Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size.
OBJECTIVE
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke.


METHODS
Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later.


RESULTS
MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05).


CONCLUSIONS
These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.
Romanic
A M
AM
Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA. anne_romanic-1@sbphrd.com
White
R F
RF
Arleth
A J
AJ
Ohlstein
E H
EH
Barone
F C
FC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001157:Arterial Occlusive Diseases / Q000201:enzymology / Q000503:physiopathology; D001930:Brain Injuries / Q000188:drug therapy / Q000201:enzymology; D002545:Brain Ischemia / Q000201:enzymology*; D002536:Cerebral Arteries / Q000473:pathology / Q000503:physiopathology; D002544:Cerebral Infarction / Q000201:enzymology / Q000473:pathology; D017364:Collagenases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D004790:Enzyme Induction; D018093:Gelatinases / Q000032:analysis / Q000378:metabolism; D007150:Immunohistochemistry; D008264:Macrophages / Q000166:cytology / Q000201:enzymology; D008297:Male; D020781:Matrix Metalloproteinase 1; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis*; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology; D011480:Protease Inhibitors / Q000008:administration & dosage / Q000276:immunology; D051381:Rats; D011918:Rats, Inbred SHR; D012967:Sodium Dodecyl Sulfate; D013997:Time Factors; D014020:Tissue Extracts / Q000737:chemistry; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000008:administration & dosage / Q000276:immunology
D000911:Antibodies, Monoclonal; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D014020:Tissue Extracts; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; D012967:Sodium Dodecyl Sulfate; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1