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Vasodilator phosphostimulated protein (VASP) protects endothelial barrier function during hypoxia.

ABSTRACT: The endothelial barrier controls the passage of solutes from the vascular space. This is achieved through active reorganization of the actin cytoskeleton. A central cytoskeletal protein involved into this is vasodilator-stimulated phosphoprotein (VASP). However, the functional role of endothelial VASP during hypoxia has not been thoroughly elucidated. We determined endothelial VASP expression through real-time PCR (Rt-PCR), immunhistochemistry, and Western blot analysis during hypoxia. VASP promoter studies were performed using a PGL3 firefly luciferase containing plasmid. Following approval by the local authorities, VASP ( -/- ) mice and littermate controls were subjected to normobaric hypoxia (8% O(2), 92% N(2)) after intravenous injection of Evans blue dye. In in vitro studies, we found significant VASP repression in human microvascular and human umbilical vein endothelial cells through Rt-PCR, immunhistochemistry, and Western blot analysis. The VASP promoter construct demonstrated significant repression in response to hypoxia, which was abolished when the binding of hypoxia-inducible factor 1 alpha was excluded. Exposure of wild-type (WT) and VASP ( -/- ) animals to normobaric hypoxia for 4 h resulted in an increase in Evans blue tissue extravasation that was significantly increased in VASP ( -/- ) animals compared to WT controls. In summary, we demonstrate here that endothelial VASP holds significant importance for endothelial barrier properties during hypoxia.

SUBMITTER: Schmit MA

PROVIDER: S-EPMC3314830 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAfter cerebral injury blood-brain barrier disruption significantly impairs brain homeostasis. Volatile anesthetics have been shown to be protective in ischemia-reperfusion injury scenarios. Their impact on brain endothelial cells after hypoxia-reoxygenation (H/R) has not yet been studied in detail.MethodsRat brain endothelial cells (RBE4) were exposed to severe hypoxia and reoxygenated in air in the presence or absence of sevoflurane. Changes in dextran permeability and architecture of the cellular junctional proteins ZO-1 and ?-catenin were measured. To determine necrosis and apoptosis rate DNA content, LDH release and caspase activity were quantified. The role of vascular endothelial growth factor (VEGF) as an inflammatory mediator increasing vascular permeability was assessed. At the same time, it was evaluated if sevoflurane effects are mediated through VEGF. Results were analyzed by unpaired t-tests or one way-analysis of variance followed by Bonferroni's correction.ResultsH/R led to a 172% increase in permeability (p<0.001), cell swelling and qualitatively but not quantitatively modified expression of ZO-1, ?-catenin and F-actin. In the presence of sevoflurane during reoxygenation, barrier function improved by 96% (p = 0.042) in parallel to a decrease of the cell size and less re-arranged junction proteins and F-actin. Sevoflurane-induced improvement of the barrier function could not be explained on the level of necrosis or apoptosis as they remained unchanged independent of the presence or absence of the volatile anesthetic. Increased expression of VEGF after H/R was attenuated by sevoflurane by 34% (p = 0.004). Barrier protection provided by sevoflurane was similar to the application of a blocking VEGF-antibody. Furthermore, the protective effect of sevoflurane was abolished in the presence of recombinant VEGF.ConclusionsIn H/R-induced rat brain endothelial cell injury sevoflurane maintains endothelial barrier function through downregulation of VEGF, which is a key player not only in mediating injury, but also with regard to the protective effect of sevoflurane.

Project description:BackgroundCell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier.MethodsTwo different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs-CMECs) and blood-brain barrier (BBB; brain-like ECs-BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM-control). CMECs or BLECs were exposed (6 h) to oxygen-glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control-5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h).ResultsThe analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity.ConclusionOur results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.

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Vasodilator phosphostimulated protein (VASP) protects endothelial barrier function during hypoxia.

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