Inflammatory Cytokines and Oxidized Low Density Lipoproteins Increase Endothelial Cell Expression of Membrane Type 1-Matrix Metalloproteinase

Tripathi B Rajavashisth,James K Liao,Zorina S Galis,Sangeetika Tripathi,Ulrich Laufs,Jagannath Tripathi,Ning-Ning Chai,Xiao-Ping Xu,Stefan Jovinge,Prediman K Shah,Peter Libby

doi:10.1074/jbc.274.17.11924

Abstract

We investigated whether inflammatory cytokines or oxidized low density lipoproteins (Ox-LDL) present in human atheroma modulate extracellular matrix degradation by inducing membrane type 1-matrix metalloproteinase (MT1-MMP) expression. Cultured human endothelial cells (EC) constitutively expressed MT1-MMP mRNA and protein with enzymatic activity. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha, or interleukin-1beta caused a time-dependent increase in the steady-state MT1-MMP mRNA levels within 4 h of exposure, peaking about 4-fold by 6 h, and remaining elevated for 12 h. Increased MT1-MMP mRNA correlated with a 2.5-fold increase in MT1-MMP protein in EC membranes. Ox-LDL also increased MT1-MMP mRNA levels that varied with the duration of exposure and degree of LDL oxidation. The increase in MT1-MMP mRNA occurred within 6 h of exposure to Ox-LDL and peaked over 3-fold by 6 h. Ox-LDL, but not native LDL, increased MT1-MMP protein by 2-fold in EC membranes. A combination of TNF-alpha and Ox-LDL was additive in increasing MT1-MMP expression. Nuclear run-on assays showed that TNF-alpha or Ox-LDL augmented steady-state mRNA levels by increased transcription of the MT1-MMP gene. These findings indicate that activation of EC by inflammatory cytokines and/or Ox-LDL increase MT1-MMP expression. Since MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokines or Ox-LDL may influence extracellular matrix remodeling.

Highlights

We investigated whether inflammatory cytokines or oxidized low density lipoproteins (Ox-LDL) present in human atheroma modulate extracellular matrix degradation by inducing membrane type 1-matrix metalloproteinase (MT1-matrix metalloproteinases (MMPs)) expression
Northern blotting assays using this cloned cDNA fragment as a probe showed that endothelial cells (EC) contain a single mRNA species of 4.5 kb (Figs. 1 and 2), a size similar to that of membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA observed in normal lung tissue and tumor cells
The effect of Ox-LDL on the levels of MT1-MMP mRNA depended on the degree of LDL oxidation as measured by presence of the thiobarbituric acid-reactive substances (TBARS)

Summary

EXPERIMENTAL PROCEDURES

Materials—All tissue culture medium and supplements were purchased from Life Technologies, Inc. Hybridization of probes to 10 ␮g of total RNA, prepared from unstimulated EC or EC stimulated with cytokines or Ox-LDL, was performed essentially as described in the ribonuclease protection manual of Ambion, Inc. RNase digestion of hybridized probe was carried out using a mixture of RNase A and RNase T1. Affinity-purified mouse monoclonal antibodies (10 ␮g of IgG/ml) to human MT1-MMP were incubated with the blots overnight at 4 °C in PBS buffer containing 0.1% Tween 20 [23]. The cells were pelleted by centrifugation and incubated with 20 ␮g of goat IgG in PBS containing 1% fetal calf serum and 0.1% sodium azide on ice for 15 min. Equal amounts of the supernatants were added to culture media harvested from human smooth muscle cells containing pro-MMP-2 and assayed for gelatinolytic activity essentially as described by Galis et al [24]. A significance difference was considered for p values equal to or less than 0.05

Inflammatory Activation of EC and Matrix Remodeling

DISCUSSION

Shah and Peter Libby