Metalloproteinase mediated occludin cleavage in the cerebral microcapillary endothelium under pathological conditions

Reactive oxygen species (ROS) as well as matrix metalloproteinases (MMPs) induce modifications in the tight junction (TJ) protein occludin which is crucial for the blood–brain barrier (BBB) function. We investigated the role of ROS and MMPs in endothelial autoregulatory response on oxidative stress with respect to occludin and the BBB integrity. The ROS hydrogen peroxide (H2O2) was applied to our well-established BBB cell culture model based on primary porcine brain capillary endothelial cells (PBCEC). At low concentrations (2.5 mM), H2O2-induced barrier impairment correlated with an altered occludin phosphorylation. At high, cell toxic H2O2 concentrations (≥ 10 mM) occludin cleavage occurred and elevated levels of active MMP-2 were detected. Under those conditions intercellular gaps were formed within the monolayer visualizing the barrier breakdown also determined by impedance analysis. A primary structure sequence analysis revealed potential type IV collagenase sensitive motives in the first extracellular loop, thus providing evidence that occludin might be an MMP-2 substrate. MMP inhibition by the metalloproteinase inhibitor GM6001 prevented occludin degradation and reduced the intercellular gap formation. However, the barrier function quantified by impedance measurement could not be maintained despite MMP inhibition. When we applied an enzymatic activity level which caused occludin cleavage in injured PBCEC to intact PBCEC, neither occludin cleavage nor barrier impairment was observed. Thus, in our model occludin cleavage is not an autoregulatory mechanism of microcapillary endothelium in barrier modulation under oxidative stress, but only occurs upon endothelial damage.