Effect of Normal Pregnancy Followed by Lactation on Long-Term Maternal Health in a Mouse Model.

Although it has been widely accepted that pregnancies with complications are associated with increased maternal cardiovascular risk later in life, there is no consensus if noncomplicated pregnancy followed by lactation plays a protective role or is a risk factor. The objective of this study was to investigate the effects of normal pregnancy and lactation on long-term maternal health in a mouse model. CD-1 mice were allocated to breeding (primigravid [PG]) and nonbreeding (nulligravid [NG]) groups. The PG group proceeded through normal pregnancy and delivery. Using a telemetry system, blood pressure (BP) was analyzed in the PG group at 6 months postpartum and in age-matched NG mice. Serum analytes, gene expressions, and protein levels were determined using appropriate analysis methods. Primigravid mice had significantly lower systolic and diastolic BP and fasting glucose levels. Circulating oxytocin (OXT) levels were significantly higher in PG mice. Oxt gene expression was significantly higher in the heart and aorta and lower in visceral adipose tissue (VAT) from PG mice. The oxytocin receptor ( Oxtr) gene expression was significantly higher in the heart, aorta, and VAT from PG animals. The level of Oxtr DNA hypermethylation and the expression of mmu-miR-29a were significantly lower in the hearts of PG mice. In PG VAT, glucose transporter-4 expression was significantly higher. Our study demonstrates that a history of normal pregnancy followed by lactation was associated with lower maternal cardiovascular risk factors later in life in female mouse.

High-fructose diet in pregnancy leads to fetal programming of hypertension, insulin resistance, and obesity in adult offspring.

BACKGROUND: Consumption of fructose-rich diets in the United States is on the rise and thought to be associated with obesity and cardiometabolic diseases. OBJECTIVE: We sought to determine the effects of antenatal exposure to high-fructose diet on offspring's development of metabolic syndrome-like phenotype and other cardiovascular disease risk factors later in life. STUDY DESIGN: Pregnant C57BL/6J dams were randomly allocated to fructose solution (10% wt/vol, n = 10) or water (n = 10) as the only drinking fluid from day 1 of pregnancy until delivery. After weaning, pups were started on regular chow, and evaluated at 1 year of life. We measured percent visceral adipose tissue and liver fat infiltrates using computed tomography, and blood pressure using CODA nonivasive monitor. Intraperitoneal glucose tolerance testing with corresponding insulin concentrations were obtained. Serum concentrations of glucose, insulin, triglycerides, total cholesterol, leptin, and adiponectin were measured in duplicate using standardized assays. Fasting homeostatic model assessment was also calculated to assess insulin resistance. P values <.05 were considered statistically significant. RESULTS: Maternal weight, pup number, and average weight at birth were similar between the 2 groups. Male and female fructose group offspring had higher peak glucose and area under the intraperitoneal glucose tolerance testing curve compared with control, and higher mean arterial pressure compared to control. Female fructose group offspring were heavier and had higher percent visceral adipose tissue, liver fat infiltrates, homeostatic model assessment of insulin resistance scores, insulin area under the intraperitoneal glucose tolerance testing curve, and serum concentrations of leptin, and lower concentrations of adiponectin compared to female control offspring. No significant differences in these parameters were noted in male offspring. Serum concentrations of triglycerides or total cholesterol were not different between the 2 groups for either gender. CONCLUSION: Maternal intake of high fructose leads to fetal programming of adult obesity, hypertension, and metabolic dysfunction, all risk factors for cardiovascular disease. This fetal programming is more pronounced in female offspring. Limiting intake of high fructose-enriched diets in pregnancy may have significant impact on long-term health.

Fetal DNA methylation of autism spectrum disorders candidate genes: association with spontaneous preterm birth.

OBJECTIVE: Autism spectrum disorder (ASD) is associated with preterm birth (PTB), although the reason underlying this relationship is still unclear. Our objective was to examine DNA methylation patterns of 4 ASD candidate genes in human fetal membranes from spontaneous PTB and uncomplicated term birth. STUDY DESIGN: A literature search for genes that have been implicated in ASD yielded 14 candidate genes (OXTR, SHANK3, BCL2, RORA, EN2, RELN, MECP2, AUTS2, NLGN3, NRXN1, SLC6A4, UBE3A, GABA, AFF2) that were epigenetically modified in relation to ASD. DNA methylation in fetal leukocyte DNA in 4 of these genes (OXTR, SHANK3, BCL2, and RORA) was associated with PTB in a previous study. This study evaluated DNA methylation, transcription (reverse transcription polymerase chain reaction), and translation patterns (immunostaining and Western blot) in fetal membrane from term labor (n = 14), term not in labor (TNIL; n = 29), and spontaneous preterm birth (PTB; n = 27). Statistical analysis was performed with analysis of variance; a probability value of < .05 was significant. RESULTS: Higher methylation of the OXTR promoter was seen in fetal membranes from PTB, compared with term labor or TNIL. No other gene showed any methylation differences among groups. Expression of OXTR was not different among groups, but the 70 kDa OXTR protein was seen only in PTB, and immunostaining was more intense in PTB amniocytes than term labor or TNIL. CONCLUSION: Among the 4 genes that were studied, fetal membranes from PTB demonstrate differences in OXTR methylation and regulation and expression, which suggest that epigenetic alteration of this gene in fetal membrane may likely be indicating an in utero programing of this gene and serve as a surrogate in a subset of PTB. The usefulness of OXTR hypermethylation as a surrogate for a link to ASD should be further evaluated in longitudinal and in vitro studies.

The role of NADPH oxidase in a mouse model of fetal alcohol syndrome.

OBJECTIVE: Fetal alcohol syndrome (FAS) is the most common cause of nongenetic mental retardation. Oxidative stress is one of the purported mechanisms. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is an enzyme involved in the production of reactive oxygen species. Our objective was to evaluate NOX in the fetal brain of a well-validated mouse model of FAS. STUDY DESIGN: Timed, pregnant C57BL/6J mice were injected intraperitoneally with 0.03 mL/g of either 25% ethyl alcohol or saline. Fetal brain, liver, and placenta were harvested on gestational day 18. The unit of analysis was the litter; tissue from 6-8 litters in the alcohol and control group was isolated. Evaluation of messenger ribonucleic acid (mRNA) expression of NOX subunits (DUOX1, DUOX2, NOX1, NOX2, NOX3, NOX4, NOXA1, NOXO1, RAC1, p22phox, and p67phox) was performed using quantitative real-time polymerase chain reaction; alcohol vs placebo groups were compared using a Student t test or a Mann-Whitney test (P < .05). RESULTS: Alcohol exposed fetal brains showed significant up-regulation in subunits DUOX2 (1.61 ± 0.28 vs 0.84 ± 0.09; P = .03), NOXA1 (1.75 ± 0.27 vs 1.09 ± 0.06; P = .04), and NOXO1 (1.59 ± 0.10 vs 1.28 ± 0.05; P = .02). Differences in mRNA expression in the placenta were not significant; p67phox was significantly up-regulated in alcohol-exposed livers. CONCLUSION: Various NOX subunits are up-regulated in fetal brains exposed to alcohol. This effect was not observed in the fetal liver or placenta. Given the available evidence, the NOX system may be involved in the causation of FAS through the generation of reactive oxygen species and may be a potential target for preventative treatment in FAS.

Effects of pravastatin on angiogenic and placental hypoxic imbalance in a mouse model of preeclampsia.

In order to determine the effects of pravastatin (Pra) on angiogenic and placental hypoxic imbalance in a model of preeclampsia induced by overexpression of soluble fms-like tyrosine kinase 1 (sFlt-1), we randomly allocated pregnant CD1 mice to injection with adenovirus-carrying sFlt-1 or mFc (control). The sFlt-1 group received either Pra (sFlt-1 + Pra) or water (sFlt-1). Mice were sacrificed at day 18, and serum levels of sFlt-1 and soluble endoglin (sEng) were measured. Placental expression of placental (PLGF) and vascular endothelial (VEGF) growth factors and other markers of angiogenesis and hypoxia were assayed. We observed that Pra treatment in sFlt-1 mice reduced sFlt-1 and sEng concentrations at day 18 to levels similar to control group. Placental PLGF and VEGF expression were upregulated, and markers of hypoxia downregulated to levels similar to control group. Hence, Pra prevents the rise in circulating antiangiogenic factors in a mouse model of preeclampsia. Statins may represent a novel approach to prevention of preeclampsia.

Single-nucleotide polymorphisms in genes involved in placental function and unexplained stillbirth.

OBJECTIVE: The purpose of this study was to evaluate the association between unexplained stillbirth (SB) and single-nucleotide polymorphisms (SNPs) in genes involved in placental function using a well-characterized cohort. STUDY DESIGN: Placentas were obtained from 50 unexplained SB and 46 live birth controls. Classification of stillbirth was by Wigglesworth criteria. SBs were stratified by weight: appropriate (AGA-SB) and small for gestational age (SGA-SB, less than the 10th percentile) and gestational age: before 32 and after 32 weeks. Placental DNA was extracted and various SNPs in the endothelial nitric oxide synthase (eNOS), Klotho, hypoxic inducible factor-1α, and and tumor necrosis factor-α genes were evaluated. RESULTS: None of the SNPs were associated with SB overall. Significantly different genotype distribution emerged for eNOS-SNP rs1800783 when comparing AGA-SB with SGA-SB and control (P = .004). Its allele-A was more frequent in AGA-SB compared with both controls (P = .03) and SGA-SB (P = .001). No differences were seen accordingly to gestational age. CONCLUSION: Unexplained stillbirth in the setting of adequate growth is associated with carrier of allele A of rs1800783 eNOS gene in the placenta.

The expression of antioxidant enzymes in a mouse model of fetal alcohol syndrome.

OBJECTIVE: Superoxide dismutase, glutathione peroxidase, and catalase prevent cellular damage produced by free radicals. Our objective was to evaluate if prenatal alcohol exposure decreased the expression of antioxidant enzymes in the brain, liver, or placenta of fetal mice. STUDY DESIGN: Timed, pregnant C57BL6/J mice were treated on gestational day 8 with intraperitoneal injection of alcohol (0.03 mL/g) or saline (control). Fetuses were harvested on gestational day 18. Fetal brain, liver, and placenta were analyzed for mRNA expression of superoxide dismutase, glutathione peroxidase, and catalase by real-time polymerase chain reaction, with 18S RNA used as reference. RESULTS: Superoxide dismutase, glutathione peroxidase, and catalase expression was lower in fetal brains exposed to alcohol with no differences detected in the liver or placenta between the 2 groups. CONCLUSION: Maternal alcohol consumption causes a decrease in superoxide dismutase, glutathione peroxidase, and catalase expression in the fetal brain. This may explain the long-term neurologic findings in fetal alcohol syndrome.

The role of oxidative stress in the developmental origin of adult hypertension.

OBJECTIVE: To determine whether oxidative stress plays a role in the development of hypertension using a mouse model of fetal programming induced by endothelial nitric oxide synthase deficiency. STUDY DESIGN: Homozygous nitric oxide synthase knockout and wild type mice were cross-bred producing maternal (endothelial nitric oxide synthase+pat/-mat) and paternal (endothelial nitric oxide synthase+mat/-pat) heterozygous offspring. RNA from liver and kidney tissues of female pups were obtained at 14 weeks of age. Relative expression of the heat shock protein-B6, peroxiredoxin-3, superoxide dismutase-1, peroxisome proliferator-activated receptor gamma, nitric oxide synthase-1 and -2 were determined. RESULTS: In the kidneys, expression of nitric oxide synthase-2, peroxiredoxin-3, heat shock protein-B6, and superoxide dismutase-1 was up-regulated in endothelial nitric oxide synthase+pat/-mat but not in endothelial nitric oxide synthase+mat/-pat compared with wild type offspring. In the liver, there were no significant differences in the expression of nitric oxide synthase-1, nitric oxide synthase-2, peroxiredoxin, superoxide dismutase-1, or peroxisome proliferator-activated receptor gamma; however, heat shock protein-B6 was down-regulated in both heterozygotes offspring compared with wild type. CONCLUSION: The intrauterine environment alters oxidative pathways gene expression in the kidneys of offspring, which may be a mechanism in the development of adult hypertension.

A peripheral neuroimmune link: glutamate agonists upregulate NMDA NR1 receptor mRNA and protein, vimentin, TNF-alpha, and RANTES in cultured human synoviocytes.

Human primary and clonal synovial cells were incubated with glutamate receptor agonists to assess their modulating influence on glutamate receptors N-methyl-d-aspartate (NMDA) NR1 and NR2 and inflammatory cytokines to determine potential for paracrine or autocrine (neurocrine) upregulation of glutamate receptors, as has been shown for bone and chondrocytes. Clonal SW982 synoviocytes constitutively express vimentin, smooth muscle actin (SMA), and NMDA NR1 and NR2. Coincubation (6 h) with glutamate agonists NMDA (5 microM), and the NMDA NR1 glycine site activator (+/-)1-aminocyclopentane-cis-1,3-dicarboxylic acid (5 muM), significantly increases cellular mRNA and protein levels of glutamate receptors, as well as increasing vimentin, SMA, tumor necrosis factor-alpha, and RANTES (regulated on activation, normal T-cell expressed and secreted), assessed qualitatively and quantitatively with nucleotide amplification, image analysis of immunocytochemical staining, fluorescein-activated cell sorting, Western blotting, and immunoassays. Human primary synovial cells harvested from patients with arthritic conditions also constitutively expressed NMDA NR1 with increases after agonist treatment. Glutamate receptor agonist-induced increases were blocked by the noncompetitive glutamate antagonist MK-801 (8 microg/ml) and NR1 blocking antibody. Coincubation with glutamate agonists and phorbol 12-myristate 13-acetate, a protein kinase C activator, significantly enhanced mean levels of TNF-alpha and RANTES in SW982 cell supernatants compared with incubation with either agent alone. Increases were diminished with protein kinase inhibitor and NR1 blocking antibody. The functional activation of glutamate receptors on human synoviocytes establishes a neurogenic cell signaling link between neurotransmitter glutamate released from nerve terminals and target cells in the joint capsule. The influence of glutamate on subsequent release of cellular proinflammatory mediators in non-neural tissue for activation of downstream immune events supports a peripheral neuroimmune link in arthritis.

Tumor necrosis factor-alpha (TNF-alpha) enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes.

BACKGROUND: We have shown functional expression of several TRP channels on human synovial cells, proposing significance in known calcium dependent proliferative and secretory responses in joint inflammation. The present study further characterizes synoviocyte TRP expression and activation responses to thermal and osmotic stimuli after pre-treatment with proinflammatory mediator tumor necrosis factor alpha (TNF-alpha, EC50 1.3221 x 10(-10) g/L). RESULTS: Fluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1) TRPV1 agonists capsaicin and resiniferatoxin (20-40% of cells), (2) moderate and noxious temperature change, and (3) osmotic stress TRPV4 activation (11.5% of cells). TNF-alpha pre-treatment (1 ng/ml, 8-16 hr) significantly increases (doubles) capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca2+]i responses. With TNF-alpha pre-treatment for 8, 12, and 16 hr, activation with 36 or 45 degree bath solution induces bimodal [Ca2+]i increase (temperature controlled chamber). Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr) is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3-4 fold increase). TNF-alpha increases TRPV1 (8 hr peak) and TRPV4 (12 hr peak) immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions. CONCLUSION: TNF-alpha provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca2+]i response dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.

Maternal hypercholesterolemia leads to activation of endogenous cholesterol synthesis in the offspring.

OBJECTIVE: The purpose of this study was to determine the effect of maternal hypercholesterolemia on hepatic cholesterol metabolism in the offspring in a mouse model. STUDY DESIGN: Male and female wild type and apoE(-/-KO) (knockout for the apoprotein E [apoE]) gene) mice were crossbred to obtain all 4 possible genetic offspring types. The litters were maintained on regular chow and sacrificed at 8 months of age. Liver samples were collected and the mRNA expression levels for SCAP, SREBP-1a, SREBP-2, HMGCR, and LDLR determined using real-time RT-PCR. RESULTS: We found a significant activation of the transcriptional activity of genes involved in endogenous cholesterol synthesis, as well as LDLR, in the liver of adult mice born to hypercholesterolemic dams. CONCLUSION: Reprogramming of hepatic cholesterol homeostasis may be the basis for an increased predisposition to hypercholesterolemia and atherosclerosis found in offspring of mice exposed to a high cholesterol environment during early life.

Transient receptor potential vanilloid 1 mediates hyperalgesia and is up-regulated in rats with chronic pancreatitis.

BACKGROUND & AIMS: The neurobiologic basis of pancreatic hyperalgesia in chronic pancreatitis (CP) is understood poorly and there is a need to identify novel therapeutic targets. Our aim was to study the role of the transient receptor potential vanilloid 1 (TRPV1), a key integrator of noxious stimuli, in the pathogenesis of pancreatic pain in a rat model of CP. METHODS: CP was induced in rats by intraductal injection of trinitrobenzene sulfonic acid. TRPV1 currents in pancreas-specific DRG neurons were measured using perforated patch-clamp techniques. Reverse-transcription polymerase chain reaction was used to measure mRNA expression of TRPV1 in these neurons after laser capture microdissection. Immunofluorescence and Western blot analysis, using TRPV1-specific antibodies, also were performed. Pancreatic hyperalgesia was assessed by rat's nocifensive behavior to electrical stimulation of the pancreas. RESULTS: CP was associated with a 4-fold increase in capsaicin-induced current density (P < .02), along with an increase in the proportion of pancreas-specific DRG neurons that responded to capsaicin (52.9% in controls vs 79.0% in CP; P < .05). CP also was associated with a significant increase in TRPV1 expression both at the messenger RNA and protein level in whole thoracic DRGs and pancreas-specific sensory neurons. Systemic administration of the TRPV1 antagonist SB-366791 markedly reduced both visceral pain behavior and referred somatic hyperalgesia in rats with CP, but not in control animals. CONCLUSIONS: TRPV1 up-regulation and sensitization is a specific molecular mechanism contributing to hyperalgesia in CP and represents a useful target for treating pancreatic hyperalgesia caused by inflammation.

Enhanced excitability and suppression of A-type K+ current of pancreas-specific afferent neurons in a rat model of chronic pancreatitis.

Chronic pancreatitis (CP) is a relatively common disorder, characterized by glandular insufficiency and chronic, often intractable, pain. The mechanism of pain in CP is poorly understood. We have previously developed a model of trinitrobenzene sulphonic acid (TNBS)-induced CP that results in nociceptive sensitization in rats. This study was designed to examine changes in the excitability and alteration of voltage-gated K(+) currents of dorsal root ganglia (DRG) neurons innervating the pancreas. CP was induced in adult rats by an intraductal injection of TNBS. DRG neurons innervating the pancreas were identified by 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate fluorescence labeling. Perforated patch-clamp recordings were made from acutely dissociated DRG neurons from control and TNBS-treated rats. Pancreas-specific DRG neurons displayed more depolarized resting potentials in TNBS-treated rats than those in controls (P < 0.02). Some neurons from the TNBS-treated group exhibited spontaneous firings. TNBS-induced CP also resulted in a dramatic reduction in rheobase (P < 0.05) and a significant increase in the number of action potentials evoked at twice rheobase (P < 0.05). Under voltage-clamp conditions, neurons from both groups exhibited transient A-type (I(A)) and sustained outward rectifier K(+) currents (I(K)). Compared with controls, the average I(A) but not the average I(K) density was significantly reduced in the TNBS-treated group (P < 0.05). The steady-state inactivation curve for I(A) was displaced by approximately 20 mV to more hyperpolarized levels after the TNBS treatment. These data suggest that TNBS treatment increases the excitability of pancreas-specific DRG neurons by suppressing I(A) density, thus identifying for the first time a specific molecular mechanism underlying chronic visceral pain and sensitization in CP.

Antigenic diversity in maxadilan, a salivary protein from the sand fly vector of American visceral leishmaniasis.

The salivary protein maxadilan (MAX) is a vasodilator and immunomodulator from the sand fly vector of the protozoan parasite Leishmania chagasi. Vaccinating BALB/c mice with sand fly salivary gland extracts or with MAX protects the host against L. major infection. Because of the potential use of MAX in an anti-Leishmania vaccine, we characterized the vertebrate host IgG response to MAX in the present study. Our immunochemical analysis indicated that antibodies to MAX were detected in BALB/c mice, as well as in pigs and humans, from a area in Nicaragua endemic for Lutzomyia longipalpis. Previous studies demonstrate that the MAX protein is polymorphic on the amino acid level. Our findings suggested that naturally occurring MAX variants were recognized specifically by the host immune system and antigenicity appeared to be associated with amino-acid sequence variability. Thus, antigenic diversity of MAX and possibly of other arthropod salivary proteins may dictate the development of vector-based vaccines(s).