HIPPO-Integrin-linked Kinase Cross-Talk Controls Self-Sustaining Proliferation and Survival in Pulmonary Hypertension.

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 HIF1α, Notch3 intracellular domain and ß-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 up-regulated 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 down-regulation 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.

Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity.

AIMS: Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions. METHODS AND RESULTS: It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels. CONCLUSIONS: 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.

Nox-derived ROS are acutely activated in pressure overload pulmonary hypertension: indications for a seminal role for mitochondrial Nox4.

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.

Direct sGC activation bypasses NO scavenging reactions of intravascular free oxy-hemoglobin and limits vasoconstriction.

AIMS: Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation. RESULTS: Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 µM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770. INNOVATION AND CONCLUSION: These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling.