[Effect of erythropoietin on the caspase-3 subfamily in preventing apoptosis of endothelial cell].
OBJECTIVE
To explore effect of erythropoietin on the caspase-3 subfamily in preventing apoptosis of human umbilical vein endothelial cells (HUVECs) induced by oxidized-low density lipoprotein (ox-LDL).


METHODS
Third-sixth passages of HUVECs were used. Two experiments were conducted. In the first experiment, there was a blank control group, ox-LDL control group (100 mg/L, incubated for 48 hours), and low, medium, and high recombinant human erythropoietin (rhEPO) groups (6.25, 25.00, 100.00 kU/L rhEPO incubated for 24 hours+100 mg/L ox-LDL incubated for 48 hours). Another experimental protocol consisted of groups of the cells pretreated with either caspase-3 inhibitor DEVD-CHO, or caspase-8 inhibitor z-IETD-fmk, or caspase-9 inhibitor z-LEHD-fmk of 25 micromol/L for 24 hours, then HUVECs were exposed ox-LDL (100 mg/L) incubated for 48 hours. The activity of caspase-3, caspase-8, or caspase 9 was determined by caspase colorimetric assay. The cell survival rate was assessed with methyl thiazolyl tetrazolium (MTT) method. The positive expression rate of caspase-3 and apoptotic rate were measured by flow cytometer.


RESULTS
The activity of caspase-3 was significantly decreased and cell survival rate was increased in the caspase-3 inhibitor group (both P<0.05). The activity of caspase-8 was decreased in the caspase-8 inhibitor group (P<0.05), but the cell survival rate was not significantly different from that of ox-LDL group (P>0.05). The activity of caspase-3 or caspase-9 was lower and cell survival rate was higher in the caspase-9 inhibitor group than that of ox-LDL group (all P<0.05). The pretreatment with rhEPO led to decreased activity of caspase-3, caspase-9, positive expression rate of caspase-3 and apoptotic rate in a dose-dependent manner compared with ox-LDL group (all P<0.05), but the activity of caspase-8 showed no significant difference from rhEPO pretreatment groups (all P>0.05).


CONCLUSIONS
These results demonstrate that rhEPO can significantly inhibit the activity of caspase-3 or caspase-9 in endothelial cell apoptosis in a dose-dependent manner. Activation of caspase-3 or caspase-9 is involved in ox-LDL-induced HUVECs apoptotic signaling pathway, but caspase-8 is not involved.
Wang
Zhi-lu
ZL
Department of Cardiology, First Hospital of Lanzhou University, Key Lab of Preclinical Study for New Drugs of Gansu Province, Lanzhou 730000, Gansu, China. wangzhilu@medmail.com.cn
Xiong
Jian-wen
JW
Yang
Xiao-fang
XF
Li
Yan
Y
Xu
Zhe-tong
ZT
Cui
Li-jun
LJ
Wang
Feng
F
Tan
Li-li
LL
Zhang
Li
L
D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D004730:Endothelium, Vascular / Q000166:cytology; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D008077:Lipoproteins, LDL / Q000633:toxicity; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000187:drug effects; D014471:Umbilical Veins / Q000166:cytology
D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; C099252:oxidized low density lipoprotein; D004921:Erythropoietin; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9