MMP-mediated disruption of claudin-5 in the blood-brain barrier of rat brain after cerebral ischemia

Abstract

The blood-brain barrier (BBB) has become a major focus of attention in cerebral pathophysiology and disease progression in the central nervous system. Endothelial tight junctions, the basal lamina, and perivascular astrocytes are jointly referred to as BBB or neurovascular unit. Around the cerebral endothelial cells is the basal lamina composed primarily of laminin, fibronectin, and heparan sulfate. The basal lamina provides a structural barrier to extravasation of cellular blood elements and anchors endothelial cells to astrocytes. Barriers limiting transport into and out of the brain are found at the tight junction proteins and at the basal lamina. The relative contribution of these two sites has not been studied, but it is likely that both are disrupted to some extent in various injury scenarios. We have shown that activation of matrix metalloproteinases (MMPs) opens the BBB by degrading tight junction proteins (claudin-5 and occludin) and increases BBB permeability after stroke, and that an MMP inhibitor prevents degradation of tight junction proteins and attenuates BBB disruption.

Fig. 1

Confocal micrographs showing claudin-5 immunoreactivity in the sham-operated animals and ischemic and nonischemic hemispheres after 3 h of reperfusion (a–c). The sham-operated animals and the nonischemic side show that the claudin-5 (Cy-3, red) in blood vessels is separated from the astrocytes (GFAP-FITC, green) surrounding them (a and b). The merged images show that the claudin-5 and astrocytes are separate. In the ischemic hemisphere (c), there is fragmentation and degeneration of the claudin-5 immunoreactivity. Co-localization of claudin-5 and GFAP was seen in the ischemic hemisphere. Confocal micrographs showing claudin-5 immunoreactivity in the ischemic and nonischemic hemispheres after 24 h of reperfusion (d–f). Immunoreactivity of claudin-5 with GFAP in brain vessels in nonischemic hemisphere (d). Ischemia caused a loss of claudin-5 from the blood vessels at 24 h (e). There was co-localization of the claudin-5 in the GFAP-positive astrocytes (f). These photos were taken from penumbral areas in the caudate and lateral cortex. Scale bars indicate 10 μm in (a–e). Scale bars show 5 μm in (f) (reproduced from ref. 23).

Fig. 2

(a) Western blots for claudin-5. The Western blots were performed with whole-cell extracts from rat brains with a 90-min MCAO followed by 3 hours reperfusion (3hr RP). The Western blot showed significant reductions (p < 0.01, n = 6) of claudin-5 (22 kDa) at 3 h in the ischemic piriform cortex (PFC). In addition, a new 17-kDa band for claudin-5 was found in the ischemic hemisphere at 3 h post-reperfusion. The 17-kDa protein showed a significant increase (p < 0.01). Treatment with BB-1101, a broad-spectrum MMP inhibitor (3hr RP/BB1101), significantly inhibited degradation of claudin-5 (22 kDa) (p < 0.02, n = 6). (b) Western blots showing degradation of claudin-5 after a 90-min MCAO followed by 24-h reperfusion. A decrease in the higher molecular weight form (22 kDa) of claudin-5 was observed in both the ischemic and nonischemic hemispheres compared to sham-operated animals (p < 0.05, p < 0.01, respectively, n = 6). A significant increase in the 17-kDa claudin-5 was seen in the ischemic hemisphere (p < 0.002) (reproduced from ref. 23).