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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 68  |  Issue : 3  |  Page : 215-220

Association of vascular endothelial growth factor and soluble fms-like tyrosine kinase-1 polymorphisms with their circulating protein levels in preeclampsia


1 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India
2 Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India
3 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India

Date of Submission15-Oct-2019
Date of Acceptance05-Nov-2019
Date of Web Publication07-Jan-2020

Correspondence Address:
Prof. Renu Dhingra
Department of Anatomy, All India Institute of Medical Sciences, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JASI.JASI_145_19

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  Abstract 


Introduction: Normal development of placental vascular tree requiring angiogenesis and vasculogenesis is structurally and functionally indispensable for both adequate placental growth and delivery of nutrients from mother to the fetus. Impaired placental angiogenesis has been implicated in the pathophysiology of pregnancy complications which have immediate and long-lasting effects on the mother and her child, such as preeclampsia (PE) and fetal growth restriction. The mechanisms underlying the deregulation of placental angiogenesis in PE include a misbalance between the secretion and activity of pro-angiogenic (vascular endothelial growth factor [VEGF]) and anti-angiogenic (soluble fms-like tyrosine kinase-1 [sFlt-1]) factors. Considering the important roles of VEGF and sflt1 in pregnancy, functional polymorphisms in these genes may be potentially important as genetic markers for susceptibility to PE. Thus, the aim of the study was to screen for the presence of VEGF and sFlt-1 gene polymorphisms and to measure their levels in PE patients and controls of Indian origin. Material and Methods: Fifty each of clinically diagnosed patients and gestational and maternal age-matched normotensive, nonproteinuric controls were recruited after taking informed consent. DNA isolated from blood samples was processed for polymerase chain reaction amplification followed by restriction fragment length polymorphism to screen for the presence of VEGF + 936C/T, sFlt-1 (+4244G/A, −4771G/T, −523C/G) polymorphisms. Serum levels of VEGF-A and sFlt-1 were measured by Sandwich enzyme-linked immunosorbent assay (ELISA). Results: Decreased frequency of wild type genotype with respect to VEGF + 936C/T and sFlt-1 (+4244G/A, −4771G/T, and −523C/G) polymorphisms was seen in patients. ELISA results showed lower VEGF-A (198.43 ± 14.63 pg/ml vs. 235.08 ± 16.72 pg/ml [mean ± standard error of mean]) and higher sFlt-1 levels (2932.81 [1802.33–5760.46] pg/ml vs. 1114.94 [655.03–2694.35] Median [Range]; P < 0.05) in patients as compared to controls. Preeclamptic women with increased frequency of VEGF + 936CT genotype had lower serum levels of VEGF-A. However, preeclamptic women with increased frequency of GA, AA, GT, TT, CG, and GG genotypes of sFlt-1 (+4244G/A, −4771G/T, −523C/G) polymorphisms had increased serum levels of sFlt-1. Discussion and Conclusion: The present study shows, for the first time, a possible association of VEGF and sFlt-1 polymorphisms with gene expression and altered protein levels in preeclamptic patients of Indian origin.

Keywords: Preeclampsia, sFlt-1, single nucleotide polymorphism, vascular endothelial growth factor


How to cite this article:
Arora P, Sangwan SK, Gupta R, Mochan S, Gupta SK, Saxena S, Rani N, Bhatla N, Dwivedi S, Sharma A, Dhingra R. Association of vascular endothelial growth factor and soluble fms-like tyrosine kinase-1 polymorphisms with their circulating protein levels in preeclampsia. J Anat Soc India 2019;68:215-20

How to cite this URL:
Arora P, Sangwan SK, Gupta R, Mochan S, Gupta SK, Saxena S, Rani N, Bhatla N, Dwivedi S, Sharma A, Dhingra R. Association of vascular endothelial growth factor and soluble fms-like tyrosine kinase-1 polymorphisms with their circulating protein levels in preeclampsia. J Anat Soc India [serial online] 2019 [cited 2020 Aug 15];68:215-20. Available from: http://www.jasi.org.in/text.asp?2019/68/3/215/275272




  Introduction Top


Preeclampsia (PE) is a life-threatening pregnancy-specific syndrome characterized by hypertension and proteinuria after the 20th week of gestation.[1] It is one of the four leading causes of maternal mortality and morbidity worldwide.[2] In developing countries, the prevalence of PE is much higher due to the lack of prenatal care, access to hospital care, resources, appropriate diagnosis and management of patients.[2] The cause of PE remains unclear but is believed to result from a combination of insufficient blood flow to the uterus, damage to the blood vessels, a problem with the immune system, dietary and genetic factors that lead to the failure of normal trophoblastic invasion and improper remodeling of the uterine spiral arteries.[3] The process of PE may thus begin with impaired trophoblast invasion which produces an increase in oxidative stress that results in a systemic inflammatory response and endothelial dysfunction.[4],[5] The inadequate placentation is followed by an increase in the placenta-derived anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1).[6] sFlt1, a splice variant of VEGF receptor Flt1(VEGFR-1) lacking the transmembrane and cytoplasmic domains, acts as a potent VEGF and placental growth factor (PlGF) antagonist.[6] Vascular endothelial growth factor (VEGF) is a major angiogenic factor and a prime regulator of endothelial cell proliferation. It plays a crucial role in physiological vasculogenesis and vascular permeability.[7] Soluble Flt-1 binds VEGF-A with a higher affinity resulting in extremely low circulating levels of free VEGF-Ato bind with the VEGFR-1 thus reducing the beneficial effects of VEGF. Studies have shown that the imbalance in angiogenic/anti-angiogenic factors affects vasculogenesis, angiogenesis, and placental development and is strongly associated with signs and symptoms of PE.[8],[9],[10],[11] Family studies have shown that genetic factors play a role in PE, but the exact inheritance pattern is still unknown.[12],[13] Candidate genes (VEGF, Flt-1) have been studied worldwide for PE, and single nucleotide polymorphisms (SNPs) in these genes have been found to be associated with PE. Various genetic studies conducted worldwide suggest the polymorphisms of VEGF and sFlt-1to be significantly associated with PE.[14],[15],[16],[17],[18],[19] However, there is no data available on the VEGF and sFlt-1 polymorphisms and their association with protein levels in PE mothers of Indian origin. Thus, the present study was planned to screen for the presence of VEGF (+936 C/T) and sFlt-1 ((+4244G/A,-4771G/T,-523C/G) gene polymorphisms and also measure their protein levels in PE patients and healthy controls.


  Material and Methods Top


Subjects

It was designed as a case–control study where clinically diagnosed patients (n = 50) and gestational and maternal age-matched normotensive, nonproteinuric controls (n = 50) from the antenatal clinic and the inpatient ward of the Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India, were recruited in the study after taking written, informed consent. A protocol of the study was approved by the Institute Ethics Committee. PE was defined according to ACOG guidelines: Blood pressure (BP) ≥140 mm Hg systolic and ≥ 90 mm Hg diastolic (mild) or ≥160 mm Hg systolic and ≥110 mm Hg diastolic (severe) on 2 occasions at least 4 h apart after 20 weeks of gestational age in women with a previously normal BP, confirmed within a short interval to facilitate timely antihypertensive therapy; proteinuria >300 mg/24 h urine collection or protein/creatinine ratio >0.3 mg/dl or dipstick reading of >1+ or in the absence of proteinuria, newonset hypertension with new onset of one or more of the following: thrombocytopenia: platelet count <100,000/μl, renal insufficiency: serum creatinine >1.1 mg/dl or doubling of serum creatinine in the absence of other renal disease, impaired liver function: elevated blood levels of liver transaminases to twice normal concentrations, pulmonary edema and cerebral edema. Pregnant women with chronic hypertension, chorioamnionitis, diabetes, renal disease, and cardiac disease were excluded from the study.

Clinical information from all the patients was recorded on a predesigned questionnaire and detailed pedigree information up to at least three generations was taken.

Sample collection

A volume of 5 ml peripheral blood was drawn (2.5 ml in ethylenediaminetetraacetic acid [EDTA] and 2.5 ml in serum vials) under aseptic conditions. The blood specimen was given a personal identifier number that was used to link and maintain the biological information derived from cases as well as controls. The study was done in two parts.

Part I

Blood sample (2.5 ml) in EDTA vial was used for DNA isolation using the salting-out method (Sambrook et al., 1989). Genomic DNA was amplified by polymerase chain reaction which was carried out in 25 μl volume and included 80–120 ng/μl DNA, 1X buffer (Thermo), 1.5 mM MgCl2(Thermo), 10 μM DNTPs (Thermo), 0.5 U Taq polymerase (Thermo). 20 pM each of forward and reverse primers were used (Sigma) [Table 1]. The conditions for PCR amplification of VEGF (+936 C/T) were initial denaturation at 95°C for 7 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 10 min. The conditions for PCR amplification of sFlt-1 (+4244G/A, −4771G/T, −523C/G) were initial denaturation at 95°C for 7 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 10 min using a Thermal Cycler (BIO-RAD, California, USA). PCR products were separated by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide (EtBr) staining under UV (Syngene USA Inc.). The amplified products were digested using the different restriction enzymes and incubated overnight at 37°C (restriction fragment length polymorphism). The amplified products of VEGF (+936 C/T) were digested with the enzyme Nla III, whereas for sFlt-1 (+4244G/A), sFlt-1 (−4771G/T), sFlt-1 (−523C/G), Ava II, Taq 1 and BsiHKai restriction enzymes (New England Biolabs) were used. Fragments were separated on 3% agarose gel and visualized by EtBr staining under ultraviolet light.
Table 1: Polymerase chain reaction primer sequences of the polymorphisms

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Part II

Serum was separated from the other 2.5 ml venous blood, aliquoted, and stored at −80°C until analysis. The serum levels of VEGF-A and sFlt-1 proteins were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (R and D Systems Inc., Minneapolis, MN, USA). The optical density was read on ELISA reader at 450 nm (TECAN, Mannedorf, Switzerland).

Statistical analysis

Data were analyzed by STATA 14.0 software (Texas, USA) and Graph Pad Prism 7. Data were presented as mean ± standard deviation, mean ± standard error of mean and median (range) as appropriate. The genotypic frequencies were analyzed/compared using McNemar's test. Protein levels of VEGF and sFlt-1 were analyzed by paired t-test. P < 0.05 was considered statistically significant.


  Results Top


The clinical characteristics of the preeclamptic patients and gestational and maternal age-matched normotensive, nonproteinuric controls are presented in [Table 2].
Table 2: Clinical characteristics of the study participants

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Results of the screening of vascular endothelial growth factor + 936C/T (rs 3025039), sFlt-1 [+4244G/A (rs 722503),-4771G/T (rs 7335588),-523C/G (rs 12584067)] polymorphisms

The analysis of VEGF + 936C/T showed the CC (wild type) genotype to be significantly higher in the controls while the CT (heterozygous) genotype was higher in the patients.

Genotyping of sFlt1 + 4244G/A (rs722503) showed GG (wild type) genotype to be more in controls whereas both GA (heterozygous) and AA (homozygous) genotypes were found to be more in patients while analysis of sFlt-1-4771G/T (rs 7335588) showed GG genotype was found to be more in controls and GT, TT genotypes were found to be more in patients. However, these differences were not statistically significant.

Genotyping of sFlt-1- 523C/G (rs 12584067) showed CC (wild type) genotype was more in controls and CG, GG genotypes were more in patients showing a significant difference.

Reduced vascular endothelial growth factor-A and elevated sFlt-1 levels in preeclamptic patients sera

A decrease in VEGF-A levels in PE patients (198.43 ± 14.63) pg/ml was observed as compared to normotensive, nonproteinuric controls (235.08 ± 16.72) pg/ml.


The sFlt-1 levels in patients' era were 2932.81 (1802.33–5760.46) pg/ml whereas normotensive, nonproteinuric pregnant women had 1114.94 (655.03–2694.35) pg/ml which were significantly different (P< 0.05).

Analysis of vascular endothelial growth factorand sFlt-1 polymorphisms and their levels

Patients with increased frequency of VEGF + 936CT genotype had lower levels of VEGF-A.

sFlt-1 + 4244G/A with the GA, AA genotype, sFlt-1-4771G/T with the GT, TT genotype, sFlt-1-523 C/G with the CG, GG genotype showed increased serum levels of sFlt-1 in patients than controls.


  Discussion Top


VEGF-A promotes angiogenesis, vasodilatation, increases vascular permeability, maintains the integrity of glomerular filtration barrier, and reduces apoptosis.[20],[21] VEGF and its two receptors (VEGFR-1/Flt-1 and VEGFR-2/Flk) play an essential role during placentation. sFlt-1 is the soluble form of VEGF receptor. It acts as a potent antagonist of VEGF-A and PlGF by inhibiting their binding to the intact cell surface receptors and is thus considered an anti-angiogenic factor.[22],[23] Soluble Flt-1 is secreted by endothelial cells, monocytes, and the placenta.[24] A critical balance between angiogenic and anti-angiogenic factors is required for the fine-tuning of vasculogenesis and angiogenesis during pregnancy.[25],[26] Considering the important roles of these two molecules (VEGF and sFlt-1) during pregnancy, the polymorphisms in their genes may be potentially important as genetic markers for susceptibility to the disease. Worldwide studies reported SNPs inVEGF and sFlt-1 genes have been found to be associated with PE.[14],[15],[16],[17],[18],[19] However, there is no data available on the VEGF and sFlt-1 polymorphisms and their association with protein levels in PE mothers of Indian origin. Thus, the aim of the present study was to screen for the presence of VEGF + 936 C/T and sFlt-1 (+4244G/A,-4771G/T,-523C/G) polymorphisms, and to measure the levels of VEGF-A and sFlt-1 proteins and correlate the polymorphisms of VEGF and sFlt-1 with their serum levels in PE patients and healthy controls. The present study indicated a possible association between VEGF + 936 C/T polymorphism and PE. The frequency of CT (heterozygous) being significantly higher among patients than controls [Figure 1], [Table 3], which is similar to the findings of Papazoglou et al.[14] Similarly, Shim et al. also found a higher frequency of CT genotype among Korean women with PE than controls. They reported that the 936 T was more frequent in patients than controls.[15] Although Cunha et al. also suggested that a higher frequency of the T allele of the VEGF + 936C/T polymorphism was observed in patients with PE, the difference was not significant statistically.[16] Cheng et al. also reported the T allele carriers to have an increased risk of developing PE.[17] A recent study conducted by Procopciuc et al. reported that the risk to develop PE was higher in association with the 936 C/T-VEGF heterozygous genotype and increased further in severe PE women who were carriers of the homozygous 936-TT VEGF genotype. They also reported that pregnant women with gestational hypertension and severe PE delivered at a significantly earlier gestational age and neonates having low birth weight if both the preeclamptic mothers and their newborns were carriers of the VEGF-T936 allele.
Figure 1: RFLP electrophoretogram with respect to (a) VEGF+ 936C/T (L1-L8) (b) sFlt-1 + 4244G/A (L1-L5) (c) sFlt-1 - 4771G/T (L1-L4) (d) sFlt-1 - 523C/G (L1-L6) showing the respective genotypes. Lanes (l): samples, M: 50bp DNA ladder, N: 100bp DNA ladder

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Table 3: Distribution and frequency of four loci with susceptibility to preeclampsia (n=50)

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The analysis of sFlt-1 + 4244G/A (rs722503) showed the frequency of GG (wild type) genotype was more in controls, but the frequency of both GA (heterozygous) and AA (homozygous) genotypes was found to be more in patients. Screening of sFlt-1-4771G/T (rs 7335588) showed the GG (wild type) genotype was found to be more in controls. The analysis of sFlt-1- 523C/G (rs 12584067) showed the CC (wild type) genotype to be more in controls, but CG (heterozygous) and GG (homozygous) genotype were more in patients, and the difference was statistically significant [Figure 1], [Table 3]. A case-control study on 606 American pregnant women focusing on 124 tag SNPs in 6 genes found a significant association between sFlt-1- 523C/Gand sFlt-1-4771G/T and PE in black women and sFlt-1 + 4244G/A and PE in white women.[19] Our study on Indian pregnant women showed a similar trend.

We reported lower VEGF-A levels in serum samples of PE patients as compared to controls [Table 4]. Similar to the studies done by Procopciuc et al. and Lyall et al.[18],[27] In contrast, several studies reported increase in VEGF-A levels in the sera of patients.[28],[29],[30] The discrepancy could be due to the type of VEGF-A (free or total) measured. The trapping of VEGF by sFlt-1 reduces the availability of free circulating VEGF, whereas the total VEGF may increase or remain the same. The present study reported significantly higher sFlt-1 levels in the sera of patients as compared to controls [Table 4]. Similar to many previous studies.[31],[32],[33] Inadequate perfusion of the fetoplacental unit causing hypoxia may lead to increase in the production of sFlt-1 by placental trophoblasts, which may later reflect as raised sFlt-1 levels in maternal circulation.[34],[35] The present study points to a possible association of VEGF and sFlT-1 polymorphisms with gene expression leading to altered levels in PE. However, studies on a larger sample size are required to confirm the results.
Table 4: Maternal serum levels of vascular endothelial growth factor-A and soluble fms-like tyrosine kinase-1 in preeclamptic and normotensive, nonproteinuric pregnant women

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  Conclusion Top


Increased frequency of VEGF + 936CT genotype had lower levels of VEGF-A and sFlt-1 + 4244G/A with the GA, AA genotype, sFlt-1-4771G/T with the GT, TT genotype, sFlt-1- 523 C/G with the CG, GG genotype showed increased serum levels of sFlt-1 in patients than controls indicating their (VEGF and sFlt-1) potential role as future biomarkers in the diagnosis of PE. However, association studies on large samples are required to validate the results.

Financial support and sponsorship

This study was supported by Institute Research Grant for Intramural Project, All India Institute of Medical Sciences, New Delhi - 110 029, India.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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