Background. Hypertensive disorder generally complicates 5–10 percent of all pregnancies. Angiogenic growth factors may be helpful for the diagnosis and prediction of preeclampsia. Therefore, in this study we attempted to determine the serum levels of asymmetric dimethylarginine (ADMA), vascular endothelial growth factor (VEGF), and nitric oxide (NO) metabolite (nitrite) in preeclampsia patients and compared the levels with those obtained from normal pregnant women. Methods. Ninety pregnant women (19–33 years old) in two groups of preeclampsia and normal were considered during 2012. The levels of ADMA, VEGF, and nitrite were measured in maternal serum samples using ELISA kits. Results. Significant increase of VEGF and nitrite levels was observed in preeclampsia patients when compared with other groups (P<0.05). The serum level of ADMA demonstrated a similar increased trend in preeclampsia patients; however, the increase was not statistically significant (P=0.08). Conclusion. The findings reveal that the elevation of serum levels of VEGF and nitrite and possibly ADMA may be involved in the pathogenesis of preeclampsia.
1. Introduction
Hypertensive disorder is generally the most common disease and cause of death in pregnancy and complicates 5–10% of all pregnancies. Furthermore, the pregnancy syndrome preeclampsia complicates approximately 3–7% of all pregnancies, and the syndrome becomes a major contributor of death [1–4]. Preeclampsia generally is recognized by deficient uteroplacental perfusion [5, 6], and some risk factors such as obesity, multifetal gestations, maternal age above 35, and African-American ethnicity are associated with this syndrome [7–9]. The association between pregnancy and hypertension and its effects on mother and child life remain a subject of intensive researches for many years, and no definite solution was achieved yet. Preeclampsia often affects young and nulliparous women, and its incidence is influenced by race and genetic predispositions [10]. The role of the renin-angiotensin system and angiogenic and antiangiogenic biomarkers of placental origin has been described in pathogenesis of preeclampsia [11]. However, there is no definite biomarker to be considered as a sensitive parameter to control the disease severity. Nitric oxide (NO) is a potent vasodilator that is synthesized from L-arginine by the NO synthase and endothelial cells [12] and regulates the vascular tone and blood flow in the vascular smooth muscles. The endothelium-released NO is influenced by hypertension, and it is considered as a factor of endothelial functions [13, 14]. NO could promote the release of an important angiogenesis mediator; vascular endothelial growth factor (VEGF). VEGF is a potent endothelial cell-specific mitogen that promotes vascular hyperpermeability, and vasodilation [15–18]. Asymmetric dimethylarginine (ADMA) is an antiangiogenesis factor [19, 20] that decreases the expression of VEGF in endothelial cells [21] and may interrupt the NO-producing activity of NO synthase.
Chedraui et al. measured the serum levels of NO, ADMA, and VEGF in severe preeclampsia and found positive correlation between NO and ADMA among preeclampsia cases, and the NO levels were significantly different in the artery and vein of umbilical vessels. Moreover, the level of VEGF was significantly lower in the artery but not in the vein in preeclampsia cases [22]. Other studies indicated an increase in ADMA level and a decrease in NO levels [23] or decrease in the level of both factors [24] in preeclampsia. Furthermore, the importance of NO has been emphasized in preeclampsia [25, 26]. ADMA may have a clinical significance [27, 28], and it is a potential biomarker to identify pregnant women at the risk of developing preeclampsia [29]. Angiogenic growth factors such as VEGF may be helpful for the diagnosis and prediction of preeclampsia [30], and it may be responsible for impaired vascular development [31]. Still, there is no well-documented data that explains how NO, ADMA, and VEGF levels vary in preeclampsia patients. So, we attempted to determine the serum levels of these biomarkers in serum samples of preeclampsia patients and compared them with those obtained from normal pregnant women.
2. Materials and Methods2.1. Patients
This study included patients with and without preeclampsia referred to obstetrics and gynecology clinics of Isfahan University of Medical Sciences during 2012. This study was approved by the Ethical Committee of Isfahan University of Medical Sciences.
All pregnant subjects were nulliparous healthy women without any known preexisting medical complications. Exclusion criteria included multiple gestation, prior preeclampsia, and preexisting medical conditions such as diabetes, chronic hypertension, and renal disease. Forty-five women with preeclampsia (aged 24.7±0.6 years) and 45 women without preeclampsia (aged 24.5±0.5 years) were selected. Preeclampsia was diagnosed by the presence of gestational hypertension, proteinuria, and hyperuricemia beginning after the 20th week of pregnancy with resolution of gestational hypertension and postpartum proteinuria. Gestational hypertension was defined as a new onset increase in blood pressure including an absolute systolic blood pressure (BP) ≥140 mmHg and/or diastolic BP ≥ 90 mmHg after 20 weeks of gestation. Proteinuria was defined as ≥300 mg per 24-hour urine collection, ≥2 + protein on voided urine sample, ≥1 + protein on catheterized urine specimen, or a protein-creatinine ratio of ≥0.3. The official informed consent was obtained from all subjects, and the demographic data were collected.
2.2. Measurements
Maternal blood sample was obtained and centrifuged. The serum samples were collected and stored at −20°C for later analysis. The serum levels of VEGF were measured using an enzyme immunoassay kit (Immuno-Biological Laboratory Co., Japan). Briefly, the kit is a solid-phase sandwich ELISA using specific polyclonal and monoclonal antibodies, and the coloring agent was tetramethylbenzidine (TMB). The absorbance at 450 nm is in proportion to the VEGF concentration using the standard curve.
The serum levels of nitrite (stable NO metabolite) were measured using a colorimetric assay kit (Promega Corporation, USA) that involves the Griess reaction. Briefly, after adding sulphanilamide solution and incubation, N-1-naphtylethylenediamine dihydrochloride solution was added. Then, the absorbance was measured by a microreader at 540 nm wavelength. The nitrite concentrations of the samples were determined by comparison to the nitrite standard reference curve.
The serum levels of ADMA were measured using an enzyme immunoassay kit (DLD Diagnostika GmbH, Germany). Briefly, ADMA in the samples competes with solid phase-bound ADMA for a fixed number of rabbit anti-ADMA. The anti-rabbit/peroxidase was used to detect the antibody bound to the solid-phase ADMA, which is inversely proportional to the ADMA concentration of serum or peritoneal fluid.
2.3. Statistical Analyses
The data are reported as mean ± SEM. The parameters between case and control groups were compared using unpaired t-tests, and P value less than 0.05 was considered statistically significant.
3. Results
The data of serum levels of VEGF, nitrite, and ADMA for the patients with preeclampsia (case) and pregnant women without preeclampsia (control) is demonstrated in Figure 1. A significant increase in VEGF and nitrite levels was observed in preeclampsia patients when compared with the control group (P<0.05). The serum level of ADMA demonstrated a similar increased trend in preeclampsia patients, but the difference was not statistically significant (P=0.08).
Serum levels of VEGF, nitrite, and ADMA in patients with PIH with normal matched pregnant women. The data is shown as mean ± SEM and was compared using two-tailed Student’s t-test analysis.
4. Discussion
Our observation indicated that serum levels of VEGF and nitrite were higher in patients with preeclampsia (case) than normotensive pregnant women (control). In addition, the serum level of ADMA was nonsignificantly different between the case and control groups. The increase of VEGF in preeclampsia patients was reported before [32, 33] due to endothelial activation. However, the level of free VEGF decreased in preeclampsia subjects possibly because of endothelial dysfunction [34]. In the current study, we measured the total VEGF; bounding and free levels in serum, which was detectable by ELISA assay. Therefore, higher levels of VEGF in preeclampsia subject were expected which is consistent with the findings reported in previous studies [32, 33]. Moreover, NO level increases in hypertension condition, and about 50% of our patients had a blood pressure above 160/90 mmHg, which may increase the serum level of NO. However, there are some controversies for alteration of NO in preeclampsia patients. Some reports indicated decreased NO level [35–39], and no change in NO level was detected by others [40–44]. The reason for such observation may be related to increase of nitrite excretion in the urine or decrease of placental NO synthase activation [45] in pregnant women with preeclampsia. In addition, it is also reported that the serum level of NO is related to gestational age, which become at maximum level in gestational age of 35 to 36 weeks [46]. We measured the serum level of nitrite as one of the stable NO metabolite, which was increased in the case group. Two reasons may contribute to such finding. First, the gestational age of our patients was 34.8±2.4 weeks, and possibly at this age the serum level of NO was high [46], and second, we measured one of the NO metabolites, nitrite. Different results would possibly be obtained if other metabolite; nitrate was also measured. One limitation for our study was lack of gestational age matching between the two groups because the preeclampsia patients were included in the study as soon as the sign of disease appeared.
ADMA is an antiangiogenesis factor that decreases VEGF expression in endothelial cells and prevents the formation of NO from NOS. Increase of ADMA in endothelium-dependent vascular dysfunction was detected in diabetic, hypertensive, and hypercholesterolemic patients [20, 47, 48]. It is reported that ADMA may decrease at the beginning of normal pregnancy [49], but under the preeclampsia condition its concentration increases [50–52], which may be associated with the disease intensity [50]. It seems that ADMA is involved in preeclampsia pathogenesis because inhibition of NO synthesis in rodents during pregnancy produces signs similar to those of preeclampsia [53–55]. In the current study, the serum level of ADMA nonsignificantly increased in the case group. The reason for such nonsignificant elevation may related to the increase of NO level in these patients.
5. Conclusion
The findings reveal that elevation of serum levels of VEGF and nitrite and possibly ADMA may be involved in the pathogenesis of preeclampsia, and these markers may be helpful for the diagnosis of the disease.
Conflict of Interests
The authors do not have any direct financial relationship with the commercial identities mentioned in this paper. So, the authors have no conflict of interests.
Acknowledgment
This research was supported by the Isfahan University of Medical Sciences (Grant no. 290203).
WagnerL. K.Diagnosis and management of preeclampsia20047012231723242-s2.0-10644259539SibaiB. M.Diagnosis and management of gestational hypertension and preeclampsia200310211811922-s2.0-003770788910.1016/S0029-7844(03)00475-7ACOG practice bulletinDiagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. American College of Obstetricians and Gynecologists2002771677510.1016/S0020-7292(02)80002-9LindheimerM. D.KatzA. I.Preeclampsia: pathophysiology, diagnosis, and management1989402332502-s2.0-0024381630KingdomJ. C.KaufmannP.Oxygen and placental vascular development19994742592752-s2.0-0034466027RobertsJ. M.Objective evidence of endothelial dysfunction in preeclampsia19993359929972-s2.0-0032959186Conde-AgudeloA.BelizánJ. M.Risk factors for pre-eclampsia in a large cohort of Latin American and Caribbean women2000107175832-s2.0-0033986747SibaiB. M.EwellM.LevineR. J.KlebanoffM. A.EsterlitzJ.CatalanoP. M.GoldenbergR. L.JoffeG.Risk factors associated with preeclampsia in healthy nulliparous women. The Calcium for Preeclampsia Prevention (CPEP) Study Group19971775100310102-s2.0-0030782497WalkerJ. J.Pre-eclampsia20003569237126012652-s2.0-0034619029SargentI. L.BorzychowskiA. M.RedmanC. W. G.Immunoregulation in normal pregnancy and pre-eclampsia: an overview20061356806862-s2.0-33751074998ShahD. M.Preeclampsia: new insights2007163213220SladekS. M.MagnessR. R.ConradK. P.Nitric oxide and pregnancy19972722, part 2R441R4632-s2.0-0030950528MyattL.BrewerA. S.LangdonG.BrockmanD. E.Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation19921661, part 12242302-s2.0-0026547315WeinerC. P.ThompsonL. P.LiuK. Z.HerrigJ. E.Endothelium-derived relaxing factor and indomethacin-sensitive contracting factor alter arterial contractile responses to thromboxane during pregnancy19921664117111812-s2.0-0026545452Charnock-JonesD. S.SharkeyA. M.Rajput-WilliamsJ.BurchD.SchofieldJ. P.FountainS. A.BoocockC. A.SmithS. K.Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines1993485112011282-s2.0-0027399789HouckK. A.FerraraN.WinerJ.CachianesG.LiB.LeungD. W.The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA1991512180618142-s2.0-0026395163PepperM. S.FerraraN.OrciL.MontesanoR.Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells199118129029062-s2.0-0026321129UnemoriE. N.FerraraN.BauerE. A.AmentoE. P.Vascular endothelial growth factor induced interstitial collagenase expression in human endothelial cells199215335575622-s2.0-002644685910.1002/jcp.1041530317CookeJ. P.Does ADMA cause endothelial dysfunction?20002092032203710.1161/01.ATV.20.9.2032CookeJ. P.Asymmetrical dimethylarginine: the Über marker?200410915181318182-s2.0-194243852110.1161/01.CIR.0000126823.07732.D5SmithC. L.BirdseyG. M.AnthonyS.ArrigoniF. I.LeiperJ. M.VallanceP.Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype200330849849892-s2.0-004316193010.1016/S0006-291X(03)01507-9ChedrauiP.SolisE. J.BocciG.Feto-placental nitric oxide, asymmetric dimethylarginine and vascular endothelial growth factor (VEGF) levels and VEGF gene polymorphisms in severe preeclampsia201326322623210.3109/14767058.2012.733760DemirB.DemirS.PasaS.The role of homocysteine, asymmetric dimethylarginine and nitric oxide in pre-eclampsia201232652552810.3109/01443615.2012.693985DikensoyE.BalatO.PenceS.BalatA.CekmenM.YurekliM.The changes of plasma malondialdehyde, nitric oxide, and adrenomedullin levels in patients with preeclampsia20092843833892-s2.0-7035030950410.3109/10641950802629634MatsubaraK.MatsubaraY.HyodoS.KatayamaT.ItoM.Role of nitric oxide and reactive oxygen species in the pathogenesis of preeclampsia20103622392472-s2.0-7795020496610.1111/j.1447-0756.2009.01128.xPoniedzialek-CzajkowskaE.MarciniakB.Kimber-TrojnarZ.Leszczyńska-GorzelakB.OleszczukJ.Nitric oxide in normal and preeclamptic pregnancy20111257437492-s2.0-79955590400PetterssonA.HednerT.MilsomI.Increased circulating concentrations of asymmetric dimethyl arginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, in preeclampsia19987788088132-s2.0-0031661048SiroenM. P.TeerlinkT.NijveldtR. J.PrinsH. A.RichirM. C.van LeeuwenP. A.The clinical significance of asymmetric dimethylarginine2006262032282-s2.0-3375013780410.1146/annurev.nutr.26.061505.111320BögerR. H.DiemertA.SchwedhelmE.LüneburgN.MaasR.HecherK.The role of nitric oxide synthase inhibition by asymmetric dimethylarginine in the pathophysiology of preeclampsia20106911132-s2.0-7034989413410.1159/000245940VerlohrenS.StepanH.DechendR.Angiogenic growth factors in the diagnosis and prediction of pre-eclampsia2012122243522-s2.0-8055514306810.1042/CS20110097TranquilliA. L.BezzeccheriV.GiannubiloS. R.ScagnoliC.MazzantiL.GarzettiG. G.Amniotic vascular endothelial growth factor (VEGF) and nitric oxide (NO) in women with subsequent preeclampsia2004113117202-s2.0-144235996410.1016/S0301-2115(03)00369-5BakerP. N.KrasnowJ.RobertsJ. M.YeoK. T.Elevated serum levels of vascular endothelial growth factor in patients with preeclampsia19958658158212-s2.0-002887942010.1016/0029-7844(95)00259-TSharkeyA. M.CooperJ. C.BalmforthJ. R.MclarenJ.ClarkD. E.Charnock-JonesD. S.MorrisN. H.SmithS. K.Maternal plasma levels of vascular endothelial growth factor in normotensive pregnancies and in pregnancies complicated by pre-eclampsia19962612118211852-s2.0-0030456654VarugheseB.BhatlaN.KumarR.DwivediS. N.DhingraR.Circulating angiogenic factors in pregnancies complicated by pre-eclampsia201023277812-s2.0-77953295600BrenneckeS. P.GudeN. M.Di IulioJ. L.KingR. G.Reduction of placental nitric oxide synthase activity in pre-eclampsia199793151552-s2.0-0030753151ChoiJ. W.ImM. W.PaiS. H.Nitric oxide production increases during normal pregnancy and decreases in preeclampsia20023232572632-s2.0-0036022311FaxénM.NisellH.KublickieneK.-R.Altered mRNA expression of ecNOS and iNOS in myometrium and placenta from women with preeclampsia2001265145502-s2.0-003508242810.1007/s004040000152RoweJ.CampbellS.GalleryE. D.Plasma from preeclamptic women stimulates decidual endothelial cell growth and prostacyclin but not nitric oxide production: close correlation of prostacyclin and thromboxane production20018132382-s2.0-003579303110.1016/S1071-5576(00)00117-9SteinertJ. R.WyattA. W.PostonL.JacobR.MannG. E.Preeclampsia is associated with altered Ca2+ regulation and NO production in human fetal venous endothelial cells20021677217232-s2.0-0036580745AyukP. T.TheophanousD.D'SouzaS. W.SibleyC. P.GlazierJ. D.L-arginine transport by the microvillous plasma membrane of the syncytiotrophoblast from human placenta in relation to nitric oxide production: effects of gestation, preeclampsia, and intrauterine growth restriction20028727477512-s2.0-003616929110.1210/jc.87.2.747Di IorioR.MarinoniE.EmilianiS.VillaccioB.CosmiE. V.Nitric oxide in preeclampsia: lack of evidence for decreased production199876165702-s2.0-003196253410.1016/S0301-2115(97)00159-0DiejomaohF. M.OmuA. E.Al-BusiriN.TaherS.Al-OthmanS.FatinikunT.FernandesS.Nitric oxide production is not altered in preeclampsia200426942372432-s2.0-2442681525ScaleraF.SchlembachD.BeinderE.Production of vasoactive substances by human umbilical vein endothelial cells after incubation with serum from preeclamptic patients20019921721782-s2.0-003570417510.1016/S0301-2115(01)00412-2YanikF. F.AmanvermezR.KoçakI.YanikA.ÇelikC.Serum nitric oxide and glutathione levels in preeclamptic and normotensive women during labor20015121101152-s2.0-003511187710.1159/000052904GarmendiaJ. V.GutiérrezY.BlancaI.BiancoN. E.de SanctisJ. B.Nitric oxide in different types of hypertension during pregnancy19979354134212-s2.0-0030717398JoT.TakauchiY.NakajimaY.FukamiK.KosakaH.TeradaN.Maternal or umblical venous levels of nitrite/nitrate during pregnancy and at delivery19981255235262-s2.0-0031739074AchanV.BroadheadM.MalakiM.WhitleyG.LeiperJ.MacAllisterR.VallanceP.Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase2003238145514592-s2.0-004173306510.1161/01.ATV.0000081742.92006.59BögerR. H.Bode-BögerS. M.SzubaA.TsaoP. S.ChanJ. R.TangphaoO.BlaschkeT. F.CookeJ. P.Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia19989818184218472-s2.0-0242322346HoldenD. P.FicklingS. A.WhitleyG. S.NusseyS. S.Plasma concentrations of asymmetric dimethylarginine, a natural inhibitor of nitric oxide synthase, in normal pregnancy and preeclampsia199817835515562-s2.0-003197859110.1016/S0002-9378(98)70437-5EllisJ.WennerholmU. B.BengtssonA.LiljaH.PetterssonA.SultanB.WennergrenM.HagbergH.Levels of dimethylarginines and cytokines in mild and severe preeclampsia20018076026082-s2.0-003494097610.1034/j.1600-0412.2001.800703.xBraekkeK.UelandP. M.HarsemN. K.StaffA. C.Asymmetric dimethylarginine in the maternal and fetal circulation in preeclampsia20096644114152-s2.0-7035046433410.1203/PDR.0b013e3181b33392SavvidouM. D.HingoraniA. D.TsikasD.FrölichJ. C.VallanceP.NicolaidesK. H.Endothelial dysfunction and raised plasma concentrations of asymmetric dimethylarginine in pregnant women who subsequently develop pre-eclampsia20033619368151115172-s2.0-003863778410.1016/S0140-6736(03)13177-7BahtiyarM. O.BuhimschiC.RavishankarV.CopelJ.NorwitzE.JulienS.GullerS.BuhimschiI. A.Contrasting effects of chronic hypoxia and nitric oxide synthase inhibition on circulating angiogenic factors in a rat model of growth restriction2007196172.e172.e62-s2.0-3384619281510.1016/j.ajog.2006.07.048BuhimschiI.YallampalliC.ChwaliszK.GarfieldR. E.Pre-eclampsia-like conditions produced by nitric oxide inhibition: effects of L-arginine, D-arginine and steroid hormones19951010272327302-s2.0-0028878984YallampalliC.GarfieldR. E.Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia19931695131613202-s2.0-0027140304