Dataset Information

Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells.

ABSTRACT:

Introduction

Hypoxia induces dilatation of the umbilical vein by releasing autocoids from endothelium; prostaglandins (PGs), adenosine and nitric oxide (NO) have been implicated. ATP is vasoactive, thus we tested whether hypoxia releases ATP from primary Human Umbilical Vein Endothelial Cells (HUVEC).

Methods

HUVEC were grown on inserts under no-flow conditions. ATP was assayed by luciferin-luciferase and visualised by quinacrine labeling. Intracellular Ca(2+) ([Ca(2+)]i) was imaged with Fura-2.

Results

ATP release occurred constitutively and was increased by hypoxia (PO2: 150-8 mmHg), ?10-fold more from apical, than basolateral surface. Constitutive ATP release was decreased, while hypoxia-induced release was abolished by brefeldin or monensin A, inhibitors of vesicular transport, and LY294002 or Y27632, inhibitors of phosphoinositide 3-kinases (PI3K) and Rho-associated protein kinase (ROCK). ATP release was unaffected by NO donor, but increased by calcium ionophore, by >60-fold from apical, but <25% from basolateral surface. Hypoxia induced a small increase in [Ca(2+)]i compared with ATP (10 ?M); hypoxia inhibited the ATP response. Quinacrine-ATP fluorescent loci in the perinuclear space, were diminished by hypoxia and monensin, whereas brefeldin A increased fluorescence intensity, consistent with inhibition of anterograde transport.

Discussion

Hypoxia within the physiological range releases ATP from HUVEC, particularly from apical/adluminal surfaces by exocytosis, via an increase in [Ca(2+)]i, PI3K and ROCK, independently of NO. We propose that hypoxia releases ATP at concentrations sufficient to induce umbilical vein dilation via PGs and NO and improve fetal blood flow, but curbs amplification of ATP release by autocrine actions of ATP, so limiting its pro-inflammatory effects.

SUBMITTER: Lim To WK

PROVIDER: S-EPMC4502406 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Publications

Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells.

Lim To W K WK Kumar P P Marshall J M JM

Placenta 20150418 7

<h4>Introduction</h4>Hypoxia induces dilatation of the umbilical vein by releasing autocoids from endothelium; prostaglandins (PGs), adenosine and nitric oxide (NO) have been implicated. ATP is vasoactive, thus we tested whether hypoxia releases ATP from primary Human Umbilical Vein Endothelial Cells (HUVEC).<h4>Methods</h4>HUVEC were grown on inserts under no-flow conditions. ATP was assayed by luciferin-luciferase and visualised by quinacrine labeling. Intracellular Ca(2+) ([Ca(2+)]i) was imag ...[more]

PMID: 25956988

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Project description:BackgroundMicroparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b - wrapped within EMPs - stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells.MethodsHuman umbilical vein endothelial cells (HUVECs) were cultured in vitro and arranged to harvest EMPs in two parts: the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle, EMPNC mimic, and EMPmiR-19b mimic. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student's t-test was used when two groups were compared.ResultsCompared with EMPcontrol- and EMPhypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40, P < 0. 001) and transwell chamber migration assay (83.00 ± 3.46 vs. 235.00 ± 16.52, P < 0.01), the number of tube formations was markedly reduced by 70% in the EMPhypoxia group (P < 0.001) in vitro analysis of HUVECs. Meanwhile, a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMPmiR-19b mimic was observed. This effect might be due to the delivery of miR-19b in EMPs. Transforming growth factor-?2 (TGF?2) was predicted to be one of the target genes of miR-19b, and we further confirmed that TGF?2 was a direct target gene of miR-19b using the luciferase assay. The expression of TGF?2 in HUVECs was inhibited by treatment with EMPhypoxia and EMPmiR-19b mimic.ConclusionsMiR-19b in EMPs induced by hypoxia could reduce endothelial cell migration and angiogenesis by downregulating TGF?2 expression, which may have inhibited the progression of atherosclerosis.

Dataset Information

Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells.

Introduction

Methods

Results

Discussion

Publications

Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells.

Similar Datasets

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