Theodosios Kyriakou

Pre-eclampsia and offspring cardiovascular health: mechanistic insights from experimental studies.

Pre-eclampsia is increasingly recognized as more than an isolated disease of pregnancy. Women who have had a pregnancy complicated by pre-eclampsia have a 4-fold increased risk of later cardiovascular disease. Intriguingly, the offspring of affected pregnancies also have an increased risk of higher blood pressure and almost double the risk of stroke in later life. Experimental approaches to identify the key features of pre-eclampsia responsible for this programming of offspring cardiovascular health, or the key biological pathways modified in the offspring, have the potential to highlight novel targets for early primary prevention strategies. As pre-eclampsia occurs in 2–5% of all pregnancies, the findings are relevant to the current healthcare of up to 3 million people in the U.K. and 15 million people in the U.S.A. In the present paper, we review the current literature that concerns potential mechanisms for adverse cardiovascular programming in offspring exposed to pre-eclampsia, considering two major areas of investigation: first, experimental models that mimic features of the in utero environment characteristic of pre-eclampsia, and secondly, how, in humans, offspring cardiovascular phenotype is altered after exposure to pre-eclampsia. We compare and contrast the findings from these two bodies of work to develop insights into the likely key pathways of relevance. The present review and analysis highlights the pivotal role of long-term changes in vascular function and identifies areas of growing interest, specifically, response to hypoxia, immune modification, epigenetics and the anti-angiogenic in utero milieu.

Adiponectin gene ADIPOQ SNP associations with serum adiponectin in two female populations and effects of SNPs on promoter activity

AbstractAdiponectin is an insulin sensitiser in muscle and liver, and low serum levels characterise obesity and insulin resistance. Eight tagging single nucleotide polymorphisms (tSNPs) in the ADIPOQ gene and promoter were selected, and association with serum adiponectin was tested, in two independent samples of Caucasian women: the Chingford Study (n = 808, mean age 62.8 ± 5.9 years) and Twins UK (n = 2,718, mean age 47.4 ± 12.6 years). In the Chingford cohort, −11391 G/A, −10066 G/A (rs182052), −7734 C/A (rs16861209), +276 G/T (rs1501299) and +3228 C/T (rs1063537) were significantly associated with fasting serum adiponectin (Ps = 1.00 × 10−4 to 1.40 × 10−2). Associations with all except +3228 C/T were replicated in the Twins UK cohort (Ps = 3.19 × 10−9 to 6.00 × 10−3). In Chingford subjects, the 12 most common 8-SNP haplotypes (frequency 1.90%) explained 2.85% (p = 5.00 × 10−2) and in Twins UK subjects, the four most common 5-SNP haplotypes (frequency > 5.00%) explained 1.66% of the variance (p = 5.83 × 10−7). To investigate effects of −11391 G/A (rs17300539) and −11377 C/G (rs266729) on promoter activity, 1.2 kb of the ADIPOQ promoter region was cloned in a luciferase reporter plasmid, and the four haplotypes were transfected in differentiated 3T3-L1 adipocytes. No significant allelic effects on promoter activity were found.

Effect of high-fat feeding on expression of genes controlling availability of dopamine in mouse hypothalamus

Objective Hypothalamic centers integrate external signals of nutrient availability and energy status and initiate responses to maintain homeostasis. Quantifying changes in hypothalamic gene expression in the presence of nutrient excess may identify novel responsive elements. Methods Affymetrix Mouse Genome 430 2.0 oligonucleotide microarrays containing 45 102 probe sets were used to interrogate differential expression of genes in dietary-induced obesity model C57BL6 inbred mice fed a high-fat (35% fat; n = 8) or standard (4% fat; n = 6) diet from 3 to 15 wk of age. Ontologies of regulated genes were examined and expression of selected genes was validated by quantitative real-time polymerase chain reaction. Results One thousand two hundred twelve unique gene transcripts showed altered expression on the microarrays. Gene ontology analysis revealed changes in neuropeptide genes responding to leptin, Pomc, Cart, Npy, and Agrp, compatible with a homeostatic response to high-fat intake, although mean weight increased 2.3-fold compared with standard fed mice (P < 0.001). Neurotransmitter system ontologies revealed upregulation of five genes controlling availability of dopamine. Changes in Th tyrosine hydroxylase (2.1-fold) and Slc18a2 solute carrier family 18 (vesicular monoamine), member 2 (4.4-fold) controlling synthesis and release, and Slc6a3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 (4.8-fold), Snca α-synuclein (1.3-fold), and Maoa monoamine oxidase (1.9-fold) limiting availability were confirmed by quantitative real-time polymerase chain reaction. Conclusion Expression of five genes involved in availability of dopamine was increased after a high-fat diet. Failure to reduce dopamine availability sufficiently, to counter the feeding reward effect, could contribute to diet-induced obesity in these mice.

Genotypic Effect of the −565C>T Polymorphism in the ABCA1 Gene Promoter on ABCA1 Expression and Severity of Atherosclerosis

Objective— Loss-of-function mutations of the ATP-binding cassette transporter A1 (ABCA1) gene cause Tangier disease, a rare genetic disorder with accumulation of lipid-laden macrophages and increased risk of atherosclerosis. Common variants of this gene may be a genetic factor for atherosclerosis in the general population. This study was performed to test the reported association between the −565C>T polymorphism and atherosclerosis severity and to investigate whether this variant per se had an effect on promoter activity of the ABCA1 gene. Methods and Results— A cohort of patients with coronary atherosclerosis were genotyped for the −565C>T polymorphism. Logistic regression analyses showed that homozygotes of the −565T allele had greatest mean number of diseased coronary arteries, particular in nonsmokers. Real-time reverse-transcriptase polymerase chain reaction showed that in atherosclerotic plaques removed from patients undergoing endarteretomy, ABCA1 expression levels were lowest in those who had the T/T genotype and highest in those of the C/C genotype. Transfection and reporter assays demonstrated that in cultured macrophages, the −565T allelic promoter had a lower activity in driving gene expression than the −565C allelic promoter. Electrophoretic mobility shift assays displayed differential binding of nuclear proteins to the 2 alleles. Conclusions— These results indicate that the −565C>T polymorphism has an allele-specific effect on ABCA1 gene expression and provide further evidence of a genotypic effect on coronary atherosclerosis severity.

Association of the lymphotoxin-alpha gene Thr26Asn polymorphism with severity of coronary atherosclerosis.

A recent large-scale, genome-wide association study of single nucleotide polymorphisms showed a strong association between susceptibility to myocardial infarction and the Thr26Asn polymorphism in the lymphotoxin-α (LTA) gene. In the present study, we investigated whether the LTA Thr26Asn polymorphism was associated with the extent of coronary atherosclerosis in a large cohort (n=1082) of well-documented coronary artery disease patients. Thr26Asn genotypes showed a significant different distribution in male patients, when stratified according to the number of diseased coronary arteries, with an odds ratio of 1.98 (95% CI 1.22–3.22) for multiple-vessel disease in patients of the Asn/Asn genotype, compared with patients of the Thr/Thr or Thr/Asn genotype (P=0.006). Thus, further to the recent finding that LTA gene variation is associated with susceptibility to coronary heart disease, the present study provides evidence of an association between LTA genotype and the extent of coronary atherosclerosis.

Association of Maternal Antiangiogenic Profile at Birth With Early Postnatal Loss of Microvascular Density in Offspring of Hypertensive Pregnancies.

Offspring of hypertensive pregnancies are more likely to have microvascular rarefaction and increased blood pressure in later life. We tested the hypothesis that maternal angiogenic profile during a hypertensive pregnancy is associated with fetal vasculogenic capacity and abnormal postnatal microvascular remodeling. Infants (n=255) born after either hypertensive or normotensive pregnancies were recruited for quantification of postnatal dermal microvascular structure at birth and 3 months of age. Vasculogenic cell potential was assessed in umbilical vein endothelial cells from 55 offspring based on in vitro microvessel tube formation and proliferation assays. Maternal angiogenic profile (soluble fms-like tyrosine kinase-1, soluble endoglin, vascular endothelial growth factor, and placental growth factor) was measured from postpartum plasma samples to characterize severity of pregnancy disorder. At birth, offspring born after hypertensive pregnancy had similar microvessel density to those born after a normotensive pregnancy, but during the first 3 postnatal months, they had an almost 2-fold greater reduction in total vessel density (−17.7±16.4% versus −9.9±18.7%; P=0.002). This postnatal loss varied according to the vasculogenic capacity of the endothelial cells of the infant at birth (r=0.49; P=0.02). The degree of reduction in both in vitro and postnatal in vivo vascular development was proportional to levels of antiangiogenic factors in the maternal circulation. In conclusion, our data indicate that offspring born to hypertensive pregnancies have reduced vasculogenic capacity at birth that predicts microvessel density loss over the first 3 postnatal months. Degree of postnatal microvessel reduction is proportional to levels of antiangiogenic factors in the maternal circulation at birth.

Association of Common JAK2 Variants With Body Fat, Insulin Sensitivity and Lipid Profile

The leptin signal is transduced via the JAK2‐STAT3 (Janus kinase 2‐signal transducer and activator of transcription‐3) pathway at the leptin receptor. JAK2 also phosphorylates insulin receptor substrate, integral to insulin and leptin action and is required for optimum adenosine triphosphate–binding cassette transporter A1 (ABCA1)‐dependent transport of lipids from cells to apolipoprotein A‐1 (apoA‐I). We hypothesized that common variation in the JAK2 gene may be associated with body fat, insulin sensitivity, and modulation of the serum lipid profile in the general population. Ten tagging single‐nucleotide polymorphisms (SNPs) spanning the gene were genotyped in 2,760 white female twin subjects (mean age 47.3 ± 12.6 years) from the St Thomas’ UK Adult Twin Registry. Minor allele frequencies were between 0.170 and 0.464. The major allele of rs7849191 was associated with higher central fat (P = 0.030), percentage of central fat (P = 0.014) and waist circumference (P = 0.027) the major allele of rs3780378 with higher serum apoA (P = 0.026), total cholesterol (P = 0.014), low‐density lipoprotein (LDL) cholesterol (P = 0.012) and lower triglyceride (P = 0.023). However, no associations were significant at a level which took account of multiple testing. Although JAK2 is a critical element in leptin and insulin signaling and has a role in cellular cholesterol transport, we failed to establish associations of common SNPs with relevant phenotypes in this human study.

Common STAT3 Variants Are Not Associated With Obesity or Insulin Resistance in Female Twins

In animal models, STAT3 action in the hypothalamus and liver appears essential for normal body weight and glucose homeostasis in response to insulin. We hypothesized that variation in the STAT3 gene may be associated with body fat and/or insulin resistance in the general population. Five tagging SNPs spanning the STAT3 gene, rs8074524, rs2293152, rs2306580, rs6503695, and rs7211777 were genotyped in 2776 white female twins (mean age, 47.4 ± 12.5 yrs) from the St Thomas’ United Kingdom Adult Twin Registry (Twins UK). Minor allele frequencies were as follows: rs8074524 (0.19), rs2293152 (0.37), rs2306580 (0.06), rs6503695 (0.35), and rs7211777 (0.34). The minor allele of rs2293152 was associated with higher homeostasis model assessment index of insulin resistance (p = 0.013) in the full cohort and confirmed in sib‐transmission/disequilibrium test (TDT): (p = 0.015; n = 60). However, there were no associations with fasting serum insulin or glucose or with obesity variables. Although defective STAT3 action results in obesity and insulin resistance in animal models, we failed to establish any indicative associations with common SNPs in this human study.

Functional Single-Nucleotide Polymorphism in Acetyl-CoA Carboxylase ACACB Gene Promoter

The acetyl-CoA carboxylase isoform ACC2 expressed in the liver generates malonyl-CoA, which primarily regulates fatty acid oxidation through inhibition of the mitochondrial carrier carnitine palmitoyl-CoA transferase-I. Activity is initiated by sterol regulatory element-binding protein-1 (SREBP-1) binding to steroid response elements SRE in ACACB gene promoter P-II. We proposed that sequence variation in the promoter might affect expression. We investigated the effect of a single-nucleotide polymorphism -368 C/T (rs16939972) in ACACB P-II on activity in transfected HepG2 hepatoma cells. The T-allele construct showed significantly lower activity than the C-allele (p = 0.016) but only in the presence of SREBP-1a overexpression. Electrophoretic mobility shift assays showed that HepG2 nuclear proteins bound specifically to both allele probes, but with higher affinity to the T-allele. We tested competition for nuclear protein binding between the T-allele probe and unlabeled probes containing consensus sequences for six candidate transcription factors plus SREBP-1a. The SREBP-1a competitor probe had no effect on the shifted complex. GATA, c-Myb, and GR competitor probes abolished the complex; however, these proteins were undetectable in mass spectrometry of gel extracts from shifted bands. In conclusion, the -368 C/T single-nucleotide polymorphism in ACACB P-II binds HepG2 nuclear proteins that affect promoter activity in an allele-specific fashion.