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

Abstract

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.

Introduction

The blood–brain barrier (BBB) plays an essential role for the maintenance of brain homeostasis. It is constituted by the neurovascular unit formed by the microcapillary endothelial cells, pericytes, astrocytes and neurons as well as the basal lamina. In cerebral ischemia (CI) and reperfusion (CI/R), the BBB can be disrupted with severe consequences such as vasogenic edema formation and/or hemorrhagic transformation. The importance of the BBB in such pathological processes is underlined by a study of focal CI in which regional neuronal injury statistically correlates with local loss of microvascular integrity (Tagaya et al., 2001). Restoration of brain perfusion (reperfusion) is the ultimate aim in therapeutic strategies in CI. However, reperfusion provokes injury by emerging reactive oxygen species (ROS) which further impair the BBB (Rosenberg et al., 1998) not only by reversible mechanisms, but also by irreversible damage leading to apoptotic microvascular endothelial cell injury (Lee and Lo, 2003, Pober et al., 2009). In severe CI/R injury, the pathology also involves necrotic events not only in neurons and astrocytes, but also in the microvascular capillaries. Moreover, recombinant tissue plasminogen activator (rt-PA) treatment, the only US Food and Drug Administration (FDA) approved clot lysis method for CI treatment, increases the risk of brain edema formation and haemorrhagic transformation due to elevated proteolytic activity within the capillaries.

Reactive oxygen species (ROS) may result from mitochondrial damage in case of oxygen deficiency and after reoxygenation. The ROS hydrogen peroxide (H₂O₂) is not only a significant mediator in oxidative neuronal damage (Crack et al., 2003, Weisbrot-Lefkowitz et al., 1998) but moreover mediates a significant increase in BBB permeability along with a decrease of barrier function in the cerebral vascular endothelium in CI/R injury (Kevil et al., 2000, Kondo et al., 1996, Schreibelt et al., 2007).

Tight junctions (TJs) are complex structures of integral membrane proteins and associated cytoplasmatic proteins which are located at the lateral apical side of the cell membrane in regions of close cell–cell contacts of the cerebral microcapillary endothelium. Since they form the functional basis for BBB integrity by sealing the intercellular clefts, the morphological as well as molecular modifications occurring at TJs during CI/R are of particular interest as possible targets for the prevention or treatment of BBB injury (Rosenberg and Yang, 2007). Integral TJ membrane proteins include occludin, the members of the claudin family and the junctional adhesion molecule family (JAMs). Occludin and claudin-5 were identified as key components of BBB integrity in the microvascular endothelium. Both consist of four transmembrane domains, intracellular N- and C-termini, and two extracellular domains that might interact with cell membranes of vicinal cells (Anderson and Van Itallie, 1995, Tsukita and Furuse, 1999) thus sealing the intercellular clefts. The long intracellular carboxy termini connect both proteins to members of the peripheral zonula occludens (ZO) proteins which are associated to the actin cytoskeleton.

Occludin phosphorylation has been identified as a mechanism for BBB regulation under physiological conditions as well as in CI/R injury. Lately, it was found that inhibition of occludin tyrosine phosphorylation coincided with reduced BBB leakage as well as infarct size (Takenaga et al., 2009). Occludin and claudin-5 cleavage by matrix metalloproteinases (MMPs) is another possible mechanism underlying BBB impairment and has been detected in in vivo models of focal CI (Liu et al., 2009, Rosenberg and Yang, 2007). Based on the observation that MMP-2 activation systems are upregulated after ischemia it was suggested that MMP-2 activity is associated with the initial opening of the BBB (Chang et al., 2003), while in a recent study MMP-9 seemed to be the major player involved (Liu et al., 2009).

The type IV collagenases MMP-2 (latent form 72 kDa, active forms of 58–66 kDa) and MMP-9 (latent form 92 kDa, active forms 84–86 kDa) have a widely overlapping substrate spectrum. Due to their proteolytic potential, their activity is controlled not only transcriptionally and post-transcriptionally but also on the level of secretion, zymogen activation from pro-MMP to active MMP, and by endogenous tissue inhibitors of metalloproteinases (TIMPs). One of their physiological functions is the remodelling of the extracellular matrix (ECM) at the mature BBB as well as in angiogenesis and wound healing. They also play an important pathological role in post-ischemic events such as vasogenic edema formation and hemorrhagic transformation by proteolytically degrading BBB components (Candelario-Jalil et al., 2008, Rosenberg, 1995). Moreover, it has been reported that MMP levels are elevated by ROS in the ischemic brain (Gasche et al., 2001) and heart (Fert-Bober et al., 2008).

The preferred cleavage sites of type IV collagenases are located in glycine-rich sequences (Seltzer et al., 1990). While this is a typical feature of many ECM components such as collagens, a variety of further substrates have been identified. In human umbilical vein endothelial cells (HUVEC) a metalloproteinase caused occludin cleavage to a C-terminal fragment of approximately 50 kDa (Wachtel et al., 1999). The molecular weight of the fragment corresponds to a C-terminally truncated form cleaved in the first extracellular loop, but the exact cleavage site remains to be determined. MMP-dependent occludin cleavage was observed in porcine brain capillary endothelial cells (PBCEC) (Lohmann et al., 2004) as well as in apoptotic epithelial cells (Bojarski et al., 2004).

Since both factors, an alteration of occludin phosphorylation and occludin cleavage, are discussed as important mechanisms controlling BBB function in CI/R pathology, we intended to further elucidate their role in the proceeding injury of the microvascular endothelium under the impact of ROS. In order to mimic endothelial CI/R injury by ROS-induced oxidative stress we applied H₂O₂ to our BBB cell culture model based PBCEC, and monitored occludin and claudin-5 integrity together with MMP-2 and MMP-9 levels. We observed a H₂O₂ concentration dependent alteration of the occludin phosphorylation and occludin cleavage at different degrees of endothelial injury. Barrier impairment at lower H₂O₂ concentrations correlated with an altered phosphorylation state. At high H₂O₂ concentrations, occludin cleavage occurred when active MMP-2 was released as a consequence of endothelial cell death. Neither occludin cleavage nor barrier impairment was observed when this enzymatic activity level was applied to intact PBCEC layers.