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bioRxiv · 10.1101/2021.03.25.437097

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrity via the ROCK pathway

Nwafor, D. C.·Brichacek, A. L.·Wang, W.·Bidwai, N.·Lilly, C. L.·Millan, J. L.·Brown, C. M.
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Abstract

Blood-brain barrier (BBB) dysfunction is a key feature in many neuroinflammatory diseases. Yet, no therapies exist to effectively mitigate BBB dysfunction. A strategy to bridge this knowledge gap requires an examination of proteins localized to brain microvascular endothelial cells (BMECs) and evaluating their role in preserving barrier integrity. Tissue-nonspecific alkaline phosphatase (TNAP) is highly abundant in brain microvascular endothelial cells (BMECs); however, its function in BMECs remains unclear. We hypothesized that a loss or inhibition of TNAP activity on BMECs would impair barrier integrity through increased cytoskeletal remodeling driven by the Rho-associated protein kinase (ROCK) pathway. First, we examined barrier integrity in hCMEC/D3 cells treated with a TNAP inhibitor (TNAPi) and in primary BMECs (pBMECs) via the conditional deletion of TNAP in endothelial cells. Our results showed that both pharmacological inhibition and genetic conditional loss of TNAP significantly worsened endothelial barrier integrity compared to controls. Next, we examined the mechanisms through which TNAP activity exerts a protective phenotype on BMECs. Our results showed that hCMEC/D3 cells treated with TNAPi displayed remarkable phalloidin and vimentin cytoskeletal remodeling compared to control. We then examined the role of ROCK, a key player in cytoskeletal remodeling. Our results showed that TNAPi increased the expression of ROCK 1/2. Furthermore, inhibition of ROCK 1/2 with fasudil mitigated TNAPi-induced and VE-cKO barrier dysfunction. Collectively, our results support a novel mechanism through which loss of TNAP activity results in cerebrovascular dysfunction, and selective modulation of TNAP activity in BMECs may be a therapeutic strategy to improve BBB function.

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Nwafor, D. C., Brichacek, A. L., Wang, W., Bidwai, N., Lilly, C. L., Millan, J. L., Brown, C. M.. 2021-03-26. Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrity via the ROCK pathway. https://doi.org/10.1101/2021.03.25.437097

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