Ralph Telgmann

G/T Substitution in Intron 1 of the UNC13B Gene Is Associated With Increased Risk of Nephropathy in Patients With Type 1 Diabetes

OBJECTIVE— Genetic and environmental factors modulate the susceptibility to diabetic nephropathy, as initiating and/or progression factors. The objective of the European Rational Approach for the Genetics of Diabetic Complications (EURAGEDIC) study is to identify nephropathy susceptibility genes. We report molecular genetic studies for 127 candidate genes for nephropathy. RESEARCH DESIGN AND METHODS— Polymorphisms were identified through sequencing of promoter, exon, and flanking intron gene regions and a database search. A total of 344 nonredundant SNPs and nonsynonymous variants were tested for association with diabetic nephropathy (persistent albuminuria ≥300 mg/24 h) in a large type 1 diabetes case/control (1,176/1,323) study from three European populations. RESULTS— Only one SNP, rs2281999, located in the UNC13B gene, was significantly associated with nephropathy after correction for multiple testing. Analyses of 21 additional markers fully characterizing the haplotypic variability of the UNC13B gene showed consistent association of SNP rs13293564 (G/T) located in intron 1 of the gene with nephropathy in the three populations. The odds ratio (OR) for nephropathy associated with the TT genotype was 1.68 (95% CI 1.29–2.19) (P = 1.0 × 10−4). This association was replicated in an independent population of 412 case subjects and 614 control subjects (combined OR of 1.63 [95% CI 1.30–2.05], P = 2.3 × 10−5). CONCLUSIONS— We identified a polymorphism in the UNC13B gene associated with nephropathy. UNC13B mediates apopotosis in glomerular cells in the presence of hyperglycemia, an event occurring early in the development of nephropathy. We propose that this polymorphism could be a marker for the initiation of nephropathy. However, further studies are needed to clarify the role of UNC13B in nephropathy.

Alcohol intake modulates the genetic association between HDL cholesterol and the PPARgamma2 Pro12Ala polymorphism.

The peroxisome proliferator-activated receptor γ (PPARγ) Pro12Ala polymorphism affects plasma lipids, but to what extent alcohol intake interferes with this association remains unknown. We randomly recruited 251 nuclear families (433 parents and 493 offspring) in the framework of the European Project on Genes in Hypertension study and genotyped 926 participants in whom all serum lipid variables and information on alcohol consumption were available for PPARγ2 Pro12Ala. Genotype-phenotype relations were assessed using generalized estimating equations (GEE) and a quantitative transmission disequilibrium test (QTDT). The Ala12 allele was more frequent in Novosibirsk (0.17) than in Cracow (0.12) and Mirano (0.11) (P < 0.01). Using GEE (P = 0.03) or QTDT (P = 0.007), Italian offspring carrying the Ala12 allele had higher serum HDL cholesterol than noncarriers. HDL cholesterol levels were on average 0.086 mmol/l (P = 0.001) higher in drinkers than in nondrinkers. Compared with Pro12 homozygotes, Ala12 allele carriers consuming alcohol had higher serum total and HDL cholesterol, with the opposite trend occurring in nondrinkers. This genotype-alcohol interaction was independent of the type of alcoholic beverage and more pronounced in moderate than in heavy drinkers. We conclude that alcohol intake modulates the relation between the PPARγ2 Pro12Ala and HDL cholesterol level and that, therefore, the Pro12Ala polymorphism, pending confirmation of our findings, might affect cardiovascular prognosis.

Apolipoprotein E Interrupts Interleukin-1β Signaling in Vascular Smooth Muscle Cells

Objectives—Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1&bgr; (IL-1&bgr;), a proinflammatory cytokine present in atherosclerotic lesions. Methods and Results—IL-1&bgr;–induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-&kgr;B and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1&bgr; signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1&bgr; signaling intermediates revealed that NF-&kgr;B transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1&bgr; signaling were abolished after silencing LDL receptor–related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1&bgr; signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-&agr; or polyinosinic:polycytidylic acid. Conclusion—ApoE targets IRAK-1 activation and thereby interrupts IL-1&bgr; and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Nebivolol decreases endothelial cell stiffness via the estrogen receptor beta: a nano-imaging study.

Background Nebivolol (NEB) is a β1-receptor blocker with nitric oxide-dependent vasodilating properties. NEB-induced nitric oxide release is mediated through the estrogen receptor. Method Here, we tested the hypothesis that NEB decreases endothelial cell stiffness and that these effects can be abolished by both endothelial nitric oxide synthase and estrogen receptor blockade. Human endothelial cells (EAHy-926) were incubated with vehicle, NEB 0.7 nmol/l, metoprolol 200 nmol/l, 17β-estradiol (E2) 15 nmol/l, the estrogen receptor antagonists tamoxifen 100 nmol/l and ICI 182780 (ICI) 100 nmol/l, the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester 1 mmol/l and combinations of NEB and E2 with either tamoxifen, ICI or Nω-nitro-L-arginine methyl ester as well as metoprolol and ICI. Atomic force microscopy was performed to measure cellular stiffness, cell volume and apical surface. Presence of estrogen receptor protein in EAHy-926 was confirmed by western blot analysis; quantification of ERα and ERβ total RNA was performed by semiquantitative PCR. Results Both NEB as well as E2 decreased cellular stiffness to a similar extent (NEB: 0.83 ± 0.03 pN/nm, E2: 0.87 ± 0.03 pN/nm, vehicle: 2.19 ± 0.07 pN/nm), whereas metoprolol had no effect on endothelial stiffness (2.07 ± 0.04 pN/nm, all n = 60, P < 0.01). The decrease in stiffness occurred as soon as 5 min after starting NEB incubation. The effects are mediated through nongenomic ERβ pathways, as ERα is not translated into measurable protein levels in EAHy-926. Furthermore, NEB increased cell volume by 48 ± 4% and apical surface by 34 ± 3%. E2 had comparable effects. Tamoxifen, ICI and Nω-nitro-L-arginine methyl ester substantially diminished the effects of NEB and E2. Conclusion NEB decreases cellular stiffness and causes endothelial cell growth. These effects are nitric oxide-dependent and mediated through nongenomic ERβ pathways. The morphological and functional alterations observed in endothelial cells may explain improved endothelial function with NEB treatment.

Molecular genetic analysis of a human insulin-like growth factor 1 promoter P1 variation

Insulin‐like growth factor 1 (IGF1) exerts important endocrine and paracrine functions in the cardiovascular system. We identified the common variant – 1411C>T in the IGF1 upstream promoter P1, located within several overlapping transcription factor binding sites. Using transient transfection assays, we identified this site as a functional enhancer. The T allele‐carrying enhancer, compared with the C allelic portion, exerts significantly reduced or even abrogated activity, respectively, inSaOs‐2 and HepG2 (all p<0.0001) as well as in differentiatedTHP‐1 macrophages. Electrophoretic mobility shift assay and subsequent supershift experiments in HepG2 identified c‐Jun as the binding partner exclusively to the T allele, whereas CCAAT/enhancer‐binding protein 8 and interferon consensus site‐binding protein/interferon‐regulating factor 8 interacted only with the C allelic promoter portion. Furthermore, genotyping in a case‐control study for essential hypertension (n=745 hypertensive patients; n=769 normotensive control subjects) for this variant revealed an odds ratio for hypertension of 0.73 (95% confidence interval 0.58‐0.91, 7=0.006) associated with the T allele, and normotensive subjects carrying the protective T allele displayed a significant decrease in diastolic (7=0.036) and systolic (7=0.024) blood pressure levels. We here report detection of a functional enhancer module in the upstream IGF1 promoter region, which might play a key role in local IGF1 bioavailability. Whether – 1411C>T is also associated with other IGF1‐related disease phenotypes should be evaluated further in population studies.— Telgmann, R., Dordelmann, C., Brand, E., Nicaud, V., Hagedorn, C., Pavenstadt, H., Cambien, F., Tiret, L., Paul, M., Brand‐Herrmann, S.‐M. Molecular genetic analysis of a human insulin growth factor 1 promoter P1 variation. FASEBJ. 23, 1303–1313 (2009)

Hypertension in mice lacking the CXCR3 chemokine receptor

The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3-/- mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3-/- mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3-/- mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased AT1R expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression.

Characterization and Functional Analyses of the Human G Protein–Coupled Receptor Kinase 4 Gene Promoter

The G protein–coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5′-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The −1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein–coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type– and stimulation-dependent transcription and may affect the expression balance of renal G protein–coupled receptor kinase 4.

SAH gene variants are associated with obesity-related hypertension in Caucasians: the PEGASE Study.

Objective The SAH gene locus has recently been proposed to be involved in obesity-related hypertension in Japanese individuals. Methods To replicate independently the initial findings in another ethnic group, we scanned the entire SAH gene in 190 Caucasian chromosomes. A total of 651 patients with essential hypertension and 776 controls (PEGASE Study) were genotyped for all identified variants using allele-specific oligonucleotides, and single nucleotide polymorphism as well as haplotype analyses were carried out. We also performed transient transfection experiments, northern and western blots, immunoprecipitation, and acyl-coenzyme A synthetase activity assays. Results We identified five polymorphisms in the promoter region (C−1808T, G−1606A, −962ins/del, G−451A, T−67C), two in introns 5 and 7 (T+9/In5C, A+20/In7T), and one missense variant (K359N). Carriage of the −1606A allele was significantly associated with hypertension [odds ratio (OR) 1.28, P = 0.049] as was 359N (OR 1.35, P = 0.048) compared with non-carriers. Conversely, for −962del, the OR for hypertension was 0.80 (P = 0.042). The SAH alleles −1606A and 359N, but not −962ins/del, displayed a raising effect on body mass index (BMI; P = 0.004 and P = 0.030, respectively) in hypertensive as well as in control individuals. After adjustment for BMI in hypertensive individuals, only the OR associated with −962ins/del remained significant (OR 0.77, P = 0.028). Functional analyses in BHK did not reveal differences for SAH 359N or 359K-containing constructs, formally excluding K359N as the functional variant. Conclusion We confirm recent evidence that the SAH locus is associated with obesity-related hypertension, in which pathophysiological context SAH variants affecting blood pressure remain, however, to be shown.

Apolipoprotein E Interrupts Interleukin-1 Signaling in Vascular Smooth Muscle Cells

Objectives—Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1&bgr; (IL-1&bgr;), a proinflammatory cytokine present in atherosclerotic lesions. Methods and Results—IL-1&bgr;–induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-&kgr;B and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1&bgr; signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1&bgr; signaling intermediates revealed that NF-&kgr;B transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1&bgr; signaling were abolished after silencing LDL receptor–related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1&bgr; signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-&agr; or polyinosinic:polycytidylic acid. Conclusion—ApoE targets IRAK-1 activation and thereby interrupts IL-1&bgr; and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Functional and Structural Profiling of the Human Thrombopoietin Gene Promoter

Human thrombopoietin (TPO) is involved in cardiovascular disease as it regulates megakaryocyte development and enhances platelet adhesion/aggregation. The THPO promoter structure is still controversial. By reverse transcription-PCR, we confirm that THPO transcription is cell line-dependently initiated at two alternative promoters, which we newly designated P1a and P1. We subsequently electrophoretically scanned and resequenced these portions in 95 and 57 patients with cardiovascular disease, respectively, and identified seven variants (–1450/del58bp, C-920T [rs2855306], A-622G, C-413T [rs885838], C+5A, G+115A, and C+135T). After subcloning of 1032 bp of THPO P1 in pGL3-basic vector, five molecular haplotypes (MolHaps1–5) were observed: [A–622-C–413-C+5-G+115; wild type (wt)], [A–622-T–413-C+5-G+115], [G–622-T–413-C+5-G+115], [A–622-C–413-A+5-G+115], [A–622-C–413-C+5-A+115], and analyzed in reporter gene assays in HEK293T and HepG2 cells. MolHaps 2, 4, and 5 were significantly more active than wt (all p values ≤0.01) in HEK293T cells, MolHap3 exerted a substantial loss of promoter activity (p < 0.0001 in HEK293T and p < 0.01 in HepG2, compared with wt). Electrophoretic mobility shift assays revealed that A-622G and C-413T individually differed from MolHaps in their DNA-protein interaction patterns. Supershift and chromatin immunoprecipitation assays identified CCAAT/enhancer-binding protein δ as the binding protein exclusively for the –622A allelic portion.