Cannabidiol Disrupts Mitochondrial Respiration and Metabolism and Dysregulates Trophoblast Cell Differentiation.

Trophoblast differentiation is a crucial process in the formation of the placenta where cytotrophoblasts (CTs) differentiate and fuse to form the syncytiotrophoblast (ST). The bioactive components of cannabis, such as Δ9-THC, are known to disrupt trophoblast differentiation and fusion, as well as mitochondrial dynamics and respiration. However, less is known about the impact of cannabidiol (CBD) on trophoblast differentiation. Due to the central role of mitochondria in stem cell differentiation, we evaluated the impact of CBD on trophoblast mitochondrial function and differentiation. Using BeWo b30 cells, we observed decreased levels of mRNA for markers of syncytialization (GCM1, ERVW1, hCG) following 20 µM CBD treatment during differentiation. In CTs, CBD elevated transcript levels for the mitochondrial and cellular stress markers HSP60 and HSP70, respectively. Furthermore, CBD treatment also increased the lipid peroxidation and oxidative damage marker 4-hydroxynonenal. Mitochondrial membrane potential, basal respiration and ATP production were diminished with the 20 µM CBD treatment in both sub-lineages. mRNA levels for endocannabinoid system (ECS) components (FAAH, NAPEPLD, TRPV1, CB1, CB2, PPARγ) were altered differentially by CBD in CTs and STs. Overall, we demonstrate that CBD impairs trophoblast differentiation and fusion, as well as mitochondrial bioenergetics and redox homeostasis.

Modeling the Progression of Placental Transport from Early- to Late-Stage Pregnancy by Tuning Trophoblast Differentiation and Vascularization.

The early-stage placental barrier is characterized by a lack of fetal circulation and by a thick trophoblastic barrier, whereas the later-stage placenta consists of vascularized chorionic villi encased in a thin, differentiated trophoblast layer, ideal for nutrient transport. In this work, predictive models of early- and late-stage placental transport are created using blastocyst-derived placental stem cells (PSCs) by modulating PSC differentiation and model vascularization. PSC differentiation results in a thinner, fused trophoblast layer, as well as an increase in human chorionic gonadotropin secretion, barrier permeability, and secretion of certain inflammatory cytokines, which are consistent with in vivo findings. Further, gene expression confirms this shift toward a differentiated trophoblast subtype. Vascularization results in a molecule type- and size-dependent change in dextran and insulin permeability. These results demonstrate that trophoblast differentiation and vascularization have critical effects on placental barrier permeability and that this model can be used as a predictive measure to assess fetal toxicity of xenobiotic substances at different stages of pregnancy.

Protocol for the COVID-19 Wellbeing and Stress Study: a longitudinal study of parent distress, biological stress and child biopsychosocial development during the pandemic and beyond.

INTRODUCTION: The COVID-19 pandemic has had a unique impact on the mental health and well-being of pregnant individuals and parents of young children. However, the impact of COVID-19-related stress during pregnancy on early child biopsychosocial development, remains unclear. The COVID-19 Wellbeing and Stress Study will: (1) investigate the impact of different forms of prenatal stress experienced during the pandemic (including objective hardship, perceived psychological distress and biological stress) on child stress biology, (2) examine the association between child stress biology and child developmental outcomes, (3) determine whether child stress biology acts as a mechanism linking prenatal stress to adverse child developmental outcomes and (4) assess whether gestational age at the onset of the COVID-19 pandemic or child sex, moderate these associations. METHODS AND ANALYSES: The COVID-19 Wellbeing and Stress Study is a prospective longitudinal study, consisting of six time points, spanning from pregnancy to 3 years postpartum. The study began in June 2020, consisting of 304 pregnant people from Ontario, Canada. This multimethod study is composed of questionnaires, biological samples, behavioural observations and developmental assessments ETHICS AND DISSEMINATION: This study was approved by the Hamilton Integrated Research Ethics Board (#11034) and the Mount Saint Vincent University Research Ethics Board (#2020-187, #2021-075, #2022-008). Findings will be disseminated through peer-reviewed presentations and publications, community presentations, and electronic forums (social media, newsletters and website postings).

The Implications of Cannabinoid-Induced Metabolic Dysregulation for Cellular Differentiation and Growth.

The endocannabinoid system (ECS) governs and coordinates several physiological processes through an integrated signaling network, which is responsible for inducing appropriate intracellular metabolic signaling cascades in response to (endo)cannabinoid stimulation. This intricate cellular system ensures the proper functioning of the immune, reproductive, and nervous systems and is involved in the regulation of appetite, memory, metabolism, and development. Cannabinoid receptors have been observed on both cellular and mitochondrial membranes in several tissues and are stimulated by various classes of cannabinoids, rendering the ECS highly versatile. In the context of growth and development, emerging evidence suggests a crucial role for the ECS in cellular growth and differentiation. Indeed, cannabinoids have the potential to disrupt key energy-sensing metabolic signaling pathways requiring mitochondrial-ER crosstalk, whose functioning is essential for successful cellular growth and differentiation. This review aims to explore the extent of cannabinoid-induced cellular dysregulation and its implications for cellular differentiation.

Cannabis use during lactation may alter the composition of human breast milk.

BACKGROUND: Cannabis is often used by women to manage symptoms of morning sickness during pregnancy, and postpartum stress and anxiety. While exclusive breastfeeding has been recommended for the first 6 months of an infant's life, the presence of cannabinoids in the milk of cannabis users complicates this recommendation. The objective of this study was to investigate the effect of maternal cannabis use on changes in the levels of macronutrients and bioactive factors in breast milk. METHODS: Milk was collected from women who were 6-8 weeks postpartum and were either using cannabis post-delivery, had used cannabis during pregnancy, or were non-users. Levels of cannabinoids, macronutrients, lactose, and SIgA were assessed in the milk of all subjects. RESULTS: THC was detected in the milk of women who reported cannabis use during lactation (n = 13, median: 22 ng/mL). Carboxy-THC, 11-hydroxy-THC, CBD, and CBN were also detected in the milk of women who used cannabis postpartum. Relative to non-users (n = 17), lactose levels were higher and SIgA levels were significantly lower in the milk of subjects who used cannabis during lactation (n = 14). CONCLUSIONS: The presence of cannabinoids, along with altered lactose and SIgA levels in the milk of cannabis users, may have implications for infant health. IMPACT: Metabolites of cannabis are found in breast milk and can accumulate in higher concentrations with ongoing consumption, which is concerning for potential exposure among infants born to mothers who consume cannabis. This work reports that lactose levels are increased and SIgA levels are decreased in the breast milk of cannabis users, relative to the milk of non-users. Change in levels of lactose and SIgA in the milk of cannabis users may have significant implications on infant health, which must be investigated in the future to better inform mothers.

Effect of Delta-9-tetrahydrocannabinol and cannabidiol on milk proteins and lipid levels in HC11 cells.

Pregnant and lactating women have been discouraged from using cannabis by Health Canada. However, the increasing rate of cannabis use among pregnant women has presented an urgent need to investigate its physiological effects during the perinatal period. During pregnancy, the mammary gland (MG) undergoes remodeling, which involves alveolar differentiation of mammary epithelial cells (MECs), which is essential for breast milk production and secretion. Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on MG development or MEC differentiation. In this study, we investigated the effects of THC and CBD on the differentiation of MECs by assessing changes in cellular viability, lipid accumulation, and gene and protein expression of major milk protein and lipid synthesizing markers. using the HC11 cells as a model. We hypothesized that THC and CBD will negatively impact the synthesis of milk proteins and lipids, as well as lipid markers in HC11 cells. Our results demonstrated that THC and CBD reduced cellular viability at concentrations above 30µM and 20µM, respectively. Relative to control, 10µM THC and 10µM CBD reduced mRNA levels of milk proteins (CSN2 and WAP), lipid synthesizing and glucose transport markers (GLUT 1, HK2, FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, co-treatment of a CB2 antagonist with THC, and a CB2 agonist with CBD, reversed the impact of THC and CBD on the mRNA levels of key markers, respectively. In conclusion, 10µM THC and CBD altered the differentiation of HC11 cells, in part via the CB2 receptor.

Se(XY) matters: the importance of incorporating sex in microphysiological models.

The development of microphysiological models is currently at the forefront of preclinical research. Although these 3D tissue models are being developed to mimic physiological organ function and diseases, which are often sexually dimorphic, sex is usually neglected as a biological variable. For decades, national research agencies have required government-funded clinical trials to include both male and female participants as a means of eliminating male bias. However, this is not the case in preclinical trials, which have been shown to favor male rodents in animal studies and male cell types in in vitro studies. In this Opinion, we highlight the importance of considering sex as a biological variable and outline five approaches for incorporating sex-specific features into current microphysiological models.

The Impact of Early Life Exposure to Cannabis: The Role of the Endocannabinoid System.

Cannabis use during pregnancy has continued to rise, particularly in developed countries, as a result of the trend towards legalization and lack of consistent, evidence-based knowledge on the matter. While there is conflicting data regarding whether cannabis use during pregnancy leads to adverse outcomes such as stillbirth, preterm birth, low birthweight, or increased admission to neonatal intensive care units, investigations into long-term effects on the offspring's health are limited. Historically, studies have focused on the neurobehavioral effects of prenatal cannabis exposure on the offspring. The effects of cannabis on other physiological aspects of the developing fetus have received less attention. Importantly, our knowledge about cannabinoid signaling in the placenta is also limited. The endocannabinoid system (ECS) is present at early stages of development and represents a potential target for exogenous cannabinoids in utero. The ECS is expressed in a broad range of tissues and influences a spectrum of cellular functions. The aim of this review is to explore the current evidence surrounding the effects of prenatal exposure to cannabinoids and the role of the ECS in the placenta and the developing fetus.

Matrigel® enhances 3T3-L1 cell differentiation.

Culturing cells on bio-gels are believed to provide a more in vivo-like extracellular matrix. 3T3-L1 cells cultured on Matrigel® significantly alteregd their proliferation and differentiation as compared to growth on tissue culture-coated polystyrene surfaces. Growth on a 250-µm thick layer of Matrigel® facilitated the formation of cellular aggregates of 3T3-L1 cells. Differentiation of 3T3-L1 cells cultured on Matrigel® demonstrated increased levels of mRNA levels for key adipogenic transcription factors (PPARγ, C/EBPα, SREBP1), lipogenic markers (FAS, FABP4, LPL, PLIN1) and markers of adipocyte maturity (LEP), compared to cells cultured directly on a polystyrene tissue culture surface. The gene expression of extracellular matrix proteins (FN1, COL1A1, COL4A1, COL6, LAM) was decreased in 3T3-L1 cells cultured on Matrigel®. Furthermore, growth on Matrigel® increased lipid accumulation in 3T3-L1 cells in the presence and absence of rosiglitazone, a thiazolidinedione routinely used to optimize differentiation in these cells. These changes in adipocyte gene expression and lipid accumulation patterns may be a result of the increased cell-cell and cell-ECM interactions occurring on the Matrigel®, a scenario that is more reflective of an in vivo model. Taken together, our data advance the understanding of the value of culturing 3T3-L1 cells on Matrigel®.

Cannabinoid Signalling in Immune-Reproductive Crosstalk during Human Pregnancy.

Despite the intricate involvement of the endocannabinoid system in various physiological processes, it remains one of the most under-studied biological systems of the human body. The scope of endocannabinoid signalling is widespread, ranging from modulation of immune responses in innate and adaptive immunity to gestational processes in female physiology. Cannabinoid receptors are ubiquitously distributed in reproductive tissues and are thought to play a role in regulating the immune-reproductive interactions required for successful pregnancy, specifically among uterine natural killer cells and placental extravillous trophoblasts. The use of cannabis during pregnancy, however, can perturb endocannabinoid homeostasis through effects mediated by its major constituents, Δ-9-tetrahydrocannabinol and cannabidiol. Decidualization of the endometrium, invasion, and angiogenesis may be impaired as a consequence, leading to clinical complications such as miscarriage and preeclampsia. In this review, the crosstalk between endocannabinoid signalling in uterine natural killer cells and placental extravillous trophoblasts will be examined in healthy and complicated pregnancies. This lays a foundation for discussing the potential of targeting the endocannabinoid system for therapeutic benefit, particularly with regard to the emerging field of synthetic cannabinoids.

The Effects of Exercise Serum From Prepubertal Girls and Women on In Vitro Myoblast and Osteoblast Proliferation and Differentiation.

PURPOSE: In girls and women, the authors studied the effects of an acute bout of low-impact, moderate-intensity exercise serum on myoblast and osteoblast proliferation in vitro. METHODS: A total of 12 pre/early pubertal girls (8-10 y old) and 12 women (20-30 y old) cycled at 60% VO2max for 1 hour followed by 1-hour recovery. Blood samples were collected at rest, mid-exercise, end of exercise, mid-recovery, and end of recovery. C2C12 myoblasts and MC3T3E1 osteoblasts were incubated with serum from each time point for 1 hour, then monitored for 24 hours (myoblasts) or 36 hours (osteoblasts) to examine proliferation. Cells were also monitored for 6 days (myoblasts) to examine myotube formation and 21 days (osteoblasts) to examine mineralization. RESULTS: Exercise did not affect myoblast or osteoblast proliferation. Girls exhibited lower cell proliferation relative to women at end of exercise (osteoblasts, P = .041; myoblasts, P = .029) and mid-recovery (osteoblasts, P = .010). Mineralization was lower at end of recovery relative to rest (P = .014) in both girls and women. Myotube formation was not affected by exercise or group. CONCLUSION: The systemic environment following one acute bout of low-impact moderate-intensity exercise in girls and women does not elicit osteoblast or myoblast activity in vitro. Differences in myoblast and osteoblast proliferation between girls and women may be influenced by maturation.

Delta-9-tetrahydrocannabinol inhibits invasion of HTR8/SVneo human extravillous trophoblast cells and negatively impacts mitochondrial function.

Prenatal cannabis use is a significant problem and poses important health risks for the developing fetus. The molecular mechanisms underlying these changes are not fully elucidated but are thought to be attributed to delta-9-tetrahydrocannabinol (THC), the main bioactive constituent of cannabis. It has been reported that THC may target the mitochondria in several tissue types, including placental tissue and trophoblast cell lines, and alter their function. In the present study, in response to 48-h THC treatment of the human extravillous trophoblast cell line HTR8/SVneo, we demonstrate that cell proliferation and invasion are significantly reduced. We further demonstrate THC-treatment elevated levels of cellular reactive oxygen species and markers of lipid damage. This was accompanied by evidence of increased mitochondrial fission. We also observed increased expression of cellular stress markers, HSP70 and HSP60, following exposure to THC. These effects were coincident with reduced mitochondrial respiratory function and a decrease in mitochondrial membrane potential. Taken together, our results suggest that THC can induce mitochondrial dysfunction and reduce trophoblast invasion; outcomes that have been previously linked to poor placentation. We also demonstrate that these changes in HTR8/SVneo biology may be variably mediated by cannabinoid receptors CB1 and CB2.

Metabolic Flexibility during Exercise in Children with Obesity and Matched Controls.

BACKGROUND: Impaired metabolic flexibility (MetFlex) could contribute to ectopic fat accumulation and pathological conditions, such as type 2 diabetes. MetFlex refers to the ability to adapt substrate oxidation to availability. To the best of our knowledge, no studies have examined MetFlex under exercise conditions in children with obesity (OB) compared with a control group (CON) without obesity. Therefore, the primary objective was to compare MetFlex during exercise in children with OB compared with CON matched for chronological age, sex, and biological maturation. A better understanding of MetFlex could help elucidate its role in the pathogenesis of childhood obesity and insulin resistance. METHODS: Children with obesity and without obesity age 8 to 17 yr attended two visits, which included anthropometric measurements, blood work (OB group only), a maximal aerobic fitness (V˙O2max) test, and MetFlex test with a C-enriched carbohydrate (1.75 g per kg of body mass, up to 75 g) ingested before 60 min of exercise at 45% V˙O2max. Breath measurements were collected to calculate exogenous CHO (CHOexo) oxidative efficiency as a measure of MetFlex. RESULTS: CHOexo oxidative efficiency (CHOexo oxidized/CHO ingested × 100) during exercise was significantly lower in OB (17.3% ± 4.0%) compared with CON (22.6% ± 4.7%, P < 0.001). CHOexo contributed less to total energy expenditure during exercise in OB compared with CON (P < 0.001), whereas the contribution of endogenous CHO (P = 0.19) and total fat was not significantly different (P = 0.91). CONCLUSIONS: The ability to oxidize oral CHO, an indicator of MetFlex, was 23.5% lower in children with obesity compared with controls, independent of age, sex, and pubertal effects. Thus, obesity at a young age could be associated with reduced MetFlex and future comorbidities.

Delta-9-tetrahydrocannabinol disrupts mitochondrial function and attenuates syncytialization in human placental BeWo cells.

The psychoactive component in cannabis, delta-9-tetrahydrocannabinol, can restrict fetal growth and development. Delta-9-tetrahydrocannabinol has been shown to negatively impact cellular proliferation and target organelles like the mitochondria resulting in reduced cellular respiration. In the placenta, mitochondrial dysfunction leading to oxidative stress prevents proper placental development and function. A key element of placental development is the proliferation and fusion of cytotrophoblasts to form the syncytium that comprises the materno-fetal interface. The impact of delta-9-tetrahydrocannabinol on this process is not well understood. To elucidate the nature of the mitochondrial dysfunction and its consequences on trophoblast fusion, we treated undifferentiated and differentiated BeWo human trophoblast cells, with 20 µM delta-9-tetrahydrocannabinol for 48 hr. At this concentration, delta-9-tetrahydrocannabinol on BeWo cells reduced the expression of markers involved in syncytialization and mitochondrial dynamics, but had no effect on cell viability. Delta-9-tetrahydrocannabinol significantly attenuated the process of syncytialization and induced oxidative stress responses in BeWo cells. Importantly, delta-9-tetrahydrocannabinol also caused a reduction in the secretion of human chorionic gonadotropin and the production of human placental lactogen and insulin growth factor 2, three hormones known to be important in facilitating fetal growth. Furthermore, we also demonstrate that delta-9-tetrahydrocannabinol attenuated mitochondrial respiration, depleted adenosine triphosphate, and reduced mitochondrial membrane potential. These changes were also associated with an increase in cellular reactive oxygen species, and the expression of stress responsive chaperones, HSP60 and HSP70. These findings have important implications for understanding the role of delta-9-tetrahydrocannabinol-induced mitochondrial injury and the role this might play in compromising human pregnancies.

The Systemic Effects of Exercise on Regulators of Muscle and Bone in Girls and Women.

PURPOSE: To assess the systemic effects of an acute bout of moderate-intensity exercise on factors that are known to regulate muscle and bone growth in prepubertal girls and women. METHODS: A total of 12 prepubertal girls (8-10 y) and 12 women (20-30 y) cycled at 60% maximal oxygen uptake for 1 hour followed by 1 hour recovery. Blood samples were collected at rest, mid-exercise, end of exercise, mid-recovery, and end of recovery. Plasma was analyzed for interleukin-6, chemokine ligand 1, fibroblast growth factor-2, total insulin growth factor-1 (IGF-1), and free IGF-1 using enzyme-linked immunosorbent assays assays. RESULTS: Both groups had similar concentrations of systemic factors at baseline with the exception of free IGF-1, which was higher in girls (P = .001). Interleukin-6 response was lower in girls versus women (P = .04), with a difference of +105.1% at end of exercise (P < .001), +113.5% at mid-recovery (P = .001), and +93.2% at end of recovery (P = .02). Girls and women exhibited significant declines in chemokine ligand 1, fibroblast growth factor-2, and total IGF-1 during recovery. CONCLUSION: Compared with women, an acute bout of moderate-intensity exercise in girls elicits a lower inflammatory response, suggesting that other mechanisms may be more important for driving the anabolic effects of exercise on muscle and bone in girls.

Establishment of an in vitro placental barrier model cultured under physiologically relevant oxygen levels.

The human placental barrier facilitates many key functions during pregnancy, most notably the exchange of all substances between the mother and fetus. However, preclinical models of the placental barrier often lacked the multiple cell layers, syncytialization of the trophoblast cells and the low oxygen levels that are present within the body. Therefore, we aimed to design and develop an in vitro model of the placental barrier that would reinstate these factors and enable improved investigations of barrier function. BeWo placental trophoblastic cells and human umbilical vein endothelial cells were co-cultured on contralateral sides of an extracellular matrix-coated transwell insert to establish a multilayered barrier. Epidermal growth factor and forskolin led to significantly increased multi-nucleation of the BeWo cell layer and increased biochemical markers of syncytial fusion, for example syncytin-1 and hCGß. Our in vitro placental barrier possessed size-specific permeability, with 4000-Da molecules experiencing greater transport and a lower apparent permeability coefficient than 70 000-Da molecules. We further demonstrated that the BeWo layer had greater resistance to smaller molecules compared to the endothelial layer. Chronic, physiologically low oxygen exposure (3-8%) increased the expression of hypoxia-inducible factor 1α and syncytin-1, further increased multi-nucleation of the BeWo cell layer and decreased barrier permeability only against smaller molecules (457 Da/4000 Da). In conclusion, we built a novel in vitro co-culture model of the placental barrier that possessed size-specific permeability and could function under physiologically low oxygen levels. Importantly, this will enable future researchers to better study the maternal-fetal transport of nutrients and drugs during pregnancy.

Reactive oxygen species from mitochondria impacts trophoblast fusion and the production of endocrine hormones by syncytiotrophoblasts.

The placenta, a tissue that is metabolically active and rich in mitochondria, forms a critical interface between the mother and developing fetus. Oxidative stress within this tissue, derived from the dysregulation of reactive oxygen species (ROS), has been linked to a number of adverse fetal outcomes. While such outcomes have been associated with mitochondrial dysfunction, the causal role of mitochondrial dysfunction and mitochondrially generated ROS in altering the process of placentation remains unclear. In this study, mitochondrial complex I activity was attenuated using 10 nM rotenone to induce cellular oxidative stress by increasing mitochondrial ROS production in the BeWo choriocarcinoma cell line. Increased mitochondrial ROS resulted in a significant decrease in the transcripts which encode for proteins associated with fusion (GCM1, ERVW-1, and ERVFRD-1) resulting in a 5-fold decrease in the percentage of BeWo fusion. This outcome was associated with increased indicators of mitochondrial fragmentation, as determined by decreased expression of MFN2 and OPA1 along with an increase in a marker of mitochondrial fission (DRP1). Importantly, increased mitochondrial ROS also resulted in a 5.0-fold reduction of human placental lactogen (PL) and a 4.4-fold reduction of insulin like growth factor 2 (IGF2) transcripts; hormones which play an important role in regulating fetal growth. The pre-treatment of rotenone-exposed cells with 5 mM N-acetyl cysteine (NAC) resulted in the prevention of these ROS mediated changes in BeWo function and supports a central role for mitochondrial ROS signaling in the maintenance and function of the materno-fetal interface.

Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids.

Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ9-tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.