[Proliferation-inhibiting effect of advanced glycation end products modified human serum albumin to vascular endothelial cell ECV304].
OBJECTIVE
To study the proliferation-inhibiting and apoptosis-inducing effects of advanced glycation end products (AGE) modified human serum albumin (AGE-HSA) on human vein endothelial cells.


METHODS
Human umbilical vein endothelial cells ECV304 were cultured in vitro with AGE-HSA of the concentrations of 12.5, 25, 50, 100, and 200 micro g/ml for 6, 12, 24, or 48 hour, then 20 micro l of 5 mg/ml MTT were added and the optical density (OD) at each time point was determined. Another ECV304 cells were cultured with AGE-HAS for 2, 4, or 8 days and then were stained with trypan blue to calculate the number of dead cells so as to calculate the proliferation-inhibiting rate. Another ECV304 cells were cultured with AGE-HAS for 6, 12, 24, or 48 hours and then stained with annexin V Fitc and propidium iodide (PI). Flow cytometry was used to calculate the annexin V Fitc positive cells (early and middle stage apoptotic cells) and Annexin V Fitc/PL positive cells (late apoptotic cells). Inverted microscope, transmission electron microscope, and fluorescence microscope were used to observe the histological changes of apoptotic cells. FCV304 cells incubated with HSA of the above-mentioned and without addition of the other agents concentrations were used as controls.


RESULTS
The OD values of ECV304 cells cultured for 48 h with low concentrations (12.5, 25, and 50 micro g/ml) of AGE-HSA were not significantly different from those of the control (1.104 +/- 0.080, 1.098 +/- 0.097 and 1.059 +/- 0.122 VS. 1.159 +/- 0.088, all P > 0.05). The OD values of ECV304 cells cultured with low concentrations of AGE-HSA for 4 days and 6 days were significantly lower than those in the control group. The OD values of ECV304 cells cultured with high concentrations (100 and 200 micro g/ml) of AGE-HSA for 6 - 48 hours decreased to 0.117 +/- 0.033 and 0.081 +/- 0.020 in comparison with that of the control group (P < 0.01). Flow cytometry and fluorescence microscopy showed higher proportions of apoptotic cells among the ECV304 cells cultured with high concentrations of AGE-HAS than among the control cells at each time point (P < 0.01). The numbers of cells in the control group exponentially increased after culture for 2, 4, and 6 days. The number of cells cultured with low concentrations of AGE-HAS for 2 days was not significantly different from that of the control group (P > 0.05), however, the numbers of cells cultured with low concentrations of AGE-HAS for 4 and 6 days were significantly lower than those of the control group (both P < 0.01). The numbers of cells cultured with 100 or 200 micro g/ml AGE-HAS for 2 days were significantly lower than those of the control group (both P < 0.01) with a proliferation-inhibiting rate of 39.56% +/- 2.82% and 60.32% +/- 4.51% respectively. The apoptotic rates in cells cultured with low concentrations of AGE-HAS for 48 hours were not significantly different from those in the control group. The apoptotic rates in cells cultured with 100 or 200 micro g/ml AGE-HAS for 6, 12, 24, or 48 hours were significantly higher than those in the control group (all P < 0.01). The apoptotic rates in 200 micro g/ml group at different time points were significantly higher than those in the 100 micro g/ml group (P < 0.05 or 0.01). The apoptotic rate and number of apoptotic cells increased along with the increase of culture time and concentration of AGE-HAS. Microscopy showed morphological changes among the cells cultured with 100 micro g/ml AGE-HAS for 6, 12, 24, and 48 hours and the numbers of apoptotic cells, mainly late apoptotic cells, and dead cells increased remarkably since the cells were cultured for 48 hours.


CONCLUSIONS
AGE-HSA inhibits the proliferation of vascular endothelial cells and induces apoptosis in dose and time dependent manner. AGE modification-induced pathobiological cascade may be involved in the pathogenesis of impaired wound healing in diabetes by the mechanism of angiogenesis retardation.
Lin
Wei-dong
WD
Shanghai Burn Institute, Burn Department of Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Lu
Shu-liang
SL
Qing
Chun
C
Yao
Min
M
Chen
Xiang-fang
XF
Xiang
Jun
J
Qiao
Liang
L
Liu
Ying-kai
YK
Dong
Jiao-yun
JY
Shi
Gui-ying
GY
Liao
Zhen-jiang
ZJ
Shi
Ji-xiang
JX
D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology*; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D012709:Serum Albumin / Q000494:pharmacology*; D013997:Time Factors; D014945:Wound Healing / Q000187:drug effects
D017127:Glycation End Products, Advanced; D012709:Serum Albumin