Low-density lipoprotein augments interleukin-1-induced vascular adhesion molecule expression in human endothelial cells

Abstract

In this study, the effect of low density lipoproteins (LDL) on the ability of the vascular endothelium to respond to vascular cell adhesion molecule 1 (VCAM-1) activation by a cytokine was investigated. After a 4-day pre-exposure to 240 mg/dl of LDL, human umbilical vein endothelial cells (HUVECs) were hyperresponsive to minute amounts of interleukin 1α (IL-1α) as demonstrated by an augmentation of VCAM-1 gene expression. Furthermore, in response to LDL exposure, endothelial recruitment of monocytes induced by minute amounts of IL-1α was increased. This enhancing effect was blocked by an anti-VCAM antibody. The increased response appears not to be due to changes in IL-1 binding affinity or induction of endogenous IL-1α. Transient transfection of HUVECs with a reporter driven by the VCAM promoter showed that LDL increased cellular response to IL-1α by 46%. LDL itself does not increase NF-κB binding in endothelial cells (ECs). However, after a 2-day LDL incubation, NF-κB binding could be induced by over 63% with a very low dose of IL-1α. IL-1α at this dose (which activates NF-κB, but not AP-1) also enhanced LDL-activated AP-1 binding. This cross-enhanced effect may be an important intracellular signaling mechanism for EC activation. The results from this study provide new clues to understanding the mechanisms governing combined risk factors for atherosclerosis.

Introduction

The attachment and migration of leukocytes into the vessel wall is an early event in atherogenesis [1], [2]. Endothelial cells (ECs) play a major role in determining and maintaining the inflammatory reactions occurring in vascular tissues [1]. In response to various inflammatory stimuli, ECs express and activate specific adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or E-selectin, to define the types of leukocytes recruited, such as monocytes, lymphocytes, or neutrophils [3]. VCAM-1, a 110-kDa member of the immunoglobulin gene superfamily, was first identified as an adhesion molecule in Ecs, inducible by cytokines e.g. IL-1 and TNFα [4]. The importance of VCAM-1 in atherogenesis has been demonstrated in vivo both in the development of human coronary atherosclerotic plaques [5] and animal models [6]. The exact mechanisms governing VCAM-1 induction in either experimental models of atherogenesis or in clinical specimens of atherosclerotic lesions remain to be determined. An oxidation-sensitive mechanism has been proposed as mainly responsible for the VCAM-1 induction in ECs through activating the transcription factor, NF-κB [7].

Low-density lipoprotein (LDL) is a well-established risk factor for atherosclerosis. In addition, in pathophysiological concentrations, LDL provides a potent backdrop for other pathological conditions such as diabetes, nicotine abuse, and hypertension, by cumulatively increasing the incidence of coronary artery disease in these conditions [8], [9]. In the context of studying the cellular basis of LDL’s pathophysiological mechanisms, most laboratories have focused on the role of modified LDL as a primary regulator of cellular function and gene expression [10], [11], [12], but fewer studies have concentrated on effects of native LDL [13], [14], [15], [16]. Little is known of the role of LDL in modulating the response of ECs to important factors resident in atherosclerotic plaques, such as cytokines and endotoxin [17]. Endothelial recruitment of monocytes appears to be induced by LDL via VCAM-1 activation [13], [18]. This event is, in part, mediated by increased AP-1 binding, but not increased NF-κB binding [19]. Thus, LDL may activate VCAM-1 promoter activity predominantly through the activation of an AP-1 pathway [13]. This suggests that in addition to the cytokine-activated redox sensitive mechanism involving NF-κB, other types of regulatory signaling, such as the AP-1 pathway, also activate ECs and increase VCAM-1 expression. Hence, different EC activation mechanisms may interact and augment one another in the induction of certain genes. Particularly, the chronic atherogenic effect of LDL may augment the ability of the vascular endothelium to respond to the acute challenge of cytokines in VCAM-1 induction.

To further explore the concept derived principally from the findings of epidemiological studies examining the cellular basis of LDL’s effects, the present study was undertaken to study the mechanism underlying the combined effects of LDL and other atherogenic insults on vascular endothelial cells [20]. Specifically, the enhancing action of LDL on VCAM-1 induction by proinflammatory cytokines was examined. An augmented response of LDL-exposed cells to very low levels of IL-1α in VCAM-1 expression has been observed. This coordinated effect between LDL and IL-1α was observed at different levels of VCAM-1 gene regulation. An increase in both AP-1 and NF-κB binding in both LDL and IL-1 treated cells has been observed. The effects of LDL on endogenous IL-1α transcription and on IL-1 binding in ECs were also examined.