Download PDF
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)

    Permissions and Reprints

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
 

  • References

  • 1 Hornig CR, Dorndorf W, Agnoli AL. Haemorrhagic cerebral infarct - a prospective study. Stroke 1986; 24: 465-8.
    PubMedGoogle Scholar
  • 2 Nagy Z. Blood-brain barrier and the cerebral endothelium. In: Pathophysiology of the blood-brain barrier. Johanson BB, Owman C, Widner H. eds Elsevier Science 1990; 11-29.
    PubMedGoogle Scholar
  • 3 Webersinke G, Bauer H, Amberger A, Zach O, Bauer HC. Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes. Biochem Biophys Res Commun 1992; 189: 877-84.
    CrossrefPubMedGoogle Scholar
  • 4 Nagy Z, Kolev K, Csonka É. et al. Contraction of human brain endothelial cells induced by thrombogenic and fibrinolytic factors. Stroke 1995; 26: 265-70.
    CrossrefPubMedGoogle Scholar
  • 5 Nagy Z, Kolev K, Csonka É. et al. Perturbation of the integrity of the blood-brain barrier by fibrinolytic enzymes. Blood Coagul Fibrinolysis 1998; 9: 471-8.
    CrossrefPubMedGoogle Scholar
  • 6 Carmeliet P, Collen D. Development and disease in proteinase-deficient mice: role of the plasminogen, matrix metalloproteinase and coagulation system. Thromb Res 1998; 91: 255-85.
    CrossrefPubMedGoogle Scholar
  • 7 Mun-Bryce S, Rosenberg GA. Matrix metalloproteinases in cerebrovascular disease. J Cerebr Blood Flow Metab 1998; 18: 1163-72.
    PubMedGoogle Scholar
  • 8 Rosenberg GA, Navratil M. Metalloproteinase inhibition blocks edema in intracerbral hemorrhage in the rat. Neurology 1997; 48: 921-6.
    CrossrefPubMedGoogle Scholar
  • 9 Rosenberg GA, Estrada EY, Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain. Stroke 1998; 29: 2189-95.
    CrossrefPubMedGoogle Scholar
  • 10 Gasche Y, Fujimura M, Morita-Fujimura Y. et al. Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction. J Cerebr Blood Flow Metab 1999; 19: 1020-8.
    PubMedGoogle Scholar
  • 11 Lapchak PA, Chapman DF, Zivin JA. Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)-induced hemorrhage after thromboembolic stroke. Stroke 2000; 31: 3034-40.
    CrossrefPubMedGoogle Scholar
  • 12 Romanic AM, White RF, Arleth AJ. et al. Matrix metalloproteinase expression increases after cerebral focal ischemia in rats. Stroke 1998; 29: 1020-30.
    CrossrefPubMedGoogle Scholar
  • 13 Heo JH, Lucero J, Abumiya T. et al. Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J Cerebr Blood Flow Metab 1999; 19: 624-33.
    PubMedGoogle Scholar
  • 14 Rosenberg GA, Cunningham LA, Wallace J. et al. Immunohistochemistry of matrix metal-loproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures. Brain Res 2001; 893: 104-12.
    CrossrefPubMedGoogle Scholar
  • 15 Duhamel-Clerin E, Orvain C, Lanza F. et al. Thrombin receptor-mediated increase of two matrix metalloprotenases, MMP-1 and MMP-3, in human endothelial cells. Arterioscler Thromb Vasc Biol 1997; 17: 1931-8.
    CrossrefPubMedGoogle Scholar
  • 16 Haas TL, Davis SJ, Madri JA. Three-dimensional type I collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells. J Biol Chem 1998; 273: 3604-10.
    CrossrefPubMedGoogle Scholar
  • 17 Harkness KA, Adamson P, Sussman JD. et al. Dexamethasone regulation of matrix metal-loproteinase expression in CNS vascular endothelium. Brain 2000; 123: 698-709.
    CrossrefPubMedGoogle Scholar
  • 18 Pagenstecher A, Stalder AK, Kincaid CL. et al. Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metal-loproteinase genes in the mouse central nervous system in normal and inflammatory states. Am J Pathol 1998; 152: 729-41.
    PubMedGoogle Scholar
  • 19 Baldwin AS. The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol 1996; 14: 649-81.
    CrossrefPubMedGoogle Scholar
  • 20 Sato H, Seiki M. Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells. Oncogene 1993; 8: 395-405.
    PubMedGoogle Scholar
  • 21 Haddad JJE, Olver RE, Land SC. Anti-oxidant/pro-oxidant equilibrium regulates HIF-1α and NF-κB redox sensitivity. Evidence for inhibition by glutathione oxidation in alveolar epithelial cells. J Biol Chem 2000; 275: 21130-9.
    CrossrefPubMedGoogle Scholar
  • 22 Kokura S, Wolf RE, Yoshikawa T. et al. Molecular mechanisms of neutrophil-endothelial cell adhesion induced by redox imbalance. Circ Res 1999; 84: 516-24.
    CrossrefPubMedGoogle Scholar
  • 23 Kokura S, Rhoads CA, Wolf RE. et al. NFkB signaling in posthypoxic endothelial cells: relevance to E-selectin expression and neutro-phil adhesion. J Vasc Res 2001; 38: 47-58.
    CrossrefPubMedGoogle Scholar
  • 24 Vastag M, Nagy Z. Method of isolation and culture of human brain microvessel endothelium. In: Drug Transport across the Blood Brain Barrier. de Boer A, Suanto W. eds Harwood Academic Publishers GmbH 1997; 109-13.
    PubMedGoogle Scholar
  • 25 Csonka E, Szemenyei K, Miskulin M. et al. Morphological examinations of aortic endothelial and smooth muscle cells grown in vitro on collagen membranes. Artery 1980; 8: 253-8.
    PubMedGoogle Scholar
  • 26 Baird AE, Warach S. Magnetic resonance imaging of acute stroke. J Cereb Blood Flow Metab 1998; 18: 583-609.
    CrossrefPubMedGoogle Scholar
  • 27 Adams J, Stein R. Novel inhibitors of the proteasome and their therapeutic use in inflammation. Annu Rep Med Chem 1996; 31: 279-88.
    PubMedGoogle Scholar
  • 28 Chaturvedi MM, Mukhopadhyay A, Aggarwal BB. Assay for redox-sensitive transcription factor. Meth Enzymol 2000; 319: 585-602.
    PubMedGoogle Scholar
  • 29 Murphy G, Crabbe T. Gelatinases A and B. Meth Enzymol 1995; 248: 470-84.
    PubMedGoogle Scholar
  • 30 Politis DN. Computer-intensive methods in statistical analysis. IEEE Signal Proc Mag 1998; 15: 39-55.
    CrossrefPubMedGoogle Scholar
  • 31 Kim CH, Kim JH, Moon SJ. et al. Biphasic effects of dithiocarbamates on the activity of nuclear factor-κB. Eur J Pharmacol 2000; 392: 133-6.
    CrossrefPubMedGoogle Scholar
  • 32 Russel J, Epstein CJ, Grisham MB. et al. Regulation of E-selectin expression in postischemic intestinal microvasculature. Am J Physiol 2000; 278: G878-G885.
    PubMedGoogle Scholar
  • 33 Cao W, Carney JM, Duchon A. et al. Oxygen free radical involvement in ischemia and reperfusion injury to brain. Neurosci Lett 1988; 88: 233-8.
    CrossrefPubMedGoogle Scholar
  • 34 Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem 1992; 59: 1609-23.
    CrossrefPubMedGoogle Scholar
  • 35 Chinopoulos C, Tretter L, Rozsa A. et al. Exacerbated responses to oxidative stress by an Na+ load in isolated nerve terminals: the role of ATP depletion and rise of [Ca2+]i. J Neurosci 2000; 20: 2094-103.
    CrossrefPubMedGoogle Scholar
  • 36 Hyslop PA, Zhang Z, Pearson DV. et al. Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H2O2 in vitro. Brain Res 1995; 671: 181-6.
    CrossrefPubMedGoogle Scholar
  • 37 Ying W, Han SH, Miller JW. et al. Acidosis potentiates oxidative neuronal death by multiple mechanisms. J Neurochem 1999; 73: 1549-56.
    PubMedGoogle Scholar
  • 38 Zhang ZG, Zhang L, Tsang W. et al. Dynamic platelet accumulation at the site of the occluded middle cerebral artery and in downstream microvessels is associated with loss of microvascular integrity after embolic middle cerebral artery occlusion. Brain Res 2001; 912: 181-94.
    CrossrefPubMedGoogle Scholar
  • 39 Olson MW, Gervasi DC, Mobashery S. et al. Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. J Biol Chem 1997; 272: 29975-83.
    CrossrefPubMedGoogle Scholar
  • 40 Itoh Y, Nagase H. Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. J Biol Chem 1995; 270: 16518-21.
    CrossrefPubMedGoogle Scholar
  • 41 Sun SC, Ganchi PA, Ballard DW. et al. NF-κB controls expression of inhibitor IκBα: evidence for an inducible autoregulatory pathway. Science 1993; 259: 1912-5.
    CrossrefPubMedGoogle Scholar
  • 42 Hoffmann A, Levchenko A, Scott ML. et al. The IκB-NF-κB signaling module: temporal control and selective gene activation. Science 2002; 298: 1241-5.
    CrossrefPubMedGoogle Scholar
  • 43 Bini A, Wu D, Schnuer J. et al. Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7) and membrane type 1 matrix metal-loproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments. Biochemistry 1999; 38: 13928-36.
    CrossrefPubMedGoogle Scholar
  • 44 Lelongt B, Bengatta S, Delauche M. et al. Matrix metalloproteinase 9 protects mice from anti-glomerular basement membrane nephritis through its fibrinolytic activity. J Exp Med 2001; 193: 793-802.
    CrossrefPubMedGoogle Scholar