L5, the most electronegative subfraction of plasma LDL, induces endothelial vascular cell adhesion molecule 1 and CXC chemokines, which mediate mononuclear leukocyte adhesion

Abstract

We have previously reported that L5, the most negatively charged subfraction of plasma low-density lipoprotein (LDL), induces mononuclear leukocyte (MNC) adhesion under flow conditions in vitro when endothelial cells are incubated with L5. The present study was undertaken to identify responsible adhesion molecules and chemokines. LDL isolated from patients with homozygous familial hypercholesterolemia was separated into five distinct subfractions by high-capacity ion-exchange chromatography. Differentially expressed mRNA between human umbilical vein endothelial cells (HUVEC) incubated (for 22h) with the earliest subfraction (L1: 20 microg/ml) and the latest and most negatively charged subfraction (L5: 20 microg/ml) was identified by DNA microarray analysis using three independent sets of RNA. mRNA consistently upregulated by L5 included VCAM-1 (2.3-fold) and CXC chemokines GRO-alpha (2.3), GRO-beta (4.6), IL-8 (2.5), ENA-78 (2.3), GRO-gamma (1.6) and GCP-2 (1.5). These results were validated by Northern analysis, semi-quantitative RT-PCR or ELISA. Blocking studies using monoclonal antibodies revealed that both primary capture and stable adhesion of MNC to HUVEC and human aortic endothelial cells (HAEC) incubated with L5 was mediated by VCAM-l/alpha4 integrin, whereas GRO and its receptor CXCR2 were involved in the stable adhesion of MNC to L5-treated HAEC.