Laura L. Coe

Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: Implications for tumor angiogenesis

Studies aimed at elucidating the function of the protein synthesis factor eukaryotic initiation factor 4E (elF‐4E) have demonstrated that overexpression of this protein results in marked cell phenotypic and proliferative changes, including neoplastic transformation of cells. These data suggest that elF‐4E may somehow participate in the development and progression of tumors in vivo. In order to determine how elF‐4E exerts its transforming effects, we examined vascular permeability factor (VPF) levels in cells transfected with an elF‐4E vector. Cells overexpressing elF‐4E showed an increase in intracellular, and an average 130‐fold increase in secreted VPF protein levels (CHO 0.13 ± 0.12 ng/ml; CHO‐4E 20.5 ± 12.5 ng/ml) over control cells. HUVEC growth induction revealed these VPF levels to be biologically active. Northern analysis revealed no difference in VPF transcript between the 2 cell lines. Polysome analysis showed that the VPF message in elF‐4E‐transfected cells was associated with the heavy polysomal regions, whereas the VPF message was associated with light polysomes in control cells. These data strongly suggest that enhanced VPF expression is achieved through translational regulation rather than transcriptional regulation in cells overexpressing elF‐4E. This indicates that elF‐4E‐induced VPF expression may be an important factor in some forms of tumor angiogenesis and development.

Expression of Zonula Occludens and Adherens Junctional Proteins in Human Venous and Arterial Endothelian Cells: Role of Occludin in Endothelial Solute Barriers

Objective: The purpose of this study was to correlate the expression of occludin and VE‐cadherin with the solute barrier properties of arterial and venous endothelial monolayers.

Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells

We compared U-937 cell adhesion and adhesion molecule expression in human umbilical venous (HUVECs) and arterial (HUAECs) endothelial cells exposed to tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS). TNF and LPS stimulated vascular cell adhesion molecule (VCAM)-1 surface expression and adhesion of U-937 monocyte-like cells to HUVECs but not to HUAECs. Antibody studies demonstrated that in HUVECs at least 75% of the adhesion response is VCAM-1 mediated. Interleukin-1 stimulated U-937 cell adhesion to and VCAM-1 surface expression in both HUVECs and HUAECs. Pyrrolidinedithiocarbamate and the proteasome inhibitor MG-132 blocked TNF- and LPS-stimulated U-937 cell adhesion to HUVECs. These agents also significantly decreased TNF- and LPS-stimulated increases in HUVEC surface VCAM-1. TNF increased VCAM-1 protein and mRNA in HUVECs that was blocked by pyrrolidinedithiocarbamate. However, neither TNF or LPS stimulated VCAM-1 expression in HUAECs. TNF stimulated expression of both intercellular adhesion molecule-1 and E-selectin in HUVECs, but in HUAECs, only intercellular adhesion molecule-1 was increased. Electrophoretic mobility shift assays demonstrated no difference in the pattern of TNF-stimulated nuclear factor-κB activation between HUVECs and HUAECs. These studies demonstrate a novel and striking insensitivity of arterial endothelium to the effects of TNF and LPS and indicate a dissociation between the ability of HUAECs to upregulate nuclear factor-κB and VCAM-1.

SOLUBLE SELECTINS AND ICAM-1 MODULATE NEUTROPHIL-ENDOTHELIAL ADHESION AND DIAPEDESIS IN VITRO

We observed that normal plasma dramatically reduces neutrophil-endothelial adhesion. Therefore, we identified factors in plasma which might limit PMN adhesion in vitro. We found that the anti-adhesive effect was not mediated by vasoactive lipids present in plasma. Immunoprecipitation of soluble adhesion molecules, P and E-selectins and ICAM-1 restored PMN adhesion to control values. We further examined whether soluble adhesion molecules in plasma might also regulate PMN endothelial migration in response to fMLP (10−6 M). Plasma significantly reduced PMN migration, and this effect was prevented only by the simultaneous removal of soluble P and E selectins and ICAM-1 together, but not individually. These data show that soluble selectins and ICAM-1 may regulate PMN adhesion and diapedesis, and that alterations in the levels of these molecules may regulate PMN-endothelial interactions in vivo.

Exogenous NO enhances hydrogen peroxide-mediated neutrophil adherence to cultured endothelial cells

One important aspect of oxidant injury is the enhancement of neutrophil-endothelial adhesion by oxidants such as hydrogen peroxide. Recent studies suggest that nitric oxide (NO) can limit oxidant-mediated tissue injury, since inhibitors of endogenous NO synthesis often promote neutrophil-endothelial adhesion. However, less is known about the direct role of exogenous NO in modulating proadhesive effects of oxidants. The objective of this study was to examine how an NO donor modifies hydrogen peroxide-mediated adhesion of neutrophils to cultured endothelial cells. Human umbilical vein endothelial cell monolayers were exposed for 30 min to 0-0.1 mM hydrogen peroxide with or without the NO donor spermine-NONOate (SNO; 0-0.5 mM), and the adhesion of 51Cr-labeled polymorphonuclear neutrophils (PMNs) was measured in a static adhesion assay. PMN adherence was not altered by either peroxide (up to 0.1 mM) or SNO (up to 0.5 mM) alone but was significantly increased by over 300% by coadministration of both 0.1 mM peroxide and 0.5 mM SNO. This increase in adhesion with these two agents was correlated with an increase in the presentation of surface P-selectin but not intercellular adhesion molecule-1. Both PMN adhesion and P-selectin presentation were blocked by 0.1 mM desferrioxamine (an iron chelator) and 1 mM methionine (an oxyradical scavenger). WEB-2086, a platelet-activating factor-receptor antagonist (10 microM), also prevented PMN adhesion but not P-selectin expression. An antibody directed against either P-selectin or intercellular adhesion molecule-1 also blocked adhesion. These data indicate that NO may actually exacerbate rather than protect against the inflammatory effects of peroxide in some models of inflammation through the synthesis of platelet-activating factor and the mobilization of P-selectin.One important aspect of oxidant injury is the enhancement of neutrophil-endothelial adhesion by oxidants such as hydrogen peroxide. Recent studies suggest that nitric oxide (NO) can limit oxidant-mediated tissue injury, since inhibitors of endogenous NO synthesis often promote neutrophil-endothelial adhesion. However, less is known about the direct role of exogenous NO in modulating proadhesive effects of oxidants. The objective of this study was to examine how an NO donor modifies hydrogen peroxide-mediated adhesion of neutrophils to cultured endothelial cells. Human umbilical vein endothelial cell monolayers were exposed for 30 min to 0-0.1 mM hydrogen peroxide with or without the NO donor spermine-NONOate (SNO; 0-0.5 mM), and the adhesion of51Cr-labeled polymorphonuclear neutrophils (PMNs) was measured in a static adhesion assay. PMN adherence was not altered by either peroxide (up to 0.1 mM) or SNO (up to 0.5 mM) alone but was significantly increased by over 300% by coadministration of both 0.1 mM peroxide and 0.5 mM SNO. This increase in adhesion with these two agents was correlated with an increase in the presentation of surface P-selectin but not intercellular adhesion molecule-1. Both PMN adhesion and P-selectin presentation were blocked by 0.1 mM desferrioxamine (an iron chelator) and 1 mM methionine (an oxy-radical scavenger). WEB-2086, a platelet-activating factor-receptor antagonist (10 μM), also prevented PMN adhesion but not P-selectin expression. An antibody directed against either P-selectin or intercellular adhesion molecule-1 also blocked adhesion. These data indicate that NO may actually exacerbate rather than protect against the inflammatory effects of peroxide in some models of inflammation through the synthesis of platelet-activating factor and the mobilization of P-selectin.

Exogenous Nitric Oxide Increases Neutrophil Adhesion to Cultured Human Endothelial Monolayers through a Protein Kinase G Dependent Mechanism

Endothelial-neutrophil adhesion is a critical step in acute inflammatory diseases, which is mediated in part by P-selectin and platelet-activating factor (PAF). Nitric oxide (NO) is well known as an endogenous second messenger derived from endothelial cells, and regulates many important physiological events, however, the direct effects of NO on endothelial-neutrophil adhesion is less well understood. The objective of this study was to examine whether, and how relatively high levels of exogenous NO increases neutrophil adhesion with respect to P-selectin and PAF. Endothelial monolayers were exposed to chemical agents for 30 min, and the adhesion of 5lCr-labeled neutrophils measured in a static adhesion assay. Spermine-NONOate (SNO), an NO donor, significantly increased neutrophil adhesion and expression of P-selectin at a concentration of 1 mM. SNO (1 mM)-mediated neutrophil adhesion was significantly inhibited by a protein kinase G inhibitor, KT5823 (0.5 μM), but not by a classical protein kinase C inhibitor, Gö6976 (10 nM), a tyrosine kinase inhibitor, genistein (1 μM), or a protein kinase A inhibitor, H-89 (0.1 μM). P-selectin surface expression induced by 1 mM SNO was also significantly inhibited by 0.5 μM KT5823. Conversely, a cytoplasm calcium chelator, TMB-8 (0.1 mM), significantly exacerbated both the neutrophil adhesion and P-selectin expression induced by SNO. WEB 2086 (10 μM), a PAF receptor antagonist, blocked neutrophil adhesion, but did not block P-selectin expression induced by SNO. These data suggest that NO increases endothelial-neutrophil adhesion through protein kinase G-mediated P-selectin mobilization to the cell surface and endothelial PAF synthesis.

Polynitroxylated starch/TPL attenuates cachexia and increased epithelial permeability associated with TNBS colitis

Free radicals play an important role in the initiation and progression of inflammatory bowel disease (IBD). Therefore, the reduction or elimination of adverse oxidant effects can provide novel therapy for IBD. Here, the antioxidant capacity and protective effects of a new class of chemically modified hetastarch (polynitroxyl starch, or PNS) plus 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol or TPL) (PNS/TPL) were assessed in a model of colitis. The superoxide scavenging capacity of PNS/TPL—that is, the inhibition of the reduction of cytochrome c in the presence of xanthine/xanthine oxidase (X/XO)—was evaluated in vitro. The effects of PNS/TPL on X/XO–induced neutrophil endothelial adhesion in vitro were investigated. Also, this study tested the protection produced by PNS/TPL in a mouse model of trinitrobenzene sulfonic acid (TNBS)–induced colitis. PNS/TPL was given intravenously immediately before (<30 min) and intraperitoneally at 24 and 72 hr after TNBS induction. The body weight and survival rate of the mice were checked daily. Colonic mucosal damage was assessed on the 7th day by measuring intestinal permeability to Evans blue (EB) in vivo. The ability of PNS to reoxidize bioreduced TPL was documented by whole-body electron paramagnetic resonance (EPR) detection. We found that PNS or TPL exhibits superoxide dismutase (SOD)–like activity, with approximately 2% of SOD activity occurring on a molar basis. The endothelial–neutrophil adherence induced by X/XO was significantly inhibited by PNS/TPL but not by TPL alone. PNS/TPL protected against cachexia and mortality, both usually induced by TNBS. Epithelial permeability was increased significantly in TNBS mice but was ameliorated by the administration of PNS/TPL. In conclusion, PNS/TPL may be beneficial in the treatment or prevention of IBD through its antioxidant effects, which inhibit oxidant-mediated leukocyte adhesion and injury to endothelial cells.

Intracellular mechanisms of nitric oxide plus hydrogen peroxide-mediated neutrophil adherence to cultured human endothelial cells.

Abstract.Objective and Design: We investigated signal-transduction in nitric oxide/hydrogen peroxide (NO/H2O2) mediated neutrophil-endothelial adhesion and P-selectin mobilization.¶Materials and Methods: Human endothelial monolayers (HUVEC) were exposed to 0.1 mM H2O2 plus an NO donor, 0.5 mM spermine-NONOate, and second message inhibitors and neutrophil adhesion and P-selectin expression measured.¶Results: Neutrophil adherence induced by NO/H2O2 was blocked by a PKG inhibitor, (KT5823, 0.5 μM), a PKC inhibitor, (Gö6976, 10 nM), a calcium chelator, TMB-8 (0.1 mM) and a K+ channel blocker, glibenclamide, (10 μM), but not by a PKA inhibitor, (H-89, 0.1 μM) or a tyrosine kinase inhibitor, (genistein, 1 μM). P-selectin expression induced by NO/H2O2 was blocked by KT5823 and Gö6976, but not by TMB-8 or glibenclamide.¶Conclusions: These data demonstrate that NO/ H2O2 promotes neutrophil-endothelial adhesion through PKG, PKC, calcium, and K+ channels, but not PKA or tyrosine kinase. Conversely, P-selectin mobilization requires only PKG and PKC.