A Link Between Gene Polymorphisms Of Endothelial Nitric Oxide Synthase And The Heterogeneity Of LPS-lnduced Endothelial Cell Injury

Purpose：To investigate the impact of endothelial nitric oxide synthase(eNOS) genepolymorphisms on eNOS expressionand the production of nitric oxide (NO), whichlinked to the heterogeneityof lipopolysaccharide (LPS)-induced cell activation andinjury in primary human umbilical vein endothelial cells (HUVEC).Methods：Endothelial cells were isolated from human umbilical veins, cultured and passed invitro. Polymerase chain reaction (PCR) was used for amplifying the target fragmentof genomic DNA. Variable number of tendem repeats in intron4(4a/b VNTR) andrestrictive long fragment polymorphisms in extron (894G�'T,RLFP) of eNOS genewere analyzed and confirmed by sequencing. RT-PCR、Western Blot、ELISA andchromatometry were used for measuring the expression of eNOS mRNA and protein;production of NO; release of TNF-α and IL-6；and vWF, VCAM-1,VE-Cadherin,biomarkers of endothelial cell damage in primary HUVEC treated with or withoutLPS. Results：(1) The frequencies ofgeneotypeGG, GT and TT in eNOS894G�'TRLFP were181/222(81.5%),38/222(17.1%) and3/222(1.4%).Frequencies ofVNTR genotypeaa, ab and bb in eNOS intron4were3/274(1.1%),35/274(12.8%)and236/274(86.1%) respectively.Three dominant hepanotypeswereeNOS GG/bb(67.3%), GT/bb (17.1%), and GG/ab (15.2%).(2) Significant low expression of thebasic levels of eNOSmRNA and protein, andproduction of NO were detected inHUVEC with genotype894GT in comparison with894GG. LPS-induced significantdownregulation of eNOS expression and decrease of NO production were found incells with both GG and GT genotypes,but these changes were greater in cells with genotypeGT than genotype GG (p<0.05).Higher basic level of TNF-α, IL-6release,expression of vWFand VCAM-1, and lower expression of VE-cadherinwerefound incells with894GTthancells with894GG. Furtherly, LPS stimulation induced markeddecrease of the eNOS mRNA/proteinexpression and production of NO, increase ofTNF-α and IL-6release and vWF, VCAM-1expression, lowed expression ofVE-cadherin in HUVEC withboth genotype GT and GG. However, the change of IL-6was significantly greater in HUVEC with894GT than894GG (p <0.05).The impactsofgenotypes ab and bb in eNOS intron4VNTR on eNOS expression, NO production,inflammatory cytokines release as well as the changes of endothelial cell damagebiomarkers, vWF, VCAM-1and VE-cadherin were similar to genotypes of eNOS894GG and894GT.In the threedominant hepanotypes, hepanotypeGT/bbbasicallyexpressed the lowest eNOS mRNA and protein, producedthe least NO (p<0.05).These impacts were significantly enhanced by LPS stimulation,which leaded togreater increases ofthe inflammatory mediators (TNF-α, IL-6)， biomarkers ofendothelial injury (vWF, VCAM-1), and decrease of VE-cadherin, animportant cellstructure integrity indicator.Conclusions:BotheNOS894G�'T and eNOS introns4a/bVNTR gene polymorphisms affectedthe eNOSexpressionand NOproduction inprimary HUVEC. The lowerbasic levels ofeNOSexpressionand NOproduction werefound in genotypes of eNOS894GT, eNOS4bb VNTR and hepanotypeeNOSGT/4bb.Compared with the othergenotypes and hepanotypes, the changes ondecreasing expression of eNOS and production of NOwere greater in those HUVECwith genotypes of eNOS894GT, eNOS4bb VNTR and hepanotypeeNOSGT/4bbunder stimulation of LPS, which likely accounted forLPS-reducedheterogenetic activation and injury in endothelial cells.