| Objective To investigate the roles of protein kinase C (PKC)β2 and PPARαin the mRNA expression of vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule-1 (VCAM-1) played in human umbilical vein endothelial cells (HUVECs) exposed to high glucose, and to explore their relationship.Methods The HUVECs were divided into eight groups: normal glucose (NG, 5 mmol/L D-glucose) group, high glucose (HG, 25 mmol/L D-glucose) group, osmotic control (L, NG + 20 mmol/L L-Glucose) group, normal glucose transfected with empty vector (NN, NG + Ad5-null) group, high glucose transfected with PKCβ2 (HB, HG + Ad5-PKCβ2) group, high glucose plus fenofibrate (HF, HG + 40μmol/L fenofibrate) group, high glucose transfected with PKCβ2 plus fenofibrate (HBF, HB + 40μmol/L fenofibrate) group. HUVECs were incubated with fenofibrate for 20 minutes as HF20 group. All cells in various groups were cultured for 6 days. The expressions of VEGF and VCAM-1 mRNA were detected by RT-PCR. PPARαprotein expression was tested by Western blot. The expression and translocation of PKCβ2 protein was observed by confocal laser scanning microscopy.Results (1) HG increased VEGF and VCAM-1 mRNA expressions, with 1.91 and 1.56 folds of NG group (both P <0.05). Meanwhile, HG induced PKCβ2 translocation from cytosol to nucleus and quantitative analysis showed the ratio of cytosol/nucleus fluorescence intensity in HG group decreased by 37% compared with NG group (P <0.05); Compared with HG group, PKCβ2 in HB group translocated more obviously from cytosol to nucleus and the mRNA expressions of VEGF and VCAM-1 in HB group further increased, with 2.59 and 2.07 folds of NG group (all P<0.05). (2)The protein expression of PPARαdecreased by 20% in HG group compared with NG group (P <0.05); Compared with HG group, PPARαexpression in HF group was 13% increased and VEGF and VCAM-1 mRNA expression in HF group decreased, with 68% and 74% of HG group (all P <0.05); There was no significant differences in the expressions of VEGF and VCAM-1 mRNA between HF20 and HG groups. (3) PPARαexpression further decreased in HB group, being 78% of HG group (P <0.05).Conclusion High glucose stimulates VEGF and VCAM-1 mRNA expressions in HUVECs via PKCβ2 activation-dependent PPARαpathway. PART 2'HYPERGLYCEMIC MEMORY'IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS MEDIATED BY INTERACTING LOOP OF PROTEIN KINASE CΒ2- REACTIVE OXYGEN SPECIESObjective To investigate the roles of protein kinase C (PKC)β2 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, 5 mmol/L D-glucose×3 weeks) group, high glucose (HG, 25 mmol/L D-glucose×3 weeks) group, memory (M, 25 mmol/L D-glucose×2 weeks + 5 mmol/L D-glucose×1 week) group, memory transfected with PKCβ2 (MB, M + Ad5-PKCβ2) group, memory plusα-lipoic acid (MA, M + 62.5μmol/L ALA) group. The mRNA expression of VEGF and VCAM-1 was detected by RT-PCR. ROS levels was tested with fluorescence microscopy and fluorescence microplate reader. The expression and translocation of PKCβ2 protein was observed by 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% (all P <0.05). (3) PKCβ2 was activated and translocated from cytosol to nucleus in group HG and group M. Quantitative analysis showed the ratio of cytosol/nuclear fluorescence intensity in group HG and group M decreased to 70% and 74% of those in group NG (both P <0.05); As compared with group M, PKCβ2 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 PKCβ2 in group MA was inhibited and the ratio of cytosol/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).Conclusion Persistence of'hyperglycemic memory'exists after glucose levels are normalized in HUVECs. This may be related with the interacting loop of PKCβ2–ROS.PART 3 INTERMITTENT HIGH GLUCOSE ENHANCES HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INJURY THROUGH PKCΒ2 ACTIVATION AND THE EFFECT OF METFORMINObjective To investigate the role of protein kinase C (PKC)β2 in vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1(VCAM-1) mRNA expression and reactive oxygen species (ROS) leves in human umbilical vein endothelial cells (HUVECs) exposed to intermittent high glucose and the effect of Metformin. Methods The HUVECs were divided into five groups: normal glucose (NG, 5 mmol/L D-glucose) group, stable high glucose (SHG, 15 mmol/L D-glucose) group, Intermittent high glucose (IHG, alternating 5 mmol/L D-glucose and 25 mmol/L D-glucose every 24h) group, Intermittent high glucose transfected with PKCβ2 (IHGB, IHG+Ad5-PKCβ2) group, Intermittent high glucose plus metformin (IHGM, IHG+20μmol/L metformin) group. After 14 days, the mRNA expression of VEGF and VCAM-1 were detected with RT-PCR. ROS levels was tested with fluorescence microplate reader. The expression and translocation of PKCβ2 protein was observed by confocal laser scanning microscopy.Results (1) VEGF, VCAM-1 mRNA expression and ROS levels in group IHG and SHG increased, being 1.29, 2.32, 3.21 and 1.17, 1.63, 1.75 folds of those in group NG (all P <0.05) and this increase was more marked in group IHG (P<0.05). (2) PKCβ2 was activated and translocated from cytosol to nucleus in group IHG and group SHG. Quantitative analysis showed the ratio of cytosol/nucleus fluorescence intensity in group IHG and SHG was 21% and 26% decreased as compared with those in group NG, but in group IHG the decrease was more marked (all P<0.05). As compared with group IHG, PKCβ2 in group IHGB translocated more obviously and VEGF, VCAM-1 mRNA expression and ROS levels in group IHGB increased 1.16, 1.26 and 1.25 folds of those in group IHG (all P<0.05). (3) As compared with group IHG, VEGF, VCAM-1 mRNA expression and ROS levels in group IHGM decreased significantly to 87%, 76% and 39%, and the activation of PKCβ2 in group IHGM was inhibited with the ratio of cytosol/nuclear fluorescence intensity increase 1.09 fold as compared with that in group IHG (all P<0.05).Conclusion Intermittent high glucose and stable high glucose induce severe injury of HUVECs, especially IHG, which is closely associated with PKCβ2 activation. Metformin could alleviate HUVECs injury induced by intermittent high glucose by blocking PKCβ2 activation. |
PDF Full Text Request