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- Materials and methods
- Statistical analysis
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.
Antiphospholipid (aPL) antibodies are associated with aPL syndrome (APS), which is a syndrome of thrombosis, pregnancy loss, and thrombocytopenia . aPL antibodies have been shown to enhance thrombus formation, but the mechanisms involved are not completely understood [2–4].
Several studies have shown that aPL antibodies activate endothelial cells (ECs) in vitro, as demonstrated by enhanced expression of adhesion molecules (intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 VCAM-1, and E-selectin, on human umbilical vein endothelial cells (HUVECs) and enhanced monocyte adherence to ECs in vitro[2–6] and in vivo[7,8].
Our group recently showed that the thrombogenic effects of aPL antibodies are mediated by ICAM-1, VCAM-1 and P-selectin . As additional support for the hypothesis that aPL antibodies activate ECs and may create an hypercoagulable state in APS patients, two recent studies indicated that the levels of soluble ICAM-1 and VCAM-1 were significantly increased in the plasma of patients with APS and recurrent thrombosis [10,11].
E-Selectin is an adhesion molecule, expressed on activated endothelium and has a terminal lectin domain which binds carbohydrate ligands expressed on leukocytes serving to slow leukocytes in the first phase of migration . Neutrophils appear early at sites of acute inflammation and this is in part controlled by the cytokine induction of E-selectin on the surface of endothelium in these areas. Stimulation in vitro of endothelium with tumor necrosis factor-alpha (TNF-α), induces expression of E-selectin over a period of 4–12 h, and this molecule appears early during inflammatory reactions in vivo, but this expression wanes by 24 h . While rapidly induced by various proinflammatory signals, E-selectin may mediate initial attachment and rolling of leukocytes before firm adhesion through interaction with carbohydrate ligands. TNF-α, LPS and other cytokines have been shown to up-regulate expression of VCAM-1, ICAM-1 and E-selectin [14,15], by increasing the transcription of specific mRNA  and by inducing the translocation of nuclear factor NF-κB to the nuclei of treated cells .
Although there is a wealth of data suggesting that aPL antibodies activate ECs in vitro and in vivo and they enhance thrombosis, and these effects seem to be mediated by ICAM-1, VCAM-1 and P-selectin [6–9], it is unclear what is the relative role of E-selectin in this process. To examine this question several experiments were performed both in vitro and in vivo. E-selectin-deficient [E-sel (−/−)] mice were utilized to determine the role of E-selectin on aPL-mediated enhancement of thrombosis and increased leukocyte adhesion to ECs in the microcirculation of mouse cremaster muscle. Thrombogenic effects were examined by using a previously described mouse model of induced thrombosis. We also examined whether the effects observed in vivo correlated with effects in vitro. Utilizing human umbilical endothelial cells (HUVECs) we found that aPL increased expression of E-selectin to HUVEC in vitro. The effects of aPL antibodies on activation of NF-κB were also examined.
Our findings indicate that E-selectin plays a role in aPL-induced EC activation and thrombus formation in vivo. APL antibodies also enhanced expression of E-selectin and significantly activated NF-κB.
- Top of page
- Materials and methods
- Statistical analysis
Although there is a wealth of data suggesting that aPL antibodies activate ECs in vitro and in vivo and enhance thrombosis, it was unclear until recently what the relative roles of the individual adhesion molecules in these processes, are [5–8,25]. In a recent study, we reported  that the thrombogenic effects of aPL antibodies are mediated by ICAM-1, VCAM-1 and P-selectin. In this study, the data indicate that aPL antibodies up-regulate the expression of E-selectin, causing increased adhesion of leukocytes to ECs and activation NF-κB in vitro. aPL enhanced leukocyte adhesion and enhanced thrombus formation in wild type (C57BL/6 J mice), as has been observed previously [7–9]. In the absence of E-selectin expression, there is no aPL-induced enhancement of WBC adhesion to ECs nor is thrombus size enhanced. The latter observation suggests a role of E-selectin in aPL-induced thrombosis.
Under normal conditions, vascular endothelium maintains an anticoagulant surface of blood vessels . The influence of ECs on the thombosis pathway is complex and involves multiple cell-surface and secreted compounds. Key elements in this process are the expression of tissue factor, tissue factor-pathway inhibitor, and thrombomodulin and cell adhesion molecules on the surface of ECs. The up-regulation of adhesion molecules on ECs induces increased monocyte adherence to endothelium with increased production of tissue factor and the generation of a hypercoagulable state in ECs as a consequence [27,28]. Hence, our mouse model of microcirculation, the adhesion of leukocytes to endothelium, can be used as an indication of EC activation in vivo.
It is known that the rolling and adhesion of leukocytes to the endothelium involves several sequential steps. Initially the interaction of selectins (E- and P-selectin) allows leukocytes to adhere reversibly to the vessel wall, so that circulating leukocytes can be seen to roll along the endothelium [12,14,15]. The first adhesive interaction permits the stronger interaction mediated by ICAM-1 and VCAM-1. In this study, leukocytes that remain stationary for 30 s or more in the ECs of the cremaster muscle are counted as adhering leukocytes. Thus, early disruption of the interaction of leukocytes to ECs in E-sel (−/−) mice may lead to a complete abrogation of the adhesion of leukocytes to the vessel wall, as observed in our studies. What is relevant in this study is that significant increase of adhesion of leukocytes to endothelium induced by aPL was abrogated in knockout mice. Most importantly, this study shows that the enhancement of thrombus size mediated by aPL was abrogated in E-sel (−/−) indicating that E-selectin expression may be at least one of the important factors in aPL-mediated thrombosis. This observation regarding E-selectin in aPL-mediated thrombosis have not previously been reported in vivo.
We cannot exclude the possibility that mechanisms other than EC activation may be involved in the pathogenesis of the thrombotic events associated with aPL antibodies. Studies clearly indicate that abnormalities in platelet function or aPL interfere with phospholipid–protein complexes may play a critical role in regulation of coagulation [29–31]. Such molecules as protein C , thrombomodulin , or tissue factor , are undoubtedly important.
The adhesion molecule E-selectin belongs to a family of adhesive receptors found in leukocytes. Stimuli commonly found in inflammatory processes, such as the cytokines TNF-α or IL-1, concurrently induce the expression of E-selectin and also VCAM-1 and ICAM-1 on ECs in a concentration and time-dependent fashion, by increasing the transcription of specific mRNA [13–16]. The intracellular signals activated during this process are now known. For example it is known that those cytokines induce the translocation of NF-κB to the nuclei of treated cells . Inactive NF-κB is localized in the cytoplasm of most unstimulated cells in complex with its inhibitor, I kappa B. Phosphorylation of I kappa B, followed by its degradation, allows the translocation of NF-κB into the nucleus to activate NF-κB-dependent gene transcription. In our study, IgG-APS activated NF-κB on ECs to a comparable degree as TNF-α (used as a positive control). This was concomitant with an increased expression of E-selectin on HUVECs and increased adhesion of leukocytes to HUVECs.
The conversion of the normal antithrombotic endothelial phenotype to a prothrombotic surface might be one of the pathological events that causes the hypercoagulable state of the antiphospholipid syndrome . Studies by Del Papa and others showed that aPL antibodies bind to ECs through interaction with β2GPI and induce activation of ECs as measured by increased adhesion molecule expression and up-regulation of cytokine secretion and arachidonic acid metabolism [36–38]. Simantov et al. showed that the up-regulation of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin) on HUVECs when cells were treated with aPL antibodies in the presence of β2GPI in vitro, were mediated through the F(ab)′2 fragment of the antibody . In another study by George et al. up-regulation of expression of ICAM-1, VCAM-1 and E-selectin by some murine monoclonal antibodies in vitro on HUVECs, correlated with increased fetal resorption in mice in vivo. Our group showed that affinity purified aPL antibodies from patients with APS and murine monoclonal aPL antibodies up-regulated the expression of ICAM-1, VCAM-1 and E-selectin in vitro, and these effects correlated with enhanced thrombosis and leukocyte adhesion in vivo[7,8]. In another recent study by our group, we demonstrated that those effects were abrogated in ICAM-1 knockout, ICAM-1/P-selectin double knockout mice and by infusions with monoclonal anti-VCAM-1 antibodies . Furthermore recent studies suggested increased levels of soluble ICAM-1 and VCAM-1 are observed in patients with APS and this correlated with recurrences in thrombosis [10,11]).
In summary, this study provides strong evidence that E-selectin expression on activated ECs is important in mediating thrombus formation in patient with APS and based on this study and our previous results , we propose an integrated model of aPL-induced endothelial cell activation in which its thrombogenic effects are mediated by adhesion molecules. These new findings show that not only ICAM-1, VCAM-1 and P-selectin but also E-selectin is involved. This study and data from our previous publication  did not enable conclusions to be drawn about the relative importance of any one adhesion molecule compared with the other three. Abrogation of any one molecule negates the aPL effect, suggesting that either all four adhesion molecules are required or that aPL stimulation of the expression of any of three molecules does not compensate for the abrogation of a fourth molecule. We do not discard the possibility that other molecules such as tissue factor may also play an important role in aPL-mediated thrombogenesis.
The observations made in this study may have important implications in determining new approaches to the treatment and prevention of thrombosis in APS.