Abstract
Background: The extracellular matrix (ECM) plays an important role for normal brain functions and homeostasis, and contributes to the inflammatory response and mechanisms of brain repair after acute brain injury. We have previously reported that the ECM laminin-10 (LM-10) is a key regulator of blood-brain barrier (BBB) integrity, and is involved in BBB repair after hypoxic injury and interleukin-1 (IL-1)-induced inflammation in vitro. To further investigate the role of LM-10 in BBB inflammation and repair, we investigated for the first time the signalling mechanisms regulated by LM-10 in brain endothelial cells in response to IL-1β-induced inflammation in vitro. Methods: Human brain endothelial cell line hCMEC/D3 cultured on Matrigel- or LM-10-coated tissue culture plates were left untreated or were treated with human recombinant IL-1β at various concentrations and/or for various periods of time. In vitro hallmarks of angiogenesis were assessed using a scratch injury model and tube formation assay. Expression of cell adhesion molecules ICAM-1 and VCAM-1, as well as IL-8 was measured using ELISA. Activation of signalling pathways ERK1/2, p38, NF-κB and YAP was assessed by quantitative ELISA or Western blot. Activation of genes downstream of YAP signalling was assessed by quantitative polymerase chain reaction.Results: LM-10 promoted endothelial proliferation and subsequent repair of an endothelial monolayer after scratch injury, induced tube formation, and upregulated IL-1β-induced ICAM-1 and VCAM-1 expression in vitro. Classical IL-1β-induced signalling pathway ERK1/2 and p38 were not modulated by LM-10, whilst LM-10 upregulated IL-1β-induced NF-κB activation. Importantly, we demonstrate for the first time a role of the YAP signalling pathway in endothelial cell activation, in that LM-10 significantly downregulates p-YAP (S397) activation without affecting phosphorylation of YAP (S127), leading to differential expression of YAP target genes, ctgf and serpine-1 involved in endothelial cell activation. Conclusion: Our study provides for the first time evidence that the YAP signalling pathway is an important regulator of endothelial cell activation, and could be a new therapeutic target for the treatment of cerebrovascular inflammatory diseases.
Endothelial Cell Activation by Interleukin-1 and Extracellular Matrix Laminin-10 Occurs via the Yap Signalling Pathway
