Mouse Pulmonary Artery Smooth Muscle Cells Research Articles

Therapeutic potential and protective role of GRK6 overexpression in pulmonary arterial hypertension

Abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key mechanism in the development of pulmonary arterial hypertension (PAH). Signal transducer and activator of transcription 3 (STAT3) signalling plays a critical role in modulating PASMC proliferation, and G-protein-coupled receptor kinase 6 (GRK6) regulates the STAT3 pathway. However, the mechanism underlying the relationship between GRK6 and PAH remains unclear. In this study, we aimed to investigate the role of GRK6 in PAH and determine its potential as a therapeutic target. We utilised hypoxia- and SU5416-induced PAH mouse models and a monocrotaline-induced PAH rat model to analyse the involvement of GRK6. We conducted gain- and loss-of-function experiments using mouse PASMCs. Modulation of GRK6 expression was achieved via a lentiviral vector in vitro and an adeno-associated virus serotype 1 encoding GRK6 in vivo. GRK6 was significantly downregulated in the lung tissues of PAH mice and rats, predominantly in PASMCs. Knockout of GRK6 exacerbated PAH, while both therapeutic and prophylactic overexpression of GRK6 alleviated PAH, as evidenced by a reduction in right ventricular systolic pressure, right ventricular wall to left ventricular wall plus ventricular septum ratio, pulmonary vascular media thickness, and pulmonary vascular muscularisation. Mechanistically, GRK6 overexpression attenuated hypoxia-induced PASMC proliferation and STAT3 phosphorylation. Conversely, knockdown of GRK6 promoted hypoxia-induced proliferation, which was mitigated by a STAT3 inhibitor. Our findings highlight the potential protective and beneficial roles of GRK6 in PAH; we propose a lung-targeted GRK6 gene therapy utilizing adeno-associated virus serotype 1 as a potential treatment approach for patients with PAH.

Nicotine promotes e-cigarette vapour-induced lung inflammation and structural alterations.

Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear. In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations (e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL-1; control: 37.0±21.1 cells·mL-1; NF ECV: 198.6±94.9 cells·mL-1) and in lung tissue (ECV: 25.7±3.3 cells·mm-3; control: 4.8±1.1 cells·mm-3; NF ECV: 14.1±2.2 cells·mm-3). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function (e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV. NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations.

JMJD1C promotes smooth muscle cell proliferation by activating glycolysis in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a chronic disorder characterized by hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs). JMJD1C, a member of the Jumonji domain containing C (JMJC) histone demethylase family, contributes to cardiovascular dysfunction. However, the role of JMJD1C in PAH remains unknown. Mice were exposed to hypoxia to mimic several features associated with PAH clinically. We found that JMJD1C was highly expressed in the lungs of mice after hypoxia exposure. JMJD1C knockdown ameliorated hypoxia-induced right ventricular remodeling and thickening of the pulmonary arterial wall. PASMC hyperproliferation and resistance to apoptosis in mice exposed to hypoxia were suppressed by JMJD1C inhibition. We demonstrated that JMJD1C silencing reduced glycolytic enzymes (HK2, PGK1 and LDHA) and lactate overaccumulation in the lungs of mice exposed to hypoxia. In vitro, hypoxia-induced hyperproliferation and activated glycolytic processes in mouse PASMCs were impaired by JMJD1C knockdown. In addition, the activation of STAT3 signaling by hypoxia was suppressed by JMJD1C silencing both in vivo and in vitro. The overexpression of STAT3 reversed the inhibitory effect of JMJD1C depletion on proliferation and glycolysis in PASMCs under hypoxia. Thus, JMJD1C induces glycolytic processes by activating STAT3 signaling to promote PASMC proliferation and pulmonary vascular remodeling, suggesting the potential role of JMJD1C in regulating the metabolic program and vascular remodeling in PAH.

Up-regulation of nPKC contributes to proliferation of mice pulmonary artery smooth muscle cells in hypoxia-induced pulmonary hypertension

This study is designed to investigate the role of novel protein kinases C (nPKC) in mediating pulmonary artery smooth muscle cells (PASMCs) proliferation in pulmonary hypertension (PH) and the underlying mechanisms. Mouse PASMCs was isolated using magnetic separation technology. The PASMCs were divided into 24 h group, 48 h group and 72 h group according to different hypoxia treatment time, then detected cell proliferation rate and nPKC expression level in each group. We treated PASMCs with agonists or inhibitors of PKCdelta (PKCδ) and PKCepsilon (PKCε) and exposed them to hypoxia or normoxia for 72 h, then measured the proliferation of PASMCs. We also constructed a lentiviral vector containing siRNA fragments for inhibiting PKCδ and PKCε to transfected PASMCs, then examined their proliferation. PASMCs isolated successfully by magnetic separation method and were in good condition. Hypoxia promoted the proliferation of PASMCs, and the treatment for 72 h had the most significant effect. Hypoxia upregulated the expression of PKCδ and PKCε in mouse PASMCs, leading to PASMCs proliferation. Moreover, Our study demonstrated that hypoxia induced upregulation of PKCδ and PKCε expression resulting to the proliferation of PASMCs via up-regulating the phosphorylation of AKT and ERK. Our study provides clear evidence that increased nPKC expression contributes to PASMCs proliferation and uncovers the correlation between AKT and ERK pathways and nPKC-mediated proliferation of PASMCs. These findings may provide novel targets for molecular therapy of pulmonary hypertension.

Abstract 17220: Functional Roles and Molecular Mechanisms of Calcium Release Signaling in Hypoxic Cellular Responses in Pulmonary Artery Smooth Muscle Cells

Introduction: Hypoxia causes contractile and proliferative responses in pulmonary arterial smooth muscle cells (PASMCs), but not systemic artery SMCs, thereby leading to pulmonary hypertension (PH), but not systemic hypertension. However, the underlying mechanisms remain unclear. Hypothesis: Ca 2+ release signaling in PASMCs may mediate hypoxic pulmonary artery vasoconstriction, remodeling and hypertension. Methods: Single cell RT-PCR and Western blot analysis, FRET and immunoprecipitation, tissue contraction assay, immunohistochemistry staining and pressure-volume loop analysis, and genetic and pharmacological approaches, were, respectively, used to determine targeted molecular expression, interaction and function. Results: All ryanodine receptor (RyR1, RyR2 and RyR3) Ca 2+ release channels are expressed in mouse PASMCs. RyR1 or RyR3 knockout (KO) partially inhibits, while RyR2 KO completely blocks, hypoxic cellular responses. SMC-specific RyR2 KO restores abolishes hypoxic pulmonary artery vasoconstriction, remodeling and hypertension in mice. RyR2 KO also inhibits hypoxic nuclear factor (NF)-κB and downstream cyclin D1 activation. FK506 binding protein 12.6 (FKBP12.6) is an endogenous RyR2 inhibitor; its KO or FK506 treatment enhances, and treatment of S107 (a specific RyR2/FKBP12.6 stabilizer) inhibits hypoxic PH. Lentiviral shRNA-mediated SMC-specific Rieske iron-sulfur protein (RISP) knockdown diminishes mitochondrial reactive oxygen species (ROS) generation, RyR2/FKBP12.6 complex dissociation, and RyR2 activation, thereby blocking hypoxic PH. Conclusions: RyR1, RyR2 and RyR3 are all involved in hypoxic contractile and proliferative responses in PASMCs, but RyR2 is a most important player. RISP-dependent mitochondrial ROS - FKBP12.6/RyR2 complex dissociation - RyR2 hyperfunction signaling mediate PA vasoconstriction. RyR2 hyperfunction also activates Ca2+-dependent NF-κB/cyclinD1 pathway to provoke PA remodeling; PA vasoconstriction and remodeling together cause PH. Furthermore, RISP-mediated ROS inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be novel and effective options for the treatment of PH.

Effects of apolipoprotein E on proliferation of mouse pulmonary arterial smooth muscle cells induced by hypoxia

To investigate the effects of apolipoprotein E (apoE) on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia. Primary culture of mouse PASMCs was prepared from male C57BL/6 mouse pulmonary artery by the method of tissue block anchorage. PASMCs were divided into four groups: normoxia group, normoxia with apoE administration group, hypoxia group and hypoxia with apoE administration group. The proliferation of PASMCs was observed by EdU incorporation. The protein levels of apoE, proliferating cell nuclear antigen (PCNA), protein kinase C (PKC) and phosphorylated protein kinase C (p-PKC) were analyzed by Western blot. The percentage of PASMCs proliferation of hypoxia group was significantly higher than that of normoxia group by 64.7% (P＜0.05), and the protein expression levels of PCNA and p-PKC of hypoxia group were up-regulated than those of normoxia group by 69.0% and 120.0%, while the protein expression of apoE was down-regulated by 51.0% (P＜0.05), respectively. The percentage of PASMCs proliferation of hypoxia with apoE administration group was significantly lower than that of hypoxia group by 19.6% (P＜0.05), and the protein expression levels of PCNA and p-PKC of hypoxia with apoE administration group were down-regulated than those of hypoxia group by 19.8% and 103.2% (P＜0.05), respectively. There was no significant difference among each group in the protein expression of PKC, nor do there any significant difference between normoxia group and hypoxia group in the protein expression of p-PKC (P＞0.05). ApoE can inhibit the proliferation of PASMCs induced by hypoxia, and the mechanism of its effect may be attributed to blocking PKC pathway.

Type 2 inositol 1,4,5-trisphosphate receptor inhibits the progression of pulmonary arterial hypertension via calcium signaling and apoptosis.

Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca2+ signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca2+ signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IP3R2), which is an intracellular Ca2+ release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IP3R2-/- mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IP3R2-/- murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca2+ entry (SOCE) was significantly enhanced in IP3R2-/- PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca2+ imaging. Furthermore, the enhancement of SOCE in IP3R2-/- PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IP3R2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IP3R in the development of PAH and may contribute to the development of targeted therapies.

Smooth muscle cell-specific FoxM1 controls hypoxia-induced pulmonary hypertension

RationaleForkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known. MethodsWe determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O2, 90% N2) or normoxia (Room air, 21% oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins. ResultsWe showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling. ConclusionsOur studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC.

PLCγ1-PKCε-IP3R1 signaling plays an important role in hypoxia-induced calcium response in pulmonary artery smooth muscle cells.

Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs). We have reported that phospholipase C-γ1 (PLCγ1) plays a significant role in the hypoxia-induced increase in [Ca2+]i in PASMCs and attendant HPV. In this study, we intended to determine molecular mechanisms for hypoxic Ca2+ and contractile responses in PASMCs. Our data reveal that hypoxic vasoconstriction occurs in pulmonary arteries, but not in mesenteric arteries. Hypoxia caused a large increase in [Ca2+]i in PASMCs, which is diminished by the PLC inhibitor U73122 and not by its inactive analog U73433 . Hypoxia augments PLCγ1-dependent inositol 1,4,5-trisphosphate (IP3) generation. Exogenous ROS, hydrogen peroxide (H2O2), increases PLCγ1 phosphorylation at tyrosine-783 and IP3 production. IP3 receptor-1 (IP3R1) knock-down remarkably diminishes hypoxia- or H2O2-induced increase in [Ca2+]i. Hypoxia or H2O2 increases the activity of IP3Rs, which is significantly reduced in protein kinase C-ε (PKCε) knockout PASMCs. A higher PLCγ1 expression, activity, and basal [Ca2+]i are found in PASMCs, but not in mesenteric artery smooth muscle cells from mice exposed to chronic hypoxia (CH) for 21 days. CH enhances H2O2- and ATP-induced increase in [Ca2+]i in PASMCs and PLC-dependent, norepinephrine-evoked pulmonary vasoconstriction. In conclusion, acute hypoxia uniquely causes ROS-dependent PLCγ1 activation, IP3 production, PKCε activation, IP3R1 opening, Ca2+ release, and contraction in mouse PASMCs; CH enhances PASM PLCγ1 expression, activity, and function, playing an essential role in pulmonary hypertension in mice.

RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for PAH treatment.

Chronic hypoxia increases pro‐inflammatory TH17 cells via NFATc3‐induced interleukin 6 production in lung (1175.5)

We have demonstrated that the adaptive immune system along with nuclear factor of activated T cells isoform c3 (NFATc3) contribute to chronic hypoxia (CH)‐induced pulmonary hypertension (PH). NFATc3 regulates IL‐6 expression in multiple cells types. Considering that IL‐6 contributes to hypoxic PH and is required for polarization of naïve CD4+ T helper cells to the TH17 subset, we hypothesized that NFATc3 drives IL‐6 expression in pulmonary arterial smooth muscle cells (PASMC) contributing to CH‐induced increases in lung TH17 cells. Since TH17 cells are implicated in several cardiovascular diseases, these cells may play an unexplored role in PH. In support of our hypothesis, we found that IL‐6 transcripts as determined by real‐time PCR were increased in cultured mouse PASMC in which NFAT was activated with ionomycin and phorbol 12‐myristate 13‐acetate. This response was prevented by the calcineurin/NFAT inhibitor cyclosporine A and is consistent with our previous report demonstrating that CH activates NFATc3 in PASMC. Furthermore, lung TH17 cells as determined by flow cytometry were increased in male C57BL/6 mice exposed to 5 days of CH (PB=380 mmHg) compared to normoxic mice. This response was prevented by i.p. administration of a selective NFAT inhibitor (A‐285222, AbbVie). Our data suggest that CH‐induced PASMC NFATc3 activation and subsequent IL‐6 production contribute to increases in lung TH17 cells.Grant Funding Source: Supported by T32 HL07736, R01HL088151, R01 HL088192, R01 AI097202

Mir-206 Regulates Pulmonary Artery Smooth Muscle Cell Proliferation and Differentiation

Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.

TRPC1 and Orai1 interact with STIM1 and mediate capacitative Ca2+entry caused by acute hypoxia in mouse pulmonary arterial smooth muscle cells

Previous studies in pulmonary artery smooth muscle cells (PASMCs) showed that acute hypoxia activates capacitative Ca(2+) entry (CCE) but the molecular candidate(s) mediating CCE caused by acute hypoxia remain unclear. The present study aimed to determine if transient receptor potential canonical 1 (TRPC1) and Orai1 interact with stromal interacting molecule 1 (STIM1) and mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura-2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. Acute hypoxia also increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+), and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive rise in [Ca(2+)](i) and the increase in Mn(2+) quench rate were both inhibited in cells treated with TRPC1 antibody or TRPC1 small interfering (si)RNA, in STIM1 siRNA-transfected cells and in Orai1 siRNA-transfected cells. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca(2+)](i) and Mn(2+) quench rate caused by acute hypoxia, and they were reduced in cells treated with TRPC1 antibody and in cells transfected with Orai1 siRNA. Furthermore, TRPC1 and Orai1 coimmunoprecipitated with STIM1 and the precipitation levels of TRPC1 and Orai1 were increased in cells exposed to acute hypoxia. Immunostaining showed colocalizations of TRPC1-STIM1 and Orai1-STIM1, and the colocalizations of these proteins were more apparent in acute hypoxia. These data provide direct evidence that TRPC1 and Orai1 channels mediate CCE through activation of STIM1 in acute hypoxic mouse PASMCs.

Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

IntroductionPulmonary Arterial Hypertension (PAH) is a progressively devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. The proliferation of cancer cells is mediated by increased expression of Enhancer of Zeste Homolog 2 (EZH2), a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes. However, the role of EZH2 in PAH has not been studied. In this study, it is hypothesized that EZH2 could play a role in the proliferation of PASMCs.MethodsIn the present study, the expression patterns of EZH2 were investigated in normal and hypertensive mouse PASMCs. The effects of EZH2 overexpression on the proliferation of human PASMCs were tested. PASMCs were transfected with EZH2 or GFP using nucleofector system. After transfection, the cells were incubated for 48 hours at 37°C. Proliferation and cell cycle analysis were performed using flow cytometry. Apoptosis of PASMCs was determined using annexin V staining and cell migration was tested by wound healing assay.ResultsEZH2 protein expression in mouse PASMCs were correlated with an increase in right ventricular systolic pressure and Right Ventricular Hypertrophy (RVH). The overexpression of EZH2 in human PASMCs enhances proliferation, migration, and decrease in the rate of apoptosis when compared to GFP-transfected cells. In the G2/M phase of the EZH2 transfected cells, there was a 3.5 fold increase in proliferation, while there was a significant decrease in the rate of apoptosis of PASMCs, when compared to control.ConclusionThese findings suggest that EZH2 plays a role in the migration and proliferation of PASMCs, which is a major hallmark in PAH. It also suggests that EZH2 could play a role in the development of PAH and can serve as a potential target for new therapies for PAH.

BMP type II receptor deficiency confers resistance to growth inhibition by TGF-β in pulmonary artery smooth muscle cells: role of proinflammatory cytokines

Mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (HPAH) and a significant proportion of sporadic cases. Pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) not only exhibit attenuated growth suppression by BMPs, but an abnormal mitogenic response to transforming growth factor (TGF)-β1. We sought to define the mechanism underlying this loss of the antiproliferative effects of TGF-β1 in BMPR-II-deficient PASMCs. The effect of TGF-β1 on PASMC proliferation was characterized in three different models of BMPR-II dysfunction: 1) HPAH PASMCs, 2) Bmpr2(+/-) mouse PASMCs, and 3) control human PASMCs transfected with BMPR-II small interfering RNA. BMPR-II reduction consistently conferred insensitivity to growth inhibition by TGF-β1. This was not associated with altered canonical TGF-β1/Smad signaling but was associated with a secreted factor. Microarray analysis revealed that the transcriptional responses to TGF-β1 differed between control and HPAH PASMCs, particularly regarding genes associated with interleukins and inflammation. HPAH PASMCs exhibited enhanced IL-6 and IL-8 induction by TGF-β1, an effect reversed by NF-κB inhibition. Moreover, neutralizing antibodies to IL-6 or IL-8 restored the antiproliferative effect of TGF-β1 in HPAH PASMCs. This study establishes that BMPR-II deficiency leads to failed growth suppression by TGF-β1 in PASMCs. This effect is Smad-independent but is associated with inappropriately altered NF-κB signaling and enhanced induction of IL-6 and IL-8 expression. Our study provides a rationale to test anti-interleukin therapies as an intervention to neutralize this inappropriate response and restore the antiproliferative response to TGF-β1.

Contractile and electrophysiological properties of pulmonary artery smooth muscle are not altered in TASK‐1 knockout mice

The acid-sensitive, two-pore domain K+ channel, TASK-1, contributes to the background K+ conductance and membrane potential (Em) of rat and human pulmonary artery smooth muscle cells (PASMCs), but its role in regulating tone remains elusive. This study aimed to clarify the role of TASK-1 by determining the functional properties of pulmonary artery (PA) from mice in which the TASK-1 gene was deleted (TASK-1/3 KO), in comparison with wild-type (WT) C57BL/6 controls. Small vessel wire myography was used to measure isometric tension developed by intact PA. Em and currents were recorded from freshly isolated PASMCs using the perforated patch-clamp technique. Reverse transcription-polymerase chain reaction (RT-PCR) was used to estimate K+ channel expression. We could find no difference between PA from WT and TASK-1/3 KO mice. They showed similar constrictor responses to a range of agonists and K+ concentrations, the K+ channel blockers 4-aminopyridine, tetraethylammonium ions and XE991. Treprostinil, proposed to dilate by activating TASK-1, was just as effective in TASK-1/3 KO arteries. Blocking Ca2+ influx with nifedipine (1 μM) or levcromakalim (10 μM) had no effect on resting tone in either strain. The resting Em of PASMCs and its responses to K+ channel blockers were unchanged in TASK-1/3 KO mice as were voltage-activated K+ currents, including the non-inactivating K+ current (IKN) measured at 0 mV. The Em was, however, depolarised in comparison with other species.Mouse IKN was much smaller than in rat and showed no sensitivity to pH. The results imply that TASK-1 does not form a functional channel in mouse PASMCs.

TRPC1, STIM1 and Orai1 Mediate Capacitative Calcium Entry Activated by Acute Hypoxia in Mouse Pulmonary Arterial Smooth Muscle Cells

Previous studies in pulmonary artery smooth muscle cells (PASMCs) showed that acute hypoxia activates capacitative Ca2+ entry (CCE) but the molecular channel(s) and the molecular signal(s) mediating CCE caused by acute hypoxia remain unclear. The present study aimed to determine if TRPC1, STIM1 and Orai1 mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura-2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). The transient but not sustained rise in [Ca2+]i was partially inhibited by nifedipine. In addition, acute hypoxia increased the rate of Mn2+ quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni2+, La3+ and Gd3, exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells pretreated with antibodies raised against extracellular epitopes of TRPC1, in cells transfected with STIM1 siRNA and in cells transfected with Orai1 siRNA. Taken together, acute hypoxia causes activation of voltage-operated Ca2+ entry and CCE. These data suggest that TRPC1, STIM1 and Orai1 mediate CCE activated by acute hypoxia in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca(2+) entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.

Orai1 and STIM1 Mediate Capacitative Ca2+ Entry in Mouse Pulmonary Arterial Smooth Muscle Cells

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 and STIM1 mediates the sustained component of capacitative Ca2+ entry (CCE) but the molecular candidate(s) that mediate the transient component of CCE remains unknown. The aim of the present study was to further examine if Orai1 mediates the transient component of CCE through activation of STIM1 protein in mouse PASMCs.In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i).The transient but not the sustained rise in [Ca2+]i was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 siRNA. Moreover, over-expression of STIM1 enhanced the rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to co-immunoprecipitate with STIM1 and immunostaining showed co-localization of Orai1 and STIM1 proteins. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that the TRPC1 channel mediates capacitative Ca(2+) entry (CCE), but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediates CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. In addition, CPA increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+) and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive sustained rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both inhibited in cells pretreated with antibody raised against an extracellular epitope of TRPC1. Moreover, STIM1 siRNA reduced the rise in [Ca(2+)](i) and Mn(2+) quench of fura-2 fluorescence caused by CPA, whereas overexpression of STIM1 resulted in a marked increase in these responses. RT-PCR revealed TRPC1 and STIM1 mRNAs, and Western blot analysis identified TRPC1 and STIM1 proteins in mouse PASMCs. Furthermore, TRPC1 was found to co-immunoprecipitate with STIM1, and the precipitation level of TRPC1 was increased in cells subjected to store depletion. Taken together, store depletion causes activation of voltage-operated Ca(2+) entry and CCE. These data provide direct evidence that CCE is mediated by TRPC1 channel through activation of STIM1 in mouse PASMCs.