Background Enhanced proliferation, resistance to apoptosis and metabolic shift to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMC) are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH). The role of distinct mTOR complexes mTORC1 (mTOR-raptor) and mTORC2 (mTOR-rictor) in PAVSMC proliferation and survival in PAH and their therapeutic relevance is unknown. Methods and Results Immunohistochemical and immunoblot analyses revealed that mTORC1 and mTORC2 pathways are markedly up-regulated in small remodeled PAs and isolated distal PAVSMC from IPAH subjects that have increased ATP levels, proliferation and survival that depend on glycolytic metabolism. siRNA- and pharmacological-based analysis showed that while both mTORC1 and mTORC2 contributing to proliferation, only mTORC2 is required for ATP generation and survival of IPAH PAVSMC. mTORC2 down-regulated energy sensor AMPK allowing activation of mTORC1-S6 and increased proliferation, and deficiency of pro-apoptotic protein Bim and IPAH PAVSMC survival. Nox4 protein levels were increased in IPAH PAVSMC that was necessary for mTORC2 activation, proliferation and survival. Nox4 levels and mTORC2 signaling were significantly up-regulated in small PAs from hypoxia-exposed rats at days 2-28 of hypoxia. Treatment with the mTOR kinase inhibitor PP242 at days 15-28 suppressed mTORC2, but not Nox4, induced SM-specific apoptosis in small PAs and reversed hypoxia-induced pulmonary vascular remodeling in rats. Conclusions These data provide a novel mechanistic link of Nox4-dependent activation of mTORC2 via energy sensor AMPK to increased proliferation and survival of PAVSMC in PAH suggesting a new potential pathway for the therapeutic interventions.

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.

Background Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in eNOS null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in pre-clinical models of PH. Methods and Results In order to evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a “two-hit” PH-HFpEF model in rats with multiple features of metabolic syndrome due to double leptin receptor defect (obese ZSF1) with the combined treatment of VEGF receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose lowering effect of nitrite was abolished in SIRT3 deficient human skeletal muscle cells, as well as in SIRT3 knockout mice fed a high-fat diet. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMPK. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMPK. Conclusions These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.

Although the cellular functions of TSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of LAM patients compared with smooth muscle cells from normal human blood vessels and trachea. Smooth muscle (SM) cells derived from these lesions (LAMD-SM) also exhibited S6 hyperphosphorylation, constitutive activation of p70 S6 kinase (p70S6K), and increased basal DNA synthesis. In parallel, TSC2؊/؊ smooth muscle cells (ELT3) and TSC2؊/؊ epithelial cells (ERC15) also exhibited hyperphosphorylation of S6, constitutive activation of p70S6K, and increased basal DNA synthesis. Re-introduction of wild type tuberin into LAMD-SM, ELT3, and ERC15 cells abolished phosphorylation of S6 and significantly inhibited p70S6K activity and DNA synthesis. Rapamycin, an immunosuppressant, inhibited hyperphosphorylation of S6, p70S6K activation, and DNA synthesis in LAMD-SM cells. Interestingly, the basal levels of phosphatidylinositol 3-kinase, Akt/protein kinase B, and p42/p44 MAPK activation were unchanged in LAMD-SM and ELT3 cells relative to levels in normal human tracheal and vascular SM. These data demonstrate that tuberin negatively regulates the activity of S6 and p70S6K specifically, and suggest a potential mechanism for abnormal cell growth in LAM.

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; chi(2) analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.

Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

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SummaryIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease associated with aging. However, the molecular mechanisms of the aging process that contribute to the pathogenesis of IPF have not been elucidated. IPF is characterized by abundant foci of highly active fibroblasts and myofibroblasts resistant to apoptosis. Remarkably, the role of aging in the autophagy activity of lung fibroblasts and its relationship with apoptosis, as adaptive responses, has not been evaluated previously in this disease. In the present study, we analyzed the dynamics of autophagy in primary lung fibroblasts from IPF compared to young and age-matched normal lung fibroblasts. Our results showed that aging contributes for a lower induction of autophagy on basal conditions and under starvation which is mediated by mTOR pathway activation. Treatment with rapamycin and PP242, that target the PI3K/AKT/mTOR signaling pathway, modified starvation-induced autophagy and apoptosis in IPF fibroblasts. Interestingly, we found a persistent activation of this pathway under starvation that contributes to the apoptosis resistance in IPF fibroblasts. These findings indicate that aging affects adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts. The activation of this pathway also contributes to the resistance to cell death in IPF lung fibroblasts.