title: PKC-β exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway.
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.
authors: Division of Stroke, University of Nottingham, Nottingham, UK.
authors: Kirtiman
authors: K
authors: Srivastava
authors: Beili
authors: B
authors: Shao
authors: Ulvi
authors: U
authors: Bayraktutan
mesh_terms: D001812:Blood-Brain Barrier / Q000378:metabolism / Q000473:pathology*; D024722:Cardiac Myosins / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000473:pathology; D006801:Humans; D006943:Hyperglycemia / Q000150:complications* / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D064546:Protein Kinase C beta / Q000378:metabolism*; D015398:Signal Transduction; D019108:Tight Junctions / Q000378:metabolism / Q000473:pathology; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D018994:Myosin Light Chains; C107184:myosin light chain 2; D054460:rho-Associated Kinases; D064546:Protein Kinase C beta; D024722:Cardiac Myosins; D020742:rhoA GTP-Binding Protein
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