Synthesis of phospholipids in human placenta.

INTRODUCTION: Placental phospholipid synthesis is critical for the expansion of the placental exchange surface area and for production of signaling molecules. Despite their importance, it is not yet established which enzymes involved in the de novo synthesis and remodeling of placental phospholipids are expressed and active in the human placenta. METHODS: We identified phospholipid synthesis enzymes by immunoblotting in placental homogenates and immunofluorescence in placenta tissue sections. Primary human trophoblast (PHT) cells from term healthy placentas (n = 10) were cultured and exposed to 13C labeled fatty acids (16:0, 18:1 and 18:2 n-6, 22:6 n-3) for 2 and 24 h. Three phospholipid classes; phosphatidic acid, phosphatidylcholine, and lysophosphatidylcholine containing 13C fatty acids were quantified by Liquid Chromatography with tandem mass spectrometry (LC/MS-MS). RESULTS: Acyl transferase and phospholipase enzymes were detected in human placenta homogenate and primarily expressed in the syncytiotrophoblast. Three representative 13C fatty acids (16:0, 18:1 and 18:2 n-6) were incorporated rapidly into phosphatidic acid in trophoblasts, but 13C labeled docosahexaenoic acid (DHA; 22:6 n-3) incorporation was not detected. 13C DHA was incorporated into phosphatidylcholine. Lysophosphatidylcholine containing all four 13C labeled fatty acids were found in high abundance. CONCLUSIONS: Phospholipid synthesis and remodeling enzymes are present in the syncytiotrophoblast. 13C labeled fatty acids were rapidly incorporated into cellular phospholipids. 13C DHA was incorporated into phospholipids through the remodeling pathway rather than by de novo synthesis. These understudied pathways are highly active and critical for structure and function of the placenta.

Knockdown of Placental Major Facilitator Superfamily Domain Containing 2a in Pregnant Mice Reduces Fetal Brain Growth and Phospholipid Docosahexaenoic Acid Content.

INTRODUCTION: Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid critical for fetal brain development that is transported to the fetus from the mother by the placenta. The lysophosphatidylcholine (LPC) transporter, Major Facilitator Superfamily Domain Containing 2a (MFSD2a), is localized in the basal plasma membrane of the syncytiotrophoblast of the human placenta, and MFSD2a expression correlates with umbilical cord blood LPC-DHA levels in human pregnancy. We hypothesized that placenta-specific knockdown of MFSD2a in pregnant mice reduces phospholipid DHA accumulation in the fetal brain. METHODS: Mouse blastocysts (E3.5) were transduced with an EGFP-expressing lentivirus containing either an shRNA targeting MFSD2a or a non-coding sequence (SCR), then transferred to pseudopregnant females. At E18.5, fetuses were weighed and their placenta, brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR in the brain, liver and placenta and phospholipid DHA was quantified by LC-MS/MS. RESULTS: MFSD2a-targeting shRNA reduced placental mRNA MFSD2a expression by 38% at E18.5 (n = 45, p < 0.008) compared with SCR controls. MFSD2a expression in the fetal brain and liver were unchanged. Fetal brain weight was reduced by 13% (p = 0.006). Body weight, placenta and liver weights were unaffected. Fetal brain phosphatidyl choline and phosphatidyl ethanolamine DHA content was lower in fetuses with placenta-specific MFSD2a knockdown. CONCLUSIONS: Placenta-specific reduction in expression of the LPC-DHA transporter MFSD2a resulted in reduced fetal brain weight and lower phospholipid DHA content in the fetal brain. These data provide mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA, which is critical for brain growth.

Characterization of the Primary Human Trophoblast Cell Secretome Using Stable Isotope Labeling With Amino Acids in Cell Culture.

The placental villus syncytiotrophoblast, the nutrient-transporting and hormone-producing epithelium of the human placenta, is a critical regulator of fetal development and maternal physiology. However, the identities of the proteins synthesized and secreted by primary human trophoblast (PHT) cells remain unknown. Stable Isotope Labeling with Amino Acids in Cell Culture followed by mass spectrometry analysis of the conditioned media was used to identify secreted proteins and obtain information about their relative rates of synthesis in syncytialized multinucleated PHT cells isolated from normal term placental villus tissue (n = 4/independent placenta). A total of 1,344 proteins were identified, most of which have not previously been reported to be secreted by the human placenta or trophoblast. The majority of secreted proteins are involved in energy and carbon metabolism, glycolysis, biosynthesis of amino acids, purine metabolism, and fatty acid degradation. Histone family proteins and mitochondrial proteins were among proteins with the slowest synthesis rate whereas proteins associated with signaling and the plasma membrane were synthesized rapidly. There was a significant overlap between the PHT secretome and proteins known be secreted to the fetal circulation by the human placenta in vivo. The generated data will guide future experiments to determine the function of individual secreted proteins and will help us better understand how the placenta controls maternal and fetal physiology.

Effect of type 2 diabetes mellitus on placental expression and activity of nutrient transporters and their association with birth weight and neonatal adiposity.

AIMS: Infants born to women with Type 2 Diabetes Mellitus (T2DM) are at risk of being born large for gestational age due to excess fetal fat accretion. Placental nutrient transport determines fetal nutrient availability, impacting fetal growth. The aims of the study were to evaluate the effect of T2DM on placental insulin signaling, placental nutrient transporters and neonatal adiposity. METHODS: Placentas were collected from BMI-matched normoglycemic controls (NGT, n = 9) and T2DM (n = 9) women. Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated. Expression of glucose (GLUT1, -4), fatty acid (FATP2, -4, -6, FAT/CD36), amino acid (SNAT1, -2, -4, LAT1, -2) transporters, insulin signaling, and System A transporter activity was determined. Neonatal fat mass (%) was measured in a subset of neonates born to T2DM women. RESULTS: GLUT1 protein expression was increased (p = 0.001) and GLUT4 decreased (p = 0.006) in BM from T2DM. MVM FATP6 expression was increased (p = 0.02) and correlated with birth weight in both T2DM and NGT groups (r = 0.65, p = 0.02). BM FATP6 expression was increased (p = 0.01) in T2DM. In MVM of T2DM placentas, SNAT1 expression was increased (p = 0.05) and correlated with birth weight (r = 0.84, p = 0.004); SNAT2 was increased (p = 0.01), however System A transporter activity was not different between groups. MVM LAT1 expression was increased (p = 0.01) in T2DM and correlated with birth weight (r = 0.59, p = 0.04) and neonatal fat mass (r = 0.76, p = 0.06). CONCLUSION: In pregnancies complicated by T2DM placental protein expression of transporters for glucose, amino acids and fatty acids is increased, which may contribute to increased fetal growth and neonatal adiposity.

Placental Insulin/IGF-1 Signaling, PGC-1α, and Inflammatory Pathways Are Associated With Metabolic Outcomes at 4-6 Years of Age: The ECHO Healthy Start Cohort.

An adverse intrauterine environment is associated with the future risk of obesity and type 2 diabetes. Changes in placental function may underpin the intrauterine origins of adult disease, but longitudinal studies linking placental function with childhood outcomes are rare. Here, we determined the abundance and phosphorylation of protein intermediates involved in insulin signaling, inflammation, cortisol metabolism, protein glycosylation, and mitochondrial biogenesis in placental villus samples from healthy mothers from the Healthy Start cohort. Using MANOVA, we tested the association between placental proteins and offspring adiposity (fat mass percentage) at birth (n = 109) and infancy (4-6 months, n = 104), and adiposity, skinfold thickness, triglycerides, and insulin in children (4-6 years, n = 66). Placental IGF-1 receptor protein was positively associated with serum triglycerides in children. GSK3ß phosphorylation at serine 9, a readout of insulin and growth factor signaling, and the ratio of phosphorylated to total JNK2 were both positively associated with midthigh skinfold thickness in children. Moreover, peroxisome proliferator-activated receptor γ coactivator (PGC)-1α abundance was positively associated with insulin in children. In conclusion, placental insulin/IGF-1 signaling, PGC-1α, and inflammation pathways were positively associated with metabolic outcomes in 4- to 6-year-old children, identifying a novel link between placental function and long-term metabolic outcomes.

Associations between the activity of placental nutrient-sensing pathways and neonatal and postnatal metabolic health: the ECHO Healthy Start cohort.

OBJECTIVE: Our hypothesis was that the activity of placental nutrient-sensing pathways is associated with adiposity and metabolic health in childhood. RESEARCH DESIGN AND METHODS: Using placental villus samples from healthy mothers from the Healthy Start Study, we measured the abundance and phosphorylation of key intermediates in the mTOR, insulin, AMPK, and ER stress signaling pathways. Using multivariate multiple regression models, we tested the association between placental proteins and offspring adiposity (%fat mass) at birth (n = 109), 4-6 months (n = 104), and 4-6 years old (n = 64), adjusted for offspring sex and age. RESULTS: Placental mTORC1 phosphorylation was positively associated with adiposity at birth (R2 = 0.13, P = 0.009) and 4-6 years (R2 = 0.15, P = 0.046). The mTORC2 target PKCα was positively associated with systolic blood pressure at 4-6 years (ß = 2.90, P = 0.005). AMPK phosphorylation was positively associated with adiposity at birth (ß = 2.32, P = 0.023), but the ratio of phosphorylated to total AMPK was negatively associated with skinfold thickness (ß = -2.37, P = 0.022) and body weight (ß = -2.92, P = 0.005) at 4-6 years. CONCLUSIONS: This is the first report of associations between key placental protein activity measures and longitudinal child outcomes at various life stages. Our data indicate that AMPK and mTOR signaling are linked to cardiometabolic measures at birth and 4-6 years, providing novel insight into potential mechanisms underpinning how metabolic signaling in the placenta is associated with future risk of cardiovascular disease.

Decreased placental folate transporter expression and activity in first and second trimester in obese mothers.

Obese women have an approximately twofold higher risk to deliver an infant with neural tube defects (NTDs) despite folate supplementation. Placental transfer of folate is mediated by folate receptor alpha (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC). Decreased placental transport may contribute to NTDs in obese women. Serum folate levels were measured and placental tissue was collected from 13 women with normal BMI (21.9±1.9) and 11 obese women (BMI 33.1±2.8) undergoing elective termination at 8-22 weeks of gestation. The syncytiotrophoblast microvillous plasma membranes (MVM) were isolated using homogenization, magnesium precipitation, and differential centrifugation. MVM expression of FR-α, PCFT and RFC was determined by western blot. Folate transport capacity was assessed using radiolabeled methyl-tetrahydrofolate and rapid filtration techniques. Differences in expression and transport capacity were adjusted for gestational age and maternal age in multivariable regression models. P<.05 was considered statistically significant. Serum folate levels were not significantly different between groups. Placental MVM folate transporter expression did not change with gestational age. MVM RFC (-19%) and FR-α (-17%) expression was significantly reduced in placentas from obese women (P<.05). MVM folate transporter activity was reduced by-52% (P<.05) in obese women. These differences remained after adjustment for gestational age. There was no difference in mTOR signaling between groups. In conclusion, RFC and FR alpha expression and transporter activity in the placental MVM are significantly reduced in obese women in early pregnancy. These results may explain the higher incidence of NTDs in infants of obese women with adequate serum folate.

The effects of protein-coated surfaces on passaged porcine TMJ disc cells.

OBJECTIVE: Implantation of synthetic temporomandibular joint (TMJ) disc replacements aimed to alleviate pain and restore functional losses caused by TMJ disorders. Unfortunately, these synthetic replacements have been largely unsuccessful and in some instances have incited severe immune responses. Tissue engineering, however, may provide viable TMJ disc replacements. Towards this end, we have studied TMJ disc gene expression as a measure of protein production potential. With passage, collagen type I and aggrecan gene expression decrease in TMJ disc cell cultures. We hypothesize that surfaces coated with TMJ disc proteins may rapidly recover the lost gene expression in passaged TMJ disc cells. DESIGN: To study these effects, passages 0, 1, and 2 TMJ disc cells were plated in wells coated with aggrecan, collagen type I, collagen type II, or decorin. Safranin O staining was conducted to visualize cell aggregation. RESULTS: At passage 0, cultures appeared similar on each surface; however, by passages 1 and 2, aggrecan-coated and decorin-coated surfaces appeared to have more cell aggregates. Gene expression data did not correspond to these visual changes. No treated surface offered a significant change in aggrecan, collagen type I, or decorin expression relative to untreated controls. Furthermore, aggrecan and collagen type I gene expression dropped relative to samples taken prior to plating. CONCLUSIONS: These results indicate that, despite visual changes described by cell aggregates, protein coatings have limited effects for recovering TMJ disc gene expression in monolayer cultures.

Biomaterials for stem cell differentiation.

The promise of cellular therapy lies in the repair of damaged organs and tissues in vivo as well as generating tissue constructs in vitro for subsequent transplantation. Unfortunately, the lack of available donor cell sources limits its ultimate clinical applicability. Stem cells are a natural choice for cell therapy due to their pluripotent nature and self-renewal capacity. Creating reserves of undifferentiated stem cells and subsequently driving their differentiation to a lineage of choice in an efficient and scalable manner is critical for the ultimate clinical success of cellular therapeutics. In recent years, a variety of biomaterials have been incorporated in stem cell cultures, primarily to provide a conducive microenvironment for their growth and differentiation and to ultimately mimic the stem cell niche. In this review, we examine applications of natural and synthetic materials, their modifications as well as various culture conditions for maintenance and lineage-specific differentiation of embryonic and adult stem cells.

Vascular permeability in ocular disease and the role of tight junctions.

Vascular permeability is closely linked with angiogenesis in a number of pathologies. In the retina, the normally well-developed blood-retinal barrier is altered in a host of eye diseases preceding or commensurate with angiogenesis. This review examines the literature regarding the tight junction complex that establishes the blood-retinal barrier focusing on the transmembrane proteins occludin and the claudin family and the membrane associated protein zonula occludens. The changes observed in these proteins associated with vascular and epithelial permeability is discussed. Finally, novel literature addressing the link between the tight junction complex and angiogenesis is considered.