Sequential steps during vasculogenesis and angiogenesis in the very early human placenta

Development of blood vessels takes place via two subsequent processes, vasculogenesis and angiogenesis. During vasculogenesis, formation of first blood vessels is achieved by differentiation of hemangiogenic stem cells from pluripotent mesenchymal cells, while during angiogenesis new blood vessels form from already existing vessels. The combination of our data with those from the literature leads us to depict the chronological steps of cell differentiation in the mesenchymal core of placental villi during vasculogenesis and angiogenesis. This current opinion will focus on the temporal and spatial expression of VEGF and its receptors VEGFR-1 and VEGFR-2, and the angiopoietin receptors Tie-1 and Tie-2 in parallel to vascular maturation in human placental villi during very early stages of placental development. There is evidence that the interplay of a variety of growth factors secreted from different cell types during development is needed to trigger as well as maintain placental vasculogenesis and angiogenesis.     

Hofbauer cells in early human placenta: possible implications in vasculogenesis and angiogenesis

The stroma of the placental villi contain numerous macrophages, so-called Hofbauer cells which are of mesenchymal origin and are thought to function in many processes. Although there are many studies concerning placental vasculogenesis and angiogenesis, there has been a lack of evidence on the possible roles of Hofbauer cells in these processes. In this study we hypothesized that Hofbauer cell locations and numbers might be correlated with the vascular structures within the placental villi core and therefore may be implicated to play roles in placental vasculogenesis and angiogenesis. Placental tissues were obtained from normal first-trimester pregnancies. Tissues were prepared for light microscopic investigations. Double immunohistochemistry staining with CD31/PECAM1 and CD68 was applied to placental tissues. In placental villous core, majority of the Hofbauer cells were found to be either in close contact with angiogenic cell cords and primitive vascular tubes or located in between them. Moreover, the number of Hofbauer cells and vasculogenic structures were found to be significantly correlated. The findings of this study suggest for the first time that Hofbauer cells might be involved in the processes of vasculogenesis and angiogenesis in the placenta.     

Spatial and temporal distribution of Tie-1 and Tie-2 during very early development of the human placenta

Vasculogenesis in the human placenta comprises differentiation and growth of newly forming blood vessels derived from hemangiogenic stem cells within the mesenchymal core of villi. In a second stage, angiogenesis leads to the expansion and remodeling of the already existing vessels. At present, relatively little is known about the regulatory mechanisms of vasculogenesis and angiogenesis during very early placentation. Using placental villous tissues from days 22 to 48 of pregnancy, we analyzed the spatial and temporal expression of Tie-1 and Tie-2 in parallel to vascular maturation in the human placenta. In immunohistochemistry both receptors, Tie-1 and Tie-2 show a cell and villous type specific expression during this early phase of placental development. Especially, cytotrophoblast and hemangiogenic cell cords in mesenchymal villi and Hofbauer cells in immature intermediate villi have the strongest immunoreactivities. Western blot analysis showed that no significant changes were detected for Tie-1 and Tie-2 as pregnancy advanced. Moreover, phospho-Tie-2 levels did not change significantly in parallel to pregnancy ages. We conclude that both receptors are involved in angiogenesis as well as vascular modulation of early vessels. Due to their spatial distribution we speculate on an additional role in regulation of villous and extravillous trophoblastic behavior.     

Co-localization of vascular endothelial growth factor and its two receptors flt-1 and kdr in the mink placenta

Placental angiogenesis plays an important role in placental development and morphogenesis. Vascular endothelial growth factor (VEGF) is a well-known angiogenic growth factor, which has previously been localized in different epitheliochorial and haemochorial placenta types. In the present study VEGF and its Flt-1(VEGFR-1) and KDR (VEGFR-2) receptors were immunolocalized in the endotheliochorial mink placenta throughout gestation. VEGF, Flt-1 and KDR co-localized to fetal and maternal microvascular endothelial cells, but with a temporal difference, displaying KDR in endothelial cells throughout gestation, whereas the VEGF and Flt-1 maternal endothelial cell staining was most intense during late gestation. Additionally, KDR was found in vascular related mesenchymal cells. The VEGF-receptors were also localized in non-endothelial cells, e.g. the uterine luminal and glandular epithelium as well as the trophoblast. Our results are in agreement with former studies, showing the different effects of the Flt-1-and KDR receptors in respect of angiogenesis. More importantly, the present study of the endotheliochorial placenta localizes the VEGF-ligand-receptor system in non-endothelial cells, and thereby strengthen the hypothesis that VEGF, apart from its well-established angiogenic properties, must also have additional functional roles in the establishment and development of the placenta.     

MiR 20a,-20b and -200c are involved in hydrogen sulfide stimulation of VEGF production in human placental trophoblasts

Hydrogen sulfide (H₂S) has been implicated to angiogenesis in various tissues. We sought to investigate the role of hydrogen sulfide (H₂S) in regulating production of vascular endothelial growth factor (VEGF) proteins, the key factors of angiogenesis and vasculogenesis, in placenta.MethodsPlacental tissues were obtained from pregnant women with preeclampsia and healthy pregnant women who underwent elective cesarean section. Explants and trophoblasts were isolated from healthy placentas and treated with H₂S donor and precursor. Western blotting was used to determine the levels of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The levels of VEGF mRNA, miR miR-200c,-20a and -20b were determined by quantitative real time PCR.ResultsNaHS and l-cysteine increased VEGF but not placenta growth factor (PlGF) production in cultured explants and trophoblasts. Transfection of CBS and CSE siRNA reversed the stimulatory effect of l-cysteine on VEGF production in placental cells. H₂S prolonged the half-life of VEGF mRNA and decreased the expression of miR-200c,-20a and -20b in placental cells. MiR-200c mimic and inhibitor affected VEGF mRNA and protein level, whereas miR-20a or -20b mimic and inhibitor affect VEGF protein release but not mRNA expression. The expression level of miR-200c,-20a and -20b as well as the level of CBS, CSE and VEGF were downregulated in preeclamptic placentas.ConclusionH₂S produced via CSE and CBS plays a critical role in VEGF production in human placenta. Reduced expression of CSE and CBS may contribute to the abnormal production of angiogenic factors in preeclamptic placenta.     

Aspects of human fetoplacental vasculogenesis and angiogenesis. II. Changes during normal pregnancy

In this second review, we describe the main morphological events which accompany the development of the fetoplacental vascular system throughout normal human pregnancy and summarize findings on the expression of angiogenic growth factors and their receptors. Fetoplacental vasculogenesis starts at day 21 after conception by formation of haemangioblastic cords. In the following phase of branching angiogenesis (day 32 to week 25 post conception), haemangioblastic cords develop into a richly branched villous capillary bed with low fetoplacental blood flow impedance. This period is characterized by high placental levels of VEGF but moderate PlGF expression. In week 15, large centrally located villi show regression of peripheral capillary nets. In parallel, some remaining central capillaries acquire a tunica media and transform into arteries and veins. Beginning at about week 25 in the newly formed peripheral villi, angiogenesis switches from branching to non-branching and this period is accompanied by a steep drop in VEGF and a slower decline in PlGF expression. As a consequence of this switch, long poorly branched capillary loops are formed in the periphery of the fetoplacental vascular trees. These increase fetoplacental impedance but blood flow still increases due to rising fetal blood pressure. The possible interactions between (a). the biphasic development of intraplacental oxygen tensions, (b). changes in VEGF and PlGF levels and (c). developing vascular geometry are discussed. Special attention is given to the obvious discrepancy between sudden elevation of intervillous oxygen tensions which is not coincident with the appearance of angiogenic growth factor peaks and the switch from branching to non-branching angiogenesis. Finally, we deal with methods of quantifying aspects of angiogenesis in the villous vascular system and summarize the main findings during uncomplicated human pregnancy.     

Increased placental angiogenesis in late and early onset pre-eclampsia is associated with differential activation of vascular endothelial growth factor receptor 2

Introduction

Placentas from both early-onset (EOPE) and late-onset pre-eclampsia (LOPE) exhibit signs of underperfusion, which in turn, may be associated with altered angiogenesis. Tyrosine 951 (Y951) and Y1175 phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2) induced by VEGF triggers the angiogenesis process. Endothelial markers such as CD31 and CD34 have been used for estimating angiogenic processes in several tissues, including placenta. We asked whether vascular density in placental villi was related to Y951/Y1175 phosphorylation of VEGFR2 in LOPE or EOPE.

Methods

We obtained placental samples from women with normal pregnancies (n = 22), LOPE (n = 13), EOPE (n = 15) and preterm deliveries (n = 10). Slices from placental tissue were used for CD31 immunostaining. We estimated the expression of CD31, CD34, VEGF, and VEGFR2 by western blot and quantitative PCR. Y951 phosphorylation of VEGFR2 was estimated by western blot, whereas Y1175 phosphorylation was analyzed by ELISA.

Results

Vessel density in terminal villi and CD31 and CD34 protein abundance were increased in LOPE and EOPE compared to normal pregnancy. However, mRNA levels for CD31 and CD34 were lower in LOPE than in normal pregnancy and VEGF mRNA was higher in EOPE. VEGFR2 protein concentration was not different among the studied groups. Y951 and Y1175 phosphorylation of VEGFR2 was higher in LOPE than in the normotensive group, but only Y951 exhibited greater phosphorylation in EOPE compared to normal pregnancy.

Discussion

Changes in vessel formation in the pre-eclamptic placenta are controversial. Our study suggests a pro-angiogenic state in both LOPE and EOPE. These changes are however, associated with differential expression of endothelial markers and VEGFR2 activation.

Conclusion

There is evidence of increased placental angiogenesis in LOPE and EOPE that is associated with differential activation of VEGFR2.     

Cyclic changes in expression of mRNA of vascular endothelial growth factor, its receptors Flt-1 and KDR/Flk-1, and Ets-1 in human corpora lutea

OBJECTIVE: To evaluate the expression of mRNA of vascular endothelial growth factor (VEGF), its receptors Flt-1 and KDR/Flk-1, and Ets-1 in human corpora lutea. DESIGN: Prospective laboratory study. SETTING: University hospital in Japan. PATIENT(S): Women with regular menstrual cycles who underwent hysterectomy. INTERVENTION(S): Fifteen corpora lutea were obtained during hysterectomy (5 in the early luteal phase, 5 in the mid-luteal phase, and 5 in the late luteal phase). MAIN OUTCOME MEASURE(S): Expression of VEGF, Flt-1, KDR/Flk-1, and Ets-1 in human corpora lutea on northern blot analysis or immunohistochemistry. RESULT(S): Human corpora lutea in early luteal phase and mid-luteal phase had high VEGF mRNA expression. Expression of VEGF mRNA was significantly reduced in the late luteal phase. Immunohistochemistry showed that VEGF protein was expressed mainly in granulosa lutein cells and faintly in thecal lutein cells. Staining of VEGF protein was decreased in human corpora lutea in the late luteal phase. Expression of Flt-1 and KDR/Flk-1 mRNA was increased in the early luteal phase and mid-luteal phase and decreased in the late luteal phase. Immunohistochemistry showed that Flt-1 and KDR/Flk-1 proteins were expressed mainly in granulosa lutein cells and faintly in thecal lutein cells and endothelial cells in the early luteal phase and mid-luteal phase; their protein staining was reduced in the late luteal phase. Expression of Ets-1 mRNA changed similarly to VEGF and its receptor mRNA in human corpora lutea during the luteal phase. CONCLUSION(S): Levels of mRNA of VEGF and its receptors Flt-1 and KDR/Flk-1 in human luteal cells may be related to luteal function.     

Imbalance of expression of bFGF and PK1 is associated with defective maturation and antenatal placental insufficiency

Objective

Defective placental maturation is associated with restricted functional capacity and adverse perinatal fetal outcomes. The aim of the study was a comparative analysis of the role of mRNA expression of various angiogenic factors in placental maturation defects.

Study design

We examined the mRNA expression patterns of prokineticin 1 (PK1), its receptors (PKRs), basic-fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in tissue from third-trimester placentae that exhibited delayed or accelerated villous maturation.

Results

The expression of PK1 and PKR2 was elevated in placental tissue exhibiting accelerated maturation and a predominant differentiation of terminal villi. The opposite was found in tissue exhibiting delayed maturation and deficiency of the terminal villi. In addition, low expression of bFGF correlated with the predominant differentiation of terminal villi, whereas the opposite was observed when terminal villi were deficient. The expression of VEGF, PIGF, and PKR1 showed no significant differences between the groups.

Conclusion

Defective placental maturation is associated with an imbalance of expression of bFGF and PK1. Our results demonstrate an involvement of the PK1/PKR2-signalling pathway in the regulation of the functional adequate capillarization in late pregnancy. We propose the bFGF/PK1-ratio as a monitor of placental function and a possible indicator of latent clinical problems, such as placental dysfunction leading to fetal hypoxia.     

The distribution of angiopoietin-1, angiopoietin-2 and their receptors tie-1 and tie-2 in the very early human placenta

Angiopoietins are integral to vasculogenesis and angiogenesis, which play crucial roles in the growth and development of the placenta. The current study assessed expression of angiopoietins (Ang-1 and Ang-2) and their receptors (Tie-1 and Tie-2) during development of the early human placenta. First-trimester placental tissues were obtained from women undergoing curettage during normal pregnancies. The use of immunohistochemistry (IHC) showed that Ang-1 was primarily localized to syncytiotrophoblasts where it displayed moderate immunoreactivity, whereas weak immunoreactivity for Ang-1 was observed in endothelial cells and angiogenic cell cords (ACC). Strong immunoreactivity for Ang-2 was also found predominantly in syncytiotrophoblasts with lower immunostaining levels evident in cytotrophoblasts. Moderate immunoreactivity for Ang-2 was observed in endothelial cells, ACC and Hofbauer cells. By contrast, the trophoblastic shell, as well as endothelial cells and ACC exhibited strong staining intensity for Tie-1 with the strongest immunoreactivity for Tie-2 observed in cytotrophoblasts, ACC and endothelial cells. Western blotting of tissue extracts confirmed the IHC results. Previous studies focused on VEGF and its receptors in controlling vasculogenesis and angiogenesis in human placenta. However, the specific localization patterns of angiopoietins and their receptors revealed by the current study emphasize the importance of these molecules in placental vascular development. Functional studies aimed at identifying the molecular mechanisms of actions of these factors and receptors may prove essential in elucidating the pathophysiology of placental disorders such as intrauterine growth restriction and pre-eclampsia.     

胎盘绒毛血管生成状况及VEGF、MMP-2和MMP-9表达与早期自然流产的关系

目的:探讨VEGF、MMP2和MMP9表达变化对胎盘绒毛血管生成的影响及与早期自然流产的关系。方法:选用CD34作为微血管标记物,应用免疫组化检测30例早期自然流产病例(自然流产组)和20例早期人工流产病例(人工流产组)胎盘绒毛微血管密度(MVD);RT-PCR检测胎盘绒毛VEGFmRNA的表达;酶谱分析法测定MMP-2和MMP-9的表达。结果:自然流产组VEGF121mRNA表达及MVD均明显低于人工流产组(P<0.01)。相关分析显示,两组中VEGF121mRNA相对表达量与MVD值均呈显著正相关(rθ=0.9794,rλ=0.9183)。自然流产组酶原型MMP-9的表达量显著低于人工流产组(P<0.01),激活型MMP-2的表达量显著高于人工流产组(P<0.01)。结论:孕期胎盘绒毛内VEGF121mRNA低表达所致的血管生成障碍以及MMP-2和MMP-9表达失调可能共同参与了早期自然流产的发生发展过程。     

胎儿宫内生长迟缓患者脐动脉波形异常与胎盘病理改变的关系

目的　探讨胎儿宫内生长迟缓（IUGR）患者胎儿脐动脉波形异常与胎盘重量、体积及各级绒毛血管数量的关系。方法　选择分娩前1周内,患IUGR或B超检查有胎儿脐动脉波形异常的患者40例,分为脐动脉波形异常伴IUGR10例为研究组;脐动脉波形异常不伴IUGR（AN）;脐动脉波形正常伴IUGR（NA）,脐动脉波形正常不伴IUGR（NN）各10例为对照组。用免疫组织化学SP法,检测以上各组胎盘干血管中α-平滑肌肌动蛋白（α-smoothmuscleactin,α-SMA）表达,以确定各级绒毛和各级血管,比较研究组和各对照组间胎盘重量、体积大小、各级绒毛及血管数量。结果　（1）研究组胎盘重量、体积分别为（283.40±23.82)g及（234.60±52.08)cm     

Ontogenesis of villi and fetal vessels in the human placenta

The ontogenesis of the villous and vascular arborescence in normal pregnancy is reviewed. The emergence of the villi and the fetal vessels is described from early pregnancy to term together with the specializations of the villi, vessels and capillaries observed in the last trimester. The expression, localization and role of the angiogenic growth factors (FGF, VEGF, PLGF, HGF) are described and discussed. Pathological pregnancies with hyper- and hypocapillarization are related to altered oxygenation. The potential roles of growth factors are presented and hypotheses proposed which may provide a molecular basis for the development of the most frequent placental pathologies.     

Animal models of placental angiogenesis

The study of the development of the fetal membranes is an ancient one, and the importance of placental vascular development to placental function has long been recognized. Animal models have been important in these studies, as they allow for controlled experiments and analysis of multiple time-points during pregnancy. Since the demonstration nearly 20 years ago that the placenta produces angiogenic factors, the major factors regulating placental angiogenesis have been identified. These major factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), the angiopoietins (ANG), and their receptors. Recently, sophisticated computerized image analysis methods have been developed to establish the pattern of placental vascular development in sheep. The maternal placental capillary bed develops primarily by increased size of capillaries, with only small increases in capillary number or surface densities. In contrast, the microvasculature of the fetal placenta develops primarily by increased branching, resulting in a large increase in capillary number and surface densities. These observations help to explain the relatively large increase in umbilical blood flow and nutrient delivery to the fetus that occurs during the last half of gestation. In addition, expression of mRNAs for VEGF, bFGF, ANG, and their receptors have recently been correlated with normal placental vascular development in sheep, and further refinement of these mathematical models is warranted. Lastly, the recent development of animal models of compromised pregnancies, including those resulting from maternal nutrition (both restriction and excess), multiple fetuses, environmental stress (heat stress and high altitude), and fetal and maternal breed effects, has already indicated that reductions in placental vascular development and expression of angiogenic factors are probably a root cause of fetal growth restriction. With these methods and models now in place, we should soon be able to establish the mechanisms involved in both normal and abnormal placental angiogenesis.     

Angiogenesis and placental growth in normal and compromised pregnancies

Research on the subject of pre-eclampsia has revolved around placental growth and angiogenesis, as both are central to the aetiology of the disease. Vascular angiogenic growth factor (VEGF) is elevated in pre-eclampsia and correlates with the severity of disease. Its actions in vitro mimic the actions of plasma from women with pre-eclampsia. This chapter examines the available evidence that implicates VEGF in the maternal systemic effects seen in pre-eclampsia, and discusses how an understanding of this growth factor could lead to diagnostic and therapeutic options. Oxygenation status is the unifying concept that surrounds the discussion of placental growth and angiogenesis. The concept that 'hypoxia' is too simplistic a notion to describe pre-eclampsia is discussed. Maldevelopment of the angiogenic process can be assessed by Doppler ultrasound. The future may see a role for magnetic resonance imaging in the identification of poorly perfused placenta.     

VEGF, bFGF and their receptors at the fetal-maternal interface of the rhesus monkey

Placental development involves trophoblast outgrowth and a coordinated angiogenesis in the implantation site. In this study, expression of angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), VEGF receptors, kinase insert domain-containing region (KDR), and bFGF receptor Flg was characterized at the maternal-embryonic boundary of the rhesus monkey on Day 17, 19, 28 and 34 of pregnancy. Immunohistochemistry and in situ hybridization showed that VEGF mRNA and protein were both strongly expressed in the cytotrophoblast, the blood vessels and certain immunocytes. These sites were also immunopositive for KDR. In addition to the vascular endothelial cells and the vascular smooth muscle cells, the protein and mRNA for bFGF were also detected in cyto/syncytiotrophoblast bilayer, whereas the staining for Flg protein was mainly localized in the cytotrophoblast cells. The staining degree of VEGF and bFGF in the villi gradually decreased with the development of placenta. Strong expression of bFGF, Flg and KDR was also detected in the decidual cells. These data suggest that VEGF and bFGF may be involved in angiogenesis, cytotrophoblast proliferation and migration during early stage of placentation in the rhesus monkey.