Hans G. Richter

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.

Maternal melatonin selectively inhibits cortisol production in the primate fetal adrenal gland

We tested the hypothesis that in primates, maternal melatonin restrains fetal and newborn adrenal cortisol production. A functional G‐protein‐coupled MT1 membrane‐bound melatonin receptor was detected in 90% gestation capuchin monkey fetal adrenals by (a) 2‐[125I] iodomelatonin binding (Kd, 75.7 ± 6.9 pm; Bmax, 2.6 ± 0.4 fmol (mg protein)−1), (b) cDNA identification, and (c) melatonin inhibition of adrenocorticotrophic hormone (ACTH)‐ and corticotrophin‐releasing hormone (CRH)‐stimulated cortisol but not of dehydroepiandrosterone sulphate (DHAS) production in vitro. Melatonin also inhibited ACTH‐induced 3β‐hydroxysteroid dehydrogenase mRNA expression. To assess the physiological relevance of these findings, we next studied the effect of chronic maternal melatonin suppression (induced by exposure to constant light during the last third of gestation) on maternal plasma oestradiol during gestation and on plasma cortisol concentration in the 4‐ to 6‐day‐old newborn. Constant light suppressed maternal melatonin without affecting maternal plasma oestradiol concentration, consistent with no effect on fetal DHAS, the precursor of maternal oestradiol. However, newborns from mothers under constant light condition had twice as much plasma cortisol as newborns from mothers maintained under a normal light–dark schedule. Newborns from mothers exposed to chronic constant light and daily melatonin replacement had normal plasma cortisol concentration. Our results support a role of maternal melatonin in fetal and neonatal primate cortisol regulation.

Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light.

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a ‘time giver’ for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.

The circadian timing system: making sense of day/night gene expression.

The circadian time-keeping system ensures predictive adaptation of individuals to the reproducible 24-h day/night alternations of our planet by generating the 24-h (circadian) rhythms found in hormone release and cardiovascular, biophysical and behavioral functions, and others. In mammals, the master clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. The molecular events determining the functional oscillation of the SCN neurons with a period of 24-h involve recurrent expression of several clock proteins that interact in complex transcription/translation feedback loops. In mammals, a glutamatergic monosynaptic pathway originating from the retina regulaltes the clock gene expression pattern in the SCN neurons, synchronizing them to the light:dark cycle. The emerging concept is that neural/humoral output signals from the SCN impinge upon peripheral clocks located in other areas of the brain, heart, lung, gastrointestinal tract, liver, kidney, fibroblasts, and most of the cell phenotypes, resulting in overt circadian rhythms in integrated physiological functions. Here we review the impact of day/night alternation on integrated physiology; the molecular mechanisms and input/output signaling pathways involved in SCN circadian function; the current concept of peripheral clocks; and the potential role of melatonin as a circadian neuroendocrine transducer.

Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats.

•  High‐altitude pregnancy is associated with reduced oxygenation and placental complications, which can affect maternal and fetal outcome. However, most high‐altitude populations are also impoverished and because maternal undernutrition itself is known to promote placental problems, the extent to which complications during high‐altitude pregnancy could be due to maternal oxygen and/or nutrient restriction remains unclear. •  The aim of the study was to investigate whether reduced placental oxygenation, independent of maternal undernutrition, increases maternal and placental oxidative stress and whether maternal treatment with vitamin C is protective. •  The study shows that hypoxic pregnancy increased maternal circulating and placental molecular indices of oxidative stress. •  Maternal vitamin C treatment was protective and increased birth weight. •  The study offers insight to mechanism and intervention against the effects of high altitude on pregnancy.

Clock gene expression in adult primate suprachiasmatic nuclei and adrenal: is the adrenal a peripheral clock responsive to melatonin?

The circadian production of glucocorticoids involves the concerted action of several factors that eventually allow an adequate adaptation to the environment. Circadian rhythms are controlled by the circadian timing system that comprises peripheral oscillators and a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus, driven by the self-regulatory interaction of a set of proteins encoded by genes named clock genes. Here we describe the phase relationship between the SCN and adrenal gland for the expression of selected core clock transcripts (Per-2, Bmal-1) in the adult capuchin monkey, a New World, diurnal nonhuman primate. In the SCN we found a higher expression of Bmal-1 during the h of darkness (2000-0200 h) and Per-2 during daytime h (1400 h). The adrenal gland expressed clock genes in oscillatory fashion, with higher values for Bmal-1 during the day (1400-2000 h), whereas Per-2 was higher at nighttime (about 0200 h), resulting in a 9- to 12-h antiphase pattern. In the adrenal gland, the oscillation of clock genes was accompanied by rhythmic expression of a functional output, the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase. Furthermore, we show that adrenal explants maintained oscillatory expression of Per-2 and Bmal-1 for at least 36 h in culture. The acrophase of both transcripts, but not its overall expression along the incubation, was blunted by 100 nm melatonin. Altogether, these results demonstrate oscillation of clock genes in the SCN and adrenal gland of a diurnal primate and support an oscillation of clock genes in the adrenal gland that may be modulated by the neurohormone melatonin.

Redox modulation of the fetal cardiovascular defence to hypoxaemia

Episodes of hypoxia in utero present a potentially serious challenge to the fetus, but are counteracted by defence responses including marked redistribution of blood flow from peripheral circulations to the brain. Here, we report the novel observation that the oxidant tone is an important modulator of this cardiovascular defence. Using pregnant Welsh Mountain sheep surgically prepared for long‐term recording, we investigated in vivo the effects on the fetal cardiovascular defence to acute hypoxaemia of fetal treatment with the antioxidant vitamin C. The mechanisms via which vitamin C may affect the vascular oxidant tone were investigated by monitoring fetal plasma concentrations of nitrates and nitrites, by determining changes in the activity of superoxide dismutase (SOD) in fetal plasma, and by investigating the effect of vitamin C treatment on the fetal cardiovascular defence to hypoxaemia following nitric oxide (NO) synthase blockade. Fetal treatment with vitamin C markedly depressed the normal femoral constrictor response to acute hypoxaemia in the fetus (5.2 ± 1.0 vs. 1.1 ± 0.3 mmHg (ml min−1)−1, mean ±s.e.m.; P < 0.05) an effect which was completely restored following NO synthase blockade (6.2 ± 1.3 mmHg (ml min−1)−1). Compared to saline infusion, fetal treatment with vitamin C during acute hypoxaemia also significantly increased fetal plasma SOD activity from normoxic baseline (−8.9 ± 6.5 vs. 15.0 ± 6.6% inhibition, P < 0.05) and decreased the plasma concentration ratio of nitrate:nitrite from normoxic baseline (ΔNO3−:NO2−; 0.15 ± 0.30 vs. −0.29 ± 0.11, P < 0.05). The data provide in vivo evidence of redox modulation of redistribution of blood flow in the fetus, part of the fetal brain sparing during acute hypoxaemic stress.

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.

Environmental Control of Biological Rhythms: Effects on Development, Fertility and Metabolism

Internal temporal organisation properly synchronised to the environment is crucial for health maintenance. This organisation is provided at the cellular level by the molecular clock, a macromolecular transcription‐based oscillator formed by the clock and the clock‐controlled genes that is present in both central and peripheral tissues. In mammals, melanopsin in light‐sensitive retinal ganglion cells plays a considerable role in the synchronisation of the circadian timing system to the daily light/dark cycle. Melatonin, a hormone synthesised in the pineal gland exclusively at night and an output of the central clock, has a fundamental role in regulating/timing several physiological functions, including glucose homeostasis, insulin secretion and energy metabolism. As such, metabolism is severely impaired after a reduction in melatonin production. Furthermore, light pollution during the night and shift work schedules can abrogate melatonin synthesis and impair homeostasis. Chronodisruption during pregnancy has deleterious effects on the health of progeny, including metabolic, cardiovascular and cognitive dysfunction. Developmental programming by steroids or steroid‐mimetic compounds also produces internal circadian disorganisation that may be a significant factor in the aetiology of fertility disorders such as polycystic ovary syndrome. Thus, both early and late in life, pernicious alterations of the endogenous temporal order by environmental factors can disrupt the homeostatic function of the circadian timing system, leading to pathophysiology and/or disease.