Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating β1 integrin
Peggy T. Shih, Mariano J. Elices, Zhuang T. Fang, Tatiana P. Ugarova, Dana Strahl, Mary C. Territo, Joy S. Frank, Nicholas L. Kovach, Carlos Cabanas, Judith A. Berliner, Devendra K. Vora
Peggy T. Shih, Mariano J. Elices, Zhuang T. Fang, Tatiana P. Ugarova, Dana Strahl, Mary C. Territo, Joy S. Frank, Nicholas L. Kovach, Carlos Cabanas, Judith A. Berliner, Devendra K. Vora
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Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating β1 integrin

Abstract

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic α4β1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial α4β1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of β1integrins, particularly those associated with α5 integrins.Activation of β1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated β1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated β1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.

Authors

Peggy T. Shih, Mariano J. Elices, Zhuang T. Fang, Tatiana P. Ugarova, Dana Strahl, Mary C. Territo, Joy S. Frank, Nicholas L. Kovach, Carlos Cabanas, Judith A. Berliner, Devendra K. Vora

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MM-LDL stimulates surface expression of CS-1 but not VCAM-1. HAEC were p...
MM-LDL stimulates surface expression of CS-1 but not VCAM-1. HAEC were plated into 96 wells and then stimulated with 250 μg/ml of MM-LDL or 2 ng/ml LPS for 4 h at 37°C. After the treatment, cells were rinsed and then fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were rinsed and then blocked for 1 h with 3% APBS. Primary antibodies were diluted to 1 μg/ml in the blocking solution and then incubated overnight at 4°C. Cells were then rinsed and blocked with GNS before incubation with either the IgM (90.45) or IgG (VCAM-1) secondary antibodies conjugated to HRP. Development was with o-phenylenediamine dihydrochloride in a phosphate citrate buffer. The plate was read on a microtiter plate reader at an OD of 450 nm. A representative experiment from four separate studies is shown. Values represent mean ± SD (n = 4). *P < 0.0001. HRP, horseradish peroxidase.