PKC-β Exacerbates in vitro Brain Barrier Damage in Hyperglycemic Settings via Regulation of RhoA/Rho-kinase/MLC2 Pathway

Srivastava, Kirtiman; Shao, Beili; Bayraktutan, Ulvi

Abstract

Stroke patients with hyperglycemia (HG) develop higher volumes of brain edema emerging from disruption of blood–brain barrier (BBB). This study explored whether inductions of protein kinase C-β (PKC-β) and RhoA/Rho-kinase/myosin-regulatory light chain-2 (MLC2) pathway may account for HG-induced barrier damage using an in vitro model of human BBB comprising human brain microvascular endothelial cells (HBMEC) and astrocytes. Hyperglycemia (25 mmol/L D-glucose) markedly increased RhoA/Rho-kinase protein expressions (in-cell westerns), MLC2 phosphorylation (immunoblotting), and PKC-β (PepTag assay) and RhoA (Rhotekin-binding assay) activities in HBMEC while concurrently reducing the expression of tight junction protein occludin. Hyperglycemia-evoked in vitro barrier dysfunction, confirmed by decreases in transendothelial electrical resistance and concomitant increases in paracellular flux of Evan's blue-labeled albumin, was accompanied by malformations of actin cytoskeleton and tight junctions. Suppression of RhoA and Rho-kinase activities by anti-RhoA immunoglobulin G (IgG) electroporation and Y-27632, respectively prevented morphologic changes and restored plasma membrane localization of occludin. Normalization of glucose levels and silencing PKC-β activity neutralized the effects of HG on occludin and RhoA/Rho-kinase/MLC2 expression, localization, and activity and consequently improved in vitro barrier integrity and function. These results suggest that HG-induced exacerbation of the BBB breakdown after an ischemic stroke is mediated in large part by activation of PKC-β.

Citation

Srivastava, K., Shao, B., & Bayraktutan, U. (2013). PKC-β Exacerbates in vitro Brain Barrier Damage in Hyperglycemic Settings via Regulation of RhoA/Rho-kinase/MLC2 Pathway. Journal of Cerebral Blood Flow and Metabolism, 33(12), 1928-1936. https://doi.org/10.1038/jcbfm.2013.151

• Journal Article Type: Article
• Online Publication Date: Aug 21, 2013
• Publication Date: 2013-12
• Deposit Date: Dec 19, 2024
• Journal: Journal of Cerebral Blood Flow & Metabolism
• Print ISSN: 0271-678X
• Electronic ISSN: 1559-7016
• Publisher: SAGE Publications
• Peer Reviewed: Peer Reviewed
• Volume: 33
• Issue: 12
• Pages: 1928-1936
• DOI: https://doi.org/10.1038/jcbfm.2013.151
• Keywords: cytoskeletal remodeling, hyperglycemia, protein kinase C, RhoA, Rho-kinase, tight junction
• Public URL: https://nottingham-repository.worktribe.com/output/41537269
• Publisher URL: https://journals.sagepub.com/doi/10.1038/jcbfm.2013.151