Metalloproteinase Processing of HBEGF Is a Proximal Event in the Response of Human Aortic Endothelial Cells to Oxidized Phospholipids

• DOI: 10.1161/atvbaha.111.241257
• Publication Date: 2012-03-09T05:54:45Z
• Authors: Sangderk Lee; James R. Springstead; Brian W. Parks; Casey E. Romanoski; Roland Palvolgyi; Tiffany Ho; Phuc Nguyen; Aldons J. Lusis; Judith A. Berliner
• Keywords: 0303 health sciences; Interleukin-8; Protein Array Analysis; Aorta, Thoracic; Receptors, Cell Surface; DNA; Atherosclerosis; 3. Good health; 03 medical and health sciences; Gene Expression Regulation; Metalloproteases; Humans; Intercellular Signaling Peptides and Proteins; Endothelium, Vascular; Oxidation-Reduction; Cells, Cultured; Phospholipids; Heparin-binding EGF-like Growth Factor; Signal Transduction

Objective—

Atherosclerosis is a chronic inflammatory disease initiated by monocyte recruitment and retention in the vessel wall. An important mediator of monocyte endothelial interaction is the chemokine interleukin (IL)-8. The oxidation products of phospholipids, including oxidized 1-palmitoyl-2-arachidonyl- sn -glycerol-3-phosphocholine (Ox-PAPC), accumulate in atherosclerotic lesions and strongly induce IL-8 in human aortic endothelial cells (HAECs). The goal of this study was to identify the proximal events leading to induction of IL-8 by Ox-PAPC in vascular endothelial cells.

Methods and Results— In a systems genetics analysis of HAECs isolated from 96 different human donors, we showed that heparin-binding EGF-like growth factor (HBEGF) transcript levels are strongly correlated to IL-8 induction by Ox-PAPC. The silencing and overexpression of HBEGF in HAECs confirmed the role of HBEGF in regulating IL-8 expression. HBEGF has been shown to be stored in an inactive form and activation is dependent on processing by a dysintegrin and metalloproteinases (ADAM) to a form that can activate the epidermal growth factor (EGF) receptor. Ox-PAPC was shown to rapidly induce HBEGF processing and EGF receptor activation in HAECs. Using siRNA we identified 3 ADAMs that regulate IL-8 induction and directly demonstrated that Ox-PAPC increases ADAM activity in the cells using a substrate cleavage assay. We provide evidence for one mechanism of Ox-PAPC activation of ADAM involving covalent binding of Ox-PAPC to cysteine on ADAM. Free thiol cysteine analogs showed inhibition of IL-8 induction by Ox-PAPC, and both a cysteine analog and a cell surface thiol blocker strongly inhibited ADAM activity induction by Ox-PAPC. Using microarray analyses, we determined that this ADAM pathway may regulate at least 30% of genes induced by Ox-PAPC in HAECs.

Conclusion— This study is the first report demonstrating a role for the ADAM-HBEGF-EGF receptor axis in Ox-PAPC induction of IL-8 in HAECs. These studies highlight a role for specific ADAMs as initiators of Ox-PAPC action and provide evidence for a role of covalent interaction of Ox-PAPC in activation of ADAMs.