The Relationship Of Catechol-O-methyltransferase And Preeclampsia

Preeclampsia (PE), a pregnancy-specific disorder characterized clinically by new onset hypertension(≥140/90mmHg on two occasions longer than6hours) and proteinuria (≥300mg/day) after20weeks of gestation, is a systemic syndrome and can affect maternal multiple organs.According to the severity of the condition, preeclampsia can be further differentiated into mild and severe forms. Severe preeclampsia is diagnosed if there are more severe elevations of blood pressure or proteinuria or evidence of other end-organs dysfunction. Severe preeclampsia often associated with liver and renal disfunction. Meternal serum uric acid can be used as severity index in severe preeclampsia. Preeclampsia also can be classified into2different entities, namely early-onset preeclampsia and late-onset preeclampsia. The early-onset preeclampsia tends to develop before34weeks of gestation, which is associated with increased fetal and maternal morbidity and mortality. The late-onset preeclampsia occurs beyond34weeks of gestation, and most of fetuses were in good condition. The etiology and pathogenesis may be different between early-onset and late-onset serere preeclampsia. Additionally, it has been suggested that preeclampsia constitutes a clinical spectrum that includes two distinct conditions:"maternal preeclampsia"and "placental preeclampsia". In this model, placental preeclampsia refers to otherwise healthy women who develop preeclampsia primarily because of abnormal placentation, whereas maternal preeclampsia refers to women who develop preeclampsia because of a pre-existing condition such as cardiovascular disease, chronic arterial hypertension, or diabetes, without abnormal placentation. Preeclampsia complicates roughly5%of pregnancies worldwide, and is a major source of both maternal and fetal morbidity. Despite intensive investigation (more than25000articles published annully on preelampsia), our current understanding of the pathophysiology is still incomplete. Several theories like gene, immunology, oxidative response and other factors have been advanced to explain the pathogenesis of preeclampsia, but none has yet accounted for the vast number of risk factors associated with the disease. And although numerous animal models of preeclampsia exist, the ideal animal model has not found to study preeclampsia. The pathophysiology that triggers the disease is still not clearly elucidated. Preeclmapsia originates in the placenta, as it may occur only in the presence of placenta or a hydatiform mole and resolves dramatically postpartum after the delivery of the placenta. It is widely accepted that the pathoghysiological process of preeclampsia begins with placental hypoxia. Various factors can cause placental hypoxia (maternal hypoxia, maternal hypotension and placental ischemia). But the context of preeclampsia, placental hypoxia is often considered secondary to defective placental vasculature. The placenta is an organ that mediates the exchange of waste and nutrients between mother and fetus. Proper development of the placenta and its vasculature is dependent on an embryonic cell type known as the cytotrophoblast. One cytotrophoblast derivative, the extravillous cytotrophoblast, invades the uterine matrix, makes contact with the maternal blood supply, and then replaces the smooth muscle cell layer of the maternal vasculature. If the extravillous cytotrophoblast invasion is too shallow, the resultant maternal vasculature could be incapable of providing the fetus with adequate nutrition and oxygen, conditions that might lead to placental hypoxia. In this manner, shallow extravillous cytotrophoblast invasion is thought to contribute significantly to the development of preeclampsia. It is thought that hypoxia placenta can trigger preeclampsia by releasing multiple factors and initiating a chain of events that ultimately results in the maternal syndrome of hypertension and proteinuria. Search for a unique circulating factor resulted in an identification of multiple factors of endothelial dysfunction, activation and oxidative stress. A major breakthrough was achieved during the past years by a series of experimental and clinical studies implicating that changes in circulating angiogenic factors play a decisive role in the pathogenesis of preeclampsia. The’angiogenic imbalance’theory which hypothesizes that preeclampsia results from an imbalance of factors that promote or antagonize angiogenesis was address. Soluble fins-like tyrosine kinase1(sFlt1), vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) are postulated to cause endothelial dysfunction, producing the clinical characteristics of preeclampsia. Increased expression of sFlt1, a variant of the membrane-bound VEGF-receptor1that consists of the extracellular ligand-binding domain, was associated with decreased PIGF and VEGF signaling and consecutive defective angiogenesis. The relative expressions of these angiogenic markers and receptors were found to change during the process of uterine invasion. Failure to undergo either set of changes was associated with the development of preelampsia. Soluble Flt1acts as a natural antagonist of the circulating VEGF and increases from mid-gestation onwards, to reach the peak5-8weeks before the onset of the maternal syndrome.The first study suggesting that sFlt1was associated with preeclampsia was published in2000, when sFlt1levels were found to be increased in the amniotic fluid of women with preeclampsia. Later, sFltl levels were found to be elevated in the placentas and maternal serum of patients with preeclampsia. An independent study confirmed serum sFlt1elevation in patients with preeclampsia, and also demonstrated significant decreases in the circulating levels of the proangiogenic factors VEGF and PIGF. The sFlt1concentrations increased beginning approximately five weeks before the onset of preeclampsia. At the onset of clinical disease, the mean serum level in the women with preeclampsia was threefold higher as compared to controls with fetuses of similar gestational age. The PIGF expression was lower in the women who later had preeclampsia than in the controls already at13to16weeks of gestation. The greatest difference occurred during the weeks before the onset of preeclampsia, coincident with the increases in sFlt1. The findings strongly suggested that increased levels of sFlt1and reduced levels of PlGF as excellent and remarkably specific biomarkers for the diagnosis and prediction of preeclampsia. A potentially pathogenic role for sFlt1was suggested in a study showing that injection of sFlt1into mice induced proteinuria. And adenoviral administration of sFlt1to male, pregnant female and non-pregnant female rats resulted in glomerular endotheliosis and hypertension. Based on this evidence, a pathogenic model was put forward, which suggested that in normal pregnancy, sFlt1levels are low in the second trimester but increase steadily in the third trimester of pregnancy. This rise in sFlt1is concurrent with decreases in VEGF and PIGF, changes that are speculated to be a normal physiological mechanism designed to rein in placental vascular growth. And in preeclampsia, it is thought that this natural antiangiogenic process is exaggerated. But not all women with preeclampsia have increased sFlt1concentration. And further upstream, the mechanisms at work in the primary placental defect are unknown. And one in vivo report using sFlt1knockout mice suggested that trophoblastic sFlt1had no obvious effects on placental vascular formation. Thus, there are several unanswered questions concerning the present view of preeclampsia pathogenesis, implicating that additional synergistic factors elaborated by the placenta may be capable of inducing a generalized endothelial dysfunction. Further research might be required to elucidate the initiate factors involved in the angiogenic imbalance.The recent work of K. Kanasaki and associates performed under the aegis of R. Kalluri and published recently in Nature seems to provide a foundation for the unifying hypothesis on the pathogenesis of preeclampsia. The authors demonstrated that pregnant mice deficient in Catechol-O-Methyltransferase (COMT) develop multiple functional and structural features of preeclampsia-like phenotype. COMT-deficient mice delivered preterm with higher wastage of fetuses and showed a higher blood pressure and a higher urinary albumin excretion in comparison with wild-type mice. The arteriopathy of the placenta in the COMT-deficient mice resembled human decidual vascular lesion, and glomerular endotheliosis was also present. Levels of hypoxia-inducible factor-1Alpha (HIF-la) protein were higher in the placenta and the sFlt1plasma concentrations were significantly higher in pregnant COMT-deficient mice compared to controls. Administration of2-methoxyestradiol (2-ME) rescued the COMT-deficient mice from the preeclampsia-like syndrome without toxicity. Moreover,2-ME ameliorated placental hypoxia by inhibition of HIF-la expression and precluded sFltl elevation. However, under hypoxic conditions, it has been shown to facilitate the development of the placenta and embryo by promoting the establishment of the uteroplacental circulation. The site of action of2-ME could be upstream from the uncontrolled production of sFltl and sEng by the ischemic placenta, since COMT-deficient mice also exhibit defective placental vascularization.2-ME is a naturally occurring metabolite of estradiol. It is generated by hydroxylation at the2-position of17-β-esteradiol by Cytochrome P450enzymes, and subsequently O-methylation of the catechol ring by COMT. But in human preeclampsia, what’s the upstream for reduced placental COMT expression? A recent report demonstrated that one possible explanation for this observation is the presence of a G-to-A transition in codon158of the COMT gene that results in a valine to methionine substitution at position108/158in soluble-bound and membrane-bound COMT proteins, respectively. This single-nucleotide polymorphism (SNP) affects protein abundance and enzyme activity. Functional COMT polymorphisms are associated with endometriosis risk and the timing of menopause, and they could also affect preeclampsia.Although these studies provide an attractive hypothesis implicating reduced COMT expression in the pathogenesis of preeclampsia, evidence confirming that reduced COMT expression occurs in human disease has been limited. So in this case-control study we aimed to examine the expression of placental COMT between preeclampsia group and control group to explore the relationship between them, examine plasma2-ME level in case and control group to explore the association with preeclampsia, and analyse the relationship between placental COMT expression and plasma2-ME level and to investigate the relationship between placental COMT polymorphisms and the risk of preeclampsia. This study is meaningful to provide new evidence of preeclampsia etiology and pathophysiology, reduce morbidity, reduce infant and maternal complications and reduce maternal and perinatal mortality. CHAPTER1EXPRESSION OF PLACANTAL COMT IN SEVERE PREECLMAPSIAOBJECTIVETo determine the expression of placental COMT mRNA and protein by realtime quantitative PCR and immunohistochemical technique in severe preeclampsia patients and healthy pregnant women and to explore the relationship between placental COMT and preeclampsia.METHODS1Research subjects:This study was a case-control study. We recruited37severe preeclampsia patients (including15early-onset preeclampsia) and40healthy pregnant women as control (including30gestational age≥34weeks). Severe preeclampsia was defined according to<Obstetrics and Gynecology>(People’s Medical Publishing House, Seventh Edition, edited by Le Jie). Both groups consisted of Han Chinese women, singleton without chronic hypertension, heart disease, kidney disease, diabetes or other complications.2Sample collection:Placental tissues were obtained within lOmin of delivery from all of the subjects. Placental samples (≈0.5*0.5*0.5cm3) were obtained from the maternal side of the placenta under aseptic conditions, avoiding tissue from areas showing placental infarcts. The tissue was washed in ice-cold PBS to remove maternal blood contamination. Samples for collected for RNA extraction were immersed in RNAlater (Ambion) overnight at4℃and transferred to-20℃for long-term storage. Samples for immunohistochemistry were fixed in4%paraformaldehyde.3Real-time PCR:Total RNA from placental samples (50mg) was obtained using1ml of Trizol (Invitrogen) according to the manufacturer’s instructions. Total RNA was reverse-transcribed to cDNA in a20ul reaction using oligo-dT primers according to manufacturer’s protocol(MBI). Real-time PCR analysis was then performed in a fluorescent temperature cycler (SYBR Green dye) to examine the mRNA level of COMT and HIF-la in placental tissue with LightCycler480(Roche) according to the manufacturer’s instructions. 4Immunohistochemistry:The placentas were fixed in4%paraformaldehyde for12to24hours. The localization and semi-quantitative detection of placental COMT were carried out using the one-step Max Vision solution.5Statistical analysis:Statistical analyses were performed with SPSS software version13.0. Measurement data were presented as mean±standard deviation (SD). The statistical significance of the results was assessed by t test. The ranked data from the semiquantification of immunohistochemical staining was analysed using a Mann-Whitney Test. The associations between COMT mRNA and clinical parameters were analyzed by partial correlation. All probability values were2-sided and differences were considered statistically significant when P<0.05.RESULTS1There were no significant difference in maternal age, gestational age and body mass index (BMI) between normal pregnancies (n=40) and pregnancies with severe preeclampsia (n=37)(p>0.05). The birth weight of infants was lower in the preeclamptic group than in the control group (p<0.05). Serum uric acid level and MAP were significantly higher in pregnant women with preeclampsia (p<0.05).2COMT-mRNA of all placentas was detected. The mRNA level of COMT in preeclamptic placentas was significantly decreased compared to that in gestation-matched normal placentas (p<0.001). Further subgroup analyses showed the placental mRNA level of COMT was no significant difference between early-onset preeclamptic group and gestational matched control group (p>0.05). The mRNA level of COMT in late-onset preeclamptic group was significantly decreased compared to that in gestational matched control group (p<0.001). And in the control group, the mRNA level of placental COMT in<34weeks subgroup was significantly lower than that in≥34weeks subgroup.3When controlling the gestational age, maternal age and BMI, the level of COMT-mRNA was negatively correlated with HIF-1a mRNA, serum uric acid, MAP and24-hour urine protein (all P<0.05). Further subgroup analyses showed the placental mRNA level of COMT in late-onset preeclampsia group was significantly negatively correlated with serum uric acid, MAP and24-hour urine protein (all P<0.05). But in the early-onset preeclamptic subgroup things were different (p>0.05).4Protein expression and immunolocalization of COMT in placentas:The cells that produced COMT in the placenta were identified with immunohistochemistry. COMT was primarily detected in the syncytiotrophoblasts of the placentas from preeclamptic women. A similar but more intense staining pattern was observed in the same cell types of the placentas from the normal pregnant women. Semiquantitative analysis indicated this reduced expression of COMT in preeclamptic placentas was significant (p<0.05). Further subgroup analyses showed the placental COMT expression was no significant difference between early-onset preeclamptic group and gestational matched control group (p>0.05). The placental COMT expression in late-onset preeclamptic group was significantly decreased compared to that in gestational matched control group (p<0.05).CONCLUSION1Using real-time quantitative PCR and immunohistochemical technique, we found that reduced placental expression of COMT in preeclampsia patients compared with control pregnancy at both the mRNA and protein levels, suggesting that COMT may be involved in the pathogenesis of preeclampsia. It provided new evidence for the study of preeclampsia etiology and pathophysiology.2It was also demonstrated COMT-mRNA expression was negatively correlated with MAP, serum uric acid and24-hour urine protein, suggesting placental COMT mRNA level can reflect the severity of preeclampsia.3The placental COMT mRNA level in<34weeks control subgroup was significantly lower than that in≥34weeks control subgroup, demonstrating that in different gestational age there was distinct placental COMT expression.4The placental mRNA level of COMT was no significant difference between early-onset preeclamptic group and gestational matched control group, suggesting that the role of placental COMT involved in late-onset preeclampsia may be different from the early-onset preeclampsia. CHAPTER2PLASMA2-ME LEVEL IN PATIENTS WITH LATE-ONSET SEVERE PREECLAMPSIAOBJECTIVETo determine the plasma2-ME level in late-onset preeclampsia patients by the enzyme immunoassay to explore the relationship between plasma2-ME and placental COMT and its possible role involved in preeclampsia.METHODS1Research subjects:22late-onset severe preeclampsia patients and30healthy control women with gestatinal age≥34weeks (as in chapter1).2Sample collection:Individual maternal blood samples were obtained within60min before delivery into EDTA anticoagulation tube from all of the subjects. The samples were then centrifuged with a relative centrifugal force of1000X g for15min. the supernatants (plasma) were stored at-80℃until use.32-ME measurement:Measurement of EDTA-tube plasma2-ME was performed with a Cayman EIA Kit. As2-ME is mostly as glucuronide and sulfate conjugates, all the plasma samples were hydrolyzed with β-glucuronidase prior to assay.4Statistical analysis:Statistical analyses were performed with SPSS software version13.0. Measurement data were presented as mean±standard deviation (SD). The statistical significance of the results was assessed by t test. The associations between plasma2-ME and placental COMT mRNA and clinical parameters were analyzed by bivariate correlation. All probability values were2-sided and differences were considered statistically significant when P<0.05.RESULTS1There was no significant difference in2-ME levels in plasma between patients with late-onset severe preeclampsia and gestational age matched normal pregnant women (P>0.05).2There was no significant correlation between plasma2-ME and placental COMT mRNA, serum uric acid,24-hour urine protein or MAP (all P>0.05).CONCLUSION1We were unable to find the difference of plasma2-ME level between the late-onset severe preeclampsia and control group, suggesting that the role of plasma2-ME in the late-onset severe preeclampsia may be limited.2There was no significant correlation between plasma2-ME and placental COMT expression, indicating the level of plasma2-ME may be mostly affected by its precursor level, cytochrome P450enzymes and other factors.3There was no significant correlation between plasma2-ME and serum uric acid,24-hour urine protein and MAP, suggesting plasma2-ME concentration have little significance in diagnosis and evaluation the severity of preeclampsia.CHAPTER3THE ASSOCIATION OF PLACENTAL COMT GENE POLYMORPHISMS AND PREECLAMPSIAOBJECTIVETo examine the placental COMT rs4680and rs6269polymorphisms by the method of Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALI-TOF-MS) to explore the association between placental COMT gene polymorphisms and preeclampsia.METHODS1Research subjects:We recruited92preeclampsia patients (including37severe preeclampsia in chaper1) and125healthy pregnant women as control(including40healthy pregnant women in chapter1). Preeclampsia was defined according to <Obstetrics and Gynecology>(People’s Medical Publishing House, Seventh Edition, edited by Le Jie).2Sample collection:Placental tissues were obtained as in chapter1.3SNP detection:Placental tissue DNA was isolated from samples by using Trizol following the manufacturer’s recommendations. The method of MALI-TOF-MS was applied to detect the SNPs of rs4680and rs6269in COMT gene.4Statistical analysis:Statistical analyses were performed with SPSS software version13.0. Concordance with Hardy-Weinberg proportions was tested using a Chi-square goodness-of-fit statistic. Genotype distribution and allele frequencies were compared between groups using the R×C Crosstabs Chi-square test and correlations. A threshold of a=0.05was set for statistical significance of all computed analyses.RESULTS1Genotypes of rs4680and rs6269were found to be in Hardy-Weinberg equilibrium in both the preeclampsia and control groups(all P>0.05).2There were no significant differences in rs4680genotype and allele frequencies between patients with preeclampsia and controls (P=0.931and P=0.713, respectively).3There were significant differences in rs6269genotype and allele frequencies between patients with preeclampsia and controls (P=0.019and P=0.012, respectively). The odds ratio for the risk of preeclampsia was1.707(95%CI:1.125-2.590) in women with G allele.CONCLUSION1No association was observed between placental COMT rs4680genotype and preeclampsia in Chinese Han women.2Placental COMT rs6269genotype contributes to the genetic susceptibility of preeclampsia in Han Chinese women.SUMMARY1We found that reduced placental expression of COMT in especially late-onset preeclampsia patients compared with control pregnancy at both the mRNA and protein levels, suggesting that COMT may be involved in the pathogenesis of late-onset preeclampsia. It provided new evidence for the study of preeclampsia etiology and pathophysiology.2It was also demonstrated COMT-mRNA expression was negatively correlated with MAP, serum uric acid and24-hour urine protein, suggesting placental COMT mRNA level can reflect the severity of preeclampsia.3The placental COMT mRNA level in<34weeks control subgroup was significantly lower than that in≥34weeks control subgroup, demonstrating that in different gestational age there was distinct placental COMT expression.4We were unable to find the difference of plasma2-ME level between the late-onset severe preeclampsia and control group, suggesting that the role of plasma2-ME in the diagnosis and evaluation for late-onset severe preeclampsia may be limited.5Placental COMT rs6269genotype contributes to the genetic susceptibility of preeclampsia in Han Chinese women.In conclusion, preeclampsia is a complicated systemic condition that may involve multiple factors in the pathogenesis. Our study taken together with placental COMT expression, plasma2-ME and placental COMT gene polymorphisms demonstrated COMT could play a role in the pathophysiology of late-onset preeclampsia. Placental COMT rs6269polymorphism may be attributed to the role. More investigations are needed to elucidate the role of2-ME in the development of PE.