The Effects And Mechanisms Of Intrauterine Growth Restriction On Function Of Human Umbilical Vein Endothelial Cells

Intrauterine growth restriction(IUGR)or fetal growth restriction(FGR)refers to a pathological state in which fetal growth rate is lower than normal fetal growth potential.Internationally,there is no uniform diagnostic criteria for IUGR.For the diagnostic purpose,clinicians often adopt the concept of small for gestational age(SGA),namely the birth weight is lower than the same gestational age and gender of the fetus average"birth weight of the tenth percentile or two standard deviations".IUGR is a major risk factor for stillbirth and neonatal complications,significantly increasing perinatal morbidity and mortality.Numerous epidemiological evidences have linked reduced fetal growth to cardiovascular diseases(CVDs)in later life.The correlation between the feto-placental vascular dysfunction and the increasing susceptibility to CVDs in later life has been validated.The impaired vasorelaxation and structural remodeling found in the umbilical cord vessels from IUGR newborns may partly account for the deeper reason for the vascular dysfunction.Besides,increased aortic intima-media thickness is commonly seen in fetuses and newborns with IUGR,and this may be correlated with the elevated blood pressure when they grow into infancy.Our previous studies conducted on rats showed similar vascular dysfunction,as we found that IUGR rats presented more susceptible to hypoxic insult and suffered a high risk of pulmonary arterial hypertension(PAH)and pulmonary vascular remodeling in later life.Given the critical role of endothelium in maintaining the vascular structure and function,the above alterations of vasorelaxation,structural remodeling,and increased arterial wall thickness reflect the endothelial dysfunction.However,the mechanisms underlying impaired endothelial function following IUGR remain to be elucidated.Endothelial dysfunction is characterized by impaired vasodilation,which is associated with various cardiovascular diseases.Reduced bioavailability of nitric oxide(NO)is essential for endothelial dysfunction.Endothelial nitric oxide synthase(eNOS)plays a key role in the regulation of vascular tone and homeostasis.NO is synthesized by eNOS via L-arginine/NO pathway.Enhanced arginase impairs vascular endothelial function by competing with eNOS for L-arginine,while arginase inhibition improves endothelial function in patients with type 2 diabetes mellitus and familial hypercholesterolaemia.As an endogenous NOS inhibitor,the elevation of asymmetric dimethyl-arginine(ADMA)is supposed to be a prospective marker of cardiovascular risk and mortality.ADMA is synthesized by protein-arginine methyltransferase(PRMT)and metabolized mainly by dimethyl-arginine dimethylaminohydrolase(DDAH).Endothelin-1(ET-1)is a potent vasoconstrictor synthesized by vascular endothelial cells and plays a crucial role in vascular dysfunction in hypertension.The endothelium-derived ET-1 causes vasoconstriction through acting on endothelin receptor type-A(ETAR)and type-B(ETBR)in vascular smooth muscle cells(VSMCs).ETBR inside the endothelial cells can mediate the release of NO,and ET-1 clearance.In addition,hypoxia-inducible factor-1?(HIF-1?),as a subunit of the transcription factor HIF-1,can induce transcription of ET-1 gene.Our previous study suggested that hypoxia-induced pulmonary arterial hypertension following IUGR is correlated with increased level of HIF-1? binding in the promoter of the vascular endothelial ET-1 gene.IUGR related studies have found that NO system plays an important role in endothelial dysfunction.Whether the vascular endothelial dysfunction associated with IUGR should be attributed to the dysregulation of arginase,ADMA,and the ET-1 system requires further elucidation.In summary,we hypothesize that:(1)IUGR affects expression of genes related to vascular function in endothelial cells,causing vascular endothelial dysfunction;(2)arginase-2,ADMA,and ET-1 pathway participate in the regulation of vascular endothelial dysfunction associated with IUGR.In the present study,human umbilical vein endothelial cells(HUVECs)were derived from IUGR and normal newborns.The proliferation and transcription of genes related to vasodilatation,oxidative stress and angiogenesis were analyzed in HUVECs.To explore the effect of IUGR on vascular endothelial function and its potential mechanism,we evaluated NO production,ADMA metabolism,expression of eNOS,Arg-2 and ET-1 system under basal and hypoxic conditions.Part I The effects of intrauterine growth restriction on function of human endothelial vein endothelial cellsObjective:1.To analyze clinical characteristics of intrauterine growth restriction(IUGR)newborns and their mothers.2.To establish vascular endothelial cell lines by havesting and culturing primary human umbilical vein endothelial cells(HUVECs)derived from IUGR and normal newborns.3.To evaluate the proliferation ability of HUVECs derived from IUGR newborns.4.To investigate the effect of IUGR on the vascular endothelial function by determining the transcriptional level of genes related to vascular function in HUVECs.Methods:1.The neonatal and maternal medical records were obtained from three maternity hospitals.IUGR was defined as birth weight below the tenth percentile according to the fetal growth chart.The birth weight between the tenth percentile and the 90th percentile was identified as the Control group.The clinical data of neonates and their mothers were collected and analyzed.2.Primary HUVECs were isolated using the digestion method with collagenase from the fresh umbilical cord tissue,which were from IUGR and normal newborns.Primary HUVECs were identified by the immunofluorescence staining and cultured in vitro.3.The ability of cell proliferation was evaluated using Cell Counting Kit-8.4.The transcripts of genes related to vascular function such as regulation of vascular tone,oxidative stress,and angiogenesis were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR).Results:1.Fifteen subjects were included in IUGR and control group,respectively.The neonatal and maternal characteristics showed that the birth weight and ponder index of IUGR group were lower than that of the Control group.The median birth weight in IUGR group was the 3th percentile.There was significant difference in non-reassuring state between the two groups.2.Typical phenotype of vascular endothelial cell was observed in cultured primary cells,which were polygon or fusiform,with uniform size,big nucleus,and abundant cytoplasm.Cultured cells presented a cobblestone-like appearance at confluency under phase-contrast microscopy.3.Immunofluorescence assay showed that over 95%of cells expressed endothelial markers CD31 and vWF.4.The cumulative growth curves showed that proliferation of the HUVECs from IUGR group was significantly higher than the Control group at the time point of 96 h(P = 0.003).5.The transcripts of EDN1(P = 0.018)and ARG2(P = 0.007)gene related to vasoconstriction were increased,while the NOS3(P = 0.035),EDNRB(P = 0.023)and AGTR1(P<0.001)gene which mediated vasodilation decreased in IUGR-HUVECs.The HIF1A gene(P = 0.008)related to oxidative stress was up-regulated,and the anti-oxidative SOD1 gene(P = 0.005)was down-regulated in HUVECs from IUGR.The ANG gene(P<0.001)which mediated angiogenesis was up-regulated,anti-angiogenic AKAP12 gene(P<0.001)was down-regulated in the IUGR group.Conclusion:1.The cultured cells were identified as HUVECs,and vascular endothelial cell lines of the IUGR group and Control group were successfully established.2.IUGR promotes proliferation of HUVECs.3.Gene profiles related to vascular function in IUGR-HUVECs were dysregulated and charactered by imbalances of vasoconstriction and vasodilation,oxidative stress and antioxidation,pro-angiogenesis and anti-angiogenesis,indicating vascular endothelial dysfunction.Part ? The mechanisms underlying intrauterine growth restriction-related vascular endothelial dysfunctionObjective:1.To verify the role of reduced NO bioavailability of HUVECs and the dysregulation of eNOS and Arg-2 in IUGR related vascular endothelial dysfunction.2.To investigate whether endogenous NOS inhibitor ADMA and the key enzymes(PRMT and DDAH)for synthesis and degradation of ADMA participate in IUGR related vascular endothelial dysfunction.3.To determine the role of ET-1,ETBR and transcription factor HIF-1? in the IUGR related dysregulation of vascular endothelial dysfunction.Methods:1.Intracellular NO production was determined by the Nitrate/Nitrite Assay Kit based on the Griess colorimetric assay.2.Arginase activity was detected by the Arginase Activity Kit.3.HUVECs were treated with 100 ?M CoCl2 for 24 h to induce hypoxia.4.The levels of L-arginine and ADMA in HUVECs were determined by ELISA.5.The spatial distributions of ET-1 and ETBR in HUVECs were analyzed with immunofluorescence.6.The expression levels of eNOS,Arg-2,PRMT1,DDAH1,DDAH2,ET-1,ETBR and transcription factor HIF-1? were determined by RT-qPCR and western blot.Results:1.Compared with normal newborns,NO production was reduced(P = 0.002)with decreased eNOS(P = 0.021)and increased bioactivity of arginine(P = 0.012)and expression of Arg-2(P = 0.009)in IUGR-HUVECs.2.Compared with normal newborns,ADMA(P = 0.001)and ADMA/L-arginine(P =0.046)in HUVECs in the IUGR group were significantly increased,while the expression of DDAH1 was significantly decreased(P = 0.010).3.ADMA in HUVECs in Control group was significantly elevated In hypoxia conditions compared with in normoxia conditions(P = 0.042).Compared with Control group,the ratio of ADMA to L-arginine(P = 0.003)and PRMT1 expression(P = 0.006)were significantly increased in IUGR group.4.The co-localization of ETBR and ET-1 was observed in HUVECs,indicating an interaction between ET-1 and ETBR.The expression of ET-1(P = 0.004)and HIF-la(P = 0.007)were increased,and ETBR(P = 0.027)was reduced in the IUGR group.Under hypoxia,expression of HIF-1?(P = 0.027)in the IUGR group was higher than the control group.Conclusion:1.IUGR caused the imbalance of eNOS and Arg-2 expression in HUVECs,and reduced NO production and bioavailability,resulting in vascular endothelial dysfunction.2.The reduction of DDAHI expression and ADMA degradation in HUVECs in IUGR led to the accumulation of ADMA and decrease of NO production,further causing vascular endothelial dysfunction.3.Under hypoxic condition,expression of PRMT1 and synthesis of ADMA in HUVECs in IUGR increased,which led to decreased bioavailability of L-arginine,thus causing vascular endothelial dysfunction.4.Increased expression of HIF-la in HUVECs in IUGR promoted the transcription of ET-1 and decreased expression of ETBR reduced NO release,together resulting in vascular endothelial dysfunction.