Concentration of free vascular endothelial growth

Journal of Maternal-Fetal and Neonatal Medicine, 2012; Early Online: 1–5 © 2012 Informa UK, Ltd. ISSN 1476-7058 print/ISSN 1476-4954 online DOI: 10.3109/14767058.2012.667462

ORIGINAL PAPER

Concentration of free vascular endothelial growth factor and its soluble receptor, sFlt-1 in the maternal and fetal circulations of normal term pregnancies at high and low altitudes J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by JHU John Hopkins University on 06/19/12 For personal use only.

Kamal Z.M. Ali1, Graham J. Burton2, Ali M. Al-BinAli3, Mamdoh A. Eskandar4, Awad A. El-Mekki5, Riyad A. Moosa5, Salah A. Abd-Alla5, Ali G.A. Salih1, Abulqasim M.B. Sideeg1 & Ahmed A. Mahfouz6 1Department of Anatomy, College of Medicine, King Khalid University, Abha, Saudi Arabia, 2Centre for Trophoblast Research,

University of Cambridge, Cambridge, UK, 3Department of Pediatrics, College of Medicine, King Khalid University, Abha, Saudi Arabia,

4Department of Obstetrics and Gynaecology, College of Medicine, King Khalid University, Abha, Saudi Arabia, 5Department of

Microbiology and Immunology, College of Medicine, King Khalid University, Abha, Saudi Arabia, and 6Department of Community Medicine, College of Medicine, King Khalid University, Abha, Saudi Arabia In the adult, angiogenesis is also essential during pregnancy [1]. Vascular endothelial growth factor (VEGF-A, also called VEGF or vascular permeability factor) is the most important regulator of blood vessel formation in health and disease. It is essential for embryonic vasculogenesis and angiogenesis, and is a key mediator of neovascularization in disease states [1,5]. VEGF is acutely regulated by hypoxia at both the transcriptional level and translational levels [6,7]. Bioavailability of VEGF is further modulated by binding to a soluble form of one of its receptors, VEGFR-1or sFlt-1. Therefore, it is important to distinguish between the free and total forms of VEGF, as the latter may give a false impression of bioactivity. VEGF binding to sFlt-1 reduces its angiogenic potency [8,9], whereas free VEGF is clinically important in mediating angiogenesis [10]. Hence, the balance between VEGF and sFlt-1 is critical. Long-term exposure of the placental tissues to hypoxic conditions results in a pro-angiogenic state, with hypercapillarization of the organ [11,12]. In pregnancy at high altitude, hypobaric hypoxia stimulates expression of VEGF [13], which in turn stimulates angiogenesis and leads to an increase in vessel profile number and vascular volume, surface area and permeability [14–18]. However, hypoxia also stimulates expression of sFlt-1, which thereby potentially reduces the bioavailability of VEGF and its angiogenic potency [8,19]. All these studies relate to changes in the maternal circulation, and little is known about changes in the levels of VEGF and sFlt-1 in the fetal circulation. In a study comparing the serum levels of VEGF in males and females throughout their life cycle, serum levels of VEGF in cord blood (fetuses) and neonatal blood were significantly higher compared with those of adults [20]. However, these studies did not indicate clearly whether they measured the total or free VEGF. In our recently published study, we measured free VEGF, and found it to be undetectable in maternal samples, but present in the cord blood of babies born at high altitude [21]. We speculated that these levels would be beneficial for both mother and the baby, but did not have the opportunity to investigate levels of sFlt-1. Therefore, the purpose of this study was to measure the levels of free plasma VEGF and sFlt-1 in the maternal and the cord bloods, and to determine whether these are affected by exposure to chronic hypoxia at high altitude.

Objective: Vascular endothelial growth factor (VEGF) is regulated by hypoxia that is essential for placental development. It is antagonized by a soluble form of its receptor (sFlt-1). The purpose of this study was to measure these factors in the maternal and the cord bloods, at low and high altitude. Methods: Samples were collected from full term births normal pregnant women. Free (unbound) VEGF and sFlt-1 levels were measured in plasma samples from cord and maternal blood for each subject by enzyme-linked immunosorbent assay (ELISA) using commercially available kits from R&D systems, UK (Cat # DVE00 and Cat # SVR100B, respectively). Results: At high altitude, the average maternal free VEGF in pg/ml was significantly (p < 0.001) lower than that of the cord level (71.30 ± 282.14 and 431.35 ± 424.31, respectively). On the other hand, the average maternal sFlt-1 was significantly (p < 0.001) higher than that of the cord level (8205.41 ± 6244.72 and 1811.74 + 3469.30, respectively). At low altitude, the average maternal free VEGF was significantly lower than that of the cord level (0.47 ± 0.89 and 483.44 ± 457.31, respectively, p < 0.001). On the other hand, the average maternal sFlt-1 was significantly higher than that of the cord level (9267.82 ± 6345.68 and 958.66 ± 1359.92, respectively, p < 0.001). There were no significant differences by altitude. Conclusion: Secretion of sFlt-1 appears to be polarized, in that concentrations are higher in the maternal compartment than on the fetal side at both high and low altitudes. This may be a normal physiological phenomenon to permit angiogenesis in the placenta and fetus while protecting the mother. Chronic exposure to hypobaric hypoxia at high altitude does not affect these distributions. Keywords: altitude, cord blood, maternal blood, VEGF and sFlt-1

Introduction The formation of a vascular system is an essential requirement during embryogenesis and involves two basic processes: vasculogenesis, defined as the differentiation of endothelial cell progenitors into the primary capillary plexus, and angiogenesis, the formation of new blood vessels from pre-existing vessels [1–4].

Correspondence: Dr. Kamal Z.M. Ali, Department of Anatomy, College of Medicine, King Khalid University, P.O. Box 641, Abha, Saudi Arabia. E-mail: [email protected] (Received 25 October 2011; revised 09 December 2011; accepted 14 February 2012)

1

2 K. Z. M. Ali et al. We hypothesized that levels of free plasma VEGF would be increased and those of the soluble receptor, sFlt-1, decreased in the cord blood in comparison to the maternal blood at both low and high altitude.

J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by JHU John Hopkins University on 06/19/12 For personal use only.

Material and methods 69 normal term pregnancies of Saudi pregnant women, living permanently in Abha City (elevation 3000 m) and Muhail City (elevation 500 m) in Southwestern Saudi Arabia were randomly selected from Abha Maternity Hospital and Muhail General Hospital and included in the present study. Informed consent was obtained from all mothers, after ethical approval of the Research Ethics Committee at the College of Medicine, King Khalid University. Sixty-nine blood samples were collected; 45 of maternal and cord blood from Abha City, and 24 from Muhail City. All samples were from full-term pregnancies uncomplicated by congenital anomalies, diabetes mellitus, multiple gestations, preeclampsia, or small for gestational age. Birth weights, and placenta weights and sizes were measured and recorded immediately after normal vaginal delivery. Maternal blood was collected immediately after delivery by vein puncture into K3EDTA vacutainers, and centrifuged at 1000 × g for 15 min within 30 min of collection. Plasma was collected and aliquoted into cryogenic freezing vials and stored at −80°C until used. The levels of VEGF were determined using an enzyme-linked immunosorbent assay (ELISA) kit commercially available from R&D systems, UK (Cat # DVE00). Briefly, 100 µl of assay diluent was added to all microtitre plate wells, then maternal and cord plasma samples along with standards were added to assigned wells and incubated for 2 h at room temperature. After that the plates were washed three times using automatic ELISA plate washer (Stat fax -2600, Awareness Technology Inc. Miami, FL). Then, 200 µl of polyclonal antibody against VEGF conjugated to horseradish peroxidase was added to all wells and incubated for 2 h at room temperature. After washing the plates, 200 µl of substrate solution was added to all wells, and the reaction was stopped after 25 min by addition of 50 µl of 2N sulphuric acid. The VEGF levels were determined using a standard curve generated by ELISA plate reader (Humareader, Germany).

The sFlt-1 levels were determined in a similar fashion using an ELISA kit commercially available from R&D systems, UK (Cat # SVR100B). The VEGF kit from R&D systems has been shown to measure only VEGF not bound to sFlt-1 [22–24]. Literature from R&D systems in regard to VEGF ELISA kit DVE-00 reports no interference with sFlt-1 until greater than 1.250 pg/ml. Previous reports using this kit have referred to plasma VEGF measured using this method as “free VEGF [10,24,25]”. Therefore, we refer to circulating VEGF measured by this ELISA kit as free plasma VEGF. The ELISA for sFlt-1 measures total plasma sFlt-1, both that which is bound to VEGF and unbound. Statistical analysis was performed using an unpaired ‘t’ test to compare the clinical data between the two altitude levels. Mann– Whitney and Wilcocxon signed rank non-parametric tests and a two-way analysis of variance (two-way ANOVA) were used to test for differences in the levels of free plasma VEGF and of sFLT, with cord/maternal blood and low/high altitude as the principal effects. Correlations between the values of free VEGF and sFLT were tested for using Pearson’s correlation coefficient. A value of p < 0.05 was considered significant.

Results Clinical data Clinical data pertaining to the pregnancies are presented in Table I. As can be seen, there was no difference in the mean age of the mothers or the birth weight. None of the study sample was diagnosed or receiving any treatment for hypertension and/or diabetes mellitus (GDM or for type 2 diabetes). Placental weight and volume were both significantly increased at high altitude, but there was no significant difference in the placenta/fetal weight ratio at birth. Gestational age at delivery was increased at high altitude, but there was no significant difference between the percentage of male and female newborns. Free plasma VEGF and sFlt-1 level The concentrations of both free plasma VEGF and sFlt-1 for both maternal and cord blood at high and low altitudes are presented in Table II, and Figures 1–3.

Table I. Clinical Data of the Study sample of normal pregnant women at high and low altitude areas in Southwestern Saudi Arabia. Altitude N Mean ± s.d. t Maternal age (years) High altitude 42 28.21 ± 6.8 0.868 Low altitude 20 26.7 ± 5.5 Birth weight (kg) High altitude 38 3.097 ± 0.41 1.45 Low altitude 24 2.933 ± 0.46 Placental weight (g) High altitude 43 437.72 ± 85.4 2.736 Low altitude 24 377.46 ± 88.4 Placenta/Fetal weight ratio: High altitude 38 0.134 + 0.026 1.651 Low altitude 24 0.131 + 0.032 Placental volume High altitude 43 397.53 ± 86.7 3.455 Low altitude 24 320.754 ± 88.9 Gestational age (weeks) High altitude 41 39.27 ± 1.05 3.495 Low altitude 23 38.39 ± 0.8

PSig.(2-tailed) 0.389 0.151 0.008 0.104 0.001 0.001

Systolic blood pressure of both groups ranged from 110 to 120 mmHg. Diastolic blood pressure of both groups ranged from 70 to 80 mmHg.

Journal of Maternal-Fetal and Neonatal Medicine

VEGF and sFlt-1 in the maternal and fetal circulations 3


Table II. Free plasma VEGF and sFlt-1 levels of the study sample of normal pregnant women at high and low altitude areas in Southwestern Saudi Arabia. Data High altitude (n = 45) Low altitude (n = 24) Mann–Whitney (P) Free plasma VEGF (pg/ml): mean ± s.d.: Maternal 71.3 ± 282.1 0.5 ± 0.9 0.101 Cord 431.4 ± 424.3 483.4 ± 457.3 0.753 Wilcoxon Signed Rank (P) 0.001 0.001 Free plasma sFlt-1 (pg/ml): mean ± s.d.: Maternal 8205.4 ± 6244.7 9267.8 ± 6345.7 0.262 Cord 1811.7 ± 3469.3 958.6 ± 1359.9 0.450 Wilcoxon Signed Rank (P) 0.001 0.001

Comparison of maternal and cord levels Table II shows that at high altitude, the mean value for maternal free VEGF level (71.30 ± 282.14 pg/ml) was significantly (p = 0.001) lower than that of the cord level (431.35 ± 424.31 pg/ ml). Similarly, at low altitude the maternal value of free VEGF is significantly lower than the cord value. On the other hand at high altitude, the mean value for maternal sFlt-1 level (8205.4 ± 6244.7 pg/ml) was significantly (p = 0.001) higher than that of the cord level (1811.7 ± 3469.3 pg/ml). Similarly, at low altitude the maternal value of sFlt-1 was significantly higher than the cord value.

Figure 1. Free vascular endothelial growth factor (VEGF) concentration in maternal and cord plasma at high altitude (n = 45) and low altitude areas (n = 24) of Aseer Region, Southwestern Saudi Arabia.

Correlation of VEGF and sFlt-1 Overall, the maternal values of free VEGF were significantly (p = 0.027) inversely correlated (r = −0.266) with values of sFlt-1. The scatter plot shown in Figure 3 reveals that even low concentrations of sFLT are able to bind and inactive plasma VEGF. Similarly, the cord values of free VEGF were significantly (p = 0.017) inversely correlated (r = −0.286) with cord values of sFlt-1. Measuring the effect of altitude and maternal cord relation on VEGF and sFlt-1 level In a two-way independent analysis of variance, there was a significant main effect of maternal-cord relation on levels of free VEGF (F = 59.9, p = 0.001). On the other hand the effect of altitude was not significant (F = 0.028, p = 0.867). Similarly in a two-way independent ANOVA, there was a significant main effect of maternal-cord relation on levels of sFlt-1 (F = 70.5, p = 0.001). On the other hand the effect of altitude was not significant (F = 0.014, p = 0.905).

Discussion

Figure 2. Soluble vascular endothelial growth factor receptor (sFlt1) concentration in maternal and cord plasma at high altitude (n = 45) and low altitude areas (n = 24) of Aseer Region, Southwestern Saudi Arabia.

Comparison of low altitude and high altitude levels Table II shows that the cord free plasma VEGF at high altitude (431.4 ± 424.3 pg/ml) was not statistically different (p = 0.638) from that at low altitude (483.4 ± 457.3 pg/ml). Similarly, there were no significant differences between the high and low altitude values of free maternal VEGF, maternal sFlt-1 and cord sFlt-1. Copyright © 2012 Informa UK, Ltd.

The present study has shown that the concentration of free VEGF is significantly higher in the cord blood than in maternal blood at the end of normal pregnancy at both altitudinal levels, whereas the reverse is true for the antagonistic soluble receptor, sFlt-1. Although there is considerable individual variation in the values of free VEGF and sFlt-1, a strong negative correlation exists between their levels at both altitudes. Hence, the presence of this receptor appears to be the principal regulator of VEGF bioactivity. Altitude did not have a significant effect on the concentrations of either free VEGF or sFlt-1, although there was a trend for higher concentrations of free VEGF in the maternal blood at high altitude. This most probably reflects the high variability in concentrations between individuals. These findings are consistent with previous studies [20,21], and indicate that the soluble receptor must be secreted in a highly polarized fashion into the maternal blood rather than into the fetal and placental compartments. VEGF is an essential growth factor for vasculogenesis and angiogenesis. Equally, development of the placental and fetal vascular beds is a key process, and


4 K. Z. M. Ali et al.

Figure 3. Scatter plot of soluble vascular endothelial growth factor receptor (sFlt1) concentration in maternal and cord plasma against free vascular endothelial growth factor (VEGF) concentration in maternal and cord plasma at high altitude and low altitude areas of Aseer Region, Southwestern Saudi Arabia.

deletion of even one allele for VEGF in the mouse is embryonic lethal [26]. The human placenta is a highly vascular organ, and by the end of gestation it has developed a large surface area of capillary network [27] that is essential for effective materno-fetal exchange. Therefore, it is logical that the trophoblast should produce VEGF to stimulate placental angiogenesis. However, this poses a danger to the mother if concentrations rise too high in her circulation, and stimulate excessive neovascularisation. Secretion of sFLT-1 by the trophoblast in a polarized fashion may resolve these conflicting demands. This was hypothesized by Clark et al. [28], but, to our knowledge, the current study provides the first proof that it does take place. Both villous and extravillous trophoblast contain very high levels of sFlt-1 [29,30]. In the placenta, the villi are in direct contact with maternal blood, and hence sFlt-1 is detectable in the peripheral maternal circulation [29]. Moreover, it makes sense for sflt-1 to be higher in maternal blood because it is released from the syncytiotrophoblast surface, and is thus, for example, released from explanted placental villi into the media [31]. Consequently, at low altitude bioactive free VEGF is almost undetectable in maternal blood, whereas high levels are present within the fetal circulation at delivery. This finding is consistent with quantitative studies that have revealed that the development of the placental capillary network increases continuously until term [32]. Concerning our findings at high altitude, it might be expected that the concentrations of VEGF and sFLT-1 will be increased, as they are both regulated by hypoxia. However, it has previously been reported that the concentration of free VEGF is low in the maternal circulation in severe hypobaric hypoxia of high altitude [33]. This can be explained by the high concentration of sFlt-1, which is also secreted by the trophoblast and is also stimulated at high altitude [34] or when trophoblast cells are cultured under hypoxic conditions in vitro [35]. Moreover, an in vitro study has shown that VEGF and sFLT are regulated by hypoxia (1% O2 [36]), which is a more severe insult than the hypobaric hypoxia experienced in vivo by the women in the present study. This milder stimulus, together with the variability in our data, could explain why we found altitude had no significant effect on the concentrations of either factor. Nonetheless, there was a trend toward an

increase in free VEGF at 3000 m, and it is possible that elevated concentrations of free VEGF could account for the increased incidence of placental chorangiomas that has been reported at more extreme altitudes [17,37]. Regardless of the effect of altitude, it is clear that the balance of pro-angiogenic and anti-angiogenic factors must be carefully regulated during pregnancy [38]. On the placental side, excessive stimulation may lead to chorangioma, whereas inadequate stimulation can lead to hypovascularisation and terminal villus deficiency [39]. On the fetal side, the level of free VEGF must also be carefully controlled, and disturbance may lead to intrauterine growth restriction [40]. On the maternal side, the concentration of sFlt-1 in the maternal blood increases steadily during normal pregnancy toward term [41]. Recently, excessive placental secretion of sFLT-1 has been associated with the pathophysiology of pre-eclampsia [25]. By binding VEGF and reducing its bioavailability, sFlt-1 sensitizes endothelial cells to pro-inflammatory cytokines that are also released by the placenta in these cases [42]. As a result, the endothelial cells become activated, leading to hypertension and proteinuria.

Conclusions The detection of a high concentration of free plasma VEGF and a low concentration of sFlt-1 in the umbilical circulation is a normal physiological phenomenon, regardless of the altitude, which is necessary to promote angiogenesis in the placenta and fetus. On the other hand, the low level of free VEGF on the maternal side is likely a physiological protective mechanism against neovascularisation. Hypobaric hypoxia of high altitude does not play a major role in these distributions.

Acknowledgement K.Z.M. conceived of the study, its design and coordination and draft the manuscript and was ultimately responsible for this work., K.Z.M., G.B., A.M., M.A., A.A. have made substantial contributions to analysis and interpretation of data, R.A., S.A., A.G.A., A.M.B. collected samples, results and carried out the laboratory procedures, A.A. Mahfouz carried out the statistical analysis of the data and G.B. have given final approval of the version to be published. All authors read and approved the final manuscript. Declaration of interest: The authors report no conflict of interest.

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