Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and NF-kappaB as a signal transducer.
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 (pO(2)=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 micro mol/L H(2)O(2) caused a 6-fold increase in the specific activity of MMP-9 over the normoxic cells and a comparable effect was exerted by thrombin (50 nmol/L) and leukocyte elastase (10 nmol/L). The role of NF-kappaB, 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-kappaB 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-kappaB activation. The reported regulation of endothelium-derived MMP-9 supports its potential involvement in the post-hypoxic disturbances of the cerebral microcirculation.
Kolev
Krasimir
K
Semmelweis University, Department of Medical Biochemistry, Puskin u.9., 1088 Budapest, Hungary.
Skopál
Judit
J
Simon
László
L
Csonka
Eva
E
Machovich
Raymund
R
Nagy
Zoltán
Z
D001921:Brain / Q000098:blood supply; D002196:Capillaries / Q000201:enzymology / Q000473:pathology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000473:pathology*; D004790:Enzyme Induction; D006801:Humans; D002534:Hypoxia, Brain / Q000201:enzymology* / Q000473:pathology; D020925:Hypoxia-Ischemia, Brain / Q000201:enzymology / Q000473:pathology; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000096:biosynthesis*; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology; D010084:Oxidation-Reduction
D016328:NF-kappa B; D020780:Matrix Metalloproteinase 9