The physiological function of the purinergic receptor P2Y2 in pulmonary arterial endothelial cells and its role in pulmonary hypertension

2020 ◽  
Author(s):  
J Stockburger ◽  
M Shihan ◽  
S Offermanns ◽  
HA Ghofrani ◽  
F Grimminger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Physiological Function ◽  
Purinergic Receptor ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging

2007 ◽  
Vol 292 (3) ◽  
pp. H1373-H1389 ◽  
Author(s):  
Somshuvra Mukhopadhyay ◽  
Fang Xu ◽  
Pravin B. Sehgal
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Plasma Membrane ◽  
Live Cell ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Pulmonary Arterial ◽  
And Function ◽  
Endothelial Nitric Oxide ◽  
Arterial Endothelial Cells

We previously reported the disruption of caveolae/rafts, dysfunction of Golgi tethers, N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptor proteins (SNAREs), and SNAPs, and inhibition of anterograde trafficking in endothelial cells in culture and rat lung exposed to monocrotaline pyrrole (MCTP) as a prelude to the development of pulmonary hypertension. We have now investigated 1) whether this trafficking block affects subcellular localization and function of endothelial nitric oxide (NO) synthase (eNOS) and 2) whether Golgi blockade and eNOS sequestration are observed after hypoxia and senescence. Immunofluorescence data revealed that MCTP-induced “megalocytosis” of pulmonary arterial endothelial cells (PAEC) was accompanied by a loss of eNOS from the plasma membrane, with increased accumulation in the cytoplasm. This cytoplasmic eNOS was sequestered in heterogeneous compartments and partially colocalized with Golgi and endoplasmic reticulum (ER) markers, caveolin-1, NOSTRIN, and ER Tracker, but not Lyso Tracker. Hypoxia and senescence also produced enlarged PAEC, with dysfunctional Golgi and loss of eNOS from the plasma membrane, with sequestration in the cytoplasm. Live-cell imaging of caveolar and cytoplasmic NO with 4,5-diaminofluorescein diacetate (DAF-2DA) as probe showed a marked loss of caveolar NO after MCTP, hypoxia, and senescence. Although ionomycin stimulated DAF-2DA fluorescence in control PAEC, this ionophore decreased DAF-2DA fluorescence in MCTP-treated and senescent PAEC, suggesting localization of eNOS in an aberrant cytoplasmic compartment that was readily discharged by Ca2+-induced exocytosis. Thus monocrotaline, hypoxia, and senescence produce a Golgi blockade in PAEC, leading to sequestration of eNOS away from its functional caveolar location and providing a mechanism for the often-reported reduction in pulmonary arterial NO levels in experimental pulmonary hypertension, despite sustained eNOS protein levels.

NF-κB pathway is involved in CRP-induced effects on pulmonary arterial endothelial cells in chronic thromboembolic pulmonary hypertension

2013 ◽  
Vol 305 (12) ◽  
pp. L934-L942 ◽  
Author(s):  
Marijke Wynants ◽  
Leanda Vengethasamy ◽  
Alicja Ronisz ◽  
Bart Meyns ◽  
Marion Delcroix ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Serotonin Receptor ◽  
Molecular Mechanisms ◽  
Inflammatory Marker ◽  
Pulmonary Arteries ◽  
Chronic Thromboembolic Pulmonary Hypertension ◽  
Thromboembolic Pulmonary Hypertension ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thrombofibrotic obstruction of proximal pulmonary arteries. The cellular and molecular mechanisms underlying the pathogenesis remain incompletely understood, although we recently evidenced the potential involvement of the inflammatory marker C-reactive protein (CRP). We aimed to investigate the intracellular mechanisms induced by CRP in proximal pulmonary arterial endothelial cells (PAEC). PAEC were isolated from vascular material obtained during pulmonary endarterectomy. RNA was extracted from CRP-stimulated PAEC, and first-stand cDNA was generated. A RT2 profiler PCR Array was used to evaluate the expression of 84 key genes related to NF-κB-mediated signal transduction. CRP-induced NF-κB activation was studied. The effects of pyrrolidine-dithio-carbamate ammonium (PDTC), an inhibitor of the NF-κB pathway, were investigated on CRP-induced adhesion of monocytes to PAEC, adhesion molecule expression, endothelin-1 (ET-1), interleukin-6 (IL-6), and von Willebrand factor (vWF) secretion. Compared with nonstimulated PAEC, serotonin receptor 2B was downregulated by 25%, inhibitor of NF-κB kinase subunit epsilon (IKBKE) by 30%, and toll-like receptor-4 and -6 by 18 and 39%, respectively, in CRP-stimulated PAEC. The transcription factor FOS was threefold upregulated. CRP induced RelA/NF-κBp65 phosphorylation. PDTC dose dependently inhibited the adhesion of monocytes to CRP-stimulated PAEC. PDTC also inhibited the CRP-induced expression of ICAM-1 at the surface of PAEC. PDTC impaired the secretion of ET-1 by 18% and tended to inhibit the secretion of IL-6 by CRP-stimulated PAEC by 46%. PDTC did not inhibit the CRP-induced secretion of vWF. These results suggest an involvement of the NF-κB pathway in mediating different effects of CRP on proximal CTEPH-PAEC.

Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide

1998 ◽  
Vol 274 (5) ◽  
pp. L833-L841 ◽  
Author(s):  
A. Macduff Sheehy ◽  
Michael A. Burson ◽  
Stephen M. Black
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Enzyme Activity ◽  
Xanthine Oxidase ◽  
Disulfonic Acid ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells ◽  
Inhaled No

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy for pulmonary hypertension, suggesting that inhaled NO may downregulate basal NO production. However, the exact mechanism of this rebound pulmonary hypertension remains unclear. The objectives of these studies were to determine the effect of NO exposure on endothelial NO synthase (eNOS) gene expression, enzyme activity, and posttranslational modification in cultured pulmonary arterial endothelial cells. Sodium nitroprusside (SNP) treatment had no effect on eNOS mRNA or protein levels but did produce a significant decrease in enzyme activity. Furthermore, although SNP treatment induced protein kinase C (PKC)-dependent eNOS phosphorylation, blockade of PKC activity did not protect against the effects of SNP. When the xanthine oxidase inhibitor allopurinol or the superoxide scavenger 4,5-dihydroxy-1-benzene-disulfonic acid were coincubated with SNP, the inhibitory effects on eNOS activity could be partially alleviated. Also, the levels of superoxide were found to be elevated 4.5-fold when cultured pulmonary arterial endothelial cells were exposed to the NO donor spermine/NO. This suggests that NO can stimulate xanthine oxidase to cause an increase in cellular superoxide generation. A reaction between NO and superoxide would produce peroxynitrite, which could then react with the eNOS protein, resulting in enzyme inactivation. This mechanism may explain, at least in part, how NO produces NOS inhibition in vivo and may delineate, in part, the mechanism of rebound pulmonary hypertension after withdrawal of inhaled NO.

Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L60-L68 ◽  
Author(s):  
Louis G. Chicoine ◽  
Michael L. Paffett ◽  
Tamara L. Young ◽  
Leif D. Nelin
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Urea Production ◽  
Pulmonary Arterial ◽  
Factor Α ◽  
Arterial Endothelial Cells ◽  
Regular Medium ◽  
Necrosis Factor

Nitric oxide (NO) is produced by NO synthase (NOS) from l-arginine (l-Arg). Alternatively, l-Arg can be metabolized by arginase to produce l-ornithine and urea. Arginase (AR) exists in two isoforms, ARI and ARII. We hypothesized that inhibiting AR with l-valine (l-Val) would increase NO production in bovine pulmonary arterial endothelial cells (bPAEC). bPAEC were grown to confluence in either regular medium (EGM; control) or EGM with lipopolysaccharide and tumor necrosis factor-α (L/T) added. Treatment of bPAEC with L/T resulted in greater ARI protein expression and ARII mRNA expression than in control bPAEC. Addition of l-Val to the medium led to a concentration-dependent decrease in urea production and a concentration-dependent increase in NO production in both control and L/T-treated bPAEC. In a second set of experiments, control and L/T bPAEC were grown in EGM, EGM with 30 mM l-Val, EGM with 10 mM l-Arg, or EGM with both 10 mM l-Arg and 30 mM l-Val. In both control and L/T bPAEC, treatment with l-Val decreased urea production and increased NO production. Treatment with l-Arg increased both urea and NO production. The addition of the combination l-Arg and l-Val decreased urea production compared with the addition of l-Arg alone and increased NO production compared with l-Val alone. These data suggest that competition for intracellular l-Arg by AR may be involved in the regulation of NOS activity in control bPAEC and in response to L/T treatment.

Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension

2021 ◽  
Author(s):  
Ziyi Wang ◽  
Jiyuan Chen ◽  
Aleksandra Babicheva ◽  
Pritesh P. Jain ◽  
Marisela Rodriguez ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Cyclic Stretch ◽  
Mechanosensitive Channel ◽  
Pathogenic Role ◽  
Membrane Stretch ◽  
Mrna And Protein Expression ◽  
Resultant Increase ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells ◽  
Notch Ligands

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared to normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, while downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

Intracellular redox status affects transplasma membrane electron transport in pulmonary arterial endothelial cells

2002 ◽  
Vol 282 (1) ◽  
pp. L36-L43 ◽  
Author(s):  
Marilyn P. Merker ◽  
Robert D. Bongard ◽  
Nicholas J. Kettenhofen ◽  
Yoshiyuki Okamoto ◽  
Christopher A. Dawson
Keyword(s):  
Endothelial Cells ◽  
Electron Transport ◽  
Control Level ◽  
Redox Status ◽  
Cellular Mechanisms ◽  
Control Activity ◽  
Transplasma Membrane Electron Transport ◽  
Cell Extracts ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Pulmonary arterial endothelial cells possess transplasma membrane electron transport (TPMET) systems that transfer intracellular reducing equivalents to extracellular electron acceptors. As one aspect of determining cellular mechanisms involved in one such TPMET system in pulmonary arterial endothelial cells in culture, glycolysis was inhibited by treatment with iodoacetate (IOA) or by replacing the glucose in the cell medium with 2-deoxy-d-glucose (2-DG). TPMET activity was measured as the rate of reduction of the extracellular electron acceptor polymer toluidine blue O polyacrylamide. Intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were determined by high-performance liquid chromatography of KOH cell extracts. IOA decreased TPMET activity to 47% of control activity concomitant with a decrease in the NADH/NAD+ ratio to 34% of the control level, without a significant change in the NADPH/NADP+ ratio. 2-DG decreased TPMET activity to 53% of control and decreased both NADH/NAD+ and NADPH/NADP+ ratios to 51% and 55%, respectively, of control levels. When lactate was included in the medium along with the inhibitors, the effects of IOA and 2-DG on both TPMET activity and the NADPH/NADP+ ratios were prevented. The results suggest that cellular redox status is a determinant of pulmonary arterial endothelial cell TPMET activity, with TPMET activity more highly correlated with the poise of the NADH/NAD+redox pair.

Matrix Stiffening Induces a Pathogenic QKI-miR-7-SRSF1 Signaling Axis in Pulmonary Arterial Endothelial Cells

2021 ◽  
Author(s):  
Chen-Shan Chen Woodcock ◽  
Neha Hafeez ◽  
Adam Handen ◽  
Ying Tang ◽  
Lloyd D Harvey ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rna Binding ◽  
Vascular Diseases ◽  
Vascular Stiffness ◽  
Artery Stiffness ◽  
Signaling Axis ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells ◽  
Splicing Factor 1

Pulmonary arterial hypertension (PAH) refers to a set of heterogeneous vascular diseases defined by elevation of pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR), leading to right ventricular (RV) remodeling and often death. Early increases in pulmonary artery stiffness in PAH drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling. Dysregulation of microRNAs can drive PAEC dysfunction. However, the role of vascular stiffness in regulating pathogenic microRNAs in PAH is incompletely understood. Here, we demonstrated that extracellular matrix (ECM) stiffening downregulated miR-7 levels in PAECs. The RNA binding protein Quaking (QKI) has been implicated in the biogenesis of miR-7. Correspondingly, we found that ECM stiffness up-regulated QKI, and QKI knockdown led to increased miR-7. Downstream of the QKI-miR-7 axis, the serine and arginine rich splicing factor 1 (SRSF1) was identified as a direct target of miR-7. Correspondingly, SRSF1 was reciprocally up-regulated in PAECs exposed to stiff ECM and was negatively correlated with miR-7. Decreased miR-7 and increased QKI and SRSF1 were observed in lungs from PAH patients and PAH rats exposed to SU5416/hypoxia. Lastly, miR-7 upregulation inhibited human PAEC migration, while forced SRSF1 expression reversed this phenotype, proving that miR-7 depended upon SRSF1 to control migration. In aggregate, these results define the QKI-miR-7-SRSF1 axis as a mechanosensitive mechanism linking pulmonary arterial vascular stiffness to pathogenic endothelial function. These findings emphasize implications relevant to PAH and suggest the potential benefit of developing therapies that target this miRNA-dependent axis in PAH.

Abstract 19126: Endothelial-specific Deletion of Prolyl Hydroxylase Domain Protein 2 (PHD2) Results in Severe Pulmonary Hypertension in Mice by Enhancing the HIF2alpha Signaling Pathway

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Haiyang Tang ◽  
Yali Gu ◽  
Ramon Aryon ◽  
Shanshan Song ◽  
Ruby A Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Remodeling ◽  
Normoxic Condition ◽  
Severe Pulmonary Hypertension ◽  
Pulmonary Arterial ◽  
Domain Protein ◽  
Specific Deletion

Rationale: Sustained pulmonary vasoconstriction and excessive vascular remodeling are major causes of elevated pulmonary vascular resistance which leads to increased pulmonary arterial pressure in patients with pulmonary hypertension. Hypoxic-inducible factor (HIF) and its upstream regulators have been linked to the hypoxia response in vascular remodeling and the development of pulmonary hypertension. In this study, we aimed at defining whether increased HIF1α and/or HIF2α, due to endothelial cell specific deletion of prolyl hydroxylase domain protein 2 (PHD2) under normoxic condition are involved in or required for the initiation and progression of pulmonary hypertension. Methods: PHD2, HIF1α and HIF2α conditional knockout mice were created. Right ventricle systolic pressures (RVSP), right ventricular hypertrophy by RV/(LV+S) ratios, and small pulmonary artery smooth muscle layer thickness were measured. Pulmonary arterial smooth muscle cells (PASMCs) and pulmonary arterial endothelial cells (PAECs) were isolated from wild type (WT) or knockout (KO) mice, followed with cell-based assays. Results: We report here that mice with targeted deletion of PHD2 developed severe pulmonary hypertension under normoxic condition. Conditional and inducible deletion of HIF2α in endothelial cells, but not smooth muscle cells, dramatically protected mice from hypoxia-induced pulmonary hypertension. HIF2α KO mice had significantly lower RVSP, RV/(LV+S) ratios, and displayed less pulmonary vascular remodeling when exposed to hypoxia compared to their WT mice. Conclusion: This work shows that the endothelium is responsible for the development of pulmonary hypertension and it demonstrates a crucial role of PHD2/HIF signaling for hypoxic response in pulmonary hypertension. These findings unveil temporally and spatially distinct functions for HIFs in the development of pulmonary hypertension.

Endothelin-1 upregulates activin receptor-like kinase-1 expression via Gi/RhoA/Sp-1/Rho kinase pathways at transcriptional and post-transcriptional levels in human pulmonary arterial endothelial cells

2020 ◽  
Author(s):  
Koichi Sugimoto ◽  
Tetsuro Yokokawa ◽  
Tomofumi Misaka ◽  
Takashi Kaneshiro ◽  
Shinya Yamada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Rho Kinase ◽  
Luciferase Assay ◽  
Activin Receptor ◽  
Rhoa Activation ◽  
Pulmonary Vasoconstriction ◽  
Receptor Like Kinase ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Abstract BackgroundsPulmonary arterial hypertension (PAH) is a disease with poor prognosis that is characterized by pulmonary vasoconstriction and organic stenosis due to abnormal proliferation of pulmonary vascular cells. It has been demonstrated that endothelin (ET)-1 induces pulmonary vasoconstriction through activation of RhoA. Moreover, we previously demonstrated that Gi, a heterotrimeric G protein, functions upstream of RhoA activation. A gene mutation of activin receptor-like kinase (ACVRL)-1 is recognized in idiopathic or heritable PAH patients. However, little is known about the association between ET-1 and ACVRL-1. In the present study, we investigated the effect of ET-1 on ACVRL-1 expression and aimed to delineate the involvement of the Gi/RhoA/Rho kinase pathway.MethodsET-1 was added to culture medium of human pulmonary arterial endothelial cells (PAECs), and ACVRL-1 expression levels were analyzed using western blotting and quantitative polymerase chain reaction. The promoter activity of ACVRL-1 was evaluated by dual luciferase assay. Before adding ET-1 to the PAECs, pretreatment with pertussis toxin (PTX) or exoenzyme C3 transferase (C3T) was performed for the inhibition of Gi or RhoA, respectively. Rho kinase was inhibited by Y27632. Active form of RhoA (GTP-RhoA) was assessed by pull-down assay.ResultsACVRL-1 expression was increased by ET-1 in the PAECs. Pull-down assay revealed that ET-1 rapidly induced a GTP-loading of RhoA. The ET-1-induced RhoA activation was suppressed by pretreatment with PTX or C3T. Further, PTX, C3T, and Y27632 suppressed the ET-1-induced ACVRL-1 expression. The activity of ACVRL-1 promotor and the lifespan of ACVRL-1 mRNA was increased by ET-1. Sp-1, which is one of the transcriptional factors of ACVRL-1, peaked 15 min after adding ET-1 to the PAECs. PTX and C3T prevented the increase of Sp-1 induced by ET-1.ConclusionThe present study demonstrated that ET-1 increases ACVRL-1 expression at the transcriptional and post-transcriptional levels in human PAECs via the Gi/RhoA/Rho kinase pathway with involvement of Sp-1.

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