Chronic Hypoxic Pulmonary Hypertension Research Articles

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

The Effect of SOD Mimetic Treatment on Interstitial Macrophage Accumulation in Chronic Hypoxic Pulmonary Hypertension

The Effect of SOD Mimetic Treatment on Interstitial Macrophage Accumulation in Chronic Hypoxic Pulmonary Hypertension

Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signalling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signalling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH). Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1), and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMCs), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. In the present study, we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signalling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH.

PDE9A deficiency does not prevent chronic-hypoxic pulmonary hypertension in mice.

Inhibition of cyclic guanosine monophosphate (cGMP)‐specific phosphodiesterases (PDEs) is a cornerstone of pulmonary arterial hypertension (PAH)‐specific therapy. PDE9A, expressed in the heart and lung tissue, has the highest affinity for cGMP of all known PDEs. PDE9A deficiency protects mice against chronic left ventricular (LV) pressure overload via increased natriuretic peptide (NP)‐dependent cGMP signaling. Chronic‐hypoxic pulmonary hypertension (CH‐PH) is a model of chronic right ventricular (RV) pressure overload, and previous studies have demonstrated a protective role for NPs in the murine model. Therefore, we hypothesized that PDE9A deficiency would promote NP‐dependent cGMP signaling and prevent RV remodeling in the CH‐PH model, analogous to findings in the LV. We exposed wild‐type and PDE9A‐deficient (Pde9a −/−) C57BL/6 mice to CH‐PH for 3 weeks. We measured RV pressure, hypertrophy, and levels of lung and RV cGMP, PDE9A, PDE5A, and phosphorylation of the protein kinase G substrate VASP (vasodilatory‐stimulated phosphoprotein) after CH‐PH. In wild‐type mice, CH‐PH was associated with increased circulating ANP and lung PDE5A, but no increase in cGMP, PDE9A, or VASP phosphorylation. Downstream effectors of cGMP were not increased in Pde9a −/− mice exposed to CH‐PH compared with Pde9a +/+ littermates, and CH‐PH induced increases in RV pressure and hypertrophy were not attenuated in knockout mice. Taken together, these findings argue against a prominent role for PDE9A in the murine CH‐PH model.

Increased intracellular Cl- concentration in pulmonary arterial myocytes is associated with chronic hypoxic pulmonary hypertension.

Chloride channels play an important role in regulating smooth muscle contraction and proliferation, and contribute to the enhanced constriction of pulmonary arteries (PAs) in pulmonary hypertension (PH). The intracellular Cl- concentration ([Cl-]i), tightly regulated by various Cl- transporters, determines the driving force for Cl- conductance, thereby the functional outcome of Cl- channel activation. This study characterizes for the first time the expression profile of Cl- transporters/exchangers in PA smooth muscle and provides the first evidence that the intracellular Cl- homeostasis is altered in PA smooth muscle cells (PASMCs) associated with chronic hypoxic PH (CHPH). Quantitative RT-PCR revealed that the endothelium-denuded intralobar PA of rats expressed Slc12a gene family-encoded Na-K-2Cl cotransporter 1 (NKCC1), K-Cl cotransporters (KCC) 1, 3, and 4, and Slc4a gene family-encoded Na+-independent and Na+-dependent Cl-/HCO3- exchangers. Exposure of rats to chronic hypoxia (10% O2, 3 wk) caused CHPH and selectively increased the expression of Cl--accumulating NKCC1 and reduced the Cl--extruding KCC4. The intracellular Cl- concentration ([Cl-]i) averaged at 45 mM and 47 mM in normoxic PASMCs as determined by fluorescent indicator MEQ and by gramicidin-perforated patch-clamp technique, respectively. The ([Cl-]i was increased by ∼10 mM in PASMCs of rats with CHPH. Future studies are warranted to further establish the hypothesis that the altered intracellular Cl- homeostasis contributes to the pathogenesis of CHPH.

Effects of Intermittent Normoxia on Chronic Hypoxic Pulmonary Hypertension and Right Ventricular Hypertrophy in Rats.

Liu, Chunlei, Xu Chen, Ge Guo, Xiang Xu, Xin Li, Qingxia Wei, Yanying Shen, Hanlu Li, Jianxiu Hao, Ya Ping Tian, and Kunlun He. Effects of intermittent normoxia on chronic hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. High Alt Med Biol. 22: 184-192, 2021. Background: Individuals with chronically low arterial oxygen tension owing to high altitude develop elevated rates of pulmonary hypertension (PH) and right ventricular (RV) hypertrophy. However, the effects of the frequency and duration of normoxic exposure on PH and RV hypertrophy have not been adequately assessed; thus, we aimed to analyze the same. Materials and Methods: PH and RV hypertrophy were induced in 60 rats using a hypobaric chamber. Of these 60 rats, every 10 were exposed to normoxic conditions for 30 minutes once (1T/D), three times (3T/D), or five times daily (5T/D), or for one 150-minute recovery daily (1LT/D). Furthermore, 10 rats were housed in a normoxic environment, and another 10 were subjected to continuous hypoxia. After 4 weeks, hemodynamic measurements were recorded, and the hearts were harvested for pathomorphological observations. Results: Average pulmonary arterial pressures (PAP) of control rats and those exposed to hypobaric hypoxia were 14.1 and 32.3 mmHg, respectively. After 30 minutes of exposure to normoxia 3T/D, 5T/D, or 1LT/D, PAP values were reduced to 27.1, 27.9, or 26.8 mmHg, respectively. Four weeks of hypoxic exposure elevated the RV/heart weight (HW) ratios, while exposure to normoxia 3T/D, 5T/D, and 1LT/D significantly reduced RV/HW. In addition, exposure to normoxia 3T/D, 5T/D, 1LT/D reduced the percentage wall thickness of the pulmonary artery as well as the hypertrophy indices of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain 7 (MYH-7). Conclusions: Thirty-minute exposure to normoxic conditions of 3T/D, 5T/D, or 1LT/D effectively ameliorates PH and RV thickening.

Serotonin and chronic hypoxic pulmonary hypertension activate a NADPH oxidase 4 and TRPM2 dependent pathway for pulmonary arterial smooth muscle cell proliferation and migration

5-Hydroxytryptamine (5-HT)-dependent signaling mediated through its transporters and receptors plays important roles in chronic hypoxic pulmonary hypertension (CHPH), which is associated with aberrant reactive oxygen species (ROS) production. NADPH oxidase 4 (NOX4) is one of the major sources of ROS in pulmonary vasculature, and has been implicated in the development of PH. NOX4 generates H2O2, which can activate the transient receptor potential melastatin 2 (TRPM2) channels, providing Ca2+ signals for cell proliferation and migration. However, the connection between 5-HT, NOX4, ROS and TRPM2 in the context of PH has not been established. Here we examined the level of 5-HT and expression of NOX4 and TRPM2, and their roles in pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration. NOX4 and TRPM2 were upregulated in pulmonary arteries of CHPH rats, which were associated with elevated levels of 5-HT and ROS, and enhanced proliferation and migration in PASMCs. The increase in ROS, and the enhanced proliferation and migration of PASMCs from CHPH rats were mimicked by treating normoxic PASMCs with 5-HT. 5-HT; and CH-induced ROS production were reversed by catalase, the NOX1/NOX4 inhibitor GKT137831, and Nox4 siRNA. 5-HT and H2O2 elicited Ca2+ responses were significantly augmented in CHPH PASMCs; and the augmented Ca2+ responses were obliterated by the 2-Aminoethoxydiphenyl borate (2-APB) and Trpm2-specific siRNA. Moreover, 5-HT and CH-induced proliferation and migration were suppressed by Nox4 or Trpm2 siRNA; and simultaneous transfection of both siRNA did not cause further inhibition. These results suggest that the 5-HT and CH-induced PASMC proliferation and migration were mediated, at least in part, by TRPM2 via activation of NOX4-dependent ROS production; and revealed a novel NOX4-ROS-TRPM2 signaling pathway for the pathogenesis of CHPH.

A Hemolytic Event in a Heartmate 3 Patient in the Setting of Polycythemia

Introduction The Heartmate 3 (HM3) LVAD has significantly reduced hemocompatibility events. We present a case of a hemolytic event in a HM3 patient likely related to underling polycythemia. Case Report A 49-year-old man with multisystem sarcoidosis (pulmonary and cardiac) complicated by stage D systolic heart failure status post HeartMate 3 left ventricular assist device (LVAD) implantation and secondary polycythemia attributed to chronic hypoxic respiratory disease and pulmonary hypertension presented with symptoms of left arm numbness and tingling followed by severe substernal chest pain. Initial examination was unremarkable. EKG revealed nonspecific intraventricular conduction delay. Troponin I was elevated at 6.1. Hemoglobin was elevated to 19. LDH was elevated to LDH 568 from baseline of 300s. INR was 1.6 upon arrival. LVAD interrogation revealed speed 5600 rpm, flow 4.5 L/min, power 4.4 Watts and pulsatility index of 4.8. Heparin drip, aspirin and statin were initiated for presumed NSTEMI. LDH remained elevated to 600s. Left heart catherization revealed an acute clot in mid-circumflex associated with 50% stenosis consistent with partial resolution of thrombosis. Further workup revealed normal haptoglobin, erythropoietin and total bilirubin levels however plasma free hemoglobin was elevated to 63.1. Urine hemoglobin was not detected. A JAK2 mutation was excluded. Slow therapeutic phlebotomies with replacement of 125cc of blood with equivalent normal saline were initiated given concern for hemolytic event in the setting of worsening polycythemia. Renal imaging was negative and pretest probability for hematologic malignancies remained low. Of note, the patient reported non-compliance with home oxygen therapy used only at the time of exertion. Compliance was reinforced. Subsequently, LDH trended down to baseline following successful heparin bridge to therapeutic warfarin level. The patient was discharged in stable condition. Summary An acute hemolytic event (and possible cardioembolic NSTEMI) was seen in our patient evidenced by an elevation of LDH, plasma hemoglobin and new coronary thrombus in the setting of worsening polycythemia. Despite a remarkable low rate of pump thrombosis, HM3 recipients with acquired pro-thrombotic states such as polycythemia maybe at risk for adverse hemocompatibility events. Close surveillance is warranted for these special populations.

Abstract 15303: Dimethylarginine Dimethylaminohydrolase-1 is Not Involved in Chronic Hypoxic Pulmonary Hypertension and Right Ventricular Hypertrophy in Mice

Introduction: Chronic hypoxia causes persistent pulmonary vasoconstriction and leads to pulmonary hypertension and right ventricular hypertrophy. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis; its level increases in hypoxia concomitantly with reduced activity of dimethylarginine dimethylaminohydrolases (DDAH-1 and DDAH-2), the enzymes metabolizing ADMA. DDAH knockout models may therefore help to understand the pathophysiological roles of this enzyme and its substrate, ADMA, in the development of hypoxia-associated pulmonary hypertension. Hypothesis: We hypothesized that DDAH1 knock-out mice have an attenuated hypoxia-induced elevation of ADMA and reduced right ventricular hypertrophy. Methods: DDAH1 knock-out mice (KO) and their wild-type littermates (WT) were subjected to normoxia (NX) or hypoxia (HX) during 21 days. We measured ADMA concentration in plasma, DDAH1 and DDAH2 expression in the lung, right ventricular hypertrophy by the Fulton index, cardiomyocyte hypertrophy by dystrophin staining of heart tissues, and muscularization of pulmonary arterioles by CD31 and α-actin staining of lung sections. Results: DDAH1 KO mice had higher ADMA concentration than WT under NX (2.31±0.33 μmol/l vs. 1.20±0.17 μmol/l; p < 0.05). ADMA significantly increased in WT-HX (to 1.74±0.86 μmol/l; p < 0.05 vs. normoxia), whilst it did not further increase in KO-HX (2.58±0.58 μmol/l; p = n.s.). This was paralleled by a 38±13% reduction in DDAH1 mRNA but not DDAH2 mRNA expression, and reduced DDAH protein expression. We observed right ventricular hypertrophy under hypoxia in both, WT and KO mice, with no significant differences between both genotypes. Further, cardiomyocyte hypertrophy and pulmonary arteriolar muscularization were significantly increased by hypoxia, but not significantly different between WT and KO mice. Conclusions: We conclude that chronic hypoxia causes an elevation of ADMA, which impairs NO production and leads to endothelial dysfunction and vasoconstriction. Downregulation of DDAH expression and activity may be involved in this; however, knockout of DDAH1 does not modify the pathophysiological changes in remodeling of the pulmonary vasculature and the right ventricle.

Preventive but nontherapeutic effect of danshensu on hypoxic pulmonary hypertension.

ObjectivesDanshensu is a traditional Chinese medicine that is used for treatment of cardiovascular diseases. We previously demonstrated its preventive effect against early-stage hypoxic pulmonary hypertension (HPH) in a rat model. To determine whether danshensu treatment might be useful for patients with chronic HPH, we examined its therapeutic effect in rats with prolonged HPH.MethodsAdult Sprague-Dawley rats received danshensu (80, 160, and 320 mg/kg) during or after hypoxia exposure to assess preventive and therapeutic effects, respectively. Right ventricle systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), and mean left carotid artery pressure (mCAP) were measured in each group. Western blotting was used to assess transforming growth factor (TGF)-β expression levels in rats and cultured cells exposed to hypoxia.ResultsPreventive danshensu treatment significantly reduced the elevation of RVSP and RVHI in rats exposed to hypoxia, whereas therapeutic danshensu treatment did not; mCAP did not change in any treatment group. The increased expression levels of TGF-β induced by hypoxia were inhibited by preventive danshensu treatment, but not by therapeutic danshensu treatment.ConclusionsAlthough danshensu treatment could prevent HPH, it had no obvious therapeutic effect after development of HPH. Therefore, danshensu might be suitable for clinical treatment of early-stage HPH.

Role of TG2-Mediated SERCA2 Serotonylation on Hypoxic Pulmonary Vein Remodeling.

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pumps take up Ca2+ from the cytoplasm to maintain the balance of intracellular Ca2+. A decline in expression or activity of SERCA results in persistent store-operated calcium entry (SOCE). In cardiomyocytes as well as vascular smooth muscle cells (SMCs), SERCA2 acts as an important regulator of calcium cycling. The purpose of this study is to identify and better understand the role of transglutaminases2 (TG2) as a key factor involved in SERCA2 serotonination (s-SERCA2) and to elucidate the underlying mechanism of action. Human pulmonary venous smooth muscle cell in normal pulmonary lobe were isolated and cultured in vitro. Establishment of hypoxic pulmonary hypertension model in wild type and TG2 knockout mice. SERCA2 serotonylation was analyzed by co-(immunoprecipitation) IP when the TG2 gene silenced or overexpressed under normoxia and hypoxia in vivo and in vitro. Intracellular calcium ion was measured by using Fluo-4AM probe under normoxia and hypoxia. Real-time (RT)-PCR and Western blot analyzed expression of TG2, TRPC1, and TRPC6 under normoxia and hypoxia. Bioactivity of cells were analyzed by using Cell Counting Kit (CCK)-8, flow cytometry, wound healing, RT-PCR, and Western blot under PST-2744 and cyclopiazonic acid. We confirmed that 1) hypoxia enhanced the expression and activity of TG2, and 2) hypoxia increased the basal intracellular Ca2+ concentration ([Ca2+]i) and SOCE through activating TRPC6 on human pulmonary vein smooth muscle cells (hPVSMC). Then, we investigated the effects of overexpression and downregulation of the TG2 gene on the activity of SERCA2, s-SERCA2, basal [Ca2+]i, and SOCE under normoxia and hypoxia in vitro, and investigated the activity of SERCA2 and s-SERCA2 in vivo, respectively. We confirmed that SERCA2 serotonylation inhibited the activity of SERCA2 and increased the Ca2+ influx, and that hypoxia induced TG2-mediated SERCA2 serotonylation both in vivo and in vitro. Furthermore, we investigated the effect of TG2 activity on the biological behavior of hPVSMC by using an inhibitor and agonist of SERCA2, respectively. Finally, we confirmed that chronic hypoxia cannot increase vessel wall thickness, the right ventricular systolic pressure (RVSP), and right ventricular hypertrophy index (RVHI) of vascular smooth muscle-specific Tgm2−/− mice. These results indicated that hypoxia promoted TG2-mediated SERCA2 serotonylation, thereby leading to inhibition of SERCA2 activity, which further increased the calcium influx through the TRPC6 channel. Furthermore, tissue-specific conditional TG2 knockout mice prevents the development of pulmonary hypertension caused by hypoxia. In summary, we uncovered a new target (TG2) for treatment of chronic hypoxic pulmonary hypertension (CHPH).

Cytochrome P450 Epoxygenase-Dependent Activation of TRPV4 Channel Participates in Enhanced Serotonin-Induced Pulmonary Vasoconstriction in Chronic Hypoxic Pulmonary Hypertension.

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic trpv4−/− mice. These results suggest that TRPV4 contributes to the enhanced 5-HT-induced vasoconstriction in chronic hypoxic PAs, in part via the CYP-EET-TRPV4 pathway. Our results further support the notion that manipulation of TRPV4 function may offer a novel therapeutic strategy for the treatment of hypoxia-related pulmonary hypertension.

Consequence of Desmin gene mutation on development of chronic hypoxic pulmonary hypertension in mice

Desmin deficiency is responsible for various forms of skeletal and cardiac disorders. However, role of desmin in the normal and pathological pulmonary vasculature remains unknown. To investigate whether desmin deficiency affects pulmonary circulation in normoxic and hypoxic conditions. Desmin-deficient mice (Des-/-) and their littermate Des+/+ (controls) aged 2–4 months were exposed to chronic hypoxia (9% O2, 2 weeks) or normoxia. Cardiac function was assessed by closed-chest echocardiography and right ventricle (RV) hemodynamics combined with measurements of Fulton Index and muscularization of pulmonary arteries (PA). Contractile properties of intrapulmonary arteries were measured by using an arteriography. Heart and lung phenotype were determined by using standard techniques of histology, immunostaining, immunoblot and RT-qPCR analysis. Under normal conditions Des-/- mice developed cardiomyopathy with alteration of both left ventricle (LV) and RV functions. Left Heart Disease was characterized by ventricular dilatation, decreased Ejection Fraction and Strain Rate in agreement with extreme transmural fibrosis and cardiomyocytes hypertrophy. RV dysfunction was evidenced by an increase of RV systolic pressure (RVSP) and PA muscularization. After exposure to hypoxia, LV function was globally not modified in both Des-/- and Des+/+ mice. RV was similarly affected in both strains, though the increase of RV weight, RVSP and PA muscularization were higher in Des-/- mice. Conversely, contractile capacities of PA were not modified in Des-/- mice in normoxia. Under hypoxia, we recorded modifications of several contractile, structural and Ca2 ± handling proteins in PA - smooth muscle cells (SMC). Our results indicate that desmin can be replaced by vimentin in SMC without loss of function. However, clinical features of desminopathies can also include pulmonary hypertension with PA remodeling and higher RV dysfunction.

Magnesium attenuates endothelin-1-induced vasoreactivity and enhances vasodilatation in mouse pulmonary arteries: Modulation by chronic hypoxic pulmonary hypertension.

What is the central question of this study? The central goal of this study was to elucidate the role of magnesium in the regulation of pulmonary vascular reactivity in relationship to hypoxic pulmonary hypertension. What is the main finding and its importance? We found that magnesium is essential for normal vasoreactivity of the pulmonary artery. Increasing the magnesium concentration attenuates vasoconstriction and improves vasodilatation via release of nitric oxide. Pulmonary hypertension is associated with endothelial dysfunction resulting in the suppression of magnesium modulation of vasodilatation. These results provide evidence that magnesium is important for the modulation of pulmonary vascular function. Pulmonary hypertension (PH) is characterized by enhanced vasoreactivity and sustained pulmonary vasoconstriction, arising from aberrant Ca2+ homeostasis in pulmonary arterial (PA) smooth muscle cells. In addition to Ca2+ , magnesium, the most abundant intracellular divalent cation, also plays crucial roles in many cellular processes that regulate cardiovascular function. Recent findings suggest that magnesium regulates vascular functions by altering the vascular responses to vasodilator and vasoactive agonists and affects endothelial function by modulating endothelium-dependent vasodilatation in hypertension. Administration of magnesium also decreased pulmonary arterial pressure and improved cardiac output in animal models of PH. However, the role of magnesium in the regulation of pulmonary vascular function related to PH has not been studied. In this study, we examined the effects of magnesium on endothelin-1 (ET-1)-induced vasoconstriction, ACh-induced vasodilatation and the generation of NO in PAs of normoxic mice and chronic hypoxia (CH)-treated mice. Our data showed that removal of extracellular magnesium suppressed vasoreactivity of PAs to both ET-1 and ACh. A high concentration of magnesium (4.8mm) inhibited ET-1-induced vasoconstriction in endothelium-intact or endothelium-disrupted PAs of normoxic and CH-treated mice, and enhanced the ACh-induced production of NO in PAs of normoxic mice. Moreover, magnesium enhanced ACh-induced vasodilatation in PAs of normoxic mice, and the enhancement was completely abolished after exposure to CH. Hence, in this study we demonstrated that increasing the magnesium concentration can attenuate the ET-1-induced contractile response and improve vasodilatation via release of NO from the endothelium. We also demonstrated that chronic exposure to hypoxia can cause endothelial dysfunction resulting in suppression of the magnesium-dependent modulation of vasodilatation.

Abstract 266: Phosphodiesterase 9A Deficiency Does Not Attenuate Chronic-hypoxic Pulmonary Hypertension

Background: Phosphodiesterase (PDE) inhibition is a cornerstone of therapy for pulmonary arterial hypertension. A novel role for PDE9A in natriuretic peptide-mediated left ventricular cyclic guanosine monophosphate (cGMP) signaling and remodeling has recently been described. Since cGMP-signaling can promote vaso-relaxation and prevent vascular smooth muscle cell proliferation and cardiac myocyte hypertrophy, we hypothesized that PDE9A deficiency would attenuate right ventricular (RV) and pulmonary vascular remodeling during chronic RV pressure overload. Methods: Chronic-hypoxic pulmonary hypertension (CH-PH) was induced in mice lacking PDE9A expression ( Pde9a -/- ) and wild-type littermates ( Pde9a +/+ ) by exposure to normobaric hypoxia (FiO2 = 10%) for 3 weeks. RV systolic pressures were measured to quantify PH, and RV hypertrophy was quantified as the ratio of RV free wall mass to LV/septal mass (RV/LV+S). Heart, lung, and serum were flash frozen for biochemical analyses. Results: In wild-type mice, exposure to chronic hypoxia resulted in significant increases in RV pressure (30 ± 2.4 vs. 20 ± 3.2 mm Hg; P = 0.0006) and RV hypertrophy (0.34 ± 0.04 vs. 0.25 ± 0.02; P < 0.0001). Serum ANP levels were increased 5-fold with CH-PH ( P = <0.0001), associated with a 72% reduction in lung Npr3 receptor expression (responsible for ANP clearance; P < 0.0001) but no increase in RV Nppa expression. After 3 weeks of CH-PH, tissue levels of cGMP were not increased in lung (993 ± 502 pg/mg vs. 667 ± 210 pg/mg; P = 0.27) or RV (47 ± 18 pg/mg vs. 49 ± 13 pg/mg; P = 0.88) homogenates. Accordingly, serine-239 phosphorylation of vasodilatory-stimulated phosphoprotein (VASP; a surrogate for protein kinase G activation) was not increased by CH-PH in lung or RV. Expression of Pde9a RNA was not increased by CH-PH in lung or RV. Pde9a -/- mice did not have attenuated CH-PH induced increases in RVH ( P = 0.27, 2-way ANOVA) or RVSP ( P = 0.23, 2-way ANOVA) when compared to Pde9a +/+ mice. In both Pde9a -/- and Pde9a +/+ mice, CH-PH was associated with significant increases in lung PDE5A expression. Conclusions: In a murine model of chronic RV pressure overload, PDE9A deficiency does not attenuate RV remodeling or PH, despite robust increases in circulating natriuretic peptide levels.

Calpain-2 activates Akt via TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells.

Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism.

The effects of the 5-HT2A receptor antagonist sarpogrelate hydrochloride on chronic hypoxic pulmonary hypertension in rats

ABSTRACTPurpose of the Study: This study investigated whether sarpogrelate hydrochloride (SPG), a 5-HT2A receptor antagonist, alleviates chronic hypoxic pulmonary hypertension (CH-PH) in rats by stimulating apoptosis and inhibiting proliferation in pulmonary artery smooth muscle cells (PASMCs). Materials and Methods: Forty male Sprague-Dawley rats were pretreated with SPG (50 mg/kg/day by oral gavage) or saline vehicle and then subjected to chronic hypoxia (CH) (hypobaric chamber set to 380 mmHg, 10% oxygen) or normoxia for 14 days. Mean pulmonary artery pressure (PAP) and right ventricular hypertrophy (RVH) were measured. Hypertensive pulmonary vascular remodeling was assayed by light microscopy. Terminal deoxynucletidyl transferase dUTP nick end ligase (TUNEL) assays, western blotting, and real-time polymerase chain reaction were used to assess apoptosis, proliferation and underlying signaling pathways in PASMCs from lung tissue and isolated pulmonary artery rings. Results: CH increased mean PAP and RVH. CH increased the percentage of muscularized arteries in the peripheral pulmonary vasculature and medial wall thickness in small muscular arteries. CH increased pulmonary protein and mRNA levels of the B-cell lymphoma protein 2 (Bcl-2), pyruvate dehydrogenase kinase (PDK), phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2), cyclin D1, proliferating cell nuclear antigen (PCNA) and decreased protein and mRNA levels of Bcl-2-associated X protein (BAX), cleaved caspase-3. Pretreatment with SPG, which has been shown previously to inhibit ERK1/2 phosphorylation and PDK, countered all of these effects. Isolated pulmonary artery rings incubated with 5-HT increased pERK1/2, PDK, and Bcl-2 expression, and decreased Bax expression. Conclusion: Administration of SPG ameliorated the development of CH-PH by stimulating apoptosis in and inhibiting proliferation of PASMCs.

TRPC6 is required for hypoxia-induced basal intracellular calcium concentration elevation, and for the proliferation and migration of rat distal pulmonary venous smooth muscle cells.

Hypoxia induces pulmonary vasoconstriction and reconstruction in the pulmonary arteries and pulmonary veins (PVs), and elevation of intracellular calcium concentration ([Ca2+]i) is a primary factor of these processes. In the present study, the role of transient receptor potential cation channels (TRPCs) in mediating the hypoxia-induced elevation of [Ca2+]i in rat distal pulmonary venous smooth muscle cells (PVSMCs) was investigated. Rats with chronic hypoxic pulmonary hypertension (CHPH) were used for in vivo experiments, and PVSMCs were isolated for in vitro experiments. [Ca2+]i was measured using fura-2-based fluorescence calcium imaging. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of TRPCs. Methyl thiazolyl tetrazolium and Transwell assays were used to investigate the proliferation and migration of PVSMCs, respectively. The results of the present study demonstrated that TRPC6 was increased in the distal PVs of CHPH rats, and in PVSMCs exposed to hypoxic conditions (4% O2, 72 h); however, TRPC1 was not. The 1-oleoyl-2-acetyl-sn-glycerol-induced [Ca2+]i elevation was increased in PVSMCs isolated from CHPH rats and in PVSMCs cultured under hypoxic conditions (4% O2, 72 h). Hypoxia induced PVSMC [Ca2+]i elevation, proliferation and migration. These alterations were inhibited following TRPC6 knockdown. Results from the present study suggest that TRPC6 expression is increased during chronic hypoxia, which contributes to Ca2+ entry into the cell, thus promoting proliferation and migration of PVSMCs.

Effect of panax notoginseng saponins on pulmonary artery pathology of pulmonary hypertension rats

Objective To observe the effect of panax notoginseng saponins on pulmonary artery pathology in chronic hypoxic pulmonary hypertensionp (HPH) rats models, discuss the role and possible mechanisms of panax notoginseng saponins in prevention of chronic hypoxic pulmonary hypertension. Methods Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n=10). Group C: control group; Group H: rats were treated with hypoxia only; Group HC: rats were treated with hypoxia and captopril; and Group HP: rats were treated with hypoxia and panax notoginseng saponins. To observe the effect of panax notoginseng saponins prophylactic treatment in chronic hypoxic pulmonary hypertension rats, after the establishment of the model of hypoxic rat animal pulmonary hypertension models, transthoracic direct pulmonary artery intubation was measured in rat pulmonary arterial systolic pressure and mean pulmonary artery pressure, at the end of separation and cut for pulmonary artery to observe the pathological changes of pulmonary artery, results were recorded and statistically analyzed. Results ⑴Transthoracic direct pulmonary artery intubation was measured in rat pulmonary arterial systolic pressure and mean pulmonary artery pressure: pulmonary artery systolic pressure (PASP) and mean pulmonary arterial pressure (MPAP) were significantly higher in Group H than in Group C (P<0.05), Group HP was no significant difference relative to Group C. ⑵Pathological section shew that the primary pathological change of group H is vascular intimal hyperplasia and the proliferation of smooth muscle cells, medial hypertrophy, extracellular matrix increased, thickening of the vessel wall and the vascular stenos. Conclusions Panax notoginseng saponins inhibit the formation of chronic hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats, and have the effect of prevention and treatment of pulmonary hypertension. Key words: SANCHINOSIDE/PD; Hypertension, pulmonary/DT/PA; Pulmonary artery/PA; Anoxia

Abstract 378: Endothelial Cell Hypoxia-inducible Factor (HIF) Activity is Necessary for Right Ventricular Remodeling in a Murine Model of Chronic-hypoxic Pulmonary Hypertension

Introduction: Pulmonary hypertension induced myocardial remodeling is required to minimize RV wall stress as pulmonary arterial pressures increase. Myocardial angiogenesis is presumed to play an integral role in early RV remodeling, though molecular regulators remain undefined. Endothelial cell (EC) expression of HIF-1 is necessary for adaptive LV remodeling during chronic pressure overload, with loss of EC-specific HIF-1 activity associated with reduced LV capillary density. We hypothesized that EC HIF activity may also be necessary for adaptive RV remodeling and angiogenesis in a model of chronic RV pressure overload. Methods: We used a murine model of chronic hypoxic pulmonary hypertension (CH-PH) to induce RV remodeling. Conditional transgenic mice lacking expression of the obligate HIF-1α/HIF-2α co-factor ARNT (aryl hydrocarbon receptor nuclear translocator) in Tie2+ lineage cells ( ARNT fl/fl ; Tie2-Cre +/- ) and ARNT fl/fl ;Tie2-Cre -/- controls were exposed to normobaric hypoxia (10% FiO 2 ) for 1-5 weeks. We measured RV hypertrophy (RVH), RV systolic pressure (RVSP), and myocardial EC proliferation (flow cytometry) as a marker for sprouting angiogenesis. Results: After three weeks of CH-PH, RVH was observed in ARNT fl/fl ;Tie2-Cre -/- control mice, as anticipated (0.332±0.02 vs. 0.231±0.02; P &lt; 0.05). CH-PH induced RVH in ARNT fl/fl ;Tie2-Cre -/- mice was associated with an early (1 week) increase in RV EC proliferation (vs. LV, 4.5±1.3% vs.1.8±0.5%; P &lt; 0.05). Conversely, ARNT fl/fl ; Tie2 - Cre +/- mice did not develop RVH after 3 weeks of CH ( 0.222±0.01 vs. 0.241±0.02; P = NS), and early RV EC proliferation was attenuated (vs. LV, 2.9±1.2% vs. 1.5±0.3%; P = NS). Despite similar increases in CH-PH induced RVSP after 3 weeks, after 5 weeks RVSP normalized in ARNT fl/fl ; Tie2 - Cre +/- mice, but remained elevated in ARNT fl/fl ;Tie2-Cre -/- controls. Conclusions: These preliminary findings confirm that EC-specific HIF activity is necessary for CH-PH induced RVH, and suggest that reduced EC proliferation (and potentially angiogenesis) may contribute to abrogated RV remodeling in ARNT fl/fl ; Tie2 - Cre +/- mice. We hypothesize that late normalization of RVSP in these mice may be indicative of premature RV failure.