Jeremy A. Hansell

Developmental Programming of Cardiovascular Dysfunction by Prenatal Hypoxia and Oxidative Stress

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O2) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.

Melatonin improves placental efficiency and birth weight and increases the placental expression of antioxidant enzymes in undernourished pregnancy

Abstract:  Melatonin participates in circadian, seasonal and reproductive physiology. Melatonin also acts as a potent endogenous antioxidant by scavenging free radicals and upregulating antioxidant pathways. The placenta expresses melatonin receptors and melatonin protects against oxidative damage induced in rat placenta by ischemia‐reperfusion. One of the most common complications in pregnancy is a reduction in fetal nutrient delivery, which is known to promote oxidative stress. However, whether melatonin protects placental function and fetal development in undernourished pregnancy is unknown. Here, we investigated the effects of maternal treatment with melatonin on placental efficiency, fetal growth, birth weight and protein expression of placental oxidative stress markers in undernourished pregnancy. On day 15 of pregnancy, rats were divided into control and undernourished pregnancy (35% reduction in food intake), with and without melatonin treatment (5 μg/mL drinking water). On day 20 of gestation, fetal biometry was carried out, the placenta was weighed and subsequently analyzed by Western blot for xanthine oxidase, heat shock protein (HSP) 27 and 70, catalase, manganese superoxide dismutase (Mn‐SOD) and glutathione peroxidase 1 (GPx‐1). A separate cohort was allowed to deliver to assess effects on birth weight. Maternal undernutrition led to a fall in placental efficiency, disproportionate intrauterine growth retardation and a reduction in birth weight. Maternal treatment with melatonin in undernourished pregnancy improved placental efficiency and restored birth weight, and it increased the expression of placental Mn‐SOD and catalase. The data show that in pregnancy complicated by undernutrition, melatonin may improve placental efficiency and birth weight by upregulating placental antioxidant enzymes.

Partial contributions of developmental hypoxia and undernutrition to prenatal alterations in somatic growth and cardiovascular structure and function

OBJECTIVE The objective of the study was to compare and contrast the effects of developmental hypoxia vs undernutrition on fetal growth, cardiovascular morphology, and function. STUDY DESIGN On day 15 of gestation, Wistar dams were divided into control, hypoxic (10% O(2)), or undernourished (35% reduction in food intake) pregnancy. On day 20, fetal thoraces were fixed, and the fetal heart and aorta underwent quantitative histological analysis. In a separate group, fetal aortic vascular reactivity was determined via wire myography. RESULTS Both hypoxic and undernourished pregnancy was associated with asymmetric fetal growth restriction. Pregnancy complicated by hypoxia promoted fetal aortic thickening without changes in cardiac volumes when expressed as a percentage of total heart volume. In contrast, maternal undernutrition affected fetal cardiac morphology without changes in aortic structure. Fetal aortic vascular reactivity was also differentially affected by hypoxia or undernutrition. CONCLUSION Developmental hypoxia or undernutrition in late gestation has differential effects on fetal cardiovascular morphology and function.

Prenatal hypoxia independent of undernutrition promotes molecular markers of insulin resistance in adult offspring.

Molecular mechanisms predisposing people to insulin resistance are starting to emerge. Altered insulin signaling for hepatic gluconeogenesis and muscle glucose uptake is thought to play a central role. Development under suboptimal conditions is also known to increase the risk of insulin resistance in adulthood. However, the partial contributions of reduced oxygen vs. nutrient delivery to the fetus, two common adverse conditions in utero, to developmental programming of insulin resistance remain unknown. The aim of this study was to determine the effects of developmental hypoxia or undernutrition on the expression of insulin‐signaling proteins in liver and skeletal muscle in adult rat offspring. We show that the expression of hepatic phospho‐Akt and muscle Akt2 were significantly reduced in offspring of hypoxic, relative to offspring from normoxic or undernourished, pregnancies. Hepatic Akt‐1, Akt‐2, and PKCζ protein expression was reduced in offspring from both hypoxic and undernourished pregnancies. Muscle GLUT4 expression was decreased in undernourished, and further decreased in hypoxic, offspring. These findings link prenatal hypoxia to down‐regulation of components of hepatic and muscle Akt expression in adult offspring. Akt may represent a pharmaceutical target for clinical intervention against the developmental programming of metabolic disease resulting from prenatal hypoxia.—Camm, E. J., Martin‐Gronert, M. S., Wright, N. L., Hansell, J. A., Ozanne, S. E., Giussani, D. A. Prenatal hypoxia independent of undernutrition promotes molecular markers of insulin resistance in adult offspring. FASEB J. 25, 420–427 (2011). www.fasebj.org

Statin treatment depresses the fetal defence to acute hypoxia via increasing nitric oxide bioavailability

Non‐technical summary  The fetal cardiovascular defence to episodes of reduced oxygenation, or acute hypoxia, includes redistribution of the cardiac output away from peripheral and towards essential circulations, such as those perfusing the brain – the so called brain‐sparing effect. The peripheral vasoconstriction is triggered by a chemoreflex and maintained by constrictor hormones. Nitric oxide (NO) synthesis during hypoxia opposes these mechanisms, but the balance of all effects favours constriction. Statins are drugs commonly used to lower cholesterol. Since women are delaying pregnancy until later in life, there is increasing clinical interest in treating pregnant women with statins. However, statins have other effects, including increasing NO levels, and their effects on the physiology of the fetus are completely unknown. Here, we show that fetal exposure to statins depresses the fetal peripheral constrictor response to acute hypoxia via increasing NO bioavailability. Use of statins in pregnancy should be viewed with caution.

Heart Disease Link to Fetal Hypoxia and Oxidative Stress

The quality of the intrauterine environment interacts with our genetic makeup to shape the risk of developing disease in later life. Fetal chronic hypoxia is a common complication of pregnancy. This chapter reviews how fetal chronic hypoxia programmes cardiac and endothelial dysfunction in the offspring in adult life and discusses the mechanisms via which this may occur. Using an integrative approach in large and small animal models at the in vivo, isolated organ, cellular and molecular levels, our programmes of work have raised the hypothesis that oxidative stress in the fetal heart and vasculature underlies the mechanism via which prenatal hypoxia programmes cardiovascular dysfunction in later life. Developmental hypoxia independent of changes in maternal nutrition promotes fetal growth restriction and induces changes in the cardiovascular, metabolic and endocrine systems of the adult offspring, which are normally associated with disease states during ageing. Treatment with antioxidants of animal pregnancies complicated with reduced oxygen delivery to the fetus prevents the alterations in fetal growth, and the cardiovascular, metabolic and endocrine dysfunction in the fetal and adult offspring. The work reviewed offers both insight into mechanisms and possible therapeutic targets for clinical intervention against the early origin of cardiometabolic disease in pregnancy complicated by fetal chronic hypoxia.

A role for xanthine oxidase in the control of fetal cardiovascular function in late gestation sheep

Key points  •  There is growing physiological and clinical interest in the role of the enzyme xanthine oxidase in the regulation of fetal cardiovascular function. •  The xanthine oxidase inhibitor allopurinol is undergoing human clinical trials in complicated pregnancy to protect the fetal brain from injury by decreasing excessive generation of reactive oxygen species (ROS) and increasing nitric oxide (NO) availability. However, the effects on fetal cardiovascular physiology of xanthine oxidase inhibition are largely unknown. •  We have previously reported that the balance between ROS and NO plays an important physiological role in the control of fetal cardiovascular function. Therefore, it seems likely that allopurinol might perturb this balance and alter fetal cardiovascular homeostasis. •  Here, we report that maternal allopurinol treatment in late gestation ovine pregnancy has significant in vivo effects on umbilical blood flow and the cardiovascular system of the mother and fetus by altering NO and β1‐adrenergic mechanisms. •  The evidence suggests that xanthine oxidase has an important role in basal cardiovascular function in the fetus during late gestation. Therefore, further research is warranted before safe clinical application of maternal allopurinol during pregnancy in humans.

Xanthine oxidase and the fetal cardiovascular defence to hypoxia in late gestation ovine pregnancy

Periods of impaired oxygenation or acute hypoxia in the fetus can be common during labour and how the fetus withstands these challenges is of interest. During hypoxia, the fetus shunts blood flow away from peripheral and towards essential vascular beds: the so called brain‐sparing effect. Part of the peripheral vasoconstriction is driven by reactive oxygen species (ROS) that inactivate nitric oxide (NO), thereby limiting its vasodilator action. Here, we investigate the source of ROS generation contributing to fetal peripheral vasoconstriction during hypoxia, and show that xanthine oxidase (XO) is fundamentally involved. Fetal exposure to the XO inhibitor allopurinol markedly diminished the peripheral vasoconstriction during hypoxia via NO‐dependent mechanisms. The data increase our understanding of the physiological control of fetal cardiovascular function during stress. The findings are also of significant clinical relevance as allopurinol is being administered to pregnant women in clinical obstetric trials.

Oxidative Stress in the Developing Brain: Effects of Postnatal Glucocorticoid Therapy and Antioxidants in the Rat

In premature infants, glucocorticoids ameliorate chronic lung disease, but have adverse effects on long-term neurological function. Glucocorticoid excess promotes free radical overproduction. We hypothesised that the adverse effects of postnatal glucocorticoid therapy on the developing brain are secondary to oxidative stress and that antioxidant treatment would diminish unwanted effects. Male rat pups received a clinically-relevant tapering course of dexamethasone (DEX; 0.5, 0.3, and 0.1 mg.kg−1.day−1), with or without antioxidant vitamins C and E (DEXCE; 200 mg.kg−1.day−1 and 100 mg.kg−1.day−1, respectively), on postnatal days 1–6 (P1–6). Controls received saline or saline with vitamins. At weaning, relative to controls, DEX decreased total brain volume (704.4±34.7 mm3 vs. 564.0±20.0 mm3), the soma volume of neurons in the CA1 (1172.6±30.4 µm3 vs. 1002.4±11.8 µm3) and in the dentate gyrus (525.9±27.2 µm3 vs. 421.5±24.6 µm3) of the hippocampus, and induced oxidative stress in the cortex (protein expression: heat shock protein 70 [Hsp70]: +68%; 4-hydroxynonenal [4-HNE]: +118% and nitrotyrosine [NT]: +20%). Dexamethasone in combination with vitamins resulted in improvements in total brain volume (637.5±43.1 mm3), and soma volume of neurons in the CA1 (1157.5±42.4 µm3) and the dentate gyrus (536.1±27.2 µm3). Hsp70 protein expression was unaltered in the cortex (+9%), however, 4-HNE (+95%) and NT (+24%) protein expression remained upregulated. Treatment of neonates with vitamins alone induced oxidative stress in the cortex (Hsp70: +67%; 4-HNE: +73%; NT: +22%) and in the hippocampus (NT: +35%). Combined glucocorticoid and antioxidant therapy in premature infants may be safer for the developing brain than glucocorticoids alone in the treatment of chronic lung disease. However, antioxidant therapy in healthy offspring is not recommended.

Investigation of the Use of Antioxidants to Diminish the Adverse Effects of Postnatal Glucocorticoid Treatment on Mortality and Cardiac Development

Background: In premature infants, glucocorticoids ameliorate chronic lung disease, but have adverse effects on growth and the cardiovascular system. Glucocorticoid excess promotes free radical overproduction and vascular dysfunction. Objectives: We hypothesized that the adverse effects of postnatal glucocorticoid therapy are secondary to oxidative stress and that antioxidant treatment would diminish unwanted effects. Methods: Male rat pups received a clinically relevant course of dexamethasone (Dex), or Dex with vitamins C and E (DexCE), on postnatal days 1–6 (P1–6). Controls received saline (Ctrl) or saline with vitamins (CtrlCE). Results: At P21, Dex reduced survival (Ctrl: 96 vs. Dex: 70%) and promoted asymmetric growth restriction (ponderal index, Ctrl: 6.3 ± 0.1 g · mm–3 × 10–5 vs. Dex: 7.4 ± 0.2 g · mm–3 × 10–5), both p < 0.05. Dex increased cardiac oxidative stress (protein expression: 4-HNE +20%, Hsp90 –42% and eNOS –54%), induced left ventricle (LV) wall thinning (LV wall volume: Ctrl: 47.2 ± 1.2 mm3 vs. Dex: 38.9 ± 1.7 mm3) and decreased the ratio of the aortic lumen:total vessel area (Ctrl: 0.74 ± 0.01 vs. Dex: 0.66 ± 0.02), all p < 0.05. DexCE restored towards control values survival, growth symmetry the aortic lumen:total vessel area, and increased the cardiac expression of Hsp90 relative to Dex. In addition, relative to controls, the decrease in the cardiac expression of eNOS was no longer significant in DexCE animals (–20.3 ± 14.4%, p > 0.05). However, DexCE did not prevent growth retardation, cardiac 4-HNE upregulation (DexCE: +29%) or LV thinning (DexCE: 40.1 ± 1.1 mm3). Treatment of neonates with vitamins alone affected somatic growth and promoted thinner LV walls (CtrlCE: 39.9 ± 0.5 mm3, p < 0.05). Conclusions: Combined glucocorticoid and antioxidant therapy in premature infants may be safer than glucocorticoids alone in the treatment of chronic lung disease. However, antioxidant therapy in healthy offspring is not recommended.