ABSTRACT
There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.
Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms/metabolism , Signal Transduction/drug effects , Animals , Apoptosis , Autophagy , Benzamides/pharmacology , Cell Line, Tumor , Cell Respiration/drug effects , Cell Survival , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Lipid Droplets/metabolism , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Neoplasms/enzymology , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phospholipase A2 Inhibitors/pharmacology , Phospholipids/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/pharmacology , Tumor Cells, CulturedABSTRACT
The degree that maternal glycemia affects placental metabolism of trophoblast cell types [cytotrophoblast (CTB) and syncytiotrophoblast (SCT)] in pregnant persons with gestational diabetes mellitus (GDM) is unknown. We tested the hypotheses that (a) hyperglycemia suppresses the metabolic rates of CTB and SCT; and (b) low placental metabolic activity from GDM placentas is due to decreased oxygen consumption of CTB. Trophoblast cells isolated from GDM and non-GDM term placentas were cultured for 8-hour (CTB) and following syncytialization at 72-hour (SCT) in 5 mM of glucose or 25 mM of glucose. Oxygen consumption rates, glycolysis, ATP levels, and lipid droplet morphometries were determined in CTB and SCT. In CTB from GDM placentas compared to control CTB: (a) oxidative phosphorylation was decreased by 44% (41.8 vs 74.2 pmol O2 /min/100 ng DNA, P = .002); (b) ATP content was 39% lower (1.1 × 10-7 vs 1.8 × 10-7 nM/ng DNA, P = .046); and (c) lipid droplets were two times larger (31.0 vs 14.4 µm2 /cell, P < .001) and 1.7 times more numerous (13.5 vs 7.9 lipid droplets/cell, P < .001). Hyperglycemia suppressed CTB glycolysis by 55%-60% (mean difference 20.4 [GDM, P = .008] and 15.4 [non-GDM, P = .029] mpH/min/100 ng DNA). GDM SCT was not metabolically different from non-GDM SCT. However, GDM SCT had significantly decreased expression of genes associated with differentiation including hCG, GCM1, and syncytin-1. We conclude that suppressed metabolic activity by the GDM placenta is attributable to metabolic dysfunction of CTB, not SCT. Critical placental hormone expression and secretion are decreased in GDM trophoblasts.
Subject(s)
Diabetes, Gestational/metabolism , Hyperglycemia/metabolism , Lipids , Mitochondria/metabolism , Cell Differentiation , Female , Glucose/metabolism , Glycolysis/physiology , Humans , Oxidative Phosphorylation/drug effects , Oxygen Consumption/physiology , Placenta/metabolism , Pregnancy , Trophoblasts/metabolismABSTRACT
BACKGROUND & AIMS: Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have demonstrated similar efficacy in removal of neoplastic esophageal lesions. However, significant controversy exists over the preferred resection technique. Our primary aim was to compare the pathologic specimens produced via EMR and ESD and secondarily gauge their effect on clinical decision making and patient outcomes. METHODS: Using a retrospective cohort study design, all esophageal Barrett's-associated neoplastic lesions resected by a single provider from 2012 to 2017 were reviewed. The pathology was re-reviewed by two blinded authors for diagnosis, margins, and adverse outcomes and recurrence rates were also collected. RESULTS: Thirty-one EMR and 20 ESD cases were identified. Baseline demographics and lesion characteristics were similar. ESD produced more R0 resections and more en bloc resections compared to EMR. EMR produced more equivocal lateral (13/31, 41.9% vs 1/20, 5.0%) and vertical margins (13/31, 41.9% vs. 0/20, 0%, both P < 0.05). This led to an inability to reach a definitive diagnosis in 13/31 EMR vs 0/20 ESD pathology specimens (P = 0.003). Of the 13 EMR specimens with equivocal pathology, 11 were noted to have 'at least intramucosal adenocarcinoma'. Four of the 11 patients chose to undergo elective esophagectomy with final surgical pathology demonstrating ≤T1a disease in 2, and ≥T1b disease in two. CONCLUSION: Compared to ESD, EMR was associated with greater pathologic uncertainty in Barrett's-associated neoplasia.
Subject(s)
Adenocarcinoma/pathology , Barrett Esophagus/pathology , Barrett Esophagus/surgery , Dissection , Endoscopic Mucosal Resection , Esophageal Neoplasms/pathology , Adenocarcinoma/surgery , Aged , Clinical Decision-Making , Esophageal Neoplasms/surgery , Esophagectomy , Female , Humans , Male , Middle Aged , Neoplasm Staging , Retrospective StudiesABSTRACT
Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.
ABSTRACT
Somatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of "outlier" candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53-/- oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53-/- esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.
Subject(s)
Neoplasms , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Oncogenes , Cell Transformation, Neoplastic/genetics , Neoplasms/genetics , Carcinogenesis/genetics , Gene AmplificationABSTRACT
So-called primary yolk sac tumors of the vulva are very rare and often have an aggressive disease course. Their molecular features have not been previously characterized. There is also a well-documented group of SMARCB1 (INI-1)-deficient vulvar neoplasms, which includes proximal-type epithelioid sarcoma and myoepithelial carcinoma. Until now, "vulvar yolk sac tumors" and SMARCB1-deficient neoplasms were considered unrelated diseases. After reviewing an index case of a vulvar yolk sac tumor with loss of SMARCB1 by immunohistochemistry, we retrospectively identified 2 additional cases diagnosed as vulvar yolk sac tumors. Patient ages were 34, 32, and 25 years old, and 2 tumors were associated with a pregnancy. All 3 cases showed morphology typical of a yolk sac tumor, and by immunohistochemistry all were positive for SALL4, glypican-3, keratins, and lacked CD34 positivity. All tumors also demonstrated loss of SMARCB1 in tumor cells. Targeted molecular profiling was performed in 2 cases and identified 2 copy deletion of SMARCB1, without genomic alterations typically seen in gonadal yolk sac tumors. In the third case, isochromosome 12p was not identified by fluorescence in situ hybridization. All 3 patients had either local recurrences or distant metastases, and 2 died of disease. One patient had progressive disease while receiving the enhancer of zeste homolog 2 inhibitor tazemetostat. Overall, these findings suggest that vulvar tumors with pure yolk sac-like morphology may represent morphologic variants of SMARCB1-deficient tumors and not veritable germ cell neoplasia. This potential reclassification may have both prognostic and treatment implications and warrants study of additional extragonadal yolk sac tumors.
Subject(s)
Biomarkers, Tumor/deficiency , Endodermal Sinus Tumor/chemistry , SMARCB1 Protein/deficiency , Vulvar Neoplasms/chemistry , Adult , Biomarkers, Tumor/genetics , Chromosome Deletion , Chromosomes, Human, Pair 12 , Disease Progression , Endodermal Sinus Tumor/genetics , Endodermal Sinus Tumor/secondary , Endodermal Sinus Tumor/surgery , Female , Gene Deletion , Humans , Neoplasm Recurrence, Local , Retrospective Studies , SMARCB1 Protein/genetics , Treatment Outcome , Vulvar Neoplasms/genetics , Vulvar Neoplasms/pathology , Vulvar Neoplasms/surgeryABSTRACT
Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy.
Subject(s)
Immune Tolerance , Killer Cells, Natural/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms, Cystic, Mucinous, and Serous/immunology , Ovarian Neoplasms/immunology , Tumor Escape , Tumor Microenvironment/immunology , Antineoplastic Agents/pharmacology , Carboplatin/pharmacology , Cell Line, Tumor , Coculture Techniques , Cytokines/metabolism , Cytotoxicity, Immunologic , Female , Humans , Immune Tolerance/drug effects , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/metabolism , Neoplasms, Cystic, Mucinous, and Serous/drug therapy , Neoplasms, Cystic, Mucinous, and Serous/metabolism , Neoplasms, Cystic, Mucinous, and Serous/pathology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Phenotype , Receptors, Natural Killer Cell/metabolism , Tetraspanin 29/metabolism , Trogocytosis , Tumor Escape/drug effectsABSTRACT
Mutations in ARID1A rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout (KO) in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/ß-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein-Barr virus-associated subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFICANCE: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of ARID1A-deficient gastric cancers.See related commentary by Zafra and Dow, p. 1327.This article is highlighted in the In This Issue feature, p. 1307.
Subject(s)
CRISPR-Cas Systems , Cell Transformation, Neoplastic , DNA-Binding Proteins/genetics , Stomach Neoplasms/genetics , Transcription Factors/genetics , Humans , Models, Biological , MutationABSTRACT
Two recent papers in Cell Stem Cell and Nature Medicine (Yao et al. [2019] and Ganesh et al. [2019]) demonstrate the successful use of rectal cancer patient-derived organoids to predict patient responses to neoadjuvant chemoradiation therapy, paving the way toward a new paradigm for precision medicine.
Subject(s)
Organoids , Rectal Neoplasms , Humans , Neoadjuvant Therapy , Precision MedicineABSTRACT
Tenosynovial giant cell tumors (TGCTs) are benign neoplasms that arise from the synovium of tendon sheaths, bursae, and joints. We report a rare presentation of TGCT involving the suboccipital spine.
Subject(s)
Brain Neoplasms/pathology , Giant Cell Tumors/pathology , Synovial Membrane/pathology , Giant Cell Tumor of Tendon Sheath/pathology , Humans , Occipital Lobe/pathology , Spinal Neoplasms/pathologyABSTRACT
Filamins are actin binding proteins that contribute to cytoskeletal integrity and biochemical scaffolds during mechanochemical signal transductions. Structurally, human filamins are dimers composed of an actin-binding domain with 24 immunoglobulin (Ig)-like repeats. In this study, we focus on the recently solved high-resolution crystal structure of Ig-like repeats 19-21 of filamin-A (IgFLNa-R19-R21). IgFLNa-R19-21 is of marked importance because it contains the binding site for integrins and facilitates the dynamic ability of filamin-A to communicate with the extracellular environment. However, the structure of filamin-A shows an interesting domain arrangement where the integrin binding site on IgFLNa-R21 is hindered sterically by IgFLNa-R20. Thus, a number of hypotheses on the regulation of filamin-A exist. Using molecular dynamics simulations we evaluated the effects of two primary regulators of filamin-A, force and phosphorylation. We find that a tensile force of 40 pN is sufficient to initiate the partial removal of the autoinhibition on the integrin binding site of IgFLNa-R21. Force coupled to phosphorylation at Ser(2152), however, affords complete dissociation of autoinhibition with a decreased force requirement. Phosphorylation seems to decrease the threshold for removing the IgFLNa-R20 beta-strand inhibitor within 300 ps with 40 pN tensile force. Furthermore, the molecular dynamic trajectories illustrate phosphorylation of Ser(2152) without force is insufficient to remove autoinhibition. We believe the results of this study implicate filamin-A as a tunable mechanosensor, where its sensitivity can be modulated by the degree of phosphorylation.
Subject(s)
Contractile Proteins/metabolism , Integrins/metabolism , Microfilament Proteins/metabolism , Biomechanical Phenomena , Contractile Proteins/antagonists & inhibitors , Contractile Proteins/chemistry , Filamins , Humans , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/chemistry , Molecular Dynamics Simulation , Phosphorylation , Protein Binding , Protein Denaturation , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid , Serine , Solvents/chemistryABSTRACT
Rearrangement of the actin cytoskeleton is integral to cell shape and function. Actin-binding proteins, e.g., filamin, can naturally contribute to the mechanics and function of the actin cytoskeleton. The molecular mechanical bases for filamin's function in actin cytoskeletal reorganization are examined here using molecular dynamics simulations. Simulations are performed by applying forces ranging from 25 pN to 125 pN for 2.5 ns to the rod domain of filamin. Applying small loads ( approximately 25 pN) to filamin's rod domain supplies sufficient energy to alter the conformation of the N-terminal regions of the rod. These forces break local hydrogen bond coordination often enough to allow side chains to find new coordination partners, in turn leading to drastic changes in the conformation of filamin, for example, increasing the hydrophobic character of the N-terminal rod region and, alternatively, activating the C-terminal region to become increasingly stiff. These changes in conformation can lead to changes in the affinity of filamin for its binding partners. Therefore, filamin can function to transduce mechanical signals as well as preserve topology of the actin cytoskeleton throughout the rod domain.
Subject(s)
Contractile Proteins/chemistry , Contractile Proteins/ultrastructure , Microfilament Proteins/chemistry , Microfilament Proteins/ultrastructure , Models, Chemical , Models, Molecular , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/ultrastructure , Computer Simulation , Elasticity , Filamins , Mechanics , Protein Conformation , Protein Structure, Tertiary , Stress, MechanicalABSTRACT
INTRODUCTION: The placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates. METHODS: We employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30â¯min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen. RESULTS: Long- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not. DISCUSSION: Fatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.
Subject(s)
Fatty Acids/metabolism , Microscopy, Confocal/methods , Placenta/metabolism , Trophoblasts/metabolism , Adult , Biological Transport , Boron Compounds , Capillaries/metabolism , Cells, Cultured , Fatty Acids/chemistry , Female , Fetus/blood supply , Fluorescent Antibody Technique , Fluorescent Dyes , Humans , Kinetics , PregnancyABSTRACT
The syncytiotrophoblast (SCT) at the maternal-fetal interface has been presumed to be the primary driver of placental metabolism, and the underlying progenitor cytotrophoblast cells (CTB) an insignificant contributor to placental metabolic activity. However, we now show that the metabolic rate of CTB is much greater than the SCT. The oxygen consumption and extracellular acidification rate, a measure of glycolysis, are both greater in CTB than in SCT in vitro (CTB: 96 ± 16 vs SCT: 46 ± 14 pmol O2 × min-1 × 100 ng DNA-1, p < 0.001) and (CTB: 43 ± 6.7 vs SCT 1.4 ± 1.0 ∆mpH × min-1 × 100 ng DNA-1, p < 0.0001). Mitochondrial activity, as determined by using the mitochondrial activity-dependent dye Mitotracker CM-H2TMRosa, is higher in CTB than in SCT in culture and living explants. These data cast doubt on the previous supposition that the metabolic rate of the placenta is dominated by the SCT contribution. Moreover, differentiation into SCT leads to metabolic suppression. The normal suppression of metabolic activity during CTB differentiation to SCT is prevented with a p38 MAPK signaling inhibitor and epidermal growth factor co-treatment. We conclude that the undifferentiated CTB, in contrast to the SCT, is highly metabolically active, has a high level of fuel flexibility, and contributes substantially to global metabolism in the late gestation human placenta.
Subject(s)
Glycolysis , Oxidative Phosphorylation , Placenta/metabolism , Adenosine Triphosphate/metabolism , Adult , Cell Differentiation/drug effects , Cells, Cultured , Epidermal Growth Factor/pharmacology , Fatty Acids, Nonesterified/metabolism , Female , Glycolysis/drug effects , Humans , Imidazoles/pharmacology , Metabolic Flux Analysis , Microscopy, Fluorescence , Mitochondria/metabolism , Oxidative Phosphorylation/drug effects , Oxygen Consumption/drug effects , Pregnancy , Pyridines/pharmacology , Term Birth , Trophoblasts/cytology , Trophoblasts/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C12, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.
Subject(s)
Boron Compounds/metabolism , Fatty Acids/metabolism , Fluorescent Dyes/metabolism , Lipids/physiology , Placenta/metabolism , Trophoblasts/metabolism , Adult , Biological Transport/physiology , Fatty Acid-Binding Proteins/metabolism , Female , Fetal Development/physiology , Fetus/metabolism , Humans , Lipid Metabolism/physiology , Pregnancy , Term Birth/metabolismABSTRACT
The placenta is a key organ in programming the fetus for later disease. This review outlines nine of many structural and physiological features of the placenta which are associated with adult onset chronic disease. 1) Placental efficiency relates the placental mass to the fetal mass. Ratios at the extremes are related to cardiovascular disease risk later in life. 2) Placental shape predicts a large number of disease outcomes in adults but the regulators of placental shape are not known. 3) Non-human primate studies suggest that at about mid-gestation, the placenta becomes less plastic and less able to compensate for pathological stresses. 4) Recent studies suggest that lipids have an important role in regulating placental metabolism and thus the future health of offspring. 5) Placental inflammation affects nutrient transport to the fetus and programs for later disease. 6) Placental insufficiency leads to inadequate fetal growth and elevated risks for later life disease. 7) Maternal height, fat and muscle mass are important in combination with placental size and shape in predicting adult disease. 8) The placenta makes a host of hormones that influence fetal growth and are related to offspring disease. Unfortunately, our knowledge of placental growth and function lags far behind that of other organs. An investment in understanding placental growth and function will yield enormous benefits to human health because it is a key player in the origins of the most expensive and deadly chronic diseases that humans face.
Subject(s)
Fetal Development/physiology , Maternal-Fetal Exchange/physiology , Placenta/physiology , Animals , Female , Humans , Placental Insufficiency/physiopathology , Pregnancy , Prenatal Exposure Delayed Effects/physiopathologyABSTRACT
The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.
Subject(s)
Carrier Proteins/biosynthesis , Ectogenesis , Embryo Transfer/adverse effects , Fertilization in Vitro/adverse effects , Metabolic Diseases/etiology , Obesity/etiology , Thioredoxins/biosynthesis , Up-Regulation , Acetylation , Adipose Tissue/embryology , Adipose Tissue/growth & development , Adipose Tissue/metabolism , Animals , Biomarkers/blood , Biomarkers/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Disease Susceptibility , Epigenesis, Genetic , Female , Histones/metabolism , Male , Metabolic Diseases/blood , Metabolic Diseases/metabolism , Metabolic Diseases/pathology , Mice , Mice, Inbred C57BL , Obesity/blood , Obesity/metabolism , Obesity/pathology , Oxidative Stress , Promoter Regions, Genetic , Protein Processing, Post-Translational , Thioredoxins/genetics , Thioredoxins/metabolism , Transcription, GeneticABSTRACT
More than 4.5 million children have been conceived by in vitro fertilization (IVF). Interestingly, singleton IVF offspring born at term have an increased incidence of low birth weight. The mechanism responsible for the lower birth weight is unknown, but alterations in placental function are possible. Hence, the goal of our study was to examine placental growth and function in mice generated in vivo or in vitro. To assess placental function, blastocysts were generated by IVF or produced by natural mating (control group); both IVF and control blastocysts were transferred to pseudopregnant recipients. Placental weights did not differ at embryonic d 15.5 (E15.5) but were increased at E18.5 in the IVF group (25.4%, P < 0.001) compared with control. Proliferation was increased in IVF placentae, whereas overall placental gross morphology and apoptosis were not affected. Both fetal weights (16.4% lower at E15.5 and 8.8% lower at E18.5, P < 0.05) and fetal to placental ratios were lower (P < 0.001) in the IVF compared with the control group at both time points, whereas birth weights did not differ. At E18.5, the mRNA for selected glucose, system A amino acid transporters, and imprinted genes were down-regulated in IVF placentae. GLUT3 protein level was decreased in the IVF group (P < 0.05). Importantly, intrajugular injections of (14)C-methyl-D-glucose or (14)C-MeAIB tracers (n = 6 litters per group) showed that placental transport of glucose and amino acids were 24.8% (not significant) and 58.1% (P < 0.05) lower in the IVF group. Fetal accumulation of glucose was not different, but amino acid accumulation was significantly (36 %) lower in IVF fetuses (P < 0.05). We conclude that IVF alters both fetal and placental growth and, importantly, decreases placental transport efficiency in mice conceived by IVF.
Subject(s)
Fertilization in Vitro/methods , Placenta/physiology , Amino Acids/metabolism , Animals , Apoptosis , Biological Transport , Blastocyst/metabolism , Embryo Transfer , Female , Fetal Weight , Glucose/metabolism , Mice , Models, Animal , Organ Size , Placenta/metabolism , Pregnancy , Pregnancy, Animal , Time FactorsABSTRACT
It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68 ± 15% vs. 59 ± 18%), blastocyst (64 ± 9.1% vs. 50 ± 18%) and hatching blastocyst stages (54 ± 25% vs. 21 ± 16%) and an increase in the number of trophectodermal cell (TE,65 ± 13 vs. 49 ± 12 cells) compared to control embryos cultured in PD (mean ± S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.