Abstract
Nitric oxide serves essential roles in normal vascular physiology, but paradoxically contributes to vascular pathology in disease. During brain ischemia, aberrant nitric oxide levels can cause cellular injury through induction of nitrosative/oxidative stress and post-translational activation of matrix-metalloproteinase-9 (MMP-9). We recently demonstrated that brain pericyte somata were associated with very early and localized MMP-9 activation along capillaries during cerebral ischemia, leading to focal blood-brain barrier disruption. Here, we tested whether this effect was dependent upon nitric oxide production. In vivo two-photon imaging was used to directly visualize MMP9 activity using a FITC-gelatin probe and leakage of intravenous dye during photothrombotically induced capillary ischemia. Results showed that the NOS inhibitor, L-NIL, at concentrations affecting both iNOS and constitutive NOS isoforms, attenuated capillary leakage at pericyte soma-specific locations and substantially reduced FITC-gelatin cleavage. We also found that combined administration of L-NIL and anisomycin, an inhibitor of protein synthesis, led to near complete elimination of FITC-gelatin cleavage and vascular leakage. These results indicate that both nitric oxide synthase and new protein synthesis are involved in the rapid activation of MMP-9 at somata of capillary pericytes during ischemia.
Highlights
During stroke, rapid activation of the proteolytic enzyme, matrix-metalloproteinase-9 (MMP9) leads to degradation of the neurovascular unit and compromise of the blood-brain barrier (BBB) (Gasche et al, 1999; Planas et al, 2001; Montaner, 2003; Fukuda et al, 2004)
We extend these prior studies by examining Nitric oxide (NO) production as a potential upstream signal that leads to this Matrix metalloproteinases (MMPs)-9 activation
We show that pharmacological inhibition of nitric oxide synthase (NOS) with L-NIL significantly reduces the number of sites of BBB breakdown
Summary
Rapid activation of the proteolytic enzyme, matrix-metalloproteinase-9 (MMP9) leads to degradation of the neurovascular unit and compromise of the blood-brain barrier (BBB) (Gasche et al, 1999; Planas et al, 2001; Montaner, 2003; Fukuda et al, 2004). To study rapid MMP-9 activity in vivo, we used two-photon microscopy to image mice with labeled pericytes, in combination with a well established probe of MMP-9 activity (FITC-gelatin) This was coupled with a photothrombotic model of capillary occlusion to induce ischemia during imaging, and rapidly track its pathological consequence. These studies revealed extensive overlap between the somata of capillary pericytes, FITC-gelatin cleavage and leakage of blood plasma. The prior studies did not delineate the upstream signals involved in pericyte-associated MMP-9 activation
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