First identification and functional analysis of the human xylosyltransferase II promoter.

Recently, we demonstrated that the human xylosyltransferase II (XT-II) has enzymatic activity and is able to catalyze the initial and rate-limiting step in the biosynthesis of glycosaminoglycans (GAGs) like chondroitin and dermatan sulfate, as well as heparan sulfate and heparin. Therefore, this enzyme also very likely assumes a crucial regulatory role in the biosynthesis of proteoglycans (PGs). In this study, we identified and characterized for the first time the XYLT2 gene promoter region and transcription factors involved in its regulation. Several binding sites for members of the Sp1 family of transcription factors were identified as being necessary for transcriptional regulation of the XYLT2 gene. This was determined by mithramycin A treatment, electrophoretic mobility shift and supershift assays, as well as numerous site-directed mutagenesis experiments. Different 5' and 3' deletion constructs of the predicted GC rich promoter region, which lacks a canonical TATA and CAAT box, revealed that a 177 nts proximal promoter element is sufficient and indispensable to drive the constitutive transcription in full strength in HepG2 hepatoma cells. In addition, we also detected the transcriptional start site using 5'-RACE (rapid amplification of cDNA ends). Our results provide an insight into transcriptional regulation of the XYLT2 gene and may contribute to understanding the manifold GAG-involving processes in health and disease.

Analysis of MMP2 promoter polymorphisms in patients with pseudoxanthoma elasticum.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder predominantly affecting the skin, the eyes and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling and are likely implied in PXE pathology. The aim of the present study was to investigate the association of single nucleotide polymorphisms (SNPs) in the promoter of the MMP2 gene, and PXE. METHODS: We evaluated the allelic distribution of five SNPs in the MMP2 promoter in DNA samples from 168 German patients affected by PXE and in 168 healthy, age- and sex-matched control subjects using restriction fragment length polymorphism analysis. RESULTS: The alleles c.-1575G, c.-1306C, and c.-790T were more abundant in the PXE patients' group. Furthermore, the haplotype GCTCG was significantly associated with PXE (OR 1.56, 95% CI 1.14-2.12, P(corrected)=0.026). CONCLUSIONS: Our results may indicate an involvement of MMP2 in the pathology of PXE. The promoter polymorphisms associated with PXE may lead to increased MMP2 expression and thereby contribute to the elevated proteolytic activity observed in PXE in vitro and in vivo.

Involvement of a cysteine protease in the secretion process of human xylosyltransferase I.

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.

Differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans.

The xylosyltransferase (XT) isoforms XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis. Here, we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression, while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines. Additionally, to shed light on putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined. Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans. Kinetic analysis showed that K(m) and V(max) values for XT-I mediated xylosylation were slightly higher than those for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence. Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here, mutations on the N-terminal side and the attachment site were found to be more susceptible to a loss of acceptor function than mutations in the C-terminus. Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp or Glu) for XT-I mediated xylosylation could be approved and additionally extended to apply to XT-II as well.

Identification and characterization of the human xylosyltransferase I gene promoter region.

Human xylosyltransferase I catalyzes the initial and rate-limiting step in the biosynthesis of glycosaminoglycans and proteoglycans. Furthermore, this enzyme has been shown to play a major role in the physiological development of bone and cartilage as well as in pathophysiological processes such as systemic sclerosis, dilated cardiomyopathy, or fibrosis. Here, we report for the first time the identification and characterization of the XYLT1 gene promoter region and important transcription factors involved in its regulation. Members of the activator protein 1 (AP-1) and specificity protein 1 (Sp1) family of transcription factors are necessary for the transcriptional regulation of the XYLT1 gene, which was proven by curcumin, tanshinone IIA, mithramycin A, and short interference RNA treatment. A stepwise 5' and 3' deletion of the predicted GC-rich promoter region, which lacks a TATA and/or CAAT box, revealed that a 531-bp core promoter element is able to drive the transcription on a basal level. A binding site for transcription factors of the AP-1 family, which is essential for full promoter activity, was identified by site-directed mutagenesis located 730 bp 5' of the translation initiation site. The ability of this site to bind members of the AP-1 family was further verified by electrophoretic mobility shift assays. A promoter element containing this binding site was able to drive the transcription to about 79-fold above control in SW1353 chondrosarcoma cells. Our findings provide a first insight into the regulation of the XYLT1 gene and may contribute to understanding the processes taking place during extracellular matrix formation and remodeling in health and disease.

Vascular endothelial growth factor gene polymorphisms as prognostic markers for ocular manifestations in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder affecting the skin, eyes and cardiovascular system. It is caused by mutations in the ABCC6 gene and its clinical picture is highly variable. PXE often leads to severe visual impairment due to the development of choroidal neovascularisation (CNV). CNV in PXE-associated retinopathy is believed to be mediated by the action of vascular endothelial growth factor (VEGF). The objective of the present study was to evaluate a possible impact of variations in the VEGFA gene on ocular manifestations of PXE. For this purpose, we evaluated the distribution of 10 single nucleotide polymorphisms (SNPs) in the promoter and coding region of the VEGFA gene in DNA samples from 163 German patients affected by PXE and in 163 healthy control subjects. Haplotype analysis of SNPs c.-1540A>C, c.-460C>T, c.-152G>A, c.405C>G, c.674C>T, c.1032C>T, c.4618C>T and c.5092C>A revealed that the haplotype CTGGCCCC was associated with PXE (OR 2.05, 95% CI 1.33-3.15, P(corrected) = 0.01). Furthermore, five SNPs showed significant association with severe retinopathy. The most significant single SNP association was c.-460C>T (OR 3.83, 95% CI 2.01-7.31, P(corrected) = 0.0003). Logistic regression analysis identified the c.-460T and the c.674C alleles as independent risk factors for development of severe retinopathy. Our findings suggest an involvement of VEGF in the pathogenesis of ocular PXE manifestations. VEGF gene polymorphisms might prove useful as prognostic markers for the development of PXE-associated retinopathy and permit earlier therapeutic intervention in order to prevent loss of central vision, one of the most devastating consequences of this disease.

Quantitative determination and comparison of the glycosaminoglycan Delta-disaccharide composition in 22 different human cell lines.

Changes in proteoglycan and glycosaminoglycan (GAG) content and distribution may play an important role in the development of many diseases, atherosclerosis, cancer and diabetes. Human cell lines act as models for the underlying pathomechanisms. Despite the importance of proteoglycans for cell functioning, information on the GAG composition of most human cell lines is limited. Comparative analysis of the GAG Deltadisaccharide amount in 22 human cell lines yielded a mean value of 94 +/- 58 pmol/10(6) cells (mean+/-SEM). Total GAG amount and heparan sulfate/heparin Deltadisaccharide composition, but not chondroitin sulfate/dermatan sulfate Deltadisaccharide composition, differed significantly between the investigated adherent and suspension cell lines. We provide a novel overview of GAG Deltadisaccharide composition in 22 different human cell lines.

Circulating calcitriol concentrations and total mortality.

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.

Vitamin D supplementation enhances the beneficial effects of weight loss on cardiovascular disease risk markers.

BACKGROUND: High blood concentrations of parathyroid hormone and low concentrations of the vitamin D metabolites 25-hydroxyvitamin D [25(OH)D] and calcitriol are considered new cardiovascular disease risk markers. However, there is also evidence that calcitriol increases lipogenesis and decreases lipolysis. OBJECTIVE: We investigated the effect of vitamin D on weight loss and traditional and nontraditional cardiovascular disease risk markers in overweight subjects. DESIGN: Healthy overweight subjects (n = 200) with mean 25(OH)D concentrations of 30 nmol/L (12 ng/mL) received vitamin D (83 microg/d) or placebo in a double-blind manner for 12 mo while participating in a weight-reduction program. RESULTS: Weight loss was not affected significantly by vitamin D supplementation (-5.7 +/- 5.8 kg) or placebo (-6.4 +/- 5.6 kg). However, mean 25(OH)D and calcitriol concentrations increased by 55.5 nmol/L and 40.0 pmol/L, respectively, in the vitamin D group but by only 11.8 nmol/L and 9.3 pmol/L, respectively, in the placebo group (P < 0.001), whereas a more pronounced decrease occurred in the vitamin D group than in the placebo group in blood concentrations of parathyroid hormone (-26.5% compared with -18.7%; P = 0.014), triglycerides (-13.5% compared with +3.0%; P < 0.001), and the inflammation marker tumor necrosis factor-alpha (-10.2% compared with -3.2%; P = 0.049). The beneficial biochemical effects were independent of the loss in body weight, fat mass, and sex. However, compared with placebo, vitamin D supplementation also increased LDL-cholesterol concentrations (+5.4% compared with -2.5%; P < 0.001). CONCLUSIONS: The results indicate that a vitamin D supplement of 83 microg/d does not adversely affect weight loss and is able to significantly improve several cardiovascular disease risk markers in overweight subjects with inadequate vitamin D status participating in a weight-reduction program. This trial was registered at clinicaltrials.gov as NCT00493012.

Characterization of the ATP-binding cassette transporter gene expression profile in Y79: a retinoblastoma cell line.

Chemotherapy failure was reported in treatment of retinoblastoma suggesting a role for ATP-binding cassette (ABC) proteins. Little is known about the expression pattern of ABC proteins in this cancer type. We investigated the gene expression profile of 47 ABC proteins in the human retinoblastoma cell line Y79 by TaqMan low-density array. Analysis revealed 31 ABC transporter genes expressed in this tumor cell line. Y79 cells demonstrate high gene expression of ABCA7, ABCA12, ABCB7, ABCB10, ABCC1, ABCC4, ABCD3, ABCE1, ABCF1, ABCF2, and ABCF3 (more than twofold compared to pooled RNA from different tissues). Moreover, we show that Y79 cells exhibit an active calcein efflux pointing to multidrug resistance protein (MRP)-like transporter activity. In summary, we present for the first time an ABC transporter gene expression profile in cells derived from retinoblastoma. Most of the highly expressed ABC transporter genes are typical markers of cancer cells and might exhibit potential targets for medical treatment of retinoblastoma.

Calcitriol deficiency and 1-year mortality in cardiac transplant recipients.

BACKGROUND: Administration of the vitamin D hormone calcitriol improves survival in solid-organ transplanted experimental animals. We investigated whether lower serum calcitriol concentrations are associated with increased 1-year mortality in cardiac transplant recipients. METHODS: We prospectively recruited 171 patients who underwent cardiac transplantation at out institution between May 2004 and April 2006. We assessed calciotropic hormones, inflammation markers, and renal function preoperatively and on postoperative days 6 (t1) and 21 (t2). RESULTS: Serum creatinine and C-reactive protein increased, whereas calcitriol decreased significantly after transplantation (P<0.001). As determined by multivariable Cox regression analysis, the calcitriol level at t2 was an independent predictor of 1-year mortality. One-year mortality was 3.7 per 100 person-years in the tertile with the highest calcitriol concentrations at t2 (> 18 pg/mL), 13.2 per 100 person-years in the intermediate tertile (11-18 pg/mL), and 32.1 per 100 person-years in the tertile with the lowest calcitriol concentrations at t2 (< 11 pg/mL) (P<0.001). 25-Hydroxyvitamin D deficiency (serum concentrations below 10 ng/mL), renal insufficiency (serum creatinine > or = 1.6 mg/dL), and high serum concentrations of the inflammation markers C-reactive peptide and tumor necrosis factor-alpha were predictors of a serum calcitriol concentration below 11 pg/mL (P=0.037-0.001). CONCLUSIONS: Low postoperative calcitriol concentrations are independently associated with high 1-year mortality in cardiac transplant recipients. A causal relationship has yet to be proven by intervention trials using active vitamin D.

Lipopolysaccharide-binding protein: a new biomarker for infectious endocarditis?

BACKGROUND: Infectious endocarditis (IE) is a bacterial infection of the endocardium. Diagnosis is based on results obtained from echocardiography, blood cultures, and molecular genetic screening for bacteria and on data for inflammatory markers such as the leukocyte (WBC) count and the C-reactive protein (CRP) concentration. The aim of the present study was to evaluate lipopolysaccharide-binding protein (LBP) as a supportive biomarker for the diagnosis and therapeutic monitoring of IE. METHODS: We measured LBP and CRP concentrations and WBC counts in 57 IE patients at hospital admission, 40 patients with noninfectious heart valve diseases (HVDs), and 55 healthy blood donors. The progression of these 3 markers and the influence of cardiac surgery on them were evaluated in 29 IE patients and 21 control patients. RESULTS: Serum LBP concentrations were significantly higher in IE patients [mean (SD), 33.41 (32.10) mg/L] compared with HVD patients [6.67 (1.82) mg/L, P < 0.0001] and healthy control individuals [5.61 (1.20) mg/L]. The progression in the LBP concentration during therapy of IE patients correlated with the changes in the CRP concentration. The 2 markers were equally influenced by antibiotic treatment and surgical intervention. CONCLUSIONS: Serial LBP measurement may provide an effective and useful tool for evaluating the response to therapy in IE patients. We found a strong correlation between LBP and CRP concentrations; LBP has a tendency to increase earlier in cases of reinfection.

Prevalent platelet dysfunction in patients with aortic valve disease.

BACKGROUND AND AIM OF THE STUDY: In patients with heart valve disease, the valve leaflets display a gapped, rough endothelial lining often covered with calcified areas. As a consequence, blood flow is disturbed and a stimulation of components of the hemostasis system is assumed. The possible mechanisms of this process are, however, unclear at present. METHODS: Platelet function was studied in 660 patients considered for isolated coronary artery bypass graft (CABG) surgery, and in 421 patients considered for single aortic valve replacement (AVR). Platelet function was monitored preoperatively using the platelet function analyzer device (PFA-100). The test results were reported as closure time of the membrane hole at the end of a capillary tube. The von Willebrand factor antigen, and its collagen-binding activity, were also determined among subgroups of 40 AVR and 50 CABG candidates. RESULTS: Platelet dysfunction was displayed by only 22% of CAD patients, but by 83% of AVR candidates. The mean PFA closure time in AVR patients was considerably higher than in CAD patients (231 +/- 59 s versus 153 +/- 60 s, respectively; p < 0.01). The mean platelet volume, platelet distribution width and von Willebrand factor collagen binding and antigen levels did not differ between the patient groups. CONCLUSION: It is assumed that, due to disturbed flow and shear exposition, following an initial activation, the platelets are partially degranulated, shed microparticles, and might become involved in the pathogenesis of microvascular dysfunction and thrombotic events in patients with aortic valve disease.

Complement factor H variant p.Y402H in pseudoxanthoma elasticum patients.

Pseudoxanthoma elasticum (PXE) is a hereditary disorder predominantly affecting the eyes, the skin, and the vascular system. The subretinal neovascularization and retinal hemorrhages leading to the loss of central vision in PXE are similar to the process observed in age-related macular degeneration (AMD). The complement factor H (CFH) variant c.1277T > C (p.Y402H) is a recently discovered risk factor for AMD. The aim of this study was to analyze whether this CFH variant is a secondary genetic risk factor for PXE. Therefore, the genotypes of CFH c.1277T > C (p.Y402H) were determined in 189 German PXE patients and 189 age- and sex-matched controls. The allelic frequencies of the investigated variant did not differ between patients and controls. The frequencies were 33%, 56%, and 11% for wild-type, heterozygous, and homozygous genotypes in the PXE patients and 36%, 51%, and 13% in the control cohort, respectively. Further, no significant associations were identified when allele carriers were analyzed or after adjustment for sex, age, smoking, organ involvement, hypertension, or age at disease onset. No significant genotype-phenotype correlation was detected. In conclusion, our data reliably show that the CFH variant c.1277T > C (p.Y402H) is not a genetic risk factor for PXE.

The local calcification inhibitor matrix Gla protein in pseudoxanthoma elasticum.

OBJECTIVES: Recent studies have revealed the involvement of calcification inhibitory proteins in the pathogenesis of pseudoxanthoma elasticum (PXE). DESIGN AND METHODS: We analyzed serum concentrations of the calcification inhibitor matrix Gla protein (MGP) in a large cohort of patients suffering from PXE (n=101), 34 first-degree relatives and 67 healthy controls. Moreover, we determined the distribution of the two MGP promoter polymorphisms c.-7G>A and c.-138T>C in the three cohorts. RESULTS: We found significantly lower total MGP concentrations in the sera of PXE patients compared to healthy controls (p=0.0002). Furthermore, higher serum MGP concentrations could be correlated with a later PXE onset. Analysis of MGP promoter polymorphism frequencies revealed one MGP haplotype to be a potential protective co-factor in PXE. CONCLUSIONS: Our findings point to a role of the local calcification inhibitor MGP in PXE manifestation.

Increased levels of xylosyltransferase I correlate with the mineralization of the extracellular matrix during osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable to differentiate into osteoblasts. Therefore, they represent attractive cell sources for tissue engineering applications, especially for bone replacement. Proteoglycans (PGs) exhibit a crucial role for matrix assembly and remodeling. Nevertheless, since bone development is a highly dynamic and complex process, the regulation of the extracellular matrix (ECM) formation remains elusive. Consequently, the aim of this study was to investigate the mRNA expression levels of genes involved in PG assembly in different stages of osteogenesis. For the rate-limiting enzyme in glycosaminoglycan (GAG) biosynthesis xylosyltransferase I (XT-I), maximal mRNA expression levels (3.89 +/- 0.83-fold increase) and elevated enzyme activities (285 +/- 17 dpm/mug DNA) were observed 10 days after osteogenic induction, simultaneously to the beginning mineralization of the ECM, whereas the highly homologous protein XT-II showed no specific alterations. The differential expression of chondroitin sulfate, dermatan sulfate and heparan sulfate chains was determined by analyzing the mRNA expression of EXTL2 (alpha-1,4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5-epimerase) as they represent crucial enzymes in GAG biosynthesis. Besides GlcAC5E, all key enzymes showed upregulated mRNA contents (up to 3.6-fold) around day 10. Except for decorin, which exhibited heightened mRNA levels even in the early stages of osteogenesis, we found similar upregulated mRNA contents (up to 14.6-fold) for all investigated PG core proteins. The synchronized expression profiles demonstrate the coordinated biosynthesis of the PGs during bone formation and osteogenic stem cell differentiation occurring in parallel to the mineralization of the extracellular matrix.

Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy.

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Proteoglycan biosynthesis during chondrogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells are multipotent progenitor cells that can differentiate into the chondrogenic lineage. To date, only limited knowledge about the formation and remodeling of the cartilaginous extracellular matrix is available. We recently analyzed the coordinated expression of proteins involved in the biosynthesis of proteoglycans and collagens, the two major components of cartilage matrix, to understand matrix formation and to provide potential tools to improve the quality of tissue-engineered cartilage.

Relative quantification of glycosaminoglycan-induced upregulation of TFPI-mRNA expression in vitro.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial (Ea.hy 926) and in chondrosarcoma (SW 1353) cells. METHODS: We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene. RESULTS: Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect. CONCLUSION: Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Measurement of fibrosis marker xylosyltransferase I activity by HPLC electrospray ionization tandem mass spectrometry.

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.