Serotonin-deficient neonatal mice are not protected against the development of experimental bronchopulmonary dysplasia or pulmonary hypertension.

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor and contributes to high pulmonary vascular resistance in the developing ovine lung. In experimental pulmonary hypertension (PH), pulmonary expression of tryptophan hydroxylase-1 (TPH1), the rate limiting enzyme in 5-HT synthesis, and plasma 5-HT are increased. 5-HT blockade increases pulmonary blood flow and prevents pulmonary vascular remodeling and PH in neonatal models of PH with bronchopulmonary dysplasia (BPD). We hypothesized that neonatal tph1 knock-out (KO) mice would be protected from hypoxia-induced alveolar simplification, decreased vessel density, and PH. Newborn wild-type (WT) and tph1 KO mice were exposed to normoxia or hypoxia for 2 weeks. Normoxic WT and KO mice exhibited similar alveolar development, pulmonary vascular density, right ventricular systolic pressures (RVSPs), and right heart size. Circulating (plasma and platelet) 5-HT decreased in both hypoxia-exposed WT and KO mice. Tph1 KO mice were not protected from hypoxia-induced alveolar simplification, decreased pulmonary vascular density, or right ventricular hypertrophy, but displayed attenuation to hypoxia-induced RVSP elevation compared with WT mice. Tph1 KO neonatal mice are not protected against hypoxia-induced alveolar simplification, reduction in pulmonary vessel density, or RVH. While genetic and pharmacologic inhibition of tph1 has protective effects in adult models of PH, our results suggest that tph1 inhibition would not be beneficial in neonates with PH associated with BPD.

Maxingxiongting mixture attenuates hypoxia pulmonary arterial hypertension to improve right ventricular hypertrophy by inhibiting the rho-kinase signaling pathway.

OBJECTIVE: To explore the mechanism of Maxingxiongting mixture (MXXTM) on pulmonary hypertension in a rat model established by intraperitoneal injection of monocrotaline solution, smoking and forced swimming. METHODS: A total of 30 male Sprague-Dawley rats were randomly divided into five groups: control group, model group, high-dose of MXXTM group (HM), low-dose of MXXTM group (LM), and fasudil group. The mean pulmonary artery pressure (mPAP) was measured by using a miniature catheter. Lung tissue and right ventricular tissue sections were stained with hematoxylin-eosin. The right ventricle (RV) and left ventricle + septum (LV + S) were weighted. RV/(LV+S) was calculated to reflect the degree of right ventricular hypertrophy. Rho/Rho-kinase signaling pathway key proteins (RhoA, ROCK Ⅰ and ROCK Ⅱ) in rat right ventricular tissue were measured by Western blot analysis. The levels of serum hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and the levels of plasma renin activity (PRA), angiotensin Ⅱ (ANG-Ⅱ), aldosterone (ALD) in rat anticoagulated plasma were all measured by enzyme-linked immunosorbent assay. RESULTS: Compared with the control group, the mPAP and RV/(LV+S) in the model group were significantly increased. Administration of fasudil resulted in a significant decrease of mPAP and RV/ (LV+S). In the HM group and LM group, mPAP and RV/ (LV+S) were significantly lower than the model group. Compared with the control group, the contents of HIF-1α, VEGF, PRA, ANG-Ⅱ and ALD in the model group were significantly increased. The administration of fasudil and high-dose MXXTM significantly reduced the contents of HIF-1α, VEGF, PRA, ANG-II and ALD. Compared with the control group, the expression of RhoA, ROCK Ⅰ and ROCK Ⅱ in the right ventricle of the model group were significantly increased. The administration of fasudil and high-dose MXXTM significantly reduced the expression of RhoA and Rock Ⅱ proteins. Our results indicated that high-dose of MXXTM had similar effects on reducing pulmonary artery pressure and improving right ventricular remodeling to fasudil. However, MXXTM was unable to restore parameters above to control levels. CONCLUSIONS: MXXTM attenuates hypoxia pulmonary arterial hypertension to improve right ventricular hypertrophy by inhibiting the Rho-kinase signaling pathway.

Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-N-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH.

Therapeutic Benefit of the Association of Lodenafil with Mesenchymal Stem Cells on Hypoxia-induced Pulmonary Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. This work investigated the benefit of the association of human umbilical cord mesenchymal stem cells (hMSCs) with lodenafil, a phosphodiesterase-5 inhibitor, in an animal model of PAH. Male Wistar rats were exposed to hypoxia (10% O2) for three weeks plus a weekly i.p. injection of a vascular endothelial growth factor receptor inhibitor (SU5416, 20 mg/kg, SuHx). After confirmation of PAH, animals received intravenous injection of 5.105 hMSCs or vehicle, followed by oral treatment with lodenafil carbonate (10 mg/kg/day) for 14 days. The ratio between pulmonary artery acceleration time and RV ejection time reduced from 0.42 ± 0.01 (control) to 0.24 ± 0.01 in the SuHx group, which was not altered by lodenafil alone but was recovered to 0.31 ± 0.01 when administered in association with hMSCs. RV afterload was confirmed in the SuHx group with an increased RV systolic pressure (mmHg) of 52.1 ± 8.8 normalized to 29.6 ± 2.2 after treatment with the association. Treatment with hMSCs + lodenafil reversed RV hypertrophy, fibrosis and interstitial cell infiltration in the SuHx group. Combined therapy of lodenafil and hMSCs may be a strategy for PAH treatment.

Effect of p53 activation on experimental right ventricular hypertrophy.

The leading cause of death in Pulmonary Arterial Hypertension (PAH) is right ventricular (RV) failure. The tumor suppressor p53 has been associated with left ventricular hypertrophy (LVH) and remodeling but its role in RV hypertrophy (RVH) is unclear. The purpose of this study was to determine whether pharmacological activation of p53 by Quinacrine affects RV remodeling and function in the pulmonary artery banding (PAB) model of compensated RVH in mice. The effects of p53 activation on cellular functions were studied in isolated cardiomyocytes, cardiac fibroblasts and endothelial cells (ECs). The expression of p53 was examined both on human RV tissues from patients with compensated and decompensated RVH and in mouse RV tissues early and late after the PAB. As compared to control human RVs, there was no change in p53 expression in compensated RVH, while a marked upregulation was found in decompensated RVH. Similarly, in comparison to SHAM-operated mice, unaltered RV p53 expression 7 days after PAB, was markedly induced 21 days after the PAB. Quinacrine induced p53 accumulation did not further deteriorate RV function at day 7 after PAB. Quinacrine administration did not increase EC death, neither diminished EC number and capillary density in RV tissues. No major impact on the expression of markers of sarcomere organization, fatty acid and mitochondrial metabolism and respiration was noted in Quinacrine-treated PAB mice. p53 accumulation modulated the expression of Heme Oxygenase 1 (HO-1) and Glucose Transporter (Glut1) in mouse RVs and in adult cardiomyocytes. We conclude that early p53 activation in PAB-induced RVH does not cause substantial detrimental effects on right ventricular remodeling and function.

Protection against pressure overload-induced right heart failure by uncoupling protein 2 silencing.

AIMS: The role of uncoupling protein 2 (UCP2) in cardiac adaptation to pressure overload remains unclear. In a classical model of left ventricular pressure overload genetic deletion of UCP2 (UCP2-/-) protected against cardiac hypertrophy and failure. However, in UCP2-/- mice increased proliferation of pulmonary arterial smooth muscle cells induces mild pulmonary hypertension, right ventricular (RV) hypertrophy, and reduced cardiac output. This suggests a different role for UCP2 in RV and left ventricular adaptation to pressure overload. To clarify this situation in more detail UCP2-/- and wild-type mice were exposed to pulmonary arterial banding (PAB). METHODS AND RESULTS: Mice were analysed (haemodynamics, morphometry, and echocardiography) 3 weeks after PAB or sham surgery. Myocytes and non-myocytes were isolated and analysed separately. Cell shortening of myocytes and fura-2 loading of cardiomyocytes were used to characterize their function. Brd assay was performed to study fibroblast proliferation. Isolated mitochondria were analysed to investigate the role of UCP2 for reactive oxygen species (ROS) production. UCP2 mRNA was 2.7-fold stronger expressed in RV myocytes than in left ventricular myocytes and stronger expressed in non-myocytes compared with myocytes. Three weeks after PAB, cardiac output was reduced in wild type but preserved in UCP2-/- mice. UCP2-/- had increased RV wall thickness, but lower RV internal diameters and displayed a significant stronger fibrosis. Cardiac fibroblasts from UCP2-/- had reduced proliferation rates but higher collagen-1 expression. Myocytes isolated from mice after PAB banding showed preserved function that was further improved by UCP2-/-. Mitochondrial ROS production and respiration was similar between UCP2-/- or wild-type hearts. CONCLUSION: Despite a mild pulmonary hypertension in UCP2-/- mice, hearts from these mice are well preserved against additional pressure overload (severe pulmonary hypertension). This-at least in part-depends on different behaviour of non-myocytes (fibroblasts).

Inhibition of Src activation reverses pulmonary vascular remodeling in experimental pulmonary arterial hypertension via Akt/mTOR/HIF-1 signaling pathway.

Pulmonary arterial hypertension (PAH) is a diffuse pulmonary microvascular remodeling disease accompanied by malignant proliferation of pulmonary artery smooth muscle cells (PASMCs), which causes persistent pulmonary artery pressure elevation, right ventricular hypertrophy (RVH) and death. However, current therapies targeting pulmonary vascular remodeling and RVH remain poorly effective in reversing PAH. Overactivation of the protein tyrosine kinase Src plays an important role in tumor cell growth, proliferation and invasion; we thus hypothesized that inhibitors targeting Src activation could reverse experimental PAH. We demonstrated that Src was markedly activated in hypoxia-stimulated PASMCs from donors and PASMCs isolated from PAH patients. We investigated the effects of the Src-selective inhibitor 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP1) and berberine (BBR) on PAH-PASMC proliferation and migration by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU) and wound-healing assays. Our in vitro results showed that inhibition of Src (Tyr416) phosphorylation repressed PAH-PASMC proliferation and migration by inhibiting hypoxia-inducible factor-1α (HIF-1α) expression through Akt/mTOR signal pathway. In vivo, PP1 and BBR significantly alleviated distal pulmonary vascular remodeling and decreased right ventricular systolic pressure (RVSP) and RVH in Sugen (SU) 5416/hypoxia (SU-PAH) mice. These findings demonstrate that pharmacological (PP1 or BBR) inhibition of Src activation could be a novel means of treating severe pulmonary vascular remodeling and RVH in PAH patients.

CB2-deficiency is associated with a stronger hypertrophy and remodeling of the right ventricle in a murine model of left pulmonary artery occlusion.

AIMS: Pulmonary hypertension (PH) leads to right ventricular (RV) adaptation and remodeling and has deleterious long-term effects on RV function. The endocannabinoid receptor CB2 has been associated with protective effects in adaptation and remodeling of the left ventricle after ischemia. Therefore, we investigated the role of CB2 receptor in RV adaptation after occlusion of the left pulmonary artery (LPA) in a murine model. MAIN METHODS: C57/Bl6 (WT)- and CB2 receptor-deficient (Cnr2-/-)-mice underwent paramedian sternotomy and LPA was occluded using a metal clip. Right heart hemodynamic study (Millar®) preceded organ harvesting for immunohistochemistry and mRNA analysis 7 and 21 days (d) post-occlusion. KEY FINDINGS: LPA occlusion led to higher RV systolic pressure in Cnr2-/--hearts, while hemodynamics were comparable with WT-hearts after 21d. Cnr2-/--hearts showed higher macrophage infiltration and lower interleukin-10 expression after 7 d, but otherwise a comparable inflammatory mediator expression profile. Cardiomyocyte-hypertrophy was stronger in Cnr2-/--mice, presenting with higher tenascin-C expression than WT-hearts. Planimetry revealed higher collagen area in Cnr2-/--hearts and small areas of cardiomyocyte-loss. Surrounding cardiomyocytes were cleaved caspase-3- and TUNEL positive in Cnr2-/--hearts. This was associated by maladaptation of myosin heavy-chain isoforms and lower reactive oxygen scavenger enzymes induction in Cnr2-/--hearts. We found comparable morphological changes in both lungs between the two genotypes. SIGNIFICANCE: LPA occlusion led to increased systolic pressure and adaptation of RV in CB2-deficient mice. CB2 receptor seems to modulate RV adaptation through expression of contractile elements, reactive oxygen scavenger enzymes, and inflammatory response in order to prevent cardiomyocyte apoptosis.

Right ventricular involution: What can we learn from nature's model of compensated hypertrophy?

BACKGROUND: Right ventricular (RV) failure (RVF) is a vexing problem facing patients with various disease processes and carries a high mortality. RVF is a poorly understood phenomenon with limited treatment options. In mammalian fetal circulation, the right ventricle is the systemic ventricle. In neonates, however, the left ventricle assumes that role and gradually thickens compared with the right ventricle. This process, known as right ventricular involution (RVI), is poorly understood. We sought to define the time course and identify mechanisms involved in RVI. METHODS: Wild-type mice were bred and sacrificed on day of life (DOL) 1, 4, 8, 16, and 30 to evaluate left ventricular (LV) and RV wall thickness and apoptosis. A terminal deoxynucleotidyl transferase nick-end labeling assay and RNA sequencing were performed to measure changes during RVI. RESULTS: Morphometric analysis demonstrated the changes in RV and LV wall thickness occurring between DOL 1 and DOL 16 (RV:LV, 0.53:0.44; P = .03). In addition, apoptosis was most active early, with the highest percentage of apoptotic cells on DOL 1 (1.0%) and a significant decrease by DOL 30 (0.23%) (P = .02). Similarly, expression of the proapoptotic genes BCL2l11 and Pawr were increased at DOL 1, and the antiapoptotic genes Nol3 and Naip2 were significantly increased at DOL 30. CONCLUSIONS: RVI is a misnomer, but significant changes occur early (by DOL 16) in neonatal mouse hearts. Apoptosis plays a role in RVI, but whether manipulation of apoptotic pathways can prevent or reverse RVI is unknown and warrants further investigation.

MicroRNAs in right ventricular remodelling.

Right ventricular (RV) remodelling is a lesser understood process of the chronic, progressive transformation of the RV structure leading to reduced functional capacity and subsequent failure. Besides conditions concerning whole hearts, some pathology selectively affects the RV, leading to a distinct RV-specific clinical phenotype. MicroRNAs have been identified as key regulators of biological processes that drive the progression of chronic diseases. The role of microRNAs in diseases affecting the left ventricle has been studied for many years, however there is still limited information on microRNAs specific to diseases in the right ventricle. Here, we review recently described details on the expression, regulation, and function of microRNAs in the pathological remodelling of the right heart. Recently identified strategies using microRNAs as pharmacological targets or biomarkers will be highlighted. Increasing knowledge of pathogenic microRNAs will finally help improve our understanding of underlying distinct mechanisms and help utilize novel targets or biomarkers to develop treatments for patients suffering from right heart diseases.

Activation of LXRα improves cardiac remodeling induced by pulmonary artery hypertension in rats.

Inflammatory factors regulated by NF-κB play a significant role in PAH and myocardial hypertrophy. LXR activation may inhibit myocardial hypertrophy via suppressing inflammatory pathways; it is unknown whether LXR is also involved in PAH-induced myocardial hypertrophy or remodeling. To further explore the protective effect of LXR in PAH-induced cardiac hypertrophy and remodeling, a PAH model was developed, and T0901317, an agonist of LXR, was used to examine the effect of LXR activation. PAH rats demonstrated obvious cardiac hypertrophy and remodeling in the right ventricle, but significant improvement of cardiac hypertrophy and remodeling was observed in PAH rats treated with T0901317. Through RT-PCR, Western blot and ELISA examination, NF-κB, IL-6, TNF-α, and iNOS were found to be significantly reduced in PAH rats treated with T0901317 compared to PAH rats treated with DMSO. Apoptosis was also significantly reduced in PAH rats treated with T0901317. Thus, LXR activation may inhibit PAH-induced cardiac hypertrophy and remodeling by inhibiting NF-κB-mediated inflammatory pathways.

Peroxisome Proliferator-Activated Receptor γ Regulates the V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1/microRNA-27a Axis to Reduce Endothelin-1 and Endothelial Dysfunction in the Sickle Cell Mouse Lung.

Pulmonary hypertension (PH), a serious complication of sickle cell disease (SCD), causes significant morbidity and mortality. Although a recent study determined that hemin release during hemolysis triggers endothelial dysfunction in SCD, the pathogenesis of SCD-PH remains incompletely defined. This study examines peroxisome proliferator-activated receptor γ (PPARγ) regulation in SCD-PH and endothelial dysfunction. PH and right ventricular hypertrophy were studied in Townes humanized sickle cell (SS) and littermate control (AA) mice. In parallel studies, SS or AA mice were gavaged with the PPARγ agonist, rosiglitazone (RSG), 10 mg/kg/day, or vehicle for 10 days. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with vehicle or hemin for 72 hours, and selected HPAECs were treated with RSG. SS mice developed PH and right ventricular hypertrophy associated with reduced lung levels of PPARγ and increased levels of microRNA-27a (miR-27a), v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1), endothelin-1 (ET-1), and markers of endothelial dysfunction (platelet/endothelial cell adhesion molecule 1 and E selectin). HPAECs treated with hemin had increased ETS1, miR-27a, ET-1, and endothelial dysfunction and decreased PPARγ levels. These derangements were attenuated by ETS1 knockdown, inhibition of miR-27a, or PPARγ overexpression. In SS mouse lung or in hemin-treated HPAECs, activation of PPARγ with RSG attenuated reductions in PPARγ and increases in miR-27a, ET-1, and markers of endothelial dysfunction. In SCD-PH pathogenesis, ETS1 stimulates increases in miR-27a levels that reduce PPARγ and increase ET-1 and endothelial dysfunction. PPARγ activation attenuated SCD-associated signaling derangements, suggesting a novel therapeutic approach to attenuate SCD-PH pathogenesis.

Monocrotaline-Induced Pulmonary Hypertension Involves Downregulation of Antiaging Protein Klotho and eNOS Activity.

The objective of this study is to investigate whether stem cell delivery of secreted Klotho (SKL), an aging-suppressor protein, attenuates monocrotaline-induced pulmonary vascular dysfunction and remodeling. Overexpression of SKL in mesenchymal stem cells (MSCs) was achieved by transfecting MSCs with lentiviral vectors expressing SKL-green fluorescent protein (GFP). Four groups of rats were treated with monocrotaline, whereas an additional group was given saline (control). Three days later, 4 monocrotaline-treated groups received intravenous delivery of nontransfected MSCs, MSC-GFP, MSC-SKL-GFP, and PBS, respectively. Ex vivo vascular relaxing responses to acetylcholine were diminished in small pulmonary arteries (PAs) in monocrotaline-treated rats, indicating pulmonary vascular endothelial dysfunction. Interestingly, delivery of MSCs overexpressing SKL (MSC-SKL-GFP) abolished monocrotaline-induced pulmonary vascular endothelial dysfunction and PA remodeling. Monocrotaline significantly increased right ventricular systolic blood pressure, which was attenuated significantly by MSC-SKL-GFP, indicating improved PA hypertension. MSC-SKL-GFP also attenuated right ventricular hypertrophy. Nontransfected MSCs slightly, but not significantly, improved PA hypertension and pulmonary vascular endothelial dysfunction. MSC-SKL-GFP attenuated monocrotaline-induced inflammation, as evidenced by decreased macrophage infiltration around PAs. MSC-SKL-GFP increased SKL levels, which rescued the downregulation of SIRT1 (Sirtuin 1) expression and endothelial NO synthase (eNOS) phosphorylation in the lungs of monocrotaline-treated rats. In cultured endothelial cells, SKL abolished monocrotaline-induced downregulation of eNOS activity and NO levels and enhanced cell viability. Therefore, stem cell delivery of SKL is an effective therapeutic strategy for pulmonary vascular endothelial dysfunction and PA remodeling. SKL attenuates monocrotaline-induced PA remodeling and PA smooth muscle cell proliferation, likely by reducing inflammation and restoring SIRT1 levels and eNOS activity.

The von Hippel-Lindau Chuvash mutation in mice alters cardiac substrate and high-energy phosphate metabolism.

Hypoxia-inducible factor (HIF) appears to function as a global master regulator of cellular and systemic responses to hypoxia. HIF pathway manipulation is of therapeutic interest; however, global systemic upregulation of HIF may have as yet unknown effects on multiple processes. We used a mouse model of Chuvash polycythemia (CP), a rare genetic disorder that modestly increases expression of HIF target genes in normoxia, to understand what these effects might be within the heart. An integrated in and ex vivo approach was employed. Compared with wild-type controls, CP mice had evidence (using in vivo magnetic resonance imaging) of pulmonary hypertension, right ventricular hypertrophy, and increased left ventricular ejection fraction. Glycolytic flux (measured using [(3)H]glucose) in the isolated contracting perfused CP heart was 1.8-fold higher. Net lactate efflux was 1.5-fold higher. Furthermore, in vivo (13)C-magnetic resonance spectroscopy (MRS) of hyperpolarized [(13)C1]pyruvate revealed a twofold increase in real-time flux through lactate dehydrogenase in the CP hearts and a 1.6-fold increase through pyruvate dehydrogenase. (31)P-MRS of perfused CP hearts under increased workload (isoproterenol infusion) demonstrated increased depletion of phosphocreatine relative to ATP. Intriguingly, no changes in cardiac gene expression were detected. In summary, a modest systemic dysregulation of the HIF pathway resulted in clear alterations in cardiac metabolism and energetics. However, in contrast to studies generating high HIF levels within the heart, the CP mice showed neither the predicted changes in gene expression nor any degree of LV impairment. We conclude that the effects of manipulating HIF on the heart are dose dependent.

Purinergic dysregulation in pulmonary hypertension.

Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.

Exosomes induce and reverse monocrotaline-induced pulmonary hypertension in mice.

AIMS: Extracellular vesicles (EVs) from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) induce PH in healthy mice, and the exosomes (EXO) fraction of EVs from mesenchymal stem cells (MSCs) can blunt the development of hypoxic PH. We sought to determine whether the EXO fraction of EVs is responsible for modulating pulmonary vascular responses and whether differences in EXO-miR content explains the differential effects of EXOs from MSCs and mice with MCT-PH. METHODS AND RESULTS: Plasma, lung EVs from MCT-PH, and control mice were divided into EXO (exosome), microvesicle (MV) fractions and injected into healthy mice. EVs from MSCs were divided into EXO, MV fractions and injected into MCT-treated mice. PH was assessed by right ventricle-to-left ventricle + septum (RV/LV + S) ratio and pulmonary arterial wall thickness-to-diameter (WT/D) ratio. miR microarray analyses were also performed on all EXO populations. EXOs but not MVs from MCT-injured mice increased RV/LV + S, WT/D ratios in healthy mice. MSC-EXOs prevented any increase in RV/LV + S, WT/D ratios when given at the time of MCT injection and reversed the increase in these ratios when given after MCT administration. EXOs from MCT-injured mice and patients with idiopathic pulmonary arterial hypertension (IPAH) contained increased levels of miRs-19b,-20a,-20b, and -145, whereas miRs isolated from MSC-EXOs had increased levels of anti-inflammatory, anti-proliferative miRs including miRs-34a,-122,-124, and -127. CONCLUSION: These findings suggest that circulating or MSC-EXOs may modulate pulmonary hypertensive effects based on their miR cargo. The ability of MSC-EXOs to reverse MCT-PH offers a promising potential target for new PAH therapies.

The Succinate Receptor GPR91 Is Involved in Pressure Overload-Induced Ventricular Hypertrophy.

BACKGROUND: Pulmonary arterial hypertension is characterized by increased pressure overload that leads to right ventricular hypertrophy (RVH). GPR91 is a formerly orphan G-protein-coupled receptor (GPCR) that has been characterized as a receptor for succinate; however, its role in RVH remains unknown. METHODS AND RESULTS: We investigated the role of succinate-GPR91 signaling in a pulmonary arterial banding (PAB) model of RVH induced by pressure overload in SD rats. GPR91 was shown to be located in cardiomyocytes. In the sham and PAB rats, succinate treatment further aggravated RVH, up-regulated RVH-associated genes and increased p-Akt/t-Akt levels in vivo. In vitro, succinate treatment up-regulated the levels of the hypertrophic gene marker anp and p-Akt/t-Akt in cardiomyocytes. All these effects were inhibited by the PI3K antagonist wortmannin both in vivo and in vitro. Finally, we noted that the GPR91-PI3K/Akt axis was also up-regulated compared to that in human RVH. CONCLUSIONS: Our findings indicate that succinate-GPR91 signaling may be involved in RVH via PI3K/Akt signaling in vivo and in vitro. Therefore, GPR91 may be a novel therapeutic target for treating pressure overload-induced RVH.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. METHODS AND RESULTS: By Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. CONCLUSION: Our data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.

Lack of EC-SOD worsens alveolar and vascular development in a neonatal mouse model of bleomycin-induced bronchopulmonary dysplasia and pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) worsens clinical outcomes in former preterm infants with bronchopulmonary dysplasia (BPD). Oxidant stress disrupts alveolar and vascular development in models of BPD. Bleomycin causes oxidative stress and induces BPD and PAH in neonatal rats. Disruption in the vascular endothelial growth factor (VEGF) and nitric oxide signaling pathways contributes to BPD. We hypothesized that loss of EC-SOD would worsen PAH associated with BPD in a neonatal mouse model of bleomycin-induced BPD by disrupting the VEGF/NO signaling pathway. METHODS: Neonatal wild-type mice (WT), and mice lacking EC-SOD (EC-SOD KO) received intraperitoneal bleomycin (2 units/kg) or phosphate-buffered saline (PBS) three times weekly and were evaluated at weeks 3 or 4. RESULTS: Lack of EC-SOD impaired alveolar development and resulted in PH (elevated right ventricular systolic pressures, right ventricular hypertrophy (RVH)), decreased vessel density, and increased small vessel muscularization. Exposure to bleomycin further impaired alveolar development, worsened RVH and vascular remodeling. Lack of EC-SOD and bleomycin treatment decreased lung total and phosphorylated VEGFR2 and eNOS protein expression. CONCLUSION: EC-SOD is critical in preserving normal lung development and loss of EC-SOD results in disrupted alveolar development, PAH and vascular remodeling at baseline, which is further worsened with bleomycin and associated with decreased activation of VEGFR2.