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

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.

Extra domain A and type III connecting segment of fibronectin in assembly and cleavage.

To determine the role of the extra domain A (EDA) and type III connecting segment (IIICS) of fibronectin in fiber assembly, topographical distribution and proteolytic cleavage, eight full-length human fibronectin cDNA variants (aa0, aa64, aa89, and aa120 variations in the IIICS with or without the EDA) tagged with the V5 epitope were cloned from human endothelial cells and were expressed in CHO-K1 cells. All eight variants were assembled on cell surfaces. However, only the EDA(+) variants, regardless of the type of the IIICS domain, formed extensive fibrous networks. In contrast, the EDA(-)/aa64 and EDA(-)/aa89 variants were present predominantly as a soluble form. Western analysis of both soluble and cell-associated fibronectin/V5 variants showed that aa64, aa89, and aa120 variants with or without the EDA domain produced the major 50- to 62-kDa C-terminal fragments, whereas the aa0 variants did not, suggesting that the IIICS domain provides proteolytic cleavage sites.