Rationale: Enhanced proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiologic components of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH).Objectives: To determine the role and therapeutic relevance of HIPPO signaling in PAVSMC proliferation/apoptosis imbalance in PAH.Methods: Primary distal PAVSMCs, lung tissue sections from unused donor (control) and idiopathic PAH lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Measurements and Main Results:Immunohistochemical and immunoblot analyses demonstrated that the HIPPO central component large tumor suppressor 1 (LATS1) is inactivated in small remodeled pulmonary arteries (PAs) and distal PAVSMCs in idiopathic PAH. Molecular-and pharmacology-based analyses revealed that LATS1 inactivation and consequent up-regulation of its reciprocal effector Yes-associated protein (Yap) were required for activation of mammalian target of rapamycin (mTOR)-Akt, accumulation of HIF1a, Notch3 intracellular domain and b-catenin, deficiency of proapoptotic Bim, increased proliferation, and survival of human PAH PAVSMCs. LATS1 inactivation and up-regulation of Yap increased production and secretion of fibronectin that upregulated integrin-linked kinase 1 (ILK1). ILK1 supported LATS1 inactivation, and its inhibition reactivated LATS1, down-regulated Yap, suppressed proliferation, and promoted apoptosis in PAH, but not control PAVSMCs. PAVSM in small remodeled PAs from rats and mice with SU5416/hypoxia-induced PH showed downregulation of LATS1 and overexpression of ILK1. Treatment of mice with selective ILK inhibitor Cpd22 at Days 22-35 of SU5416/hypoxia exposure restored LATS1 signaling and reduced established pulmonary vascular remodeling and PH.Conclusions: These data report inactivation of HIPPO/LATS1, self-supported via Yap-fibronectin-ILK1 signaling loop, as a novel mechanism of self-sustaining proliferation and apoptosis resistance of PAVSMCs in PAH and suggest a new potential target for therapeutic intervention.

These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.

Pulmonary arterial hypertension is a severe progressive disease with marked morbidity and high mortality in which right ventricular (RV) failure is the major cause of death. Thus knowledge of the mechanisms underlying RV failure is an area of active interest. Previous studies suggest a role of NADPH oxidase in cardiomyocyte dysfunction in the left heart. Here we postulate that acute pressure overload induced by pulmonary artery banding (PAB) leads to a Nox4-initiated increase in reactive oxygen species (ROS) in mouse RV that may lead to feed-forward induction of Nox2. To test our hypothesis, ROS production was measured in RV and left ventricle homogenates. The data show that hydrogen peroxide (H2O2), but not superoxide anion (O2(·-)), was increased in the early phases (within 6 h) of PAB in RV and that this increase was diminished by catalase and diphenyleneiodonium chloride but not by SOD, N(ω)-nitro-l-arginin methyl ester, febuxostat, or indomethacin. H2O2 production in RV was not attenuated in Nox2 null mice subjected to 6 h PAB. Moreover, we observed an upregulation of Nox4 mRNA after 1 h of PAB and an increase in mitochondrial Nox4 protein 6 h post-PAB. In contrast, we observed an increase in Nox2 mRNA 1 day post-PAB. Expression of antioxidant enzymes SOD, catalase, and glutathione peroxidase did not change, but catalase activity increased 6 h post-PAB. Taken together, these findings show a role of mitochondria-localized Nox4 in the early phase of PAB and suggest an involvement of this isozyme in early ROS generation possibly contributing to progression of RV dysfunction and failure.

Pulmonary arterial hypertension (PAH; World HealthOrganization group 1) is a disease of the small pulmonary arteries, characterized by vasoconstriction, vascular proliferation, and remodeling. Although at present there are 14 drugs approved by the U.S. Food and Drug Administration for the treatment of PAH available on the U.S. market, the morbidity and mortality of PAH remain high. Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) (also referred to as combined pre-and postcapillary PH or CpcPH; World Health Organization group 2) is known to occur secondary to the left ventricular diastolic dysfunction and is recognized as a clinical complication of the metabolic syndrome (1). At present, there are no approved therapies for PH-HFpEF. We have recently reported that metformin, the first-line antidiabetic drug and the canonical AMP-activated protein kinase (AMPK) activator, exhibits therapeutic efficacy in the early treatment of PH-HFpEF in preclinical rat models (2). Because PH-HFpEF shares many pathophysiological characteristics with PAH, and many patients with PAH exhibit signs of insulin resistance and glucose intolerance in the absence of obesity and diabetes (3, 4), we evaluated metformin in the treatment of group 1 PH.Although metformin has been found to prevent the development of PAH in hypoxia and monocrotaline rat models (5) and reverse PAH in SU5416/hypoxia (SuHx) rats (6), and is currently in a phase 2 clinical trial for the treatment of PAH (NCT01352026), our data showed that metformin treatment failed to reverse pulmonary pressures and vascular remodeling in mice with SuHx-induced PAH (Figure 1). In this experimental model, 6-week-old male C57BL/6J mice were injected subcutaneously with SU5416 (20 mg/kg) or vehicle buffer once per week during the first 3-week exposure to hypoxia (10% oxygen). Metformin (100 mg/kg) was given in drinking water and continued for 2-week exposure to hypoxia (Figure 1A). We observed no changes in right ventricular systolic pressure (RVSP) in metformin-