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Many diseases of the brain are associated with dysfunction of the BBB, where hypoxia is a common stressor. However, the contribution of hypoxia to BBB dysfunction is challenging to study due to the complexity of the brain microenvironment. In this study, we used a BBB model with brain microvascular endothelial cells and pericytes differentiated from iPSCs to investigate the effect of hypoxia on barrier function. We found that hypoxia\u2010induced barrier dysfunction is dependent upon increased actomyosin contractility and is associated with increased fibronectin fibrillogenesis. We propose a role for actomyosin contractility in mediating hypoxia\u2010induced barrier dysfunction through modulation of junctional claudin\u20105. 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