Fuxia Xiong

Norepinephrine causes epigenetic repression of PKCε gene in rodent hearts by activating Nox1-dependent reactive oxygen species production

Heart disease is the leading cause of death in the United States. Recent studies demonstrate that fetal programming of PKCε gene repression results in ischemia‐sensitive phenotype in the heart. The present study tests the hypothesis that increased norepinephrine causes epigenetic repression of PKCε gene in the heart via Nox1‐dependent reactive oxygen species (ROS) production. Prolonged norepinephrine treatment increased ROS production in fetal rat hearts and embryonic ventricular myocyte H9c2 cells via a selective increase in Nox1 expression. Norepinephrine‐induced ROS resulted in an increase in PKCε promoter methylation at Egr‐1 and Sp‐1 binding sites, leading to PKCε gene repression. N‐acetylcysteine, diphenyleneiodonium, and apocynin blocked norepinephrine‐induced ROS production and the promoter methylation, and also restored PKCε mRNA and protein to control levels in vivo in fetal hearts and in vitro in embryonic myocyte cells. Accordingly, norepinephrine‐induced ROS production, promoter methylation, and PKCε gene repression were completely abrogated by knockdown of Nox1 in cardiomyocytes. These findings provide evidence of a novel interaction between elevated norepinephrine and epigenetic repression of PKCε gene in the heart mediated by Nox1‐dependent oxidative stress and suggest new insights of molecular mechanisms linking the heightened sympathetic activity to aberrant cardioprotection and increased ischemic vulnerability in the heart.—Xiong, F., Xiao, D., Zhang, L. Norepinephrine causes epigenetic repression of PKCε gene in rodent hearts by activating Nox1‐dependent reactive oxygen species production. FASEB J. 26, 2753–2763 (2012). www.fasebj.org

Perinatal Nicotine Exposure Increases Vulnerability of Hypoxic–Ischemic Brain Injury in Neonatal Rats Role of Angiotensin II Receptors

Background and Purpose— Maternal cigarette smoking increases the risk of neonatal morbidity. We tested the hypothesis that perinatal nicotine exposure causes heightened brain vulnerability to hypoxic–ischemic (HI) injury in neonatal rats through aberrant expression patterns of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors in the developing brain. Methods— Nicotine was administered to pregnant rats through subcutaneous osmotic minipumps. HI brain injury was determined in 10-day-old pups. AT1R and AT2R expression patterns were assessed through Western blotting, quantitative polymerase chain reaction, immunofluorescence, and confocal imaging. Results— Perinatal nicotine exposure significantly increased HI brain infarct size in male, but not female, pups. In fetal brains, nicotine caused a decrease in mRNA and protein abundance of AT2R but not AT1R. The downregulation of AT2R persisted in brains of male pups, and nicotine treatment resulted in a significant increase in methylation of CpG locus 3 bases upstream of TATA-box at the AT2R gene promoter. In female brains, there was an increase in AT2R but a decrease in AT1R expression. Both AT1R and AT2R expressed in neurons but not in astrocytes in the cortex and hippocampus. Central application of AT1R antagonist losartan or AT2R antagonist PD123319 increased HI brain infarct size in both male and female pups. In male pups, AT2R agonist CGP42112 abrogated nicotine-induced increase in HI brain infarction. In females, PD123319 uncovered the nicotines effect on HI brain infarction. Conclusion— Perinatal nicotine exposure causes epigenetic repression of the AT2R gene in the developing brain resulting in heightened brain vulnerability to HI injury in neonatal male rats in a sex-dependent manner.

Fetal hypoxia increases vulnerability of hypoxic–ischemic brain injury in neonatal rats: Role of glucocorticoid receptors☆

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 days 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.

Epigenetic Upregulation of Large-Conductance Ca2+-Activated K+ Channel Expression in Uterine Vascular Adaptation to Pregnancy

Our previous study demonstrated that pregnancy increased large-conductance Ca2+-activated potassium channel &bgr;1 subunit (BK&bgr;1) expression and large-conductance Ca2+-activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BK&bgr;1 expression patterns in uterine arteries. Ovine BK&bgr;1 promoter of 2315 bp spanning from −2211 to +104 of the transcription start site was cloned, and an Sp1−380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BK&bgr;1 promoter activity. Estrogen receptor-&agr; bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BK&bgr;1. The Sp1 binding site at BK&bgr;1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BK&bgr;1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BK&bgr;1 promoter and BK&bgr;1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BK&bgr;1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca2+-activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

Hypoxia inhibits cardiomyocyte proliferation in fetal rat hearts via upregulating TIMP-4

Maternal hypoxia inhibits cardiomyocyte proliferation in the heart of fetal and neonatal rats. The present study tested the hypothesis that hypoxia has a direct effect inhibiting cardiomyocyte proliferation via upregulating tissue inhibitors of metalloproteinases (TIMP) in fetal rat hearts. Isolated fetal rat hearts and rat embryonic ventricular myocyte H9c2 cells were treated ex vivo with 20% or 1% O(2) for 48 or 24 h, respectively. Hypoxia caused a significant reduction in cardiomyocyte Ki-67 expression and bromodeoxyuridine incorporation in fetal hearts and H9c2 cells. In both fetal hearts and H9c2 cells, hypoxia resulted in a significant decrease in a cell division marker cyclin D2 but an increase in a cell division inhibitor p27. Additionally, hypoxia caused an upregulation of TIMP-3 and TIMP-4 in fetal hearts and H9c2 cells. Knockdown of TIMP-3 in H9c2 cells significantly increased cyclin D2 and Ki-67 and partially blocked the hypoxia-induced inhibition of cyclin D2 and Ki-67 in H9c2 cells. Unlike TIMP-3, TIMP-4 knockdown had no significant effects on the basal levels of cell proliferation but completely abrogated the hypoxia-mediated effects. These findings provide evidence of a novel causal role of TIMP-4 and TIMP-3 in the direct inhibitory effect of hypoxia on cardiomyocyte proliferation in the developing heart.

Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart

Fetal hypoxia causes protein kinase Cε (PKCε) gene repression in the heart resulting in heightened ischemic injury in male offspring in a sex-dependent manner. The present study tested the hypothesis that heightened methylation of the early growth response factor-1 (Egr-1) binding site at PKCε gene promoter contributes to sex dimorphism of hypoxia-induced programming of PKCε gene repression in the developing heart. Pregnant rats were divided into normoxic and hypoxic (10.5% O2 from day 15 to 21 of gestation) groups. Hypoxia selectively decreased PKCε mRNA and protein abundance in the heart of male, but not female, near-term (21 days) fetuses. Methylation of the CpG site at the Egr-1 binding site of PKCε promoter was significantly increased in the male hearts by hypoxia, resulting in decreased Egr-1 binding affinity and reduced Egr-1 binding to the PKCε promoter. Nuclear Egr-1 levels were not affected by hypoxia. There was significantly higher abundance of estrogen receptor α (ERα) and β (ERβ) isoforms in female than in male fetal hearts, which were not significantly altered by hypoxia. Both ERα and ERβ bind to the Egr-1 binding site with significant greater levels in the female fetal hearts. The increased methylation with reduced Egr-1 binding and PKCε gene repression persisted in 3-mo-old adult male hearts in a sex-dependent manner. The results indicate a key role for heightened methylation of the Egr-1 binding site in hypoxia-mediated programming of PKCε gene repression in the developing heart and suggest a novel protective mechanism of ER by binding to the Egr-1 binding site in epigenetic regulation of PKCε gene expression patterns in the early developmental stage.

Gestational hypoxia and epigenetic programming of brain development disorders

Adverse environmental conditions faced by an individual early during its life, such as gestational hypoxia, can have a profound influence on the risk of diseases, such as neurological disorders, in later life. Clinical and preclinical studies suggest that epigenetic programming of gene expression patterns in response to maternal stress have a crucial role in the fetal origins of neurological diseases. Herein, we summarize recent studies regarding the role of epigenetic mechanisms in the developmental programming of neurological diseases in offspring, primarily focusing on DNA methylation/demethylation and miRNAs. Such information could increase our understanding of the fetal origins of adult diseases and help develop effective prevention and intervention against neurological diseases.