Adverse Fetal and Neonatal Outcomes Associated with a Life-Long High Fat Diet: Role of Altered Development of the Placental Vasculature

Maternal obesity results in a number of obstetrical and fetal complications with both immediate and long-term consequences. The increased prevalence of obesity has resulted in increasing numbers of women of reproductive age in this high-risk group. Since many of these obese women have been subjected to hypercaloric diets from early childhood we have developed a rodent model of life-long maternal obesity to more clearly understand the mechanisms that contribute to adverse pregnancy outcomes in obese women. Female Sprague Dawley rats were fed a control diet (CON - 16% of calories from fat) or high fat diet (HF - 45% of calories from fat) from 3 to 19 weeks of age. Prior to pregnancy HF-fed dams exhibited significant increases in body fat, serum leptin and triglycerides. A subset of dams was sacrificed at gestational day 15 to evaluate fetal and placental development. The remaining animals were allowed to deliver normally. HF-fed dams exhibited a more than 3-fold increase in fetal death and decreased neonatal survival. These outcomes were associated with altered vascular development in the placenta, as well as increased hypoxia in the labyrinth. We propose that the altered placental vasculature may result in reduced oxygenation of the fetal tissues contributing to premature demise and poor neonatal survival

Vascular endothelial growth factor and its receptor, FIk-1/KDR, are cytoprotective in the extravascular compartment of the ovarian follicle

Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/ KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the f

Production and Purification of High-Titer Newcastle Disease Virus for Use in Preclinical Mouse Models of Cancer

Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus in the Paramyxoviridae family. Although primarily an avian pathogen, NDV is a potent oncolytic virus that has been shown to be safe and effective in a variety of preclinical cancer models and human clinical trials. To produce virus for oncolytic trials, NDV is commonly amplified in embryonated chicken eggs and purified from the allantoic fluid. Conventional methods for purifying virus from allantoic fluid often result in relatively low-titer preparations containing high levels of impurities, including immunogenic chicken host cell proteins from allantoic fluid. However, large quantities of virus need to be delivered intravenously to administer oncolytic NDV systemically to mice. This route of administration requires virus preparations that are both highly concentrated (to enable delivery of small volumes) and highly pure (to limit toxic effects from contaminants). Given the accumulation of promising preclinical and clinical data demonstrating the efficacy of NDV as an oncolytic agent, strategies for increasing the titer and purity of NDV preparations are sorely needed to allow for effective intravenous administration in mice. Here, we describe an optimized protocol for the rescue, production, and purification of high-titer in vivo-grade NDV for preclinical studies in mouse models. Keywords: Newcastle disease virus, oncolytic virus, tangential flow filtration, preclinical grade, intravenous, allantoic flui

Pre-pregnancy metabolic parameters of fasted dams.

<p>Values are mean ± SEM,</p>*<p>significantly different between CON-fed and HF-fed dams.</p

Markers of tissue specific oxidative damage are not significantly increased in placentas from HF-fed dams at GD15.

<p>A. 10 µg of total whole placental homogenate was separated on a 12.5% SDS-PAGE and subjected to Western blot analysis. The average content of 4-HNE was normalized to total protein (using Ponceau-S staining) in CON (hatched bars) or HF-fed (solid black bars) dams. B. Representative lanes containing 10 µg placental homogenates, developed using the 4-HNE monoclonal antibody. C. The relative content of protein carbonyls was quantified using a polyclonal antibody directed towards 2,4-dinitrophenylhydrazine (DNPH). This quantification was carried out using 5 µg of placental homogenate enriched for mitochondria. D. Representative lanes containing 5 µg of protein, enriched for mitochondria, prepared from CON or HF-fed dam placenta were separated on a 12.5% SDS-PAGE and developed using the anti DNPH polyclonal antibody. Values represent mean ± SEM; ^†p<0.10, n = 6 per group.</p

The labyrinth layers of placentas from HF-fed dams exhibit increased expression of endothelial cell markers at GD15.

<p>Representative images, acquired at 100× magnification, of GD15 placenta from CON-fed (A) and HF-fed dams (B) immunostained with CD31 antibody are shown. Scale bar = 50 µm; the labyrinth (L) and junctional zone (JZ) are indicated for reference. Four distinct regions from each histological section were quantified and averaged in determining percent immunopositive area. Images from an individual dam represent a single statistical unit. C. The percentage of area immunopositive for CD31 in the labyrinth of GD15 placenta based on the analysis of cross sections from CON-fed and HF-fed dams sacrificed at GD15. D. The number of blood vessels per field for CON-fed or HF-fed dams at GD15. All values mean ± SEM, ^*p<0.05; n = 5 dams for each group.</p

Neonatal health outcomes for pups born to CON or HF-fed dams.

<p>A. The body weight of offspring of CON-fed (hatched bar) or HF-fed (solid black bar) dams on postnatal day 1 (PND1). B. The average number of pups per litter born to CON-fed or HF-fed dams. C. The percentage of CON-fed or HF-fed dams giving birth to at least one stillborn pup. D. The percentage of the total number of live pups in the litter that survived to PND4. E. The fetal/placental weight ratio was calculated for CON-fed and HF-fed dams. F. The average number of resorption sites for CON-fed and HF-fed dams at GD15. Values represent mean ± SEM; ^*p<0.05; n≥12 dams per group.</p