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

J Cereb Blood Flow Metab. 2013 Dec;33(12):1928-36. doi: 10.1038/jcbfm.2013.151. Epub 2013 Aug 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Myosin Light Chains
  • myosin light chain 2
  • rho-Associated Kinases
  • Protein Kinase C beta
  • Cardiac Myosins
  • rhoA GTP-Binding Protein