The Effect of Normoxic and Hypoxic U-87 Glioblastoma Paracrine Secretion on the Modulation of Brain Endothelial Cells

Background: Glioblastoma multiforme (GBM) is a highly invasive brain tumour, characterized by its ability to secrete factors promoting its virulence. Brain endothelial cells (BECs) in the GBM environment are physiologically modulated. The present study investigated the modulatory effects of normoxically and hypoxically induced glioblastoma U-87 cell secretions on BECs. Methods: Conditioned media (CM) were derived by cultivating U-87 cells under hypoxic incubation (5% O2) and normoxic incubation (21% O2). Treated bEnd.3 cells were evaluated for mitochondrial dehydrogenase activity, mitochondrial membrane potential (ΔΨm), ATP production, transendothelial electrical resistance (TEER), and endothelial tight-junction (ETJ) gene expression over 96 h. Results: The coculture of bEnd.3 cells with U-87 cells, or exposure to either hypoxic or normoxic U-87CM, was associated with low cellular viability. The ΔΨm in bEnd.3 cells was hyperpolarized after hypoxic U-87CM treatment (p < 0.0001). However, normoxic U-87CM did not affect the state of ΔΨm. BEC ATP levels were reduced after being cocultured with U-87 cells, or with hypoxic and normoxic CM (p < 0.05). Suppressed mitochondrial activity in bEnd.3 cells was associated with increased transendothelial permeability, while bEnd.3 cells significantly increased the gene expression levels of ETJs (p < 0.05) when treated with U-87CM. Conclusions: Hypoxic and normoxic glioblastoma paracrine factors differentially suppressed mitochondrial activity in BECs, increasing the BECs’ barrier permeability.

Altered Moesin and Actin Cytoskeleton Protein Rearrangements Affect Transendothelial Permeability in Human Endothelial Cells upon Dengue Virus Infection and TNF-α Treatment

It has been hypothesized that the host, viral factors, and secreted cytokines (especially TNF-α) play roles in the pathogenesis of secondary dengue infections. Mass spectrometry-based proteomic screening of cytoskeleton fractions isolated from human endothelial (EA.hy926) cells upon dengue virus (DENV) infection and TNF-α treatment identified 450 differentially altered proteins. Among them, decreased levels of moesin, actin stress fiber rearrangements, and dot-like formations of vinculin were observed with western blot analyses and/or immunofluorescence staining (IFA). In vitro vascular permeability assays using EA.hy926 cells, seeded on collagen-coated transwell inserts, showed low levels of transendothelial electrical resistance in treated cells. The synergistic effects of DENV infection and TNF-α treatment caused cellular permeability changes in EA.hy926 cells, which coincided with decreasing moesin levels and the production of abnormal organizations of actin stress fibers and vinculin. Functional studies demonstrated moesin overexpression restored transendothelial permeability in DENV/TNF-α-treated EA.hy926 cells. The present study improves the understanding of the disruption mechanisms of cytoskeleton proteins in enhancing vascular permeability during DENV infection and TNF-α treatment. The study also suggests that these disruption mechanisms are major factors contributing to vascular leakage in severe dengue patients.

A venous-specific purinergic signaling cascade initiated by Pannexin 1 regulates TNFα-induced increases in endothelial permeability

The endothelial cell barrier regulates the passage of fluid between the bloodstream and underlying tissues, and barrier function impairment exacerbates the severity of inflammatory insults. To understand how inflammation alters vessel permeability, we studied the effects of the proinflammatory cytokine TNFα on transendothelial permeability and electrophysiology in ex vivo murine veins and arteries. We found that TNFα specifically decreased the barrier function of venous endothelium without affecting that of arterial endothelium. On the basis of RNA expression profiling and protein analysis, we found that claudin-11 (CLDN11) was the predominant claudin in venous endothelial cells and that there was little, if any, CLDN11 in arterial endothelial cells. Consistent with a difference in claudin composition, TNFα increased the permselectivity of Cl− over Na+ in venous but not arterial endothelium. The vein-specific effects of TNFα also required the activation of Pannexin 1 (Panx1) channels and the CD39-mediated hydrolysis of ATP to adenosine, which subsequently stimulated A2A adenosine receptors. Moreover, the increase in vein permeability required the activation of the Ca2+ channel TRPV4 downstream of Panx1 activation. Panx1-deficient mice resisted the pathologic effects of sepsis induced by cecal ligation and puncture on life span and lung vascular permeability. These data provide a targetable pathway with the potential to promote vein barrier function and prevent the deleterious effects of vascular leak in response to inflammation.

Fenestral diaphragms and PLVAP associations in liver sinusoidal endothelial cells are developmentally regulated

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.

Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein–coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9–based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/β-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/β-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.

Phosphorylation of endothelial nitric oxide synthase by atypical PKCζ contributes to angiopoietin-1–dependent inhibition of VEGF-induced endothelial permeability in vitro

Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine that also increases vascular permeability. Nitric oxide (NO) released from endothelial cells, after activation of endothelial NO synthase (eNOS), contributes to proangiogenic and permeability effects of VEGF. Angiopoietin-1 (Ang-1), via Tie2 receptors, shares many of the proangiogenic properties of VEGF on endothelial cells. However, in contrast to VEGF, Ang-1 protects blood vessels from increased plasma leakage, which contributes to their stabilization. Because eNOS-derived NO is central to increased permeability in response to VEGF, we investigated whether Ang-1 interferes with VEGF signaling to eNOS. We demonstrate that Ang-1 stimulation of endothelial cells inhibits VEGF-induced NO release and transendothelial permeability. In contrast to VEGF stimulation, Ang-1 causes a marked protein kinase C (PKC)–dependent increase in phosphorylation of eNOS on the inhibitory Thr497. Furthermore, using pharmacologic inhibitors, overexpression studies, and small interfering RNA-mediated gene silencing, we demonstrate that atypical PKCζ is responsible for phosphorylation of eNOS on Thr497 in response to Ang-1. In addition, PKCζ knockdown abrogates the capacity of Ang-1 to inhibit VEGF-induced NO release and endothelial permeability. Thus, inhibition of NO production by Ang-1, via phosphorylation of eNOS on Thr497 by PKCζ, is responsible, at least in part, for inhibition of VEGF-stimulated endothelial permeability by Ang-1.