Transcript Objective
MATERNAL OBESITY MAY CONTRIBUTE TO INCREASED PLACENTAL AND FETAL INFLAMMATION Molecular indicators of stress as indicators of immune status AMANDA JONES April 22, 2015 MCB5255 Background • Obesity • Maternal obesity • Pre-pregnancy obesity • Gestational diabetes • High prevalence of obesity in women of reproductive age Causes: • Maternal Age • Pre-pregnancy obesity • Pre-existing chronic medical conditions http://www.cdc.gov/reproductivehealth/MaternalInfantHealth/SevereMaternalMorbidity.html Background • Maternal Impact • Complications • Cesarean delivery • Gestational diabetes • Fetal Consequences • Fetal programming- Thrifty Phenotype (Barker) Hypothesis • Fetal stress- Intrauterine Growth Restriction (IUGR) http://www.frieslandcampinainstitute.com/en/maternal-nutrition/fetal-programming-andthe-risk-of-cardiometabolic-disorders.aspx http://www.medscape.com/viewarticle/776504 http://www.medscape.com/viewarticle/776504 Paper 1 • Objective: Evaluate how the placenta may display exaggerate inflammation in response to maternal obesity. Methods • Lean mothers (n=15) • Obese mothers (n=20) • Pre-gravid BMI • Singleton pregnancies • Cesarean delivery • Maternal blood draw • Fluorescence activated cell sorter (FACS) analysis • Umbilical blood draw • Glucose, insulin, cytokine- plasma assay • Placental Biopsy • RNA extraction • Primary cell isolation- FACS analysis • Immunohistochemistry- MP’s Results Results: Plasma Assays Results: Increased gene expression of maternal cytokines and monocytes in maternal PBMC’s Results: Increased localization of macrophages in obese placental tissue CD68+ Lean Obese CD14+ Results: Accumulation of monocyte heterogeneity in placenta of obese women Results: Higher expression of macrophages in the placenta, along with increased inflammatory cytokine expression Paper 1 Conclusion • Evidence of macrophage accumulation in the placenta of OB mothers • Macrophages in the placenta produce pro-inflammatory molecules • Obesity exacerbates basal inflammatory state of pregnancy, and may influence the in utero environment for fetal development Sheep versus Rat model • • • • • • • Sheep Monotocous Comparable birth weight Similar maternal:fetal ratio Similar organogenesis and growth rate Lack of available antibodies, reagents, etc. Labor intensive Ruminants • • • • • Rat Litter bearing High growth and metabolic rates Short gestation Cost effective Easier genetic manipulation Paper 2 • Objective: Evaluate effects of maternal obesity on adipogenesis, inflammatory signaling, and insulin pathways at late gestation when ovine fetal skeletal muscle matures Methods • Control Diet: 100% NRC requirement (n=6) • Obesogenic Diet: 150% NRC requirement (n=6) • 2 singleton, 4 twins = 3 M, 3F Start Diet -60d Conception 0d • Necropsy Collection • Fetal blood (umbillical vein) • Glucose and Insulin assay • Semitendenosus (St) muscle • Histology • mRNA extraction, RT-PCR • Immunoblot Analysis Necropsy (Full Term) 135d (148d) Results: Obese model achieved Results: Evidence of increased adipogenesis in OB offspring CON OB • OB Offspring • # fat cells • fat cell size • Fat cell infiltration • # muscle cells (G) • muscle cell diameter (H) PPARϒ mRNA and protein • Results: Gene expression of TLR4 and co-receptors is higher in OB offspring NFkB and JNK Pathway http://www.jci.org/articles/view/29069/figure/1 Results: Phosphorylation of key NFkB components are increased Results: Phosphorylation of key JNK components are increased Zhu et al 2010 Results: Disregulation of PKC, Akt and AMPK suggest insulin insensitivity and promoteds adipogenesis Conclusion, Paper 1 • Enhanced adipogenesis in OB offspring during late gestation • Increased TLR4 expression may contribute to activation of NFkB and JNK pathway, leading to inflammation in the ST • Increase of p-PKC and reduction of the insulin receptor and p-AKT indicate decreased insulin signaling • Reduction in AMPK activity is indicative of lipogenesis and adipogenesis • Obesity during gestation is associated with evidence of inflammation fetal muscle, indicative of fetal immune stress during important developmental stages Grant Proposal • Objective: Establish maternal and fetal systemic inflammatory profile • Sheep model • Control • Overfed • Restricted • Isolate RNA from white blood cells • PCR Array • Bovine Inflammatory Cytokines and Receptors • Maternal: Longitudinal analysis • Fetal: pre- versus post- parturition Grant Schematic Maternal Conception 0d Diet 30d 45d 90d Fetal 135d 148d