Daliao Xiao

Chronic Prenatal Hypoxia Induces Epigenetic Programming of PKCε Gene Repression in Rat Hearts

Rationale: Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus and results in decreased protein kinase C epsilon (PKCϵ) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats. Objectives: The present study tested the hypothesis that fetal hypoxia-induced methylation of cytosine-phosphate-guanine dinucleotides at the PKCϵ promoter is repressive and contributes to PKCϵ gene repression in the heart of adult offspring. Methods and Results: Hypoxic treatment of pregnant rats from days 15 to 21 of gestation resulted in significant decreases in PKCϵ protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCϵ promoter at SP1 binding sites, −346 and −268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O2 for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2′-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCϵ mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCϵ mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor &agr; and &bgr; isoforms in females than in males. Both estrogen receptor &agr; and &bgr; interacted with the SP1 binding sites in the fetal heart, which may explain the sex differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCϵ restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring. Conclusions: The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCϵ gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.

Prenatal Gender-Related Nicotine Exposure Increases Blood Pressure Response to Angiotensin II in Adult Offspring

Epidemiological studies suggest that maternal cigarette smoking is associated with an increased risk of elevated blood pressure (BP) in postnatal life. The present study tested the hypothesis that prenatal nicotine exposure causes an increase in BP response to angiotensin II (Ang II) in adult offspring. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps throughout the gestation. BP and vascular responses to Ang II were measured in 5-month–old adult offspring. Prenatal nicotine had no effect on baseline BP but significantly increased Ang II–stimulated BP in male but not female offspring. The baroreflex sensitivity was significantly decreased in both male and female offspring. Prenatal nicotine significantly increased arterial media thickness in male but not female offspring. In male offspring, nicotine exposure significantly increased Ang II–induced contractions of aortas and mesenteric arteries. These responses were not affected by inhibition of endothelial NO synthase activity. Losartan blocked Ang II–induced contractions in both control and nicotine-treated animals. In contrast, PD123319 had no effect on Ang II–induced contractions in control but inhibited them in nicotine-treated animals. Nicotine significantly increased Ang II type 1 receptor but decreased Ang II type 2 receptor protein levels, resulting in a significant increase in the ratio of Ang II type 1 receptor/Ang II type 2 receptor in the aorta. Furthermore, the increased contractions of mesenteric arteries were mediated by increases in intracellular Ca2+ concentrations and Ca2+ sensitivity. These results suggest that prenatal nicotine exposure alters vascular function via changes in Ang II receptor–mediated signaling pathways in adult offspring in a gender-specific manner, which may lead to an increased risk of hypertension in male offspring.

Prenatal Nicotine Exposure Increases Heart Susceptibility to Ischemia/Reperfusion Injury in Adult Offspring

In the present study we tested the hypothesis that prenatal nicotine exposure increases heart susceptibility to ischemia/reperfusion (I/R) injury in adult offspring. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps throughout gestation. Nicotine treatment resulted in a rapid and transient decrease in food-intake and a moderate decrease in maternal body weight gain. Hearts were isolated from adult male and female offspring and subjected to I/R in a Langendorff preparation. Nicotine significantly attenuated left ventricle (LV) developed pressure, heart rate, and coronary flow rate in female but not male hearts at baseline. Additionally, nicotine significantly increased LV infarct size and attenuated postischemic recovery of LV function in both male and female offspring with more pronounced effects in females. In female but not male hearts, nicotine significantly decreased the postischemic coronary flow rate. However, coronary nitric oxide release was decreased in male but not female hearts. Caspase-3, -8, and -9 levels were not significantly changed in either female or male hearts. However, nicotine caused a significant decrease in protein levels of protein kinase (PK) Cϵ in both male and female hearts and a decrease in PKCδ levels in female hearts only. Control studies of maternal food restriction showed that a moderate decrease in maternal body weight gain had no effect on female hearts but significantly improved postischemic recovery of LV function in male hearts. The results suggest that prenatal nicotine exposure causes in utero programming of the PKC isozyme gene expression pattern in the developing heart and increases heart susceptibility to I/R injury in adult offspring.

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

Gestational Hypoxia Induces Preeclampsia-Like Symptoms via Heightened Endothelin-1 Signaling in Pregnant Rats

Preeclampsia is a life-threatening pregnancy disorder. However, its pathogenesis remains unclear. We tested the hypothesis that gestational hypoxia induces preeclampsia-like symptoms via heightened endothelin-1 (ET-1) signaling. Time-dated pregnant and nonpregnant rats were divided into normoxic and hypoxic (10.5% O2 from the gestational day 6–21) groups. Chronic hypoxia had no significant effect on blood pressure or proteinuria in nonpregnant rats but significantly increased blood pressure on day 12 (systolic blood pressure, 111.7±6.1 versus 138.5±3.5 mm Hg; P=0.004) and day 20 (systolic blood pressure, 103.4±4.6 versus 125.1±6.1 mm Hg; P=0.02) in pregnant rats and urine protein (&mgr;g/&mgr;L)/creatinine (nmol/&mgr;L) ratio on day 20 (0.10±0.01 versus 0.20±0.04; P=0.04), as compared with the normoxic control group. This was accompanied with asymmetrical fetal growth restriction. Hypoxia resulted in impaired trophoblast invasion and uteroplacental vascular remodeling. In addition, plasma ET-1 levels, as well as the abundance of prepro–ET-1 mRNA, ET-1 type A receptor and angiotensin II type 1 receptor protein in the kidney and placenta were significantly increased in the chronic hypoxic group, as compared with the control animals. Treatment with the ET-1 type A receptor antagonist, BQ123, during the course of hypoxia exposure significantly attenuated the hypoxia-induced hypertension and other preeclampsia-like features. The results demonstrate that chronic hypoxia during gestation induces preeclamptic symptoms in pregnant rats via heightened ET-1 and ET-1 type A receptor–mediated signaling, providing a molecular mechanism linking gestational hypoxia and increased risk of preeclampsia.

Fetal and neonatal nicotine exposure differentially regulates vascular contractility in adult male and female offspring.

Epidemiologic studies suggest that prenatal exposure to maternal cigarette smoking is associated with an increased risk of elevated blood pressure in postnatal life. The present study was designed to test the hypothesis that fetal and neonatal nicotine exposure increased vascular contractility in adult offspring. Nicotine was administered to pregnant rats via s.c. osmotic minipumps throughout gestation and up to 10 days after delivery. Aortas were isolated from adult male and female offspring at the age of 3 months old. Nicotine significantly increased KCl- and norepinephrine-induced contractions of the aorta in male, but not female, offspring. Inhibition of endothelial nitric oxide synthase (eNOS) with NG-nitro-l-arginine (l-NNA) significantly increased norepinephrine-induced contractions in control male offspring but showed no effect in nicotine-treated male offspring. In the presence of l-NNA, there was no significant difference in norepinephrine-induced contractions between control and nicotine-treated males. In contrast, nicotine caused a significant increase in l-NNA-mediated potentiation of norepinephrine-induced contractions in female offspring. Nicotine had no effect on sodium nitroprusside-induced endothelium-independent relaxations of aortas from either male or female offspring. However, it decreased endothelium-dependent relaxations induced by acetylcholine in male offspring but increased them in females. There were no differences in eNOS protein levels in aortas between the control and nicotine-treated animals in either male or female offspring. The results suggest that fetal and neonatal nicotine exposure alters vascular functions in adult offspring in a gender-specific manner, which may lead to an increased risk of cardiovascular dysfunction in later life.

Pregnancy Upregulates Large-Conductance Ca 2+ -Activated K + Channel Activity and Attenuates Myogenic Tone in Uterine Arteries

Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca2+-activated K+ (BKCa) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K+ current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K+ currents to the same extent in uterine arterial myocytes. The BKCa channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BKCa channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17&bgr;-estradiol and progesterone caused a significant increase in the BKCa channel current density. Western blot analyses demonstrated a significant increase of the &bgr;1, but not &agr;, subunit of BKCa channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the &bgr;1, but not &agr;, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the &bgr;1 subunit and BKCa channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.

Antenatal nicotine induces heightened oxidative stress and vascular dysfunction in rat offspring

BACKGROUND AND PURPOSE Antenatal nicotine exposure causes aberrant vascular reactivity and increased blood pressure in adult male rat offspring in a sex‐dependent manner. The present study tested the hypothesis that maternal nicotine administration increases the production of reactive oxygen species resulting in the vascular hypertensive reactivity in male offspring.

Direct Effects of Nicotine on Contractility of the Uterine Artery in Pregnancy

Recent studies indicate that smoking/nicotine increases maternal blood pressure and decrease in uterine blood flow in pregnancy. However, the mechanisms are not fully understood. The present study was designed to test the hypothesis that nicotine exposure decreases endothelium-dependent relaxation and increases vascular contractility of the uterine artery in pregnancy. Uterine arteries were isolated from near-term (∼140 days gestation) pregnant ewes. Arteries were subjected to acute (20 min) or chronic (48 h) nicotine treatment, and agonist-induced contractions and relaxations were measured in tissue bath. Endothelial eNOS was detected by immunohistochemistry in situ in arteries and by Western blotting in isolated endothelial cells. Chronic nicotine treatment produced a concentration-dependent increase in α1-adrenoceptor agonist phenylephrine-induced contractions. In contrast, the acute treatment showed no effect. Inhibition of eNOS with NG-nitro-l-arginine (l-NNA) significantly increased phenylephrine-induced contractions, which was abolished in uterine arteries after chronic nicotine treatment. In the presence of l-NNA, there was no significant difference in phenylephrine-induced contractions between control and nicotine-treated vessels. Chronic, but not acute, nicotine treatment significantly attenuated the calcium ionophore A23187-induced relaxations. Unlike A23187, the endothelium-independent relaxation mediated by sodium nitroprusside was not affected by nicotine. Endothelial eNOS protein levels and the phosphorylation levels of eNOSSer1179 were significantly decreased in nicotine-treated uterine arteries. The results suggest that nicotine impairs uterine vascular function in pregnancy, which may lead to an increased vascular resistance and a decrease in uterine blood flow.

Chronic Hypoxia and Developmental Regulation of Cytochrome C Expression in Rats

Objective: To test the hypothesis that chronic hypoxia upregulates cytochrome c expression in heart, brain, and liver of fetal and maternal rats. Methods: Time-dated pregnant Sprague-Dawley rats were divided into normoxic and hypoxic (48 hours of 10.5% oxygen from days 19 to 21) groups, and were killed on day 21. Tissue levels of cytochrome c in heart, brain, and liver were determined by using monoclonal antiserum for cytochrome c. Results: Chronic hypoxia caused a decrease in fetal body weight (5.3 ± 0.1 to 4.7 ± 0.1 g) and an increase in heart/body weight ratio (0.0048 ± 0.0001 to 0.0061 ± 0.0002). Cytochrome c levels were 4-, 2.6-, and 13-fold higher in heart, liver, and brain, respectively, of the mother than of the fetus. Chronic hypoxia did not change cytochrome c levels in maternal tissues but caused a 70% increase and 54% decrease in cytochrome c levels in the fetal heart and liver, respectively. No difference was observed in the fetal brain. Conclusions: The results suggest that expression of cytochrome c is tissue specific and developmentally regulated. Chronic hypoxia showed differential regulation of cytochrome c levels both developmentally and tissue specifically. The increased sensitivity of cytochrome c in fetal tissue to chronic hypoxia is likely to represent a fetal adaptive mechanism to the stress of chronic hypoxia.