The Prenatal Hypoxic Pathology Associated with Maternal Stress Predisposes to Dysregulated Expression of the chrna7 Gene and the Subsequent Development of Nicotine Addiction in Adult Offspring.

INTRODUCTION: Previous studies have shown that fetal hypoxia predisposes individuals to develop addictive disorders in adulthood. However, the specific impact of maternal stress, mediated through glucocorticoids and often coexisting with fetal hypoxia, is not yet fully comprehended. METHODS: To delineate the potential effects of these pathological factors, we designed models of prenatal severe hypoxia (PSH) in conjunction with maternal stress and prenatal intrauterine ischemia (PII). We assessed the suitability of these models for our research objectives by measuring HIF1α levels and evaluating the glucocorticoid neuroendocrine system. To ascertain nicotine dependence, we employed the conditioned place aversion test and the startle response test. To identify the key factor implicated in nicotine addiction associated with PSH, we employed techniques such as Western blot, immunohistochemistry, and correlational analysis between chrna7 and nr3c1 genes across different brain structures. RESULTS: In adult rats exposed to PSH and PII, we observed increased levels of HIF1α in the hippocampus (HPC). However, the PSH group alone exhibited reduced glucocorticoid receptor levels and disturbed circadian glucocorticoid rhythms. Additionally, they displayed signs of nicotine addiction in the conditioned place aversion and startle response tests. We also observed elevated levels of phosphorylated DARPP-32 protein in the nucleus accumbens (NAc) indicated compromised glutamatergic efferent signaling. Furthermore, there was reduced expression of α7 nAChR, which modulates glutamate release, in the medial prefrontal cortex (PFC) and HPC. Correlation analysis revealed strong associations between chrna7 and nr3c1 expression in both brain structures. CONCLUSION: Perturbations in the glucocorticoid neuroendocrine system and glucocorticoid-dependent gene expression of chrna7 associated with maternal stress response to hypoxia in prenatal period favor the development of nicotine addiction in adulthood.

Hypoxia in utero increases the risk of pulmonary hypertension in rat offspring and is associated with vasopressin type­2 receptor upregulation.

Pulmonary hypertension (PH) in newborns and adults is a disease that can lead to right heart failure and result in a shorter lifespan. PH was induced by maintaining pregnant rats in a hypoxic chamber for 4 h twice a day, from days 7­21 of pregnancy. Hypoxia was confirmed by a decrease in the partial pressure of oxygen (PaO2) and the oxygen saturation (SaO2) of arterial blood in the aorta. The body weight of newborns from hypoxic rats was ~20% decreased compared with the control newborns of normoxic rats. The vascular wall thickness/vascular diameter values of hypoxia treated pubs were increased compared with that of control newborns 7 days after birth; however, it decreased to similar levels than in the control group after 3 months, and then further decreased to significantly lower levels than in the control group at 6 months after birth. At birth, the lung tissues of newborns from hypoxic rats exhibited an increase in the levels of mRNA and proteins associated with PH such as HIF­1α, HIF­2α, V2R, TGF­ß, TNF­α, Ang­2 and α­SMA. At 3 and 6 months after birth, the levels of both V2R mRNA and protein in offspring from hypoxic rats were at least 2­fold higher, whereas the expression of all other factors decreased compared with the control offspring. By contrast, HIF­2α and Ang­2 expression levels were significantly increased in the 6­month­old control offspring from normoxic rats. V2R overexpression in pups induced by hypoxia in maternal rats was sustained until their adulthood. V2R may be a marker for detecting PH.

[Morphological characteristics of fetal testes in chronic intrauterine hypoxia in different gestation periods]. / Morfologicheskie osobennosti iaichek ploda pri khronicheskoi vnutriutrobnoi gipoksii na raznykh srokakh gestatsii.

OBJECTIVE: To study the morphological characteristics and expression of vascular endothelial growth factor (VEGF) in the fetal testes exposed to chronic intrauterine hypoxia during pathological pregnancy in different gestation periods. MATERIAL AND METHODS: The testes from 48 male fetuses that had died in the antenatal or early neonatal period in mothers with pathological pregnancy were morphologically evaluated. RESULTS: Chronic intrauterine hypoxia was shown to be a powerful damaging factor and leads to delayed gonadal development. Histological examination of testicular tissue showed a significant reduction in the number of tubular cells per vision field, a decrease in tubular diameter and area, with the simultaneously increased area of the stroma and a larger number of vessels. Immunohistochemical study revealed the pronounced cytoplasmic expression of VEGF in testicular tissue in different gestation periods in the spermatogenic epitheliocytes, vessels, Leydig interstitial cells, while the maximal expression of this receptor was observed at 19-25 weeks' gestation, the degree of expression decreased at 26-29 weeks' gestation. CONCLUSION: Intrauterine hypoxia has a destabilizing effect on the processes of proliferation and differentiation of the spermatogenic epithelium, interstitial endocrinocytes, activates the processes of angiogenesis and the growth of connective tissue. All this can involve not only gonadal dysgenesis, but also future reproductive dysfunction. Hypoxia stimulates the expression of VEGF, whose receptors are present in almost all testicular cell populations. It can be assumed that VEGF can act as a paracrine regulator of Leydig cell activity, also as an inducer of angiogenesis, and thus play a certain role in the development of male fertility.

Assessing the association between hypoxia during craniofacial development and oral clefts

Abstract Objectives To evaluate the association between hypoxia during embryo development and oral clefts in an animal model, and to evaluate the association between polymorphisms in the HIF-1A gene with oral clefts in human families. Material and Methods The study with the animal model used zebrafish embryos at 8 hours post-fertilization submitted to 30% and 50% hypoxia for 24 hours. At 5 days post-fertilization, the larvae were fixed. The cartilage structures were stained to evaluate craniofacial phenotypes. The family-based association study included 148 Brazilian nuclear families with oral clefts. The association between the genetic polymorphisms rs2301113 and rs2057482 in HIF-1A with oral clefts was tested. We used real time PCR genotyping approach. ANOVA with Tukey's post-test was used to compare means. The transmission/disequilibrium test was used to analyze the distortion of the inheritance of alleles from parents to their affected offspring. Results For the hypoxic animal model, the anterior portion of the ethmoid plate presented a gap in the anterior edge, forming a cleft. The hypoxia level was associated with the severity of the phenotype (p<0.0001). For the families, there was no under-transmitted allele among the affected progeny (p>0.05). Conclusion Hypoxia is involved in the oral cleft etiology, however, polymorphisms in HIF-1A are not associated with oral clefts in humans.

Neonatal vascularization and oxygen tension regulate appropriate perinatal renal medulla/papilla maturation.

Congenital medullary dysplasia with obstructive nephropathy is a common congenital disorder observed in paediatric patients and represents the foremost cause of renal failure. However, the molecular processes regulating normal papillary outgrowth during the postnatal period are unclear. In this study, transcriptional profiling of the renal medulla across postnatal development revealed enrichment of non-canonical Wnt signalling, vascular development, and planar cell polarity genes, all of which may contribute to perinatal medulla/papilla maturation. These pathways were investigated in a model of papillary hypoplasia with functional obstruction, the Crim1(KST264/KST264) transgenic mouse. Postnatal elongation of the renal papilla via convergent extension was unaffected in the Crim1(KST264/KST264) hypoplastic renal papilla. In contrast, these mice displayed a disorganized papillary vascular network, tissue hypoxia, and elevated Vegfa expression. In addition, we demonstrate the involvement of accompanying systemic hypoxia arising from placental insufficiency, in appropriate papillary maturation. In conclusion, this study highlights the requirement for normal vascular development in collecting duct patterning, development of appropriate nephron architecture, and perinatal papillary maturation, such that disturbances contribute to obstructive nephropathy.

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 ß (ERß) isoforms in female than in male fetal hearts, which were not significantly altered by hypoxia. Both ERα and ERß 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.

Hypoxia and fetal heart development.

Fetal hearts show a remarkable ability to develop under hypoxic conditions. The metabolic flexibility of fetal hearts allows sustained development under low oxygen conditions. In fact, hypoxia is critical for proper myocardial formation. Particularly, hypoxia inducible factor 1 (HIF-1) and vascular endothelial growth factor play central roles in hypoxia-dependent signaling in fetal heart formation, impacting embryonic outflow track remodeling and coronary vessel growth. Although HIF is not the only gene involved in adaptation to hypoxia, its role places it as a central figure in orchestrating events needed for adaptation to hypoxic stress. Although "normal" hypoxia (lower oxygen tension in the fetus as compared with the adult) is essential in heart formation, further abnormal hypoxia in utero adversely affects cardiogenesis. Prenatal hypoxia alters myocardial structure and causes a decline in cardiac performance. Not only are the effects of hypoxia apparent during the perinatal period, but prolonged hypoxia in utero also causes fetal programming of abnormality in the heart's development. The altered expression pattern of cardioprotective genes such as protein kinase c epsilon, heat shock protein 70, and endothelial nitric oxide synthase, likely predispose the developing heart to increased vulnerability to ischemia and reperfusion injury later in life. The events underlying the long-term changes in gene expression are not clear, but likely involve variation in epigenetic regulation.

Chronic prenatal hypoxia induces epigenetic programming of PKC{epsilon} 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 (PKCepsilon) 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 PKCepsilon promoter is repressive and contributes to PKCepsilon 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 PKCepsilon 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 PKCepsilon promoter at SP1 binding sites, -346 and -268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O(2) 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 PKCepsilon 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 PKCepsilon mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor alpha and beta isoforms in females than in males. Both estrogen receptor alpha and beta 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 PKCepsilon 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 PKCepsilon gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.

Prenatal exposure to nicotine with associated in utero hypoxia decreased fetal brain muscarinic mRNA in the rat.

Prenatal exposure to nicotine can be associated with fetal abnormal development and brain damage. This study determined the effect of administration of nicotine with associated in utero hypoxia in maternal rats from early, middle, and late gestation on fetal blood hemoglobin, and expression of cholinergic receptor subtypes in the fetal brain. Our results demonstrated that maternal subcutaneous nicotine from the early gestation increased fetal hemoglobin and hematocrit, associated with reduction of PO(2). Although exposure to nicotine during late gestation had no effects on fetal brain weight, nicotine administration from the early gestation significantly decreased fetal brain muscarinic receptor (M1, M2, M3, and M4) mRNA expression, associated with restricted brain growth. Nicotine-altered muscarinic receptor subtype expression in the fetal forebrain and hindbrain showed regional differences. In addition, there were gestational differences for fetal brain muscarinic suppression by prenatal nicotine. Together, the results demonstrate that nicotine-induced in utero hypoxia is associated with poor development of muscarinic receptors in the fetal brain and restricted brain growth, and that either prolonged prenatal exposure to nicotine or critical "window" period for the brain development during pregnancy may play a role in prenatal nicotine-induced fetal muscarinic-receptor deficiency in the fetal brain.