Role of vascular endothelial growth factor and ...

doi:10.1111/jog.12751

J. Obstet. Gynaecol. Res. 2015

Role of vascular endothelial growth factor and placental growth factor expression on placenta structure in preeclamptic pregnancy Emel Kurtoglu1, Berrin Z. Altunkaynak2, Isinsu Aydin2, Ayse Z. Ozdemir1, Gamze Altun2, Arif Kokcu1 and Suleyman Kaplan2 Departments of 1Obstetrics and Gynecology, 2Histology and Embryology, Ondokuz Mayis University, Samsun, Turkey

Abstract Aim: Pre-eclampsia is a hypertensive disease that is characterized by high blood pressure and proteinuria after 20 gestational weeks and complicates 3–8% of all pregnancies. It is classified as either mild or severe pre-eclampsia according to severity, and the aim of this study was to investigate the structural differences between these two classifications. Methods: Placenta samples were collected from 68 women who underwent cesarean delivery. Total volume of villi and numerical density of vascular endothelial growth factor (VEGF)- and placental growth factor (PIGF)-positive cells were estimated on stereology and evaluated using one-way ANOVA. Results: There was no significantly difference in total villi volumes between the groups (P > 0.05). In contrast, on immunohistochemistry, the numerical density of VEGF-positive cells in severe pre-eclampsia was significantly different to the control and mild pre-eclampsia groups (P 0.05; Figs 5,7).

Discussion It has long been known that pregnancies complicated by pre-eclampsia have macroscopic and microscopic alterations in placental structure. In normal placentation, the cytotrophoblast cells invade the uterine spiral arteries and transform them from small-caliber resistance vessels into high-caliber capacitance vessels in order to

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provide adequate perfusion for both villous and fetal growth.28,29 In pre-eclampsia, this invasion is limited to the proximal decidua, and spiral arteries fail to dilate and remain responsive to vasomotor influences, which leads to high-resistance chorioplacental circulation.30,31 As a result, the average diameter of the blood vessels decreases and, owing to the placental ischemia accelerating villous branching, causes an increase in total villi volume, and occurrence of numerous syncytial knots (apoptotic cell clusters) associated with diffusion and villitis.26,32–37 In contrast to the previous findings, there was no significant difference in mean volume of villi between the control and the pre-eclamptic groups. On the other hand, mean capillary volume in the villi was found to be increased in both mild and severe pre-eclamptic groups compared with the controls. Chronic hypoxia may have induced the growth of capillaries, increasing mean volume, but capillary loops with narrow lumens may have led to insufficient maternal–fetal perfusion. In addition, perivillous fibrin deposits and increased syncytial knot density owing to reduced perfusion and oxidative stress injury may have obstructed the hyperramification of the villi and decreased maternofetal exchange.

© 2015 Japan Society of Obstetrics and Gynecology

VEGF and PIGF in pre-eclamptic placenta

Figure 3 (a–c) H&E-stained placental sections in the (a) control group; (b) mild pre-eclampsia group; and (c) severe pre-eclampsia group. White arrows, syncytial knots; black arrows, perivillous fibrinoid deposition; black arrowheads, thrombosis areas. Although there was no significantly difference in terms of total villi volume between the groups (P > 0.05), thrombosis was noted in (b,c) the mild and severe pre-eclampsia groups on histopathology. Additionally, (a) perivillous fibrin deposition was observed in the control group. (Scale bars, 500 μm; insets, 250 μm).

Figure 4 Mean numerical density of placental growth factor- and vascular endothelial growth factorpositive cells per mm2 was estimated using unbiased counting frame for each section. X, Cells touching the red edges were excluded from the counting; √, cells that touched the green edges were included in the counting.

Figure 5 Numerical density of (a) placental growth factor- and (b) vascular endothelial growth factor-positive cells in the control and mild and severe pre-eclampsia groups. Error bars, SEM. *P < 0.05, **,##P < 0.01.

Given that defects in angiogenesis and remodeling of the trophoblast lead to maldevelopment, and inadequate placental mass, placental dysfunction, and chronic hypoxemia play key roles in the pathogenesis of preeclampsia, VEGF and PIGF and pro-angiogenic proteins produced by villous syncytiotrophoblasts have been the focus of many studies.17,18,38,39 Maternal serum and placenta levels of these pro-angiogenic factors have been


investigated in many studies, but there has been little interest in the immunohistochemistry of the expression of these factors in the placenta. Some of the previous studies comparing placental expression of the VEGF system, which is known to be regulated in response to mainly placental hypoxia, noted increased placental VEGF and PIGF immunostaining in pre-eclampsia pregnancies.14,17,18,40 Induction and

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Figure 6 Immunohistochemical localization of vascular endothelial growth factor (VEGF) in the (a) control group; (b) mild preeclampsia group; and (c) severe pre-eclampsia group. There are more VEGF-specific cells (white arrows) in the (c) severe preeclampsia group than in the other groups. Black stars, vessels; white arrows, syncytial knots. Scale bars, 25 μm.

Figure 7 Immunohistochemical localization of placental growth factor (PIGF) in the (a) control group; (b) mild pre-eclampsia group; and (c) severe pre-eclampsia group. The number of PIGF-specific cells (black arrows) in the (b) mild and (c) severe pre-eclampsia groups was significantly higher than in the (a) control group (P < 0.01). White stars, vessels; black arrowheads, syncytial knots. Scale bars, 25 μm.

upregulation of the VEGF system by chronic hypoxia and endothelial activation were suggested as possible mechanisms of this increase. In contrast, most of the studies reported decreased placental VEGF and PIGF immunostaining.12,13,38,41,42 In one of these investigations, VEGF was thought to be decreased due to insufficient compensation of the placenta.43 In addition, PIGF was thought to be lower in the placentas of pre-eclamptic patients due to reduced development of placental structures.44 Moreover, disregulation in the VEGF family and two possible mechanisms were suggested to explain the defective angiogenesis: one was downregulation of membrane-bound VEGF receptor-1 (VEGFR-1) in placentas leading to defective uteroplacental development; and the second was overproduction of competitive soluble VEGFR-1, which may lead to suppression of VEGF and PIGF effects.45 In the present study, VEGF was found to be significantly increased in the placentas of severe pre-eclamptic patients when compared with mild pre-eclampsia and the controls. It is possible that the hypoxic environment

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that induces VEGF production has different features in terms of severity and enhancing factors in mild and severe cases, such that VEGF remained unchanged in mild cases. But, despite elevated levels, disregulation at the receptor level leading to dysfunction of VEGF and defective angiogenesis may have occurred. In this study, PIGF was also found to have increased in both mild and severe pre-eclampsia when compared with the controls, whereas there was no significant difference in PIGF between mild and severe pre-eclampsia. Despite the fact that this pro-angiogenic factor is induced by chronic hypoxia, the severity of hypoxia and oxidative injury and structural maldevelopment of the placenta may have affected the level of PIGF in the preeclampsia groups. Also, elevated levels seemed to be ineffective to supply adequate maternofetal circulation, which may have been caused by a defective function of the ligand–receptor complex of PIGF. Furthermore, in contrast to the previous studies, PIGF was found to be elevated in mild pre-eclamptic patients whose duration of pregnancy was 38.1±1.7 weeks and in severe pre-eclamptic


VEGF and PIGF in pre-eclamptic placenta

patients whose duration of pregnancy was 34.0±4.3 weeks. It may be that increase in PIGF does not depend on preterm delivery. In conclusion, placenta structure, particularly vascularization, reflects a complex interaction of regulatory factors including predominantly growth factors. Given that pre-eclampsia has been shown to have placentarelated vascular insufficiencies that lead to alterations in structure, understanding the features of the regulatory system will provide insight into the pathogenesis of preeclampsia.

Disclosure The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.

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