Pulmonary hypertension (PH) is a fatal and incurable disease defined by an elevated resting mean pulmonary artery pressure (mPAP) > 20 mmHg measured by right heart catheterization (Simonneau et al., 2019). This increase in mPAP can result from pre-capillary (arterial) or post-capillary (cardiac/venous) pathophysiological mechanisms. The current clinical classification divides PH into five groups based on pathophysiology and clinical features in order to optimize therapeutic approaches, predict patient outcomes and facilitate research strategies (Simonneau et al., 2019). Nevertheless, current evidence suggests that endothelial dysfunction and the progressive accumulation of vascular cells in the wall of small to medium-sized distal pulmonary arteries are two common denominators of these different forms of PH (Humbert et al., 2019). However, the pathogenic mechanism underlying these processes have not yet been elucidated.

AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor that plays a central role in the maintenance of endothelial integrity and vascular homeostasis (Salt and Hardie, 2017). Metformin, an AMPK activator, is the first line treatment for type 2 diabetes and has been on the market in Europe for over 60 years with few notable side effects (Deng et al., 2020). Recent studies have reported that metformin protected against the development of experimental PH in several rodent models (Agard et al., 2009; Dean et al., 2016; Liu et al., 2019; Omura et al., 2016; Ranchoux et al., 2019; Yoshida et al., 2020; Zhai et al., 2018; Zhang et al., 2018). Knockout of AMPK in mice led to reduced phosphorylation of endothelial NO synthase (eNOS) and aggravated features of hypoxia-induced PH (Omura et al., 2016), while metformin enhanced eNOS phosphorylation and reversed chronic hypoxia-induced PH in rats (Agard et al., 2009). Even though metformin can prevent monocrotaline (MCT)-induced PH (Agard et al., 2009; Li et al., 2016; Sun et al., 2019; Yoshida et al., 2020; Zhai et al., 2018), its treatment potential for established PH and its effect on eNOS phosphorylation in this rat model of severe PH have not been studied. Furthermore, the effect of AMPK on the production of PGI2, an essential endothelium derived vasodilator, has not been investigated in PH. In addition to its effect on the vascular endothelium, AMPK activation was shown to reduce the contractility of rat and mouse aorta (Davis et al., 2012; Pyla et al., 2014) and mesenteric arteries (Chen et al., 2019; Schneider et al., 2015) in an endothelium independent manner. Since vasoconstriction is one pathological feature of PH, AMPK activation may be beneficial in disease management. Nevertheless, the effect of AMPK activation on pulmonary vascular tone in absence of endothelium has not been tested.

While treprostinil was shown to protect against PH associated with left heart disease (Group 2 PH) in rats partially by activating AMPK in skeletal muscles (Wang et al., 2020b) and to improve exercise capacity in patients with PH due to interstitial lung disease (Waxman et al., 2021), its effect (or that of other PGI2 analogues) on AMPK activity and subsequent eNOS activation in pulmonary artery smooth muscle cells (PASMCs) has also not been investigated.

Therefore, in this study, we used MCT-injected rats with established PH to investigate the effect of metformin treatment on the vasorelaxant pathways (NO and/or PGI2) and to consequently prevent the progression of the disease. In addition, we examined the effect of two different AMPK activators (metformin and/or AICAR) on the contractility of human pulmonary arteries (HPA) from Non-PH and Group 3 PH patients (PH due to lung diseases and/or hypoxia). Finally, we tested the effect of treprostinil on AMPK and eNOS activation in human hPASMCs.