Hypoxia-ischemia or excitotoxin-induced tissue plasminogen activator- dependent gelatinase activation in mice neonate brain microvessels.
Hypoxia-ischemia (HI) and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9) activity links in HI and excitotoxicity lesion models in 5 day-old pups in wild type and in t-PA or its inhibitor (PAI-1) genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O₂). Excitotoxic lesions were produced by intra parenchymal cortical (i.c.) injections of 10 µg ibotenate (Ibo). Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA⁻/⁻ and enhanced in PAI-1⁻/⁻ mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA⁻/⁻ mice. In PAI-1⁻/⁻ mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1⁻/⁻ and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM) induced DQ-gelatin activation in vessels. The effect was not detected in t-PA⁻/⁻ mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL). In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have neuroprotection potential against neonatal brain injuries.
Haute-Normandie-INSERM ERI-28, Institute for Research and Innovation in Biomedicine of Rouen University, Rouen, France.
Priscilla L
PL
Omouendze
Vincent J
VJ
Henry
Baptiste
B
Porte
Nicolas
N
Dupré
Peter
P
Carmeliet
Bruno J
BJ
Gonzalez
Stéphane
S
Marret
Philippe
P
Leroux
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000098:blood supply* / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D018093:Gelatinases / Q000378:metabolism*; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology*; D058258:Serpin E2 / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000502:physiology*
D009498:Neurotoxins; D058258:Serpin E2; C511869:Serpine2 protein, mouse; D010959:Tissue Plasminogen Activator; D018093:Gelatinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9