PKC-β exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway

Kirtiman Srivastava, Beili Shao, Ulvi Bayraktutan

Research output: Contribution to journalArticle

34 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1928-36
Number of pages9
JournalJournal of Cerebral Blood Flow and Metabolism
Volume33
Issue number12
DOIs
Publication statusPublished - Dec 2013

Keywords

  • Blood-Brain Barrier
  • Cardiac Myosins
  • Cell Line
  • Cytoskeleton
  • Humans
  • Hyperglycemia
  • Myosin Light Chains
  • Protein Kinase C beta
  • Signal Transduction
  • Tight Junctions
  • rho-Associated Kinases
  • rhoA GTP-Binding Protein

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