Methods to assess pericyte-endothelial cell interactions in a coculture model.

The blood-brain barrier (BBB) comprises the microvascular endothelial cells, pericytes, and astrocytes, which are connected by the extracellular matrix (ECM). Current BBB models focus solely on the microvascular endothelial cells which constitute a physical barrier by formation of tight junctions (TJs), while the impact of pericytes on barrier regulation is poorly understood. We established a coculture model from primary porcine brain capillary endothelial cells (PBCECs) and pericytes (PBCPs) to approach the in vivo situation. This model allows the examination of pericyte impact on pharmacological, transport, migration, and metabolic activity of the BBB. In vivo the interaction between pericytes and endothelial cells is partly controlled by the ECM which is remodeled by matrix metalloproteinases (MMPs). Both endothelial cells and pericytes secrete MMPs which are important not only for ECM remodeling but also for TJ cleavage. In this chapter, current methods to study the interactions of these cell types by ECM signaling as well as MMP secretion are described.

Regulation of the blood-brain barrier integrity by pericytes via matrix metalloproteinases mediated activation of vascular endothelial growth factor in vitro.

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes, and neurons. In this study, we analyzed the influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells in a species-consistent in vitro coculture model. We were able to show a barrier integrity-decreasing impact of pericytes by transendothelial electrical resistance (TEER) and (14)C-sucrose permeability measurements. The morphology analysis revealed serrated cell borders and a shift of the endothelial morphology towards a cobblestone shape under the influence of pericytes. The analysis of the two major barrier integrity modulators vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) displayed higher MMP activity and higher levels VEGF, MMP-2, and MMP-9 in the coculture, whereas VEGF levels were decreased by the MMP inhibitor GM6001, indicating a complex interplay of both. Inhibition experiments with neutralizing VEGF antibody and GM6001 increased the TEER, which proves the involvement of VEGF and MMPs in the barrier-decreasing process. Analysis of occludin yielded decreased protein content and discontinuous expression at the endothelial cell borders under the influence of pericytes. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity via MMPs and VEGF.

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 (H(2)O(2)) 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), H(2)O(2)-induced barrier impairment correlated with an altered occludin phosphorylation. At high, cell toxic H(2)O(2) concentrations (>or=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.

TIMP-3: a novel target for glucocorticoid signaling at the blood-brain barrier.

Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood-brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.