Elsevier

Atherosclerosis

Volume 136, Issue 2, February 1998, Pages 297-303
Atherosclerosis

Monocytic cell adhesion to endothelial cells stimulated by oxidized low density lipoprotein is mediated by distinct endothelial ligands

https://doi.org/10.1016/S0021-9150(97)00223-2Get rights and content

Abstract

Treatment of human umbilical vein endothelial cells (HUVEC) with oxidized low density lipoprotein (ox-LDL, 100 μg/ml) for 24 h increased adhesion of human monocytic Mono Mac 6 cells from 4.8±0.9% to 17.6±2.5% (P<0.001). The effect was dose dependent and first evident at 10 μg/ml ox-LDL. In contrast, adhesion of U937 cells was not significantly increased. Mac-1 (CD11b/CD18), a monocytic counter-receptor for intercellular adhesion molecule-1 (ICAM-1), that also binds to heparin, is present on Mono Mac 6 but not on U937 cells, and may thus explain these differences in adhesion. Consistently, ox-LDL induced a 2-fold upregulation of ICAM-1 surface expression on HUVEC. The presence of maltose-1-phosphate or heparin but not monoclonal antibodies (mAbs) to ICAM-1 reduced adhesion of Mono Mac 6 cells to untreated HUVEC. Combinations of mAbs to ICAM-1 with either maltose-1-phosphate or heparin inhibited Mono Mac 6 adhesion to ox-LDL-stimulated HUVEC by more than 50%, while either alone had no effect. This suggests that two distinct endothelial ligands for Mac-1, inducible ICAM-1 and carbohydrate-decorated heparin-like proteoglycan structures mediate monocytic cell interaction with ox-LDL-treated HUVEC. The stimulating activity in ox-LDL could partly be transfered to bovine serum albumin, while lysophosphatidylcholine or 8-epi prostaglandin F2α produced no stimulatory effects. The inhibition of ox-LDL effects with the antioxidant PDTC indicates radicals as possible mediators. In conclusion, we show that oxidatively modified LDL induces adhesion of monocytic cells, which utilize at least two distinct adhesive receptors on endothelium, one being identified as ICAM-1.

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.

Section snippets

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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