Monocytic cell adhesion to endothelial cells stimulated by oxidized low density lipoprotein is mediated by distinct endothelial ligands
Introduction
Oxidized low density lipoprotein (ox-LDL) has been implicated in the process of atherogenesis. Extensive oxidative modification of LDL may occur in an antioxidant-depleted subendothelial microenvironment [1]. As an initial step, adhesion and transmigration of human blood monocytes to vascular endothelium can be induced by ox-LDL [2]. It is known that ox-LDL enhances the recruitment, retention [3]and adhesiveness of human monocytes and monocytic cell lines 4, 5to endothelium. On the other hand, stimulation of endothelial cells with ox-LDL has been reported to increase adhesion of human blood monocytes [6]. However, the mechanisms involved in this effect remains to be completely elucidated. Studies in human umbilical vein endothelial cells (HUVEC) demonstrated the upregulation of ICAM-1 expression by ox-LDL or by long term incubation with native LDL 6, 7. Moreover, two endothelial adhesion proteins for monocytic cells have recently been described to be induced by minimally modified LDL 8, 9. In studies of cytokine-stimulated endothelial-monocyte binding we have found that the human monocytic cell line Mono Mac 6 10, 11is an appropriate in vitro model to study monocyte-endothelial interactions [12]. In this study, we therefore investigated the adhesion of Mono Mac 6 cells to ox-LDL-stimulated HUVEC, attempting to characterize the endothelial ligands involved in this adhesion and to identify responsible active components in the ox-LDL preparations used.
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Cell culture and cell lines
HUVEC were obtained from human umbilical cord veins by digestion with α-chymotrypsin and cultured in low-serum EGM (PromoCell, Heidelberg, Germany) 12, 13. Cell purity was assessed by morphology and factor VIII staining. Confluent HUVEC passage 2 were detached by 0.05% trypsin/0.02% EDTA and grown in T-25 flasks or 24 well plates for treatment with ox-LDL. U937 cells were grown in RPMI 1640 medium with 2 mM L-glutamine and 10% FCS in suspension. Mono Mac 6 cells (provided by Prof. H.W.L.
Effects of oxidatively modified ldl on monocytic cell adhesion
We found that preincubation of HUVEC with washed ox-LDL in protein concentrations of up to 100 μg/ml for 6 or 24 h did not significantly enhance adhesion of U937 cells (Fig. 1). Adhesion to untreated HUVEC was only slightly increased from 1.6±0.8% to 3.0±0.9% by pretreatment with ox-LDL (100 μg/ml) for 24 h. In contrast, Mono Mac 6 cells showed higher levels of adhesion to untreated HUVEC (4.8±0.9%) which was dose-dependently enhanced by preincubation of HUVEC with washed ox-LDL for 24 h (Fig. 1
Discussion
We have found that treatment of HUVEC with non-toxic ox-LDL induced an increase in adhesion of human Mono Mac 6 cells. Enhanced endothelial adhesiveness was associated with an upregulation of ICAM-1 expression but not of VCAM-1 or E-selectin expression, and requires additional ligands, possibly carbohydrate-decorated, heparin-like structures, e.g. endothelial proteoglycans. Using Mono Mac 6 cells as a model for mature monocytes [12]enabled us to avoid interexperimental and interindividual
Acknowledgements
This work was supported by Bundesministerium für Forschung und Technologie and Deutsche Forschungsgemeinschaft.
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