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.

Targeting IL-17 attenuates hypoxia-induced pulmonary hypertension through downregulation of β-catenin

Thorax ◽  
2019 ◽  
Vol 74 (6) ◽  
pp. 564-578 ◽  
Author(s):  
Lei Wang ◽  
Jie Liu ◽  
Wang Wang ◽  
Xianmei Qi ◽  
Ying Wang ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Lung Tissue ◽  
Interleukin 17 ◽  
Antibody Assays ◽  
Coculture Model ◽  
Potential Benefits ◽  
Pulmonary Arterial ◽  
And Migration ◽  
Arterial Endothelial Cells

BackgroundThe role of interleukin 17 (IL-17) in hypoxic pulmonary hypertension (HPH) remains unclear. This study is designed to explore whether IL-17 is a potential target for HPH treatment.MethodsClinic samples from the lung tissue and serum were obtained from qualified patients. Western blotting, immunohistochemistry and/or ELISA were used to measure the expression of relevant proteins. HPH models were established in C57BL/6 wild-type (WT) and IL-17−/− mice and were treated with exogenous recombinant mouse IL-17 (rmIL-17) or an IL-17 neutralising antibody. Assays for cell proliferation, angiogenesis and adhesion were employed to analyse the behaviours of human pulmonary arterial endothelial cells (HPAECs). A non-contact Transwell coculture model was used to evaluate intercellular interactions.ResultsExpression of IL-17 was increased in lung tissue of both patients with bronchiectasis/COPD-associated PH and HPH mouse model. Compared with WT mice, IL-17−/− mice had attenuated HPH, whereas administration of rmIL-17 aggravated HPH. In vitro, recombinant human IL-17 (rhIL-17) promoted proliferation, angiogenesis and adhesion in HPAECs through upregulation of Wnt3a/β-catenin/CyclinD1 pathway, and siRNA-mediated knockdown of β-catenin almost completely reversed this IL-17-mediated phenomena. IL-17 promoted the proliferation but not the migration of human pulmonary arterial smooth muscle cells (HPASMCs) cocultured with HPAECs under both normoxia and hypoxia, but IL-17 had no direct effect on proliferation and migration of HPASMCs. Blockade of IL-17 with a neutralising antibody attenuated HPH in WT mice.ConclusionsIL-17 contributes to the pathogenesis of HPH through upregulation of β-catenin expression. Targeting IL-17 might provide potential benefits for alternative therapeutic strategies for HPH.

scholarly journals Caveolae are involved in mechanotransduction during pulmonary hypertension

2016 ◽  
Vol 310 (11) ◽  
pp. L1078-L1087 ◽  
Author(s):  
Guillaume Gilbert ◽  
Thomas Ducret ◽  
Jean-Pierre Savineau ◽  
Roger Marthan ◽  
Jean-François Quignard
Keyword(s):  
Pulmonary Hypertension ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Osmotic Shock ◽  
Ryanodine Receptors ◽  
Membrane Microdomains ◽  
Mechanical Stimuli ◽  
Membrane Stretch ◽  
Arterial Blood ◽  
Pulmonary Arterial

Caveolae are stiff plasma membrane microdomains implicated in various cell response mechanisms like Ca2+ signaling and mechanotransduction. Pulmonary arterial smooth muscle cells (PASMC) transduce mechanical stimuli into Ca2+ increase via plasma membrane stretch-activated channels (SAC). This mechanotransduction process is modified in pulmonary hypertension (PH) during which stretch forces are increased by the increase in arterial blood pressure. We propose to investigate how caveolae are involved in the pathophysiology of PH and particularly in mechanotransduction. PASMC were freshly isolated from control rats (Ctrl rats) and rats suffering from PH induced by 3 wk of chronic hypoxia (CH rats). Using a caveolae disrupter (methyl-β-cyclodextrin), we showed that SAC activity measured by patch-clamp, stretch-induced Ca2+ increase measured with indo-1 probe and pulmonary arterial ring contraction to osmotic shock are enhanced in Ctrl rats when caveolae are disrupted. In CH rats, SAC activity, Ca2+, and contraction responses to stretch are all higher compared with Ctrl rats. However, in contrast to Ctrl rats, caveolae disruption in CH-PASMC, reduces SAC activity, Ca2+ responses to stretch and arterial contractions. Furthermore, by means of immunostainings and transmission electron microscopy, we observed that caveolae and caveolin-1 are expressed in PASMC from both Ctrl and CH rats and localize close to subplasmalemmal sarcoplasmic reticulum (ryanodine receptors) and mitochondria, thus facilitating Ca2+ exchanges, particularly in CH. In conclusion, caveolae are implicated in mechanotransduction in Ctrl PASMC by buffering mechanical forces. In PH-PASMC, caveolae form a distinct Ca2+ store facilitating Ca2+ coupling between SAC and sarcoplasmic reticulum.

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.

scholarly journals PKR deficiency alleviates pulmonary hypertension via inducing inflammasome adaptor ASC inactivation

2021 ◽  
Vol 11 (4) ◽  
pp. 204589402110461
Author(s):  
Yapei Li ◽  
Ying Li ◽  
Lijun Li ◽  
Minghui Yin ◽  
Jiangang Wang ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Interleukin 1 ◽  
High Mobility ◽  
Interleukin 1 Beta ◽  
Therapeutic Approaches ◽  
Downstream Signaling ◽  
Dependent Protein ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Pulmonary hypertension is a progressive fatal disease that currently has no specific therapeutic approaches. In this study, dsRNA-dependent protein kinase (PKR) was considered a candidate molecule in pulmonary hypertension. We demonstrated that PKR is activated in the endothelium of experimental pulmonary hypertension models. Deletion of PKR or treatment with the PKR activation inhibitor C16 inhibited the development of pulmonary hypertension. To explore the mechanism of PKR in pulmonary hypertension, we detected its downstream signaling and found that PKR knockout represses apoptosis-associated speck-like protein containing CARD (ASC) activation to inhibit high mobility group box 1 (HMGB1) and interleukin-1 beta release. To further explore whether ASC mediates the pro-pulmonary hypertension role of PKR, we used ASC deletion mice and found that ASC deletion inhibits the development of pulmonary hypertension and the release of HMGB1 and interleukin-1 beta. Furthermore, we co-cultured pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs) and found that endothelial PKR promotes PASMCs proliferation through the release of HMGB1 and interleukin-1 beta. In conclusion, these data indicate that endothelial PKR promotes the excessive proliferation of PASMCs by inducing ASC activation to release HMGB1 and interleukin-1 beta, which lead to the development of pulmonary hypertension. Our study will provide a novel insight that PKR is a potential target in the future treatment of pulmonary hypertension.

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.

scholarly journals Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

2021 ◽  
Author(s):  
Risa Ramadhiani ◽  
Koji Ikeda ◽  
Kazuya Miyagawa ◽  
Gusty Rizky Teguh Ryanto ◽  
Naoki Tamada ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Notch Signaling ◽  
Pulmonary Arteries ◽  
Hypoxia Exposure ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
And Migration ◽  
Notch Ligands

AbstractPulmonary arterial hypertension (PAH) is a fatal disease characterized by pathological pulmonary artery remodeling. Endothelial cells (EC) injury including DNA damage is critically involved in the vascular remodeling in PAH, and persistent injury leads to cellular senescence in ECs. Here, we show that EC senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling. EC-specific progeroid mice that we recently generated showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation in the distal pulmonary arteries. Mechanistically, we identified that senescent ECs highly expressed Notch ligands, and thus activated Notch signaling in PASMCs, leading to enhanced PASMCs proliferation and migration capacities. Consistently, pharmacological inhibition of Notch signaling attenuated the effects of senescent ECs on SMCs functions in vitro, and on the pulmonary hypertension in EC-specific progeroid mice in vivo. These data establish EC senescence as a crucial disease-modifying facor in PAH.

scholarly journals Reduced membrane cholesterol limits pulmonary endothelial Ca2+ entry after chronic hypoxia

2017 ◽  
Vol 312 (6) ◽  
pp. H1176-H1184 ◽  
Author(s):  
Bojun Zhang ◽  
Jay S. Naik ◽  
Nikki L. Jernigan ◽  
Benjimen R. Walker ◽  
Thomas C. Resta
Keyword(s):  
Pulmonary Hypertension ◽  
Chronic Hypoxia ◽  
Pulmonary Arteries ◽  
Barometric Pressure ◽  
Membrane Cholesterol ◽  
Potential Mechanism ◽  
Direct Role ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Chronic hypoxia (CH)-induced pulmonary hypertension is associated with diminished production of endothelium-derived Ca2+-dependent vasodilators such as nitric oxide. Interestingly, ATP-induced endothelial Ca2+ entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from CH rats (4 wk, barometric pressure = 380 Torr) compared with normoxic controls. Store-operated Ca2+ entry (SOCE) and depolarization-induced Ca2+ entry are major components of the response to ATP and are similarly decreased after CH. We hypothesized that membrane Chol facilitates both SOCE and depolarization-induced pulmonary endothelial Ca2+ entry and that CH attenuates these responses by decreasing membrane Chol. To test these hypotheses, we administered Chol or epicholesterol (Epichol) to acutely isolated pulmonary arterial endothelial cells (PAECs) from control and CH rats to either supplement or replace native Chol, respectively. The efficacy of membrane Chol manipulation was confirmed by filipin staining. Epichol greatly reduced ATP-induced Ca2+ influx in PAECs from control rats. Whereas Epichol similarly blunted endothelial SOCE in PAECs from both groups, Chol supplementation restored diminished SOCE in PAECs from CH rats while having no effect in controls. Similar effects of Chol manipulation on PAEC Ca2+ influx were observed in response to a depolarizing stimulus of KCl. Furthermore, KCl-induced Ca2+ entry was inhibited by the T-type Ca2+ channel antagonist mibefradil but not the L-type Ca2+ channel inhibitor diltiazem. We conclude that PAEC membrane Chol is required for ATP-induced Ca2+ entry and its two components, SOCE and depolarization-induced Ca2+ entry, and that reduced Ca2+ entry after CH may be due to loss of this key regulator. NEW & NOTEWORTHY This research is the first to examine the direct role of membrane cholesterol in regulating pulmonary endothelial agonist-induced Ca2+ entry and its components. The results provide a potential mechanism by which chronic hypoxia impairs pulmonary endothelial Ca2+ influx, which may contribute to pulmonary hypertension.

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.

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