Placenta (2002), 23, Supplement A, Trophoblast Research, 16, S119–S129 doi:10.1053/plac.2002.0792, available online at http://www.idealibrary.com on
Placental Development in Normal and Compromised Pregnancies— A Review T. R. H. Regnaulta, H. L. Galanb, T. A. Parkera and R. V. Anthonya,c,d a Department of Pediatrics, b Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, CO 80262 and c Department of Physiology, Colorado State University, Fort Collins, CO 80523, USA
Intrauterine growth restriction (IUGR) is a significant cause of infant mortality and morbidity. It is now clear that IUGR infants exhibit higher rates of coronary heart disease, type 2-diabetes, hypertension and stroke as adults. Therefore, fetal growth not only impacts the outcome of the perinatal period, but also impacts adult well-being. The etiologies of IUGR are numerous, but are often associated with abnormalities in placental structure and function. The process of implantation and placentation requires the production of a plethora of growth factors, cell-adhesion molecules, extracellular matrix proteins, hormones and transcription factors. Many of these exhibit altered expression within the placenta of IUGR pregnancies. However, it has been difficult to fully assess their role during the development of placental insufficiency (PI) in the human, underscoring the need for animal models. Using an ovine model of PI-IUGR we have observed changes in the expression of vascular endothelial growth factor, placental growth factor, their common receptors, as well as angiopoietin 2 and its receptor, Tie 2. We found that changes in these growth factors can be associated with both acute and chronic changes in placental vascular structure and function. These studies and others are providing needed insight into the developmental chronology of placental insufficiency. Placenta (2002), 23, Supplement A, Trophoblast Research 16, S119–S129 2002 IFPA and Elsevier Science Ltd
INTRODUCTION Intrauterine growth restriction (IUGR) is a significant cause of infant mortality and morbidity, affecting upwards to 8 per cent of all pregnancies (Brar and Rutherford, 1988; Pollack and Divon, 1992). Complications arising during IUGR pregnancies include: (1) intrauterine demise, (2) intrapartum fetal distress, and (3) perinatal asphyxia. In IUGR infants, neonatal complications include: meconium aspiration, metabolic and hematologic disturbances, cognitive dysfunction and cerebral palsy. Furthermore, epidemiological studies provide compelling evidence that IUGR predisposes individuals for coronary heart disease, hypertension, stroke and diabetes during adulthood (Barker et al., 1989, 1990, 1993a,b). While IUGR fetuses can be categorized into symmetric or asymmetric patterns of growth, the majority of cases present an asymmetric pattern of growth, often associated with abnormalities in placental structure and function. The placenta is a multifaceted organ that plays critical roles in maintaining and protecting the developing fetus. These roles include transferring nutrients from the mother to the fetus and waste secretion from the fetus to the mother, acting as a barrier for the fetus against pathogens and the maternal immune system, and serving as an active endocrine organ d
To whom correspondence should be addressed at: ARBL-Foothills Campus, Department of Physiology, Colorado State University, Fort Collins, CO 80523-1683, USA. Tel: 970-491-2586; Fax: 970-4913557; E-mail:
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capable of synthesizing and secreting a plethora of hormones, growth factors, cytokine and other bioactive products. A number of placental structural abnormalities have been associated with IUGR pregnancies, including a decrease in villous number, diameter and surface area, as well as a decrease in arterial number, lumen size and branching (Giles, Trudinger and Baird, 1985; Krebs et al., 1996; Lee and Yeh, 1986; Macara et al., 1996; Salafia et al., 1997). All of these abnormalities can adversely affect placental function, and ultimately deprive the developing fetus of the nutrients required for optimal growth.
PLACENTAL DEVELOPMENT The process of implantation and placentation requires the production of numerous angiogenic growth factors, celladhesion molecules, cytokines and growth factors, extracellular matrix metalloproteinases, hormones and transcription factors. During normal human pregnancy, extravillous trophoblast (EVT) cells migrate and invade the spiral artery vessel walls within the decidua and myometrium (Brosens, Robertson and Dixon, 1967; Meekins et al., 1994), but in pregnancies complicated by pre-eclampsia and/or IUGR, this invasion is largely restricted to the decidual portion of the spiral arteries, and failure of these arteries to become low resistance vessels (Meekins et al., 1994; Redline and Patterson, 1995; Roberson, 2002 IFPA and Elsevier Science Ltd
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Brosens and Dixon, 1975). A number of factors have been associated with EVT invasion, including angiogenic growth factors and their respective receptors. While it remains unresolved as to the exact mechanism causing deficient trophoblast invasion, angiogenic growth factors are logical candidates as regulatory molecules in placental development and function.
Fetal placenta angiogenesis A number of angiogenic growth factors have been identified in human placenta, including: basic fibroblast growth factor (bFGF; Crescimanno et al., 1995; Shams and Ahmed, 1994), hepatocyte growth factor (HGF; Kilby et al., 1996), placenta growth factor (PlGF; Shore et al., 1997; Vuorela et al., 1997) and vascular endothelial growth factor (VEGF; Ahmed et al., 1995; Sharkey et al., 1993; Shore et al., 1997; Vuorela et al., 1997). The later two (VEGF and PlGF) belong to a family of angiogenic growth factors that include VEGF (also called VEGF-A), PlGF, VEGF-B (Olofsson et al., 1996), VEGF-C (Juokov et al., 1996), VEGF-D (Yamada et al., 1997) and VEGF-E (Lyttle et al., 1994). While VEGF, PlGF, VEGF-B and VEGF-C are all expressed in the human placenta (Vuorela et al., 1997; Dunk and Ahmed, 2001), previous investigations have focused primarily on VEGF-A and PlGF. VEGF-A exists as multiple isoforms (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206) that result from alternative splicing of the VEGF primary transcript (Dvorak et al., 1995). Similar alternative splicing of the other family members (e.g. PlGF-1 and PlGF-2) also occurs. Beyond the multiple ligands in this family, there are several receptors capable of binding members of this growth factor family. These include: fms-like tyrosine kinase-1 (Flt-1 or VEGFR-1) and a soluble form of VEGFR-1 (sVEGFR-1; Thomas, 1996), kinase insert domain-containing receptor (KDR, Flk-1 or VEGFR-2; Thomas, 1996), fms-like tyrosine kinase-4 (Flt-4 or VEGFR-3; Joukov et al, 1996) and neuropilin-1 and -2 (Gluzman-Poltorak et al., 2000). While VEGF expression occurs in the villous trophoblast (Ahmed et al., 1995; Sharkey et al., 1993; Vuorela et al., 1997), PlGF expression appears confined to villous syncytiotrophoblast (Shore et al., 1997; Vuorela et al., 1997). Both VEGFR-1 and -2 are expressed in placental villous endothelium (Charnock-Jones et al., 1994; Vuckovic et al., 1996), but VEGFR-1 is also expressed in EVT (Ahmed et al., 1995). VEGFR-1 binds homodimers of VEGF, homodimers of PlGF, or VEGF/PlGF heterodimers, whereas VEGFR-2 binds only VEGF homodimers or VEGF/PlGF heterodimers (Thomas, 1996). When VEGF binds VEGFR-2, rather than VEGFR-1, efficient mitogenic and chemotactic responses occur with endothelial cells. However, the lack of a potent mitogenic response from PlGF binding VEGFR-1 led to the suggestion that it is ineffective in mediating endothelial cell mitogenesis (Thomas, 1996). Furthermore, the binding of VEGF/PlGF heterodimers to VEGFR-2 led to the suggestion that PlGF may diminish VEGF stimulated angiogenesis through heterodimer formation (Cao et al., 1996; DiSalvo et al., 1995).
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However, the presence of functional VEGFR-1 receptors on isolated human trophoblast suggests that VEGF or PlGF binding to trophoblast VEGFR-1 may play a role in EVT invasion and differentiation (Ahmed et al., 1995; Shore et al., 1997). Supporting this hypothesis is the fact that early placental development occurs in a hypoxic environment (Kingdom and Kaufmann, 1999), a known stimulator of VEGF expression (Schweiki et al., 1992; Stavri et al., 1995). Indeed, VEGF expression by placental tissues is upregulated by hypoxia (Shore et al., 1997; Wheeler, Elcock and Anthony, 1995) and downregulated by hyperoxia (Shore et al., 1997), whereas the opposite appears to occur with PlGF (Shore et al., 1997; Khaliq et al., 1999). Hypoxic regulation of VEGF expression is mediated at both the transcriptional and post-transcriptional levels (Figure 1). Increased VEGF mRNA stability during low oxygen tension is an important post-transcriptional response (Levy et al., 1998; Shima, Deutsch and D’Amore, 1995; Shih and Claffey, 1999). A hypoxia stability region in the 3 -untranslated region of the human and bovine VEGF mRNA is bound by heterogeneous nuclear ribonuceloprotein L (hnRNP L), stabilizing the mRNA (Shih and Claffey, 1999), thereby increasing its half-life. Additionally, hypoxia inducible factor-1 (HIF-1) is a basic helix-loop-helix transcription factor comprised of two subunits (HIF-1 and HIF-1) that interacts with a ‘hypoxic response element (HRE)’ in genes induced by hypoxia (Wang et al., 1995). HIF-1 is constitutively expressed, whereas HIF-1 is regulated (Figure 1) by targeted proteolysis by the von Hippel-Lindau tumor suppressor gene product (pVHL; Maxwell et al., 1999). In the presence of oxygen, HIF-1 is degraded (Maxwell et al., 1999), but under hypoxic conditions it is stabilized allowing functional HIF-1 to act via the HRE in genes such as VEGF (Figure 1). While the expression of hnRNP L and HIF-1 have not been described in preeclamptic or IUGR placenta, Caniggia et al. (2000a) reported HIF-1 expression to be high at 5–7 weeks of gestation, decreasing around 9 weeks when placental O2 tension is believed to increase, and HIF-1 was absent at 11–14 weeks. Additionally, these investigators found that transforming growth factor 3 (TGF3) expression parallels that of HIF-1, and treatment of placental villous explants with HIF-1 anti-sense oligonucleotides inhibits the expression of both HIF-1 and TGF3. From these results and others, Caniggia et al. (2000b) suggest that the developmental increase in placental O2 tension reduces HIF-1 expression, which in turn reduces TGF3 expression, thereby removing TGF3’s inhibition of trophoblast differentiation along the invasive pathway. They also suggest that a failure in this developmental switch would arrest EVT at an immature stage, resulting in a shallow invasion of the uterus that would lead to reduced utero-placental perfusion. The basic helix-loop-helix transcriptioin factor HIF-2 is also capable of heterodimerizing with HIF-1 to form functional HIF-1 (Figure 1), and was recently found to be in greater abundance in late-gestational placenta from preeclamptic pregnancies (Rajakumar et al., 2001). In these
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hnRNP-L Figure 1. Schematic representation of the regulation of VEGF gene transcription and mRNA stability. Hypoxia inducible factor-1 (HIF-1) or HIF-2 combine with HIF-1 to form the functional basic helix-loop-helix transcription factor HIF-1 to stimulate transcription of the VEGF gene, via interaction with the hypoxia response element (HRE). This occurs under hypoxic conditions, but is down regulated under normoxic conditions due to targeted proteolysis of HIF-1 and HIF-2, mediated by the von Hippel-Lindau tumor suppressor gene product (pVHL). Additionally, VEGF is regulated post-transcriptionally through stabilization of its mRNA by interaction with heterogeneous nuclear ribonuceloprotein L (hnRNP L).
studies, the concentration of HIF-1 and HIF-2 mRNA was not altered by pre-eclampsia, just the amount of HIF-2 protein. Such findings indicate that both HIF-1 and HIF-2 are important factors that appear to have a role in abnormal placental development, and that primary regulation of these transcription factors is at the protein level (Rajakumar and Conrad, 2000). Recent evidence that pVHL abundance is greater within hypoxic regions of the placenta, and is increased under hypoxic culture conditions of chorionic villi explants, along with HIF-2 indicates a complex regulation (Figure 1) of HIF-1 and HIF-2 by pVHL (Genbacev et al., 2001). While pVHL targets HIF-1 and HIF-2 for proteosome degradation (Maxwell et al., 1999), it appears that pVHL itself may be finely regulated by hypoxia (Genbacev et al., 2001) such that shuttling of HIF-1 back and forth between the cytoplasm and nucleus, coupled with cytoplasmic targeting for proteolysis, provides the exacting control of hypoxia-mediated gene expression.
Angiogenic growth factors in compromised pregnancies The concept that a lack of EVT invasion of the spiral arteries (Meekins et al., 1994; Redline and Patterson, 1995; Roberson, Brosens and Dixon, 1975) results in placental ischemia and the development of pre-eclampsia and IUGR, led to the investigation of VEGF/PlGF expression in these placental pathologies. Kingdom and Kaufmann (1997) suggested that three distinct types of hypoxia may occur in the feto-placental unit. These include: (1) Pre-placental hypoxia, where the mother, placenta and fetus are hypoxia as may occur at high altitude; (2) Utero-placental hypoxia, where maternal oxygenation is
normal but impaired utero-placental circulation results in placental and fetal hypoxia with preserved end diastolic flow in the umbilical arteries as in pre-eclampsia; and (3) Postplacental hypoxia, where only the fetus is believed to be hypoxic, with reduced, absent or reversed end diastolic flow of the umbilical arteries. Another way of classifying these pregnancies resulting in IUGR, is early- or late-onset IUGR (Kingdom et al., 2000), with late-onset generally encompassing the pre-placental and utero-placental hypoxic groups. In pre-eclamptic pregnancies, placental VEGF was reduced (Cooper et al., 1996; Lyall et al., 1997). These results do not coincide with an expectation of placental hypoxia stimulating VEGF expression. The reason VEGF expression is reduced in these pre-eclamptic pregnancies (Cooper et al., 1996; Lyall et al., 1997) is not readily apparent, but suggests the placenta may not always be able to respond to hypoxic conditions with increased VEGF production. However, supporting the concept that pre-eclamptic placenta are hypoxic is the reduction of PlGF in maternal blood observed in pre-eclamptic pregnancies (Torry et al., 1998; Tidwell et al., 2001). In cases of post-placental hypoxia associated with early-onset IUGR, the placental villi are believed to be exposed to greater oxygen tension or ‘placental hyperoxia’ (Kingdom and Kaufmann, 1997). This concept is supported by the observation that the oxygen content of uterine venous blood is close to arterial values in severe IUGR pregnancies (Pardi et al., 1992), and that VEGF expression is reduced and PlGF expression is increased (Lyall et al., 1997; Khaliq et al., 1999). Regulation of angiogenic growth factor expression by hypoxia or hyperoxia involves numerous levels of control, and to fully understand how growth factors like VEGF and PlGF may act in the development of compromised placental
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function, interaction with their receptors and/or other factors must also be considered. For example, we do not know what happens to VEGFR-1 and -2 expression at various times during the development of pre-eclamptic or IUGR pregnancies. In addition to the two more common receptors for VEGF and PlGF, neuropilin-1 and -2 (NP-1, NP-2) are in human umbilical vein endothelial cells (Gluzman-Poltorak et al., 2000), and while they have not been localized to the placenta proper yet, it would not be surprising that they are also involved in placenta vascular regulation. This could add another layer of regulation to the actions of VEGF and PlGF, since NP-1 and NP-2 act as receptors for VEGF165 and PlGF-2 but only NP-2 acts as a receptor for VEGF145 (Gluzman-Poltorak et al., 2000; Migdal et al., 1998). NP-1 not only binds VEGF165, but it also complexes with VEGFR-1 (Fuh, Garcia and deVos, 2000), which led to the hypothesis that VEGFR-1 acts as a negative regulator of angiogenesis by competing for NP-1. To further understand the regulation by endothelial growth factors, one must consider the down-stream effects of VEGF and PlGF interaction with the various receptors that might influence placental circulation. Expression of endothelial nitric oxide synthase (eNOS; Kroll and Waltenberger, 1998) and angiopoietin 2 (Ang2; Oh et al., 1999) are stimulated by VEGF. In late gestation human pregnancies complicated by pre-eclampsia or IUGR, eNOS expression within the syncytiotrophoblast is reported to increase (Myatt et al., 1997), as is the nitric oxide concentration of umbilical venous blood (Lyall et al., 1996). This may be a compensatory response to improve blood flow within the IUGR placenta. However, these data do not fit with the reports of depressed VEGF expression in IUGR placenta (Khaliq et al., 1999; Lyall et al., 1997). This apparent discrepancy between VEGF and eNOS expression may result from the type of IUGR present in these studies; i.e., did the various placentae in these reports exhibit uteroplacental hypoxia or post-placental hypoxia (see discussion above)? Alternatively, different pathways for VEGF and eNOS regulation may be in play. In addition to eNOS, both hypoxia and VEGF stimulate Ang 2 expression (Oh et al., 1999), and Ang 2 stimulates the release of NO from human trophoblast cells in vitro (Dunk et al., 2000). Angiopoietin 1 (Ang1) has been localized in the cyto/syncytiotrophoblast bilayer in firsttrimester human placenta (Dunk et al., 2000), Ang2 was found in the cytotrophoblast layer (Dunk et al., 2000) or in the syncytiotrophoblast (Goldman-Wohl et al., 2000), and their common receptor (Tie2) was found in the cyto/ syncytiotrophoblast bilayer (Dunk et al., 2000) and in endovascular invasive trophoblasts (Goldman-Wohl et al., 2000). While Ang1 acts synergistically with VEGF (Koblizek et al., 1998) to stimulate angiogenesis, Ang 2 is an antagonist to Ang1 and in the absence of VEGF it is thought to destabilize or regress blood vessels (Maisonpierre et al., 1997). At term, in severe IUGR placenta (Dunk et al., 2000), Ang 2 mRNA concentrations were not different from normal placenta, but the amount of Ang 2 protein appeared to be significantly decreased. Reduction in placental Ang 2 (Dunk et al., 2000),
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coupled with reduced VEGF expression and increased PlGF expression (Lyall et al., 1997; Khaliq et al., 1999), fits with the concept of post-placental hypoxia in severe or early-onset IUGR. Unfortunately, the studies examining vascular growth factors and their regulation or function in compromised pregnancies have, by necessity, utilized late gestational or term tissues. While these studies are extremely valuable, they do not provide insight into the role of these factors and receptors in the progressive development of a compromised placenta. Furthermore, with most of the human pregnancies examined we do not know what is being supplied to or being utilized by the utero-placental unit. Consequently, animal models of IUGR are valuable for providing a better understanding of the changes that likely take place in the development and progression of placental insufficiency leading to IUGR.
ANIMAL MODELS OF IUGR A variety of methods exist for creating IUGR pregnancies in several animal species. Of the species used for IUGR studies, the pregnant sheep provides several advantages as an animal model to examine the development and progression of IUGR pregnancies, not the least of which is the ability to instrument both mother and fetus for nutrient transfer and utilization studies. Growth restriction in fetal sheep can be induced by nutrient restriction, nutrient excess, placental embolization with microspheres, limitation of placental implantation sites, umbilical artery ligation, administration of corticosteroids and exposure to elevated environmental temperatures. Of all models of IUGR, sheep exposed to high ambient temperatures develop the most severe IUGR (Alexander and Williams, 1971; Bell, Wilkening and Meschia, 1987; Thureen et al., 1992). Exposure of pregnant ewes to hyperthermic conditions for approximately 80 days (40–120 days post coitus; dpc) results in an IUGR fetus whose placenta is also growth retarded (Bell, Wilkening and Meschia, 1987; Thureen et al., 1992). Similar results are obtained when exposure is for only 55 days (37–93 dpc), with significant reductions in fetal and placental weights (Galan et al., 1999; Regnault et al., 1999). Fetal growth in these pregnancies is asymmetric in nature, as evidenced by greater biparietal diameter/abdominal circumerance ratios (Galan et al., 1999). Furthermore, current evidence indicates that development of IUGR in chronically hyperthermic ewes occurs as a consequence of primary reduction in placental growth in early gestation (Bell et al., 1989; Galan et al., 1999; Thureen et al., 1992), making this an excellent model to examine normal and impaired placental development, i.e., placental insufficiency (PI) that results in IUGR.
Placental development in sheep While structural differences exist between human and sheep placenta, there are important similarities in function and
Regnault et al.: Placental Development in IUGR Pregnancies
functional structure. A primary structural difference in the placenta of these two species occurs as a result of raised areas of non-glandular but well vascularized endometrium, termed caruncles, that are always present within the uterus of sheep (Boyd and Hamilton, 1952; Stegeman, 1974). Intimate association of developing chorion over these raised surfaces establishes the foundation for placental development. In areas of caruncular projections, fetal vascularizatioin of the placenta is derived from the vascularized allantois, following its expansion from the hind-gut at 15 dpc allowing contact and fusion with the chorion (Stegeman, 1974). Early in the fourth week of pregnancy, interdigitation of embryonic and maternal tissues becomes apparent (Boshier, 1969), and in areas of close association between chorion and the caruncular surface, a series of ridges and grooves form which eventually give rise to fetal villi of the cotyledon (Steven, 1975). The maternal caruncles continue to grow and develop deeply branched crypts into which the cotyledonary villi project, elongate and branch forming an apposing network of fetal villi within the maternal crypts (Steven, 1975). Maximum surface area obtained by branching of the villi appears to be regulated by the size of the placentome (caruncle plus cotyledon), which is directly correlated to uterine weight in normal sheep (Stegeman, 1974). While the placenta of sheep can be divided into numerous discreet attachment sites, within each placentome the villous tree is structurally similar to that which arises in the human, as both can be divided into stem, intermediate, and terminal villi (Leiser et al., 1997; Kaufmann, Sen and Schweikhart, 1979; Kaufmann, 1982). Furthermore, the structure of fetal vessels (stem arteries and veins, intermediate arterioles and venules, and terminal capillaries) of sheep and humans are comparable (Leiser et al., 1997), allowing the sheep to serve as a model of fetal placenta vascularization throughout the period of placental development.
PI-IUGR in sheep The natural phenomenon of hyperthermia-induced PI-IUGR in sheep allows the opportunity to study PI-IUGR in a commonly used fetal physiology model. Early studies (Alexander and Williams, 1971) examined the effects of exposure to heat at various stages of gestation on placental and fetal outcomes. Pregnant ewes exposed to hyperthermic conditions in mid-gestation (50–100 dpc), mid-gestation through term (50–150 dpc), as well as only in the final third of gestation (100–150 dpc), resulted in PI-IUGR. The decrease in placental mass associated with hyperthermia during the middle and last third of gestation has been implicated as the underlying cause of IUGR (Galan et al., 1999; Regnault et al., 1999). Indeed, placental size has been linked with size of the fetus late in pregnancy when fetal demands are highest (Mellor and Murray, 1981, 1982), but placental size is a crude indicator that does not address the mechanisms responsible for IUGR. Studies of the PI-IUGR sheep model show that it has characteristics in common with human IUGR. For example,
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ewes exposed to hyperthermic conditions from 39–125 dpc had fetal and placental masses roughly one-half of their thermoneutral counterparts (1.6 vs. 3.1 kg; 0.15 vs 0.36 kg, respectively) at 135 dpc (Thureen et al., 1992). These fetuses were hypoxic and hypoglycemic, and the fetal hypoglycemia resulted in a greater maternal : fetal glucose gradient (Thureen et al., 1992). Similar results from human IUGR pregnancies have been reported (Economides and Nicolaides, 1989; Pardi et al., 1993; Nieto-Diaz et al., 1996). Transport of two essential amino acids, leucine and threonine, across the PI-IUGR placenta is also reduced, resulting in the routing of these two amino acids into fetal accretion and away from oxidation (Anderson et al., 1997; Ross et al., 1996). The reduction in transport is not simply a function of placental mass, since transport was still less when normalized on placental weight. A reduction in the fetal : maternal leucine molar percent enrichment has been observed in both sheep (Ross et al., 1996) and human (Marconi et al., 1999) IUGR. Reductions in fetal amino acid concentrations, reduced placental transport, and a reduction in the system A transporter in cases of human IUGR (Glazier et al., 1997; Jansson, Scholtbach and Powell, 1998; Marconi et al., 1999; Norberg, Powell and Jansson, 1998) agree with the results from sheep. Furthermore, reduced umbilical blood flow, and increased umbilical artery pulsatility index and placental vascular resistance have been reported for both human and sheep IUGR pregnancies (Galan et al., 1998; Thureen et al., 1992; Trudinger et al., 1987). Combined, these data highlight important functional similarities between human IUGR and sheep PI-IUGR pregnancies induced by hyperthermia (summarized in Figure 2), and provide evidence of true functional-placental insufficiency. When pregnant ewes are subjected to a hyperthermic (HT) environment (40C for 12 h/d and 35C for 12 h/d; 30–40 per cent relative humidity) or a thermoneutral (TN) environment (20C) beginning at 40 dpc, the HT exposure results in a rapid (within 2–3 d) and sustained increase (HT 39.860.1 vs. TN 39.200.1C; Pc0.001) in core body temperature (CBT) (Regnault et al., 1999). Exposure to this regimen for either 55 or 80 days results in significant reductions in placental and fetal weights near term (Galan et al., 1999). Recently, we have collected tissues from HT and TN pregnancies following treatment for 15 days (55 dpc), 50 days (90 dpc) of HT, or 80 days followed by TN exposure for an additional 15 days (135 dpc). Placental weights tended to be smaller (Pc0.10) by 55 dpc, whereas it took until 90 dpc to see a reduction in fetal weights. Interestingly, at 55 dpc the fetal/placental weight ratio is greater (Pc0.01) in HT pregnancies. The fetal/ placental weight ratio as an indicator of placental function, suggests that placental function may be acutely enhanced in HT pregnancies. Due to the changes in umbilical Dopplar velocity wave forms observed in human IUGR (Krebs et al., 1996; Salafia et al., 1997; Trudinger et al., 1989) and in our sheep model of PI-IUGR (Galan et al., 1998), vascular architecture in HT and TN ewes was examined in the developing placenta by generating vascular erosion casts at 90 dpc (Figure 3). Fetal vessels
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Figure 2. Summary of the common features of IUGR pregnancies that have been described in the human and in a sheep model of IUGR induced by hyperthermic exposure of pregnant ewes. MPE=molar per cent enrichment.
from TN placentae (Figure 3, panel B) were relatively straight and organized parallel to one another. However, fetal vessels of HT placentae were coiled and appeared in a more random orientation relative to neighboring vessels (Figure 3, panel C). Additionally, sinusoidal dilations also appeared to be more numerous in HT fetal vessels (Figure 3, panel C). Similar architectural differences are observed in fetal placenta vascular casts of ewes kept at sea level (normoxia) with those of ewes kept at high altitude (hypoxia) between 45 and 140 dpc (Krebs, Longo and Leiser, 1997). Exposure of pregnant ewes to conditions of hypoxia increased the length and degree of coiling of fetal and maternal placental vasculature. These investigators concluded that the increase in vessel length and coiling occurred as an adaptation to supply more oxygen to the fetus. The similarity in these results may indicate that alterations in placental vasculature resulting from exposure of pregnant ewes to hypoxic or hyperthermic conditions may be mediated by the same mechanisms. Using the fetal placenta cotyledons and uterine caruncles collected at 55, 90 and 135 dpc from TN and HT pregnancies, we have characterized the expression of VEGF, PlGF and their receptors (VEGFR-1 and -2). At 55 dpc, there was a significant tissue by treatment interaction for VEGF mRNA expression, as a result of increased expression of cotyledonary VEGF in HT ewes only (Pc0.01). There was no difference in VEGF mRNA expression between treatments for caruncular tissue at 55 dpc, nor was mRNA expression different between treatments or tissue types at 90 dpc. The concentration of PlGF mRNA was not effected by treatment group or tissue type at either gestational age examined. With the 135 dpc tissues, we found a reduction (Pc0.01) in VEGF mRNA within fetal cotyledons, and an increase (Pc0.05) in maternal caruncle PlGF mRNA (summarized in Figure 4). The enhanced expression of VEGF in HT pregnancies at 55 dpc may be indicative of an increased state of hypoxia within the developing HT placenta, and could be involved in driving the altered vasculature presented in Figure 3. However, it is not known if the HT environment creates a more hypoxic
environment for early placental development, or if the increased CBT in HT ewes may directly stimulate VEGF expression. To better understand the functional significance of the changes observed in VEGF expression within HT pregnancies, we examined mRNA concentrations for the VEGF/ PlGF receptors, VEGFR-1 and -2. Both VEGFR-1 and -2 mRNA concentrations in cotyledonary tissue rose significantly with advancing gestational age (Pc0.05), but no effect of gestational age was observed in uterine caruncular tissue. No effect of the HT environment was observed for either receptor at 55 dpc in either fetal placental cotyledons or maternal uterine caruncles, or in maternal uterine caruncles at 90 dpc. However, both VEGFR-1 and -2 mRNA concentration in fetal cotyledons was significantly reduced at 90 dpc. At 135 dpc, only VEGFR-1 mRNA concentration was reduced in fetal cotyledons (Figure 4). Combined (Figure 4), the results of our examination of VEGF, PlGF and their receptors at various time-points in our IUGR pregnancies, point to differing acute and chronic responses to the HT environment (Figure 4). Acute up-regulation of VEGF expression may be a compensatory mechanism aimed at maintaining normal placental development, by enhancing angiogenesis. This appears to be borne out in preliminary histomorphometry data. Within the basal plate region of the placenta, the number of maternal vessels is reduced (30.83.38 vs. 15.02.49; TN vs. HT; Pc0.01). However, within the chorionic plate, the number of fetal vessels is increased (15.61.60 vs. 22.171.66; TN vs. HT; Pc0.05). A reduction in maternal placental angiogenesis may result in fetal placental hypoxia, thereby driving increased VEGF expression and accelerated fetal placental angiogenesis. Interestingly, we have preliminary data indicating that both VEGF and eNOS mRNA concentrations are reduced (Pc0.01) in fetal lung at 55 dpc. This could result if fetal PO2 was increased. Such a scenario might exist if increased fetal placental angiogenesis and vessel number resulted in increased O2 extraction by the fetal placenta. If ‘fetal hyperoxia’ existed during early to mid-gestation, followed by fetal hypoxia
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Figure 4. Summary of changes in placental expression of vascular growth factors and their receptors in control and IUGR ovine pregnancies. Arrows indicate significant changes in the mRNA concentration within either maternal placental or fetal placental tissues, during acute (55 dpc), transitional (90 dpc) or chronic (135 dpc) progression of placental insufficiency. Horizontal lines indicate no significant changes.
Figure 3. Scanning electron micrographs of ovine fetal placental vascular casts. Panel A=low power magnification (100) of fetal placenta vasculature (90 dpc) extending from the intermediate zone (top) to the basal plate zone (bottom). Panel B=Fetal placenta vasculature from a control pregnancy, in the intermediate zone at higher magnification (1000) showing organized outgrowth of vessels which remain relatively straight. Panel C=Fetal placenta vasculature (1000 magnification) from an IUGR pregnancy in the intermediate zone demonstrating an increase in vessel coiling and overall disorganized arrangement of vessels.
(Thureen et al., 1992) during late-gestation, it is easy to envision how fetal organogenesis could be negatively impacted and could lead to fetal hypertension.
In contrast to our results following acute exposure to the HT environment, chronic exposure (50 days), or possibly a failure to adequately compensate, results in down-regulation of both receptors at 90 dpc. The consequences of the later may be an inability to respond to VEGF. The importance of the reduction in VEGFR-1 alone at 135 dpc is not clear. Since it is possible that the measured reduction in VEGFR-1 resulted from a reduction in the expression of the soluble form of VEGFR-1 mRNA, we tested our samples for the presence of sVEGFR-1 mRNA. As yet, we have not been able to detect mRNA encoding sVEGFR-1 within sheep placenta, suggesting that the observed changes result from VEGFR-1 transcripts. If only ‘full-length’ VEGFR-1 mRNA is reduced, this may be a mechanism to drive VEGF binding to VEGFR-2. This would be advantageous in regards to VEGFs ability to interact with a functional receptor, thereby stimulating angiogenesis in an attempt to overcome a deficit in placental vasculature. However, one must keep in mind that fetal placental VEGF mRNA concentration is reduced and maternal placental PlGF concentration is increased at 135 dpc. Consequently, stimulation of angiogenesis through VEGFR-2 may be impaired by a lack of VEGF. Other regulators of placental angiogenesis and placental vascular control have also been analysed in the samples collected at 55, 90 and 135 dpc. Both Ang 1 and Ang 2 mRNA concentration were greater (Pc0.05) in HT fetal cotyledons at 55 dpc, but not at other gestational ages, or in maternal caruncles (Figure 4). Interestingly, Tie2 mRNA concentration in fetal cotyledons was increased at 55 dpc, unaffected by treatment at 90 dpc, and reduced at 135 dpc. Additionally, there was a significant reduction (Pc0.05) in its mRNA
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Placenta (2002), Vol. 23, Supplement A, Trophoblast Research, Vol. 16
Control A
IUGR V
PO2 84 mmHg
PO2 52 mmHg
A
PO2 61 mmHg**
PO2 84 mmHg
Placenta
Placenta
PO2 19 mmHg**
PO2 29 mmHg a
V
g
a
g
Uterine blood flow to Umbilical blood flow ratio 2.19 + 0.84 4.38 + 0.48** Figure 5. Schematic representation of utero-placental PO2 in normal and IUGR ovine pregnancies at 135 dpc. IUGR was induced by HT treatment from 40 to 120 dpc, followed by TN conditions until 135 dpc when these measurements were taken. A=maternal femoral artery; V=uterine vein; a=fetal femoral artery; g=umbilical vein. **Pc0.01 between treatment comparisons.
concentration at 135 dpc in maternal caruncles. These results indicate that angiopoietin stimulated angiogenesis or vascular remodeling may be increased acutely in HT pregnancies, but reduced chronically. Immuno-blotting of maternal caruncle and fetal cotyledon tissue protein obtained at 55, 90 and 135 dpc revealed contrasting results. The concentrations of Ang1 and Tie 2 were not effected by treatment, but there was a significant increase in Ang2 at 55 dpc and a decrease at 135 dpc in HT fetal cotyledons. The contrasting results between the mRNA and protein concentration data are not easily explainable, but are similar in many ways with results obtained with human IUGR placenta (Dunk et al., 2000). In those studies, changes in Ang2 tissue concentration without concordant changes in Ang2 mRNA were observed. It is possible that post-transcriptional regulation of both Ang2 and Tie2, such as exists with VEGF (Shih and Claffey, 1999), could explain these discrepancies. Alternatively, disagreement between measures of tissue concentration of mRNA and protein for these growth factors and receptors could result from the fact that they exist in more than one cell type within the placenta (Dunk et al., 2000; Goldman-Wohl et al., 2000). Expression by one cell type may be increased while at the same time it is decreased in another cell type. Such a pattern of expression could easily impact the results of measuring whole tissue concentrations of mRNA or protein, making it difficult to fully ascertain the ‘functional’ amount of a given ligand or receptor. It must also be kept in mind that many things can impact tissue concentration of a given protein, including rate of synthesis, rate of secretion, interaction with extracellular matrix, amount bound by receptor and the turnover rate of bound receptor. Consequently, we believe that tissue concentration of mRNA may give a more accurate assessment of the activity of these systems within the placenta. Our results with VEGF, and the reported relationship between VEGF and endothelial nitric oxide synthase (eNOS) expression (Kroll and Waltenberger, 1998), prompted us to begin examining eNOS in our collected tissues. We have
measured eNOS protein concentration in cotyledons from TN and HT pregnancies at 90 dpc, and localized eNOS in cotyledon cross-sections by immunohistochemistry (Galan et al., 2001). The fetal placental cotyledons had greater (Pc0.01) concentrations of eNOS than did the maternal uterine caruncles, and cotyledon eNOS concentration was reduced by ]50 per cent in the HT pregnancies at 90 dpc (Pc0.05). We have yet to make these comparisons in our 55 and 135 dpc tissues to determine if eNOS expression is altered in the same manner as VEGF and Ang 2. Based on our results so far, we postulate that eNOS expression will be stimulated in the 55 dpc, and that the diminished expression of eNOS at 90 dpc is a function of the reduction in VEGFR-1 or VEGFR-2. Our immunohistochemistry data (Galan et al., 2001) localized eNOS to the vascular endothelium, but also to chorionic binucleate cells (BNCs). These cells are thought to be analogous to human syncytiotrophoblasts, but they are also migratory in nature (Wooding, 1992). It is possible that the BNC source of eNOS may provide a means for the fetal placenta to modify maternal uterine caruncle vascular tone, when the BNCs migrate and fuse with the syncytium thereby releasing their contents towards the maternal vasculature (Wooding, 1992). The reduction in eNOS that we observed in our HT pregnancies is in contrast to the increased eNOS and nitric oxide production by human placenta from IUGR and preeclamptic pregnancies (Lyall et al., 1996; Myatt et al., 1997). However, the human studies utilized late-gestation tissues, and it is possible that we will see similar results with our 135 dpc tissues. This would again highlight that measured alterations in placental development of IUGR pregnancies are gestational age dependent.
FUNCTIONAL SIGNIFICANCE The question remains as to what functional impact the altered expression of vascular growth factors and their receptors may
Regnault et al.: Placental Development in IUGR Pregnancies
have on delivery of O2 and nutrients to the rapidly growing fetus during later gestation. Earlier studies of human and HT-induced sheep IUGR pregnancies demonstrated that these fetuses are hypoxic and hypoglycemic (see discussion above; Figure 2). Yet, it has been suggested (Kingdom and Kaufmann, 1997; Kingdom et al., 2000) that early-onset IUGR may result in ‘placental hyperoxia’ during late gestation. Our observations of growth factor and receptor expression during late-gestation sheep IUGR pregnancies (Figure 4) agree with this concept, and are supported by recent data obtained with these pregnancies (Figure 5). At 135 dpc, our IUGR pregnancies exhibit increased (Pc0.01) uterine venous blood flow and P2. This coupled with reduced umbilical vein blood flow and P2, suggests that a barrier to placental O2 extraction exists during late-gestation in these pregnancies. The extent of this extraction barrier is borne out when one considers the doubling of the uterine vein : umbilical vein blood flow ratio (Figure 5). We believe that this barrier results from the changes exhibited in vascular growth factors and their receptors during the development of these IUGR pregnancies. Furthermore, the combination of our results support the concept of ‘placental hyperoxia’, at least at the maternal-fetal
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interface of the placenta, derived from examination of human IUGR placenta (Kingdom and Kaufmann, 1997; Kingdom et al., 2000). In this review, we have tried to highlight a number of the factors that may be involved in altered placental vascular development in both human and sheep IUGR pregnancies. While it is not easy to assign cause and effect from the data discussed, it is likely safe to assume that altered vascular development in the placenta may be a driving force behind many of the hallmarks of IUGR pregnancies. From the summarization of our results from sheep IUGR pregnancies, it appears that altered expression of vascular growth factors and their receptors occurs in a fashion such that acute changes may be quite different from changes observed in late-gestation. Therefore, a thorough understanding of when and why these changes take place may be paramount to our understanding of the development of IUGR-associated placenta. Such understanding will likely be obtained only from use of a consistent animal model, such as ours, in which the ex vivo examination of regulator factor expression can be compared to in vivo fluxes of O2 and nutrients across the placenta.
ACKNOWLEDGEMENTS The authors would like to thank Drs B. deVrijer, S. W. Limesand and R. B. Wilkening, as well as Ms M. Davidsen and A. S. Erickson-Hagen for their contributions to the experimental results and interpretation of those results discussed in this review. This work was supported in part by a grant from the National Institutes of Health (HD 20761).
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