Focal cerebral ischemia induces active proteases that degrade microvascular matrix.
OBJECTIVE
Focal cerebral ischemia causes microvessel matrix degradation and generates proteases known to degrade this matrix. However, proof that the proteases generated do indeed degrade vascular matrix is lacking. Here we demonstrate that active proteases derived from ischemic tissue after middle cerebral artery occlusion (MCAO) and transferred to normal tissue can degrade vascular matrix.


METHODS
In an ex vivo bioassay, the effects of supernatants from ischemic and normal basal ganglia of nonhuman primates, proteases, and control buffer on the immunoreactivity of vascular matrix constituents in normal brain tissue sections were quantified. Protease families were identified with specific inhibitors.


RESULTS
Plasmin, active matrix metalloproteinase (MMP)-2, and active MMP-9 significantly reduced microvessel-associated collagen, laminin, and heparan sulfate proteoglycans (HSPG). The vascular HSPG perlecan was more sensitive than collagen or laminin in the bioassay and in the ischemic core 2 hours after MCAO. Two-hour and 7-day ischemic tissue samples significantly degraded matrix perlecan and collagen. Inhibitor studies confirmed that while active MMPs were generated, active cysteine proteases significantly degraded microvessel perlecan. The cysteine proteases cathepsins B and L were generated in the microvasculature and adjacent neurons or glial cells 2 hours after MCAO and decreased perlecan in the bioassay.


CONCLUSIONS
This is the first direct evidence that active proteases are generated in ischemic cerebral tissues that are acutely responsible for vascular matrix degradation. Degradation of vascular perlecan, the most sensitive matrix component thus far identified, may be due to cathepsins B and L, generated very rapidly after MCAO.
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif 92037, USA.
Shunichi
S
Fukuda
Catherine A
CA
Fini
Takuma
T
Mabuchi
James A
JA
Koziol
Leonard L
LL
Eggleston
Gregory J
GJ
del Zoppo
D000818:Animals; D002545:Brain Ischemia / Q000201:enzymology*; D002540:Cerebral Cortex / Q000098:blood supply* / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000276:immunology / Q000378:metabolism; D007150:Immunohistochemistry; D020244:Infarction, Middle Cerebral Artery / Q000201:enzymology; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008833:Microcirculation / Q000378:metabolism; D010215:Papio
D019812:Heparan Sulfate Proteoglycans; C071927:perlecan; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D020782:Matrix Metalloproteinases