[Protective effects and its mechanism of lipoxins on human umbilical vein endothelial cells under hypoxia].
OBJECTIVE
To investigate protective effects and mechanisms of lipoxinA(4) (LXA(4)) on human umbilical vein endothelial cells (HUVEC) under hypoxia in vitro.


METHODS
The HUVEC culture were divided into groups as followed: added M199 culture medium as normal control groups, added CoCl2 to mimic hypoxia in vitro as hypoxia group and added different concentrations of LXA(4) (1, 10, 100 nmol/L) were added to the induced hypoxial HUVEC as agents intervention group. Morphological changes of HUVEC were observed by using inverted phase contrast microscope. The influence of LXA(4) on cell survival was investigated by methyl thiazolyl tetrazolium (MTT) assaying method after the treatment with different concentrations of LXA(4) and 100 nmol/L lipoxinA(4) according to different time (4, 8, 12 and 24 hours). The expression of von-willebrand factor (vWF) was detected by immunocytochemistry method. The changes of cytosolic Ca(2+) were measured by laser scanning confocal microscope.


RESULTS
(1) Morphological changes:the cells under hypoxia lost its normal shapes and showed necrosis, while the cells cocultured with 100 nmol/L LXA(4) were normal appropriately. (2) Survival rate: the survival rates of HUVEC under hypoxia was (40.1 +/- 3.9)% and increased to (52.9 +/- 1.4)%, (64.1 +/- 3.3)%, (76.6 +/- 1.6)% respectively when added with LXA(4) with concentration of 1, 10, 100 nmol/L into culture medium. There was significant different survival rate when compared with that of hypoxia group. (3) The level of vWF: The expression of vWF was decreased with the increasing concentrations of LXA(4) added into culture medium, the gray values were 203.9 +/- 0.7 in 1 nmol/L, 204.6 +/- 0.9 in 10 nmol/L, 191.8 +/- 0.5 in 100 nmol/L respectively, which reached statistical difference in comparison with that of hypoxia groups (P < 0.05). (4) Confocal analysis: the intracellular free Ca(2+) concentrations of HUVEC were intensified with LXA(4) treatment.


CONCLUSIONS
LXA(4) plays an important role in keeping the normal shape of HUVEC under hypoxia, can enhance survival of hypoxial HUVEC and decrease the level of vWF in cytoplasm. The protective mechanism might be via decreasing mitochondria Ca(2+) overload and increasing cytoplasm Ca(2+) by nucleus Ca(2+) transference.
Liu
Song-jun
SJ
Department of Obstetrics and Gynecology, First Affiliated Hospital, Wenzhou Medical College, Wenzhou 325000, China.
Li
Yong-sheng
YS
Zhou
Xiao-yan
XY
Cai
Lei
L
Liu
Xiao-li
XL
Huang
Yan-jun
YJ
Ye
Du-yun
DY
Huang
Yin-ping
YP
D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D003124:Colorimetry; D004305:Dose-Response Relationship, Drug; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D044045:Lipoxins / Q000008:administration & dosage / Q000494:pharmacology*; D013997:Time Factors; D014841:von Willebrand Factor / Q000378:metabolism
D044045:Lipoxins; C040527:lipoxin A4; D014841:von Willebrand Factor; D003035:Cobalt; C018021:cobaltous chloride; D002118:Calcium