Impact of maternal diet on vascular function in the offspring

Female Wistar rats were mated and fed diets that altered in protein, folate or methionine content throughout pregnancy.  In both the male and female PR offspring no difference to the control offspring in systolic blood pressure was found by tail-cuff plethysmography.  However, endothelium-dependent relaxation of the small mesenteric arteries as assessed by small wire myography was impaired (p&lt;0.05), with a specific reduction in the NO component of this relaxation response.  There was also remodelling of these arteries with a reduction in smooth muscle content.  In the PR male offspring, the mRNA expression of endothelial nitric oxide synthase (eNOS) as measured by real-time PCR was increased in the mesenteric arteries (p&lt;0.05) but the level of soluble guanylate cyclase was unchanged.  mRNA expression of the antioxidants manganese superoxide dismutase and glutamate cysteine ligase was unchanged with maternal diet but the expression of heme oxygenase-1 (HO-1) was reduced (p&lt;0.05) in the PR males’ livers.  However, the level of oxidative stress as measured by protein carbonyl concentration in the livers was not found to be altered following maternal PR.  Neither maternal folate supplementation nor the partial removal of methionine from the maternal diet prevented the endothelial dysfunction in the PR male offspring. Thus, PR in utero alters the structure and function of the resistance arteries of both the male and female offspring, which results from both an attenuated NO pathway and a reduced antioxidant defence compensated for in the males by up-regulation of eNOS mRNA expression.  This study provides a mechanism for the induction of vascular dysfunction by the pre-natal environment, which may be relevant to the developmental origins of CVD.</p

In utero protein restriction leads to a down-regulation of antioxidant gene expression in adult male offspring

In rats, restriction of dietary protein during pregnancy leads to raised blood pressure, impaired vasodilatation (Brawley et al. 2003) and increased oxidative damage (increased protein carbonyl concentration) (Langley-Evans et al. 2005) in the male offspring. The aim of the present study was to assess mRNA expression of three key endothelial antioxidant genes, heme oxygenase-1 (HO-1), glutamate cysteine ligase (GCL) and manganese superoxide dismutase (Mn-SOD), as well as, endothelial nitric oxide synthase (eNOS), in the liver and mesenteric arteries of adult male offspring of protein-restricted rat dams. Pregnant Wistar rats (240 g) were fed either a control diet (C; 18% casein, n = 6) or a protein-restricted diet (PR; 9% casein, n = 6) throughout gestation from conception. At 120 days of age, male offspring were culled and liver and small mesenteric arteries (~250 µm diameter and ~12 arteries per animal) were removed and snap frozen in liquid nitrogen. RNA was extracted and mRNA levels of HO-1, GCL, MnSOD and eNOS were measured using quantitative real-time PCR and normalised with respect to 28S RNA (Mahn et al. 2005). Data are presented as mean ± SEM, and differences assessed by two-way analysis of variance (ANOVA) with significance accepted at p&lt;0.05. The mRNA expression of all three antioxidant genes was reduced in the livers of the PR compared to C male offspring (HO-1: C; 1807 ± 439, n = 11, PR; 424 ± 109, n = 11, p = 0.006, GCL: C; 120 ± 16, n = 11, PR; 70 ± 11, n = 11, p = 0.016, MnSOD: C; 1544 ± 189, n = 11, PR; 1044 ± 101, n = 12, p = 0.027, figures expressed as gene mRNA relative to 28s RNA). Expression in the mesenteric arteries followed the same trend, being reduced by 14% for HO-1, 36% for MnSOD and 36% for GCL but did not reach significance for any of the three genes. The eNOS mRNA expression was not significantly different between the two groups in either tissue type. These findings demonstrate that the previously observed oxidative damage in the offspring of in utero protein-restricted rats (Langley-Evans et al. 2005) may be in part due to a reduced antioxidant enzyme defence. Diminished antioxidant defences may also account for the previously observed vascular dysfunction in the mesenteric arteries due to a nitric oxide–reactive oxygen species imbalance in the PR offspring

Maternal protein restriction with or without folic acid supplementation during pregnancy alters the hepatic transcriptome in adult male rats

Feeding pregnant rats a protein-restricted (PR) diet induces altered expression of candidate genes in the liver of the adult offspring, which can be prevented by supplementation of the PR diet with folic acid (PRF). We investigated the effect of maternal nutrition during pregnancy on the liver transcriptome in their adult male offspring. Pregnant rats were fed control, PR or PRF diets. Male offspring were killed on day 84. The liver transcriptome was analysed by microarray (six livers per maternal dietary group) followed by post hoc analysis of relative mRNA levels and gene ontology. These results were confirmed for selected genes by real-time RT-PCR. There were 311 genes that differed significantly ( &gt;/= 1.5-fold change; P &lt; 0.05) between PR offspring (222 increased) and control offspring, while 191 genes differed significantly between PRF offspring (forty-five increased) compared with offspring of control dams. There were sixteen genes that were significantly altered in both PR and PRF offspring compared with controls. Ion transport, developmental process, and response to reactive oxygen species (RROS) and steroid hormone response (SHR) ontologies were altered in PR offspring. Folic acid supplementation prevented changes within RROS and SHR response pathways, but not in ion transport or developmental process. There was no effect of maternal PR on mRNA expression of imprinted genes. Insulin 1 and Pleckstrin homology-like domain family A member 2 were increased significantly in PRF compared with PR offspring. The present findings show that the pattern of induced changes in the adult liver transcriptome were dependent on maternal protein and folic acid intakes during pregnancy

Endothelial dysfunction and reduced antioxidant protection in an animal model of the developmental origins of cardiovascular disease

Endothelial dysfunction underlies cardiovascular disease (CVD) in humans and is reported in animal models of developmental origins of such disease. We have investigated whether impaired antioxidant defences and NO generation underlie the genesis of endothelial dysfunction and operate as part of the normal processes of developmental plasticity regulating the induction of phenotype in the offspring. Female Wistar rats were fed either a control (C, 18% protein) or protein-restricted (PR, 9% protein) diet throughout pregnancy. Dams and pups were returned to standard laboratory chow post partum. In male offspring, PR resulted in a reduced endothelial responsiveness to acetylcholine (P &lt; 0.05) in resistance arteries, with vascular remodelling evident from a reduction in smooth muscle content. mRNA expression of endothelial NO synthase (eNOS) was increased (P &lt; 0.05) but there was no change in mRNA levels of manganese superoxide dismutase (MnSOD) or glutamate cysteine ligase (GCL) expression. Interestingly, expression of the antioxidant enzyme haem oxygenase-1 (HO-1) was reduced in the liver (P &lt; 0.05). Female PR offspring also showed a reduced endothelial responsiveness but exhibited no changes in expression of eNOS, iNOS, soluble guanylate cyclase (sGC) or antioxidant genes. Thus, in this model of the developmental origins of CVD, the structure and function of resistance arteries in offspring is altered in complex ways which cannot simply be explained by attenuation in vascular eNOS or in antioxidant protection afforded by GCL or MnSOD. The dysfunction in male offspring may partially be counteracted by an up-regulation of eNOS expression; however, PR does lead to reduced HO-1 expression in these offspring, which may affect both their growth and vascular function. Our findings have established that PR induces significant phenotypic changes in male offspring that may be indicative of an adaptive response during development<br/

Evaluation of methylation status of the eNOS promoter at birth in relation to childhood bone mineral content

Our previous work has shown associations between childhood adiposity and perinatal methylation status of several genes in umbilical cord tissue, including endothelial nitric oxide synthase (eNOS). There is increasing evidence that eNOS is important in bone metabolism; we therefore related the methylation status of the eNOS gene promoter in stored umbilical cord to childhood bone size and density in a group of 9-year-old children. We used Sequenom MassARRAY to assess the methylation status of two CpGs in the eNOS promoter, identified from our previous study, in stored umbilical cords of 66 children who formed part of a Southampton birth cohort and who had measurements of bone size and density at age 9 years (Lunar DPXL DXA instrument). Percentage methylation varied greatly between subjects. For one of the two CpGs, eNOS chr7:150315553 + , after taking account of age and sex, there were strong positive associations between methylation status and the child’s whole-body bone area (r = 0.28, P = 0.02), bone mineral content (r = 0.34, P = 0.005), and areal bone mineral density (r = 0.34, P = 0.005) at age 9 years. These associations were independent of previously documented maternal determinants of offspring bone mass. Our findings suggest an association between methylation status at birth of a specific CpG within the eNOS promoter and bone mineral content in childhood. This supports a role for eNOS in bone growth and metabolism and implies that its contribution may at least in part occur during early skeletal development

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study

Background: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms—many related to the structure or function of red blood cells—and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. Methods: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. Findings: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11–0·20; p=2·61 × 10−58), blood group O (0·74, 0·66–0·82; p=6·26 × 10−8), and –α3·7-thalassaemia (0·83, 0·76–0·90; p=2·06 × 10−6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63–0·92; p=0·001) and FREM3 (0·64, 0·53–0·79; p=3·18 × 10−14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49–0·68; p=3·22 × 10−11), as was homozygosity (0·26, 0·11–0·62; p=0·002). Interpretation: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. Funding: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative