Experiment On The Effect Of Hypoxia On The Biology Function Of HUVECs And The Expression Of VEGFR In Vitro

Objective: Hypoxia has been recognized as a primary cause ofangiogenesis, because it triggers up expression of the vascularendothelial growth factor (VEGF). VEGF is one of the most potentproangiogenic factors which promotes the proliferation and migration ofendothelial cells and increases vascular permeability. In human umbilicalvein endothelial cells (HUVEC), its potent is achieved throughcorresponding specific vascular endothelial growth factor receptor(VEGFR).The role of hypoxia on endothelial cell function and growthfactors is unknown, so the aim of this study was tried to investigatehypoxia effects on vascular endothelial growth factor (VEGF) and itsreceptors VEGFR1and VEGFR2in HUVECs and the biology functionof HUVEC.Methods: HUVECs were obtained from the Typical SpeciesPreservation Center of Wuhan University. The thrird or fourth thegeneration HUVECs are used as the research specimens. HUVEC werecultured and divide into2groups，incubated in normoxia and hypoxiarespectively. The biological behavior were assessed by cell propagation，migration and tube formation. The protein levels of VEGFR1andVEGFR2were test by immunocytochemistry. The expression of VEGF was test by RT-PCR. The statistical significance of raw data between thegroups in each experiment was evaluated by SPSS13.0, using unpairedStudent’s t-test or ANOVA followed by Student-Newman-Keulsmultiple comparison post-test. P value <0.05was considered asstatistically significant. The results were expressed as means±SEM of theindicated n values.Results:1. There is no significant difference between the hypoxiagroup and the normoxia group at24h and48h（P>0.05）,at time96h instatistically significant difference (P<0.01), The hypoxia group wasnegative under hypoxia condition.2. The normoxia group cells migratedinto the denuded area more quickly than that incubated in hypoxia, thedifference was significant difference (P<0.01).3. The normoxia groupform capillary-like structure after48hours, but the hypoxia group hadnot formed till to48hours.4. Immunocytochemical staining ofVEGFR1、VEGFR2were decreased in the experiment group, comparewith the control group in statistically significant difference (P <0.01),which VEGFR2protein significantly decreased. Whereas,VEGFR1/VEGFR2increased under hypoxia, the difference between thetwo groups was no significant difference (P<0.05).5. RT–PCR resultsshowed that VEGF expression level was obviously up-regulated at time24h and48h, as compare with the group of time at0h and12h that thedifference was significant difference (P <0.01).However, the levels of VEGF mRNA was not significant (P>0.05) between time at0h to12hand24h to48h.Conclusion: HUVEC subjected to hypoxia exhibited reduced cellmigration, proliferation and tube formation. Hypoxia stimulated VEGFmRNA expression but decreased VEGFR1and VEGFR2protein levelsin endothelial cells. A mechanistic explanation is that VEGFR1andVEGFR2protein levels were depleted whereas VEGFR1:VEGFR2ratiois substantially increased during hypoxia to block VEGF stimulated andVEGFR2regulated endothelial responses to maximize cell viability andrecovery; also create a conditions for angiogenesis.