IRP2 as a potential modulator of cell proliferation, apoptosis and prognosis in nonsmall cell lung cancer.

IREB2 is a gene that produces iron regulatory protein 2 (IRP2), which is critical to intracellular iron homeostasis and which relates to the rate of cellular proliferation. IREB2 lies in a lung cancer susceptibility locus. The aims were to assess 1) the relationship between iron loading, cell proliferation and IRP2 expression in lung cancer; 2) the potential of iron related pathways as therapeutic targets; and 3) the relevance of IRP2 in operated lung cancer patients.Cells of two nonsmall cell cancer (NSCLC) lines and primary bronchial epithelial cells (PBECs) were cultured with and without iron; and proliferation, apoptosis and migration were assessed. Reverse transcriptase PCR and Western blot were used to assess expression of iron homeostasis genes/proteins. Iron chelation and knockdown of IREB2 were used in vitro to explore therapeutics. A cohort of operated NSCLC patients was studied for markers of systemic iron status, tumour IRP2 staining and survival.Iron loading caused cell proliferation in cancer cell lines, which were less able to regulate IREB2 expression than PBECs. Iron chelation resulted in a return of proliferation rates to baseline levels; knockdown of IREB2 had a similar effect. IRP2-positive tumours were larger (p=0.045) and higher percentage staining related to poorer survival (p=0.079).Loss of iron regulation represents a poor prognostic marker in lung cancer.

Coronary Artery Disease-Associated LIPA Coding Variant rs1051338 Reduces Lysosomal Acid Lipase Levels and Activity in Lysosomes.

OBJECTIVE: Genome-wide association studies have linked variants at chromosome 10q23 with increased coronary artery disease risk. The disease-associated variants fall in LIPA, which encodes lysosomal acid lipase (LAL), the enzyme responsible for lysosomal cholesteryl ester hydrolysis. Loss-of-function mutations in LIPA result in accelerated atherosclerosis. Surprisingly, the coronary artery disease variants are associated with increased LIPA expression in some cell types. In this study, we address this apparent contradiction. APPROACH AND RESULTS: We investigated a coding variant rs1051338, which is in high linkage disequilibrium (r2=0.89) with the genome-wide association study lead-associated variant rs2246833 and causes a nonsynonymous threonine to proline change within the signal peptide of LAL. Transfection of allele-specific expression constructs showed that the risk allele results in reduced lysosomal LAL protein (P=0.004) and activity (P=0.005). Investigation of LAL localization and turnover showed the risk LAL protein is degraded more quickly. This mechanism was confirmed in disease-relevant macrophages from individuals homozygous for either the nonrisk or risk allele. There was no difference in LAL protein or activity in whole macrophage extracts; however, we found reduced LAL protein (P=0.02) and activity (P=0.026) with the risk genotype in lysosomal extracts, suggesting that the risk genotype affects lysosomal LAL activity. Inhibition of the proteasome resulted in equal amounts of lysosomal LAL protein in risk and nonrisk macrophages. CONCLUSIONS: Our findings show that the coronary artery disease-associated coding variant rs1051338 causes reduced lysosomal LAL protein and activity because of increased LAL degradation, providing a plausible causal mechanism of increased coronary artery disease risk.

Long-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene.

The chromosome 16p13 region has been associated with several autoimmune diseases, including type 1 diabetes (T1D) and multiple sclerosis (MS). CLEC16A has been reported as the most likely candidate gene in the region, since it contains the most disease-associated single-nucleotide polymorphisms (SNPs), as well as an imunoreceptor tyrosine-based activation motif. However, here we report that intron 19 of CLEC16A, containing the most autoimmune disease-associated SNPs, appears to behave as a regulatory sequence, affecting the expression of a neighbouring gene, DEXI. The CLEC16A alleles that are protective from T1D and MS are associated with increased expression of DEXI, and no other genes in the region, in two independent monocyte gene expression data sets. Critically, using chromosome conformation capture (3C), we identified physical proximity between the DEXI promoter region and intron 19 of CLEC16A, separated by a loop of >150 kb. In reciprocal experiments, a 20 kb fragment of intron 19 of CLEC16A, containing SNPs associated with T1D and MS, as well as with DEXI expression, interacted with the promotor region of DEXI but not with candidate DNA fragments containing other potential causal genes in the region, including CLEC16A. Intron 19 of CLEC16A is highly enriched for transcription-factor-binding events and markers associated with enhancer activity. Taken together, these data indicate that although the causal variants in the 16p13 region lie within CLEC16A, DEXI is an unappreciated autoimmune disease candidate gene, and illustrate the power of the 3C approach in progressing from genome-wide association studies results to candidate causal genes.

Evidence for genetic regulation of endothelial progenitor cells and their role as biological markers of atherosclerotic susceptibility.

AIMS: Endothelial progenitor cells (EPCs) are found in the peripheral circulation and are capable of endothelial repair and neovascularization. EPC number and function are reduced in subjects with cardiovascular risk factors or proven coronary artery disease (CAD). We hypothesized that EPC number and/or function may be genetically regulated and may vary in healthy adult offspring depending on parental history of CAD. METHODS AND RESULTS: We studied 102 subjects comprising 24 healthy parent-healthy offspring pairs and 27 CAD parent-healthy offspring pairs. We measured the number of circulating CD34(+)VEGFR-2(+) and AC133(+)VEGFR-2(+) EPCs, the number of EPCs grown in culture, and the migration capacity of cultured EPCs towards vascular endothelial growth factor. There was significant correlation in the number of cultured EPCs between healthy parents and their offspring (R = 0.492, P = 0.015) and CAD parents and their offspring (R = 0.751, P < 0.001). Offspring of subjects with CAD had significantly higher numbers of circulating CD34(+)VEGFR-2(+) and AC133(+)VEGFR-2(+) cells (P = 0.018 and P < 0.001, respectively). There was no difference in migration capacity between groups. CONCLUSION: Our results suggest that EPC number is, at least in part, genetically regulated. Circulating EPCs may represent biological markers of occult vascular damage in offspring with hereditary risk of CAD.

Telomere length, risk of coronary heart disease, and statin treatment in the West of Scotland Primary Prevention Study: a nested case-control study.

BACKGROUND: Inter-individual differences in biological ageing could affect susceptibility to coronary heart disease. Our aim was to determine whether mean leucocyte telomere length is a predictor of the development of coronary heart disease. METHODS: We compared telomere lengths at recruitment in 484 individuals in the West of Scotland Primary Prevention Study (WOSCOPS) who went on to develop coronary heart disease events with those from 1058 matched controls who remained event free. We also investigated whether there was any association between telomere length and observed clinical benefit of statin treatment in WOSCOPS. FINDINGS: Mean telomere length decreased with age by 9% per decade (95% CI 3.6-14.1; p=0.001) in controls; much the same trend was seen in cases (-5.9% per decade, -3.1 to 14.1; p=0.1902). Individuals in the middle and the lowest tertiles of telomere length were more at risk of developing a coronary heart disease event than were individuals in the highest tertile (odds ratio [OR] for coronary heart disease: 1.51, 95% CI 1.15-1.98; p=0.0029 in the middle tertile; 1.44, 1.10-1.90, p=0.0090 in the lowest). In placebo-treated patients, the risk of coronary heart disease was almost double in those in the lower two tertiles of telomere length compared with those in the highest tertile (1.93, 1.33-2.80, p=0.0005 in the middle tertile; 1.94, 1.33-2.84, p=0.0006 in the lowest). By contrast, in patients treated with pravastatin, the increased risk with shorter telomeres was substantially attenuated (1.12, 0.75-1.69, p=0.5755 in the middle tertile; 1.02, 0.68-1.52, p=0.9380 in the lowest). INTERPRETATION: Mean leucocyte telomere length is a predictor of future coronary heart disease events in middle-aged, high-risk men and could identify individuals who would benefit most from statin treatment. Our findings lend support to the hypothesis that differences in biological ageing might contribute to the risk--and variability in age of onset--of coronary heart disease.

Expression of renal 11beta-hydroxysteroid dehydrogenase type 2 is decreased in patients with impaired renal function.

OBJECTIVE: Renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enables selective access of aldosterone to the mineralocorticoid receptor (MR). Impaired 11beta-HSD2 activity has been suggested in patients with hypertension as well as in patients with renal disease, where it may contribute to sodium retention, oedema and hypertension. To date, these studies have relied upon urinary cortisol (F) metabolite levels as surrogate markers of renal 11beta-HSD2 activity. METHODS: We have directly analysed renal 11beta-HSD2 mRNA expression in 95 patients undergoing kidney biopsy using TaqMan real-time PCR. Serum and 24-h urine samples were used to document underlying renal function and endocrine parameters. Urinary F and cortisone (E) metabolites were analysed using gas chromatography/mass spectrometry. RESULTS: Expression of 11beta-HSD2 did not correlate with blood pressure or urinary Na/K ratio, but a significant positive correlation with creatinine clearance was observed (r = 0.284; P < 0.01). Immunofluorescence and confocal laser microscopy confirmed decreased 11beta-HSD2 expression in patients with impaired renal function. For the first time, we showed that 11beta-HSD2 mRNA expression correlated negatively with the urinary free (UF) F/E (UFF/UFE) ratio (r = 0.276; P < 0.05) as well as with the urinary tetrahydrocortisol + 5alpha-tetrahydrocortisol/tetrahydrocortisone ((THF + alphaTHF)/THE) ratio (r = 0.256; P < 0.05). No difference in 11beta-HSD2 mRNA expression or in the UFF/UFE ratio was found between groups with no proteinuria, microalbuminuria, moderate or severe proteinuria. In contrast, the urinary (THF + alphaTHF)/THE ratio increased significantly (P < 0.05) in patients with severe albuminuria, suggesting increased hepatic 11beta-HSD1 in those patients. CONCLUSIONS: These data suggest that renal 11beta-HSD2 expression may be represented only marginally better, if at all, by the UFF/UFE than by the (THF + alphaTHF)/THE ratio. Reduced renal 11beta-HSD2 expression may lead to occupancy of the MR by glucocorticoids such as cortisol and may contribute to the increased sodium retention seen in patients with impaired renal function.

Weight loss increases 11beta-hydroxysteroid dehydrogenase type 1 expression in human adipose tissue.

The global epidemic of obesity has heightened the need to understand the mechanisms that underpin its pathogenesis. Clinical observations in patients with Cushing's syndrome have highlighted the link between cortisol and central obesity. However, although circulating cortisol levels are normal or reduced in obesity, local regeneration of cortisol, from inactive cortisone, by 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) has been postulated as a pathogenic mechanism. Although levels of expression of 11betaHSD1 in adipose tissue in human obesity are debated in the literature, global inhibition of 11betaHSD1 improves insulin sensitivity. We have determined the effects of significant weight loss on cortisol metabolism and adipose tissue 11betaHSD1 expression after 10-wk ingestion of a very low calorie diet in 12 obese patients (six men and six women; body mass index, 35.9 +/- 0.9 kg/m2; mean +/- SE). All patients achieved significant weight loss (14.1 +/- 1.3% of initial body weight). Total fat mass fell from 41.8 +/- 1.9 to 32.0 +/- 1.7 kg (P < 0.0001). In addition, fat-free mass decreased (64.4 +/- 3.4 to 58.9 +/- 2.9 kg; P < 0.0001) and systolic blood pressure and total cholesterol also fell [systolic blood pressure, 135 +/- 5 to 121 +/- 5 mm Hg (P < 0.01); total cholesterol, 5.4 +/- 0.2 to 4.8 +/- 0.2 mmol/liter (P < 0.05)]. The serum cortisol/cortisone ratio increased after weight loss (P < 0.01). 11betaHSD1 mRNA expression in isolated adipocytes increased 3.4-fold (P < 0.05). Decreased 11betaHSD1 activity and expression in obesity may act as a compensatory mechanism to enhance insulin sensitivity through a reduction in tissue-specific cortisol concentrations. Inhibition of 11betaHSD1 may therefore be a novel, therapeutic strategy for insulin sensitization.