Involvement of p38 MAPK in the up‐regulation of tissue factor on endothelial cells by antiphospholipid antibodies

Abstract

Objective. To study the intracellular mechanism involved in the up-regulation of tissue factor (TF) on endothelial cells (ECs) by antiphospholipid antibodies (aPL), we examined the effects of aPL on the transcription, expression, and function of TF, the expression of interleukin-6 (IL-6) and IL-8, the induction of inducible nitric oxide synthase (iNOS), and the phosphorylation of p38 MAPK on human umbilical vein ECs (HUVECs).Methods. Cultured HUVECs were treated with IgG aPL (from patients with antiphospholipid syndrome [APS]) or with control IgG (from normal human serum). Phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS) were used as positive controls. TF expression was determined on the surface of HUVECs using an enzyme-linked immunosorbent assay (ELISA). TF activity was determined with the use of a chromogenic assay in cell lysates, and TF messenger RNA (mRNA) was determined by real-time quantitative polymerase chain reaction. Phosphorylation of p38 MAPK and induction of iNOS were determined by Western blotting, and levels of IL-6 and IL-8 were determined by ELISA.Results. PMA, LPS, and aPL significantly increased the expression of TF compared with controls. This up-regulation was significantly inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by MG132 (a specific inhibitor of NF-B). TF activity was significantly increased by treatment with IgG aPL and this effect was also inhibited by SB203580. Incubation of HUVECs with aPL increased TF mRNA 2-15-fold; these effects were abrogated by SB203580. IgG aPL induced significant phosphorylation of p38 MAPK and produced iNOS on HUVECs in a time-dependent manner. Treatment with IgG aPL also induced increased expression of IL-6 and IL-8 on HUVECs.Conclusion. Our data show that aPL induces significant increases in TF transcription, function, and expression, in IL-6 and IL-8 up-regulation, and in iNOS expression on HUVECs and that these processes involve phosphorylation of p38 MAPK and activation of NF-B.

Increased level of tumor necrosis factor-α in patients with antiphospholipid syndrome: marker not only of inflammation but also of the prothrombotic state

“…Anti-beta 2 GPI antibodies bind to beta 2 GPI autoantigen, which acts as a toll-like receptor, and induce activation of endothelium via the MyD88 pathway [41]. The other proposed mechanism for aPLinduced TF expression is the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in endothelial cells and/or platelets [42,43].…”

Receptors involved in cell activation by antiphospholipid antibodies

“…Among the important signaling intermediates that play a central role in this process are proximal mediators of innate responses, such as NF-kB [80][81][82], p38-MAP kinase [83,84], ERK [85] and IRAK [55] as well as TRIF, MyD88 and TRAF6 [86]. In addition, the PI3K/Akt pathway seems to play a specific role in platelet activation by aPLA.…”

Proteomic analysis in monocytes of antiphospholipid syndrome patients: Deregulation of proteins related to the development of thrombosis

“…Several nonexclusive mechanisms could explain the involvement of aPL in the pathogenesis of thrombosis in APS, including the induction of tissue factor (TF) expression by endothelial cells and monocytes (3,4). Intracellular mechanisms underlying aPL-induced TF gene and protein expression in endothelial cells and monocytes have also been delineated at a molecular level (5,6). Nevertheless, despite these findings, the precise pathogenesis of thrombotic diathesis associated with aPL remains unknown.…”