Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells

2002 ◽  
Vol 282 (2) ◽  
pp. C289-C301 ◽  
Author(s):  
Lisa M. Schwiebert ◽  
William C. Rice ◽  
Brian A. Kudlow ◽  
Amanda L. Taylor ◽  
Erik M. Schwiebert
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Purinergic Signaling ◽  
Primary Cultures ◽  
Atp Release ◽  
Vascular Endothelial ◽  
Multiple Blood

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X4and P2X5are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for “self-regulation” of endothelial cell function and modulation of vascular tone.

scholarly journals Exogenous H2S Regulates of Cystathionine Gamma-Lyase in HUVECs During Hypoxia

2020 ◽  
Author(s):  
Maoxian Wang
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Mrna Expression ◽  
Blood Vessels ◽  
Western Blotting ◽  
Promoter Activity ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial Cell ◽  
Luciferase Assay ◽  
Vascular Endothelial

Cystathionine gamma-lyase (CSE) is one of the essential H2S-producing enzymes, and it regulates diverse functions in connection with cardiovascular function. It is crucial how exogenous H2S regulates CSE expression of the vascular endothelial cell during hypoxia. We examined the transcription and expression of CSE in HUVECs regulated by exogenous H2S with 100 μM during hypoxia by Luciferase assay, Western blotting, and quantitative RT-qPCR. Exogenous H2S influenced on the promoter activity of CSE in HUVECs during hypoxia. The effects of 100 μM H2S on CSE mRNA expression in HUVECs is decreased compared with 0 μM H2S. The consequences of 100 μM H2S on the expression level of CSE protein in HUVECs at two h of hypoxia is reduced compared with 0 μM H2S. These findings suggest that vascular endothelial cells can respond to the signals of hypoxia in the blood, and can respond to changes in H2S concentration in the blood, thus affect the blood vessels themselves.

The Endothelial Cell and the Factor VIII Bypassing Activity of Prothrombin Complex Concentrate

1988 ◽  
Vol 60 (02) ◽  
pp. 226-229 ◽  
Author(s):  
Jerome M Teitel ◽  
Hong-Yu Ni ◽  
John J Freedman ◽  
M Bernadette Garvey
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Factor Viii ◽  
Prothrombin Complex Concentrate ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Monolayer Cultures ◽  
Coagulant Activity ◽  
Time Courses ◽  
Privileged Site

SummarySome classical hemophiliacs have a paradoxical hemostatic response to prothrombin complex concentrate (PCC). We hypothesized that vascular endothelial cells (EC) may contribute to this “factor VIII bypassing activity”. When PCC were incubated with suspensions or monolayer cultures of EC, they acquired the ability to partially bypass the defect of factor VIII deficient plasma. This factor VIII bypassing activity distributed with EC and not with the supernatant PCC, and was not a general property of intravascular cells. The effect of PCC was even more dramatic on fixed EC monolayers, which became procoagulant after incubation with PCC. The time courses of association and dissociation of the PCC-derived factor VIII bypassing activity of fixed and viable EC monolayers were both rapid. We conclude that EC may provide a privileged site for sequestration of constituents of PCC which express coagulant activity and which bypass the abnormality of factor VIII deficient plasma.

Alterations in Vascular Endothelial Cell-related Plasma Proteins In Thalassaemic Patients and their Correlation with Clinical Symptoms

1995 ◽  
Vol 74 (04) ◽  
pp. 1045-1049 ◽  
Author(s):  
P Butthep ◽  
A Bunyaratvej ◽  
Y Funahara ◽  
H Kitaguchi ◽  
S Fucharoen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Immunosorbent Assay ◽  
Plasma Proteins ◽  
Clinical Symptoms ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vascular Endothelial ◽  
Leg Ulcers

SummaryAn increased level of plasma thrombomodulin (TM) in α- and β- thalassaemia was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Nonsplenectomized patients with β-thalassaemia/ haemoglobin E (BE) had higher levels of TM than splenectomized cases (BE-S). Patients with leg ulcers (BE-LU) were found to have the highest increase in TM level. Appearance of larger platelets in all types of thalassaemic blood was observed indicating an increase in the number of younger platelets. These data indicate that injury of vascular endothelial cells is present in thalassaemic patients.

scholarly journals Shear stress augments mitochondrial ATP generation that triggers ATP release and Ca2+ signaling in vascular endothelial cells

2018 ◽  
Vol 315 (5) ◽  
pp. H1477-H1485 ◽  
Author(s):  
Kimiko Yamamoto ◽  
Hiromi Imamura ◽  
Joji Ando
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Vascular Endothelial Cells ◽  
Critical Role ◽  
Atp Release ◽  
Vascular Endothelial ◽  
The Novel ◽  
Caveolin 1 ◽  
Atp Generation

Vascular endothelial cells (ECs) sense and transduce hemodynamic shear stress into intracellular biochemical signals, and Ca2+ signaling plays a critical role in this mechanotransduction, i.e., ECs release ATP in the caveolae in response to shear stress and, in turn, the released ATP activates P2 purinoceptors, which results in an influx into the cells of extracellular Ca2+. However, the mechanism by which the shear stress evokes ATP release remains unclear. Here, we demonstrated that cellular mitochondria play a critical role in this process. Cultured human pulmonary artery ECs were exposed to controlled levels of shear stress in a flow-loading device, and changes in the mitochondrial ATP levels were examined by real-time imaging using a fluorescence resonance energy transfer-based ATP biosensor. Immediately upon exposure of the cells to flow, mitochondrial ATP levels increased, which was both reversible and dependent on the intensity of shear stress. Inhibitors of the mitochondrial electron transport chain and ATP synthase as well as knockdown of caveolin-1, a major structural protein of the caveolae, abolished the shear stress-induced mitochondrial ATP generation, resulting in the loss of ATP release and influx of Ca2+ into the cells. These results suggest the novel role of mitochondria in transducing shear stress into ATP generation: ATP generation leads to ATP release in the caveolae, triggering purinergic Ca2+ signaling. Thus, exposure of ECs to shear stress seems to activate mitochondrial ATP generation through caveola- or caveolin-1-mediated mechanisms. NEW & NOTEWORTHY The mechanism of how vascular endothelial cells sense shear stress generated by blood flow and transduce it into functional responses remains unclear. Real-time imaging of mitochondrial ATP demonstrated the novel role of endothelial mitochondria as mechanosignaling organelles that are able to transduce shear stress into ATP generation, triggering ATP release and purinoceptor-mediated Ca2+ signaling within the cells.

scholarly journals Human vascular adhesion protein 1 (VAP-1) is a unique sialoglycoprotein that mediates carbohydrate-dependent binding of lymphocytes to endothelial cells.

1996 ◽  
Vol 183 (2) ◽  
pp. 569-579 ◽  
Author(s):  
M Salmi ◽  
S Jalkanen
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Endothelial Cells ◽  
Lymphoid Tissues ◽  
Vascular Endothelial ◽  
Adhesion Protein ◽  
Culture Model ◽  
Vascular Adhesion ◽  
Vascular Adhesion Protein 1

The regulated interactions of leukocytes with vascular endothelial cells are crucial in controlling leukocyte traffic between blood and tissues. Vascular adhesion protein-1 (VAP-1) is a novel, human endothelial cell molecule that mediates tissue-selective lymphocyte binding. Two species (90 and 170 kD) of VAP-1 exist in lymphoid tissues. Glycosidase digestions revealed that the mature 170-kD form of VAP-1 expressed on the lumenal surfaces of vessels is a heavily sialylated glycoprotein. The sialic acids are indispensable for the function of VAP-1, since the desialylated form of VAP-1 no longer mediates lymphocyte binding. We also show that L-selectin is not required for binding of activated lymphocytes to VAP-1 under conditions of shear stress. The 90-kD form of VAP-1 was only seen in an organ culture model, and may represent a monomeric or proteolytic form of the larger species. These data indicate that L-selectin negative lymphocytes can bind to tonsillar venules via the VAP- 1-mediated pathway. Moreover, our findings extend the role of carbohydrate-mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins. In conclusion, VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions.

A mathematical model of plasma membrane electrophysiology and calcium dynamics in vascular endothelial cells

2007 ◽  
Vol 293 (1) ◽  
pp. C277-C293 ◽  
Author(s):  
Haroldo S. Silva ◽  
Adam Kapela ◽  
Nikolaos M. Tsoukias
Keyword(s):  
Mathematical Model ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Anion Channel ◽  
Ionic Species ◽  
Vascular Endothelial ◽  
Health And Disease ◽  
Vascular Autoregulation

Vascular endothelial cells (ECs) modulate smooth muscle cell (SMC) contractility, assisting in vascular tone regulation. Cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential ( Vm) play important roles in this process by controlling EC-dependent vasoactive signals and intercellular communication. The present mathematical model integrates plasmalemma electrophysiology and Ca2+ dynamics to investigate EC responses to different stimuli and the controversial relationship between [Ca2+]i and Vm. The model contains descriptions for the intracellular balance of major ionic species and the release of Ca2+ from intracellular stores. It also expands previous formulations by including more detailed transmembrane current descriptions. The model reproduces Vm responses to volume-regulated anion channel (VRAC) blockers and extracellular K+ concentration ([K+]o) challenges, predicting 1) that Vm changes upon VRAC blockade are [K+]o dependent and 2) a biphasic response of Vm to increasing [K+]o. Simulations of agonist-induced Ca2+ mobilization replicate experiments under control and Vm hyperpolarization blockade conditions. They show that peak [Ca2+]i is governed by store Ca2+ release while Ca2+ influx (and consequently Vm) impacts more the resting and plateau [Ca2+]i. The Vm sensitivity of rest and plateau [Ca2+]i is dictated by a [Ca2+]i “buffering” system capable of masking the Vm-dependent transmembrane Ca2+ influx. The model predicts plasma membrane Ca2+-ATPase and Ca2+ permeability as main players in this process. The heterogeneous Vm impact on [Ca2+]i may elucidate conflicting reports on how Vm influences EC Ca2+. The present study forms the basis for the development of multicellular EC-SMC models that can assist in understanding vascular autoregulation in health and disease.

CYLD regulates angiogenesis by mediating vascular endothelial cell migration

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4130-4137 ◽  
Author(s):  
Jinmin Gao ◽  
Lei Sun ◽  
Lihong Huo ◽  
Min Liu ◽  
Dengwen Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial Cell ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
Vascular Endothelial

Cylindromatosis (CYLD) is a deubiquitinase that was initially identified as a tumor suppressor and has recently been implicated in diverse normal physiologic processes. In this study, we have investigated the involvement of CYLD in angiogenesis, the formation of new blood vessels from preexisting ones. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based 3-dimensional capillary sprouting assay. Disruption of CYLD also remarkably inhibits angiogenic response in vivo, as evidenced by diminished blood vessel growth into the angioreactors implanted in mice. Mechanistic studies show that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. Furthermore, we identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. Our findings thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Rac1 activation during endothelial cell migration and angiogenesis.

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