Rationale Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction due in part to pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs, however their relationship and relevance to the development of PAH is unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate Serine-616, causes mitochondrial fission. It is however unknown whether mitochondrial fission is a prerequisite for proliferation. Objective We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential. Methods and Results Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at Serine-616. In normal PASMC, HIF-1α activation by CoCl2 or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMC and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function and hemodynamics in experimental PAH. Conclusion DRP-1-mediated mitotic fission is a cell cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Background Right ventricular hypertrophy (RVH) and RV failure contribute to morbidity and mortality in pulmonary arterial hypertension (PAH). The cause of RV dysfunction and the feasibility of therapeutically targeting the RV are uncertain. We hypothesized that RV dysfunction and electrical remodeling in RVH result, in part, from a glycolytic-shift in the myocyte, caused by activation of pyruvate dehydrogenase kinase (PDK). Methods and Results We studied 2 complementary rat models: RVH+PAH (induced by monocrotaline) and RVH+without PAH (induced by pulmonary artery banding, PAB). Monocrotaline-RVH reduced RV O2-consumption and enhanced glycolysis. RV 2-fluoro-2-deoxy-glucose uptake, Glut-1 expression and pyruvate dehydrogenase phosphorylation increased in monocrotaline-RVH. The RV monophasic action potential duration and QTc-interval were prolonged due to decreased expression of repolarizing voltage-gated K+ channels (Kv1.5, Kv4.2). In the RV working-heart model, the PDK inhibitor, dichloroacetate, acutely increased glucose oxidation and cardiac work in monocrotaline-RVH. Chronic dichloroacetate therapy improved RV repolarization and RV function in vivo and in the RV Langendorff model. In PAB-induced RVH, a similar reduction in cardiac output and glycolytic shift occurred and it too improved with dichloroacetate. In PAB-RVH the benefit of dichloroacetate on cardiac output was ~1/3 that in monocrotaline-RVH. The larger effects in monocrotaline-RVH likely reflect dichloroacetate’s dual metabolic benefits in that model: regression of vascular disease and direct effects on the RV. Conclusion Reduction in RV function and electrical remodeling in 2 models of RVH relevant to human disease (PAH and pulmonic stenosis) result, in part, from a PDK-mediated glycolytic shift in the RV. PDK inhibition partially restores RV function and regresses RVH by restoring RV repolarization and enhancing glucose oxidation. Recognition that a PDK-mediated metabolic shift contributes to contractile and ionic dysfunction in RVH offers insight into the pathophysiology and treatment of RVH.

Rationale: Pulmonary arterial hypertension (PAH) is a proliferative arteriopathy associated with glucose transporter-1 (Glut1) up-regulation and a glycolytic shift in lung metabolism. Glycolytic metabolism can be detected with the positron emission tomography (PET) tracer 18 F-fluorodeoxyglucose (FDG).Objectives: The precise cell type in which glycolytic abnormalities occur in PAH is unknown. Moreover, whether FDG-PET is sufficiently sensitive to monitor PAH progression and detect therapeutic regression is untested. We hypothesized that increased lung FDG-PET reflects enhanced glycolysis in vascular cells and is reversible in response to effective therapies. Methods: PAH was induced in Sprague-Dawley rats by monocrotaline or chronic hypoxia (10% oxygen) in combination with Sugen 5416. Monocrotaline rats were treated with oral dichloroacetate or daily imatinib injections. FDG-PET scans and pulmonary artery acceleration times were obtained weekly. The origin of the PET signal was assessed by laser capture microdissection of airway versus vascular tissue. Metabolism was measured in pulmonary artery smooth muscle cell (PASMC) cultures, using a Seahorse extracellular flux analyzer. Measurements and Main Results: Lung FDG increases 1-2 weeks after monocrotaline (when PAH is mild) and is normalized by dichloroacetate and imatinib, which both also regress medial hypertrophy. Glut1 mRNA is up-regulated in both endothelium and PASMCs, but not airway cells or macrophages. PASMCs from monocrotaline rats are hyperproliferative and display normoxic activation of hypoxia-inducible factor-1a (HIF-1a), which underlies their glycolytic phenotype. Conclusions: HIF-1a-mediated Glut1 up-regulation in proliferating vascular cells in PAH accounts for increased lung FDG-PET uptake. FDG-PET is sensitive to mild PAH and can monitor therapeutic changes in the vasculature.Keywords: hypoxia-inducible factor-1a (HIF-1a); glucose transporter-1 (Glut1); glycolysis; imatinib; Sugen 5416Pulmonary arterial hypertension (PAH) is a syndrome in which the mean pulmonary artery pressure exceeds 25 mm Hg because of obstruction and constriction of precapillary pulmonary resistance arteries. An important aspect of the pathophysiology of PAH is increased proliferation and reduced apoptosis of pulmonary arterial endothelial and smooth muscle cells (PASMCs) (1-8), leading to plexiform lesions and increased muscularization, respectively.Publications describe impaired glucose oxidation and/or a shift to glycolytic metabolism in PASMCs in rodents with experimental PAH (9), in endothelial cells of patients with PAH (10, 11), and in the pulmonary arteries of rats (12) and mice (13) exposed to chronic hypoxia. Aerobic glycolysis is also known as the Warburg effect and was shown to confer a growth advantage to proliferating cells (14). A crucial determinant of whether cells generate energy through glycolysis or aerobic mitochondrial respiration is the activity of pyruvate dehydrogenase complex (PDH). PDH catalyzes the conversion of pyruvate into acetyl coenzyme A...

Right ventricular hypertrophy (RVH) and RV failure are major determinants of prognosis in pulmonary hypertension and congenital heart disease. In RVH, there is a metabolic shift from glucose oxidation (GO) to glycolysis. Directly increasing GO improves RV function, demonstrating the susceptibility of RVH to metabolic intervention. However, the effects of RVH on fatty acid oxidation (FAO), the main energy source in adult myocardium, are unknown. We hypothesized that partial inhibitors of FAO (pFOXi) would indirectly increase GO and improve RV function by exploiting the reciprocal relationship between FAO and GO (Randle’s cycle). RVH was induced in adult Sprague-Dawley rats by pulmonary artery banding (PAB). pFOXi were administered orally to prevent (trimetazidine, 0.7 g/L for 8 weeks) or regress (ranolazine 20 mg/day or trimetazidine for 1 week, beginning 3 weeks post-PAB) RVH. Metabolic, hemodynamic, molecular, electrophysiologic, and functional comparisons with sham rats were performed 4 or 8 weeks post-PAB. Metabolism was quantified in RV working hearts, using a dual-isotope technique, and in isolated RV myocytes, using a Seahorse Analyzer. PAB-induced RVH did not cause death but reduced cardiac output and treadmill walking distance and elevated plasma epinephrine levels. Increased RV FAO in PAB was accompanied by increased carnitine palmitoyl-transferase expression; conversely, GO and pyruvate dehydrogenase (PDH) activity were decreased. pFOXi decreased FAO and restored PDH activity and GO in PAB, thereby increasing ATP levels. pFOXi reduced the elevated RV glycogen levels in RVH. Trimetazidine and ranolazine increased cardiac output and exercise capacity and attenuated exertional lactic acidemia in PAB. RV monophasic action potential duration and QTc interval prolongation in RVH normalized with trimetazidine. pFOXi also decreased the mild RV fibrosis seen in PAB. Maladaptive increases in FAO reduce RV function in PAB-induced RVH. pFOXi inhibit FAO, which increases GO and enhances RV function. Trimetazidine and ranolazine have therapeutic potential in RVH.

Rationale Rapid growth of cancer cells is permitted by metabolic changes, notably increased aerobic glycolysis and increased glutaminolysis. Aerobic glycolysis is also evident in the hypertrophying myocytes in right ventricular hypertrophy (RVH), particularly in association with pulmonary arterial hypertension (PAH). It is unknown whether glutaminolysis occurs in the heart. We hypothesized that glutaminolysis occurs in RVH and assessed the precipitating factors, transcriptional mechanisms and physiological consequences of this metabolic pathway. Methods and Results RVH was induced in two models, one with PAH (Monocrotaline-RVH) and the other without PAH (pulmonary artery banding, PAB-RVH). Despite similar RVH, ischemia as determined by reductions in RV VEGFα, coronary blood flow and microvascular density was greater in Monocrotaline-RVH versus PAB-RVH. A 6-fold increase in 14C-glutamine metabolism occurred in Monocrotaline-RVH but not PAB-RVH. In the RV working-heart model, the glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON) decreased glutaminolysis, caused a reciprocal increase in glucose oxidation and elevated cardiac output. Consistent with increased glutaminolysis in RVH, RV expression of glutamine transporters (SLC1A5 and SLC7A5) and mitochondrial malic enzyme were elevated (Monocrotaline-RVH>PAB-RVH>Control). Capillary rarefaction and glutamine transporter upregulation also occurred in RVH in patients with PAH. cMyc and Max, known to mediate transcriptional upregulation of glutaminolysis, were increased in Monocrotaline-RVH. In vivo, DON (0.5 mg/Kg/Da×3 weeks) restored pyruvate dehydrogenase activity, reduced RVH and increased cardiac output (89±8, vs. 55±13 ml/min, p<0.05) and treadmill distance (194±71, vs. 36 ±7 m, p<0.05) in Monocrotaline-RVH. Conclusions Glutaminolysis is induced in the RV in PAH by cMyc-Max, likely as a consequence of RV ischemia. Inhibition of glutaminolysis restores glucose oxidation and has therapeutic benefit in vivo.

Rationale: Pulmonary arterial hypertension (PAH) is a lethal, femalepredominant, vascular disease. Pathologic changes in PA smooth muscle cells (PASMC) include excessive proliferation, apoptosis-resistance, and mitochondrial fragmentation. Activation of dynamin-related protein increases mitotic fission and promotes this proliferation-apoptosis imbalance. The contribution of decreased fusion and reduced mitofusin-2 (MFN2) expression to PAH is unknown. Objectives: We hypothesize that decreased MFN2 expression promotes mitochondrial fragmentation, increases proliferation, and impairs apoptosis. The role of MFN2's transcriptional coactivator, peroxisome proliferator-activated receptor g coactivator 1-a (PGC1a), was assessed. MFN2 therapy was tested in PAH PASMC and in models of PAH. Methods: Fusion and fission mediators were measured in lungs and PASMC from patients with PAH and female rats with monocrotaline or chronic hypoxia1Sugen-5416 (CH1SU) PAH. The effects of adenoviral mitofusin-2 (Ad-MFN2) overexpression were measured in vitro and in vivo. Measurements and Main Results: In normal PASMC, siMFN2 reduced expression of MFN2 and PGC1a; conversely, siPGC1a reduced PGC1a and MFN2 expression. Both interventions caused mitochondrial fragmentation. siMFN2 increased proliferation. In rodent and human PAH PASMC, MFN2 and PGC1a were decreased and mitochondria were fragmented. Ad-MFN2 increased fusion, reduced proliferation, and increased apoptosis in human PAH and CH1SU. In CH1SU, Ad-MFN2 improved walking distance (381 6 35 vs. 245 6 39 m; P , 0.05); decreased pulmonary vascular resistance (0.18 6 0.02 vs. 0.38 6 0.14 mm Hg/ml/min; P , 0.05); and decreased PA medial thickness (14.5 6 0.8 vs. 19 6 1.7%; P , 0.05). Lung vascularity was increased by MFN2. Conclusions: Decreased expression of MFN2 and PGC1a contribute to mitochondrial fragmentation and a proliferation-apoptosis imbalance in human and experimental PAH. Augmenting MFN2 has therapeutic benefit in human and experimental PAH.Keywords: mitochondrial fission; peroxisome proliferator-activated receptor gamma coactivator-1 a; hypoxia-inducible factor-1 a; optic atrophy 1; female sex Pulmonary arterial hypertension (PAH) is a syndrome characterized by obstructive vascular remodeling, inflammation, and vasoconstriction of small pulmonary arteries. PAH is predominantly a disease of females (1). Recent advances in understanding the mechanism of PAH include the identification of mutations in the bone morphogenetic protein receptor 2 in familial PAH (2-4) and the recognition that increases in proliferation and apoptosisresistance of pulmonary arterial smooth muscle cells (PASMC), that have multifactorial etiology, contribute to vascular obstruction (reviewed in [5]). These discoveries have yet to be translated into approved therapies, and most PAH treatments are vasodilators. Perhaps because of this, the 1-year mortality rates remain high (z15%) (6). Although abnormalities of platelets, the endothelium, fibroblasts, and inflammatory cells contribute critically to the path...

Rationale: The etiology of hepatopulmonary syndrome (HPS), a common complication of cirrhosis, is unknown. Inflammation and macrophage accumulation occur in HPS; however, their importance is unclear. Common bile duct ligation (CBDL) creates an accepted model of HPS, allowing us to investigate the cause of HPS. Objectives: We hypothesized that macrophages are central to HPS and investigated the therapeutic potential of macrophage depletion. Methods: Hemodynamics, alveolar-arterial gradient, vascular reactivity, and histology were assessed in CBDL versus sham rats (n 5 21 per group). The effects of plasma on smooth muscle cell proliferation and endothelial tube formation were measured. Macrophage depletion was used to prevent (gadolinium) or regress (clodronate) HPS. CD68(1) macrophages and capillary density were measured in the lungs of patients with cirrhosis versus control patients (n 5 10 per group). Measurements and Main Results: CBDL increased cardiac output and alveolar-arterial gradient by causing capillary dilatation and arteriovenous malformations. Activated CD68(1) macrophages (nuclear factor-kB1) accumulated in HPS pulmonary arteries, drawn by elevated levels of plasma endotoxin and lung monocyte chemoattractant protein-1. These macrophages expressed inducible nitric oxide synthase, vascular endothelial growth factor, and platelet-derived growth factor. HPS plasma increased endothelial tube formation and pulmonary artery smooth muscle cell proliferation. Macrophage depletion prevented and reversed the histological and hemodynamic features of HPS. CBDL lungs demonstrated increased medial thickness and obstruction of small pulmonary arteries. Nitric oxide synthase inhibition unmasked exaggerated pulmonary vasoconstrictor responses in HPS. Patients with cirrhosis had increased pulmonary intravascular macrophage accumulation and capillary density. Conclusions: HPS results from intravascular accumulation of CD68 (1) macrophages. An occult proliferative vasculopathy may explain the occasional transition to portopulmonary hypertension. Macrophage depletion may have therapeutic potential in HPS.Keywords: liver transplantation; arteriovenous malformations; clodronate; cirrhosis; portopulmonary hypertension Hepatopulmonary syndrome (HPS), characterized by hypoxemia and intrapulmonary shunting, occurs in 5 to 32% of patients with liver disease (1). HPS significantly increases mortality and worsens functional status and quality of life in patients with cirrhosis (2). The major pathological abnormalities in HPS include alveolar capillary dilatation and pulmonary arteriovenous malformations (3). Clinically, patients with HPS present with hypoxemia and signs of a hyperdynamic circulatory state that include low systemic vascular resistance, low pulmonary vascular resistance (PVR), and high cardiac output (CO) (1). The pathogenesis of HPS is unclear, and currently there are no effective medical therapies. Orthotopic liver transplantation is the only available treatment (1). It is noteworthy that some patients with cirr...

Pulmonary arterial hypertension (PAH) is a syndrome in which pulmonary vascular cross sectional area and compliance are reduced by vasoconstriction, vascular remodeling, and inflammation. Vascular remodeling results in part from increased proliferation and impaired apoptosis of vascular cells. The resulting increase in afterload promotes right ventricular hypertrophy (RVH) and RV failure. Recently identified mitochondrial-metabolic abnormalities in PAH, notably pyruvate dehydrogenase kinase-mediated inhibition of pyruvate dehydrogenase (PDH), result in aerobic glycolysis in both the lung vasculature and RV. This glycolytic shift has diagnostic importance since it is detectable early in experimental PAH by increased lung and RV uptake of 18F-fluorodeoxyglucose on positron emission tomography. The metabolic shift also has pathophysiologic and therapeutic relevance. In RV myocytes, the glycolytic switch reduces contractility while in the vasculature it renders cells hyperproliferative and apoptosis-resistant. Reactivation of PDH can be achieved directly by PDK inhibition (using dichloroacetate), or indirectly via activating the Randle cycle, using inhibitors of fatty acid oxidation (FAO), trimetazidine and ranolazine. In experimental PAH and RVH, PDK inhibition increases glucose oxidation, enhances RV function, regresses pulmonary vascular disease by reducing proliferation and enhancing apoptosis, and restores cardiac repolarization. FAO inhibition increases RV glucose oxidation and RV function in experimental RVH. The trigger for metabolic remodeling in the RV and lung differ. In the RV, metabolic remodeling is likely triggered by ischemia (due to microvascular rarefaction and/or reduced coronary perfusion pressure). In the vasculature, metabolic changes result from redox-mediated activation of transcription factors, including hypoxia-inducible factor 1α, as a consequence of epigenetic silencing of SOD2 and/or changes in mitochondrial fission/fusion. Randomized controlled trials are required to assess whether the benefits of enhancing glucose oxidation are realized in patients with PAH.