Current topicAspects of Human Fetoplacental Vasculogenesis and Angiogenesis. II. Changes During Normal Pregnancy
Introduction
Current understanding of the development of the human fetoplacental vascular system is based, almost exclusively, on descriptive structural and immunohistochemical studies. Meaningful in vitro approaches (which would help to elucidate the peculiarities of human fetoplacental angiogenesis) are under-represented due to a series of problems inherent to the placenta.
In placental villi, angiogenesis takes place in the presence of an oxygenation and nutrition gradient which extends from the maternal circulation (the supplier), via the villous trophoblast (an important consumer) and developing fetal circulation (which extracts oxygen and nutrients from the villi) to the developing fetus (the ultimate consumer). Based on this situation, increasing development of the fetoplacental vasculature diminishes rather than increases its own nutrition [1]. So far, it has been impossible to mimic this unusual situation in vitro, either in villous explant culture or by in vitro perfusion of the organ.
Cell culture experiments with human placental microvascular endothelium are practicable since suitable endothelial cell lines are available [2]and human endothelial cells of different microvascular origins, and showing placenta-specific phenotypes, have been isolated [3]. However, it has not been possible to-date to establish co-culture systems which are sophisticated enough (a) to maintain the placenta-specific phenotypes and (b) to mimic the specific microenvironmental conditions of a developing placenta.
Animal experiments designed to study the influences of various factors on chorioallantoic or fetoplacental vasculogenesis and angiogenesis are easy to perform (e.g. [4], [5], [6], [7]) but the data are difficult to extrapolate to the human condition due to substantial interspecies differences regarding pregnancy length, development of the oxygenation gradient and the final architecture of the fetoplacental vascular bed. Interspecies differences also make it difficult to transfer descriptive data obtained in animals to the human placenta. Exceptions are provided by the placentae of higher primates (e.g. rhesus monkey [8]) which display a villous structure, haemodynamic conditions similar to those of the human placenta and a pregnancy length in the same range as that in humans.
Because of these peculiar local conditions of the placenta, fetoplacental vasculogenesis and angiogenesis also differ from vessel formation in other human vascular beds. The connection between developing placental capillaries and the embryonic circulatory system is established via the connective stalk (forerunner of the umbilical cord) around day 32 post conception (for review, see [9]). From this early stage onwards, the highly immature intravillous vascular bed has to perform all placental transport functions. In doing so, it never attains a stable state but rather expands continuously to meet the oxygen and nutritional needs of the growing embryo/fetus (e.g., see [10], [11]).
Development takes place in an environment of reduced oxygen tensions relative to maternal tissues but, during the course of pregnancy, intervillous oxygen tensions increase [12]. In contrast to most other developing organs, in which oxygenation improves with advancing vascularization, tissue oxygenation in placental villi appears to be inversely related to the numerical density of fetal capillaries since, rather than merely delivering oxygen, the latter extract it from the villi [1]. Consequently, a low numerical density of fetal capillaries because of reduced oxygen extraction results in increasing intraplacental oxygen levels [13], [14]which, in turn, impact further on angiogenesis ([15], this volume). In contrast, and under otherwise constant conditions, high numerical densities of capillaries and high oxygen extraction by the fetal circulation would lower intraplacental oxygen tensions [13], [14]. When exceeding certain limits, both situations may result in vicious circles.
In any vascular bed, increased flow is achieved by a combination of physiological adjustments (e.g. increased perfusion pressure, decreased vascular impedance) and changes in vascular anatomy (e.g. increases in vessel calibre, decreases in vessel length, formation of parallel rather than serial arrangements of vessels). According to the Poiseuille equation, resistance to flow is directly proportional to vessel length and inversely proportional to the square of vessel cross-sectional area. Consequently, the advantage of parallel arrangements is that, other variables being constant, they generate multiple interconnected vessel segments of reduced mean length and, hence, reduced impedance. Thus, vascular anatomy influences vascular impedance in two main ways: by the dimensions and spatial arrangements of vessels. These principles are of particular importance in the fetoplacental vascular bed which has to adapt continuously to the increasing requirements of the growing embryo/fetus.
Doppler studies have shown that fetal vascular resistance decreases during normal gestation and may be compromised in complicated pregnancies [16]. The principal strategy for minimizing lengths and resistances is branching angiogenesis. It is now recognized [17], [18]that new branches may be created by sprouting angiogenesis (lateral sprouting from existing vessels) or by intussusceptive angiogenesis (formation of transvascular epithelial pillars which partition one lumen into two or more lumina). Both types of branching strategy [15]are capable of creating parallel vascular arrangements in which the individual vessel segments are relatively short and numerous. In contrast, non-branching angiogenesis (which is likely to increase resistance to blood flow) involves elongation of existing vessel segments by vascular endothelial cell proliferation, intercalation of endothelial progenitor cells or a combination of the two [15].
Fetoplacental angiogenesis shapes villous development [1], [9], [19], [20], [21], [22], [23]. In normal human pregnancy, capillary growth is biphasic, involving an initial phase of branching angiogenesis (formation of tightly looped capillaries) followed by one of increased non-branching angiogenesis (formation of longer capillaries). To some degree, villous morphology reflects the underlying angiogenic processes (see also, Figure 4, Figure 5). For example, during the first trimester, immature intermediatevilli are large calibre, bulbous structures covered by a thick layer of trophoblast and containing a complex capillary network surrounding central stem vessels. In contrast, villi in the third trimester are overwhelmingly filiform structures associated with a thin trophoblast and tightly looped capillaries. These relationships between capillaries and villi suggest that the villous trophoblast is a plastic layer which adapts in parallel with, or in response to, the changing structure of the underlying vasculature. This view implies that the trophoblast doesnot sculpt the vasculature but, instead, angiogenesis helps to drive villous development and differentiation. The phasic nature of gestational changes in morphometric indices of villous capillarization, notably capillary : villus length ratios [23]are consistent with this notion. Vascular patterns and villous shapes also vary in complicated pregnancies (see [24], this volume).
Development of the fetal vasculature of the placenta depends on the actions of angiogenic growth factors and their receptors produced by cells and extracellular matrix ingredients lying in or near the fetal vessels. Interestingly, the vessels lack autonomic innervation and so resistance to flow in the maturing vasculature must also be regulated by local vasoactive effectors (for reviews, see [25], [26]). Therefore, it is the purpose of this review to compare both structural aspects of human fetoplacental development and some of the molecules, cell players and other factors whose interplay sculpts vascular anatomy.
Section snippets
Vasculogenesis (days 21 to 32 post conception)
Fetal vascularization of the first generation of placental villi is the result of local de novo formation of capillaries (vasculogenesis) rather than protrusion of embryonic vessels into the placenta. Vasculogenesis starts at about 21 days post conception (21 dpc), in 4-somite embryos [27], [28]. In the closely related rhesus monkey (gestation 166 days), the onset of vasculogenesis is around day 19 [8]. At this stage, in both species, villous trees comprise primary (solid trophoblastic) and
Formation of capillary networks (day 32 to week 25 post conception)
The subsequent stages of angiogenesis can be divided into three partly overlapping periods:
- 1.
formation of capillary networks from 32 dpc to 25 weeks post conception (25 wpc) by prevalence of branching angiogenesis (Figure 2a–c);
- 2.
regression of peripheral capillary webs and formation of central stem vessels mainly through 15–32 wpc(Figure 2d);
- 3.
formation of terminal capillary loops by prevalence of non-branching angiogenesis (25 wpc until term)(Figure 2e).
From 32 dpc until the end of the first
Formation of stem vessels (week 15 until week 32 post conception)
In the third month of pregnancy, some of the centrally located endothelial tubes of immature intermediate villi achieve larger diameters of 100 μm and more (Figure 2d, e and Figure 3d, e). Within a few weeks, they establish thin media- and adventitia-like structures by concentric fibrosis in the surrounding stroma and by differentiation of precursor smooth muscle (sm) cells expressing α- and γ-sm-actins in addition to vimentin and desmin. This is followed soon afterwards by the expression of
Regression of peripheral capillary nets (weeks 15–32 post conception)
In the second half of pregnancy, the fibrotic process within the stroma of the stem villus advances in a radial manner towards the villous trophoblast. The superficial, subtrophoblastic capillaries become transformed into a rarified paravascular capillary network (Figure 2d, e). In tandem with the expanding villous fibrosis in stem villi, and the transformation of central capillaries into arteries and veins, the superficial capillary networks gradually regress. By term, very few paravascular
Prevailing non-branching angiogenesis (weeks 24–25 post conception until term)
From 25 wpc until term, patterns of villous vascular growth switch from prevailing branching angiogenesis to prevalence of non-branching angiogenesis. This is due to the development of new villous types, the mature intermediate villi, at the furthermost tips of existing villous trees. Mature intermediate villi are slender (80–120 μm diameter), elongated villi (>1000 μm long containing one or two long, poorly branched capillary loops. Analysis of proliferation markers at this stage reveals a
The timing mismatch between the oxygen switch and morphological change
At early and late stages of gestation, it is necessary to protect the fetus from the potentially harmful effects of high oxygen tensions. Development and remodelling of the fetoplacental vasculature may be part of this protection. It is clear that there is a time lag of about two months between the rises in intervillous pO2levels and perfusion rates on the one hand and the morphological changes which are evident qualitatively (a transition from mainly branching to predominantly non-branching
The mature villous vascular system
As a consequence of the growth mechanisms described above, the villous vascular system differs from that of most other human organs in two principal respects. First, the arteries and veins of this low pressure system have a rather thin tunica media and, except for a few residual paravascular capillaries, vasa vasorum are mostly absent [68]. In spite of their low intraluminal pO2(10 and 20 mmHg, respectively), adequate supply of the vascular walls is achieved since the intervillous space
Quantifying the villous vasculature during gestation
Most of the processes involved in angiogenesis can be assessed by stereological quantification of the morphological features of capillaries and villi evident on placental tissue sections [23], [87], [88], [89], [90]. Nett growth of fetal capillaries within villi (which results from the counteracting effects of angiogenesis and vascular pruning) can be described by their total volume, surface area and length. Endothelial cell proliferation can be assessed by estimating the total number of cells
Areas for future research
There is structural evidence to suggest that placental oxygenation is important in controlling fetoplacental angiogenesis and, hence, villous differentiation. However, interactions between these processes, as well as the underlying molecular mechanisms and their spatiotemporal integration, merit further investigation. In the paucity or absence of suitable animal models, more correlative data (on oxygen levels, angiogenic growth factor expression levels and morphological features) are required
Acknowledgments
PK is grateful to the Deutsche Forschungsgemeinschaft and the European Union (Biomed 2) for financial support. TMM wishes to thank The Medical Research Council (Development Grant Scheme, G9826907) and The Wellcome Trust for research funding. DSCJ is supported by The Medical Research Council (Reproductive Angiogenesis Cooperative Group, G9722567).
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