or treated with metformin doses ranging from 0.1 to 100 mg·kg⁻¹·day⁻¹. Dotted lines indicated the control values in normoxic rats (^#P < 0.001 vs. control, *P < 0.001 vs. untreated, n= 5–16). LV, left ventricle; PAH, pulmonary arterial hypertension; PAP, pulmonary arterial pressure; RV, right ventricle.

Figure 2

Metformin limits progression of PAH in hypoxic rats. (A) Mean PAP, (B) right ventricular wall thickness, (C) pulmonary artery flow acceleration and (D) [RV/(LV + S)] ratio determined in control rats (normoxia), rats treated for 7 days with metformin (100 mg·kg⁻¹·day⁻¹), rats exposed to hypoxia for 21 days (hypoxia) non-treated and treated with metformin for the last 7 days of hypoxia (^#P < 0.001 vs. control, ^§P < 0.05 vs. untreated, n= 5–9). LV, left ventricle; PAH, pulmonary arterial hypertension; PAP, pulmonary arterial pressure; RV, right ventricle.

Figure 3

Metformin prevents MCT-induced PAH. (A) Mean PAP and, (B) (RV/LV + S) ratio determined in control rats, rats chronically treated for 30 days with metformin (100 mg·kg⁻¹·day⁻¹), MCT-injected rats at day 30 post MCT injection, and MCT-injected rats at day 30 post MCT injection treated for 30 days by metformin (^#P < 0.001 vs. control, *P < 0.001 vs. untreated MCT-injected rats, n= 5–10). (C) Survival rates of metformin-treated MCT-injected rats (grey) versus untreated MCT-injected rats. LV, left ventricle; MCT, monocrotaline; PAH, pulmonary arterial hypertension; RV, right ventricle.

Figure 4

Metformin prevents PAH-associated pulmonary arterial wall remodelling. Representative sections of lung tissue (A), quantification of the relative thickness of small pulmonary artery (20–60 µm) wall (B) and percentage of distal muscularization of normally non-muscular arteries (C) in samples from control, hypoxic (21 days) and MCT-injected rats (day 30), untreated and treated by metformin (^#P < 0.001 vs. control, *P < 0.001 vs. untreated hypoxic rats, ^§P < 0.001 vs. untreated MCT-injected rats). MCT, monocrotaline; PAH, pulmonary arterial hypertension.

Figure 5

Metformin improves endothelial function and reduces pulmonary artery contractility. (A) ACC phosphorylation and expression, eNOS phosphorylation and expression, Rho kinase activity (assessed by the extent of phosphorylation of MYPT) and RhoA expression have been analysed by Western blot in pulmonary artery from control rats and rats exposed to hypoxia for 21 days, untreated and treated with metformin (three different samples representative of each condition). Equal protein loading was checked by examination of β-actin expression (B) Cumulative concentration-response curves for carbachol (CCh)-induced relaxation of phenylephrine (PhE; 1 µM)-contracted pulmonary artery rings from hypoxic (21 days) and metformin-treated hypoxic rats. Tension is expressed as percentage of the amplitude of the phenylephrine-induced contraction. (C) Cumulative concentration-response curves for the contraction induced by phenylephrine in pulmonary artery rings from hypoxic (21 days) and metformin-treated hypoxic rats. (D) Cumulative concentration-response curves for the contraction induced by phenylephrine in pulmonary artery rings from normoxic rats under control condition and in the presence of metformin (4 mM). ACC, acetyl CoA carboxylase; CCh, carbachol; eNOS, endothelial nitric oxide synthase; MYPT, myosin phosphatase target subunit.

Figure 7

Metformin inhibits PDGF-induced pulmonary artery smooth muscle cell proliferation. (A) Analysis by Western blot of the expression of PCNA in rat pulmonary artery smooth muscle cells, cultured under basal conditions or stimulated by PDGF (20 ng·mL⁻¹) for 24h, without or with metformin (4 mM). (B) Typical BrdU labelling of rat pulmonary artery smooth muscle cells under basal condition and stimulated by PDGF (20 ng·mL⁻¹) for 24h, without or with metformin (4 mM). Quantification of proliferation was expressed as the percentage of BrdU positive pulmonary arterial smooth muscle cells (^#P < 0.001 vs. control, *P < 0.001 vs. PDGF alone). BrdU, bromodeoxyuridine; PCNA, proliferating cell nuclear antigen; PDGF, platelet derived growth factor.

Figure 6

Metformin decreased pulmonary arterial cell proliferation associated with hypoxic PAH. (A) Representative PCNA staining and quantification of the percentage of PCNA-positive pulmonary arteries in lungs from normoxic rats (normoxia), hypoxic (21 days) and metformin-treated hypoxic rats (^#P < 0.001 vs. control, *P < 0.001 vs. untreated). (B) ERK, p38 and JNK phosphorylation and expression analysed by Western blot in pulmonary arteries from control rats, rats exposed to hypoxia for 21 days untreated and treated with metformin (three different samples representative of each condition). β-actin amounts were also assessed in each sample. JNK, C-Jun NH2-terminal kinase; PAH, pulmonary arterial hypertension; PCNA, proliferating cell nuclear antigen.