Mechanistic insights into the development of severe fetal growth restriction

Abstract

Fetal growth restriction (FGR), which most commonly results from suboptimal placental function, substantially increases risks for adverse perinatal and long-term outcomes. The only “treatment” that exists is delivery, which averts stillbirth but does not improve outcomes in survivors. Furthermore, the potential long-term consequences of FGR to the fetus, including cardiometabolic disorders, predispose these individuals to developing FGR in their future pregnancies. This creates a multi-generational cascade of adverse effects stemming from a single dysfunctional placenta, and understanding the mechanisms underlying placental-mediated FGR is critically important if we are to improve outcomes and overall health. The mechanisms behind FGR remain unknown. However, placental insufficiency derived from maldevelopment of the placental vascular systems is the most common etiology. To highlight important mechanistic interactions within the placenta, we focus on placental vascular development in the setting of FGR. We delve into fetoplacental angiogenesis, a robust and ongoing process in normal pregnancies that is impaired in severe FGR. We review cellular models of FGR, with special attention to fetoplacental angiogenesis, and we highlight novel integrin-extracellular matrix interactions that regulate placental angiogenesis in severe FGR. In total, this review focuses on key developmental processes, with specific focus on the human placenta, an underexplored area of research.

The survival of a species is dependent on its ability to reproduce, and different reproductive mechanisms have evolved across phyla. For eutherian mammals, the development of the placenta is a key component of reproductive success, as it is responsible for creating an optimal in utero environment for the developing fetus. Highly coordinated interactions between the mother, fetus, and placenta are essential for a successful pregnancy, and abnormalities in any or all of these factors can result in a multitude of complications.

Impaired placental development is implicated in many common pregnancy complications such as fetal growth restriction (FGR), a condition that occurs when a fetus is unable to meet their inherent growth potential. While there are various causes of FGR, the most common etiology is suboptimal placental function. This oftentimes requires preterm delivery in order to avert a stillbirth, putting a fetus at risk for perinatal complications as a result of prematurity that is further compounded by growth restriction. Furthermore, fetal exposure to an inadequate in utero environment increases risk for long-term complications such as cardiometabolic disorders, predisposing these individuals to developing FGR in their future pregnancies. This creates a multi-generational cascade of adverse effects stemming from a single dysfunctional placenta.

The mechanisms underlying placental-mediated FGR are largely unknown, and for the vast majority of FGR cases, no treatments exist other than delivery. Recent studies have shown that the placenta is not only a contributing factor but also a potential candidate for intervention (1, 2). Thus, understanding mechanisms underlying both normal and abnormal placental development is imperative for the discovery of efficacious preventative and therapeutic modalities.

Human placental development

The main function of the placenta is to support the growth of a healthy fetus, and this task is accomplished via the maternal-fetal exchange of nutrients and respiratory gases. This requires the development of two vascular systems: the uteroplacental and fetoplacental circulations. The uteroplacental circulation is derived from blood flow originating in the maternal vasculature, while the fetoplacental vessels that circulate fetal blood contribute to the surface area necessary for nutrient, gas, and waste exchange to meet the demands of a growing fetus. Should the development of either placental vasculature be disrupted, the risk of developing obstetric complications and associated adverse perinatal and long-term outcomes is substantially increased.

Establishment of the maternal uteroplacental circulation

To prepare for blastocyst implantation, the uterine endometrium undergoes decidualization, resulting in changes in hormone production, immune profile, and biochemical composition (3, 4). Ideally, after the uterus is appropriately primed for implantation, the embryonic pole of the blastocyst establishes contact with the uterine endometrial epithelium, and placental development is initiated. Implantation is complete by about 8 days post-conception (p.c.), and at this point, the conceptus is surrounded by a single layer of cytotrophoblast and a multinucleated layer of syncytiotrophoblasts, two key cell types within the definitive placenta (5, 6).

Trophoblast invasion into the decidua and myometrium is a critical aspect in formation of the maternal uteroplacental circulation. Extravillous trophoblast, which do not take part in development of the definitive placenta, are invasive trophoblasts that migrate from the anchoring villi of the cytotrophoblastic shell into the decidua and endometrial stroma. Approximately 2–3 weeks p.c., these cells continue to travel through the endometrial stroma, penetrating the inner third of the myometrium and thereby gaining access to the maternal uterine spiral arteries. Endovascular-type extravillous trophoblasts continue to migrate down the lumens of these arteries, forming plugs. Simultaneously, interstitial-type extravillous cytotrophoblasts accumulate within the muscular vessel wall (7, 8). Together with uterine natural killer cells and decidual macrophages, both extravillous trophoblast types then promote apoptosis of maternal vascular endothelium and vascular smooth muscle cells (7, 9). As the plugs dissolve, the maternal circulatory component of the placenta is established (7, 10, 11). In normal pregnancies, proper trophoblast invasion and maternal uterine spiral artery remodeling result in conversion of these high resistance, small diameter vessels into low resistance, large diameter conduits (12). This allows for blood not only to efficiently enter the intervillous spaces but to also reside in a low resistance state where it can bathe the placenta in maternal blood (13).

As development progresses, cytotrophoblasts of the chorionic villi continue to differentiate into multinucleated syncytiotrophoblasts, which are in direct contact with maternal blood within the intervillous spaces. This layer of syncytiotrophoblasts enclose the placental villous stroma, which is comprised of extracellular matrix in addition to stromal fibroblasts, myofibroblasts, Hofbauer cells (a placental-specific immune cell similar to a macrophage), and the fetoplacental vasculature that is lined by fetal endothelium (14). The syncytial-lined stroma and accompanying cell types form the chorionic villi, which act as the functional unit of the placenta where nutrient, gas, and waste exchange occurs. This topic of placental development and establishment of maternal-fetal exchange is eloquently discussed in more detail in a recent review article (15).

Development of the fetoplacental vasculature

By approximately three weeks p.c., clusters of hemangioblasts begin to differentiate into endothelial cells that undergo vasculogenesis and form villous capillary segments. These capillaries ultimately establish a connection with the intraembryonic circulation, forming the fetoplacental vasculature, which is discrete from the maternal uteroplacental circulation (Figure 1) (16, 17). Early capillary segments within the placenta fuse to form a simple capillary bed, with initial expansion occurring through a balance of non-branching and sprouting angiogenesis (18). The former allows for elongation of these vessels, while the latter results in ramification of the early vascular tree. These early vessels also exhibit low pericyte coverage, further suggestive of their high capacity for remodeling and expansion (19).

Figure 1.

Development of the fetoplacental vasculature/chorionic villi. (A) Initially, clusters of hemangioblasts differentiate into endothelial cells that undergo vasculogenesis, forming early fetal capillary segments. (B) These early vessels fuse, resulting in a simple capillary bed. (C) As the villus continues to develop, the capillary bed expands primarily through branching angiogenesis. (D) Simultaneously, as gestation progresses, the centrally located capillaries become transformed into stem vessels. Non-branching angiogenesis also ensues in order to elongate the capillary loops. (E) Throughout the second half of pregnancy, longitudinal capillary growth continues, exceeding elongation of the villus, which results in capillaries bulging and coiling along the terminal surface of the villous structure.

Entering the second trimester, stem vessels, which comprise part of the macrovasculature of the placenta, begin to form. These precursors to villous arteries and veins arise from endothelial tubes that reside in the center of developing villi (20). Starting in mid-pregnancy and continuing to term gestation, the fetoplacental vasculature switches from predominantly branching angiogenesis to non-branching angiogenesis (Figure 1). This combination of non-branching and some continued branching angiogenesis allow for increases in surface area. Whereas branching angiogenesis leads to formation of sprouts from preexisting vessels, non-branching angiogenesis drives elongation of existing capillaries, creating loops and bulges as they abut the trophoblast (18, 21). These capillaries are housed in terminal villi, and as gestation advances, the capillaries focally dilate and contribute to the thin, vasculo-syncytial membrane, the principal site for maternal-fetal exchange (18, 22, 23). These critical aspects of placental development, which are responsible for the majority of terminal villous development in the latter half of pregnancy, minimizes the distance between maternal and fetal vasculatures, thereby increasing transport efficiency as gestation progresses. By term gestation, the surface area of the chorionic villi reaches 12–14 m², while that of the fetoplacental vasculature is closer to 15 m² (24).

Severe, early-onset FGR

Clinical features

Fetal growth restriction is clinically identified when the estimated fetal weight (EFW) is less than the 10^th percentile for gestational age. This diagnosis is further stratified by severity, and approximately 20–30 percent of all FGR cases are categorized as severe (25). Criteria that lead to the classification of severe FGR include an EFW or abdominal circumference less than the 3^rd percentile for gestational age and/or abnormal umbilical artery Dopplers with onset prior to 32 weeks (26). Of these features, absent or reversed umbilical artery Doppler velocimetry are indicative of a dysfunctional placenta and associated with the worst outcomes (27, 28, 29, 30, 31, 32). Normally, because the placenta is an organ with low vascular resistance, there is forward flow within the umbilical arteries at all points of the fetal cardiac cycle. However, with increased vascular resistance, a key manifestation of placental dysfunction in severe FGR, forward velocities during cardiac diastole are progressively reduced, potentially progressing to absent or even reversed umbilical artery waveforms at end-diastole. If a growth-restricted fetus exhibits either absent or reversed end-diastolic velocimetry, delivery typically occurs no later than 30–34 weeks (26). Clinically, this is considered the optimal delivery timing for these cases, as the potential benefits of gaining more fetal maturity are thought to be outweighed by risks for fetal injury or stillbirth in the absence of delivery. Furthermore, delivery even prior to 30 weeks is oftentimes needed in order to avert risk for stillbirth, incurring additional risks of prematurity in addition to those of growth restriction.

While suboptimal placental function related to impaired uteroplacental and fetoplacental perfusion is a common pathway that often results in the phenotype of FGR (33, 34, 35), limitations in diagnosis and clinical management paradigms have significantly hampered the ability to substantially improve obstetric outcomes. In fact, clinical management paradigms of severely growth-restricted pregnancies have not substantially changed from an obstetric perspective for at least the past two decades. Thus, understanding mechanisms underlying development of severe FGR is critical if we are to make substantial strides in improving perinatal and long-term outcomes.

Uteroplacental contributions to severe FGR

Aberrant maternal uteroplacental blood flow is a pathophysiologic precursor contributing to the development of severe FGR. Inadequate extravillous trophoblast invasion of the endometrial stroma and uterine vessels, resulting in deficient spiral artery remodeling, has been well-established as one key mechanism underlying impaired maternal uteroplacental blood flow (7, 33). More recent data also demonstrate that arcuate arteries, placental bed arteriovenous anastomoses, and radial arteries contribute to the UtA waveform profile (36). There are several consequences to uteroplacental malperfusion, including the potential for aberrant hypoxia, increased oxidative stress, and nutrient dysregulation, all of which further contribute to uteroplacental dysfunction. Several recent reviews have elegantly described these processes in detail (7, 35, 37, 38).

From a mechanistic perspective, there are several obstacles to targeting abnormalities within the maternal uteroplacental vascular compartment. Clinically, while uterine artery Dopplers could technically serve as a surrogate marker of how well the spiral arteries and its distal branches have adapted to pregnancy, several studies have demonstrated limited diagnostic accuracy and clinical utility in prediction of both FGR and other adverse outcomes (39, 40, 41, 42). For these reasons, its routine use has not been recommended in diagnosis or management of FGR by various expert societies (25, 26, 43). Furthermore, although the uteroplacental circulation is fully established by the end of the first trimester, the key mechanisms establishing the bulk of maternal uteroplacental blood flow are initiated within a few weeks of conception, a timeframe when intervention may be more difficult as many women are not yet aware that they have conceived.

Role of the fetoplacental vasculature in severe FGR

In contrast, clinical data demonstrate solid evidence that assessment of fetoplacental vascular resistance through umbilical artery Dopplers in management of FGR is reliable, and it is currently standard-of-care to interrogate umbilical arteries in the setting of FGR (25, 26, 28, 44, 45). While Doppler studies show that impairment of flow can occur individually or concurrently in the maternal and fetoplacental vascular compartments, several studies suggest some degree of interdependence (46, 47, 48). For instance, in a sheep model, partial occlusion of the umbilical circulation locally reduces uterine blood flow (46). This suggests that in theory, improvement of fetoplacental blood flow could result in downstream improvement in the maternal vascular compartment.

Vasomotor tone is one mechanism by which fetoplacental vascular resistance is regulated. Unlike most vascular beds where arteriolar vascular resistance is regulated both autonomically and through humoral factors, placental chorionic plate and stem villous vessels that are analogous to small arterioles lack innervation (49, 50, 51, 52). Instead, endothelial-derived vasoactive mediators are the primary mechanism of vasomotor control within the placenta (49, 52). Within severe FGR pregnancies, intrauterine sampling of umbilical cord blood demonstrated that concentrations of vasoconstrictive endothelin-1 were significantly higher, while the stable metabolite of the vasodilator prostacyclin was lower as compared to gestational age-matched, appropriately grown fetuses (53). Similarly, others have also confirmed lower levels of the stable metabolite of prostacyclin within cord blood at the time of delivery (54). More recently, investigators have demonstrated that pulsatile flow enhanced nitric oxide production within the placental vasculature, ultimately resulting in decreased basal impedance (55). Together, these data suggest that differential regulation of vasomotor tone of the larger placental vessels may be one mechanism by which fetoplacental vascular resistance is abnormally elevated in severe FGR.

In addition to regulation of vasomotor tone, fetoplacental vascular resistance is also mediated by the degree of vascular branching. Expansion of the fetoplacental vasculature occurs throughout gestation, making it more accessible for potential therapeutic intervention. Placentas of severely growth-restricted pregnancies with abnormally elevated fetoplacental vascular resistance (absent/reversed umbilical artery Dopplers), exhibit inappropriately elongated, thin, and underdeveloped terminal villous vessels secondary to impaired angiogenesis (16, 56, 57, 58, 59). These placental alterations associated with severe FGR ultimately lead to decreased vessel surface area, thereby impairing nutrient, gas, and waste exchange between the fetal and maternal circulations, while also adding substantial strain to the fetal heart (57, 59, 60, 61). Together, these pathophysiologic consequences contribute to substantially increased risk for adverse perinatal, neonatal, and long-term outcomes. The cellular and molecular mechanisms underlying impaired placental angiogenesis, however, remain understudied.

Cellular models of the fetoplacental vasculature in severe, early-onset FGR

A paucity of scientific models that accurately recapitulate the complexity of human placentation have resulted in large gaps in our mechanistic understanding of FGR pathogenesis. While animal models provide valuable systemic context that cannot be found in ex vivo and in vitro models, commonly used rodent models have considerably different placentas than humans, making it difficult to extrapolate these findings to human FGR. Placentas of non-human primates, although more similar, are limited by expense and accessibility. Thus, in order to interrogate mechanisms specific to severe, early-onset FGR, our lab has developed a method of isolating human fetoplacental endothelial cells (ECs) from these clinically well-validated, severe FGR placentas with absent or reversed umbilical artery Doppler velocimetry along with ECs from control placentas.

Leveraging this model, we have identified different mechanisms by which fetoplacental vascular resistance is abnormally elevated in severe FGR. First, ECs from severe FGR preferentially secrete vasoconstrictive prostanoids as compared to control ECs. This effect is mediated, at least in part, through estrogen receptor-beta (ESR2). ESR2 is the sole estrogen receptor expressed in fetoplacental ECs, whereby high ESR2 expression in ECs of severe FGR placentas results in up-regulation of cyclooxygenase-1 (PTGS1) and −2 (PTGS2), leading to increased prostaglandin H2 substrate for use by downstream prostanoid synthases. ESR2 differentially regulates two key prostanoid synthases, down-regulating prostacyclin synthase (PTGIS) while simultaneously up-regulating prostaglandin F synthase (AKR1C3). The net result of this is less vasodilator production and enhanced vasoconstrictor secretion. Interestingly, this effect appears to be independent of estradiol or ESR2-specific agonists, suggesting some potential for ligand-independent ESR2 regulation of these prostaglandin synthases (62).

Effects of vasoactive mediators on fetoplacental vascular tone are often thought to be more transient, whereas deficient angiogenesis within the fetoplacental vasculature oftentimes leads to persistent and severe umbilical artery Doppler abnormalities. Within our EC model, we have consistently found impaired migratory properties in severe FGR ECs as compared to control cells, demonstrating inherent angiogenic defects in ECs of severe FGR placentas (63, 64). One mechanism that contributes to this phenotype is deficient expression of the aryl hydrocarbon receptor nuclear translocator (ARNT) transcription factor in severe FGR ECs. ARNT is a heterodimeric partner to hypoxia inducible factor 1-alpha (HIF1A), and together, this complex binds to hypoxia response elements (HREs) within various genes, including vascular endothelial growth factor A (VEGFA). In ECs of severe FGR placentas, low ARNT expression results in less ARNT/HIF1A heterodimer formation, decreased binding to HREs within the VEGFA proximal promoter, ultimately resulting in decreased EC expression of VEGFA (63). This is supported by transgenic mouse models where embryos homozygous for Arnt^−/− were uniformly embryonically lethal primarily resulting from a lack of placental vascularization (65, 66). In vitro, however, rescue of ARNT expression in severe FGR ECs only led to partial recovery of EC migration, suggesting that other factors beyond those intrinsic to ECs are likely also important in placental angiogenesis (64).

One critical element lacking in our EC cultures is consideration of the microenvironment, which is a well-established regulator of angiogenesis in a variety of tissues. For instance, the postpartum mammary gland exhibits altered extracellular matrix (ECM) stiffness, which is thought to possibly provide a tumor protective effect (67). In contrast, other groups have found that specific changes in ECM composition can promote tumor angiogenesis and subsequent metastatic process (68). The ECM is also an important regulator of embryonic vascular development. For example, fibronectin, an ECM protein, has been shown to promote vascular remodeling during development, and fibronectin knock-out mice exhibit defective vascular development and early embryonic demise (69, 70, 71, 72, 73, 74). Together, these data suggest that altered ECM may play a regulatory role in impaired fetoplacental vascular development in FGR.

Placental villous stromal ECM, however, has not been well-characterized, and there is a dearth of studies focused on normal and pathological changes in ECM during pregnancy. Limited morphological data suggest an increase in fibrosis and a reduction of villous stromal volumes associated with FGR, suggesting that FGR placental ECM is altered in composition and biophysical characteristics (75). Data extrapolated from various in vivo ultrasound studies using shear wave elastography also demonstrate that placental stiffness varies significantly across patients diagnosed with pregnancy complications as compared to normal placental tissue, with FGR placentas exhibiting increased stiffness (76, 77, 78, 79). Beyond this, knowledge of placental ECM is otherwise substantially lacking.

To address this, our lab has established a method of isolating human villous stromal fibroblasts from the same placentas from which ECs are obtained, and we utilize these cells to generate placental fibroblast-specific, cell-derived matrices (CDMs). We found that as compared to control ECs plated on control CDM, severe FGR ECs plated on severe FGR CDM exhibited significant migrational defects. More importantly, though, interaction of control ECs with severe FGR CDM resulted in significant impairment of control EC migration, whereas control CDM appeared to rescue severe FGR angiogenic properties (80). Together, incorporation of stromal matrices into our model confirms the importance of the placental microenvironment in regulation of fetoplacental angiogenesis.

Integrins

Cells interact with the surrounding microenvironment via several membrane-specific proteins. Of the different cell adhesion molecule subtypes, integrins interact with ECM, and it is possible that aberrant endothelial cell integrin-microenvironment signaling in FGR could contribute to impaired fetoplacental vascular development.

Integrin signaling occurs when alpha and beta integrin subunits bind and become activated through a steric change in position of the two subunit heads. Once in the open conformation, the beta subunit tail is then able to recruit intracellular proteins that effect downstream signaling to produce a wide range of cellular functions including survival, proliferation, and migration (81, 82). There are 18 types of alpha subunits and 8 types of beta subunits, with 24 known heterodimer pairs (81, 82, 83). Not all alpha and beta subunits are capable of binding to each other, but because there are many different combinations, integrin signaling is able to impact various aspects of cellular function. Integrin signaling complexity is further increased when integrin heterodimers form focal adhesion complexes. During this process, other integrin heterodimers and other transmembrane receptors are recruited to a focal adhesion, resulting in localized signal amplification of the recruited proteins (82, 84). Ultimately, integrin signaling is highly dynamic while being tightly regulated, allowing for specific localized signals to drive complex cellular processes such as migration. Given the role of integrins in endothelial response to the ECM, they are mechanistic candidates that could contribute to dysregulation of FGR placental endothelial cells.

Integrin outside-in signaling

Outside-in signaling plays a large role in regulating cell migration and angiogenesis. It occurs when the alpha and beta heterodimer bind to a specific ligand in the ECM. The integrin heterodimer then assumes an open conformation, making the intracellular beta tail accessible. This results in the recruitment of intracellular focal adhesion proteins such as talin, vinculin, paxillin, and zyxin (82, 84). The recruitment of focal adhesion proteins promotes signaling cascades mediated by effector proteins such as focal adhesion kinase (FAK) and Src homology and collagen (Shc) adaptor proteins (85, 86). The activation of these proteins ultimately regulates actin and microtubule-based cytoskeletal rearrangements required for cell motility. For this dynamic process to occur, integrin signaling and binding have to operate in a spatially- and temporally-coordinated fashion, as the leading edge of the migrating cell needs to anchor the membrane to the ECM, allowing cytoskeletal forces to drive the cell forward (87). At the same time, the lagging edge of the cell needs to release membrane attachments (87).

In addition to recruiting intracellular proteins to effect downstream signaling, integrin heterodimers have also been shown to recruit other membrane receptors to focal adhesion complexes. This recruitment acts to locally amplify both the integrin and the receptor signals. As an example, vascular endothelial growth factor 2 receptor (KDR) can associate with integrin heterodimers, and this association enhances VEGF signaling in a specific cellular region (88, 89). Regulating cell motility and enhancing signaling from pathways that promote or inhibit angiogenesis are two ways that integrins can regulate angiogenesis via outside-in signaling.

Integrin inside-out signaling

Inside-out signaling are the internal signals that can drive the activation or inactivation of integrin heterodimers at the cell surface. This process is largely mediated by G-coupled protein receptors that bind small heteromeric G proteins that switch between the active (GTP bound) and inactive (GDP bound) forms when acted upon by GTPases (90). The activation of integrin heterodimers with inside-out signaling causes the steric repositioning of the alpha and beta heads and intracellular tails (90, 91). In this instance, the inactive heterodimer exists in the bent conformation, unable to bind to the ECM. Once the intracellular inhibitors are inactivated, the integrin heterodimer can then adopt the extended confirmation and bind to a specific ligand, transitioning into outside-in signaling (82, 91, 92, 93).

Integrin inside-in signaling

Inside-in signaling occurs when the integrin heterodimer is internalized into an endosomal vesicle and can continue signaling from within the internalized vesicle (94). Endocytic recycling is an important part of integrin signaling. Similar to other membrane-bound receptors, endocytosis of an integrin heterodimer can result in recycling back to another location within the cell membrane or in degradation if the receptor is targeted to the lysosome. During the progression from early endosome to recycling vesicles, active integrins can continue signaling, although less is known about the role of integrin signaling in this context (95). However, there is evidence that suggests signaling from actively internalized integrins contributes to ECM remodeling, adhesion site turnover, and establishment of cell polarity (95, 96, 97, 98).

Integrin signaling in endothelial cells

In response to different physiologic conditions, cells adapt by regulating the expression and function of specific integrins. For endothelial cells, this includes integrin regulation during vessel formation and maintenance. As vessels develop, endothelial integrins are required for the cellular processes that ultimately drive angiogenesis. Integrins are also important mechanosensory proteins, and within the endothelium, are required for maintaining vessel integrity, as these cells must respond to changes in shear stress resulting from normal variations in blood flow.

In cancer, integrins αvβ3 and α5β1, the main endothelial fibronectin-binding integrins, are upregulated in metastatic invasions and angiogenic tumors (99, 100). Notably, fibronectin expression is upregulated within these tumor-promoting microenvironments (101, 102, 103). In normal developmental processes, fibronectin is also increased in regions undergoing vascular remodeling (71). Within these areas, integrins αvβ3 and α5β1 are required for driving angiogenic development of new vessels (104). In contrast, within placental fibroblast-derived CDM, fibronectin, which is a primary component of the placental villous stroma, is decreased and topographically disrupted in FGR as compared to CDM generated from control placental fibroblasts (75, 80). Together, these data suggest that integrins αvβ3 and α5β1, through their interactions with fibronectin, are essential for vessel development and maintenance.

Integrins in the placenta

It is clear that integrins play several important roles in the placenta. For instance, integrins contribute to maintaining the trophoblast stem cell niche and are implicated in trophoblast invasion (105, 106, 107, 108, 109, 110). Once cytotrophoblasts commit to invasion, expression of their adhesion receptors is altered in a stepwise fashion. There is a switch from integrin α6β4 and E-cadherin expression, which are characteristic of epithelial, polarized cytotrophoblasts, to induction of endothelial-type junctional proteins including VE-cadherin, platelet-endothelial cell adhesion molecule-1 (PECAM-1), along with integrins αvβ3 and α1β1 (108, 111). Integrins have also been shown to mediate placental vascular development. For example, the majority of integrin αv-null mice are embryonically lethal, and fetal blood vessels within the placental labyrinthine zone are sparse or absent, demonstrating inadequate intermingling of these vessels and maternal blood sinuses (112). Similarly, mice lacking integrin β8, share many similarities to those of αv knockout mice, with early defects in placental development (113). Another integrin potentially implicated in placental vascular development is α4. These null embryos display a placenta lacking the allantoic component and umbilical vessels connecting the blood circulation between the embryo and placenta (114).

Although fewer β3-knockout embryos survive as compared to wild-type, a substantial proportion withstand this mutation without significant complications. Interestingly, within mice, it is the embryos of β3-null mothers who demonstrate compromised survival as a result of both restricted blood flow and placental labyrinthine hemorrhage, suggesting the importance of integrins in maternal-fetal signaling (115). Within an ovine model, however, loss of integrin β3 in conceptuses results in decreased expression of endothelial nitric oxide synthase (NOS3), suggesting that impaired development of the allantoic and placental vasculatures underlies the concomitant finding of decreased embryonic growth (116). Although none of these models were placental-specific conditional knockouts, making it difficult to specifically ascertain the fetal- and placental-specific consequences of reduced integrin expression, these studies suggest the importance of integrins in proper development of the placenta.

Integrins in severe FGR

Sparse vasculature is a key finding in severe FGR, and several lines of evidence suggest that integrins may play an important role. First, transgenic models as described above demonstrate that at least certain integrins mediate some aspects of placental vascular development and fetal growth (112, 113, 115, 116). Second, CDM generated from control and FGR placental fibroblasts impact placental endothelial cell proliferation and migration (80). Third, fibronectin is topographically disrupted in FGR-derived CDM, further suggesting the importance of EC-ECM interactions in placental vascular development.

As noted earlier, fibronectin is a key ECM protein required for normal embryonic vascular development and is also one of the most abundant matrix proteins within human placental stroma (69, 72, 75). Fetoplacental endothelial cells also highly express αvβ3 and α5β1, two key fibronectin-binding integrins. Our recent findings, which were specifically directed at understanding endothelial cell-fibronectin interactions, demonstrated that exogenous fibronectin was insufficient to rescue FGR placental endothelial migratory function. However, inhibition of α5β1 activation led to significantly compromised migration of both control and severe FGR endothelial cells, with a proportional reduction in both cohorts. In contrast, inhibition of active αvβ3 diminished control endothelial cell migration but had no effect on growth-restricted cells, suggesting that FGR endothelial cells are not able to effectively utilize αvβ3. This dysfunction of these integrins in FGR appear to be a result of increased active αvβ3 in focal adhesions that display significant more co-staining with immature focal adhesion markers, along with more α5 co-staining with a marker of mature focal adhesions. Furthermore, global changes in intracellular trafficking with reduced early and late endosomal vesicles in FGR suggest that integrin trafficking may be abnormal in FGR endothelial cells (Figure 2) (117).

Figure 2.

Proposed mechanism contributing to impaired fetoplacental angiogenesis in severe FGR. In control placentas, endothelial cell αvβ3 and α5β1 bind to normal extracellular matrix, resulting in a proper balance of αvβ3-paxillin and α5β1-zyxin focal adhesion formation. These induce downstream signals to the actin cytoskeleton, allowing for appropriate polymerization/depolymerization and subsequent cell migration. In contrast, αvβ3 and α5β1 from FGR placentas bind to a disordered extracellular matrix. Although activation of both of these integrin heterodimers in FGR are similar to that in control endothelial cells, the focal adhesion maturation cycle is disrupted. Excessive αvβ3-paxillin and α5β1-zyxin result in erroneous downstream signaling to actin, preventing proper polymerization/depolymerization for cell migration. At the same time, there is inadequate recycling of integrins, which prevents proper extension of the leading edge and retraction of the lagging edge, further impairing migratory properties.

While this study is the first to report integrin dysfunction in placental endothelial cells isolated from severe FGR, our findings emphasize the importance of integrins in regulating fetoplacental angiogenesis. Several additional avenues of investigation warrant inquiry. For instance, how does altered ECM in severe FGR affect the mechanosensory properties of endothelial cell integrins, and does this further compound the intrinsic impairments in endothelial function? What are the effects of severe FGR ECM on endothelial cell focal adhesion composition, signaling, and cell polarity? Alternatively, does the increase in fetoplacental vascular resistance itself exacerbate an already dysfunctional villus by causing further alterations to integrin mechanotransduction? These are just a few additional questions that require investigation, which can begin to be addressed by our current model and by development of three-dimensional models that accurately recapitulate the placental vasculature and microenvironment. Further elucidation of intrinsic and microenvironment-mediated endothelial cell impairments will provide more context for identifying the critical mechanisms responsible for reduced fetoplacental vasculature and elevated fetoplacental vascular resistance in severe FGR.

Additional considerations

Cell-cell adhesions

Although not necessarily specific to early-onset, severe FGR, there are several other considerations regarding endothelial cell biology that are important to acknowledge and that warrant investigation in FGR. As one example, endothelial tight and adherens junctions, in addition to being major determinants of endothelial integrity, influence junctional stability and permeability, both of which mediate angiogenesis. Within the highly angiogenic terminal villous capillaries, investigators have demonstrated a lack of occludin, a tight junction transmembrane protein that is integral in junctional stability and barrier function (118). The adherens junction protein VE-cadherin, while expressed in terminal villous capillaries, is only anchored to peri-junctional actin via α- and β-catenin, while lacking plakoglobin (118, 119). This results in a junctional phenotype that is more “plastic,” which may facilitate more dynamic angiogenic processes. In contrast, in addition to VE-cadherin, α-catenin, and β-catenin, larger conduit placental vessels also express occludin and plakoglobin, thereby creating more stable cell-cell contacts that essentially disincentivize cellular proliferation and migration. As further evidence of the role of cell-cell adhesion molecules in angiogenesis, placentas from pregnancies complicated by diabetes, which have been shown to exhibit increased placental angiogenesis, display evidence of disrupted capillary paracellular junctions with more than 50% of the microvasculature expressing lower levels of VE-cadherin and β-catenin (120, 121, 122).

In general, regulation of endothelial cell adhesion molecules occurs via various growth factors, inflammatory stimuli, and maternal nutritional status (123, 124, 125, 126). VEGFA, through its interaction with KDR, increases phosphorylation of VE-cadherin, resulting in loss of anchorage to peri-junctional actin, disruption of cell-cell contacts, and increased permeability (127). This effect is also seen in monolayer cultures of human umbilical vein endothelial cells (HUVECs) where VEGFA (specifically, the VEGFA_165a isoform) treatment of HUVECs led to more cell-cell junctions that displayed discontinuous or absent VE-cadherin staining (128). The VEGFA splice variant VEGFA_165b, on the other hand, which is a weak KDR agonist and has also been described as “anti-angiogenic” secondary to its less effective activation of VEGF receptors, prevented the VEGFA_165a-induced disruption of VE-cadherin junctions (128, 129, 130). Together, this suggests that potential alterations in ratios of VEGFA splice variants may influence VE-cadherin clustering and thereby angiogenesis in fetoplacental vessels.

Placental villous stromal composition

The primary cell types within villous stroma are fibroblasts, myofibroblasts, pericytes, and Hofbauer cells (131, 132, 133). Although placental stromal fibroblasts are the main cell type driving ECM production, fetoplacental endothelial cells and trophoblast also contribute to stromal ECM, primarily through their deposition of basement membrane proteins. Collagen IV is the main ECM protein within the fetal vascular basement membrane, whereas the trophoblast basement membrane is also composed of laminin and heparan sulfate in addition to collagen IV (134, 135). Furthermore, in contrast to other organs, these three ECM proteins are not limited to the basement membrane but are also present, along with several other ECM proteins such as collagen I, III, VI, and fibronectin, throughout the villous stroma (132, 136, 137). ECM proteins also have multivalent integrin recognition sites, and together, this suggests that alterations in ECM protein availability may promote or inhibit functions of the various villous cell populations.

Villous stromal ECM provides structural support to the placenta and actively participates in biochemical and biophysical signaling to neighboring cells. Moreover, terminal villous endothelial cells, trophoblast, and Hofbauer cells have the ability to alter the matrix via secretion of enzymes such as matrix metalloproteinases (MMPs) or ADAMTS (A Disintegrin And Metalloprotease with Thrombospondin motifs) (138, 139). These various cell-mediated modifications of the matrix may lead to changes in integrin-mediated sensing, stiffening, and remodeling that further regulate proliferation and migration of endothelial cells (140). Simultaneously, the configuration of integrin expression on the surface of cells ultimately determines how a particular cell responds to the specific microenvironment. The details, however, of how these villous stromal cells modulate placental ECM and reciprocally, how stromal composition and stiffness signal to these cells, remain an understudied area in both uncomplicated and FGR pregnancies.

Endothelial cell state

During certain processes of embryonic development, endothelial cells have been shown to undergo endothelial-mesenchymal transition (EndMT), where they lose their constituent endothelial properties and gain mesenchymal cell features. As a result of various stimuli, including inflammation, oxidative stress, and low shear stress, endothelial cell-cell junctions loosen and the basement membrane is degraded, allowing cells to migrate into the sub-endothelial interstitia (141). While these events also transpire during angiogenesis, endothelial migration in EndMT is characterized by movement of an individual cell whereas endothelial cells migrate collectively in a coordinated manner during angiogenesis (142). Ultimately, cells that undergo EndMT lose endothelial functions and acquire mesenchymal characteristics, including production of ECM within tissue stroma. In pathologic conditions, these mesenchymal cells can overproduce matrix, causing fibrosis and leading to increased tissue stiffness (143, 144).

Among the various pathways that result in EndMT induction, transforming growth factor-beta (TGFβ) has been considered the major driver of EndMT both during development and in pathological conditions. All three isoforms (TGFβ1, TGFβ2, TGFβ3) have been shown to stimulate EndMT in cultured endothelial cells (145). Briefly, TGFβ mediates EndMT via Smad or non-Smad signaling, which induces expression of specific transcription factors such as Snail (SNAI1) and Slug (SNAI2). Snail transcription factors repress EC cell-cell adhesion by binding to the promoter of the gene encoding VE-cadherin or PECAM-1. Simultaneously, it also induces expression of mesenchymal markers such as alpha-smooth muscle actin (α−SMA) (146, 147). Details regarding the overall mechanisms governing EndMT have been comprehensively reviewed in recent publications (141, 143, 144, 145, 148).

Whether EndMT specifically plays a role within the human placenta, to our knowledge, has not been studied. However, there are several lines of evidence to suggest this process may be important in the development of FGR. First, as noted previously, existing evidence suggests higher tissue stiffness in placentas complicated by FGR, and recent single-cell transcriptomic evidence suggests that pathologic ECM stiffness promotes mesenchymal transcriptional signatures (76, 79, 149). Second, there is a well-established association between hypoxia and FGR, and more specifically hypoxia-inducible factor 2-alpha (HIF2α) has been shown to be expressed in higher quantities in FGR human placentas (150). HIF2a is also a known mediator of both Snail and Slug, resulting in EndMT induction in the setting of pulmonary hypertension (141, 151). Third, cells isolated from the basal plate of FGR placentas exhibited a more rapid shift toward mesenchymal phenotypes as compared to those from control placentas, and their ability to differentiate toward endothelial cell lineage was decreased (143). Lastly, placentas from pregnancies with severe, early-onset FGR and absent umbilical artery end-diastolic velocities exhibit significantly higher in vivo TGFβ1 concentrations as compared to control placentas (152). Furthermore, these investigators found over-expression of phospho-Smad2 with redistribution of α-SMA in severe, early-onset FGR, suggesting that this may be a mechanism contributing to high fetoplacental vascular resistance seen with absent umbilical artery end-diastolic velocities (152).

Clinical implications

Fetuses with severe FGR are at significant risk for adverse short- and long-term outcomes. Treatment modalities for this condition are significantly limited in clinical efficacy, and this stems from a lack of mechanistic understanding of both normal and pathophysiologic processes in pregnancy. Studies consistently demonstrate that despite prior attempts at intervention and increased clinical surveillance, current management paradigms are not improving outcomes for this specific population, underscoring the need to elucidate mechanisms resulting in FGR (153, 154, 155, 156, 157, 158, 159).

Our studies focus on placental angiogenesis, a process that is normally ongoing throughout pregnancy, and in fact, becomes even more robust in the latter half of gestation. Although it can be argued that focusing on this timeframe might miss critically early-pregnancy developmental milestones that result in FGR, we believe that there are specific benefits to investigating processes that are continuous throughout gestation. For instance, by the time prenatal care is initiated for the majority of women, the early insults predisposing to FGR have already occurred, making it essentially impossible to target intervention. Furthermore, without the ability to reliably predict the development of FGR in early pregnancy, it would be difficult to justify initiating a novel form of prevention or treatment prior to a definitive diagnosis. Lastly, if placental angiogenesis can be rescued in severe FGR, it is certainly possible that surface area for oxygen and nutrient exchange will be augmented, thereby improving fetal health and growth. It is also not entirely inconceivable that improved fetoplacental vascular function could then result in fetal-to-maternal signals that lead to overall improvement in placental function. Ultimately, understanding mechanisms underlying impaired placental angiogenesis, a key component of fetal health in severe FGR, is critical if we are to develop treatment modalities that promote the in utero health of a fetus, thereby reducing the risk for perinatal death and complications related to prematurity.