Interaction of endothelin-1 with vasoactive factors in mediating glucose-induced increased permeability in endothelial cells.
Alteration of endothelins (ET) and/or their receptors may be important in mediating vascular dysfunction in diabetes. We investigated mechanisms regulating ET-1 expression in human umbilical vein endothelial cells (HUVEC) in response to glucose and the functional significance of these mechanisms. Permeability across HUVEC, grown in medium containing either low (5 mmol/l) or high (25 mmol/l) D-glucose were investigated. L-glucose was used as a control. ET-1, ET(A), and ET(B) mRNA were assessed by semiquantitative RT-PCR. ET-1 immunoreactivity and F-actin microfilament assembly were investigated using confocal microscopy. Increased transendothelial permeability was noted in cells cultured in high glucose or when the cells grown in low (physiologic) glucose were incubated with ET-1, vascular endothelial growth factor (VEGF), or N (G) -nitro-L-arginine methyl ester but not when they were incubated with ET-3, N(G)-nitro-D-arginine methyl ester, or L-glucose. Increased permeability was associated with increased ET-1, ET(A), and ET(B) mRNA expression and augmented ET-1 immunoreactivity. High glucose induced increased permeability, increased ET-1, ET(A), and ET(B) mRNA expression. ET-1 immunoreactivity was blocked by the protein kinase C (PKC) inhibitor chelerythrine, the specific PKC isoform inhibitor 379196, VEGF-neutralizing antibody, or the ET(A) blocker TBC11251, but was not blocked by the specific ET(B) blocker BQ788 or by a VEGF-non-neutralizing antibody. Increased permeability was also associated with deranged F-actin assembly in the endothelial cells and by derangement of endothelial cell junctions as assessed by electron microscopy. Data from this study suggest that high glucose-induced increased permeability may be induced through increased ET-1 expression and disorganization of F-actin assembly. ET-1 expression and increased permeability may occur secondary to PKC isoform activation and may be modulated by VEGF and nitric oxide.
Chen
S
S
Department of Pathology, University of Western Ontario, London, Canada.
Apostolova
M D
MD
Cherian
M G
MG
Chakrabarti
S
S
D001483:Base Sequence; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000378:metabolism*; D019332:Endothelin-1 / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008222:Lymphokines / Q000378:metabolism*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009569:Nitric Oxide / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017931:DNA Primers; D016228:Endothelial Growth Factors; D019332:Endothelin-1; D008222:Lymphokines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009569:Nitric Oxide; D011493:Protein Kinase C; D005947:Glucose