Lubo Zhang

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.

Effect of fetal hypoxia on heart susceptibility to ischemia and reperfusion injury in the adult rat

Objective: Epidemiologic studies showed an association between adverse intrauterine environment and ischemic heart disease in the adult. We tested the hypothesis that prenatal hypoxia increased the susceptibility of adult heart to ischemia-reperfusion (I-R) injury. Methods: Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% oxygen from day 15 to 21) gbroups. Hearts of 6-month-old male progeny were studied using Langendorff preparation and were subjected to two protocols of I-R: 10 minutes of ischemia and 3 hours of reperfusion (I-R10) or 25 minutes of ischemia and 3 hours of reperfusion (I-R25). Results: Prenatal hypoxia did not change basal left ventricular (LV) function. I-R10 produced myocardial stunning and a transient decrease in LV function in control hearts but caused myocardial infarction and a persistent decrease in postischemic recovery of LV function in hypoxic hearts. I-R25 caused myocardial infarction in both control and hypoxic hearts, which was significantly higher in hypoxic hearts. The postischemic recovery of LV function was significantly reduced in hypoxic hearts. I-R25-induced activation of caspase-3 and apoptosis in the left ventricle were significantly higher in hypoxic than control hearts. there was a significant decrease in LV heat shock protein 70 and endothelial nitric oxice synthase levels in hypoxic hearts. Prenatal hypoxia did not change β1-adrenoreceptor levels but significantly increased β2-adrenoreceptor in the left ventricle. In addition, it increased Gsα but decreased Giα. Conclusion: Prenatal chronic hypoxia increases the susceptibility of adult heart to I-R injury. Several possible mechanisms may be involved, including an increase in β2-adrenoreceptor and theGsα/Giα ratio, and a decrease in heat shock protein 70 and endothelial nitric oxide synthase in the left ventricle.

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.

Fetal Stress and Programming of Hypoxic/Ischemic-Sensitive Phenotype in the Neonatal Brain: Mechanisms and Possible Interventions

Growing evidence of epidemiological, clinical and experimental studies has clearly shown a close link between adverse in utero environment and the increased risk of neurological, psychological and psychiatric disorders in later life. Fetal stresses, such as hypoxia, malnutrition, and fetal exposure to nicotine, alcohol, cocaine and glucocorticoids may directly or indirectly act at cellular and molecular levels to alter the brain development and result in programming of heightened brain vulnerability to hypoxic-ischemic encephalopathy and the development of neurological diseases in the postnatal life. The underlying mechanisms are not well understood. However, glucocorticoids may play a crucial role in epigenetic programming of neurological disorders of fetal origins. This review summarizes the recent studies about the effects of fetal stress on the abnormal brain development, focusing on the cellular, molecular and epigenetic mechanisms and highlighting the central effects of glucocorticoids on programming of hypoxic-ischemic-sensitive phenotype in the neonatal brain, which may enhance the understanding of brain pathophysiology resulting from fetal stress and help explore potential targets of timely diagnosis, prevention and intervention in neonatal hypoxic-ischemic encephalopathy and other brain disorders.

Review Article: Steroid Hormones and Uterine Vascular Adaptation to Pregnancy:

Pregnancy is a physiological state that involves a significant decrease in uterine vascular tone and an increase in uterine blood flow, which is mediated in part by steroid hormones, including estrogen, progesterone, and cortisol. Previous studies have demonstrated the involvement of these hormones in the regulation of uterine artery contractility through signaling pathways specific to the endothelium and the vascular smooth muscle. Alterations in endothelial nitric oxide synthase expression and activity, nitric oxide production, and expression of enzymes involved in PGI2 production contribute to the uterine artery endothelium—specific responses. Steroid hormones also have an effect on calcium-activated potassium channel activity, PKC signaling pathway and myogenic tone, and alterations in pharmacomechanical coupling in the uterine artery smooth muscle. This review addresses current understanding of the molecular mechanisms by which steroid hormones including estrogen, progesterone, and cortisol modulate uterine artery contractility to alter uterine blood flow during pregnancy with an emphasis on the pregnant ewe model.

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

Function and regulation of large conductance Ca(2+)-activated K+ channel in vascular smooth muscle cells.

Large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are abundantly expressed in vascular smooth muscle cells. Activation of BK(Ca) channels leads to hyperpolarization of cell membrane, which in turn counteracts vasoconstriction. Therefore, BK(Ca) channels have an important role in regulation of vascular tone and blood pressure. The activity of BK(Ca) channels is subject to modulation by various factors. Furthermore, the function of BK(Ca) channels are altered in both physiological and pathophysiological conditions, such as pregnancy, hypertension and diabetes, which has dramatic impacts on vascular tone and hemodynamics. Consequently, compounds and genetic manipulation that alter activity and expression of the channel might be of therapeutic interest.

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.

Epigenetic mechanisms in developmental programming of adult disease.

Adverse insults during intrauterine life can result in permanent changes in the physiology and metabolism of the offspring, which in turn leads to an increased risk of disease in adulthood. This is an adaptational response by the fetus to changes in the environmental signals that it receives during early life to ensure its survival and prepare itself for postnatal life. Increasing evidence suggests that the epigenetic regulation of gene expression patterns has a crucial role in the developmental programming of adult disease. This review summarizes recent studies of epigenetic mechanisms and focuses particularly on studies that explore identifiable epigenetic biomarkers in the promoters of specific disease-associated genes. Such biomarkers would enable early recognition of children who might be at risk of developing adult disease with fetal origins.