['Hyperglycaemic memory' in human umbilical vein endothelial cells mediated by interacting loop of protein kinase Cbeta(2)-reactive oxygen species].
OBJECTIVE
To investigate the roles of protein kinase C (PKC) beta(2) and reactive oxygen species (ROS) in the mRNA expression of vascular endothelial growth factor(VEGF) and vascular cell adhesion molecule-1(VCAM-1) in human umbilical vein endothelial cells (HUVECs), imitating 'hyperglycemic memory' in vitro and the relationship between them.


METHODS
The HUVECs were divided into 5 groups: normal glucose (NG, D-glucose 5 mmol/L x 3 weeks) group, high glucose (HG, D-glucose 25 mmol/L x 3 weeks) group, memory (M, 25 mmol/L D-glucose x 2 weeks + 5 mmol/L D-glucose x 1 weeks) group, memory transfected with PKCbeta(2) (MB, M + Ad5-PKCbeta(2)) group, memory plus alpha-lipoic acid (MA, M + 62.5 micromol/L alpha-lipoic acid) group. The mRNA expression of VEGF and VCAM-1 was detected with RT-PCR. ROS levels was tested with fluorescence microscopy and fluorescence microplate reader. The expression and translocation of PKCbeta(2) protein was observed with confocal laser scanning microscopy.


RESULTS
(1) VEGF and VCAM-1 mRNA expression in group HG and M increased, being 1.76, 1.35 and 1.69, 1.43 folds of those in group NG (all P < 0.05). (2) ROS levels in group HG and M increased 86% and 80% as compared with those in group NG (both P < 0.05). As compared with group M, ROS levels in group MA decreased 38%, and VEGF, VCAM-1 mRNA expression in group MA decreased significantly to 67% and 78%. (3) PKCbeta(2) was activated and translocated from cytosol to nucleus in group HG and group M. Quantitative analysis showed the ratio of plasma/nuclear fluorescence intensity in group HG and M decreased to 70% and 74% of those in group NG (both P < 0.05). As compared with group M, PKCbeta(2) in group MB translocated more obviously and the mRNA expression of VEGF and VCAM-1 in group MB significantly increased 1.29 and 1.18 folds of those in group M (both P < 0.05). (4) As compared with group M, the activation of PKCbeta(2) in group MA was inhibited and the ratio of plasma/nuclear fluorescence intensity increased 18%. Besides, ROS levels in group MB was 25% up-regulated as compared with those in group M (both P < 0.05).


CONCLUSIONS
Persistence of 'hyperglycemic memory' exists after glucose levels are normalized in HUVECs. This may be related with the interacting loop of PK Cbeta(2)-ROS.
Lin
Xue-bo
XB
Department of Endocrinology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Zhou
Bo
B
Sun
Fang
F
Chen
Fang-fang
FF
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C467484:VEGFA protein, human; D019010:Vascular Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D011493:Protein Kinase C; D005947:Glucose