Published Online
on March 4, 2004

A more recent version of this article appeared on April 1, 2004

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Submitted on November 14, 2003
Accepted on December 10, 2003

Focal Cerebral Ischemia Induces Active Proteases That Degrade Microvascular Matrix

Shunichi Fukuda MD, PhD; Catherine A. Fini BA; Takuma Mabuchi MD, PhD; James A. Koziol PhD; Leonard L. Eggleston Jr BS; and Gregory J. del Zoppo MD^*

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif.

^* To whom correspondence should be addressed. E-mail: grgdlzop{at}scripps.edu.

Background and Purpose--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.

Key words: cathepsins • brain ischemia • cysteine proteases • matrix metalloproteinases • microvessels

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