Margaret Colden-Stanfield

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.

E-Selectin mediates pathogenic effects of antiphospholipid antibodies

Summary.  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‐α) and 421‐fold when treated with aPL antibodies during 4 h. APL antibodies also induced activation of the nuclear factor‐kappa B (NF‐κB). 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.

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

Summary.  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.

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+( I dr) and Cl- currents ( I Cl) present in >75% of the cells. Inwardly rectifying K+ currents ( I ir) were present in only 14% of THP-1 cells. Adherence to unstimulated HUVECs or E-selectin for 5 h had no effect on I ir or I Cl but decreased I dr. Five hours after adherence to LPS-treated HUVECs, outward currents were unchanged, but I ir was present in 81% of THP-1 cells. A twofold increase in I ir 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 I irexpression 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.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.

Adhesion-Dependent Modulation of Macrophage K+ Channels

Integrin-mediated adhesion of monocytes not only triggers cell rolling and diapedesis, it also activates ionic permeability changes resulting in monocyte activation, maturation and differentiation. Mononuclear phagocytes possess voltage-dependent inwardly rectifying K+ (Kir) currents and delayed outwardly, rectifying K+ (Kdr) currents that are modulated by tissue origin, adherence, presence of growth factors or cytokines and the functional or differentiation state of the cells. This chapter reviews the exploration of Kir and Kdr channels in mononuclear phagocytes over the last 30 years with an emphasis on culturing conditions, modulation by substrates and role in macrophage function. It has only been recent that successful attempts have been made to study these K+ currents in monocytes/macrophages as they may be engaged in the human body which may serve as the foundation for the development of novel therapeutic agents targeting macrophage Kir/Kdr channel activity to favorably influence risk factors for hypertension, atherosclerosis and diabetes.