Abstract 13010: Endothelin-1 Increases ACVRL-1 Expression at the Transcriptional and Post-transcriptional Levels in Human Pulmonary Arterial Endothelial Cells via Gi, RhoA and Rho Kinase Pathway With Involvement of Sp-1

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Koichi Sugimoto ◽  
Tetsuro Yokokawa ◽  
Tomofumi Misaka ◽  
Sayoko Yokokawa ◽  
Kazuhiko Nakazato ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Active Form ◽  
Rho Kinase ◽  
Luciferase Assay ◽  
Serine Threonine Kinase ◽  
Pulmonary Vasoconstriction ◽  
Mrna And Protein Expression ◽  
Pulmonary Arterial ◽  
The Relationship ◽  
Arterial Endothelial Cells

Backgrounds: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and organic stenosis due to abnormal proliferation of pulmonary vascular cells. Endothelin (ET)-1 induces pulmonary vasoconstriction hyperplasia and plays a pivotal role in the pathogenesis of PAH. The germline mutations of activin receptor-like kinase (ACVRL)-1, a serine/threonine kinase and receptor for TGF-β, have been reported in idiopathic and hereditary PAH. However, the relationship between ET-1 and ACVRL-1 in the pathogenesis of PAH is largely unknown. Thus, we investigated the molecular mechanism of ACVRL-1 expression induced by ET-1 in human pulmonary arterial endothelial cells (hPAECs). Methods: ACVRL-1 expression levels were measured using Western blotting and quantitative real-time polymerase chain reaction. The promoter activity of ACVRL-1 was evaluated using dual luciferase assay. hPAECs were pretreated with pertussis toxin (PTX), cell-permeable C3 toxin (C3T) and Y27632 to inhibit Gi, RhoA and Rho kinase, respectively. GTP-RhoA, an active form of RhoA, was assessed using pull-down assay. Results: ET-1 increased mRNA and protein expression of ACVRL-1 in hPAECs (1.8±0.2 folds, P<0.05; 1.5±0.4 folds, P<0.05, respectively). The pull-down assay showed that ET-1 induced GTP-loading of RhoA. ET-1-induced RhoA activation was suppressed by PTX pretreatment. Furthermore, PTX, C3T, and Y27632 suppressed ET-1-induced ACVRL-1 expression. The transcriptional activity of the ACVRL-1 promoter was increased by ET-1 by 1.2±0.17 folds (P<0.05 vs. control). Moreover, ACVRL-1 mRNA was stabilized by ET-1 treatment, and the effect was canceled by Y27632. Finally, the expression of Sp-1, one of known transcriptional factors for ACVRL-1, was increased with a peak at 15 min after ET-1 treatment, and PTX, C3T and Y27632 significantly inhibited the Sp-1 induction by ET-1. Conclusion: These data indicate that ET-1 increases ACVRL-1 expression both at transcriptional and post-transcriptional mechanisms via the Gi/RhoA/Rho kinase/Sp-1 axis in human PAECs.

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.

scholarly journals Endothelin-1 Upregulates Activin Receptor-Like Kinase-1 Expression via Gi/RhoA/Sp-1/Rho Kinase Pathways in Human Pulmonary Arterial Endothelial Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Koichi Sugimoto ◽  
Tetsuro Yokokawa ◽  
Tomofumi Misaka ◽  
Takashi Kaneshiro ◽  
Shinya Yamada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rho Kinase ◽  
Endothelin 1 ◽  
Pulmonary Vasoconstriction ◽  
Receptor Like Kinase ◽  
Mithramycin A ◽  
Pulmonary Arterial ◽  
Pre Treatment ◽  
Arterial Endothelial Cells ◽  
Kinase Pathway

Background: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and organic stenosis. It has been demonstrated that endothelin-1 (ET-1) induces pulmonary vasoconstriction through the activation of RhoA. In addition, a gene mutation of activin receptor-like kinase (ACVRL)-1 is recognized in PAH patients. However, little is known about the association between ET-1 and ACVRL-1.Objective: In the present study, we aimed to investigate the effect of ET-1 on ACVRL-1 expression and delineate the involvement of the Gi/RhoA/Rho kinase pathway.Methods: ET-1 was added to culture medium of human pulmonary arterial endothelial cells (PAECs). Pre-treatment with pertussis toxin (PTX) or exoenzyme C3 transferase (C3T) was performed for inhibition of Gi or RhoA, respectively. Rho kinase was inhibited by Y27632. Mithramycin A was used for inhibition of Sp-1, which is a transcriptional factor of ACVRL-1. The active form of RhoA (GTP-RhoA) was assessed by pull-down assay.Results: ACVRL-1 expression was increased by ET-1 in the PAECs. Pull-down assay revealed that ET-1 induced GTP-loading of RhoA, which was suppressed by pre-treatment with PTX or C3T. Further, PTX, C3T, and Y27632 suppressed the ET-1-induced ACVRL-1 expression. ET-1 increased the activity of the ACVRL-1 promoter and stabilized the ACVRL-1 mRNA. Sp-1 peaked 15 min after adding ET-1 to the PAECs. PTX and C3T prevented the increase of Sp-1 induced by ET-1. Inhibition of Sp-1 by mithramycin A suppressed ET-1-induced ACVRL-1 upregulation.Conclusion: The present study demonstrated that ET-1 increases ACVRL-1 expression in human PAECs via the Gi/RhoA/Rho kinase pathway with the involvement of Sp-1.

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.

scholarly journals Effects of acute Rho kinase inhibition on chronic hypoxia-induced changes in proximal and distal pulmonary arterial structure and function

2011 ◽  
Vol 110 (1) ◽  
pp. 188-198 ◽  
Author(s):  
Rebecca R. Vanderpool ◽  
Ah Ram Kim ◽  
Robert Molthen ◽  
Naomi C. Chesler
Keyword(s):  
Pulmonary Artery ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Pressure Flow ◽  
Kinase Inhibition ◽  
Pulmonary Vasoconstriction ◽  
Pulsatile Pressure ◽  
Arterial Structure ◽  
Pulmonary Arterial ◽  
Induced Changes

Hypoxic pulmonary hypertension (HPH) is initially a disease of the small pulmonary arteries. Its severity is usually quantified by pulmonary vascular resistance (PVR). Acute Rho kinase inhibition has been found to reduce PVR toward control values in animal models, suggesting that persistent pulmonary vasoconstriction is the dominant mechanism for increased PVR. However, HPH may also cause proximal arterial changes, which are relevant to right ventricular (RV) afterload. RV afterload can be quantified by pulmonary vascular impedance, which is obtained via spectral analysis of pulsatile pressure-flow relationships. To determine the effects of HPH independent of persistent pulmonary vasoconstriction in proximal and distal arteries, we quantified pulsatile pressure-flow relationships before and after acute Rho kinase inhibition and measured pulmonary arterial structure with microcomputed tomography. In control lungs, Rho kinase inhibition decreased 0 Hz impedance (Z0), which is equivalent to PVR, from 2.1 ± 0.4 to 1.5 ± 0.2 mmHg·min·ml−1 ( P < 0.05) and tended to increase characteristic impedance (ZC) from 0.21 ± 0.01 to 0.22 ± 0.01 mmHg·min·ml−1. In HPH lungs, Rho kinase inhibition decreased Z0 ( P < 0.05) without affecting ZC. Microcomputed tomography measurements performed on lungs after acute Rho kinase inhibition demonstrated that HPH significantly decreased the unstressed diameter of the main pulmonary artery (760 ± 60 vs. 650 ± 80 μm; P < 0.05), decreased right pulmonary artery compliance, and reduced the frequency of arteries of diameter 50–100 μm (both P < 0.05). These results demonstrate that acute Rho kinase inhibition reverses many but not all HPH-induced changes in distal pulmonary arteries but does not affect HPH-induced changes in the conduit arteries that impact RV afterload.

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.

Activation of proMMP-2 and Src by HHV8 vGPCR in human pulmonary arterial endothelial cells

2007 ◽  
Vol 42 (3) ◽  
pp. 517-525 ◽  
Author(s):  
Bin Shan ◽  
Cindy A. Morris ◽  
Ying Zhuo ◽  
Bryan D. Shelby ◽  
Dawn R. Levy ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Characterization of glutamic acid uptake by bovine pulmonary arterial endothelial cells

1987 ◽  
Vol 63 (5) ◽  
pp. 1961-1965 ◽  
Author(s):  
V. Steiger ◽  
S. M. Deneke ◽  
B. L. Fanburg
Keyword(s):  
Endothelial Cells ◽  
Glutamic Acid ◽  
Cell Density ◽  
Inhibitory Effect ◽  
Transport Systems ◽  
Acid Uptake ◽  
Relative Contribution ◽  
Pulmonary Arterial ◽  
Cell Densities ◽  
Arterial Endothelial Cells

L-Glutamic acid uptake by bovine pulmonary arterial endothelial cells in culture increased linearly with time up to 30 min and did not show saturation with increased substrate concentration up to 6 X 10(-3) M. The uptake per cell decreased as cell density increased and was lowest when the cells became fully confluent. Most of the uptake was sodium dependent, although the relative contribution of sodium-independent uptake increased with an increase in cell density. Cysteic and aspartic acid strongly inhibited L-glutamic acid uptake, but at higher cell densities this effect was less pronounced than at low densities. Other amino acids, including leucine, glutamine, and serine, exerted a modest inhibitory effect at both high and low cell densities. Thus pulmonary arterial endothelial cells contain similar membrane transport systems for L-glutamic acid as those previously described for fibroblasts, hepatocytes, and nerve cells. However, quantitative properties of the transport systems differ depending on the state of cellular density in monolayers.

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