Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

Download Full-text

Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms221910287 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10287

Author(s):

Chih-Hsien Wu ◽

Yi-Lin Chiu ◽

Chung-Yueh Hsieh ◽

Guo-Shiang Tsung ◽

Lian-Shan Wu ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Nitric Oxide Synthase ◽

Umbilical Vein ◽

Sirtuin 1 ◽

No Production ◽

Beneficial Effects ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

Download Full-text

Abstract P198: Differential Regulation of Endothelial Nitric Oxide Synthase Phosphorylation by Protease-Activated Receptors in Adult Human Endothelial Cells

Hypertension ◽

10.1161/hyp.66.suppl_1.p198 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Lakeisha C Tillery ◽

Evangeline D Motley-Johnson

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Nitric Oxide Synthase ◽

Umbilical Vein ◽

Differential Regulation ◽

No Production ◽

Protease Activated Receptors ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Protease-activated receptors (PARs) have been shown to regulate endothelial nitric oxide synthase (eNOS) through the activation of specific sites on the enzyme. It has been established that phosphorylation of eNOS-Ser-1177 leads to the production of the potent vasodilator nitric oxide (NO), and is associated with PAR-2 activation; while phosphorylation of eNOS-Thr-495 decreases NO production, and is coupled to PAR-1 activation. In this study, we demonstrate a differential regulation of the eNOS/NO pathway by the PARs using primary adult human coronary artery endothelial cells (HCAEC). Thrombin and the PAR-1 activating peptide, TFLLR, which are known to phosphorylate eNOS-Thr-495 in bovine and human umbilical vein endothelial cells, phosphorylated eNOS-Ser-1177 in HCAECs, and increased NO production. The PAR-1 responses were blocked using SCH-79797, a PAR-1 inhibitor, and L-NAME was used to inhibit NO production. A PAR-2 specific ligand, SLIGRL, which has been shown to phosphorylate eNOS-Ser-1177 in bovine and human umbilical vein endothelial cells, primarily regulated eNOS-Thr-495 phosphorylation and suppressed NO production in the HCAECs. PAR-3, known for its non-signaling potential, was activated by TFRGAP, a PAR-3 mimicking peptide, and only induced phosphorylation of eNOS-Thr-495 with no effect on NO production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was calcium-dependent using the calcium chelator, BAPTA, and eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632. These data suggest a vascular bed specific differential coupling of PARs to the signaling pathways that regulate eNOS and NO production that may be responsible for the modulation of endothelial function associated with cardiovascular disease.

Download Full-text

Ketamine reduces nitric oxide biosynthesis in human umbilical vein endothelial cells by down-regulating endothelial nitric oxide synthase expression and intracellular calcium levels*

Critical Care Medicine ◽

10.1097/01.ccm.0000163246.33366.51 ◽

2005 ◽

Vol 33 (5) ◽

pp. 1044-1049 ◽

Cited By ~ 43

Author(s):

Ruei-Ming Chen ◽

Ta-Liang Chen ◽

Yi-Ling Lin ◽

Tyng-Guey Chen ◽

Yu-Ting Tai

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Nitric Oxide Synthase ◽

Intracellular Calcium ◽

Endothelial Nitric Oxide Synthase ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Download Full-text

Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

International Journal of Molecular Sciences ◽

10.3390/ijms19092753 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2753 ◽

Cited By ~ 6

Author(s):

Nina Zippel ◽

Annemarieke Loot ◽

Heike Stingl ◽

Voahanginirina Randriamboavonjy ◽

Ingrid Fleming ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Reactivity ◽

Umbilical Vein ◽

No Production ◽

Dependent Manner ◽

Calmodulin Binding ◽

Umbilical Vein Endothelial Cells ◽

Endothelial Nitric Oxide ◽

Specific Deletion

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

Download Full-text

Extract of Rubus coreanus Fruits Increases Expression and Activity of Endothelial Nitric Oxide Synthase in the Human Umbilical Vein Endothelial Cells

Journal of Life Science ◽

10.5352/jls.2011.21.1.44 ◽

2011 ◽

Vol 21 (1) ◽

pp. 44-55 ◽

Cited By ~ 4

Author(s):

Hyun-Joong Yoon ◽

Soo-Young Park ◽

Sung-Tack Oh ◽

Kee-Young Lee ◽

Sung-Yeul Yang

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Expression And Activity

Download Full-text

VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.274.3.h1054 ◽

1998 ◽

Vol 274 (3) ◽

pp. H1054-H1058 ◽

Cited By ~ 116

Author(s):

John D. Hood ◽

Cynthia J. Meininger ◽

Marina Ziche ◽

Harris J. Granger

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Umbilical Vein ◽

Northern Blotting ◽

No Production ◽

Vascular Endothelial ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Growth Factor ◽

Stimulation Of

Vascular endothelial growth factor (VEGF) is an endothelium-specific secreted protein that potently stimulates vasodilation, microvascular hyperpermeability, and angiogenesis. Nitric oxide (NO) is also reported to modulate vascular tone, permeability, and capillary growth. Therefore, we hypothesized that VEGF might regulate endothelial production of NO. The production of nitrogen oxides by human umbilical vein endothelial cells (HUVECs) was measured after 1, 12, 24, and 48 h of incubation with VEGF. VEGF treatment resulted in both an acute (1 h) and chronic (>24 h) stimulation of NO production. Furthermore, Western and Northern blotting revealed a VEGF-elicited, dose-dependent increase in the cellular content of endothelial cell nitric oxide synthase (ecNOS) message and protein that may account for the chronic upregulation of NO production elicited by VEGF. Finally, endothelial cells pretreated with VEGF for 24 h and subsequently exposed to A-23187 for 1 h produced NO at approximately twice the rate of cells that were not pretreated with VEGF. We conclude that VEGF upregulates ecNOS enzyme and elicits a biphasic stimulation of endothelial NO production.

Download Full-text

miR-101-3p induces vascular endothelial cell dysfunction by targeting tet methylcytosine dioxygenase 2

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmz154 ◽

2020 ◽

Vol 52 (2) ◽

pp. 180-191 ◽

Cited By ~ 3

Author(s):

Qiaoli Chen ◽

Xiaoye Li ◽

Lingjun Kong ◽

Qing Xu ◽

Zi Wang ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Vascular Endothelial Cell ◽

Nuclear Factor Κb ◽

Vascular Endothelial ◽

Ros Production ◽

Cell Dysfunction ◽

Umbilical Vein Endothelial Cells

Abstract Endothelial cell (EC) dysfunction represents an early key event in atherosclerosis. Recently, MicroRNAs have been demonstrated to regulate EC function. miR-101-3p has been discovered to regulate cell apoptosis and proliferation in cardiovascular diseases. Therefore, the aim of the current study was to clarify whether miR-101-3p regulates the dysfunction of vascular endothelial cells. In this study, the transfection of human umbilical vein endothelial cells (HUVECs) with miR-101-3p mimic induced reactive oxygen species (ROS) production, EC dysfunction, and activated nuclear factor-κB (NF-κB), whereas transfection with miR-101-3p inhibitor alleviated these events. The antioxidant N-acetylcysteine alleviated miR-101-3p-induced EC dysfunction. Moreover, we observed that miR-101-3p inhibited the expression of tet methylcytosine dioxygenase 2 (TET2) at the posttranscriptional level, resulting in increased ROS production and activated NF-κB. TET2 overexpression inhibited ROS production, EC dysfunction, and NF-κB activation in miR-101-3p-transfected HUVECs. These results indicate that miR-101-3p induces EC dysfunction by targeting TET2, which regulates ROS production, EC dysfunction, and NF-κB activation. Taken together, our current study reveals a novel pathway associated with EC dysfunction. The modulation of miR-101-3p and TET2 expression levels may serve as a potential target for therapeutic strategies for atherosclerosis.

Download Full-text

The loss of sustained Ca2+ signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00250.2013 ◽

2013 ◽

Vol 305 (7) ◽

pp. H969-H979 ◽

Cited By ~ 22

Author(s):

Jennifer Krupp ◽

Derek S. Boeldt ◽

Fu-Xian Yi ◽

Mary A. Grummer ◽

Heather A. Bankowski Anaya ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Umbilical Vein ◽

Normal Subjects ◽

Fura 2 ◽

Intracellular Ca ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca2+ concentration ([Ca2+]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 μM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 μM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca2+]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca2+]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca2+]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

Download Full-text