Gestational hypoxia is a common stress to the fetal development and increases the risk of neonatal morbidity. The present study tested the hypothesis that fetal hypoxia results in heightened brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats via down-regulation of glucocorticoid receptor (GR) in the developing brain. Time-dated pregnant rats were exposed to hypoxia (10.5% O2) from day 15 to 21 of gestation. Brain HI injury was determined in day 10 pups. Maternal hypoxia resulted in asymmetric intrauterine growth restriction in the fetus. The brain HI injury was significantly increased in maternal hypoxia-treated pups as compared with the normoxia control in both males and females. Activation of brain GR by dexamethasone injection into the right lateral ventricle produced a concentration-dependent reduction of HI-induced brain injury in control pups. Maternal hypoxia significantly decreased GR mRNA and protein abundance in the fetal brain and neonatal hippocampus and abolished the dexamethasone-mediated neuroprotective effect in pup brains. This decreased GR expression was resulted from increased DNA methylation, decreased binding of transcription factors Egr-1 and Sp1 to GR gene exon 17 and 111 promoters, and reduced expression of GR exon 17 and 111 mRNA variants. The results demonstrate that gestational hypoxia causes epigenetic repression of GR gene expression in the developing brain resulting in the heightened brain vulnerability to HI injury in neonatal rats.

Background The H19/IGF2 imprinted loci have attracted recent attention due to their role in cellular differentiation and proliferation, heritable gene regulation, and in utero or early postnatal growth and development. Expression from the imprinted H19/IGF2 locus involves a complex interplay of three means of epigenetic regulation: proper establishment of DNA methylation, promoter occupancy of CTCF and expression of microRNA-675 (miR675). We have previously demonstrated in a multigenerational rat model of intrauterine growth restriction the epigenetic heritability of adult metabolic syndrome in a F2 generation. We have further demonstrated abrogation of the F2 adult metabolic syndrome phenotype with essential nutrient supplementation of intermediates along the one-carbon pathway, and shown that alterations in the metabolome precede the adult onset of metabolic syndrome. However, the upstream molecular and epigenomic mediators underlying these observations have yet to be fully elucidated. Objective In the current study, we sought to characterize the impact of the intrauterine growth restricted lineage and essential nutrient supplementation on both levels and molecular mediators of H19 and IGF2 gene expression in the F2 generation. Study Design F2 intrauterine growth restricted and sham lineages were obtained by exposing P1 (grandmaternal) pregnant dams to bilateral uterine artery ligation or sham surgery at gestational day 19.5. F1 pups were allocated to the essential nutrient supplemented or control diet at postnatal day 21, and bred at 6–7 weeks of age. Hepatic tissues from the resultant F2 offspring at birth and at weaning (day 21) were obtained. Bisulfite modification and sequencing was employed for methylation analysis. H19 and IGF2 expression was measured by QPCR. Promoter occupancy was quantified using chromatin immunoprecipitation, or ChIP, against CTCF insulator proteins. Results Growth-restricted F2 on control diet demonstrated significant down-regulation in H19 expression as compared to sham lineage (0.7831 vs 1.287; p< 0.05); however, essential nutrient supplementation diet abrogates this difference (4.995 vs 5.100; p>0.05). Conversely, Igf2 was up regulated by essential nutrient supplemented diet on the sham lineage (2.0 fold, p=0.01), an effect that was not observed in the growth restricted offspring. A significant differential methylation was observed in the promoter region of region H19 among the intrauterine growth restricted lineage (18% vs 25%; p<0.05) on a control diet, while the essential nutrient supplemented diet was alternately associated with hypermethylation in both lineages (sham: 50%; IUGR: 84%, p<0.05). Consistent with essential nutrient supplementation impacting the epigenome, a decrease of CTCF promoter occupancy was observed in CTCF4 of the growth restricted lineage (2.45% vs 0.56%; p<0.05) on the control diet, an effect that was repressed with essential nutrient supplementaion. Conclusions Heritable growth restriction is associated with changes in H19 gene expression; the...

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Background and PurposeGlucocorticoids pretreatment confers protection against neonatal hypoxic-ischemic (HI) brain injury. However, the molecular mechanism remains poorly elucidated. We tested the hypothesis that glucocorticoids protect against HI brain injury in neonatal rat by stimulation of lipocalin-type prostaglandin D synthase (L-PGDS)-induced prostaglandin D2 (PGD2)-DP1-pERK mediated signaling pathway.MethodsDexamethasone and inhibitors were administered via intracerebroventricular (i.c.v) injections into 10-day-old rat brains. Levels of L-PGD2, D prostanoid (DP1) receptor, pERK1/2 and PGD2 were determined by Western immunoblotting and ELISA, respectively. Brain injury was evaluated 48 hours after conduction of HI in 10-day-old rat pups.ResultsDexamethasone pretreatment significantly upregulated L-PGDS expression and the biosynthesis of PGD2. Dexamethasone also selectively increased isoform pERK-44 level in the neonatal rat brains. Inhibitors of L-PGDS (SeCl4), DP1 (MK-0524) and MAPK (PD98059) abrogated dexamethasone-induced increases in pERK-44 level, respectively. Of importance, these inhibitors also blocked dexamethasone-mediated neuroprotective effects against HI brain injury in neonatal rat brains.ConclusionInteraction of glucocorticoids-GR signaling and L-PGDS-PGD2-DP1-pERK mediated pathway underlies the neuroprotective effects of dexamethasone pretreatment in neonatal HI brain injury.

AIMS: The present study tested the hypothesis that fetal hypoxia adversely affects kidney development in fetal and offspring rats and alter the expression patterns of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors.METHODS: Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O2 last period of gestation) groups. Protein expression, in the offspring, was determined using western blot.RESULTS: Hypoxic treatment significantly decreased body and kidney weight in 21-day fetuses (E21) and 7-day neonates (P7). In 3-month-old offspring there were no significant differences in body and kidney weight between hypoxic and control animals. Fetal hypoxia had no effect on kidney AT1R density in E21 or P7, but significantly decreased kidney AT1R protein and mRNA abundance in both male and female adults. In contrast, kidney AT2R density was not affected by fetal hypoxia throughout the developmental stages studied. The hypoxia-mediated reduction of nephron numbers was progressively from P7 worsened into the adulthood with females affected more than males.CONCLUSION: The results suggest that fetal hypoxia causes programming of aberrant kidney development and accelerates the aging process of the kidney during the postnatal development, which may contribute to an increased risk of cardiovascular disease.