Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and NF-κB as a signal transducer

Krasimir Kolev; Judit Skopál; László Simon; Éva Csonka; Raymund Machovich; Zoltán Nagy

doi:10.1160/TH03-02-0070

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 90(03): 528-537
DOI: 10.1160/TH03-02-0070

Cellular Proteolysis and Oncology

Schattauer GmbH

Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H₂O₂ as a trigger and NF-κB as a signal transducer

Authors

Krasimir Kolev

¹Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
Judit Skopál

²Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
László Simon

²Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
Éva Csonka

²Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
Raymund Machovich

¹Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
Zoltán Nagy

²Department of Vascular Neurology, Semmelweis University, Budapest, Hungary

Abstract

Summary

The haemorrhagic transformation in ischemic stroke involves disruption of the integrity of the microvascular beds, partially based on the action of matrix metalloproteinases (MMPs). The objective of the present study was to evaluate the contribution of microvascular endothelial cells from human brain (HBECs) to MMPs’ expression and regulation under conditions relevant to brain ischemia. MMPs and their inhibitors were examined with zymography, Western-blotting, ELISA and MMP-activity assay in cultured HBECs. Four-hour hypoxia (pO₂=60 mmHg) elevated the level of MMP-9 in the supernatant of the HBECs and this early response required collagen-matrix. Active oxygen species sustained the increased MMP-9 activity for at least 24 h. In the post-hypoxic period 20 μmol/L H₂O₂ caused a 6-fold increase in the specific activity of MMP-9 over the nor-moxic cells and a comparable effect was exerted by thrombin (50 nmol/L) and leukocyte elastase (10 nmol/L). The role of NF-κB, a redox-state sensitive transcription factor, was evaluated with immunofluorescence confocal microscopy and immunoblotting of nuclear and cytoplasmic extracts. The oxidative stress-dependent MMP-9 induction was accompanied by a significant increase in the NF-κB localized in the nuclei and these responses were blunted with a proteasome inhibitor (MG132). Consequently, according to our in vitro data HBECs are a source of MMP-9, which is under the control of triggers relevant to the ischemic/reperfused brain (reactive oxygen species, thrombus and inflammation related proteases) and this regulation is partially based on NF-κB activation. The reported regulation of endothelium-derived MMP-9 supports its potential involvement in the post-hypoxic disturbances of the cerebral micro-circulation.

Keywords

Brain ischemia - microvascular endothelium - reactive oxygen - thrombin - neutrophil leukocyte elastase

Full Text

References

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