Role of p38 mitogen-activated protein kinase in antiphospholipid antibody-mediated thrombosis and endothelial cell activation.

BACKGROUND: The purpose of this study was to examine whether SB 203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, is effective in reversing the pathogenic effects of antiphospholipid antibodies. METHODS: The adhesion of THP-1 monocytes to cultured endothelial cells (EC) treated with immunoglobulin G (IgG) from a patient with antiphospholipid syndrome (IgG-APS) or control IgG (IgG-NHS) in the presence and absence of SB 203580 was examined. The size of an induced thrombus in the femoral vein, the adhesion of leukocytes to EC of cremaster muscle, tissue factor (TF) activity in carotid artery and in peritoneal macrophages, the ex vivo expression of vascular cell adhesion molecule-1 (VCAM-1) in aorta preparations and platelet aggregation were studied in mice injected with IgG-APS or control IgG-NHS and with or without SB 203580. RESULTS: SB 203580 significantly reduced the increased adhesion of THP-1 to EC in vitro, the number of leukocytes adhering to EC, the thrombus size, the TF activity in carotid arteries and in peritoneal mononuclear cells, and the expression of VCAM-1 in aorta of mice, and completely abrogated platelet aggregation induced by IgG-APS. CONCLUSION: These data suggest that targeting the p38 MAPK pathway may be valuable in designing new therapy modalities for treating thrombosis in patients with APS.

E-Selectin mediates pathogenic effects of antiphospholipid antibodies.

Antiphospholipid (aPL) antibodies, detected in patients with antiphospholipid syndrome (APS) are associated with thrombosis, pregnancy loss and thrombocytopenia. Studies have shown that aPL are thrombogenic in vivo, but the mechanism(s) involved are not completely understood. Several studies have demonstrated that aPL antibodies activate endothelial cells (ECs) in vitro, as determined by up-regulation of adhesion molecules: E-selectin (E-sel); intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and in vivo. The objectives of these study were to determine the effects of aPL antibodies on the expression of E-selectin on ECs, on the adhesion of monocytes to ECs and to study the role of E-selectin on aPL antibodies enhanced thrombus formation and activation of ECs in vivo. We demonstrated that the surface expression of E-selectin on HUVEC by ELISA was increased 400-fold when treated with tumor necrosis factor-alpha (TNF-alpha) and 421-fold when treated with aPL antibodies during 4 h. APL antibodies also induced activation of the nuclear factor-kappa B (NF-kappaB). APL antibodies increased significantly the number of adhering leukocytes to ECs in vivo in C57BL/6 J mice when compared to IgG-NHS treated mice. This effect was abrogated in E-selectin-deficient mice. The thrombus size was significantly increased in C57BL/6 J mice treated with aPL antibodies when compared to mice treated with IgG-NHS. This enhancement in thrombus size by aPL antibodies was abrogated in E-selectin-deficient mice treated with aPL antibodies.

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes.

Hyperpolarization in human leukemia THP-1 monocytes adherent to vascular cell adhesion molecule (VCAM)-1 is due to an induction of inwardly rectifying K(+) currents (I(ir)) (Colden-Stanfield M and Gallin EK, Am J Physiol Cell Physiol 275: C267-C277, 1998). We determined whether the VCAM-1-induced hyperpolarization is sufficient to augment the increase in intracellular free calcium ([Ca(2+)](i)) produced by Ca(2+) store depletion with thapsigargin (TG) and readdition of external CaCl(2) in fura 2-loaded THP-1 monocytes. Whereas there was a 2.1-fold increase in [Ca(2+)](i) in monocytes bound to glass for 5 h in response to TG and CaCl(2) addition, adherence to VCAM-1 produced a 5-fold increase in [Ca(2+)](i). Depolarization of monocytes adherent to VCAM-1 by I(ir) blockade or exposure to high [K(+)] abolished the enhancement of the peak [Ca(2+)](i) response. In monocytes bound to glass, hyperpolarization of the membrane potential with valinomycin, a K(+) ionophore, to the level of hyperpolarization seen in cells adherent to VCAM-1 produced similar changes in peak [Ca(2+)](i). Adherence of monocytes to E-selectin produced a similar peak [Ca(2+)](i) to cells bound to glass. Thus monocyte adherence to the physiological substrate VCAM-1 produces a hyperpolarization that is sufficient to enhance Ca(2+) entry and may impact Ca(2+)-dependent monocyte function.

GDKV-induced antiphospholipid antibodies enhance thrombosis and activate endothelial cells in vivo and in vitro.

Antiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). beta2-Glycoprotein I (beta2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with beta2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of beta2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274-Cys288 in the fifth domain of human beta2GPI, which represents the phospholipid-binding site of beta2GPI. The Abs generated had aPL and anti-beta2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of beta2GPI are thrombogenic and activate endothelial cells.

Antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo.

BACKGROUND: Antiphospholipid (aPL) antibodies are associated with thrombosis in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. Although aPL antibodies have been shown to inhibit protein C activation and activate endothelial cells (ECs) in vitro, no study has examined whether these antibodies activate ECs in vivo. Therefore, human affinity-purified aPL (ap aPL) antibodies from APS patients were tested in a mouse model of microcirculation using the cremaster muscle that allows direct microscopic examination of thrombus formation and adhesion of white blood cells (WBCs) to ECs as an indication of EC activation in vivo. Adhesion molecule expression on human umbilical vein endothelial cells (HUVECs) after aPL exposure was performed to confirm EC activation in vitro. METHODS AND RESULTS: All 6 ap aPL antibodies significantly increased the expression of VCAM-1 (2.3- to 4.4-fold), with one of the antibodies also increasing the expression of E-selectin (1.6-fold) on HUVECs in vitro. In the in vivo experiments, each ap aPL antibody except for 1 preparation increased WBC sticking (mean number of WBCs ranged from 22.7 to 50.6) compared with control (14.4), which correlated with enhanced thrombus formation (mean thrombus size ranged from 1098 to 6476 versus 594 microm2 for control). CONCLUSIONS: Activation of ECs by aPL antibodies in vivo may create a prothrombotic state on ECs, which may be the first pathophysiological event of thrombosis in APS.

Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium.

Resting membrane potential (RMP) and whole cell currents were recorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs), lipopolysaccharide (LPS)-treated HUVECs, immobilized E-selectin, or vascular cell adhesion molecule 1 (VCAM-1) using the patch-clamp technique. RMP after 5 h on polystyrene was -24.3 +/- 1.7 mV (n = 42) with delayed rectifier K+ (Idr) and Cl- currents (ICl) present in >75% of the cells. Inwardly rectifying K+ currents (Iir) were present in only 14% of THP-1 cells. Adherence to unstimulated HUVECs or E-selectin for 5 h had no effect on Iir or ICl but decreased Idr. Five hours after adherence to LPS-treated HUVECs, outward currents were unchanged, but Iir was present in 81% of THP-1 cells. A twofold increase in Iir and a hyperpolarization (-41.3 +/- 3.7 mV, n = 16) were abolished by pretreatment of THP-1 cells with cycloheximide, a protein synthesis inhibitor, or herbimycin A, a tyrosine kinase inhibitor, or by pretreatment of the LPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interaction between THP-1 cells and LPS-treated HUVECs was required to induce Iir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similar conductances to cells adherent to LPS-treated HUVECs. Thus engagement of specific integrins results in selective modulation of different K+ conductances.

Ionizing radiation increases endothelial and epithelial cell production of influenza virus and leukocyte adherence.

To characterize the effect of 60Co gamma radiation on cell-cell and pathogen-cell interactions, the adherence of undifferentiated HL-60 cells to HUVEC monolayers was tested in the absence and presence of LPS or influenza virus type A. Basal HL-60 cell adherence to uninfected HUVEC monolayers (3.0 +/- 1.6%, n = 30) was not altered when HUVECs were exposed to 1- to 10-Gy gamma irradiation 4 to 72 h before the adhesion assay. LPS treatment of HUVEC monolayers (0.5 microgram/ml, 4 h) produced a 6.9-fold increase in adherence that was not altered by previous irradiation. However, when HUVEC monolayers were subjected to 1-10 Gy 41 h before influenza virus infection (10(6) pfu/ml) for 7 h, virus-induced adherence was enhanced in a dose-dependent manner. Increased virus hemagglutinin (HA) protein expression mediated the radiation-induced adherence for the following reasons: 1) HA Ag increases paralleled increases in leukocyte adherence. 2) Northern blot analysis demonstrated a time-dependent increase in mRNA HA levels. 3) Anti-HA blocked HL-60 cell adherence to irradiated and virus-infected HUVEC monolayers. These changes were associated with an increased virus titer yield and virus-induced HUVEC killing. In contrast, cytotoxicity produced by vesicular stomatitis virus, which unlike influenza virus replicates cytoplasmically, was not altered by radiation in HUVECs. In related studies, the canine kidney epithelial (MDCK) cell line showed a similar increased influenza virus production after gamma radiation, indicating that the radiation-induced increase in production of influenza virus is not cell-specific and probably involves a nuclear mechanism.

Characterization of influenza virus-induced leukocyte adherence to human umbilical vein endothelial cell monolayers.

The adherence of undifferentiated 51Cr-labeled HL-60 (0.5 x 10(6) HL-60 cells/well) cells was monitored on influenza virus-infected HUVEC monolayers. Whereas only 3.0 +/- 1.6% (n = 36) of HL-60 cells adhered to uninfected HUVEC, adherence was increased to 41.7 +/- 2.2% (n = 6), 79.7 +/- 1.2% (n = 6), 83.9 +/- 0.7% (n = 6), and 84.4 +/- 0.5% (n = 6) on HUVEC infected for 7 h at a MOI of 1, 3, 6, and 9, respectively. In comparison, HL-60 cell adherence increased to 35% when HUVEC monolayers were stimulated with LPS (0.2-20 micrograms) for 4 h. Increased adherence to infected HUVEC occurred at 5 h postinfection, peaked at 7 h, and was maintained at 24 h postinfection. Active virus and metabolically active endothelial cells were required to mediate the virus-induced adherence. E-selectin and ICAM-1 Ag were upregulated 78.3- and 4.1-fold, respectively, by LPS (0.02-20 micrograms, 4 h) whereas virus infection (7 h) only increased these proteins 2.6- and 1.4-fold with a MOI > or = 16. Although the time courses of expression for both adhesion molecules after LPS treatment of virus infection were similar, the difference in the magnitude of upregulation suggests that virus-induced adherence is not a result of upregulation of E-selectin and ICAM-1. In contrast, surface expression of HA is involved in HL-60 cell adherence to virus-infected HUVEC because (1) the time course and magnitude of HA AG expression paralleled the time course and magnitude of HL-60 cell adherence after virus infection of HUVEC; (2) HL-60 cell aggregates were absent on infected HUVEC monolayers in the presence of anti-HA; (3) HL-60 cells competed with RBC for infected endothelial cells stained for cellular HA Ag and (4) anti-HA abolished the virus-induced adherence. Furthermore, it appears that HL-60 cells are binding directly to HA because HL-60 cell adherence to a cell-free surface was increased if virus was prebound and neuraminidase treatment of HL-60 cells prevented the HL-60 cell adherence to influenza virus-infected endothelial monolayers.

Comparison of apical and basal surfaces of confluent endothelial cells: patch-clamp and viral studies.

The distribution of inwardly rectifying (Ki) and calcium-activated (KCa) potassium channels on the apical and basal surfaces of bovine aortic endothelial cells (BAECs) was examined by inverting BAEC monolayers onto polylysine-coated cover slips. To monitor cellular polarity, we examined human red blood cell adherence (hemadsorption) to the influenza virus protein, hemagglutinin (HA), and virus budding on the surface of infected BAECs. Hemadsorption and virus budding occurred on the apical surface but were not apparent on the basal surface of monolayers 1 and 5 h after inversion, although cellular HA antigen localization confirmed that all monolayers were infected. In contrast, by 9.5 and 24 h after inversion, hemadsorption was evident on the "new" apical surface. Single-channel patch-clamp analysis revealed the presence of both Ki and KCa channels on the apical surface and basal surface of BAEC monolayers 2-5 h after inversion. K channel conductance and kinetics were similar regardless of the surface monitored. This nonenzymatic mechanical technique of exposing the basal surface of endothelium provides a useful tool to study the distribution of ion channels in endothelium and in other polarized cell types grown in tissue culture.

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells.

Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]i, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with 86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2(+)-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin, noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100 nM [Ca2+]i. The whole-cell current measurements show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected by noxiustoxin or tetrabutylammonium chloride. Efflux of 86Rb+ from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and by noxiustoxin, but not by apamin or furosemide. Thus, 86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2(+)-activated membrane currents. The results confirm that bradykinin-stimulated 86Rb+ efflux occurs via Ca2(+)-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2(+)-activated K+ current in the response of BAECs to bradykinin.

Morphine induces an intracellular alkalinization in bovine aortic endothelial cells (BAECs).

The resting intracellular pH (pHi) of BAECs, by using BCECF fluorescence, at 37 degrees C in Na+ Hanks' was 7.22 +/- 0.03. Cells which had been acid-loaded recovered from the intracellular acidification in Na+ Hanks' in a [Na+]o-dependent and amiloride-sensitive manner. Recovery from acidification had an apparent Km for Na+ of 40 +/- 10 mM and Ki for amiloride of 26 +/- 4 microM. Morphine (50 microM, 20 min, 37 degrees C) increased the pHi to 7.55 +/- 0.05. Naloxone (50 microM) given 5 min before morphine (50 microM) blocked this effect, indicating that this was an opiate receptor-mediated phenomenon. To determine if morphine activated the Na+/H+ exchanger, pHi was monitored in Na+-free Hanks', acidic Na+ Hanks' or amiloride-containing Na+ Hanks'. The alkalinization produced by morphine was not observed under all these circumstances. These data suggest morphine activates the Na+/H+ exchanger via opiate receptors.

Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells.

The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 microM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 microM) and nitrendipine (1 microM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H](+)PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)