Placental development is a critical, but poorly understood process. Previous work indicated that ASB4 mediated early vascular differentiation, and was localized to the placenta. Thus I sought to fully characterize the role of ASB4 in placental vascularization and trophoblast differentiation.
The experiments described herein attempt to fully characterize the role of ASB4 during placental vascular development by uncovering its expression, molecular function, biologic consequences, and molecular regulation. Data generated here reveal that 1) Asb4 is expressed in pluripotent trophoblast cells committed to the vasculature during a narrow time window during differentiation; 2) ASB4, as a ubiquitination complex, degrades the HLH protein ID2; and promotes differentiation of the vascular lineage through, in part, its
degradation of ID2; and 4) deletion of Asb4 in mice induces a placental vascular phenotype and recapitulates pre-eclampsia in Asb4-/- mothers (Chapter 2).
The role of ASB4 in vascular commitment and patterning
Previous investigations in this laboratory elucidated a role for ASB4 in the lineage commitment step of pluripotent-to-endothelial differentiation [19]. Further, due to the high expression pattern of Asb4 in “hotbeds” of vascular activity and vasculogenesis (e.g. allantois, yolk sac, etc.) [19], we hypothesized that ASB4 would be responsible, in part, for endothelial differentiation in these areas.
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Earlier work from our laboratory on ASB4 function [19,36] led us to examine vascular development in the placenta. Ferguson, et al [19] described how Asb4 expression was restricted to a narrow spatiotemporal window in which the placenta was developing. This study also elucidated that ASB4 functioned as an oxygen sensor, further implicating its function in the placenta, where hemodynamic stresses and hypoxia/normoxia events are required for proper vascularization. Likewise, Bode, et al. [36] described how laminar shear stress repressed Asb4 expression. We therefore examined the gross morphological and physiological consequences of globally deleting Asb4 expression in mice, which was generated in [87].
Our initial investigation of Asb4-/- mice established that there was embryonic lethality in a subset of pups that was occurring at or about E9.5 (Figure 2.7A). This spontaneous abortion appeared to occur in the absence of any gross fetal defect or pathology, but the placentas of said pups resigned to intrauterine demise were dramatically less vascular or developed (Figure 2.7A, lower conceptus) compared with littermates that appeared to be morphologically “normal” (Figure 2.7A, upper conceptus). To begin understanding this pathology, a histological examination was undertaken and revealed that ASB4 expression only coincided with cells that were either pluripotent (Figure 2.1B) or cells that had committed to the vascular lineage (Figure 2.1B’). Cells that had fully differentiated into endothelium (Figure 2.1B”) and cells of different lineages (spongiotrophoblasts, trophoblast giant cells, syncytiotrophoblasts, etc.) did not express Asb4.
Though there have been recent advances in understanding mouse placental
development, the field woefully lacks a definitive and authoritative consensus with regard to markers of differentiating cells. Therefore, there are some caveats in trying to infer specific
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cell types through the use of protein expression markers in the mouse placenta. To identify pluripotent/stem cells in the context of the mouse placenta, we relied on the receptor c-Kit (also known as CD117 or mast/stem cell growth factor receptor [SCFR]). c-Kit is a receptor tyrosine kinase that binds to stem cell factor ligand and mediates proliferation,
differentiation, and serves as a marker of progenitor cells of mature endothelium [128]. However, c-kit expression is also found in trophoblasts that invade, migrate, and differentiate into the TGC lineage [118,119], indicating that it may also serve as a marker undifferentiated CTBs and TGCs, though this expression pattern has not been empirically determined.
Similarly, the use platelet endothelial cell adhesion molecule (PECAM) (also known as CD31) and cytokeratin 17 for the determination of endothelial committed and mature endothelial-like cells, respectively, was based on studies investigating their roles in human placental tissue [129,130]. While we are confident that there are few or no discrepant functions of these broad endothelial specific molecules between mice and humans, we must acknowledge that there is a dearth of data that definitively describe these protein markers in differentiating mouse trophoblasts.
These experiments illuminate the role of ASB4 in placental vascular determination and differentiation. While we are limited by the scope of markers of endothelial
differentiation in the placenta, we are convinced that ASB4 plays an important role in early placental vascular events, which correspond with earlier reports on the function of ASB4 [19]. However, to fully understand the role of ASB4, we asked how ASB4, as a ubiquitin ligase, would function in the developing endothelium, and its context in the placenta.
58 ASB4 as an E3 ligase
Like all E3 ubiquitin ligase proteins, the functional ramifications of ASB4 are dependent upon the function of the substrate protein(s) that it degrades. Multiple attempts to identify binding partners of ASB4 have not yielded the identification of ASB4 ubiquitination substrate proteins. Through the use of yeast 2-hybrid, the Patterson laboratory identified FIH as an interaction partner of ASB4, though no putative substrate proteins were identified [19]. This result not only highlights the limitations of this assay, but also that of identifying substrate proteins in general. The use of co-immunoprecipitation has also been successfully utilized to identify co-factors of ASB4 [19], but protein turn-over, lack of specificity, and difficulty in separating signal-to-noise all confound any results indicating a putative ASB4 target (data not shown and [87]).
Therefore, we took a candidate approach in our investigation into ASB4 substrates. We knew that any substrate of ASB4 must be involved in early differentiation of the
vasculature, be expressed in CTBs in vivo, and have overexpression models that mimic Asb4 deletion. Literature searches and coinciding data (Figure 2.3B) indicated that members of the ID family would fit these criteria. Specifically, studies of the retinoblastoma (Rb) protein in mice indicate that Rb-deficient mice die embryonically due to abnormal trophoblast
proliferation and placental dysplasia [82]. This effect, however, is ameliorated by the
concomitant deletion of Id2 [83], signifying that Rb-mediated overexpression of ID2 induces abnormal placental development. This Rb-induced ID2 overexpression also leads to the formation of embryonal cancer, abnormal proliferation, and impaired angiogenesis, further highlighting the importance of ID2 in early vascular and differentiation/proliferation events [77].
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Because ID2 mediates differentiation, invasion, and migration in CTBs [79], we hypothesized that ASB4 normally regulates ID2 via its role as an E3 ligase. The role of ID2 in human placental differentiation has served as the crux for our further investigation in our Asb4-/- mice. Specifically, in cells driven to overexpress ID2, invasion was abrogated, migration was abnormally enhanced, and these cells retained characteristics of
undifferentiated cells. Also of note, the abnormal maintenance of ID2 expression throughout placental development was associated with placentas from pre-eclamptic patients. Taken together, this report specifies the role of ID2 in the context of placental differentiation and pre-eclampsia [79]. Other studies have also illuminated the importance of ID2 in the context of proper placental development. Specifically, ID2 mediates transcriptional activity in the placenta [80]; ID2 inhibits extravillous trophoblast-to-TGC differentiation [131]; and Id2 is expressed and involved in trophoblast stem cell differentiation [132-134]. In conjunction with the known expression profile of Asb4, the hypothesized function of ASB4, the
consequence of Asb4 deletion in mice, and our data demonstrating that ID2 and ASB4 have reciprocal expression patterns (Figure 2.3B), we hypothesized that ID2 may be one substrate of ASB4.
In undertaking our biochemical assays measuring the interaction of ASB4 and ID2, we had to make several concessions. Foremost, we wanted to use the most representative cell line but had to concede that no placental cell line endogenously expresses both Asb4 and Id2. The CTB-derived JAR choriocarcinoma cell line best combined placental pertinence, ease of manipulation, and assay-to-assay utility [135]. Because our JAR cells lack endogenous Asb4 expression, we also utilized the 2h-11 endothelial cell line for our experiments that required
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endogenous Asb4 expression. Because these 2h-11 cells are not placental in origin, these cells were limited to biochemical assays and assays that required endothelial cells [122].
ID2 has been shown to translocate from the nucleus to the cytoplasm during nervous cell differentiation [136,137]. However, we observed a predominance of nuclear ID2
expression in vivo, as has been observed elsewhere [138], though nuclear and cytoplasmic expression appears to be dependent on the differentiative and proliferative context of the cells, such as in models of cancer [139,140]. We did not see any shifting of ID2 expression in our JAR cell fractionation assay (Figure S2.4C), though we did not examine the expression pattern during differentiation. Thus, while we show that ID2 expression decreases in the presence of ASB4 in both the cytoplasm and nucleus, we did not explicitly demonstrate that this was due to ubiquitination. That is, we cannot exclude the possibility that ASB4 may somehow simply repress (or accentuate) translocation, which in turn may mediate ID2 stability. Similarly, we did not test whether ASB4 itself is able to translocation between the cytoplasm and nucleus as is the case of another ID2 ubiquitin ligase, APC/C-Cdh1 [141]. These translocation events will have to be demonstrated in future studies to complete the
Pseudovasculogenesis and placental vascular remodeling occurs over time in a complex three-dimensional space. Thus, to recapitulate these dynamic events, we adapted various protocols to assay these complex and multifaceted in vivo events using in culture techniques. By adapting various protocols, we were able to assay placental stem cell and CTB differentiation [111,112], endothelial cell replacement by CTBs [113], and vascular stability[114].
These assays and the resulting data are the best current techniques for addressing complex questions regarding placental development; however, certain limitations are
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obvious. Trophoblast stem cell and CTB differentiation assays either occur in isolation (Figure 2.6A) or with exogenous pharmacological intervention (Figure 2.6B). Unlike true endothelial differentiation [123], the endogenous factors required for trophoblast-to- endothelial cell differentiation remain unknown. The models of CTB-mediated endothelial cell apoptosis (Figure 2.5A) and vascular stability (Figure 2.5C) also occur in the absence of exogenous factors, save for the components found in standard growth medium. However, these assays represent the best current techniques available for recreating in vivo placental defects and pathologies in cell culture.
The role of ASB4 in placental pathologies and pre-eclampsia
ASB4 is expressed in the developing placenta when the maturing vasculature is exposed to dynamic changes in environmental cues and undergoes drastic morphologic changes. Blood flow is initiated, which exerts hemodynamic stresses on the vasculature, and maternal-fetal blood-gas exchange in the placenta is initiated, which drastically changes embryonic oxygen tension from hypoxia to relative normoxia. Furthermore, the placental vasculature undergoes extensive changes during this time, including differentiating and making a vascular commitment, migration, and invasion. Links between these environmental cues and morphologic changes are just beginning to be identified in both normal and
abnormal placental development, and ASB4 is spatiotemporally expressed and regulated in such a way as to represent a potential direct link between differentiation cues and vascular pattering responses.
The data presented in Chapter 2 suggest that ASB4 mediates the early differentiation steps that promote trophoblast cells into the vascular lineage, but does not exclude the possibility that ASB4 may also be involved in other vascular processes within the placenta.
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Preliminary data in this laboratory indicate that ASB4 may mediate other aspects of vascular function, such as migration (via the Boyden Chamber and wound healing assay) and
angiogenesis (via endothelial tube formation and subcutaneous Matrigel/tumor injections). While these data do not negate the effects we observe, they add to the possibility and plausibility that ASB4 functions in different contexts and milieus throughout development and adulthood.
Though many placental pathologies are not completely idiopathic, the etiology of most remains cloudy at best. As discussed in Chapter 1, disorders of placental adherence or invasion (e.g., abruption placentae, placenta accreta/increta/percreta) are often attributed to general vascular or blood dysfunction in cases where no previous or obvious trauma has occurred [142]. Disorders where the placenta blocks the ostium of uterus (i.e., external opening of the cervix) like placenta previa are again attributed to either previous trauma, or general vascular dysfunction/dysmorphia [143]. Lastly, common disorders such as pre- eclampsia, IUGR, and HELLP syndromes are all attributed to impaired differentiation and function of the early placental vasculature [96,97,101]. Almost all cases of dysfunctional, dysmorphic, and developmentally impaired placentas share some underlying vascular component to the etiology or consequence of the pathologies.
Though most medical technologies have made enormous strides in the past decades, the diagnosis and treatment for placental pathologies has remained woefully inadequate. For the vast majority of placental disorders in which comorbidities are present, early delivery remains the only option. The effects and consequences on both the mother and baby can be chronic and severe. Children delivered before term can suffer from broad respiratory illnesses (asthma, bronchopulmonary dysplasia), neurological delay or disorders (cerebral
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palsy, autism), and physical ailments (enterocolitis, hearing loss, retinopathy) [144]. The cost to the mother can be just as great, resulting in complications from caesarean section,
hysterectomy, eclampsia, gestational diabetes, hypertension, and infertility [142,145,146]. Diagnostically, placental pathologies remain critically understudied, and can currently only be diagnosed after they have developed, precluding early interventions Typically, only the onset of sudden idiopathic and localized abdominal pain, edema, and dramatically increased blood pressure are used as indices for placental pathology. Further investigation using ultrasounds, elevated serum levels of alpha-fetoprotein or sFlt or Endoglin, and the onset of proteinurea are the only tools commonly available for diagnosis, and even then, most placental complications are little more than educated guesses. Early diagnostic biomarkers are critically needed.
Perhaps confounding this research is the fact that many placental pathologies do not present until late in gestation. Many patients appear asymptomatic until the third trimester, or even delivery, where complications can present fatal consequences if they are not prepared for. Our elucidation of ASB4’s function in early second trimester placental development may set a precedent for the earlier detection of pre-eclampsia, which may allow early
interventional strategies that would prevent serious late-term complications. While the genetic component and contribution of ASB4 in human placental pathologies remains to be studied, there are many homologies between mouse and human placental development [147]. Thus, further studies that address the role of ASB4 in human placental pathologies will be of critical importance for the health of future mothers and infants.
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Figure 3.1. Model of ASB4-mediated degradation of ID2 in trophoblast cells.
A) In the presence of ASB4 during gestation, ID2 is degraded and targeted for proteasomal degradation, allowing bHLH and E-protein transcription factors to bind to the E-box and promote pro-differentiation gene transcription, and ensures proper placental vascular development.
B) In the absence of ASB4 during pregnancy, ID2 negatively regulates E-protein and bHLH transcription factor activity. This abrogates trophoblast cell differentiation into the placental vasculature, and induces a maternal pre-eclamptic phenotype and fetal growth restriction.
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