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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

Krasimir Kolev
1   Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
,
Judit Skopál
2   Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
,
László Simon
2   Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
,
Éva Csonka
2   Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
,
Raymund Machovich
1   Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
,
Zoltán Nagy
2   Department of Vascular Neurology, Semmelweis University, Budapest, Hungary
› Author AffiliationsFinancial support: This work was supported in part by grants from the Hungarian Scientific Research Fund (T031891), the Hungarian Ministry of Health (ETT 287&288/2000), the Wellcome Trust (069520/Z/02/Z) and IC20-CT98-0206, EU-00035/98.
Further Information

Publication History

Received 03 February 2003

Accepted after resubmission 02 June 2003

Publication Date:
05 December 2017 (online)

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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
 

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