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Background: Evidence is accumulating that vitamin D supplementation of patients with low 25-hydroxyvitamin D concentrations is associated with lower cardiovascular morbidity and total mortality during long-term follow-up. Little is known, however, about the effect of low concentrations of the vitamin D hormone calcitriol on total mortality. We therefore evaluated the predictive value of circulating calcitriol for midterm mortality in patients of a specialized heart center., Methods: This prospective cohort study included 510 patients, 67.7% with heart failure (two-thirds in end stage), 64.3% hypertension, 33.7% coronary heart disease, 20.2% diabetes, and 17.3% renal failure. We followed the patients for up to 1 year after blood collection. For data analysis, the study cohort was stratified into quintiles of circulating calcitriol concentrations., Results: Patients in the lowest calcitriol quintile were more likely to have coronary heart disease, heart failure, hypertension, diabetes, and renal failure compared to other patients. They also had low 25-hydroxyvitamin D concentrations and high concentrations of creatinine, C-reactive protein, and tumor necrosis factor alpha. Eighty-two patients (16.0%) died during follow-up. Probability of 1-year survival was 66.7% in the lowest calcitriol quintile, 82.2% in the second quintile, 86.7% in the intermediate quintile, 88.8% in the fourth quintile, and 96.1% in the highest quintile (P < 0.001). Discrimination between survivors and nonsurvivors was best when a cutoff value of 25 ng/L was applied (area under the ROC curve 0.72; 95% CI 0.66-0.78)., Conclusions: Decreased calcitriol levels are linked to excess midterm mortality in patients of a specialized heart center.

Background: An accumulation of extracellular matrix molecules, such as proteoglycans, is observed in the vascular wall of hypertensive patients. Xylosyltransferases I and II (XT-I and XT-II), the chain-initiating enzymes in the biosynthesis of proteoglycans, catalyze the transfer of D-xylose from UDP-D-xylose to specific serine residues of the core protein. Because associations between XYLT polymorphisms and an altered blood pressure have been observed, genetic variations in the XYLT genes might predispose to essential hypertension. The localization of the XYLT2 gene on chromosome 17q increases its attractiveness as this region has been reported to be a potential candidate locus for essential hypertension., Methods: Genotyping of four polymorphisms in the genes XYLT1 and XYLT2 was performed in 150 unrelated essential hypertension patients and 150 age- and sex-matched normotensive controls using restriction fragment length polymorphism analysis., Results: The allele and genotype frequencies of the XYLT variants investigated did not show any significant differences between patients and controls, among allele-carriers and nonallele-carriers and among recessive and nonrecessive allele-carriers comparing patients and controls. Systolic and diastolic blood pressures did not differ significantly between the genotypes concerning all XYLT variants analyzed. Two XYLT2 variants deviated from Hardy-Weinberg equilibrium (HWE) in the hypertensive group., Conclusions: No statistically significant association was found between four XYLT variants and hypertension or blood pressure, suggesting that they do not play a significant role in the development of essential hypertension. The deviation from HWE of two XYLT2 variants might be due to gene-phenotype associations which remain to be explored, as well as the possibility of gene-gene interactions.

Background: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by progressive calcification and fragmentation of elastic fibers in connective tissues. PXE is caused by mutations in the ABCC6 gene, which encodes the membrane transporter multidrug resistance-associated protein 6. Chronic oxidative stress was recently suggested to play a crucial role in the pathogenesis of the disease. Our aim was to investigate the association of PXE with genetic variation in genes coding for antioxidant enzymes., Methods: We used restriction fragment length polymorphism and allele-specific PCR analyses to evaluate the distribution of single-nucleotide polymorphisms in the genes encoding catalase (CAT), superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 (GPX1) in DNA samples from 117 German PXE patients and 117 healthy age- and sex-matched control individuals., Results: The investigated genetic variants had previously been shown to affect the activities of these antioxidant enzymes. We found a correlation between genotype and age of disease onset for polymorphisms in CAT (c.-262C>T), SOD2 (c.47C>T), and GPX1 (c.593C>T). Furthermore, the age of disease onset was inversely correlated with the number of mutated alleles, indicating a cumulative effect on the time of disease onset [mean (SD) age of 40.9 (13.6) years, 32.4 (16.3) years, and 25.7 (15.9) years for carriers of 0, 1-2, and >2 mutated alleles, respectively; P = 0.03]., Conclusion: Our findings demonstrate that increased oxidative stress due to activity-affecting polymorphisms in genes encoding antioxidant enzymes leads to earlier PXE onset.

Background: Progressive calcification and fragmentation of elastic fibers are characteristic hallmarks of pseudoxanthoma elasticum (PXE), which is caused by mutations in ABCC6 encoding multidrug resistance-associated protein 6 (MRP6). Because of the great clinical variability of PXE, secondary genetic risk factors are suspected to exist. We investigated whether SPP1 (secreted phosphoprotein 1; previously OPN, osteopontin) promoter polymorphisms are associated with PXE., Methods: We screened an approximately 2-kb region spanning the theoretical promoter of the SPP1 gene for sequence variations by denaturing HPLC and direct sequencing in 93 PXE patients. Sequence variations with a prevalence >5% were genotyped in 93 age- and sex-matched healthy controls. Statistical and haplotype association analyses were performed using Fisher exact test, PHASE v2.1.1, and Haploview 3.2., Results: Mutational screening revealed 9 different sequence variations. Three SPP1 promoter polymorphisms (c.-1748A>G, c.-155&#95;156insG, and c.244&#95;245insTG) were significantly more frequent in PXE patients than in 93 age- and sex-matched healthy controls (P(corrected) < 0.05 each). The odds ratios (95% CI) for PXE among carriers of the 3 alleles were, respectively, 2.16 (1.34-3.48), 2.41 (1.51-3.82), and 1.97 (1.23-3.15). Haplotype analysis of 6 SPP1 promoter polymorphisms revealed 1 haplotype to be significantly reduced among PXE patients (P(corrected) = 0.035, odds ratio 1.80, 95% CI 1.19-2.71)., Conclusions: Polymorphisms in the SPP1 promoter are secondary genetic risk factors contributing to PXE susceptibility.

Background: Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients., Methods: We developed an HPLC electrospray ionization tandem mass spectrometry method to assay XT-I activity in serum by use of a synthetic peptide (Bio-BIK-F) as the XT-I substrate. On the basis of XT-I-mediated transfer of D-xylose from UDP-D-xylose to the synthetic peptide to form Bio-BIK-F-Xyl, we determined XT-I activity in human serum samples., Results: Multiple calibration curves for the analysis of Bio-BIK-F-Xyl exhibited consistent linearity and reproducibility in the range of 0.20-20 mg/L, corresponding to XT-I activity of 1.14-114 mU/L under assay conditions. The mean (SD, range) XT-I activity values in 30 blood donor sera were 18.4 (3.0, 8.7-24.8) mU/L. The limit of detection and lower limit of quantification were 8.5 microg/L (0.05 mU/L) and 163 microg/L Bio-BIK-F-Xyl (0.93 mU/L XT-I activity), respectively. Interassay imprecision (CV) was 5.4%-26.1% in the range of 0.64 to 129 mU/L, and mean recovery was 107% (range, 96%-129%). Method comparison with the radiochemical assay showed a moderate correlation (r = 0.79). The Passing-Bablok regression line was: radiochemical assay = 0.045 LC-MS/MS + 0.061 mU/L, S(y/x) = 0.186., Conclusions: This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.

In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT-I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 +/- 0.58 (mean +/- SD)-fold increase. This result correlates with significantly elevated levels of enzymatic XT-I activity (0.49 +/- 0.03 muU/1 x 10(6) cells) at this time point. Immunohistochemical staining of XT-I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT-II showed 4.7-fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (alpha-4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to -87-fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT-I, are useful markers for the developmental stages of chondrogenic differentiation.

Background: Pseudoxanthoma elasticum (PXE) is a hereditary disorder of the connective tissue affecting the skin, retina, and cardiovascular system and characterized by progressive calcification of abnormal and fragmented elastic fibers in the extracellular matrix. The aim of the present study was to investigate the association of fetuin-A, a major systemic inhibitor of calcification, with PXE., Methods: Fetuin-A was measured by quantitative sandwich enzyme immunoassay in sera from 110 German patients with PXE, 53 unaffected first-degree family members, and 80 healthy blood donors. We determined the distribution of the fetuin-A polymorphisms c.742C>T (p.T248M) and c.766C>G (p.T256S) in these same 3 groups. The occurrences of the frequent ABCC6 gene mutations c.3421C>T (p.R1141X) and c.EX23&#95;EX29del were also assessed., Results: Serum fetuin-A concentrations in male and female PXE patients were lower than in unaffected first-degree relatives and controls [mean (SD) concentrations, 0.55 (0.11) g/L in patients; 0.70 (0.23) g/L in relatives; and 0.80 (0.23) g/L in controls (P <0.0001)]. Serum fetuin-A was higher in female PXE patients with cardiovascular involvement than in the corresponding male group (P <0.05). The fetuin-A polymorphism frequencies did not differ among PXE patients, family members, and blood donors., Conclusion: A deficiency of multidrug resistance-associated protein 6 leads to alteration of circulating substrates, e.g., inhibitors of calcification as fetuin-A, leading to progressive mineralization of elastic fibers in PXE.