Vascular Endothelial Growth Factor and its Receptors in the Placenta of Gravidas with Obesity

 

Ekaterina Borovkova¹*, Alexander Schegolev²

¹Department of Obstetrics and Gynecology, Pirogov Russian National Research Medical University Moscow, Russia

²The anatomic separation Department, Federal State Budget Institution Research Center for Obstetrics, Gynecology and Perinatology V. I. Kulakov, Moscow, Russia

 

ABSTRACT:

The paper describes a comparative morphological study of placentas obtained from women with obesity and normal body weight.

The purpose of this research was to carry out immunohistochemical study VEGF and its receptors (VEGFR-1, VEGFR-2 and VEGFR-3) in the placenta of gravidas with obesity.

The study is based on the morphological analysis of 20 secundines after the timely delivery (39-40 weeks). The main group consisted of 14 women (aged 29-40), suffering from alimentary-constitutional obesity. The control group consisted of 6 patients (aged 23-37) with normal body weight and physiological pregnancy. In the main group, histological study of the placenta preparations, stained with hematoxylin and eosin, revealed that maturation degree of the villous tree corresponded to gestational age, along with moderately expressed compensatory-adaptive and involution-degenerative processes.

The identified features of immunohistochemical distribution of VEGF expression and its receptor in placental tissue of women suffering from obesity, to a certain extent, reflect the processes related to compensation and abnormal functioning of the mother-placenta-fetus system in the presence of hypoxia and hyperlipidemia.

Research results indicate unavoidable development of morphological changes in the placenta during pregnancy against the background of obesity. Decrease in body weight before pregnancy can reduce the risk of placental disorders and improve perinatal outcomes.

 

 

 

INTRODUCTION:

The paper describes a comparative morphological study of placentas obtained from women with obesity and normal body weight. Obesity is regarded as a significant risk factor of pregnancy complications and adverse perinatal outcomes^1-2.The prevalence of obesity among pregnant women in Western European countries and the USA ranges from 6 to 28%³. Obesity is considered an independent risk factor of hypertensive disorders during pregnancy.

 

The results of 13 cohort multicentre studies that included 1.4 million women showed that the risk of preeclampsia in obese patients doubled with each 5-7 kg/m²of excess body weight¹. The preeclampsia development mechanism has not been fully studied yet, although the pathogenesis in obese patients is significantly affected by insulin resistance and endothelial dysfunction in the setting of dyslipidaemia and disruption of cytotrophoblast invasion³.

 

The results of a meta-analysis that included data from nine controlled studies on the establishment of a connection between maternal obesity and risk of stillbirth showed that overweight and obese women had a significantly higher than average rate of intra- and antenatal foetal death: 1.5 times higher in overweight women (OR 1.47, 95% CI 1.08-1.94) and 2 times higher in obese women (OR 2.07, 95% CI 1.59-2.74). The frequency of operative delivery is higher in such patients, with a direct correlation between the degree of obesity and the caesarean section and perinatal outcome rate^4-5.

 

The discovery of growth factors (GF) showed a new direction in the study of angiogenesis peculiarities, in obstetrics in particular. GF are cellular polypeptides capable of stimulating or suppressing tissue growth, including that of blood vessels and the glandular tissue⁶.The growth, development, and regression of blood vessels are key mechanisms of reproductive processes. It has been proven that the female reproductive cycle (menstruation, ovulation, implantation, and placentation) is based on the formation of new vessels: vasculogenesis – the de novo formation of blood vessels in the foetus and angiogenesis – the development of new vessels from existing ones. Both processes are the foundation for the formation of the placental and foetal vascular system⁷. Angiogenesis takes place through differentiation, proliferation, and migration of endothelial cells, which is induced by various specific growth factors and intercellular interplay. In microvasculature vessels, the walls whereof are devoid of the smooth-muscle layer, the key interplay takes place between endotheliocytes and the extracellular matrix. A wide range of growth factors that stimulate or inhibit angiogenesis has been studied; the most important ones are part of the vascular endothelial growth factor (VEGF) and fibroblast growth factor family. Studies conducted over the last 10 years have proven the key role of VEGF in angiogenesis regulation. This cysteine-residue-rich homodimer is part of the VEGF superfamily of growth factors and is produced by various types of cells; it increases vascular permeability and is the main chemotactic and mitogenic stimulus for endotheliocytes⁶.

 

MATERIALS AND METHOD:

The study is based on the morphological analysis of 20 secundines after the timely delivery (39-40 weeks). The main group consisted of 14 women (aged 29-40), suffering from alimentary-constitutional obesity. The control group consisted of 6 patients (aged 23-37) with normal body weight and physiological pregnancy. In the planning phase, the study was approved by the Ethics Committee of I.M. Sechenov First Moscow Medical University.

 

Before the study, all patients were informed about research objectives and methods of operation, signed informed consent and confidentiality obligation. Patients had the right to withdraw from the study at any stage. After the macroscopic examination of secundines, tissue fragments were cut from the central zone of placenta and fixed in 10% neutral formalin. Histological examination was performed on paraffin sections stained with hematoxylin and eosin. The expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1, VEGFR-2 and VEGFR-3) was determined by immunohistochemical tests. Immunohistochemical studies were performed by the standard method using ready-to-use rabbit polyclonal antibodies to VEGF, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), VEGFR-3 (Flt-4), mouse antibodies to CD34 (clone QBEnd / 10) (and the polymer detecting system produced by Spring Bioscience (rabbit anti-human VEGF polyclonal antibody (E2611); rabbit anti-human Flt-1 / VEGFR1 polyclonal antibody (E2801); rabbit anti-human FLK-1 / KDR / VEGFR2 polyclonal antibody (E3711); rabbit anti-human FLT-4 / VEGFR3 polyclonal antibody (E3871); mouse anti-human CD34 (Endothelial Cell Marker) monoclonal antibody (Clone QBEnd / 10) (E1281).

 

Preliminary antigen unmasking was performed by boiling the samples in a citrate buffer solution with pH 6.0. Endogenous peroxidase was blocked by treatment of sections with 0.3% hydrogen peroxide solution for 15 minutes. Hematoxylin was used as the background coloring material. The character of  immunohistochemical responses to VEGF and its receptors were evaluated in syncytiotrophoblast (STB), extravillous cytotrophoblast (EVC), endotheliocytes of stem villi vessels (ESV), as well as terminal and mature intermediate villi (ETV) using a semi-quantitative scale (lack of reaction - 0, weak reaction - 1 point, moderate reaction – 2 points and the expressed reaction – 3 points). Morphometric analysis of the preparations was performed using image analysis system based on "Axio Imager M1» microscope with AxioVision software (Carl Zeiss, Germany).

 

The authors determined the area and perimeter of the terminal and mature intermediate villi, their capillaries, as well as the number of capillaries and the relative intervillous area. The obtained morphometric parameters were used to calculate villi vascularization exponent as the ratio of the total area of the villus capillaries to the cross-sectional villus area (%). Statistical appraisal of quantitative data was carried out by using “Statistica 6.0” (StatSoft, USA) software package.

 

RESULTS:

In the main group, histological study of the placenta preparations, stained with hematoxylin and eosin, revealed that maturation degree of the villous tree corresponded to gestational age, along with moderately expressed compensatory-adaptive and involution-degenerative processes. In the group of gravidas with obesity, 5 placentas showed correspondence between the villous tree development and gestational age, 9 placentas showed retardation of the villous tree development from gestation by 2-4 weeks. Thus in all cases, placentas of gravidas from the main group showed a pronounced stromal villi swelling of varying severity (Figure 1 a) and a wide intervillous space (Figure 1 b). The villous tree was largely represented by villi having large diameter with a small number of capillaries, while most villi were cut in the longitudinal section. These changes in the placentas of women with obesity show prevalence of angiogenesis without vascular branching.

 

Figure 1.а. Pronounced villi swelling in the placenta of patients with obesity. H and E stain, x200; b. Expansion of intervillous space in the placenta of patients with obesity. H and E stain, x100; c. VEGF expression in placental syncytiotrophoblast of patients with normal body weight; immunoperoxidase method, x 400; d. VEGF expression decrease in placental syncytiotrophoblast of patients with obesity; immunoperoxidase method, x 400;e. Pronounced VEGF expression in the cytoplasm of extravillous trophoblast in the placenta of women with obesity; immunoperoxidase method, x 400; f. Moderate VEGF expression in endotheliocytes of stem villus vessels in women with obesity; immunoperoxidase method, x400

 

The morphometric analysis of preparations showed that the area of intervillous space in the placentas of women with obesity exceeds the normal value by 75.7% (p < 0.05)  (Table. 1).

 

Immunohistochemical analysis of VEGF in normal placentas found that the maximum level of expression was observed in the villous syncytiotrophoblast (Figure 1). In addition, cytoplasmic staining was observed as well. The decrease in VEGF reaction intensity in syncytiotrophoblast was detected in   placentas obtained from women with obesity (Figure 1d), in endothelium of stem villi vessels (by 9% and 16.8%, respectively); higher levels of its expression was found in extravillous cytotrophoblast (Figures 1 e, 1f) and in endotheliocytes of capillaries in terminal and mature intermediate villi (15.2% and 38.0% respectively, p <0.05). As a result, maximum VEGF expression in placentas obtained from the main group of patients was found in the elements of extravillous cytotrophoblast. The most intense VEGFR-1 and VEGFR-3 reactions were found in extravillous cytotrophoblast and the most intense VEGFR-2 reaction was observed in the vascular endothelium of stem villi vessels. Minimum expression levels for all three receptors were determined in the vascular endothelium of terminal and mature intermediate villi (Figure 2 a, b, c). The placentas of gravidas with obesity showed reduced expression of VEGFR-1 and increase in VEGFR-2 and VEGFR-3. Thus, the intensity of VEGFR-1 reaction in endotheliocytes of stem and terminal villi vessels was below the normal values by 43% and 28.3% (p <0.05). At that, this intensity in syncytiotrophoblast elements and in extravillous cytotrophoblast was below normal values only by 2.7% and 2.8% (p> 0.05). In contrast, VEGFR-2 expression in the endothelium of villous capillaries exceeds the benchmarks by more than four times (p < 0.01) (Figure 2 b, c.), in the endothelium of stem villi - by 28.7% and in the extravillous trophoblast cells – by 55.3% (p < 0.05) (Figure 2 d,e). The changes in VEGFR-3 expression were less pronounced. The intensity of its reaction in syncytiotrophoblast exceeded the normal level by 33.2% (p < 0.05) (Figure 2 f), in endotheliocytes of the stem villi - by 30% (p < 0.05), and in extravillous cytotrophoblast the intensity was below the normal level by 2,8% (p > 0.05).

 

Тable 1: Morphometric parameters of placental villi

Note: *at р< 0,05, the difference between the compared figures is reliable

Figure 2. а. Pronounced VEGFR-1 expression in the cytoplasm of extravillous trophoblast in the placenta of women from the control group; immunoperoxidase method, x400; b. Pronounced VEGFR-1 expression in the cytoplasm of extravillous trophoblast in the placenta of women from the control group; immunoperoxidase method, x400; c. Moderate VEGFR-3 expression in the syncytiotrophoblast and low - in the endotheliocytes of the villi capillaries in the placenta of women from the control group; immunoperoxidase method, x400; d. Low VEGFR-1 expression in the syncytiotrophoblast and endotheliocytes of villi capillaries in the placenta of women with obesity; immunoperoxidase method, x400; e. Pronounced VEGFR-2 expression in villi syncytiotrophoblast in the placenta of women with obesity; immunoperoxidase method, x400; f. Pronounced VEGFR-1 expression in the endothelium and smooth muscle stem villi cells in the placenta of women with obesity; immunoperoxidase method, x400

 

 

Very interesting data were obtained by analyzing the expression intensity ratio of receptors and the growth factor (Table 2). The ratio of VEGFR-1 / VEGF in almost all studied structures (except for syncytiotrophoblast) was below normal values, and the ratio of VEGFR-2 / VEGF – was above the norm. The ratio of VEGFR-3 / VEGF exceeded benchmarks in the vascular endothelium of stem villi and in syncytiotrophoblast; however, it was below normal in the endotheliocytes of terminal villi and in the extravillous trophoblast. The immunohistochemical analysis of the placentas of obese women found a reduced reaction intensity of VEGF in SCT and ESV and higher levels of its expression in EVT and EIV (Diagram 1), in international enzyme activity units (IU). The analysis of the immunohistochemical distribution of VEGF receptors in normal placentas found lower levels of their expression in the endotheliocytes and trophoblast cells, when compared with the growth factor itself. The most intensive reactions of VEGFR-1 and VEGFR-3 were found in EVT, while those of VEGFR-2 – in ESV. The minimum levels of expression of all three were found in the EIV. The placental tissue of obese women had a reduced expression of VEGF-1 and increased level of VEGFR-2 and VEGFR-3. For instance, the reaction intensity of VEGFR-1 in the vascular endotheliocytes of stem and terminal villi is 43 and 28.3% lower than the normal levels (p<0.05) (Diagram 2).

 

Тable 2: Expression ratios of vascular endothelial growth factor (VEGF) and its receptors (VEGFR) in placental tissue of obese women

 

Diagram 1. VEGF expression in placental tissue

 

 

Diagram 2. VEGFR-1 expression in placental tissue

 

On the contrary, VEGFR-2 expression exceeds the control indexes by more than four times in EIV (p<0.01), by 28.7% in ESV, and by 55.3% in EVT cells (p<0.05) (Diagram 3). VEGFR-3 changes were less pronounced. The intensity of its reaction was 33.2% higher than the normal level in SCT (p<0.05), 30% higher in ESV (p<0.05), and 2.8% lower in EVT (p<0.05). In SCT and ESV, VEGFR-3 expression was 36.36% higher than the normal level, while in EVT, it was 20% lower than the normal level (Diagram 4). Interesting data were obtained during the analysis of the correlation of receptor expression intensity to that of the growth factor itself. The VEGFR-1/VEGF ratio in virtually all the studied structures (with the exception of SCT) was lower than normal, while the VEGFR-2/VEGF ratio was higher than normal. The VEGFR-3/VEGF ratio was higher than the control indexes in ESV and SCT, but lower than EIV and EVT standards.

Diagram 3. VEGFR-2 expression in placental tissue

 

Diagram 4. VEGFR-3 expression in placental tissue

 

DISCUSSION:

Thus, the obtained results of immunohistochemical study of normal placenta are generally consistent with the source data related to the distribution of VEGF and its receptors⁸. The authors of this research found that angiogenesis without vascular branching prevailed in the placentas of women with obesity. At that, a more pronounced expression of VEGF and its VEGFR-2 receptor was found in the endothelium of villous capillaries and the extravillous trophoblast in all studied structures, when compared to normal placentas. Under normal conditions, the binding of VEGF and VEGFR-2 activates the angiogenesis by enhancing endothelial cell proliferation and growth⁹.

 

In addition, under moderate hypoxia VEGF binding to its receptor VEGFR2 causes hyper- capillarization of intermediate villi according to the classical feedback mechanism, which is accompanied by a pronounced expression of VEGF and VEGFR-2 in vascular endothelial cells of the villi¹⁰. Despite the fact that obesity causes development of sub-placental hypoxia, and the study revealed combined increase in the expression of VEGF and VEGFR-2 in the capillary endothelium of the villi, histological examination of preparations revealed predominance of angiogenesis without vessel branching. According to T. M. Mayhew et al., such factors may involve high concentrations of lipids and blood glucose, which is observed in obesity and diabetes and which contribute to closing or masking of receptors¹¹.

 

We found reduced expression of VEGF in villous syncytiotrophoblast, which chemicals are released mainly in the mother's blood. The obtained results are consistent with previous studies, which found reduction in VEGF blood serum concentration after 20 weeks of pregnancy in obese women.

 

CONCLUSION:

Therefore, the identified features of immunohistochemical distribution of VEGF expression and its receptor in placental tissue of women suffering from obesity, to a certain extent, reflect the processes related to compensation and abnormal functioning of the mother-placenta-fetus system in the presence of hypoxia and hyperlipidemia. Research results indicate unavoidable development of morphological changes in the placenta during pregnancy against the background of obesity. Decrease in body weight before pregnancy can reduce the risk of placental disorders and improve perinatal outcomes.

 

CONFLICTS OF INTEREST:

The authors declare that they have no competing interests.

 

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Received on 06.10.2017         Modified on 17.11.2017

Accepted on 20.12.2017      © RJPT All right reserved