Shumei Yang

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 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.

Chronic Hypoxia During Gestation Causes Epigenetic Repression of the Estrogen Receptor-α Gene in Ovine Uterine Arteries via Heightened Promoter Methylation

Estrogen receptor-&agr; (ER&agr;) plays a key role in the adaptation of increased uterine blood flow in pregnancy. Chronic hypoxia is a common stress to maternal cardiovascular homeostasis and causes increased risk of preeclampsia. Studies in pregnant sheep demonstrated that hypoxia during gestation downregulated ER&agr; gene expression in uterine arteries. The present study tested the hypothesis that hypoxia causes epigenetic repression of the ER&agr; gene in uterine arteries via heightened promoter methylation. Ovine ER&agr; promoter of 2035 bp spanning from −2000 to +35 of the transcription start site was cloned. No estrogen or hypoxia-inducible factor response elements were found at the promoter. Two transcription factor binding sites, USF−15 and Sp1−520, containing CpG dinucleotides were identified, which had significant effects on the promoter activity. The USF element binds transcription factors USF1 and USF2, and the Sp1 element binds Sp1, as well as ER&agr; through Sp1. Deletion of the Sp1 site abrogated 17&bgr;-estradiol–induced increase in the promoter activity. In normoxic control sheep, CpG methylation at the Sp1 but not the USF site was significantly decreased in uterine arteries of pregnant as compared with nonpregnant animals. In pregnant sheep exposed to long-term high-altitude hypoxia, CpG methylation at both Sp1 and USF sites in uterine arteries was significantly increased. Methylation inhibited transcription factor binding and the promoter activity. The results provide evidence of hypoxia causing heightened promoter methylation and resultant ER&agr; gene repression in uterine arteries and suggest new insights of molecular mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

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.

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.

Chronic Hypoxia Suppresses Pregnancy-Induced Upregulation of Large-Conductance Ca2+-Activated K+ Channel Activity in Uterine Arteries

Our previous study demonstrated that increased Ca2+-activated K+ (BKCa) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BKCa channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BKCa channel activity and suppressed BKCa channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BKCa channel &bgr;1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BKCa channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the &bgr;1 subunit and BKCa channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BKCa channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BKCa channel &bgr;1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Direct Chronic Effect of Steroid Hormones in Attenuating Uterine Arterial Myogenic Tone. Role of Protein Kinase C/Extracellular Signal–Regulated Kinase 1/2

Pregnancy is associated with a significant decrease in uterine vascular tone and an increase in uterine blood flow. The present study tested the hypothesis that estrogen and progesterone differentially regulate the extracellular signal–regulated kinase (ERK)1/2 and protein kinase C (PKC) signaling pathways in vascular smooth muscle, resulting in a decrease in uterine vascular myogenic tone in pregnancy. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Chronic treatment (48 hours) of nonpregnant uterine arteries with 17β-estradiol and progesterone caused a significant decrease in PKC-mediated contractions and pressure-induced myogenic tone. In accordance, treatment of near-term pregnant uterine arteries for 48 hours with ICI 182780 and RU 486 significantly increased PKC-induced contractions and myogenic tone. In contrast, acute treatment for 30 minutes had no effect on uterine artery contractility. An ERK1/2 inhibitor, PD098059, restored the chronic effect of steroids on PKC-mediated contractions in nonpregnant sheep. ERK1/2 protein and mRNA levels were greater in near-term pregnant as compared with nonpregnant uterine arteries. 17β-Estradiol and progesterone increased ERK1/2 protein in nonpregnant sheep. In agreement, ICI 182780 and RU 486 caused significant decreases in ERK1/2 protein in near-term pregnant sheep. Western blot showed 6 PKC isozymes, α, βI, βII, Δ, ϵ, and &zgr;, in the uterine arteries. 17β-Estradiol and progesterone decreased the particulate:cytosolic ratios of PKCα, ϵ, and &zgr;, respectively, in nonpregnant sheep. ICI 182780 and RU 486 increased the ratios in near-term pregnant sheep. The results indicate a direct chronic effect of the steroid hormones in the upregulation of ERK1/2 expression and downregulation of the PKC signaling pathway, resulting in attenuated myogenic tone of the uterine artery in pregnancy.Pregnancy is associated with a significant decrease in uterine vascular tone and an increase in uterine blood flow. The present study tested the hypothesis that estrogen and progesterone differentially regulate the ERK1/2 and PKC signaling pathways in vascular smooth muscle resulting in a decrease in uterine vascular myogenic tone in pregnancy. Uterine arteries were isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Chronic treatment (48 h) of NPUA with 17β-estradiol and progesterone caused a significant decrease in PKC-mediated contractions and pressure-induced myogenic tone. In accordance, treatment of PUA for 48 h with ICI 182,780 and RU 486 significantly increased PKC-induced contractions and myogenic tone. In contrast, acute treatment for 30 min had no effects on uterine artery contractility. An ERK1/2 inhibitor PD098059 restored the chronic effect of steroids on PKC-mediated contractions in NPUA. ERK1/2 protein and mRNA levels were greater in PUA as compared with NPUA. 17β-Estradiol and progesterone increased ERK1/2 protein in NPUA. In agreement, ICI 182,780 and RU 486 caused a significant decrease in ERK1/2 protein in PUA. Western blot showed six PKC isozymes, α, βI, βII, δ, e and ζ in the uterine arteries. 17β-Estradiol and progesterone decreased the particulate-to-cytosolic ratio of PKCα, e, and ζ, respectively, in NPUA. ICI 182,780 and RU 486 increased them in PUA. The results indicate a direct chronic effect of the steroid hormones in the up-regulation of ERK1/2 expression and down-regulation of PKC signaling pathway, resulting in attenuated myogenic tone of uterine artery in pregnancy.