Potential Biochemical and Molecular Aspects in Pathophysiology of Preeclampsia: Assessment of Endothelial Nitric Oxide Synthase Gene in Preeclamptic Pakistani Women

Abstract

Background: Hypertensive disorders are a common complication of pregnancy that put women and their fetuses at disproportionate risk for further complications, as well as life-long sequelae. Preeclampsia (PE), in particular, is one of the most feared complications of pregnancy characterized by maternal endothelial cell dysfunction which causes symptoms including high blood pressure and proteinuria with maternal organ dysfunction and/or fetal growth restriction. Preeclampsia (PE) is one of the major causes of maternal and perinatal morbidity and mortality, particularly in resource-limited settings. Although the cause of PE remains largely unknown, the leading hypothesis strongly relies on disturbing placental function in early pregnancy. Assessment of hematological parameters, hormonal analysis, endothelial dysfunction, oxidative stress, and genetic screening is important for early prediction to allow close surveillance and preventive strategies. Unidentified genetic factors and vasodilation induced by impaired nitric oxide (NO) are thought to contribute to the syndrome's development. The endothelial nitric oxide synthase (eNOS) gene polymorphisms have an impact on NO production and were associated with hypertension and preeclampsia. Objectives: Current study was designed for the comprehensive assessment of biochemical and molecular risk factor in the susceptibility of PE in the Pakistani population. The objectives of the study include: ▪ Assessment of demographic, clinical and biological markers in the pathophysiology of preeclampsia in Pakistani women. ▪ To describe the potent role of oxidative stress markers in susceptibility to preeclampsia in Pakistan. ▪ Evaluation of histomorphological and histomorphometric changes of the placenta in preeclamptic Pakistani women. ▪ Determination of NO and epinephrine levels in both normal and PE patients. ▪ To analyze the placental localization and intensity of eNOS staining in normal and PE patients. ▪ Estimation of mRNA expression of eNOS in the placentas of both normal and PE patients. ▪ Screening of the mutations and polymorphisms in the DNA sequence of the eNOS gene in both normal and PE patients ▪ Computational analysis of identified variants in the coding and non-coding region of the eNOS gene. Materials and methods: Total 400 blood (PE/controls=200), 400 urine (PE/controls=200), and 100 placental tissue samples (PE/controls=50) were recruited. History and samples were collected from each subject with informed consent after diagnosis for hematological or biochemical and molecular analysis. Biochemical analysis of blood and urine routine examination was done. Histomorphological and histomorphometric analysis were performed for placental tissues. Reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) levels were analyzed through a spectrophotometer. Nitric oxide (NO), epinephrine, triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined through spectrophotometer and enzyme linked immunosorbent assay (ELISA). Immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) was done to estimate the localization and expression of eNOS in placentas of PE patients and healthy pregnant women. eNOS gene variants were screened in PE patients and control group by genotyping and sequencing. Further in silico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. Independent sample t-test, Chi-squared test (χ2) and Odds Ratio (OR), Hardy-Weinberg equilibrium model and Pearson’s correlation was determined using IBM SPSS Statistics 21 software and package of R 3.5.1 (R Development Core Team, 2018). Results: Experimental finding showed that several parameters including gestational age (p <0.001) and weight of the child (p <0.001) were significantly reduced while age (p 0.002), BMI (p=0.047) systolic blood pressure (SBP) and diastolic blood pressure (DBP) (p <0.001) were significantly elevated in PE patients as compared to control group. Pregnancy history including headache (OR: 2.42), swelling in hands and face (OR: 4.8), excessive weight gain (OR: 3.74) urination problem (OR: 2.90), abdominal pain (OR: 1.45), shortness of breath (OR: 4.49), muscular pain (OR: 1.64) and blurring of vision (OR: 2.03), history of preeclampsia in previous pregnancy and in family (OR: 7.38; OR: 8.43) were more obvious in preeclamptic women. Significantly (p<0.001) elevated levels of alkaline phosphatase, serum urea, uric acid, urine proteins, total leucocyte count (TLC) (p=0.028) and haematocrit (p=0.016) were reported in PE group. Prolonged Activated partial thromboplastin time (aPTT), prothrombin time (PT) and international normalizing ratio (INR) were recorded in both PE groups with a decrease in platelets and fibrinogen levels as compared to controls. While, total bilirubin (p=0.019), aspartate aminotransferase (AST) (p=0.012), serum calcium (p=0.002) and sodium (p= 0.010) concentrations were reduced in case patients as compared to control group. Significantly increased concentrations of ROS (p<0.001) and TBARS (p=0.04) were determined in preeclamptic patients while non-significant difference was observed in POD (p=0.11), SOD (p=0.97) and CAT (p=0.81) levels among both groups. Histological changes determined abnormal villi, more syncytial knots (SK) and a significant decrease in elongated and large villi in PE placentas as compared to normal placentas. Reduced NO (p=0.016), eNOS immunoreactivity (p≤0.001) and mRNA abundance (p≤0.001) was observed in preeclamptic group as compared to control group. Thyroid hormones demonstrated non-significant alterations in both groups. Significant positive correlation was observed in creatinine and urea levels of PE patients while SBP and DBP showed significant positive correlation with each other and with age. The frequency of c.894T (p.298Asp) was high in the PE group (p≤0.001). Likewise, a significant association of g.-786C alleles was found with the PE group (p=0.007) when compared to normotensive women. In addition, a novel homozygous variant g.2051G>A was also significantly associated with PE (P=0.02) when compared to normotensive women. Dynamic simulation studies revealed that Glu298Asp mutation destabilizes the protein molecule and decrease the overall stability of eNOS protein. Molecular docking analysis of the mutant promoter with transcription factors signal transducer and activator of transcription 3 (STAT 3) and signal transducer and activator of transcription 6 (STAT6) proposed changes in protein regulation upon these reported mutations in the upstream region of the gene. Conclusion: Considering the results of the current study, it is concluded that present study reveals the importance of demographic and clinical risk factors in early diagnosis of PE, and should be considered in the counselling, diagnostic and therapeutic interventions to improve the reproductive health of women in Pakistan. Furthermore, oxidative stress elevated epinephrine, reduced NO, reduced placental expression of eNOS and the functional alterations induced by the variants of eNOS gene results in endothelial dysfunction and histological placental alterations leading to PE in Pakistani pregnant women. These variations lead to blood pressure elevation and renal insufficiency in preeclamptic patients. However, further studies and meta-analysis are necessary to validate these findings and clarify this issue to prevent maternal and neonatal morbidity and mortality in Pakistan.