Jochen Seebach

Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function

ABSTRACT Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP1,2 has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP1,2 were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and Δ-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-α), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not Δ-peptide, induced a recovery of endothelial barrier function following treatment with TNF-α. Our results demonstrate that Ebola virus GP1,2 in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.

Endothelial barrier function under laminar fluid shear stress

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress–induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 Ωcm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of α-, β-, and γ-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress–induced junction rearrangement. Additionally, catenins were partly colocalized with β-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress–dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.

A New Ebola Virus Nonstructural Glycoprotein Expressed through RNA Editing

ABSTRACT Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP1,2) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP1,2, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

Flow-dependent regulation of angiopoietin-2.

Endothelial cells are constantly exposed to high or low shear stress in arteries and veins by the flowing blood. Angiopoietin‐2 (Ang‐2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. In this study, flow‐dependent regulation of Ang‐2 was analyzed in vitro and in vivo. Ang‐2 mRNA, protein expression and release was upregulated by 24 h of low (1 dyne/cm2), but downregulated by high flow (30 dyne/cm2) in human endothelial cells. Increased endothelial NO synthase expression and NO formation was not affecting regulation of Ang‐2 by low or high flow. Low and high flow increased VEGF‐A expression. Inhibition of VEGFR‐2 prevented upregulation of Ang‐2 by low flow, but not downregulation of Ang‐2 by high flow. Furthermore, upregulation of Ang‐2 by VEGF was reduced by application of high flow. Forkhead box O (FOXO) transcription factor FOXO1 has been shown to regulate Ang‐2 expression in endothelial cells. FOXO1 binding activity was reduced by high flow. Nuclear localization of transcription factor FOXO1 was not changed by low flow, but reduced by high flow. In vivo, Ang‐2 was higher expressed in veins compared to arteries. Arterial ligation augmented Ang‐2 expression in distal arterial low flow areas. Our results support a VEGF‐dependent induction of Ang‐2 in low flow areas, and FOXO1‐dependent downregulation of Ang‐2 in high flow areas. These data suggest a new mechanism of flow‐dependent regulation of vessel stability and differentiation. J. Cell. Physiol. 214: 491–503, 2008.

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22(phox) expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-beta no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22(phox). The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.

Caveolin-1 opens endothelial cell junctions by targeting catenins

AIMS A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown. METHODS AND RESULTS Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with β- and γ-catenin. Consequently, the association of β- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1. CONCLUSION Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulation.

Structure‐Function Analysis of the Soluble Glycoprotein, sGP, of Ebola Virus

In addition to the transmembrane protein, GP1,2, the Ebola virus glycoprotein gene encodes the soluble glycoproteins sGP and Δ‐peptide. Two more soluble proteins, GP1 and GP1,2ΔTM, are generated from GP1,2 as a result of disulfide‐bond instability and proteolytic cleavage, respectively, and are shed from the surface of infected cells. The sGP glycoprotein is secreted as a disulfide‐linked homodimer, but there have been conflicting reports on whether it is arranged in a parallel or antiparallel orientation. Off‐line HPLC‐MALDI‐TOF MS (MS/MS) was used to identify the arrangement of all disulfide bonds and simultaneously determine site‐specific information regarding N‐glycosylation. Our data prove that sGP is a parallel homodimer that contains C53–C53’ and C306–C306’ disulfide bonds, and although there are six predicted N‐linked carbohydrate sites, only five are consistently glycosylated. The disulfide bond arrangement was confirmed by using cysteine to glycine mutations at amino acid positions 53 and 306. The mutants had a reduced ability to rescue the barrier function of TNF‐α‐treated endothelial cells—a function previously reported for sGP. This indicates that these disulfide bonds are critical for the proposed anti‐inflammatory function of sGP.

ARP2/3-mediated junction-associated lamellipodia control VE-cadherin–based cell junction dynamics and maintain monolayer integrity

The ARP2/3 complex controls junction-associated intermittent lamellipodia (JAIL), which trigger VE-cadherin adhesion and dynamics. JAIL formation maintains paraendothelial barrier function under physiological conditions and depends on the local VE-cadherin concentration.

Actin filament dynamics and endothelial cell junctions: the Ying and Yang between stabilization and motion

The vascular endothelium is a cellular interface between the blood and the interstitial space of tissue, which controls the exchange of fluid, solutes and cells by both transcellular and paracellular means. To accomplish the demands on barrier function, the regulation of the endothelium requires quick and adaptive mechanisms. This is, among others, accomplished by actin dynamics that interdependently interact with both the VE-cadherin/catenin complex, the main components of the adherens type junctions in endothelium and the membrane cytoskeleton. Actin filaments in endothelium are components of super-structured protein assemblies that control a variety of dynamic processes such as endo- and exocytosis, shape change, cell–substrate along with cell–cell adhesion and cell motion. In endothelium, actin filaments are components of: (1) contractile actin bundles appearing as stress fibers and junction-associated circumferential actin filaments, (2) actin networks accompanied by endocytotic ruffles, lamellipodia at leading edges of migrating cells and junction-associated intermittent lamellipodia (JAIL) that dynamically maintain junction integrity, (3) cortical actin and (4) the membrane cytoskeleton. All these structures, most probably interact with cell junctions and cell–substrate adhesion sites. Due to the rapid growth in information, we aim to provide a bird’s eye view focusing on actin filaments in endothelium and its functional relevance for entire cell and junction integrity, rather than discussing the detailed molecular mechanism for control of actin dynamics.

Ebola sGP—The first viral glycoprotein shown to be C-mannosylated

Abstract Mass spectrometry analysis of the Ebola virus soluble glycoprotein sGP identified a rare post-translation modification, C-mannosylation, which was found on tryptophan (W) 288. This modification has not been described for any other viral protein; however, many viral transmembrane glycoproteins contain one or more of the recognition motifs (W-x-x-W). Elimination of the C-mannose on sGP did not significantly alter protein biosynthesis, processing or structure. Furthermore, the protective effect of sGP on endothelial barrier function, currently the only known activity of sGP, was unaltered. It is possible that C-mannosylation may be a common post-translational modification of viral transmembrane glycoproteins where it could play a role in particle maturation and/or entry by stabilizing the structure of these proteins. In this regard, C-mannosylation of sGP may be an anomaly resulting from the unique manner in which this protein is generated as the product of unedited transcripts from the glycoprotein gene of Ebola.