Maternal high-fat diet in mice alters immune regulation and lung function in the offspring.

PUFA modulate immune function and have been associated with the risk of childhood atopy and asthma. We investigated the effect of maternal fat intake in mice on PUFA status, elongase and desaturase gene expression, inflammatory markers and lung function in the offspring. C57BL/6J mice (n 32) were fed either standard chow (C, 20·4 % energy as fat) or a high-fat diet (HFD, 39·9 % energy as fat) for 4 weeks prior to conception and during gestation and lactation. At 21 d of age, offspring were weaned onto either the HFD or C, generating four experimental groups: C/C, C/HF, HF/C and HF/HF. Plasma and liver fatty acid composition were measured by GC and gene expression by quantitative PCR. Lung resistance to methacholine was assessed. Arachidonic acid concentrations in offspring plasma and liver phospholipids were increased by HFD; this effect was greater in the post-natal HFD group. DHA concentration in offspring liver phospholipids was increased in response to HFD and was higher in the post-natal HFD group. Post-natal HFD increased hepatic fatty acid desaturase (FADS) 2 and elongation of very long-chain fatty acid 5 expression in male offspring, whereas maternal HFD elevated expression of FADS1 and FADS2 in female offspring compared with males. Post-natal HFD increased expression of IL-6 and C-C motif chemokine ligand 2 (CCL2) in perivascular adipose tissue. The HFD lowered lung resistance to methacholine. Excessive maternal fat intake during development modifies hepatic PUFA status in offspring through regulation of gene expression of enzymes that are involved in PUFA biosynthesis and modifies the development of the offspring lungs leading to respiratory dysfunction.

Serial block-face scanning electron microscopy reveals novel intercellular connections in human term placental microvasculature.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde-fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty-seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 µm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm-3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans-endothelial channel was observed in a region of the vasculo-syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three-dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans-endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability.

Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Intrahepatic cholestasis of pregnancy (ICP) causes increased transfer of maternal bile acids to the fetus and an increased incidence of sudden fetal death. Treatment includes ursodeoxycholic acid (UDCA), but it is not clear if UDCA protects the fetus. This study explores the placental transport of the bile acid taurocholate (TC) by the organic anion-transporting polypeptide, (OATP)4A1, its effects on the placental proteome and vascular function, and how these are modified by UDCA. Various methodological approaches including placental villous fragments and Xenopus laevis oocytes were used to investigate UDCA transport. Placental perfusions and myography investigated the effect of TC on vasculature. The effects of acute TC exposure on placental tissue were investigated using quantitative proteomics. UDCA inhibited OATP4A1 activity in placental villous fragments and oocytes. TC induced vasoconstriction in placental and rat vasculature, which was attenuated by UDCA. Quantitative proteomic analysis of villous fragments showed direct effects of TC on multiple placental pathways, including oxidative stress and autophagy. The effects of TC on the placental proteome and vasculature demonstrate how bile acids may cause fetal distress in ICP. UDCA inhibition of OATP4A1 suggests it will protect the mother and fetus against the vascular effects of TC by inhibiting its cellular uptake. UDCA may protect the fetus in ICP by inhibiting OATP4A1-mediated bile acid transfer and TC-induced placental vasoconstriction. Understanding the physiologic mechanisms of UDCA may allow better therapeutic interventions to be designed specifically for the fetus in the future.-Lofthouse, E. M., Torrens, C., Manousopoulou, A., Nahar, M., Cleal, J. K., O'Kelly, I. M., Sengers, B. G., Garbis, S. D., Lewis, R. M. Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Serial block-face scanning electron microscopy of erythrocytes protruding through the human placental syncytiotrophoblast.

The syncytiotrophoblast forms a continuous barrier between the maternal and fetal circulations. Here we present a serial block-face scanning electron microscopy (SBFSEM) study, based on a single image stack, showing pooling of fetal blood underneath a region of stretched syncytiotrophoblast that has become detached from the basement membrane. Erythrocytes are protruding from discrete holes in the syncytiotrophoblast suggesting that, under specific circumstances, the syncytiotrophoblast may be permeable to fetal cells. This observation represents a pathological process but it poses questions about the physical properties and permeability of the syncytiotrophoblast and may represent an early stage in the formation of fibrin deposits in areas of syncytial denudation. This study also illustrates how the 3D images generated by SBFSEM allow the interpretation of structures that could not be understood from a single histological section.

Maternal low-protein diet during mouse pre-implantation development induces vascular dysfunction and altered renin-angiotensin-system homeostasis in the offspring.

Environmental perturbations during early mammalian development can affect aspects of offspring growth and cardiovascular health. We have demonstrated previously that maternal gestational dietary protein restriction in mice significantly elevated adult offspring systolic blood pressure. Therefore, the present study investigates the key mechanisms of blood pressure regulation in these mice. Following mating, female MF-1 mice were assigned to either a normal-protein diet (NPD; 18 % casein) or an isocaloric low-protein diet throughout gestation (LPD; 9 % casein), or fed the LPD exclusively during the pre-implantation period (3.5 d) before returning to the NPD for the remainder of gestation (Emb-LPD). All offspring received standard chow. At 22 weeks, isolated mesenteric arteries from LPD and Emb-LPD males displayed significantly attenuated vasodilatation to isoprenaline (P = 0.04 and P = 0.025, respectively), when compared with NPD arteries. At 28 weeks, stereological analysis of glomerular number in female left kidneys revealed no significant difference between the groups. Real-time RT-PCR analysis of type 1a angiotensin II receptor, Na+/K+ ATPase transporter subunits and glucocorticoid receptor expression in male and female left kidneys revealed no significant differences between the groups. LPD females displayed elevated serum angiotensin-converting enzyme (ACE) activity (P = 0.044), whilst Emb-LPD males had elevated lung ACE activity (P = 0.001), when compared with NPD offspring. These data demonstrate that elevated offspring systolic blood pressure following maternal gestational protein undernutrition is associated with impaired arterial vasodilatation in male offspring, elevated serum and lung ACE activity in female and male offspring, respectively, but kidney glomerular number in females and kidney gene expression in male and female offspring appear unaffected.

Effects of pre- and periconceptional undernutrition on arterial function in adult female sheep are vascular bed dependent.

The nutritional environment during development and even prior to conception may contribute to cardiovascular risk. In mature adult female sheep, we investigated the effect of preconceptional and periconceptional maternal nutritional restriction on the vascular reactivity of arteries from four vascular beds supplying the heart, thorax, kidney and hindlimb. Welsh Mountain ewes received 100% of nutrient requirements throughout gestation (control group, C, n = 18), or 50% of nutrient requirements for 30 days prior to conception (preconceptional group, PRE, n = 20) or for 15 days either side of conception (periconceptional group, PERI, n = 31) and 100% thereafter. In 3.5-year-old female offspring, the left anterior descending coronary (LAD), left internal thoracic (LITA), right renal and second and third order femoral arteries were dissected and their reactivity was assessed by organ bath or wire myography. Vasoconstrictor responses were greater in both LAD and LITA from PERI offspring compared with C (P < 0.01), while vasoconstriction was unaffected by maternal diet in arteries from the renal and femoral circulations (P = n.s.). Endothelium-dependent and -independent vasodilatation was attenuated in third order femoral arteries of PRE and PERI groups compared with C (P < 0.05). Endothelium-independent vasodilatation was attenuated in both the LAD and renal arteries in the PERI group compared with C (P < 0.05). These data show that moderate maternal undernutrition either prior to or around conception affects vascular function of adult offspring. The effect depends on the timing of the insult, but also on the vascular bed studied and vessel hierarchy in the vascular tree.

Maternal undernutrition leads to endothelial dysfunction in adult male rat offspring independent of postnatal diet.

Increasing evidence suggests a role for prenatal environment in the onset of cardiovascular and metabolic disease in later life. In the rat, undernutrition in utero and a postnatal high-fat diet gives rise to a phenotype similar to the metabolic syndrome. As endothelial dysfunction is a feature of both CVD and the metabolic syndrome we investigated the impact of maternal undernutrition and/or postnatal high-fat on endothelial function. Virgin Wistar rats were mated and randomly assigned to groups to receive food either ad libitum (control) or at 30% of ad libitum intake throughout gestation. At postnatal day 250, a cohort from each group was challenged with a high-fat diet (D12451, 45% energy from fat; Research Diets, Inc., New Brunswick, NJ, USA) for the remainder of the study. At 1 year of age, small mesenteric arteries were dissected and mounted on a wire myograph and responses to phenylephrine, endothelin, acetylcholine, leptin and sodium nitroprusside assessed. Vasoconstriction to endothelin was significantly enhanced in all groups compared with controls (-log effective concentration equal to 50% of the maximal response (pEC50); P < 0.001). Endothelium-dependent vasodilatation to acetylcholine was significantly blunted in all groups compared with controls (% maximum response; P < 0.01), while dilatation to leptin and sodium nitroprusside was similar in all groups. These data demonstrate that both maternal undernutrition and postnatal high fat lead to vascular alterations and suggest that maternal undernutrition alone is at least as detrimental to offspring endothelial function as a long-term exposure to a high-fat diet in the offspring.

Aortic Stenosis Prognostication in Patients With Type 2 Diabetes: Protocol for Testing and Validation of a Biomarker-Derived Scoring System.

BACKGROUND: Type 2 diabetes mellitus (T2DM) has been established as an important independent risk factor for aortic stenosis. T2DM patients present with a higher degree of valve calcification and left ventricular dysfunction compared to patients without diabetes. This may be due to an increase in incidence and severity of myocardial fibrosis. Currently, there is no reliable method of determining the optimal timing of intervention for a patient with asymptomatic aortic stenosis or predicting when a patient will become symptomatic. Research into serum biomarkers to predict subclinical onset and track progression of aortic stenosis is hampered by the multimodal nature of the pathological processes ultimately responsible for aortic stenosis. OBJECTIVE: The aim of this study is to prove that an approach using a combination of serum biomarkers and the echocardiographic parameter global longitudinal strain (GLS) can be used to establish baseline status of fibrocalcific aortic valve disease, predict rate of progression, and quantitatively assess any regression of these processes following aortic valve replacement in patients with T2DM. METHODS: Validated serum biomarkers for the separate processes of calcification, inflammation, oxidative stress and fibrosis can be used to quantify onset and rate of progression of aortic stenosis. This, in combination with the echocardiographic parameter GLS, can be compared with other objective investigations of calcification and fibrosis with the aim of developing a quick, noninvasive one-stop assessment of aortic stenosis in patients with T2DM. The serum biomarkers BNP (B-type natriuretic peptide), Gal-3 (Galectin-3), GDF-15 (growth differentiation factor-15), sST2 (soluble suppression of tumorigenicity 2), OPG (osteoprotegerin), and microRNA 19b and 21 will be sampled from patients undergoing aortic valve replacement (with and without T2DM), patients with T2DM but without aortic valve disease and healthy volunteers. These patients will also undergo computed tomography (CT) scans for calcium scoring, magnetic resonance imaging (MRI) to quantify myocardial fibrosis, and myocardial strain imaging with speckle-tracking echocardiography. Samples of calcified native aortic valve and a biopsy of ventricular myocardium will be examined histologically to determine the quantity and distribution of calcification and fibrosis, and the secretome of these tissue samples will also be analyzed for levels of the same biomarkers as in the serum samples. All patients will be followed up with in 3 months and 12 months for repeat blood sampling, echocardiography, and CT and MRI imaging to assess disease progression or regression. The results of tissue analysis and CT and MRI scanning will be used to validate the findings of the serum biomarkers and echocardiographic assessment. RESULTS: Using all of the information gathered throughout the study will yield a ranking scale for use in the clinic, which will provide each patient with a fibrocalcific profile. This can then be used to recommend an optimal time for intervention. CONCLUSION: A reliable, validated set of serum biomarkers combined with an inexpensive bedside echocardiographic examination can now form the basis of a one-stop outpatient-based assessment service, which will provide an accurate risk assessment in patients with aortic stenosis at first contact. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/13186.

Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy.

OBJECTIVES: Bicuspid aortic valve disease is common and is associated with ascending aortic aneurysms. Vascular smooth muscle cell (VSMC) apoptosis is characteristic of the ascending aorta of bicuspid patients, and NOTCH1 gene mutations have also been linked to the disease. NOTCH signalling is a fundamental cell signalling pathway, which dictates cell fate decisions including apoptosis. Our objective was to elucidate the role of NOTCH signalling in VSMC apoptosis and differentiation in bicuspid aortopathy. METHODS: Ascending aortic biopsies were obtained from 19 bicuspid and 12 tricuspid aortic valve patients and were sub-classified into 4 groups according to the maximum ascending aortic diameter (aneurysmal ≥45 mm). Apoptotic VSMCs were counted by light microscopy using a TUNEL assay. Gene expression of key regulators of NOTCH signalling (NOTCH1 and HES1), apoptosis (BAX and BCL-2) and VSMC differentiation (MYH11, CNN1 and MYH10) were quantified using quantitative real-time PCR. Primary VSMCs were cultured from 2 tricuspid aortic valve and 2 bicuspid aortic valve patients, NOTCH signalling was inhibited with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, and the gene expression was again quantified. RESULTS: The apoptotic cell count was significantly higher in bicuspid aortic valve patients (3.2 cells/50 000 µm2 vs 1.1 cells/50 000 µm2; P = 0.033). There was a trend towards lower apoptotic cell count in the aneurysmal versus non-aneurysmal tricuspid and bicuspid groups and an increased ratio of proapoptotic gene expression, which was not statistically significant. This was associated with a 2.8-fold increase in contractile gene expression (P = 0.026) and a 2.0-fold increase in NOTCH signalling gene expression in bicuspid versus tricuspid aortic valve patients (P = 0.022). NOTCH inhibition in cultured VSMCs induced a similar pattern of increased proapoptotic and procontractile gene expressions. CONCLUSIONS: This preliminary study suggests that NOTCH activation in the non-aneurysmal bicuspid aortas may underlie aortopathy by influencing VSMC apoptosis and differentiation. NOTCH signalling manipulation may provide a therapeutic target for preventing aneurysms in bicuspid patients. Further studies with larger sample sizes are needed to substantiate the present findings.

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 < 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 < 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 < 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.

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring.

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease-related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF-1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety-related behaviour in open field activities in male and female offspring (P < 0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P < 0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P < 0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P < 0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P < 0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post-fertilization preimplantation development which may reflect the relative contribution of the paternal genome.

Transmission of raised blood pressure and endothelial dysfunction to the F2 generation induced by maternal protein restriction in the F0, in the absence of dietary challenge in the F1 generation.

We have previously demonstrated that maternal protein restriction during pregnancy leads to raised blood pressure and endothelial dysfunction in the offspring (F1). Here we show that these characteristics are transmitted to the F2 offspring through the maternal line, in the absence of any additional challenges to the F1. Female Wistar rats were fed either a control (18% casein) or protein-restricted diet (PR; 9% casein) throughout pregnancy. Female F1 offspring, maintained on standard chow postpartum, were mated with breeding males to produce F2 progeny. Systolic blood pressure (SBP) in male F2 offspring was assessed by tail-cuff plethysmography at age 100 d and vascular function of small mesenteric arteries by wire myography at age 80 and 200 d. SBP was raised in PR F2 offspring compared with controls (control 122.1 (SEM 2.3) mmHg, n 7; PR 134.7 (SEM 3.2) mmHg, n 6; P<0.01) and endothelial function, assessed by vasodilatation to acetylcholine, was impaired at both age 80 d (% maximal response: control 89.7 (SEM 2.6), n 14; PR 72.7 (SEM 4.4), n 15; P<0.01) and 200 d (effective concentration equal to 50% of maximum (pEC50): control 7.67 (SEM 0.10), n 10; PR 7.33 (SEM 0.07), n 8; P<0.05). The present study demonstrates that both raised blood pressure and endothelial dysfunction are passed via the maternal line to grand-offspring in the absence of any additional dietary challenges to their F1 mothers. Risk factors for chronic disease may therefore be heritable by non-genomic processes.

Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring.

Induction of an altered phenotype by prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. We investigated the effect of feeding pregnant rats a protein-restricted (PR) diet with different amounts of folic acid on the methylation of individual CpG dinucleotides in the hepatic PPAR alpha promoter in juvenile offspring, and the effect of the maternal PR diet on CpG methylation in adult offspring. Pregnant rats (five per group) were fed 180 g/kg casein (control) or 90 g/kg casein with 1 mg/kg folic acid (PR), or 90 g/kg casein and 5 mg/kg folic acid (PRF). Offspring were killed on postnatal day 34 (five males and females per group) and day 80 (five males per group). Methylation of sixteen CpG dinucleotides in the PPAR alpha promoter was measured by pyrosequencing. Mean PPAR alpha promoter methylation in the PR offspring (4.5 %) was 26 % lower than controls (6.1 %) due to specific reduction at CpG dinucleotides 2 (40 %), 3 (43 %), 4 (33 %) and 16 (48 %) (P < 0.05). There was no significant difference in methylation at these CpG between control and PRF offspring. Methylation of CpG 5 and 8 was higher (47 and 63 %, respectively, P < 0.05) in the PRF offspring than control or PR offspring. The methylation pattern in day 80 PR offspring was comparable to day 34 PR offspring. These data show for the first time that prenatal nutrition induces differential changes to the methylation of individual CpG dinucleotides in juvenile rats which persist in adults.

Resolvin E1, resolvin D1 and resolvin D2 inhibit constriction of rat thoracic aorta and human pulmonary artery induced by the thromboxane mimetic U46619.

BACKGROUND AND PURPOSE: The ω-6 fatty acid-derived lipid mediators such as prostanoids, thromboxane and leukotrienes have well-established roles in regulating both inflammation and smooth muscle contractility. Resolvins are derived from ω-3 fatty acids and have important roles in promoting the resolution of inflammation, but their activity on smooth muscle contractility is unknown. We investigated whether resolvin E1 (RvE1), resolvin D1 (RvD1) and resolvin D2 (RvD2) can modulate contractions of isolated segments of rat thoracic aorta (RTA) or human pulmonary artery (HPA) induced by the α1 -adrenoceptor agonist phenylephrine or the stable thromboxane A2 mimetic U46619. EXPERIMENTAL APPROACH: Contractile responses in RTA and HPA were measured using wire myography. Receptor expression was investigated by immunohistochemistry. KEY RESULTS: Constriction of RTA segments by U46619, but not by phenylephrine, was significantly inhibited by pretreatment for 1 or 24 h with 10-100 nM RvE1, RvD1 or RvD2. The inhibitory effect of RvE1 was partially blocked by a chemerin receptor antagonist (CCX832). RvE1 at only 1-10 nM also significantly inhibited U46619-induced constriction of HPA segments, and the chemerin receptor, GPR32 and FPR2/ALX were identified in HPA smooth muscle. CONCLUSION AND IMPLICATIONS: These data suggest that resolvins or their mimetics may prove useful novel therapeutics in diseases such as pulmonary arterial hypertension, which are characterized by increased thromboxane contractile activity.

Candidate plasma biomarkers for predicting ascending aortic aneurysm in bicuspid aortic valve disease.

BACKGROUND: Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality affecting 1-2% of the population and is associated with a significantly increased risk of ascending aortic aneurysm. However, predicting which patients will develop aneurysms remains a challenge. This pilot study aimed to identify candidate plasma biomarkers for monitoring ascending aortic diameter and predicting risk of future aneurysm in BAV patients. METHODS: Plasma samples were collected pre-operatively from BAV patients undergoing aortic valve surgery. Maximum ascending aortic diameter was measured on pre-operative transoesophageal echocardiography. Maximum diameter ≥ 45 mm was classified as aneurysmal. Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH-MS), an advanced mass spectrometry technique, was used to identify and quantify all proteins within the samples. Protein abundance and aortic diameter were correlated using logistic regression. Levene's test was used to identify proteins demonstrating low abundance variability in the aneurysmal patients (consistent expression in disease), and high variability in the non-aneurysmal patients (differential expression between 'at risk' and not 'at risk' patients). RESULTS: Fifteen plasma samples were collected (seven non-aneurysmal and 8 aneurysmal BAV patients). The mean age of the patients was 55.5 years and the majority were female (10/15, 67%). Four proteins (haemoglobin subunits alpha, beta and delta and mannan-binding lectin serine protease) correlated significantly with maximal ascending aortic diameter (p < 0.05, r = 0.5-0.6). Five plasma proteins demonstrated significantly lower variability in the aneurysmal group and may indicate increased risk of aneurysm in non-aneurysmal patients (DNA-dependent protein kinase catalytic subunit, lumican, tetranectin, gelsolin and cartilage acidic protein 1). A further 7 proteins were identified only in the aneurysmal group (matrin-3, glucose-6-phosphate isomerase, coactosin-like protein, peptidyl-prolyl cis-trans isomerase A, golgin subfamily B member 1, myeloperoxidase and 2'-deoxynucleoside 5'-phosphate N-hydrolase 1). CONCLUSIONS: This study is the first to identify candidate plasma biomarkers for predicting aortic diameter and risk of future aneurysm in BAV patients. It provides valuable pilot data and proof of principle that could be used to design a large-scale prospective investigation. Ultimately, a more affordable 'off-the-shelf' follow-on blood assay could then be developed in place of SWATH-MS, for use in the healthcare setting.

Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (≥4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.

Developmental conditioning of endothelium-derived hyperpolarizing factor-mediated vasorelaxation.

OBJECTIVES: The endothelium maintains vascular homeostasis through the release of endothelium-derived relaxing factors (EDRF) and endothelium-derived hyperpolarization (EDH). The balance in EDH : EDRF is disturbed in cardiovascular disease and may also be susceptible to developmental conditioning through exposure to an adverse uterine environment to predispose to later risk of hypertension and vascular disease. METHODS: Developmentally conditioned changes in EDH : EDRF signalling pathways were investigated in cremaster arterioles (18-32  µm diameter) and third-order mesenteric arteries of adult male mice offspring of dams fed either a fat-rich (high fat, HF, 45% energy from fat) or control (C, 10% energy from fat) diet. After weaning, offspring either continued on high fat or were placed on control diets to give four dietary groups (C/C, HF/C, C/HF, and HF/HF) and studied at 15 weeks of age. RESULTS: EDH via intermediate (IKCa) and small (SKca) conductance calcium-activated potassium channels contributed less than 10% to arteriolar acetylcholine-induced relaxation in in-situ conditioned HF/C offspring compared with ∼60% in C/C (P < 0.01). The conditioned reduction in EDH signalling in HF/C offspring was reversed in offspring exposed to a high-fat diet both before and after weaning (HF/HF, 55%, P < 0.01 vs. HF/C). EDH signalling was unaffected in arterioles from C/HF offspring. The changes in EDH : EDRF were associated with altered endothelial cell expression and localization of IKCa channels. CONCLUSION: This is the first evidence that EDH-mediated microvascular relaxation is susceptible to an adverse developmental environment through down-regulation of the IKCa signalling pathway. Conditioned offspring exposed to a 'second hit' (HF/HF) exhibit adaptive vascular mechanisms to preserve dilator function.