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Isolation of Cerebral Capillaries from Fresh Human Brain Tissue
Published on: September 12, 2018
Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and
Krasimir Kolev1, Judit Skopál, László Simon
1Semmelweis University, Department of Medical Biochemistry, Puskin u.9., 1088 Budapest, Hungary.
Abstract:
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.
Insights
Human brain endothelial cells (HBECs) produce matrix metalloproteinase-9 (MMP-9), a key factor in hemorrhagic transformation after ischemic stroke. This production is regulated by factors like oxidative stress and inflammation, involving NF-kappaB activation.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Hemorrhagic transformation in ischemic stroke is linked to microvascular damage mediated by matrix metalloproteinases (MMPs).
- Human brain microvascular endothelial cells (HBECs) play a role in regulating MMPs, particularly MMP-9.
Purpose of the Study:
- To investigate the contribution of HBECs to MMP expression and regulation under conditions mimicking brain ischemia.
- To elucidate the mechanisms controlling MMP-9 production by HBECs.
Main Methods:
- Cultured HBECs were subjected to hypoxia and oxidative stress.
- MMP levels and activity were assessed using zymography, Western blotting, and ELISA.
- NF-kappaB activation was studied via immunofluorescence and immunoblotting.
Main Results:
- Hypoxia increased MMP-9 levels in HBECs, requiring collagen-matrix and sustained by reactive oxygen species.
- Hydrogen peroxide, thrombin, and leukocyte elastase significantly elevated MMP-9 activity.
- Oxidative stress-induced MMP-9 production correlated with NF-kappaB nuclear translocation, inhibited by MG132.
Conclusions:
- HBECs are a significant source of MMP-9, crucial in ischemic stroke.
- MMP-9 production by HBECs is regulated by ischemia-relevant triggers including oxidative stress, thrombin, and inflammatory proteases.
- NF-kappaB activation is a key pathway in the regulation of endothelium-derived MMP-9, impacting cerebral microcirculation post-hypoxia.
