Programming of cardiovascular disease across the life-course.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality, affecting both developed and developing countries. Whilst it is well recognized that our risk of CVD can be determined by the interaction between our genetics and lifestyle, this only partly explains the variability at the population level. Based on these well-known risk factors, for many years, intervention and primary prevention strategies have focused on modifying lifestyle factors in adulthood. However, research shows that our risk of CVD can be pre-determined by our early life environment and this area of research is known as the Developmental Origins of Health and Disease. The aim of this review is to evaluate our current understanding of mechanisms underlying the programming of CVD. This article is part of a special issue entitled CV Aging.H.L. Blackmore is funded by the British Heart Foundation. S.E. Ozanne is a member of the MRC Metabolic Diseases Unit and funded by MRC grantMC_UU_12012/4.This is the accepted manuscript of a paper published in the Journal of Molecular and Cellular Cardiology (Blackmore HL, Ozanne SE, Journal of Molecular and Cellular Cardiology 2014, doi:10.1016/j.yjmcc.2014.12.006)

Maternal diet-induced obesity programs cardiovascular dysfunction in adult male mouse offspring independent of current body weight.

This is the final published version. It first appeared at http://press.endocrine.org/doi/abs/10.1210/en.2014-1383?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed.Obese pregnancies are not only associated with adverse consequences for the mother but also the long-term health of her child. Human studies have shown that individuals from obese mothers are at increased risk of premature death from cardiovascular disease (CVD), but are unable to define causality. This study aimed to determine causality using a mouse model of maternal diet-induced obesity. Obesity was induced in female C57BL/6 mice by feeding a diet rich in simple sugars and saturated fat 6 weeks prior to pregnancy and throughout pregnancy and lactation. Control females were fed laboratory chow. Male offspring from both groups were weaned onto chow and studied at 3, 5, 8, and 12 weeks of age for gross cardiac morphometry using stereology, cardiomyocyte cell area by histology, and cardiac fetal gene expression using qRT-PCR. Cardiac function was assessed by isolated Langendorff technology at 12 weeks of age and hearts were analyzed at the protein level for the expression of the β1 adrenergic receptor, muscarinic type-2 acetylcholine receptor, and proteins involved in cardiac contraction. Offspring from obese mothers develop pathologic cardiac hypertrophy associated with re-expression of cardiac fetal genes. By young adulthood these offspring developed severe systolic and diastolic dysfunction and cardiac sympathetic dominance. Importantly, cardiac dysfunction occurred in the absence of any change in corresponding body weight and despite the offspring eating a healthy low-fat diet. These findings provide a causal link to explain human observations relating maternal obesity with premature death from CVD in her offspring.HLB, YN and JLTA are funded by the British Heart Foundation. DFT is supported by the MRC Metabolic Diseases Unit. DAG is a Lister Institute Fellow and Royal Society Wolfson Merit Award Holder and is supported by the British Heart Foundation. SEO is a British Heart Foundation Senior Fellow and a member of the MRC Metabolic Diseases Unit

Maternal Metformin Intervention during Obese Glucose-Intolerant Pregnancy Affects Adiposity in Young Adult Mouse Offspring in a Sex-Specific Manner.

BackgroundMetformin is commonly used to treat gestational diabetes mellitus. This study investigated the effect of maternal metformin intervention during obese glucose-intolerant pregnancy on the gonadal white adipose tissue (WAT) of 8-week-old male and female mouse offspring.MethodsC57BL/6J female mice were provided with a control (Con) or obesogenic diet (Ob) to induce pre-conception obesity. Half the obese dams were treated orally with 300 mg/kg/d of metformin (Ob-Met) during pregnancy. Gonadal WAT depots from 8-week-old offspring were investigated for adipocyte size, macrophage infiltration and mRNA expression of pro-inflammatory genes using RT-PCR.ResultsGestational metformin attenuated the adiposity in obese dams and increased the gestation length without correcting the offspring in utero growth restriction and catch-up growth caused by maternal obesity. Despite similar body weight, the Ob and Ob-Met offspring of both sexes showed adipocyte hypertrophy in young adulthood. Male Ob-Met offspring had increased WAT depot weight (p p p F4/80 (p ConclusionsMaternal metformin intervention during obese pregnancy causes excessive adiposity, adipocyte hyperplasia and WAT inflammation in male offspring, highlighting sex-specific effects of prenatal metformin exposure on offspring WAT

Poor maternal nutrition programmes a pro-atherosclerotic phenotype in ApoE-/- mice.

Numerous animal studies have consistently shown that early life exposure to LP (low-protein) diet programmes risk factors for CVD (cardiovascular disease) such as dyslipidaemia, high BP (blood pressure) and cardiac dysfunction in the offspring. However, studies on the effect of maternal under-nutrition on offspring development of atherosclerosis are scarce. Applying our LP model to the ApoE(-/-) atherosclerosis-prone mouse model, we investigated the development of atherosclerotic lesions in the aortic root of 6-month-old offspring. In addition, markers of plaque progression including SMA (smooth muscle actin) and Mac3 (macrophage marker 3) were studied. Pregnant dams were fed on a control (20% protein) or on an isocaloric LP diet (8% protein) throughout pregnancy and lactation. After weaning, male offspring were maintained on 20% normal laboratory chow. At 6 months of age, LP offspring showed a significantly greater plaque area (P<0.05) with increased cholesterol clefts and significantly higher indices of DNA damage compared with controls (P<0.05). The expression of HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) (P<0.05) and LDL (low-density lipoprotein) receptor in the liver of LP offspring were increased. Furthermore, LP offspring had higher LDL-cholesterol levels (P<0.05) and a trend towards elevated insulin. There were no differences in other lipid measurements and fasting glucose between groups. These observations suggest that early exposure to an LP diet accelerates the development and increases the progression of atherosclerotic lesions in young adult offspring. Future studies are needed to elucidate the specific mechanisms linking in utero exposure to a diet low in protein to the development of atherosclerosis.This work was supported by the British Heart Foundation [grant numbers FS/09/029/27902, FS/09/050], the Biotechnology and Biological Sciences Research Council and the Cambridge Commonwealth Trust

Maternal Exercise Intervention in Obese Pregnancy Improves the Cardiovascular Health of the Adult Male Offspring

OBJECTIVE Obesity during pregnancy is associated with an elevated risk of cardiovascular disease in the offspring. With increased numbers of women entering pregnancy overweight or obese, there is a requirement for targeted interventions to reduce disease risk in future generations. Using an established murine model of maternal obesity during pregnancy, we investigated if a treadmill exercise intervention in the mother could improve offspring cardiac health and explored potential underlying mechanisms. METHODS A 20-minute treadmill exercise intervention protocol was performed 5 days a week in diet-induced obese female C57BL/6 mice 1 week prior to, and up to E17 of pregnancy. All male offspring were weaned onto a control diet and studied at 8 weeks of age when their cardiovascular physiology was assessed by in vivo echocardiography and non-invasive tail cuff plethysmography. Cardiomyocyte cell area, re-expression of fetal genes and the expression of calcium handling and sympathetic activation proteins were determined. RESULTS At 8 weeks, there was no difference in bodyweight or fat mass between groups. Offspring of obese dams developed pathologic cardiac hypertrophy, hypertension and cardiac dysfunction characterized by reduced ejection fraction (p< 0.001). Maternal exercise prevented cardiac hypertrophy and dysfunction but failed to prevent hypertension. These offspring of exercised dams also had enhanced (p< 0.001) levels of calcium handling proteins and a sympathetic-activated inotropic response. CONCLUSIONS Exercise in obese pregnancy was beneficial to offspring cardiac function and structure but did not influence hypertension suggesting they are programmed by separate mechanistic pathways. These data suggest combination interventions in obese pregnancies will be required to improve all aspects of the cardiovascular health of the next generation.This work received funding from the British Heart Foundation, the European Union’s Seventh Framework Programme , MRC Metabolic Diseases Unit award and British Heart Foundation Studentship. Wellcome Trust

Maternal diet-induced obesity during pregnancy alters lipid supply to mouse E18.5 fetuses and changes the cardiac tissue lipidome in a sex- dependent manner

Maternal obesity during pregnancy has immediate and long-term detrimental effects on the offspring heart. In this study, we characterized the cardiac and circulatory lipid profiles in late gestation E18.5 fetuses of diet-induced obese pregnant mice and established the changes in lipid abundance and fetal cardiac transcriptomics. We used untargeted and targeted lipidomics and transcriptomics to define changes in the serum and cardiac lipid composition and fatty acid metabolism in male and female fetuses. From these analyses we observed: (1) maternal obesity affects the maternal and fetal serum lipidome distinctly; (2) female fetal heart lipidomes are more sensitive to maternal obesity than males; (3) changes in lipid supply might contribute to early expression of lipolytic genes in mouse hearts exposed to maternal obesity. These results highlight the existence of sexually dimorphic responses of the fetal heart to the same in utero obesogenic environment and identify lipids species that might mediate programming of cardiovascular health.MRC Metabolic Diseases Unit [MRC_MC_UU_00014/4] Cambridge Home and EU Student Scholarship British Heart Foundation studentship [FS/12/64/30001] II was supported by a British Heart Foundation studentship [FS/18/56/35177

Maternal Allopurinol Prevents Cardiac Dysfunction in Adult Male Offspring Programmed by Chronic Hypoxia During Pregnancy.

Integrating functional and molecular levels, we investigated the effects of maternal treatment with a xanthine oxidase inhibitor on the programming of cardiac dysfunction in adult offspring using an established rat model of hypoxic pregnancy. Female Wistar rats were divided into normoxic or hypoxic (13% O2) pregnancy±maternal allopurinol treatment (30 mg kg-1 d-1). At 4 months, hearts were isolated from 1 male per litter per outcome variable to determine cardiac function and responses to ischemia-reperfusion in a Langendorff preparation. Sympathetic dominance, perfusate CK (creatine kinase) and LDH (lactate dehydrogenase) and the cardiac protein expression of the β1-adrenergic receptor, the M2 Ach receptor (muscarinic type-2 acetylcholine receptor), and the SERCA2a (sarcoplasmic reticulum Ca2+ ATPase 2a) were determined. Relative to controls, offspring from hypoxic pregnancy showed elevated left ventricular end diastolic pressure (+34.7%), enhanced contractility (dP/dtmax, +41.6%), reduced coronary flow rate (-21%) and an impaired recovery to ischemia-reperfusion (left ventricular diastolic pressure, area under the curve recovery -19.1%; all P<0.05). Increased sympathetic reactivity (heart rate, +755.5%; left ventricular diastolic pressure, +418.9%) contributed to the enhanced myocardial contractility ( P<0.05). Perfusate CK (+431%) and LDH (+251.3%) and the cardiac expression of SERCA2a (+71.4%) were also elevated ( P<0.05), further linking molecular markers of cardiac stress and injury to dysfunction. Maternal allopurinol restored all functional and molecular indices of cardiac pathology. The data support a link between xanthine oxidase-derived oxidative stress in hypoxic pregnancy and cardiac dysfunction in the adult offspring, providing a target for early intervention in the developmental programming of heart disease.British Heart Foundatio

Maternal but not fetoplacental health can be improved by metformin in a murine diet-induced model of maternal obesity and glucose intolerance.

Maternal obesity is a global problem that increases the risk of short- and long-term adverse outcomes for mother and child, many of which are linked to gestational diabetes mellitus. Effective treatments are essential to prevent the transmission of poor metabolic health from mother to child. Metformin is an effective glucose lowering drug commonly used to treat gestational diabetes mellitus; however, its wider effects on maternal and fetal health are poorly explored. In this study we used a mouse (C57Bl6/J) model of diet-induced (high sugar/high fat) maternal obesity to explore the impact of metformin on maternal and feto-placental health. Metformin (300 mg kg-1  day-1 ) was given to obese females via the diet and was shown to achieve clinically relevant concentrations in maternal serum (1669 ± 568 nM in late pregnancy). Obese dams developed glucose intolerance during pregnancy and had reduced uterine artery compliance. Metformin treatment of obese dams improved maternal glucose tolerance, reduced maternal fat mass and restored uterine artery function. Placental efficiency was reduced in obese dams, with increased calcification and reduced labyrinthine area. Consequently, fetuses from obese dams weighed less (P < 0.001) at the end of gestation. Despite normalisation of maternal parameters, metformin did not correct placental structure or fetal growth restriction. Metformin levels were substantial in the placenta and fetal circulation (109.7 ± 125.4 nmol g-1 in the placenta and 2063 ± 2327 nM in fetal plasma). These findings reveal the distinct effects of metformin administration during pregnancy on mother and fetus and highlight the complex balance of risk vs. benefits that are weighed in obstetric medical treatments. KEY POINTS: Maternal obesity and gestational diabetes mellitus have detrimental short- and long-term effects for mother and child. Metformin is commonly used to treat gestational diabetes mellitus in many populations worldwide but the effects on fetus and placenta are unknown. In a mouse model of diet-induced obesity and glucose intolerance in pregnancy we show reduced uterine artery compliance, placental structural changes and reduced fetal growth. Metformin treatment improved maternal metabolic health and uterine artery compliance but did not rescue obesity-induced changes in the fetus or placenta. Metformin crossed the placenta into the fetal circulation and entered fetal tissue. Metformin has beneficial effects on maternal health beyond glycaemic control. However, despite improvements in maternal physiology, metformin did not prevent fetal growth restriction or placental ageing. The high uptake of metformin into the placental and fetal circulation highlights the potential for direct immediate effects of metformin on the fetus with possible long-term consequences postnatally

Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth restriction in mice

Abstract: Background: In utero exposure to obesity is consistently associated with increased risk of metabolic disease, obesity and cardiovascular dysfunction in later life despite the divergence of birth weight outcomes. The placenta plays a critical role in offspring development and long-term health, as it mediates the crosstalk between the maternal and fetal environments. However, its phenotypic and molecular modifications in the context of maternal obesity associated with fetal growth restriction (FGR) remain poorly understood. Methods: Using a mouse model of maternal diet-induced obesity, we investigated changes in the placental transcriptome through RNA sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA) at embryonic day (E) 19. The most differentially expressed genes (FDR < 0.05) were validated by Quantitative real-time PCR (qPCR) in male and female placentae at E19. The expression of these targets and related genes was also determined by qPCR at E13 to examine whether the observed alterations had an earlier onset at mid-gestation. Structural analyses were performed using immunofluorescent staining against Ki67 and CD31 to investigate phenotypic outcomes at both timepoints. Results: RNA-seq and IPA analyses revealed differential expression of transcripts and pathway interactions related to placental vascular development and tissue morphology in obese placentae at term, including downregulation of Muc15, Cnn1, and Acta2. Pdgfb, which is implicated in labyrinthine layer development, was downregulated in obese placentae at E13. This was consistent with the morphological evidence of reduced labyrinth zone (LZ) size, as well as lower fetal weight at both timepoints irrespective of offspring sex. Conclusions: Maternal obesity results in abnormal placental LZ development and impaired vascularization, which may mediate the observed FGR through reduced transfer of nutrients across the placenta

Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth restriction in mice

Abstract: Background: In utero exposure to obesity is consistently associated with increased risk of metabolic disease, obesity and cardiovascular dysfunction in later life despite the divergence of birth weight outcomes. The placenta plays a critical role in offspring development and long-term health, as it mediates the crosstalk between the maternal and fetal environments. However, its phenotypic and molecular modifications in the context of maternal obesity associated with fetal growth restriction (FGR) remain poorly understood. Methods: Using a mouse model of maternal diet-induced obesity, we investigated changes in the placental transcriptome through RNA sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA) at embryonic day (E) 19. The most differentially expressed genes (FDR < 0.05) were validated by Quantitative real-time PCR (qPCR) in male and female placentae at E19. The expression of these targets and related genes was also determined by qPCR at E13 to examine whether the observed alterations had an earlier onset at mid-gestation. Structural analyses were performed using immunofluorescent staining against Ki67 and CD31 to investigate phenotypic outcomes at both timepoints. Results: RNA-seq and IPA analyses revealed differential expression of transcripts and pathway interactions related to placental vascular development and tissue morphology in obese placentae at term, including downregulation of Muc15, Cnn1, and Acta2. Pdgfb, which is implicated in labyrinthine layer development, was downregulated in obese placentae at E13. This was consistent with the morphological evidence of reduced labyrinth zone (LZ) size, as well as lower fetal weight at both timepoints irrespective of offspring sex. Conclusions: Maternal obesity results in abnormal placental LZ development and impaired vascularization, which may mediate the observed FGR through reduced transfer of nutrients across the placenta