Metalloproteinase processing of HBEGF is a proximal event in the response of human aortic endothelial cells to oxidized phospholipids

Arterioscler Thromb Vasc Biol. 2012 May;32(5):1246-54. doi: 10.1161/ATVBAHA.111.241257. Epub 2012 Mar 8.

Abstract

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.

Conclusions: 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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aorta, Thoracic / metabolism
  • Aorta, Thoracic / pathology
  • Atherosclerosis / genetics*
  • Atherosclerosis / metabolism
  • Atherosclerosis / pathology
  • Cells, Cultured
  • DNA / genetics*
  • Endothelium, Vascular / metabolism*
  • Endothelium, Vascular / pathology
  • Gene Expression Regulation*
  • Heparin-binding EGF-like Growth Factor
  • Humans
  • Intercellular Signaling Peptides and Proteins / biosynthesis
  • Intercellular Signaling Peptides and Proteins / genetics*
  • Interleukin-8 / biosynthesis
  • Metalloproteases / metabolism*
  • Oxidation-Reduction
  • Phospholipids / metabolism*
  • Protein Array Analysis
  • Receptors, Cell Surface
  • Signal Transduction

Substances

  • HBEGF protein, human
  • Heparin-binding EGF-like Growth Factor
  • Intercellular Signaling Peptides and Proteins
  • Interleukin-8
  • Phospholipids
  • Receptors, Cell Surface
  • DNA
  • Metalloproteases