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CARDIOVASCULAR |
1 Institute of Developmental Sciences, University of Southampton, Tremona Road, Southampton SO16 6YD, UK
2
Cardiovascular Division, School of Medicine, King's College London, 150 Stamford Street, London SE1 9NH, UK
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Abstract |
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(Received 19 May 2008;
accepted after revision 30 July 2008;
first published online 31 July 2008)
Corresponding author G. F. Clough: Institute of Developmental Sciences, University of Southampton, Tremona Road, Southampton SO16 6YD, UK. Email: g.f.clough{at}soton.ac.uk
Epidemiological studies demonstrate an association between an unbalanced diet during pregnancy and long-term effects on health of the offspring, including an increased risk of cardiovascular disease (Roseboom et al. 2000; Painter et al. 2007). These data, together with those from animal studies, have given rise to the concept that the development of many organs and tissues, including those of the cardiovascular system, is affected by aspects of the prenatal environment which are transmitted to the offspring by the mother. The changes induced become permanent after the period of developmental plasticity is complete and confer protective advantages if the postnatal environment matches the prenatal one (Gluckman et al. 2005). In the face of unmatched environments, the irreversible structural and physiological changes can give rise to altered cardiovascular control and peripheral vascular dysfunction (Bertram et al. 2001; Ozaki et al. 2001; Franco et al. 2002a; Langley-Evans & Sculley, 2005). The mechanisms underlying the association between fetal development and altered postnatal vascular function and cardiovascular disease remain unclear (Armitage et al. 2005). However, there is increasing evidence that they are at least in part a result of nutrient–gene–environment interactions involving epigenetic changes which determine the development of the adult phenotype (Lillycrop et al. 2005).
Endothelial dysfunction, i.e. an altered vascular integrity, reduced vasodilator capacity and an enhanced proinflammatory state, precedes vascular patho-morphology in the early stages of atherosclerosis and hypertension. Accumulating evidence indicates that the generation of reactive oxygen species (ROS) is key to the development of much cardiovascular disease (Cai & Harrison, 2000). ROS (including superoxide and hydrogen peroxide) are produced by endothelial cells and the adjacent smooth muscle cells, adventitial fibroblasts and inflammatory cells. While low levels of ROS contribute to normal vascular function, at higher concentrations these radicals mediate cell injury and suppress endothelium-dependent dilatation by reducing bioavailability of NO through a rapid reaction between superoxide anions and NO to form peroxynitrite (Taddei et al. 2001; Pacher et al. 2007). ROS can also affect the NO pathway, particularly the activity of eNOS, by oxidation of the eNOS cofactor tetrahydrobiopterin (BH4) to uncouple eNOS and increase production of superoxide anions instead of NO (Landmesser et al. 2003). Susceptibility to alteration in vascular function via such mechanisms appears to be related to sex (Franco et al. 2002b).
Antioxidant enzymes key to the stability and function of the vasculature include haem oxygenase-1 (HO-1), which catalyses the breakdown of haem to the potent antioxidants biliverdin/bilverdin, and carbon monoxide and ferrous iron, which is induced under conditions of oxidative stress and in response to injury (Kirkby & Adin, 2006). HO-1 is also induced by NO and thereby provides protection against oxidative stress (Buckley et al. 2003; Mann et al. 2007). Superoxide dismutase (SOD), both intracellular manganese SOD and extracellular SOD, catalyses the dismutation of superoxide anions to hydrogen peroxide and enzyme activity is impaired in atherosclerotic coronary arteries (Landmesser et al. 2003). Glutamate cysteine ligase (GCL) is a key rate-limiting enzyme for glutathione synthesis and is transcriptionally regulated by factors such as H2O2, oxidized low density lipoproteins, NO, prostaglandins and TGFβ (Soltaninassab et al. 2000).
The present study examines the role that antioxidants and the NO pathway play in the genesis of endothelial dysfunction in a rodent model of the developmental origins of cardiovascular disease. Our particular focus was on the question of whether the operation of these processes could be viewed as evidence for disruption of normal development (Gluckman et al. 2005), as would be expected by toxic effects, or whether they operate as part of the normal processes of developmental plasticity which regulate the induction of phenotype in the offspring. A recent study using severe in utero nutrient restriction in the rat (50% reduction of total maternal food intake) reported that endothelial dysfunction is associated with increased superoxide production in mesenteric arteries (Franco et al. 2007). The degree of this challenge might be expected to produce disruptive effects on development. In this study, we therefore employed a less severe but more specific nutrient restriction in utero by reducing only the protein content of the maternal diet by 50%, previously shown to induce raised systolic blood pressure (Gardner et al. 1997; Jackson et al. 2002) and endothelial dysfunction (Brawley et al. 2003; Torrens et al. 2006).
The aim of the current study was to determine whether these effects are due to alterations in the expression of genes associated with the NO pathway and antioxidant protection. As sex has been shown to influence the relationship between an adverse in utero environment and later disease outcome (Franco et al. 2007), we also examined whether the impact of oxidative stress is affected by sex.
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Methods |
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Animal and dietary restriction protocol
Virgin female Wistar rats (190–220 g, Harlan, UK) were mated (the day of vaginal plug detection being defined as day 0 of pregnancy) and pregnant dams randomly assigned to either a control (18% casein, n = 6) or a protein restricted diet (PR, 9% casein, n = 6) as previously described (Torrens et al. 2008). Dams were fed on the experimental diets from confirmation of pregnancy until delivery (
22 days) and immediately postpartum dams and pups were returned to standard lab chow and continued receiving standard chow for the remainder of the experiment. To avoid rejection, pups were weighed at 48 h post partum and litters standardized to eight (4
, 4
) by cervical dislocation. Offspring were weaned at day (d) 21 and weighed weekly. At d 120 ± 7 offspring were killed by CO2 inhalation and cervical dislocation, plasma taken, and heart, lungs, liver, kidneys and adrenal glands were removed and weighed. Small mesenteric arteries and livers were dissected out and cleaned for further investigations. In addition, a subgroup of female C and PR offspring were culled at d 145 and the mesenteric arteries dissected out for assessment of vascular function.
Blood pressure measurement
At d 28 ± 1, 56 ± 1 and 112 ± 1, systolic blood pressure was assessed in the same two male and two female offspring initially randomly from selected each litter. Blood pressure was measured by tail-cuff plethysmography using an IITC blood pressure monitor (model 229, IITC, Woodhill, CA, USA) as previously described. Measurements were repeated five times, the highest and lowest of the readings discounted and a mean taken (Lewis et al. 2001).
Assessment of vascular function in resistance arteries
Third order mesenteric arteries (internal diameter 250 µm) were cleaned of connective tissue and 2 mm segments mounted on a wire myograph (Danish Myo Technology A/S, Aarhus, Denmark). Segments were bathed in physiological salt solution (PSS) of the following composition: NaCl, 119; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.17; NaHCO3, 25; KH2PO4, 1.18; EDTA, 0.026; and D-glucose, 5.5 mM; heated to 37°C and continuously gassed with 95% O2 and 5% CO2. Once mounted, the passive tension-internal circumference relationship (IC100) was determined by incremental increases in tension to achieve an internal circumference equivalent to a transmural pressure of 100 mmHg (using the Laplace relationship) and the arteries were set to a diameter equivalent to 0.9 x IC100. Functional integrity of the smooth muscle was assessed with four 2 min washes with 125 mM KPSS (PSS with an eqimolar substitution of NaCl with KCl) solution. Vessels failing to produce an active tension equivalent to 13.3 kPa were discarded from the study as previously described (Torrens et al. 2003). Following normalization, cumulative concentration–response curves to phenylephrine (PE, 1 nM to 100 µM) and the endothelium-dependent vasodilator acetylcholine (ACh, 1 nM to 10 µM) were constructed. Additionally, to assess the role of free radicals, responses to ACh were repeated following incubation (20 min) with the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (tempol; 1 mM). All drugs were obtained from Sigma (Poole, UK) unless otherwise stated.
Mesenteric artery immunohistochemistry
Second order mesenteric arteries were dissected, formalin-fixed and embedded in paraffin. Four 5 µm transverse sections were cut and stained using either Miller's method for elastin and Van Gieson's stain for assessment of wall structure, or epitope specific rabbit antibodies to eNOS (Laboratory Vision Corporation/Thermo Fisher Scientific Inc., Fremont, CA, USA, cat. no. RB-9279-R7, diluted 1 : 400) using the streptavidin–biotin–peroxidase technique. The sections were examined under a light microscope at x40 magnification and the areas of elastin, vascular smooth muscle and collagen were measured and the mean vessel diameter, luminal diameter and media + intima layer to lumen ratio calculated using Image Analysis software (AxioVision, Carl Zeiss).
Determination of plasma homocysteine
As homocysteine has been implicated in both the development of endothelial dysfunction and the generation of ROS, plasma levels of homocysteine were measured in the offspring. Blood was collected and stored on ice in heparinized tubes. Samples were centrifuged and plasma collected and snap frozen in liquid nitrogen. Plasma total homocysteine concentration was measured by high pressure liquid chromatography with fluorescence detection as previously described (Araki & Sako, 1987), and modified by the use of cysteamine as an internal standard.
Determination of protein carbonyl concentration
In order to assess the overall level of oxidative stress in the livers of the offspring, protein carbonyl concentration was measured. Approximately 250 mg of frozen liver was homogenized in water and a protein assay (Coomassie Plus, prod. no. 1856210, Pierce Biotechnology/Thermo Fisher Scientific Inc., Rockford, IL, USA) and protein carbonyl enzyme immunoassay (ALX-850-312-K101, Alexis Biochemicals AXXORA, LLC, San Diego, CA, USA) were carried out to determine protein carbonyl concentration per milligram of protein.
Analysis of gene expression
Total RNA was extracted from mesenteric arteries and livers of the offspring using TRIzol (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed into cDNA. The mRNA levels of eNOS, iNOS, sGC, MnSOD, GCL and HO-1 were analysed relative to a preselected housekeeping gene using real-time PCR (LightCycler Real-Time PCR System, Roche Applied Sciences and ABI PRISM 7700 Sequence Detector, Applied Biosystems, Foster City, CA, USA). Specific primer sequences are given in Table 1.
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Data are expressed as means ± S.E.M. Data from males and females were analysed separately, other than where stated for the analysis of the histological results. No more than two male or two female offspring from each litter were randomly selected and used for each investigation. Systolic blood pressure, gene expression and maternal data were analysed by a one-way ANOVA with Bonferroni's post hoc correction or Student's t test where appropriate. Weight gain was analysed by two-way repeated measures ANOVA, using time and dietary group as the two factors. Constrictor responses were calculated as percentage of maximum contraction induced by 125 mM KPSS and relaxant responses as percentage inhibition of PE-induced contraction. Cumulative CRCs to agonists were analysed by fitting to a four-parameter logistic equation using nonlinear regression to obtain the pEC50 and maximum response. Differences were assessed by a one-way ANOVA with Bonferroni's post hoc correction. Significance was accepted if P < 0.05. At all points the investigator was blinded to the dietary group.
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Results |
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All dams gained weight from conception to day 20 of pregnancy. Both weight gain (g; control (C) 95 ± 9, n = 6; protein restricted (PR), 90 ± 4, n = 6; not significant (n.s.)) and food intake (g day–1; C, 15.9 ± 1, n = 6; PR, 16.9 ± 0.2, n = 6; n.s.) were similar between the groups.
Offspring body and organ weights
At birth, litter size (C, 12 ± 1, n = 6; PR, 10 ± 1, n = 6; n.s.) was similar between the groups, as was pup weight at 48 h post partum (g; C, 7.1 ± 0.1, n = 6; PR, 7.2 ± 0.1, n = 6; n.s.). Postnatally, both the male and female PR offspring showed a significantly lower growth rate compared with the C offspring (P < 0.0001, two-way ANOVA, Fig. 1). No differences in the weights of the heart, lungs, liver, kidneys, adrenal glands or spleen were found at 120 days between any of the groups, whether weights were expressed as an absolute weight in grams or as a percentage of body weight.
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At all time points, systolic blood pressure was similar between the groups in both male and female offspring (Fig. 2).
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Vasoconstriction.
Response to 125 mM KPSS did not differ between the groups in either males (kPa: C, 18.7 ± 1.0, n = 10; PR, 16.7 ± 0.7, n = 8; n.s.) or females (kPa: C, 23.6 ± 1.2, n = 3; PR, 19.9 ± 1.51, n = 5; n.s.). In all arteries, the 
1-adrenoceptor agonist PE produced a concentration-dependent constriction which was similar between the groups in both the male (pEC50: C, 6.36 ± 0.03, n = 10; PR, 6.23 ± 0.03, n = 8; n.s.; % max response: C, 105.6 ± 1.8, n = 10; PR, 106.0 ± 1.9, n = 8; n.s.) and female offspring (pEC50: C, 5.98 ± 0.06, n = 3; PR, 5.90 ± 0.07, n = 5; n.s.;% max response: C, 93.1 ± 2.6, n = 3; PR, 99.2 ± 3.1, n = 5; n.s.).
Endothelium-dependent vasodilatation. The endothelium-dependent vasodilator ACh produced a concentration-dependent relaxation of PE-induced tone in all arteries. In both male (pEC50: C, 8.02 ± 0.06, n = 10; PR, 7.55 ± 0.14, n = 8; P < 0.01; Fig. 3A) and female offspring (pEC50: C, 7.61 ± 0.15, n = 3; PR, 6.91 ± 0.18, n = 5; P < 0.05; Fig. 3B) sensitivity to ACh was significantly attenuated in the PR group compared to controls with no change in the maximal response. In male offspring preincubation with the superoxide dismutase mimetic tempol (1 mM) significantly attenuated ACh responses in the controls (P < 0.05; Fig. 4A), but had no effect on the response to ACh in the PR group (Fig. 4B).
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Lumen diameter was greater in the mesenteric arteries of the PR group compared with control (µm: C, 48 ± 2; PR, 70 ± 4, n.s.). Wall thickness (µm: C, 62 ± 6; PR 60 ± 3), intima + media area (µm2: C, 10721 ± 2236; PR, 10721 ± 1244) and the ratio of intima + media to lumen diameter (C, 4.38 ± 2.09; PR, 2.12 ± 0.43) were not significantly different.
The largest component of the vessel wall was smooth muscle in all groups. This accounted for a mean area of 36 ± 2%, with collagen and elastin comprising 20 ± 1% and 17 ± 1% of the vessel wall area, respectively. When normalized to total vessel area or media + intima area, the percentage area of smooth muscle was significantly smaller in the PR compared to C mesenteric arteries (Fig. 5). Elastin and collagen were similar between the groups.
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Analysis of housekeeping genes. In order to select an appropriate housekeeping gene for the analysis of the mRNA expression by RT-PCR, the expression of three potential housekeepers (ribosomal 18S RNA, ribosomal 28S RNA and β-actin) were measured, Figs 6A and B. The RNA levels of 28S were significantly increased in the livers of the male PR offspring compared to the C (P < 0.05), with a similar trend in the female offspring (P = 0.08). However, the RNA levels of 18S were altered in the opposite direction to 28S RNA, with a significant decrease in both the male and female PR offspring (P < 0.05). As mRNA levels of β-actin were not found to be different between the PR and C groups for both male and female offspring, β-actin was chosen as a housekeeping gene.
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Levels of the downstream target of NO, sGC were similar between the groups in mesenteric arteries from both male (fold change: C, 1.00 ± 0.09; PR, 1.02 ± 0.06; n.s.) and female offspring (fold change: C, 1.00 ± 0.12; PR, 0.91 ± 0.07; n.s.). iNOS mRNA was not detectable in mesenteric arteries or livers from offspring of either group.
Antioxidant gene expression
In both males and females, mesenteric artery levels of mRNA for HO-1, MnSOD and the catalytic subunit of GCL were similar between the groups (Fig. 6C and D). Hepatic levels of HO-1 were significantly decreased in PR male, but not female offspring (P < 0.05). Hepatic levels of MnSOD and GCL were similar between the groups in both males and females (Fig. 6E and F). In addition HO-1 mRNA expression correlated with the postmortem weights of male (R2 = 0.27; Fig. 7), but not female offspring.
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In both males (nmol (mg protein)–1: C, 0.26 ± 0.02; PR, 0.22 ± 0.02; n.s.) and females (nmol (mg protein)–1: C, 0.19 ± 0.01; PR, 0.18 ± 0.01; n.s.) levels of protein carbonyls were similar between the groups. Similarly plasma levels of homocysteine in male offspring were not different between the groups (µmol l–1: C, 10.55 ± 0.63; PR, 11.02 ± 0.51; n.s.).
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Discussion |
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The lack of difference in pregnancy weight gain or food intake between the C and PR dams confirms that the PR diet, although exerting effects on the offspring, does not appear to affect the health of the mother during gestation or impact upon the viability of her pregnancy. Thus, it can be considered a moderate nutritional challenge. The growth rate of both the male and female offspring was reduced in the PR group, a finding which is supported by previous studies of growth of both male (Brawley et al. 2003; Langley-Evans & Sculley, 2006) and female (Torrens et al. 2003) offspring and suggests a long-term effect of in utero protein-restriction on the offspring.
We found no evidence for hypertension in our animals. The lack of effect of maternal PR on male or female offspring systolic blood pressure contrasts with previous studies (Langley & Jackson, 1994; Gardner et al. 1997; Nwagwu et al. 2000; Bertram et al. 2001; Jackson et al. 2002; Brawley et al. 2003; McMullen & Langley-Evans, 2005), but it is similar to reports which have recorded blood pressure by indwelling catheter (Martin et al. 2004). The optimal method for measuring blood pressure in conscious unrestrained animals is by radiotelemetry. With this method Tonkiss et al. (1998) demonstrated that blood pressure in PR offspring was only increased following exposure to stress. As the tail cuff procedure is known to induce a rise in blood pressure due to the stress of combination of restraint and heating (Kubota et al. 2006), this may have masked any difference between the groups. The lack of hypertension does not detract from our study for while blood pressure is clinically useful, it is not necessarily the most important physiological variable – measures of blood flow and nutrient delivery to the tissues may be of greater significance. We might also argue that none of the current models (except the spontaneously hypertensive rat) produce ‘hypertension’ in the clinical sense: rather they are proof of principle that an effect on CV function can be induced by the developmental environment. Furthermore, in epidemiological studies, there remains controversy about the effects of birthweight on later blood pressure (Huxley et al. 2002), which only become clearer when the outcome is clinical disease (Curhan et al. 1996).
Both male and female PR offspring exhibited a reduced sensitivity to ACh when compared to the C offspring, confirming the presence of endothelial dysfunction in the arteries of the male offspring, as shown in previous studies (Brawley et al. 2003; Torrens et al. 2006). This effect has been reported previously for arteries isolated from pregnant offspring from PR rats (Torrens et al. 2003), but our study is the first to identify the same dysfunction in non-pregnant female offspring from PR rats. As the endothelial function of resistance arteries has an influence on various different features of cardiovascular disease, such as the development of atherosclerosis and insulin sensitivity, this impairment has important implications for the cardiovascular health of the PR offspring. Therefore, taken together with the lack of raised systolic blood pressure in these young animals, our findings confirm that endothelial dysfunction may serve as an early indicator of cardiovascular disease following exposure to an adverse in utero environment (Martin et al. 2000).
This study provides the first evidence of altered ribosomal RNA levels following in utero protein restriction, with significantly increased levels of 28S and significantly reduced levels of 18S in the livers. In addition, in the male offspring there was a negative correlation between 28S and 18S expression (R2 = 0.26), suggesting that there is a specific alteration in the relative contributions of the different RNA components of the ribosome. Further work is needed to investigate whether other ribosomal components are also affected, for example ribosomal 5S RNA and 5.8S RNA.
ACh-induced relaxation is mediated by NO, PGI2 and EDHF (Shimokawa et al. 1996), and previous studies of the effect of eNOS inhibition with L-NAME on artery function using the PR model suggested an impairment of the NO–cGMP pathway in these animals (Torrens et al. 2006). The elevation of eNOS in male PR mesenteric arteries could represent an adaptive response in these arteries to endothelial dysfunction following in utero protein restriction. The umbilical cords from in utero growth restricted (IUGR) babies have raised eNOS mRNA levels but reduced NOS activity (Casanello & Sobrevia, 2002), potentially due to a negative-feedback mechanism by NO controlling eNOS expression. Another explanation for raised eNOS mRNA expression is the recent finding of an increased expression of peroxisome proliferator-activated receptor- (PPAR) in the livers of the offspring of similarly protein-restricted rat dams (Lillycrop et al. 2005), which was found alongside reduced DNA methylation of the promoter site of PPAR. As PPAR has been shown to increase the expression of eNOS (Goya et al. 2004), it is possible that an increase in mRNA expression may have occurred through this mechanism. The lack of alteration in mRNA levels of both soluble guanylate cyclase (sGC) and iNOS suggest that dysfunction in the PR resistance arteries is not due to an alteration in the levels of either of these enzymes.
As the resistance arteries of the PR offspring appeared to show an adaptive response to the endothelial dysfunction through increased eNOS expression, we investigated whether vascular remodelling also occurred. The reduction in smooth muscle area in the arteries may reflect a feedback mechanism to counteract the endothelial dysfunction observed: this finding mimics a study in the rat of offspring of high fat-fed dams, in which the smooth muscle cell number was reduced in the aorta (Armitage et al. 2005). This observation may also explain previous findings of a reduced endothelium-independent relaxation response in these vessels to the NO donor, sodium nitroprusside (Brawley et al. 2003). In addition, children of mothers exposed to the Dutch Famine during pregnancy have reduced carotid artery intima + media thickness (Painter et al. 2007), which could be explained by a reduction in smooth muscle cell volume. The changes in smooth muscle content were not, however, associated with a reduced constrictor tone to KPSS or PE.
As vascular reactivity is modulated by reactive oxygen species (ROS) and maternal PR has been reported to increase oxidative stress (Langley-Evans & Sculley, 2005), we examined whether expression of antioxidant enzymes was altered in the tissue of PR offspring. No changes were detected in the levels of MnSOD, HO-1 or the catalytic subunit of GCL, suggesting that antioxidant defences were not compromised in these resistance arteries. This was supported by the finding that the superoxide dismutase mimetic tempol did not improve the vasodilatory function of the PR arteries, as would be expected in conditions of oxidative stress. Additionally, in the PR liver, there was no evidence of oxidative stress by assaying protein carbonylation This finding agrees with a previous study which showed no difference in protein carbonyl concentrations in the livers of both male and female 18-month-old PR offspring (Langley-Evans & Sculley, 2006) but differs from earlier findings of this group showing increased levels of protein carbonyls in the livers of the male but not female PR offspring (Langley-Evans & Sculley, 2005). However, this latter finding was only significant when the carbonyl concentrations were analysed as a combined effect of diet and age in 4-, 16-, 30- and 44-week-old offspring. However, in the present study protein carbonyl concentration was significantly higher in male compared to female C livers (P < 0.05), showing that males have increased levels of oxidative stress in their livers. This finding was expected as females are known to possess protection against the effects of ROS, with heightened expression of the antioxidants ecSOD (Strehlow et al. 2003) and glutathione (Borras et al. 2003) and reduced activity of the oxidant NAD(P)H oxidase (Strehlow et al. 2003).
In contrast to our finding of no increased oxidative stress in the PR livers, mRNA expression of HO-1 was significantly decreased in PR male offspring. This finding has several relevant implications in our model, as HO-1 not only has antioxidant effects but also is believed to influence whole body growth (Yachie et al. 1999), smooth muscle cell proliferation (Duckers et al. 2001) and vascular tone in the liver (Wakabayashi et al. 1999). HO-1 has been implicated as a critical regulator of growth, with both humans and mice deficient in HO-1 displaying severe growth retardation (Poss & Tonegawa, 1997; Yachie et al. 1999). Therefore, the reduced HO-1 expression in the PR male offspring may account for the reduced growth rate found in these animals in this study as well as in previous investigations (Torrens et al. 2003), a hypothesis strengthened by the finding that HO-1 expression correlated with postmortem weight in the male animals.
HO-1 produces CO as a product of haeme breakdown and CO has been found to elicit vasorelaxation (Zhang et al. 2001). CO is able to bind to the haeme moiety of sGC and act in a similar way to NO to cause activation and subsequent production of cGMP. It has been shown to relax aorta and tail arteries of rats, an effect which can be blocked by sGC inhibitors (McLaughlin et al. 2000). The actions of HO-1-dependent CO production are thought to be particularly important in the sinusoidal microvessels of the liver, where the use of NOS inhibitors does not alter vascular resistance and therefore it is unlikely that NO is involved in vasodilatation (Wakabayashi et al. 1999). In the liver, NO and O2·– concentrations are very low and are hypothesized to counteract each other (Bautista & Spitzer, 1994), and thus it is CO which serves to up-regulate the actions of sGC and vasodilatation. Therefore, a decrease in HO-1 expression in the liver may have important implications for the vasodilatatory function of the microvessels of the liver, with an effect on the peripheral vascular resistance of the body. Indeed, levels of liver HO-1 in a model of cirrhosis increase in parallel to increases in the aorta and mesenteric artery, and have a role in altered peripheral resistance (Chen et al. 2004).
This study confirms that endothelial dysfunction can be induced by in utero protein restriction and implicates persistent changes in the NO signalling pathway. We have shown for the first time in this model that the mechanistic changes are associated with remodelling of the vascular wall. At the gene level, the effects are not due simply to a reduction in key enzymes in the NO signalling pathway (eNOS, iNOS and sGC) or antioxidants (HO-1, GCL and MnSOD) in the vasculature. The dysfunction in male offspring may in fact be partially counteracted by increased expression of eNOS. This suggests that the alteration in endothelial function is the result of a coordinated adaptive response, rather than a developmental disruption. A further novel finding is that in utero PR leads to reduced HO-1 expression in the liver, which may have consequences for both growth and vascular function in the offspring as evidenced in this study. Whether this is adaptive or disruptive merits further investigation.
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