[Effect of extracellular signal-regulated kinase pathway on nitric oxide release by human umbilical vein endothelial cell induced by placental growth factor-1].
OBJECTIVE
To investigate the effect of extracellular signal-regulated kinase (ERK) pathway on nitric oxide (NO) release by human umbilical vein endothelial cell (HUVEC) induced by placental growth factor-1 (PLGF-1).


METHODS
During January to April 2006, 50 samples of umbilical vein blood were collected from newborns delivered by cesarean section due to intrauterine distress and abnormal fetal position. HUVEC were primarily cultured by trypsin digestion. Then the following procedures were performed: (1) Cells were identified using the morphology and VIII factor immunohistochemistry methods if the culture was satisfactory. (2) Cells were collected, and fms-like tyrosin kinase (Flt-1) protein and its mRNA expression were detected with immunoprints and RT-PCR methods. (3) The protein was extracted after cells were treated with PLGF-1 (cells were collected before the treatment and 2.5, 5, 10, 20 min after the treatment). The protein levels of ERK were determined by immunoprints. (4) The cells were cultured with serum-free culture medium containing PLGF-1 only (culture media were collected 20, 40, 160, 360, 480, 720 and 1440 min after the treatment). The quantity of NO was detected with nitrate reductase method. (5) The cells were cultured with serum-free culture medium containing PD98059, the inhibitor of mitogen-activated protein kinase (MAPK)/MEK for 60 min. Then the cells were cultured continuously with the serum-free culture medium containing PLGF-1 for 60 min. The culture media were collected. The quantity of NO was detected by nitrate reductase method. The samples were divided into treatment group and control group. Control group was exactly the same in treatment time, culture condition, and time to collect the cells as the treatment group, except that it was not treated with PLGF-1 or PD 98059.


RESULTS
(1) By morphology and VIII factor immunohistochemistry the cultured cells were identified to be HUVEC. (2) Flt-1 mRNA and protein were expressed in HUVEC. (3) Expression of ERK protein started to increase at 2.5 min after treatment of HUVEC with PLGF-1, reaching the peak at 5 min, and decreased at 10 min. (4) In comparison with the control group, NO started to increase at 20 min after treatment of HUVEC with PLGF-1 (6.96 +/- 0.34) micromol/L, significantly increased at 40 min (9.45 +/- 0.59) micromol/L, and arrived at the peak at 480 min (15.82 +/- 0.69) micromol/L Comparison between the two groups showed a significant difference (P<0.05). (5) Release of NO from the cells treated with PD98059 for 1 hour and PLGF was significantly inhibited, compared with the cells treated with PLGF-1 only.


CONCLUSIONS
ERK pathways play an important role in NO release by HUVEC induced by PLGF.
Shen
Hong-ling
HL
Department of Obstetrics, Beijing Haidian District Maternal and Child Health Hospital, Beijing 100080, China. shenhongling2002@yahoo.com.cn
Chen
Han-ping
HP
D015153:Blotting, Western; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism*
D005419:Flavonoids; C000608036:PGF protein, human; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D000072483:Placenta Growth Factor; D009569:Nitric Oxide; D040281:Vascular Endothelial Growth Factor Receptor-1; D048049:Extracellular Signal-Regulated MAP Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one