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Effects of blood exposure, increased catecholamine levels and inflammatory mediators on brain microvascular endothelial cell integrity in vitro

Introduction: Recent studies have suggested the impact of increased catecholamine levels CAT and inflammatory mediators INF on blood on the integrity of the blood brain barrier (BBB), especially when occurring in combination with hemorrhagic stroke, a situation frequently observed in Taktosubo syndrome TTS. We investigated the possible effects of these stressors on the brain microvascular endothelium in vitro.

Methods: As an in vitro model of the BBB we used an immortalized murine microvascular endothelial cell line from the cerebral cortex (cEND). The cEND were treated with medium or patient blood containing supraphysiological concentrations of stress factors such as catecholamines, TNF-α and Interleukin-6 as characteristically elevated in patient blood  suffering from acute takotsubo syndrome TTS. We investigated the impact on monolayer integrity and cellular morphology by light microscopy and immunofluorescence staining. Furthermore, alterations of representative tight and adherence junction proteins as well as integrins were determined by RT-PCR and/or Western Blot.

Results: After stress factor/ patient blood treatment, reduced viability of brain capillary endothelial cells in vitro was observed. This was concomitant with a disruption of cellular monolayer integrity and changed localisation of BBB-forming tight junction proteins as determined by real-time RT-PCR (qPCR), western blot and immunofluorescence labeling. Monolayer integrity was moreover determined by measurement of transendothelial electrical resistance.

Conclusion: Cellular monolayer integrity and the expression of proteins forming the BBB are clearly affected by exposure to patient blood, catecholamines CAT and inflammatory mediators INF, respectively. Most of barrier proteins are downregulated, so a negative impact on brain endothelial cells forming the blood-brain barrier can be assumed.


Manuel Smetak1, Cora Ittner1, Malgorzata Burek1, Michiaki Nagai2, Carola Förster1

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