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.

A peptide that mimics the Vth region of beta-2-glycoprotein I reverses antiphospholipid-mediated thrombosis in mice.

Antiphospholipid (aPL) antibodies bind to beta2glycoprotein I (beta2GPI) and cause endothelial cell (EC) activation and thrombosis in mice. beta2GPI binds to EC through its Vth domain and induces their activation. TIFI is a 20 amino acid synthetic peptide that shares similarity with the Vth domain of beta2GPI. Our objectives were to examine the ability of TIFI to affect aPL-mediated thrombosis in mice and the interactions of TIFI, beta2GPI with phospholipid surfaces and target cells. CD1 mice were injected with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgG-NHS and with either TIFI or with control peptide (VITT). Size of induced thrombi was determined. Inhibition and competition studies were done using aPL antibodies, cardiolipin (CL) liposomes in the presence of varying amounts of TIFI and beta2GPI. Binding of fluorescinated beta2GPI to human ECs and to murine macrophages in the presence or absence of TIFI, was also examined. TIFI significantly decreased thrombus size in mice injected with IgG-APS. TIFI reverted the beta2GPI-dependent binding of aPL antibodies to CL liposomes in a dose-dependent fashion. This effect was abrogated by addition of beta2GPI, suggesting that TIFI displaces the binding of beta2GPI to phospholipids. TIFI inhibited the binding of fluorescinated beta2GPI to human EC and to murine macrophages. The data indicate that TIFI abrogates thrombogenic properties of aPL in mice by competing with beta2GPI and preventing its binding to target cells. This may be important in designing new modalities for the treatment of thrombosis in APS.

Fluvastatin inhibits up-regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells.

BACKGROUND: Mechanisms of thrombosis induced by antiphospholipid (aPL) antibodies include up-regulation of tissue factor (TF) expression on endothelial cells (ECs). Statins have been shown to reduce levels of TF induced by tumor necrosis factor (TNF-alpha) and lipopolysaccharide (LPS) on ECs. In a recent study, fluvastatin inhibited thrombogenic and proinflammatory properties of aPL antibodies in in vivo models. The aim of this study was to determine whether fluvastatin has an effect on aPL-induced expression of TF on ECs. METHODS: IgGs were purified from four patients with APS (IgG-APS) and from control sera (IgG-NHS). Cultured human umbilical vein endothelial cells (HUVEC) were treated with IgG-APS or IgG-NHS or with medium alone or with phorbol myristate acetate (PMA), as a positive control. In some experiments, cells were pretreated with fluvastatin (2.5, 5 or 10 micro m) with and without mevalonate (100 micro m). TF expression on HUVECs was measured by ELISA. RESULTS: PMA and the four IgG-APS preparations increased the expression of TF on EC significantly (4.9-, 2.4-, 4.2-, 3.5- and 3.1-fold, respectively), in a dose-dependent fashion. Fluvastatin (10 micro m) inhibited the effects of PMA and the four IgG-APS on TF expression by 70, 47, 65, 22 and 68%, respectively, and this effect was dose-dependent. Mevalonate (100 micro m) completely abrogated the inhibitory effects of fluvastatin on TF expression induced by aPL. CONCLUSION: Because of the suggested pathogenic role of aPL on induction of TF on ECs, our data provide a rationale for using statins as a therapeutic tool in treatment of thrombosis in APS.

Intrauterine fetal death in mice caused by cytomegalovirus-derived peptide induced aPL antibodies.

Immunization of mice with beta2 glycoprotein I (beta2GPI) and also with GDKV, a synthetic peptide representing the phospholipid (PL)-binding site of beta2GPI, induced pathogenic aPL antibodies that bind and activate endothelial cells, enhanced thrombus formation and caused fetal death in pregnant mice. TIFI is a PL-binding peptide spanning the Thr101-Thr120 of ulb0-hcmva from human cytomegalovirus (CMV), which shares structural similarity with the PL-binding site of beta2GPI. Immunization with this peptide induced pathogenic aPL and anti-beta2GPI antibodies in mice. These antibodies activated endothelial cells and enhanced thrombus formation in vivo, but whether these antibodies cause fetal death in mice is not known. The objective of this study was to examine the effects of these antibodies on pregnancy outcome in mice. Two groups of pregnant BALB/c mice were injected with either hybridoma supernatant containing D3/AC10, a CMV-peptide-induced monoclonal aPL, at days four, eight and 12 of the pregnancy, 100 microg per mouse (study group) or with culture media alone (control group). The litter size was significantly smaller in the study group (4.80 +/- 1.15 versus 7.28 +/- 0.18, t = - 2.526, P < 0.03). In conclusion, aPL induced by CMV peptides may have pathogenic properties similar to human autoimmune aPL. These findings further support the hypothesis that at least in some patients with APS, pathogenic aPL antibodies may be generated by immunization with CMV products during incidental exposure to the virus via a molecular mimicry mechanism.

Laser ablation as a function of the primary absorber in dentin.

BACKGROUND AND OBJECTIVE: Infrared transmission spectra of dentin reveal a broad absorption band between 6.0 and 7.0 microns composed of absorption peaks of water, collagen and carbonated hydroxyapatite. The nearly constant absorption and the existence of absorption peaks of different tissue components were used to investigate ablation as a function of the primary absorber. STUDY DESIGN/MATERIALS AND METHODS: Laser ablation of dentin as a function of fluence was studied in the wavelength range between 6.0 and 7.5 microns using the Vanderbilt Free-Electron Laser (FEL). Depth and volume of the ablation crater were determined with a silicon replica method and subsequent confocal laser topometry. SEM investigations were performed on the irradiated surfaces. For the description of the experimental data an ablation model is developed. RESULTS: At all applied wavelengths we found a linear increase of ablation depth as a function of fluence above a threshold fluence. The lower absorption of dentin at 7.5 microns compared to the absorption at 6.0, 6.5 and 7.0 microns results in a greater ablation threshold. At 6.0, 6.5 and 7.0 microns wavelengths the ablation thresholds are comparable. The experimental data are in good agreement with an ablation model using a mean absorption coefficient of the target material. No thermal cracking is observed after ablation in dentin. The post ablative surface structure at 6.0 and 7.0 microns looks similar whereas at 7.5 microns the surface reveals a greater roughness. CONCLUSION: The ablation efficiency and threshold depend on the mean absorption but do not depend upon the chemical identity of the primary absorber in dentin. Calculations show that heat conduction during the laser pulse leads to a thermal equalization between the heated microstructures and surrounding tissue resulting in an ablation with little dependence on the primary absorber.