Engineered models for placental toxicology: Emerging approaches based on tissue decellularization.

Recent increases in prescriptions and illegal drug use as well as exposure to environmental contaminants during pregnancy have highlighted the critical importance of placental toxicology in understanding and identifying risks to both mother and fetus. Although advantageous for basic science, current in vitro models often fail to capture the complexity of placental response, likely due to their inability to recreate and monitor aspects of the microenvironment including physical properties, mechanical forces and stiffness, protein composition, cell-cell interactions, soluble and physicochemical factors, and other exogenous cues. Tissue engineering holds great promise in addressing these challenges and provides an avenue to better understand basic biology, effects of toxic compounds and potential therapeutics. The key to success lies in effectively recreating the microenvironment. One strategy to do this would be to recreate individual components and then combine them. However, this becomes challenging due to variables present according to conditions such as tissue location, age, health status and lifestyle. The extracellular matrix (ECM) is known to influence cellular fate by working as a storage of factors. Decellularized ECM (dECM) is a recent tool that allows usage of the original ECM in a refurbished form, providing a relatively reliable representation of the microenvironment. This review focuses on using dECM in modified forms such as whole organs, scaffold sheets, electrospun nanofibers, hydrogels, 3D printing, and combinations as building blocks to recreate aspects of the microenvironment to address general tissue engineering and toxicology challenges, thus illustrating their potential as tools for future placental toxicology studies.

The implications of the purinergic signaling throughout pregnancy.

Purinergic signaling is a necessary mechanism to trigger or even amplify cell communication. Its ligands, notably adenosine triphosphate (ATP) and adenosine, modulate specific membrane-bound receptors in virtually all human cells. Regardless of the stage of the pregnancy, cellular communication between maternal, placental, and fetal cells is the paramount mechanism to sustain its optimal status. In this review, we describe the crucial role of purinergic signaling on the regulation of the maternal-fetal trophic exchanges, immune control, and endocrine exchanges throughout pregnancy. The nature of the modulation of both ATP and adenosine on the embryo-maternal interface, going through placental invasion until birth delivery depends on the general maternal-fetal health state and consequently on the selective activation of their specific receptors. In addition, an increasing number of studies have been demonstrating the pivotal role of ATP and adenosine in modulating deleterious effects of suboptimal conditions of pregnancy. Here, we discuss the role of purinergic signaling on the balance that coordinates the embryo-maternal exchanges and a promising therapeutic venue in the context of pregnancy disorders.

Disease-specific extracellular matrix composition regulates placental trophoblast fusion efficiency.

The placental syncytiotrophoblast is a multinucleated layer that regulates transport between the mother and fetus. Fusion of trophoblasts is essential to form this layer, but this process can be disrupted in pregnancy-related disorders such as preeclampsia. Disease progression is also associated with changes in the extracellular matrix (ECM), but whether disease-specific ECM compositions play any causal role in establishing syncytiotrophoblast disease phenotypes remains unknown. Here, we develop a decellularization-based platform to isolate and characterize the role of human placental ECM composition on cell function, while controlling for the confounding effects of matrix structure and mechanics that can arise in conventional tissue decellularization/recellularization experiments. Using this approach, we demonstrate that ECM compositional changes that occur in preeclampsia have a statistically significant effect on adhesion, spreading, and fusion of placental trophoblasts. Proteomic analysis of ECM content then allowed us to identify and recreate selected differences in matrix composition; indicating that replacement of normally present Type IV Collagen by Type I Collagen in preeclampsia significantly affects fusion efficiency. These results indicate that disease-specific matrix compositions can play an important role in trophoblast fusion, suggesting novel matrix-targeting therapeutic strategies for pregnancy-related disorders. More broadly, this work demonstrates the utility of a decellularization-based approach in understanding the functional contributions of matrix composition in driving cellular disease phenotypes.

Mechanobiological regulation of placental trophoblast fusion and function through extracellular matrix rigidity.

The syncytiotrophoblast is a multinucleated layer that plays a critical role in regulating functions of the human placenta during pregnancy. Maintaining the syncytiotrophoblast layer relies on ongoing fusion of mononuclear cytotrophoblasts throughout pregnancy, and errors in this fusion process are associated with complications such as preeclampsia. While biochemical factors are known to drive fusion, the role of disease-specific extracellular biophysical cues remains undefined. Since substrate mechanics play a crucial role in several diseases, and preeclampsia is associated with placental stiffening, we hypothesize that trophoblast fusion is mechanically regulated by substrate stiffness. We developed stiffness-tunable polyacrylamide substrate formulations that match the linear elasticity of placental tissue in normal and disease conditions, and evaluated trophoblast morphology, fusion, and function on these surfaces. Our results demonstrate that morphology, fusion, and hormone release is mechanically-regulated via myosin-II; optimal on substrates that match healthy placental tissue stiffness; and dysregulated on disease-like and supraphysiologically-stiff substrates. We further demonstrate that stiff regions in heterogeneous substrates provide dominant physical cues that inhibit fusion, suggesting that even focal tissue stiffening limits widespread trophoblast fusion and tissue function. These results confirm that mechanical microenvironmental cues influence fusion in the placenta, provide critical information needed to engineer better in vitro models for placental disease, and may ultimately be used to develop novel mechanically-mediated therapeutic strategies to resolve fusion-related disorders during pregnancy.

Biomimetic Micropatterned Adhesive Surfaces To Mechanobiologically Regulate Placental Trophoblast Fusion.

The placental syncytiotrophoblast is a giant multinucleated cell that forms a tree-like structure and regulates transport between mother and baby during development. It is maintained throughout pregnancy by continuous fusion of trophoblast cells, and disruptions in fusion are associated with considerable adverse health effects including diseases such as preeclampsia. Developing predictive control over cell fusion in culture models is hence of critical importance in placental drug discovery and transport studies, but this can currently be only partially achieved with biochemical factors. Here, we investigate whether biophysical signals associated with budding morphogenesis during development of the placental villous tree can synergistically direct and enhance trophoblast fusion. We use micropatterning techniques to manipulate physical stresses in engineered microtissues and demonstrate that biomimetic geometries simulating budding robustly enhance fusion and alter spatial patterns of synthesis of pregnancy-related hormones. These findings indicate that biophysical signals play a previously unrecognized and significant role in regulating placental fusion and function, in synergy with established soluble signals. More broadly, our studies demonstrate that biomimetic strategies focusing on tissue mechanics can be important approaches to design, build, and test placental tissue cultures for future studies of pregnancy-related drug safety, efficacy, and discovery.

An insight into the role of the death receptor CD95 throughout pregnancy: Guardian, facilitator, or foe.

The prototype death receptor CD95 (Fas) and its ligand, CD95L (FasL), have been thoroughly studied due to their role in immune homeostasis and elimination of infected and transformed cells. The fact that CD95 is present in female reproductive cells and modulated during embryogenesis and pregnancy has raised interest in its role in immune tolerance to the fetoplacental unit. CD95 has been shown to be critical for proper embryonic formation and survival. Moreover, altered expression of CD95 or its ligand causes autoimmunity and has also been directly involved in recurrent pregnancy losses and pregnancy disorders. The objective of this review is to summarize studies that evaluate the mechanisms involved in the activation of CD95 to provide an updated global view of its effect on the regulation of the maternal immune system. Modulation of the CD95 system components may be the immune basis of several common pregnancy disorders.

Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells.

Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor pathways. Hypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as activates Nrf2 the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a situation of H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5' adenosine monophosphate-activated protein kinase (AMPK)α, an upstream modulator of autophagy, rising to a 16-fold increase in BeWo cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94%) and ATG7 (by 97%) in BeWo cells. Moreover, H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314% in BeWo cells. In H/R conditions, melatonin reduced autophagic activity by 74% and Nrf2 expression activation by 300%, leading to BeWo cell apoptosis. In contrast, In human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.

Melatonin modulates autophagy and inflammation protecting human placental trophoblast from hypoxia/reoxygenation.

Melatonin has been proposed as a possible treatment for the deleterious effects of hypoxia/reoxygenation (H/R), such as autophagy, inflammation, and apoptosis. Pathological pregnancies, such as preeclampsia, are associated with placental H/R, and decreased placental melatonin synthesis as well as lower melatonin levels in the placenta and maternal plasma. However, the effects of exogenous melatonin on inflammation and autophagy induced by pregnancy complications associated with H/R await investigation. This study aimed to determine as to whether melatonin protects human primary villous trophoblasts against H/R-induced autophagy, inflammation, and apoptosis. Human primary villous cytotrophoblasts were isolated and immunopurified from normal term placentas. These cells were then exposed or not to 1 mmol/L melatonin for 72 hour in normoxia (8% O2 ), thereby inducing differentiation into syncytiotrophoblast that was then exposed to H/R (0.5% O2 , for 4 hour) or normoxia. H/R decreased endogenous melatonin synthesis (by 68%) and interleukin (IL)-10 levels (by 72%), coupled to increased tumor necrosis factor (TNF) (by 114%), IL-6 (by 55%), and NFκB (by 399%), compared to normoxia. Melatonin treatment reversed the H/R effect, restoring IL-10, TNF, and IL-6 levels to those of the normoxia condition. Melatonin, as well as NFκB inhibition, enhanced autophagy activation, consequently increasing syncytiotrophoblast survival in H/R conditions. This study suggests that H/R, which is present in pregnancy complications, inhibits endogenous melatonin production, thereby contributing to reduced syncytiotrophoblast viability. Results indicate that exogenous melatonin treatment may afford protection against H/R-induced damage, thereby enhancing placental cell survival, and contributing to improved fetal outcomes.

In Vitro Induction of Hypoxia/Reoxygenation on Placental Cells: A Suitable Model for Understanding Placental Diseases.

Lack of blood flow and aberrant levels of oxygenation in placentas are recurrent in pregnancy diseases, such as preeclampsia. These alterations generate situations of hypoxia and hypoxia/reoxygenation (H/R) and consequent oxidative stress, increased cell death, and inflammation in trophoblasts. The models used to understand the effects of hypoxia and H/R on trophoblasts require a rather big structure. This chapter describes the details of a suitable and reasonable approach with hypoxia chambers to expose human placental trophoblasts to variable conditions of oxygenation.

Testosterone replacement maintains smooth muscle content in the corpus cavernosum of orchiectomized rats.

OBJECTIVE: To evaluate the effects of testosterone (T) on the maintenance of corpus cavernosum (CC) structure and apoptosis. METHODS: Animals were divided into three groups: sham operation group (n = 8) underwent sham operation; Orchiectomized (Orchiec)+ oily vehicle group (n = 8) underwent bilateral orchiectomy and received a single dose of oily vehicle by intramuscular injection (i.m.) 30 days after orchiectomy; and Orchiec + T group (n = 8) underwent bilateral orchiectomy and received a single dose of T undecanoate 100 mg/kg i.m. 30 days after the surgery. Animals were euthanized 60 days after the beginning of the experiment with an anesthetic overdose of ketamine and xylazine. Blood samples and penile tissue were collected on euthanasia. Azan's trichrome staining was used to evaluate smooth muscle, Weigert's Fucsin-Resorcin staining was used to evaluate elastic fibers and Picrosirius red staining was used to evaluate collagen. Apoptosis was evaluated using TUNEL technique. RESULTS: T levels decreased in Orchiec + oily vehicle when compared to sham operation and Orchiec + T groups (p < 0.001). T deprivation reduced trabecular smooth muscle content and penile diameter and T replacement maintained both parameters (p = 0.005 and p = 0.001, respectively). No difference was observed in the content of sinusoidal space (p = 0.207), elastic fibers (p = 0.849), collagen (p = 0.216) and in apoptosis (p = 0.095). CONCLUSION: Normal testosterone levels maintain CC smooth muscle content and do not influence elastic fibers, collagen content and apoptotic index. Further studies should be performed in order to investigate the mechanisms by which androgen mediates its effects on CC structure.

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

Melatonin in Pregnancy: Effects on Brain Development and CNS Programming Disorders.

Melatonin is an important neuroprotective factor and its receptors are expressed in the fetal brain. During normal pregnancy, maternal melatonin level increases progressively until term and is highly transferred to the fetus, with an important role in brain formation and differentiation. Maternal melatonin provides the first circadian signal to the fetus. This indolamine is also produced de novo and plays a protective role in the human placenta. In pregnancy disorders, both maternal and placental melatonin levels are decreased. Alteration in maternal melatonin level has been associated with disrupted brain programming with long-term effects. Melatonin has strong antioxidant protective effects directly and indirectly via the activation of its receptors. The fetal brain is highly susceptible to oxygenation variation and oxidative stress that can lead to neuronal development disruption. Based on that, several approaches have been tested as a treatment in case of pregnancy disorders and melatonin, through its neuroprotective effect, has been recently accepted against fetal brain injury. This review provides an overview about the protective effects of melatonin during pregnancy and on fetal brain development.

Placental melatonin system is present throughout pregnancy and regulates villous trophoblast differentiation.

Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re-oxygenation-induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pM-1 mM) increases the fusion index (syncytium formation; 21% augmentation at 1 mM melatonin vs. vehicle) and ß-hCG secretion (121% augmentation at 1 mM melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well-being.