|Year : 2019 | Volume
| Issue : 3 | Page : 117-122
Altered expression of vascular endothelial growth factor, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and Soluble Fms-like Tyrosine Kinase-1 in peripheral blood mononuclear cells from normal and preeclamptic pregnancies
Zaima Ali1, Saba Khaliq2, Saima Zaki3, Hafiz Usman Ahmad2, Khalid Pervaiz Lone2
1 Department of Physiology and Cell Biology, University of Health Sciences; Department of Physiology, Lahore Medical and Dental College, Lahore, Pakistan
2 Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan
3 Department of Obstetrics and Gynecology, Jinnah Hospital, Lahore, Pakistan
|Date of Submission||13-Feb-2019|
|Date of Decision||06-May-2019|
|Date of Acceptance||14-May-2019|
|Date of Web Publication||25-Jun-2019|
Dr. Zaima Ali
Department of Physiology and Cell Biology, University of Health Sciences, Department of Physiology, Lahore Medical and Dental College, Lahore
Source of Support: None, Conflict of Interest: None
Preeclampsia (PE) is the leading cause of maternal and fetal morbidity and mortality. It complicates around 2%–10% pregnancies worldwide due to imbalance between proangiogenic and anti-angiogenic factors, leading to incomplete placentation, ischemia, and endothelial dysfunction. The study was aimed to analyze the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors, i.e., VEGF receptor-1 (VEGFR-1), VEGF receptor-2 (VEGFR-2), and soluble Fms-like tyrosine kinase-1 (sFlt-1) from maternal peripheral blood mononuclear cells (PBMCs) of PE patients. This was a cross-sectional comparative study comprising 18 normotensive and 18 PE patients; the patients were further divided as early-onset preeclampsia (EOP) and late-onset preeclampsia (LOP). The expression level of VEGF, its receptors (VEGFR-1 and VEGFR-2), and sFlt-1 was investigated using real-time polymerase chain reaction. There was a significant change in the mRNA expression with a decrease in VEGF, VEGFR-1, and VEGFR-2 and an increase in sFlt-1 in PBMCs of PE and normal pregnancies (P < 0.001). sFlt-1 mRNA expression was increased by 2.95-fold in the PE group with an inverse correlation with expression of VEGFR-2 (Spearman's rho = 0.68). Based on these findings, we conclude that PE is associated with decrease in the mRNA expression of VEGF, VEGFR-1, and VEGFR-2 as compared to an increase in sFlt-1 in PBMCs.
Keywords: Angiogenesis, normotensive pregnant women, preeclampsia, pregnancy, soluble Fms-like tyrosine, vascular endothelial growth factor
|How to cite this article:|
Ali Z, Khaliq S, Zaki S, Ahmad HU, Lone KP. Altered expression of vascular endothelial growth factor, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and Soluble Fms-like Tyrosine Kinase-1 in peripheral blood mononuclear cells from normal and preeclamptic pregnancies. Chin J Physiol 2019;62:117-22
|How to cite this URL:|
Ali Z, Khaliq S, Zaki S, Ahmad HU, Lone KP. Altered expression of vascular endothelial growth factor, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and Soluble Fms-like Tyrosine Kinase-1 in peripheral blood mononuclear cells from normal and preeclamptic pregnancies. Chin J Physiol [serial online] 2019 [cited 2019 Jul 19];62:117-22. Available from: http://www.cjphysiology.org/text.asp?2019/62/3/117/261309
| Introduction|| |
Preeclampsia (PE), a placenta-induced inflammatory disease, is a multisystem disorder affecting liver, kidneys, and hematological and nervous system causing cerebral edema, seizures, and even maternal death. According to the WHO, a woman in developing countries is seven times more likely to develop PE than a woman in a developed country, and 10%–25% of these cases result in maternal death., PE has been labeled as the “disease of theories” due to its unknown etiology and complex pathophysiology. In the early gestational phase, invasion and neovascularization are the keys to successful placentation. The uterine endometrial vasculature is invaded by extravillous trophoblast to establish local blood flow to maintain oxygen and nutrients supply from mother. In later stages, villous angiogenesis along with vascular maturation is promoted and regulated by several angiogenic and anti-angiogenic molecules. Poor placentation with inadequate cytotrophoblast invasion, resulting in widespread maternal endothelial dysfunction, has emerged as the leading cause for clinical signs and symptoms of PE. Imbalance between these factors disrupts the normal vascular changes that transform maternal vessels to become high capacitance and low resistance vessels. Defective placentation results in hypoxia and oxidative stress with the release of toxic substances in maternal circulation.
For effective angiogenesis, vasculogenesis, and adequate placental development, a balance between vascular endothelial growth factor (VEGF), its receptors, and other factors is crucial., VEGF mediates a critical role in signaling pathways in endothelial cells acting on VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2)/kinase insert domain receptor (KDR)., VEGFR-1, expressed in cytotrophoblast, has low kinase activity as compared to VEGFR-2 which is one of the mediators of signaling cascades in endothelial cell functions., Soluble Fms-like tyrosine kinase-1 (sFlt-1) is a spliced variant of VEGFR-1 which lacks the transmembrane and intracellular domain and binds to the proangiogenic VEGF and placental growth factor (PlGF), decreasing their bioavailability. Animal studies showed that increased levels of anti-angiogenic proteins such as sFlt-1 cause symptoms including proteinuria, hypertension, hematologic abnormalities, cerebral edema, and fetal growth restriction, which are observed in human PE., Due to shallow invasion and abnormal placentation which leads to hypoxia, anti-angiogenic factors such as sFlt-1 are released from placenta and neutralize VEGF and PlGF-mediated signaling, leading to endothelial dysfunction in PE patients. Mechanisms which enhance angiogenesis and vascular remodeling in utero placental unit are not completely understood. Many molecular pathways are considered to be involved in placentation defects like PE; however, among them, VEGF family-mediated angiogenic pathway is known to play a key role.
Most of the studies have measured and compared the mRNA expression of multiple genes in placental tissue,,,,, but a very few have been performed on the blood of PE and normotensive group.,, We hypothesized that mRNA transcript of VEGF family genes, highly expressed in the placenta, is also altered in peripheral blood mononuclear cells (PBMCs) in pregnancies complicated with PE and designed this study to measure and compare the mRNA using real-time polymerase chain reaction (PCR) in PE and normotensive pregnancies.
| Materials and Methods|| |
After approval from the Ethical Review Committee, University of Health Sciences, Lahore (No: UHS/Education/126-16/2754, 27/10/2016), the study was conducted following the guidelines of the Declaration of Helsinki. The samples were collected from Lady Willington Hospital, Lahore, from October 2016 to March 2017 after taking written consent from the participants. The cases included 18 pregnant women (between the ages of 18 and 40 years) in the third trimester (28–40 weeks) diagnosed as PE, and age-matched normal pregnant women at the same gestational age were considered as controls. PE was defined as onset of systolic blood pressure >140 mmHg or diastolic blood pressure ≥90 mmHg at >20 weeks of gestation accompanied by 24 h proteinuria ≥300 mg (≥1+ on dipstick), in at least two random urine samples collected 4–6 h apart. Cases were further divided into early-onset preeclampsia (EOP, 28–32 weeks) and late-onset preeclampsia (LOP, 32.1–40 weeks) groups (9 patients/each subgroup). Women with a history of smoking, diabetes, renal disease, arthritis, inflammatory bowel disease, chronic hypertension, cardiovascular illness (e.g., ischemic heart disease), or other chronic inflammatory disease were excluded. Demographic data along with complete medical, obstetric, and family history were also recorded.
Five milliliters of blood was collected in a EDTA-coated vacutainer; buffy coat was separated and stored at −20°C within an hour of sample collection. RNA was extracted using the FavorPrep Total RNA Isolation Kit (Favorgen, Taiwan) following the manufacturer's instructions. The concentrations of extracted RNA were measured using NanoDrop and stored at −80°C in RNase/DNase-free water.
Quantitative real-time polymerase chain reaction
For each sample, 2 μg of total RNA was used for cDNA synthesis by reverse transcription using RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, USA) following the manufacturer's instructions. The expression of genes was measured for cases and controls by using synthesized cDNA and gene-specific primers for real-time PCR [Table 1], on CFX 96 (BioRad, USA) using 2X SYBR Green master mix (Fermentas, USA) according to the manufacturer's instructions. All reactions were performed in 20 μl of the reaction mixture containing 1 μl of cDNA, 8 μl of 2X SYBR Green Real-Time PCR Master mix, 0.5 μl of forward and reverse primers [Table 1], and RNase-free water (Fermentas, USA). Real-time PCR conditions used were as 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, annealing at 60°C for 30 s, and extension at 70°C for 42 s in a thermal cycle followed by melt curve analysis. Samples were assayed in duplicate along with three housekeeping genes. The 2−ΔΔct method was used for the analysis of relative gene expression.
|Table 1: Sequence of primers used for quantitative real-time polymerase chain reaction|
Click here to view
All the statistical analysis was performed using Statistical Package for the Social Sciences (SPSS), Version 22.0, Armonk, New York, USA. The clinical parameters are expressed as means ± standard deviation. Expression of all genes was normalized against the mean of three housekeeping genes, i.e., glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), β-actin, and 18srRNA. Normality of the data was tested by Shapiro–Wilk test. The mRNA expression was compared using fold change values between two groups by Student's t-test and one-way ANOVA with post hoc Tukey's test for comparison between the multiple groups (EOP, LOP, and normotensive group). Differences in gene expressions were reported as fold change with standard error of the mean. Spearman correlation was used to find the relationship between expression of different genes that were nonnormally distributed and Pearson correlation for normal distributions. A P < 0.05 was considered statistically significant.
| Results|| |
Clinical characteristics of the study population presented in [Table 2] are the same as reported in another article (unpublished data). These patients were part of PhD research project. The samples in both study groups were collected from maternal and gestational age-matched female, so there was no significant difference in PE and normal group. Based on the recruitment criteria, cases had significantly higher systolic and diastolic blood pressure as compared to controls (P < 0.001). There was also a significant difference in mean body mass index of both groups (P < 0.01).
Gene expression analysis
The mRNA expression of VEGF and its receptors (VEGFR-1, VEGFR-2) was significantly decreased in the diseased group, with a more marked decrease of VEGFR-2 (0.52) fold (P < 0.001). Of all the studied genes, only sFlt-1 mRNA expression showed a significant increase (2.95-fold) in the cases (P < 0.001) [Figure 1]. To better understand the differential expression of genes in PE patients, the patients group was further divided into two groups depending upon the time of onset of disease, i.e., EOP and LOP. The expression of VEGF and VEGFR-1 was not different in EOP and LOP (P = 0.17 and 0.48, respectively) but a significant difference was observed in the mRNA expression of VEGFR-2 in the two subgroups of PE (P < 0.001). Regarding sFlt-1, 3.77- and 2.14-fold increase were observed in the EOP and in the LOP, respectively [Figure 2].
|Figure 1: Comparison of peripheral blood mononuclear cells mRNA expression between normotensive group and preeclampsia|
Click here to view
|Figure 2: Comparison of peripheral blood mononuclear cells mRNA expression of normotensive group with early-onset and late-onset preeclampsia|
Click here to view
Inverse correlation of vascular endothelial growth factor receptor-2 with soluble Fms-like tyrosine kinase 1
In addition to relative expression, an inverse correlation was observed with the increase in sFlt-1 and decrease in VEGFR-2 expression (Spearman's rho = 0.68) in blood of PE patients with a P < 0.001 [Figure 3].
|Figure 3: Scatter plot showing inverse correlation of vascular endothelial growth factor receptor-2 with soluble Fms-like tyrosine kinase 1|
Click here to view
| Discussion|| |
To date, a number of studies have been done to probe into the etiology of PE with a focus on angiogenesis because of its importance in normal placentation. Different studies have reported serum levels of VEGF as well as its placental mRNA expression in PE, but the results are controversial. In contrast to these studies, only a few have analyzed the mRNA expression of these factors in blood samples.,,, In the current study, we investigated the extraplacental expression of VEGF and related genes in PBMCs of women suffering from PE. The mRNA expression of VEGF, an angiogenic factor with high biological activity and an important role in normal placentation and fetal development, was significantly decreased in PE compared to normal pregnancy samples in our study (P < 0.01). In contrast, increased placental expression of VEGF has been reported in different studies,,, whereas few studies also found a decrease in expression similar to the current study.,, Our finding is also consistent with different studies where decreased expression of VEGF is reported from peripheral blood samples as well as placental tissue in cases of pregnancy-induced hypertension.,,
VEGFR-1 and VEGFR-2 are two important receptors essential for signaling in endothelial cells. Although both are structurally related and belong to tyrosine kinase receptor family, VEGFR-1 is well-known to have an important role in normal vascular development and exists in two isoforms, one is the soluble VEGFR-1 and the other is a transmembrane isoform. The soluble isoform (sFlt-1), a truncated variant of VEGFR-1, disrupts normal angiogenesis as it binds to both VEGF and PlGF with high affinity. VEGFR-2 is the major mediator of migration, proliferation, and differentiation of endothelial cells. Activation of VEGFR-2 results in an increase in both mitogenic cell signaling and migratory activity of endothelial cells. Differential expression of VEGF receptors in PE is documented in a number of previous studies, with a few findings of an increase in both VEGFR-1 and VEGFR-2 expression,, and others reporting no difference in VEGFR2. This study showed a significant decrease in both VEGFR-1 and VEGFR-2 expression (P < 0.01 and 0.001 respectively) in PE which is consistent with a previous study. Defective placentation in PE leads to hypoxia, one of the factors known to decrease VEGFR-2 expression in the endothelial cells. Furthermore, the low oxygen also leads to increased circulating sFlt-1, a splice variant of VEGFR-1 which lacks the transmembrane and cytoplasmic domains. sFlt-1 antagonizes the proangiogenic effects of both VEGF and PlGF by binding and thus preventing interaction with their receptors. Clinical features of PE are attributed to widespread maternal endothelial dysfunction caused by an increase in sFlt-1, an anti-angiogenic mediator, and the increased extraplacental sFlt-1 may be an additional contributor to the pathology. sFlt-1 protein and mRNA expression have been studied extensively in PE, but the focus has been placental tissue.,, In the current study, the mRNA expression of sFlt-1 in the PBMCs was studied as a source for extraplacental sFlt-1 and a 2.95-fold increased expression in the PE cases was observed compared to normotensive controls. The results are consistent with a previous report by Rajakumar et al. Moreover, a negative correlation with an increase in sFlt-1 and decrease in VEGFR-2 expression in the PE group is in accordance with a previous study by Nevo et al. sFlt-1 is known to bind VEGF leading to decreased availability of VEGF. Decreased availability of free VEGF might have resulted in decreased VEGFR-2 mRNA expression in PE as free VEGF stimulates VEGFR-2 synthesis along with its trafficking at the surface of endothelial cells. In addition to this indirect effect of binding and sequestering VEGF by sFlt-1, it is reported to directly downregulate VEGFR-2 expression and its signaling. Detailed analysis revealed no difference in the expression of VEGF and VEGFR-1 in the two subgroups of PE i.e., EOP and LOP. mRNA expression of sFlt-1 was markedly increased by 3.77-fold in EOP compared to 2.14-fold increase in LOP, indicating its role in severity of PE. The findings are in accordance with Wikström et al., who reported higher levels of sFlt-1 in the plasma of EOP as compared to LOP. EOP usually has a more aggressive course with adverse pregnancy outcomes such as intrauterine growth retardation. In addition, second-trimester Doppler studies of uterine arteries have reported more marked poor placentation in EOP as compared to LOP. Higher levels of sFlt-1 in EOP might contribute to the pathogenesis of this poor placentation because of its anti-angiogenic properties.
| Conclusion|| |
We observed that there is a decrease in mRNA expression of VEGF, VEGFR-1, and VEGFR-2 and increase in sFlt-1 in PBMCs in PE but what is the exact stage of activation of these cells is still to be probed. It is hard to get placental tissue during pregnancy while blood samples are easily accessible and can be studied extensively to understand the pathology of the disease. Extensive studies with serial blood samples during all the three trimesters might help us to answer the question. If future studies confirm our results and do find a change in mRNA expression at early stages, it might be used as a noninvasive tool to diagnose the disease at an early stage.
We are thankful to all our subjects and technical staff of hospitals for their cooperation.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Osungbade KO, Ige OK. Public health perspectives of preeclampsia in developing countries: Implication for health system strengthening. J Pregnancy 2011;2011:481095.
Uzan J, Carbonnel M, Piconne O, Asmar R, Ayoubi JM. Pre-eclampsia: Pathophysiology, diagnosis, and management. Vasc Health Risk Manag 2011;7:467-74.
Ahmed A. New insights into the etiology of preeclampsia: Identification of key elusive factors for the vascular complications. Thromb Res 2011;127 Suppl 3:S72-5.
Young BC, Levine RJ, Karumanchi SA. Pathogenesis of preeclampsia. Annu Rev Pathol 2010;5:173-92.
Can M, Sancar E, Harma M, Guven B, Mungan G, Acikgoz S. Inflammatory markers in preeclamptic patients. Clin Chem Lab Med 2011;49:1469-72.
Kar M. Role of biomarkers in early detection of preeclampsia. J Clin Diagn Res 2014;8:BE01-4.
Tripathi R, Rath G, Jain A, Salhan S. Soluble and membranous vascular endothelial growth factor receptor-1 in pregnancies complicated by pre-eclampsia. Ann Anat 2008;190:477-89.
Jia H, Bagherzadeh A, Bicknell R, Duchen MR, Liu D, Zachary I. Vascular endothelial growth factor (VEGF)-D and VEGF-A differentially regulate KDR-mediated signaling and biological function in vascular endothelial cells. J Biol Chem 2004;279:36148-57.
Zhou Y, McMaster M, Woo K, Janatpour M, Perry J, Karpanen T, et al.
Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol 2002;160:1405-23.
Gille H, Kowalski J, Li B, LeCouter J, Moffat B, Zioncheck TF, et al.
Analysis of biological effects and signaling properties of flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem 2001;276:3222-30.
Takahashi H, Shibuya M. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci (Lond) 2005;109:227-41.
Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al.
Circulating angiogenic factors and the risk of preeclampsia. N
Engl J Med 2004;350:672-83.
Lu F, Longo M, Tamayo E, Maner W, Al-Hendy A, Anderson GD, et al.
The effect of over-expression of sFlt-1 on blood pressure and the occurrence of other manifestations of preeclampsia in unrestrained conscious pregnant mice. Am J Obstet Gynecol 2007;196:396.e1-7.
Furuya M, Kurasawa K, Nagahama K, Kawachi K, Nozawa A, Takahashi T, et al.
Disrupted balance of angiogenic and antiangiogenic signalings in preeclampsia. J Pregnancy 2011;2011:123717.
Andraweera PH, Dekker GA, Laurence JA, Roberts CT. Placental expression of VEGF family mRNA in adverse pregnancy outcomes. Placenta 2012;33:467-72.
Kim SC, Park MJ, Joo BS, Joo JK, Suh DS, Lee KS. Decreased expressions of vascular endothelial growth factor and visfatin in the placental bed of pregnancies complicated by preeclampsia. J Obstet Gynaecol Res 2012;38:665-73.
Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al.
Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649-58.
Farina A, Zucchini C, Sekizawa A, Purwosunu Y, de Sanctis P, Santarsiero G, et al.
Performance of messenger RNAs circulating in maternal blood in the prediction of preeclampsia at 10-14 weeks. Am J Obstet Gynecol 2010;203:575.e1-7.
Paiva P, Whitehead C, Saglam B, Palmer K, Tong S. Measurement of mRNA transcripts of very high placental expression in maternal blood as biomarkers of preeclampsia. J Clin Endocrinol Metab 2011;96:E1807-15.
Okazaki S, Sekizawa A, Purwosunu Y, Farina A, Wibowo N, Okai T. Placenta-derived, cellular messenger RNA expression in the maternal blood of preeclamptic women. Obstet Gynecol 2007;110:1130-6.
Ren Y, Wang H, Qin H, Yang J, Wang Y, Jiang S, et al.
Vascular endothelial growth factor expression in peripheral blood of patients with pregnancy induced hypertension syndrome and its clinical significance. Pak J Med Sci 2014;30:634-7.
Zhou Q, Liu H, Qiao F, Wu Y, Xu J. VEGF deficit is involved in endothelium dysfunction in preeclampsia. J Huazhong Univ Sci Technolog Med Sci 2010;30:370-4.
Chung JY, Song Y, Wang Y, Magness RR, Zheng J. Differential expression of vascular endothelial growth factor (VEGF), endocrine gland derived-VEGF, and VEGF receptors in human placentas from normal and preeclamptic pregnancies. J Clin Endocrinol Metab 2004;89:2484-90.
Escudero C, Celis C, Saez T, San Martin S, Valenzuela FJ, Aguayo C, et al.
Increased placental angiogenesis in late and early onset pre-eclampsia is associated with differential activation of vascular endothelial growth factor receptor 2. Placenta 2014;35:207-15.
Hong F, Li Y, Xu Y. Decreased placental miR-126 expression and vascular endothelial growth factor levels in patients with pre-eclampsia. J Int Med Res 2014;42:1243-51.
Helske S, Vuorela P, Carpén O, Hornig C, Weich H, Halmesmäki E. Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies. Mol Hum Reprod 2001;7:205-10.
Munaut C, Lorquet S, Pequeux C, Coulon C, Le Goarant J, Chantraine F, et al.
Differential expression of vegfr-2 and its soluble form in preeclampsia. PLoS One 2012;7:e33475.
Nevo O, Lee DK, Caniggia I. Attenuation of VEGFR-2 expression by sFlt-1 and low oxygen in human placenta. PLoS One 2013;8:e81176.
Olszewska-Pazdrak B, Hein TW, Olszewska P, Carney DH. Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells. Am J Physiol Cell Physiol 2009;296:C1162-70.
Rajakumar A, Michael HM, Rajakumar PA, Shibata E, Hubel CA, Karumanchi SA, et al.
Extra-placental expression of vascular endothelial growth factor receptor-1, (Flt-1) and soluble flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women. Placenta 2005;26:563-73.
Wikström AK, Larsson A, Eriksson UJ, Nash P, Nordén-Lindeberg S, Olovsson M. Placental growth factor and soluble FMS-like tyrosine kinase-1 in early-onset and late-onset preeclampsia. Obstet Gynecol 2007;109:1368-74.
Odegård RA, Vatten LJ, Nilsen ST, Salvesen KA, Austgulen R. Preeclampsia and fetal growth. Obstet Gynecol 2000;96:950-5.
Aardema MW, Saro MC, Lander M, De Wolf BT, Oosterhof H, Aarnoudse JG. Second trimester Doppler ultrasound screening of the uterine arteries differentiates between subsequent normal and poor outcomes of hypertensive pregnancy: Two different pathophysiological entities? Clin Sci (Lond) 2004;106:377-82.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]