Localization of 13-hydroxyoctadecadienoic acid and the vitronectin receptor in human endothelial cells and endothelial cell/platelet interactions in vitro

Abstract Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

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Localization of 13-hydroxyoctadecadienoic acid and the vitronectin receptor in human endothelial cells and endothelial cell/platelet interactions in vitro

Blood ◽

10.1182/blood.v81.12.3303.bloodjournal81123303 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3303-3312 ◽

Cited By ~ 23

Author(s):

MR Buchanan ◽

MC Bertomeu ◽

TA Haas ◽

FW Orr ◽

LL Eltringham-Smith

Keyword(s):

Endothelial Cells ◽

Platelet Adhesion ◽

Receptor Expression ◽

Immunofluorescent Staining ◽

Apical Surface ◽

Binding Assays ◽

Vitronectin Receptor ◽

Wall Cell ◽

Hydroxyoctadecadienoic Acid

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

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Perturbation of Platelet Adhesion to Endothelial Cells by Plasminogen Activation In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657655 ◽

1997 ◽

Vol 78 (02) ◽

pp. 934-938 ◽

Cited By ~ 5

Author(s):

Hsiun-ing Chen ◽

Yueh-I Wu ◽

Yu-Lun Hsieh ◽

Guey-Yueh Shi ◽

Meei-Jyh Jiang ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Platelet Adhesion ◽

Treatment Time ◽

Shape Change ◽

Umbilical Vein ◽

Tissue Type ◽

Apical Surface ◽

Plasminogen Activation

SummaryTo investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as e-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Investigation of Wall Shear Stress in Cardiovascular Research and in Clinical Practice—From Bench to Bedside

International Journal of Molecular Sciences ◽

10.3390/ijms22115635 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5635

Author(s):

Katharina Urschel ◽

Miyuki Tauchi ◽

Stephan Achenbach ◽

Barbara Dietel

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Wall Shear Stress ◽

Cell Function ◽

Computational Techniques ◽

Apical Surface ◽

Cardiovascular Research ◽

Endothelial Cell Function ◽

Wall Shear

In the 1900s, researchers established animal models experimentally to induce atherosclerosis by feeding them with a cholesterol-rich diet. It is now accepted that high circulating cholesterol is one of the main causes of atherosclerosis; however, plaque localization cannot be explained solely by hyperlipidemia. A tremendous amount of studies has demonstrated that hemodynamic forces modify endothelial athero-susceptibility phenotypes. Endothelial cells possess mechanosensors on the apical surface to detect a blood stream-induced force on the vessel wall, known as “wall shear stress (WSS)”, and induce cellular and molecular responses. Investigations to elucidate the mechanisms of this process are on-going: on the one hand, hemodynamics in complex vessel systems have been described in detail, owing to the recent progress in imaging and computational techniques. On the other hand, investigations using unique in vitro chamber systems with various flow applications have enhanced the understanding of WSS-induced changes in endothelial cell function and the involvement of the glycocalyx, the apical surface layer of endothelial cells, in this process. In the clinical setting, attempts have been made to measure WSS and/or glycocalyx degradation non-invasively, for the purpose of their diagnostic utilization. An increasing body of evidence shows that WSS, as well as serum glycocalyx components, can serve as a predicting factor for atherosclerosis development and, most importantly, for the rupture of plaques in patients with high risk of coronary heart disease.

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Abstract 474: Effects of Cigarette Smoke on CD146 Expression and Function in Pulmonary Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a474 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Adelheid Kratzer ◽

Jonas Salys ◽

Benjy Gonzalez ◽

Hong Wei Chu ◽

Martin Zamora ◽

...

Keyword(s):

Endothelial Cells ◽

Cigarette Smoke ◽

Immunofluorescent Staining ◽

Microvascular Endothelial Cells ◽

Pulmonary Microvascular Endothelial Cells ◽

Pulmonary Endothelial Cells ◽

Membrane Bound ◽

Microvascular Endothelial ◽

Cd146 Expression

Background and Objectives: Cell adhesion molecule CD146 is a transmembrane glycoprotein constitutively expressed in all types of endothelial cells (EC). It exists in two forms: a membrane-anchored form (CD146) and a soluble, extracellular and cleaved form (sCD146). The plasma concentration of sCD146 is modulated in inflammatory diseases that involve endothelial alterations. We investigated the role of endothelial CD146 in cigarette smoke-induced emphysema in vivo and in pulmonary endothelial cells (EC) in vitro . Methods: Sprague Dawley rats exposed to cigarette smoke for 2 months developed significant emphysematous changes (measured by mean linear intercept). Levels of sCD146 were subsequently measured in the circulation as well as in the bronchoalveolar lavage fluid (BALf) via ELISA. In vitro studies were carried out in rat pulmonary microvascular endothelial cells using CSE. Results: CD146 is highly expressed in rat pulmonary microvascular endothelial cells (RPMVEC) and to a much lower extent, in pulmonary macrovascular endothelial cells (RPAEC). Treatment of RPMVEC with cigarette smoke extract (CSE) in vitro resulted in decreased expression of membrane-bound CD146 as well as a reduced gene expression and increased sCD146 levels in the culture medium after 12 hours. Moreover, CSE-induced downregulation of CD146 expression resulted in increased vascular permeability of RPMVEC, as measured by EVANs Blue assay and migration of CFSE-labeled rat alveolar macrophage. Immunofluorescent staining revealed that CSE treatment resulted in translocation of membrane-bound CD146 into the nucleus. Subsequent western blot analysis showed changes in ERK and AKT activation and signaling. Similar results were found upon siRNA silencing of CD146, implicating a role for CD146 in tissue inflammation and integrity. Circulating levels of sCD146 were also elevated in plasma and BALf of patients with COPD and correlated, in part, with the presence of anti-endothelial autoantibodies. Additionally, we found decreased expression of membrane-bound CD146 in lung tissues of COPD patients. Conclusions: Our data suggest that CD146 plays an important role in pulmonary vascular EC function. Moreover, levels of circulating soluble CD146 can be a predictor of vascular endothelial cell injury.

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Platelet Adhesion to Aortic Endothelial Cells In Vitro after Thrombin Treatment

Thrombosis Research ◽

10.1016/s0049-3848(98)00054-1 ◽

1998 ◽

Vol 91 (1) ◽

pp. 15-21 ◽

Cited By ~ 10

Author(s):

Yoshiaki Itoh ◽

Minoru Tomita ◽

Norio Tanahashi ◽

Hidetaka Takeda ◽

Masako Yokoyama ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Adhesion ◽

Aortic Endothelial Cells ◽

Aortic Endothelial

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The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding

Thrombosis and Haemostasis ◽

10.1160/th15-09-0700 ◽

2016 ◽

Vol 116 (07) ◽

pp. 87-95 ◽

Cited By ~ 3

Author(s):

D'Andra Parker ◽

Subia Tasneem ◽

Richard Farndale ◽

Dominique Bihan ◽

J. Sadler ◽

...

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Structural Features ◽

High Shear ◽

The Individual ◽

Static Conditions ◽

Von Willebrand ◽

Willebrand Factor

SummaryMultimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbD binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

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HIV-1 Tat Regulates Occludin and AβTransfer Receptor Expression in Brain Endothelial Cells via Rho/ROCK Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/4196572 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 10

Author(s):

Yanlan Chen ◽

Wen Huang ◽

Wenlin Jiang ◽

Xianghong Wu ◽

Biao Ye ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Receptor Expression ◽

Immunofluorescent Staining ◽

Mrna Levels ◽

Protein Levels ◽

Density Lipoprotein Receptor ◽

Hiv 1

HIV-1 transactivator protein (Tat) has been shown to play an important role in HIV-associated neurocognitive disorders. The aim of the present study was to evaluate the relationship between occludin and amyloid-beta (Aβ) transfer receptors in human cerebral microvascular endothelial cells (hCMEC/D3) in the context of HIV-1-related pathology. The protein expressions of occludin, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in hCMEC/D3 cells were examined using western blotting and immunofluorescent staining. The mRNA levels of occludin, RAGE, and LRP1 were measured using quantitative real-time polymerase chain reaction. HIV-1 Tat at 1 µg/mL and the Rho inhibitor hydroxyfasudil (HF) at 30 µmol/L, with 24 h exposure, had no significant effect on hCMEC/D3 cell viability. Treatment with HIV-1 Tat protein decreased mRNA and protein levels of occludin and LRP1 and upregulated the expression of RAGE; however, these effects were attenuated by HF. These data suggest that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-mediated changes in occludin, RAGE, and LRP1 in hCMEC/D3 cells. HF may have a beneficial influence by protecting the integrity of the blood-brain barrier and the expression of Aβtransfer receptors.

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