Chiranjib Dasgupta

Foetal hypoxia increases cardiac AT2R expression and subsequent vulnerability to adult ischaemic injury

AIMS Hypoxia is a common stress to the foetus and results in increased cardiac vulnerability to adult ischaemic injury. This study tested the hypothesis that foetal hypoxia causes programming of increased AT(2) receptor (AT(2)R) expression in the heart, resulting in the heightened cardiac susceptibility to adult ischaemic injury. METHODS AND RESULTS Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O(2) from days 15 to 21 of gestation) groups. Hypoxia resulted in significantly increased AT(2)R in the heart of adult offspring. Multiple glucocorticoid response elements (GREs) were identified at the AT(2)R promoter, deletion of which increased the promoter activity. Consistently, ex vivo treatment of isolated foetal hearts with dexamethasone for 48 h decreased AT(2)R expression, which was inhibited by RU 486. Hypoxia decreased glucocorticoid receptors (GRs) in the hearts of foetal, 3-week-old and 3-month-old offspring, resulting in decreased GR binding to the GREs at the AT(2)R promoter. The inhibition of AT(2)R improved postischaemic recovery of left ventricular function and rescued the foetal hypoxia-induced cardiac ischaemic vulnerability in male adult animals. In contrast, the inhibition of AT(1) receptors decreased the postischaemic recovery. CONCLUSION The results demonstrate that in utero hypoxia causes programming of increased AT(2)R gene expression in the heart by downregulating GR, which contributes to the increased cardiac vulnerability to adult ischaemic injury caused by prenatal hypoxic exposure.

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.

Angiotensin II receptors and drug discovery in cardiovascular disease

Hypertension is one of the cardiovascular diseases that might cause cardiovascular remodeling and endothelial dysfunction besides high blood pressure. Angiotensin II (Ang II) receptors are implicated in hypertension. Genetic and epigenetic manipulations of the Ang II receptors play a crucial part in the programming of cardiovascular diseases, and certain variants of the Ang II type 1 and Ang II type 2 receptors are constitutively predisposed to higher cardiovascular risk and hypertension. In this review, we focus on the expression, mode of action of Ang II receptors, and their role in programming the cardiovascular diseases in utero. In addition, we discuss possible therapeutic interventions of Ang II stimulation. Collectively, this information might lead us to new drug designs against cardiovascular diseases.

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.

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.

Perinatal Nicotine Exposure Increases Angiotensin II Receptor-Mediated Vascular Contractility in Adult Offspring

Previous studies have reported that perinatal nicotine exposure causes development of hypertensive phenotype in adult offspring. Aims The present study was to determine whether perinatal nicotine exposure causes an epigenetic programming of vascular Angiotensin II receptors (ATRs) and their-mediated signaling pathway leading to heightened vascular contraction in adult offspring. Main methods Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from day 4 of gestation to day 10 after birth. The experiments were conducted at 5 months of age of male offspring. Key Findings Nicotine treatment enhanced Angitension II (Ang II)-induced vasoconstriction and 20-kDa myosin light chain phosphorylation (MLC20-P) levels. In addition, the ratio of Ang II-induced tension/MLC-P was also significantly increased in nicotine-treated group compared with the saline group. Nicotine-mediated enhanced constrictions were not directly dependent on the changes of [Ca2+]i concentrations but dependent on Ca2+ sensitivity. Perinatal nicotine treatment significantly enhanced vascular ATR type 1a (AT1aR) but not AT1bR mRNA levels in adult rat offspring, which was associated with selective decreases in DNA methylation at AT1aR promoter. Contrast to the effect on AT1aR, nicotine decreased the mRNA levels of vascular AT2R gene, which was associated with selective increases in DNA methylation at AT2R promoter. Significance Our results indicated that perinatal nicotine exposure caused an epigenetic programming of vascular ATRs and their-mediated signaling pathways, and suggested that differential regulation of AT1R/AT2R gene expression through DNA methylation mechanism may be involved in nicotine-induced heightened vasoconstriction and development of hypertensive phenotype in adulthood.

Developmental Nicotine Exposure Results in Programming of Alveolar Simplification and Interstitial Pulmonary Fibrosis in Adult Male Rats

AIMS To determine the long-term effect of maternal nicotine intake on the lung development of the offspring in adult life, we analyzed the alveolar structure, protein expression in the adult rat offspring lungs. METHODS We determined animal body weight (BW), lung weight (LW), lung/body weight ratio (L/BWR), lung volume (LV), radial alveolar count (RAC), alveolar septal thickness (AST) and expression of collagen, AT1R, AT2R, TGF-β1, pSmad3, Smad3 and CTGF proteins. RESULTS Male offspring lung showed decreased RAC, thickened alveolar septa, increased collagen, AT1R, TGF-β1, pSmad3 and CTGF proteins. In contrast, female offspring lungs had reduced L/BWR, increased LV, and expression of AT2R, resulting in decreased AT1R to AT2R ratio. CONCLUSIONS Maternal nicotine use during development programs abnormal lung development in male rats. This finding links maternal nicotine use to increased susceptibility to interstitial pulmonary fibrosis in adult male but not female offspring, indicating sex-dependent effects of developmental nicotine exposure.

Hypoxia Represses ER-α Expression and Inhibits Estrogen-Induced Regulation of Ca2+-Activated K+ Channel Activity and Myogenic Tone in Ovine Uterine Arteries Causal Role of DNA Methylation

Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-&agr; (ER-&agr;) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-&agr; gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-&agr; gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O2 and 10.5% O2 for 48 hours. Hypoxia significantly increased ER-&agr; promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-&agr; expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2′-deoxycytidine. In addition, hypoxia abrogated steroid hormone–mediated increase in ER-&agr; expression and inhibited the hormone-induced increase in large-conductance Ca2+-activated K+ channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2′-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-&agr; gene repression and ablation of steroid hormone–mediated adaptation of uterine arterial large conductance Ca2+-activated K+ channel activity and myogenic tone in pregnancy.

Antenatal Antioxidant Prevents Nicotine-Mediated Hypertensive Response in Rat Adult Offspring

ABSTRACT Previous studies have demonstrated that perinatal nicotine exposure increased blood pressure (BP) in adult offspring. However, the underlying mechanisms were unclear. The present study tested the hypothesis that perinatal nicotine-induced programming of hypertensive response is mediated by enhanced reactive oxygen species (ROS) in the vasculature. Nicotine was administered to pregnant rats via subcutaneous osmotic mini-pumps from Day 4 of gestation to Day 10 after birth, in the absence or presence of the ROS inhibitor N-acetyl-cysteine (NAC) in the drinking water. Experiments were conducted in 8-mo-old male offspring. Perinatal nicotine treatment resulted in a significant increase in arterial ROS production in offspring, which was abrogated by NAC. Angiotensin II (Ang II)-induced BP responses were significantly higher in nicotine-treated group than in saline-treated control group, and NAC treatment blocked the nicotine-induced increase in BP response. Consistent with that, the nicotine treatment significantly increased both Ang II-induced and phorbol [12, 13]-dibutyrate (PDBu, a Prkc activator)-induced arterial contractions in adult offspring, which were blocked by NAC treatment. In addition, perinatal nicotine treatment significantly attenuated acetylcholine-induced arterial relaxation in offspring, which was also inhibited by NAC treatment. Results demonstrate that inhibition of ROS blocks the nicotine-induced increase in arterial reactivity and BP response to vasoconstrictors in adult offspring, suggesting a key role for increased oxidative stress in nicotine-induced developmental programming of hypertensive phenotype in male offspring.

Inhibition of miRNA-210 reverses nicotine-induced brain hypoxic-ischemic injury in neonatal rats.

Maternal tobacco use in pregnancy increases the risk of neurodevelopmental disorders and neurobehavioral deficits in postnatal life. The present study tested the hypothesis that perinatal nicotine exposure exacerbated brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats through up-regulation of miR-210 expression in the developing brain. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps. Experiments of HI brain injury were performed in 10-day-old pups. Perinatal nicotine treatment significantly decreased neonatal body and brain weights, but increased the brain to body weight ratio. Perinatal nicotine exposure caused a significant increase in HI brain infarct size in the neonates. In addition, nicotine enhanced miR-210 expression and significantly attenuated brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase isoform B (TrkB) protein abundance in the brain. Of importance, intracerebroventricular administration of a miR-210 inhibitor (miR-210-LNA) significantly decreased HI-induced brain infarct size and reversed the nicotine-increased vulnerability to brain HI injury in the neonate. Furthermore, miR-210-LNA treatment also reversed nicotine-mediated down-regulation of BDNF and TrkB protein expression in the neonatal brains. These findings provide novel evidence that the increased miR-210 plays a causal role in perinatal nicotine-induced developmental programming of ischemic sensitive phenotype in the brain. It represents a potential novel therapeutic approach for treatment of brain hypoxic-ischemic encephalopathy in the neonate-induced by fetal stress.