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Mutations in ABCC6, an ATP-binding cassette transporter with a so far unknown substrate mainly expressed in the liver and kidney, cause pseudoxanthoma elasticum (PXE). Symptoms of PXE in patients originate from the calcification of elastic fibers in the skin, eye, and vessels. Previous studies suggested an involvement of ABCC6 in cholesterol and lipid homeostasis. The intention of this study was to examine the influence of ABCC6 deficiency during adipogenic differentiation of human bone marrow-derived stem cells (hMSCs). Induction of adipogenic differentiation goes along with significantly elevated ABCC6 gene expression in mature adipocytes. We generated an ABCC6-deficient cell culture model using clustered regulatory interspaced short palindromic repeat Cas9 (CRISPR–Cas9) system to clarify the role of ABCC6 in lipid homeostasis. The lack of ABCC6 in hMSCs does not influence gene expression of differentiation markers in adipogenesis but results in a decreased triglyceride content in cell culture medium. Protein and gene expression analysis of mature ABCC6-deficient adipocytes showed diminished intra- and extra-cellular lipolysis, release of lipids, and fatty acid neogenesis. Therefore, our results demonstrate impaired lipid trafficking in adipocytes due to ABCC6 deficiency, highlighting adipose tissue and peripheral lipid metabolism as a relevant target for uncovering systemic PXE pathogenesis.

Remodelling of the extracellular matrix by myofibroblasts is crucial for wound repair, but if deregulated, it might contribute to the development of fibrosis. Fibroblast-to-myofibroblast differentiation is promoted by aberrant microRNA-145-5p (miR-145) expression in response to transforming growth factor β1 (TGFβ1). One of several myofibroblast markers is human xylosyltransferase-I (XT-I), which is the initial and rate-limiting enzyme of proteoglycan biosynthesis. Increased serum XT activity was quantified in patients with systemic sclerosis (SSc), but the underlying cellular mechanism of this disease remains unknown. This study aims to determine the underlying molecular basis of XT-I induction by considering the miR-mediated regulation of XT-I. We found that miR-145 is upregulated in TGFβ1-treated dermal fibroblasts and correlates with an increased cellular XYLT1 expression and XT activity. Overexpression of miR-145 in dermal fibroblasts induced XYLT1 expression and XT activity and enhanced TGFβ1-promoted XT activity increase. Since direct XYLT1 3'-UTR targeting by miR-145 could be experimentally excluded, an indirect effect of miR-145 on XT-I regulation was indicated. We identified six transcription factor-binding sites for Krueppel-like factor 4 (KLF4), a zinc-finger transcription regulator and putative miR-145 target, in the XYLT1 promoter in silico. A suppressive role of KLF4 on XYLT1 expression was confirmed by targeted gene silencing in dermal fibroblasts and the quantification of KLF4 expression in SSc fibroblasts. Taken together, this study improves the mechanistic understanding of fibrotic remodelling in SSc by identifying a hitherto unknown miR-145/KLF4 pathway mediating the fibrogenic XT-I induction. This knowledge on XYLT1 may lead to the development of novel approaches in the therapy of fibrosis.

Human xylosyltransferases-I and –II (XT-I and XT-II) catalyze the initial and rate-limiting step in proteoglycan (PG)-biosynthesis. Because PG are major components of the extracellular matrix (ECM), an alternated XT expression is associated with the manifestation of ECM-related diseases. While Drosophila melanogaster and Caenorhabditis elegans only harbor one XT-isoform, all higher organisms contain two isoforms, which are expressed in a tissue-specific manner. The reason for the appearance of two isoenzymes remains unexplained and remarkable, as all other enzymes involved in the synthesis of the tetrasaccharid linker, which connects the PG core protein with attached glycosaminoglycans, only show one isoform. In human, mutations in the XYLT genes cause diseases affecting the homeostasis of the ECM, such as skeletal dysplasias. We investigated for the first time whether already XT-I-deficient human embryonic kidney (HEK293) cells can compensate for decreased expression levels of both XT-isoforms. A siRNA-mediated XYLT2 mRNA knockdown led to reduced cellular proliferation rates and a partially increased cellular senescence of treated HEK293 cells. These results were verified by conducting a stable CRISPR/Cas9-mediated XYLT2 knockout, which revealed that only cells expressing at least partially functional XT-II proteins remain proliferative. Our study, therefore, shows for the first time that cells lacking both XT-isoforms are not viable and clearly indicates the importance of the XT concerning the cellular metabolism.

PURPOSE To characterize dark adaptation in patients with pseudoxanthoma elasticum, a systemic disease leading to calcification of elastic tissue including the Bruch membrane. METHODS In this prospective case-control study, dark adaptation thresholds were measured using a Goldmann-Weekers dark adaptometer. Additional assessments included best-corrected visual acuity testing, contrast sensitivity, low luminance deficit, and vision-related quality of life. RESULTS Dark adaptation thresholds were significantly higher, and adaptation periods were prolonged in patients with pseudoxanthoma elasticum (n = 35; 33 with 2 ABCC6 mutations) compared with controls (n = 35). The time to adapt 4 log units (20.6 ± 8.6 vs. 8.0 ± 1.3 minutes) and the mean dark adaptation threshold after 15 minutes (3.5 ± 1.1 vs. 1.8 ± 0.2 log units) were significantly different between patients and controls (both P < 0.001). Low luminance deficits (12.3 ± 6.4 vs. 6.1 ± 4.3 ETDRS letters), contrast sensitivity (1.4 ± 0.3 vs. 1.9 ± 0.1), and low luminance-related quality of life (LLQ score: 1,286 ± 355 vs. 2,167 ± 68) were also significantly worse in patients with pseudoxanthoma elasticum (all, P < 0.001). Two patients were treated with high-dose vitamin A which partially reversed impaired dark adaptation. CONCLUSION Patients with pseudoxanthoma elasticum often have impaired dark adaptation. Positive effects of vitamin A supplementation may indicate restricted retinal access of vitamin A through the Bruch membrane as one possible underlying pathogenic factor.

Fibrosis is a serious health problem often leading to accompanying organ failure. During the manifestation of the disease, an accumulation of different extracellular matrix (ECM) molecules, such as proteoglycans, takes place. There is no appropriate therapeutic option available to heal fibrosis to date. Current research focuses primarily on targets such as the cytokine transforming growth factor-β1 (TGF-β1), which is assumed to be one of the key mediators of fibrosis. Both xylosyltransferase isoforms, XT-I and XT-II, catalyze the rate-limiting step of the proteoglycan biosynthesis. Consequently, inhibiting XT activity could be a promising approach to treat fibrosis. It was shown in earlier studies that nucleotides and nucleosides have anti-fibrotic properties and decrease XT activity in cell-free systems. In contrast, we evaluated the mechanisms beyond an UDP-mediated induction of intracellular XT-activity in normal human dermal fibroblasts (NHDF). The observed pseudo-fibrotic XT increasement could be attributed to a compensation of decreased UDP-glucuronate decarboxylase 1 (UXS1) mRNA expression as well as a diminished intracellular UDP-xylose concentration. In summary, our results describe a so far unknown XT-inductive pathway and show that UDP could be a promising molecule for the development of an anti-fibrotic therapy. Nevertheless, XT activity has to be inhibited in parallel intracellularly.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder caused by mutations in the ATP-binding cassette sub-family C member 6 gene. Our previous studies revealed that PXE might be associated with premature aging. Treatment with statins showed positive effects not only for PXE but also for other diseases associated with premature aging like Hutchinson–Gilford progeria syndrome. Nevertheless, the molecular mechanisms in the case of PXE remain unclear. Thus, this study was performed to evaluate the efficiency of atorvastatin by analyzing key characteristics of the PXE phenotype in primary human dermal fibroblasts of PXE patients. Our data indicate that an atorvastatin treatment has a positive effect, especially on factors associated with cholesterol biosynthesis and prenylation processes, whereas the effect on age- and calcification-related factors was less pronounced.

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-β1 (TGF-β1) and interleukin-1β (IL-1β). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-β signaling. Upon IL-1β treatment, SScF showed an increased mRNA expression level of XT-I and TGF-β receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1β in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-β1, we showed that the pro-fibrotic effect of IL-1β may be mediated by a miRNA-21/TGF-β receptor II axis, enhancing the autocrine TGF-β signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1β-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.

Xylosyltransferases-I and –II (XT-I and –II) play an important role regarding the homeostasis of the extracellular matrix. Both enzymes catalyze the initial step of the proteoglycan (PG) biosynthesis by the transfer of xylose from their natural substrate uridine diphosphate (UDP) -xylose to a PG-core protein. The subsequent addition of further sugars, catalyzed by different glycosyltransferases, leads to the formation of a tetrasaccharide linker, which connects the PG-core protein and glycosaminoglycans. The reason for the appearance of two XT isoforms in all higher organisms is not known and remarkable, as both enzymes are able to initiate PG biosynthesis. The determination of the XT-I activity is of clinical importance because it can be used as a biomarker of several PG-associated fibrotic diseases. Since previous assays did not adequately differentiate between both XT-isoforms, the aim of this study was to develop an XT-I selective mass spectrometric (MS) assay. For this purpose, we initially used isoform-specific supernatants to successfully identify a synthetic acceptor peptide which was xylosylated much more selectively by the XT-I when compared to the XT-II isoform. The assay was further optimized concerning methodical parameters such as the injection volume and the incubation time of the reaction-mixture. By using samples covering a broad XT-activity spectrum, we successfully validated the assay to be used not only for the quantification of cell culture samples but also human serum specimens. Compared to previously used XT-activity assays, our newly developed test is more selective and sensitive, less expensive and easier to perform in high throughput.

Fibroproliferative diseases are characterized by excessive accumulation of extracellular matrix (ECM) components leading to organ dysfunction. This process is characterized by an increase in myofibroblast content and enzyme activity of xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan (PG) biosynthesis. Therefore, the inhibition of XT-I could be a promising treatment for fibrosis. We used a natural product-inspired compound library to identify non-substrate-based inhibitors of human XT-I by UPLC-MS/MS. We combined this cell-free approach with virtual and molecular biological analyses to confirm and prioritize the inhibitory potential of the compounds identified. The characterization for compound potency in TGF-&beta, 1-driven XYLT1 transcription regulation in primary dermal human fibroblasts (key cells in ECM remodeling) was addressed by gene expression analysis. Consequently, we identified amphotericin B and celastrol as new non-substrate-based XT-I protein inhibitors. Their XT‑I inhibitory effects were mediated by an uncompetitive or a competitive inhibition mode, respectively. Both compounds reduced the cellular XYLT1 expression level and XT-I activity. We showed that these cellular inhibitor-mediated changes involve the TGF‑&beta, and microRNA-21 signaling pathway. The results of our study provide a strong rationale for the further optimization and future usage of the XT-I inhibitors identified as promising therapeutic agents of fibroproliferative diseases.

Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder which is caused by ABCC6 (ATP-binding cassette subfamily C member 6) gene mutations. Characteristic hallmarks of PXE are progressive calcification and degradation of the elastic fibers in skin, cardiovascular system and ocular fundus. Since the underlying pathomechanisms of PXE remain unidentified, the aim of this study was to get new insights into PXE pathophysiology by characterizing dermal myofibroblast differentiation. Fibroblasts are the key cells of extracellular matrix (ECM) remodeling and, therefore, participate not only in physiological processes, such as calcification or wound healing, but also in pathologic events, such as fibrotization. We revealed that human dermal PXE fibroblasts possess exaggerated migration capability in wound healing and attenuated myofibroblast contractility in comparison to controls. Subsequent analyses reinforced these observations and indicated a diminished induction of the myofibroblast differentiation markers α-smooth muscle actin and xylosyltransferase-I as well as poor transforming growth factor-β1 responsiveness in PXE fibroblasts. In summary, we describe pathological deviations of dermal myofibroblast differentiation in PXE which might be mediated by aberrant supramolecular ECM organization. These results not only improve our insights into cellular PXE pathophysiology, but might also qualify us to interfere with ECM remodeling in the future.

Abstract. Background: Pseudoxanthoma elasticum (PXE) is an autosomal recessive inherited multisystem disorder of the connective tissue caused by a loss-of-function mutation of the ABCC6 gene. It can affect the cardiovascular system, presumably leading to a high prevalence of atherosclerosis. Patients and methods: 46 PXE patients and 18 controls underwent an angiological examination consisting of measurement of ankle-brachial index (ABI), strain-gauge arterial reserve (SGAR), arterial resting perfusion, pulse wave index (PWI), central pulse wave velocity, and ultrasound examination. Results: With an average age of 51.4 ± 12.4 years, 35/46 (76.1 %) of the PXE patients had atherosclerotic lesions, and 10 of them (28.6 %) had a chronic vascular occlusion of one or more peripheral vessels. 34/46 (73.9 %) had a pathologic ABI < 0.9, 15/42 (35.7 %) had a pathological SGAR < 10 mL/100 mL tissue/min, and 23/38 (60.5 %) had a pathological PWI > 180. The differences between the groups were statistically significant for ABI, arterial reserve, and PWI. Conclusions: In PXE patients atherosclerosis was found with a much higher prevalence than expected. Moreover, they were at very high risk for total vessel occlusions.

Here, we present a small Iranian family, where the index patient received a diagnosis of restrictive cardiomyopathy (RCM) in combination with atrioventricular (AV) block. Genetic analysis revealed a novel homozygous missense mutation in the DES gene (c.364T > C; p.Y122H), which is absent in human population databases. The mutation is localized in the highly conserved coil-1 desmin subdomain. In silico, prediction tools indicate a deleterious effect of the desmin (DES) mutation p.Y122H. Consequently, we generated an expression plasmid encoding the mutant and wildtype desmin formed, and analyzed the filament formation in vitro in cardiomyocytes derived from induced pluripotent stem cells and HT-1080 cells. Confocal microscopy revealed a severe filament assembly defect of mutant desmin supporting the pathogenicity of the DES mutation, p.Y122H, whereas the wildtype desmin formed regular intermediate filaments. According to the guidelines of the American College of Medical Genetics and Genomics, we classified this mutation, therefore, as a novel pathogenic mutation. Our report could point to a recessive inheritance of the DES mutation, p.Y122H, which is important for the genetic counseling of similar families with restrictive cardiomyopathy caused by DES mutations.

Pseudoxanthoma elasticum (PXE) is a rare autosomal-recessive disorder that is mainly caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Clinically PXE is characterized by a loss of skin elasticity, arteriosclerosis or visual impairments. It also shares some molecular characteristics with known premature aging syndromes like the Hutchinson&ndash, Gilford progeria syndrome (HGPS). However, little is known about accelerated aging processes, especially on a cellular level for PXE now. Therefore, this study was performed to reveal a potential connection between premature cellular aging and PXE pathogenesis by analyzing cellular senescence, a corresponding secretory phenotype and relevant factors of the cell cycle control in primary human dermal fibroblasts of PXE patients. Here, we could show an increased senescence-associated &beta, galactosidase (SA-&beta, Gal) activity as well as an increased expression of proinflammatory factors of a senescence-associated secretory phenotype (SASP) like interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP1). We further observed an increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p21, but no simultaneous induction of p53 gene expression. These data indicate that PXE is associated with premature cellular senescence, which is possibly triggered by a p53-independent p21-mediated mechanism leading to a proinflammatory secretory phenotype.

Fibrosis is a fundamental feature of systemic sclerosis (SSc) and is characterized by excessive accumulation of extracellular matrix components like proteoglycans (PG) or collagens in skin and internal organs. Serum analysis from SSc patients showed an increase in the enzyme activity of xylosyltransferase (XT), the initial enzyme in PG biosynthesis. There are two distinct XT isoforms&mdash, XT-I and XT-II&mdash, in humans, but until now only XT-I is associated with fibrotic remodelling for an unknown reason. The aim of this study was to identify new XT mediators and clarify the underlying mechanisms, in view of developing putative therapeutic anti-fibrotic interventions in the future. Therefore, we used different cytokines and growth factors, small molecule inhibitors as well as small interfering RNAs, and assessed the cellular XT activity and XYLT1 expression in primary human dermal fibroblasts by radiochemical activity assays and qRT-PCR. We identified a new function of activin A as a regulator of XYLT1 mRNA expression and XT activity. While the activin A-induced XT-I increase was found to be mediated by activin A receptor type 1B, MAPK and Smad pathways, the activin A treatment did not alter the XYLT2 expression. Furthermore, we observed a reciprocal regulation of XYLT1 and XYLT2 transcription after inhibition of the activin A pathway components. These results improve the understanding of the differential expression regulation of XYLT isoforms under pathological fibroproliferative conditions.

BiallelicIn this prospective, cross-sectional, monocentre case-control study, carriers of monoallelicThirty-eight subjects carrying monoallelicThe findings of this study indicate a possible ocular ABCC6 haploinsufficiency phenotype. Due to its late-onset and phenotypic similarities, misinterpretation as age-related macular degeneration is possible.

Diminished microRNA-29b levels have recently been revealed to provoke increased expression and accumulation of extracellular matrix molecules, such as collagens in fibrotic remodeling. Subsequently, the aim of this study was to find out whether microRNA-29b might also regulate human xylosyltransferase (XT)-I expression. XT-I has been characterized previously as a fibrosis biomarker catalyzing the key step of proteoglycan biosynthesis. While we demonstrate that XYLT1 is neither a target of microRNA-29b identified in silico nor a direct 3′ untranslated region binding partner of microRNA-29b, transfection of normal human dermal fibroblasts with microRNA-29b inhibitor strongly increased XYLT1 mRNA expression and XT activity. Combined results of the target prediction analysis and additional transfection experiments pointed out that microRNA-29b exerts indirect influence on XT-I by targeting the transcription factor specificity protein 1 (Sp1). We could confirm our hypothesis due to the decrease in XYLT1 promoter activity after Sp1 binding site mutation and the approval of occupancy of these binding sites by Sp1 in vitro. Taken together, a hitherto unidentified pathway of XT-I regulation via microRNA-29b/Sp1 was determined in this study. Our observations will facilitate the understanding of complex molecular fibrotic pathways and provide new opportunities to investigate microRNA-based antifibrotic tools.

Background Although low (but increasing) rates of lung/lung-heart transplantations of scleroderma (systemic sclerosis [SSc]) patients have been reported, exclusive heart transplantation is a rare approach for treatment of heart failure due to SSc. Cases We report on 2 cases of SSc patients receiving a heart transplantation (HTx) due to severe and progressive right heart failure without pulmonary artery hypertension. One patient received a hepatitis C virus (HCV)-positive donor heart and recovered excellently from viral transmission after administration of a direct-acting antiviral (DAA) regimen. This is the first published case of an SSc patient who underwent HTx using an HCV-positive donor heart. The clinical course of both patients was monitored by different serum SSc biomarkers. Only xylosyltransferase activity proved to be a promising biomarker for disease stage determination and therapeutic monitoring, precisely reflecting fibrotic remodeling and successful organ recovery. Conclusions Successful implementation of the 2 cases described here demonstrates that HTx is a safe and effective therapeutic option for defined SSc sub-patient groups despite the progressive character of the underlying disease. In the future, xylosyltransferase activity might be conducive to simplify the identification of patients with low systemic involvement but a strong indication for single heart transplantation. Finally, we demonstrate that treatment of HCV viral transmission from HCV-positive donor to organ recipient using DAA gives us new opportunities to consider HCV-positive donor organs for HTx and might reveal new possibilities to ease the lack of donor organs.

IMPORTANCE The diagnostic accuracy of different retinal imaging modalities to detect active choroidal neovascularization (CNV) in pseudoxanthoma elasticum (PXE) is essential to enable a correct diagnosis but is currently poorly understood. BACKGROUND Optical coherence tomography (OCT), fluorescein angiography (FA) and OCT angiography (OCT-A) are employed in daily practice, but a systematic comparison of these imaging techniques is lacking. DESIGN Retrospective, observational study. PARTICIPANTS Twenty patients (31 eyes) with PXE. METHODS OCT, FA and OCT-A imaging was performed in each eye and graded separately by independent readers. MAIN OUTCOME MEASURES Diagnostic accuracy, sensitivity and specificity to detect CNV-activity of each modality and longitudinal change of CNV size measured by OCT-A. RESULTS OCT showed the highest diagnostic accuracy (kappa = 0.57) in comparison to OCT-A or FA (kappa = 0.39 and 0.37, respectively). OCT-A, OCT and FA showed a diagnostic sensitivity of 0.9, 0.85 and 0.6, and a diagnostic specificity of 0.45, 0.72 and 0.82, respectively. Evaluation of longitudinal OCT recordings (24 eyes) resulted in optimal sensitivity and specificity (kappa = 1.0). Although median CNV size assessed using OCT-A remained stable on longitudinal measures of seven eyes, two eyes showed a distinct increase over time despite anti-vascular endothelial growth factor treatment. CONCLUSIONS AND RELEVANCE The systematic use of OCT, FA and OCT-A imaging can facilitate the diagnostic accuracy for detection and follow-up of CNV activity in PXE. While structural OCT is of high value, especially when longitudinal follow-up images are available, FA and OCT-A data might contribute to diagnostic accuracy in more complex cases.

Background Measurement of direct oral anticoagulants (DOACs) concentration in patient blood is essential in special clinical circumstances. Methods We developed a fast, selective and sensitive method for simultaneous measurement of DOACs in human plasma consisting of an automated online solid-phase extraction method coupled with ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry (online SPE-UPLC-MS/MS). Results The calibration curves of all DOACs were linear over the working range (apixaban: 0.25–760 μg/L, r > 0.99; dabigatran: 0.5–900 μg/L, r > 0.99; edoxaban: 0.6–800 μg/L, r > 0.99; rivaroxaban: 0.5–900 μg/L, r > 0.99). Limits of detection in the plasma matrix were Conclusions We developed and validated the first online SPE-UPLC-MS/MS method for fast, sensitive, specific, and reliable measurement of the new generation of DOACs and compared this method with commercial available coagulation assays.

Purpose To quantify lipofuscin-associated fundus autofluorescence in patients with pseudoxanthoma elasticum (PXE), a model disease for Bruch's membrane pathology. Methods In this prospective, monocenter, cross-sectional case-control study, 49 patients with PXE (mean age: 46 years, range 18-62) underwent quantitative fundus autofluorescence (qAF) imaging with a modified scanning laser ophthalmoscope containing an internal fluorescent reference for normalization of images. The mean qAF values of a circular ring centered on the fovea (qAF8) were measured and compared to 108 healthy controls (mean age 40 years, range 18-64). Results Overall, patients with PXE showed lower qAF8 values compared to controls (difference from controls -23%, 95% confidence interval [CI] -29% to -16%, P < 0.001). The reduction was most pronounced in patients older than 40 years (-30%, 95% CI -36% to -23%, P < 0.001) and was negatively correlated with the extent of Bruch's membrane calcification (r = -0.49, 95% CI: -0.67 to -0.22). The topographic distribution revealed a greater reduction of qAF values toward the optic disc than temporally compared to controls (P < 0.001). The phenotype of patients with reduced qAF values was characterized by pattern-dystrophy-like changes (71%; 10 of 14), reticular pseudodrusen (71%; 10 of 14) and limited areas of atrophy (29%, 4 of 14). Conclusions Reduced qAF8 values are a characteristic finding in patients with PXE, indicating that Bruch's membrane disease may result in a modification of the accumulation, distribution, or composition (or a combination thereof) of lipofuscin in retinal pigment epithelial cells.

Objectives Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by progressive calcification and fragmentation of elastic fibers. Because of the great clinical variability between PXE patients the involvement of modifier genes was recently suggested. Therefore, we investigated the association of single nucleotide variants (SNVs) in selected candidate genes known to regulate cellular pyrophosphate metabolism. Design and methods We used RLFP analyses to evaluate the distribution of SNVs in alkaline phosphatase (ALP), ectonucleotide pyrophosphatase 1 (ENPP1) and ankylosis (ANKH) in DNA samples from 190 German PXE patients and 190 age- and sex-matched healthy controls. Statistical analyses were performed using Fisher exact test and Bonferroni correction. Results The screening revealed three different SNVs in three genes, which were associated with PXE. The SNV c.1190-65C > A (rs1780329, minor allele frequency (MAF) patients: 0.17; controls: 0.11; P = 0.04) in the ALP gene was significantly more frequent in PXE patients. Furthermore, PXE was highly associated with ANKH p.A98A genotype TT (P = 0.0012), although the MAF was not different between patients and controls. After correction for multiple testing according to the Bonferroni method, one SNV in the ENPP1 gene (c.313 + 9G > T, rs7773477) remained significantly associated with PXE with significantly higher MAF values in the patient cohort (MAF: 0.04 vs. 0.00; P = 0.0024) and a high association with PXE susceptibility (OR 27.96). Conclusion Polymorphisms in ALP, ENPP1 and ANKH are important genetic risk factors contributing to PXE.

Background Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by late onset and progressive calcification of elastic fibers in skin, eyes and the cardiovascular system, exemplifying a model for conditions characterized by soft tissue calcification. Objective The aim of our study was to characterize cellular inorganic pyrophosphate (PP i ) homeostasis in PXE. Methods Gene expression of PP i metabolizing enzymes was determined by quantitative real-time PCR after incubation up to 21 days with or without addition of Na 2 HPO 4 . Extracellular and cytosolic PP i concentrations were measured by enzyme-linked bioluminescence assay. ALP and ENPP1 activity was determined spectrophotometrically. We further established a human cell culture model suitable for investigating PXE and related disorders without addition of artificial calcification triggers. Results Independently of the experimental conditions, PXE fibroblasts revealed a higher degree of matrix calcification. We observed that matrix calcification was associated with altered gene expression of PP i metabolizing enzymes in PXE fibroblasts. In this context, PXE fibroblasts exhibited significantly higher expression of ALP and OPN and reduced mRNA expression and activity of ENPP1. Here, for the first time cytosolic and extracellular PP i levels were shown to be strongly reduced in PXE fibroblasts. We further showed that PP i concentration in bovine and human sera additives had a strong impact on matrix calcification. In a last experimental line, we demonstrated that addition of PP i analogs reduced matrix calcification of PXE fibroblasts most likely by reducing ALP and OPN mRNA expression, restoring ENPP1 activity and subsequently elevating PP i concentrations. Conclusion The results of our study along with recent findings point to the essential role of PP i as the central regulatory metabolites preventing matrix calcification in PXE. But what remains to be determined is the underlying molecular mechanism leading to depletion of PP i in PXE. We further suggest that supplementation of PP i analogs might counteract pathological calcification in PXE and related disorders.

PURPOSE To characterize atrophy of the outer retina and the retinal pigment epithelium in patients with pseudoxanthoma elasticum (PXE). METHODS In this retrospective cross-sectional study, the frequency and phenotypic characteristics of manifest atrophy were investigated in 276 eyes of 139 patients using color fundus photography, fundus autofluorescence (AF) imaging, and spectral domain optical coherence tomography. Progression rates of atrophy were quantified in eyes with longitudinal AF recordings. RESULTS Atrophy was present in 90 eyes (32%; mean age, 60; range, 32-88 years). In 19 eyes (7%; mean age, 56; range, 37-77 years) atrophy occurred without any signs for an active or fibrotic choroidal neovascularization (CNV). The frequency of both, atrophy and CNV, increased with age. In those > 60 years of age, atrophy and/or CNV were almost universally present but varied considerably in severity. Eyes with emerging pure atrophy (n = 13, no signs of CNV) showed pattern dystrophy-like changes (100%), reticular pseudodrusen (82%), and reduced choroidal thickness. Advanced atrophy was multifocal, reached beyond the arcades, and was present nasal to the optic disc. The average expansion rate of atrophy was 3.3 ± 1.3 and 1.6 ± 1.1 mm2/year (mean ± SD), in those without or with signs for CNV, respectively. CONCLUSIONS Atrophy of the outer retina and the retinal pigment epithelium is a common finding in PXE patients characterized by early onset and fast progression with subsequent visual loss independent from CNV. This suggests that atrophy is the natural endpoint of Bruch's membrane disease. Phenotypic similarities with multifactorial geographic atrophy in age-related macular degeneration suggest common pathogenic pathways at the level of Bruch's membrane.

To evaluate the efficacy and safety of monthly intravitreal ranibizumab for the treatment of choroidal neovascularizations (CNV) secondary to angioid streaks (AS) in pseudoxanthoma elasticum (PXE).Twelve-month prospective, open-label, uncontrolled, nonrandomized interventional clinical trial.In 7 patients, 1 eye with an active CNV was injected with 0.5 mg ranibizumab monthly over 1 year. Distance and reading visual acuity, reading speed, angiographic findings, and central retinal thickness (CRT) on optical coherence tomography were assessed at each visit. Central retinal light increment sensitivity (LIS) was assessed by microperimetry at baseline, at 6 months, and 3 to 4 months after the last injection.Best-corrected visual acuity increased significantly from baseline to month 12 (20/63 or 61 ETDRS letters to 20/32 or 73 ETDRS letters; P = .012). The effect was maintained 3 months later (61 ETDRS letters to 72 ETDRS letters; P = .055). Reading acuity and speed could be maintained throughout the study. Central LIS improved (6.6 dB, SD ± 5.9 at baseline to 7.4 dB, SD ± 6.2 at last follow-up; P.001). Leakage from active CNVs subsided. Mean change in CRT from baseline to month 12 and 15 was -86 μm (P = .074) and -65 μm (P = .182), respectively. No serious adverse events occurred.Efficacy outcomes indicate a beneficial therapeutic effect of intravitreal ranibizumab on central visual function including retinal LIS. Both the functional and morphologic response based on angiographic and OCT findings to ranibizumab treatment implicate an important pathophysiological role of vascular endothelial growth factor in CNVs secondary to AS in PXE. Intravitreal ranibizumab appears to be a safe and efficacious treatment in these patients.

The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-β activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.

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, Pcorrected = 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.

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.

Pseudoxanthoma elasticum (PXE, OMIM 177850 and 264800) is a rare heritable disorder predominantly affecting the skin, the eyes and the vascular system. The disease is caused by mutations in the ABCC6 gene and is characterized by calcification and extracellular matrix remodeling, including alterations of the vessel walls. Here, we investigated the cell adhesion molecules ICAM-1 in PXE patients.Soluble ICAM-1 was determined in 58 non-consanguineous PXE patients by quantitative sandwich enzyme immunoassay. The allelic frequencies of the ICAM-1 variant p.K469E were analyzed in patients and age- and sex-matched controls.Soluble ICAM-1 levels were significantly elevated in male and female PXE patients (p0.02 and p0.001, respectively). In addition, the ICAM-1 concentration correlated with the ABCC6 gene status of the PXE patients. The ICAM variant p.K469E genotypes were not different in PXE patients and age- and sex-matched controls.Our data show for the first time increased ICAM-1 concentrations in PXE patients, potentially due to the chronic oxidative stress and elevated protease activity followed by extracellular matrix remodeling which have been previously observed in PXE patients.

Total knee replacement (TKR) is a common therapeutic option to restore joint functionality in chronic inflammatory joint diseases. Subsequent arthrofibrotic remodeling occurs in 10%, but the underlying pathomechanisms remain unclear. We evaluated the association of xylosyltransferases (XT), fibrotic mediators catalyzing glycosaminoglycan biosynthesis, leading to arthrofibrosis as well as the feasibility of using serum XT activity as a diagnostic marker. For this purpose, synovial fibroblasts (SF) were isolated from arthrofibrotic and control synovial biopsies. Basal α-smooth muscle actin expression revealed a high fibroblast-myofibroblast transition rate in arthrofibrotic fibroblasts. Fibrotic remodeling marked by enhanced XT activity, α-SMA protein expression as well as xylosyltransferase-I, collagen type III-alpha-1 and ACTA2 mRNA expression was stronger in arthrofibrotic than in control fibroblasts treated with transforming growth factor-β1 (TGF-β1). Otherwise, no differences between serum levels of XT-I activity or common fibrosis markers (galectin-3 and growth differentiation factor-15 levels (GDF-15)) were found between 95 patients with arthrofibrosis and 132 controls after TKR. In summary, XT-I was initially investigated as a key cellular mediator of arthrofibrosis and a target for therapeutic intervention. However, the blood-synovial-barrier makes arthrofibrotic molecular changes undetectable in serum. Future studies on monitoring or preventing arthrofibrotic remodeling should therefore rely on local instead of systemic parameters.

Importance Reticular pseudodrusen (RPD) are frequently associated with age-related macular degeneration and considered to be an independent risk factor for disease progression, but the pathophysiologic mechanisms are only incompletely understood. Therefore, it may be helpful to identify the associations of RPD with other diseases that have defined pathophysiologic mechanisms. Objective To describe the phenotype, prevalence, and topographic distribution of RPD in patients with pseudoxanthoma elasticum (PXE) and their association with a diseased Bruch membrane. Design, Setting, and Participants In this single-center, prospective, cross-sectional case series, 57 consecutive patients with PXE from a university referral center whose diagnosis has been confirmed by genetic testing and/or skin biopsy were studied from March 1, 2013, through February 28, 2014. Main Outcomes and Measures Phenotypic characteristics of RPD were evaluated with multiple imaging techniques. The RPD were defined as irregular networks of round to oval lesions that appear hyporeflective on near-infrared reflectance, hypoautofluorescent on fundus autofluorescence, and as subretinal deposits on spectral-domain optical coherence tomographic images. The presence of RPD was judged based on characteristic findings in at least 2 of the 3 imaging modalities. Results A total of 57 patients were examined, and 15 patients were excluded mainly because of large central atrophy or fibrosis. In the remaining 42 patients with PXE, RPD were detected in 22 patients (52%; 95% CI, 38%-67%). Prevalence of RPD was highest in the fifth decade at 67% (10/15; 95% CI, 42%-85%). The RPD were most frequently located within the superior quadrant and least frequently located within the central macula. The RPD were always located central to areas with peau d’orange and within an area of hypofluorescence on late-phase indocyanine green angiographic images. Conclusions and Relevance These data suggest that RPD have a high prevalence in eyes of patients with PXE. Although RPD in patients with PXE occur at a younger age, their distribution and phenotype appear to be similar to RPD associated with age-related macular degeneration. The association with diseased Bruch membrane in PXE suggests a pathogenetic role of Bruch membrane alterations for the development of RPD.

In mammals, two active xylosyltransferase isoenzymes (EC 2.4.2.16) exist. Both xylosyltransferases I and II (XT-I and XT-II) catalyze the transfer of xylose from UDP-xylose to select serine residues in the proteoglycan core protein. Altered XT activity in human serum was found to correlate directly with various diseases such as osteoarthritis, systemic sclerosis, liver fibrosis, and pseudoxanthoma elasticum. To interpret the significance of the enzyme activity alteration observed in disease states it is important to know which isoenzyme is responsible for the XT activity in serum. Until now it was impossible for a specific measurement of XT-I or XT-II activity, respectively, because of the absence of a suitable enzyme substrate. This issue has now been solved and the following experimental study demonstrates for the first time, via the enzyme activity that XT-II is the predominant isoenzyme responsible for XT activity in human serum. The proof was performed using natural UDP-xylose as the xylose donor, as well as the artificial compound UDP-4-azido-4-deoxyxylose, which is a selective xylose donor for XT-I.

Introduction The fast, precise, and accurate measurement of the new generation of oral anticoagulants such as dabigatran and rivaroxaban in patients' plasma my provide important information in different clinical circumstances such as in the case of suspicion of overdose, when patients switch from existing oral anticoagulant, in patients with hepatic or renal impairment, by concomitant use of interaction drugs, or to assess anticoagulant concentration in patients' blood before major surgery. Methods Here, we describe a quick and precise method to measure the coagulation inhibitors dabigatran and rivaroxaban using ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry in multiple reactions monitoring (MRM) mode (UPLC-MRM MS). Internal standards (ISs) were added to the sample and after protein precipitation; the sample was separated on a reverse phase column. After ionization of the analytes the ions were detected using electrospray ionization-tandem mass spectrometry. Run time was 2.5 minutes per injection. Ion suppression was characterized by means of post-column infusion. Results The calibration curves of dabigatran and rivaroxaban were linear over the working range between 0.8 and 800 mu g/L (r > 0.99). Limits of detection (LOD) in the plasma matrix were 0.21 mu g/L for dabigatran and 0.34 mu g/L for rivaroxaban, and lower limits of quantification (LLOQ) in the plasma matrix were 0.46 mu g/L for dabigatran and 0.54 mu g/L for rivaroxaban. The intraassay coefficients of variation (CVs) for dabigatran and rivaroxaban were < 4% and 6%; respectively, the interassay CVs were < 6% for dabigatran and < 9% for rivaroxaban. Inaccuracy was < 5% for both substances. The mean recovery was 104.5% (range 83.8-113.0%) for dabigatran and 87.0%(range 73.6-105.4%) for rivaroxaban. No significant ion suppressions were detected at the elution times of dabigatran or rivaroxaban. Both coagulation inhibitors were stable in citrate plasma at -20 degrees C, 4 degrees C and even at RT for at least one week. A method comparison between our UPLC-MRM MS method, the commercially available automated Direct Thrombin Inhibitor assay (DTI assay) for dabigatran measurement from CoaChrom Diagnostica, as well as the automated anti-Xa assay for rivaroxaban measurement from Chromogenix both performed by ACL-TOP showed a high degree of correlation. However, UPLC-MRM MS measurement of dabigatran and rivaroxaban has a much better selectivity than classical functional assays measuring activities of various coagulation factors which are susceptible to interference by other coagulant drugs. Conclusions Overall, we developed and validated a sensitive and specific UPLC-MRM MS assay for the quick and specific measurement of dabigatran and rivaroxaban in human plasma.

The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity. XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry. Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant. This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong interindividual variabilities in serum XT activity, our data provide increasing evidence that XT-II activity is obviously regulated by hitherto unknown complex genetic pathways, such as cis- or trans-acting enhancers, silencers or miRNAs.

Background: Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in the ABCC6 gene. Fragmentation of elastic fibres and deposition of proteoglycans result in a highly variable clinical picture. The altered proteoglycan metabolism suggests that enzymes from this pathway function as genetic co-factors in the severity of PXE. Therefore, we propose the XYLT genes encoding xylosyltransferase I (XT-I) as the chain-initiating enzyme in the biosynthesis of proteoglycans and the highly homologous XT-II as potential candidate genes. Methods: We screened all XYLT exons in 65 German PXE patients using denaturing high performance liquid chromatography and analysed the influence of the variations on clinical characteristics. Results: We identified 22 variations in the XYLT genes. The missense variation p.A115S (XT-I) is associated with higher serum XT activity (p = 0.005). The amino acid substitution p.T801R (XT-II; c.2402C>G) occurs with significantly higher frequency in patients under 30 years of age at diagnosis (43% v 26%; p = 0.04); all PXE patients with this variation suffer from skin lesions compared to only 75% of the wild type patients (p = 0.002). c.166G>A, c.1569C>T, and c.2402C>G in the XYLT-II gene were found to be more frequent in patients with higher organ involvement (p = 0.04, p = 0.01, and p = 0.02, respectively). Conclusions: Here we show for the first time that variations in the XYLT-II gene are genetic co-factors in the severity of PXE. Furthermore, the higher XT activity in patients with the exchange p.A115S (XT-I) indicates that this polymorphism is a potential marker for increased remodelling of the extracellular matrix.

Pseudoxanthoma elasticum (PXE; OMIM 177850 and 264800) is a rare heritable disorder of the connective tissue affecting the extracellular matrix of the skin, eyes, gastrointestinal system, and cardiovascular system. It has recently been found that mutations in the ABCC6 gene encoding the multidrug resistance-associated protein (MRP) 6 cause PXE. This study examined novel mutations in the ABCC6 gene in our cohort of 76 German PXE patients and 54 unaffected or not yet affected relatives with a view to expanding the known mutational spectrum of the gene. Mutational analysis was performed using denaturing high-performance liquid chromatography and direct sequencing. The mutational screening revealed a total of 22 different ABCC6 sequence variations. We identified seven novel and four previously described PXE-associated mutations as well as eight novel neutral ABCC6 sequence variants. The new PXE-associated mutations included five missense mutations, one single base pair deletion, and one larger out-of-frame deletion. We suspect that the novel missense mutations lead to an impaired function of MRP6. Both deletions are predicted to result in a dysfunctional MRP6 protein. The seven new ABCC6 mutations were not present in 200 alleles from healthy blood donors which served as a control cohort. Most of the PXE patients who were found to carry PXE-causing ABCC6 mutations were assumed to manifest the PXE phenotype because of a compound heterozygous genotype. However, a genotype-phenotype correlation could not be established for the detected ABCC6 mutations. In summary, our data give a further insight into the spectrum of ABCC6 mutations in PXE patients.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. Mutations in the ABCC6 gene could be linked to this disease and, just recently, the c.3421C>T mutation was also associated with a high risk of coronary artery disease. We have now developed new real-time PCR assays for the accurate and rapid determination of the c.3421C>T genotype. Using our new assay, we analyzed the presence of the c.3421C>T mutation in the largest collection of DNA samples from unrelated German PXE patients (n=64) and in a control cohort (n=910). For assay setup, two sets of samples with known genotype for the c.3421C>T mutation were analyzed over a period of 14 days. Results were confirmed by restriction endonuclease mapping, sequence-specific PCR and DNA sequencing. In order to ensure that no further mutations or deletions interfered with the c.3421C>T genotyping, we scanned the exon 24 of the ABCC6 gene by DHPLC and investigated the presence of the ABCC6del23–29 deletion in all patients. The assay has been set up on a group of patients with known genotype and validated on 64 PXE patients. In this group four PXE patients (6.3%) were found to be homozygous and 25 (39.0%) to be heterozygous carriers of the c.3421C>T mutation. The common ABCC6del23–29 deletion, possibly interfering with genotype determination, was searched and excluded. Furthermore, two novel mutations in the ABCC6 gene could be identified in two patients. The novel mutations c.3389C>T and c.3341G>A did not interfere with our new assay. Our new c.3421C>T genotyping assays can be used for the rapid identification of this frequent mutation in PXE patients and of the recently newly proposed cardiac risk factor in young patients with myocardial infarcts of unknown origin.

Background Human xylosyltransferase-I (XT-I) catalyzes the rate-limiting step in proteoglycan glycosylation. An increase in XYLT1 mRNA expression and serum XT activity is associated with diseases characterized by abnormal extracellular matrix accumulation like, for instance, fibrosis. Nevertheless, physiological and pathological mechanisms of transcriptional XT regulation remain elusive. Results To elucidate whether promoter variations might affect the naturally occurring variability in serum XT activity, a complete sequence analysis of the XYLT1 promoter was performed in genomic DNA of healthy blood donors. Based on promoter amplification by a specialized PCR technique, sequence analysis revealed a fragment of 238 bp, termed XYLT1238*, which has never been described in the human XYLT1 reference sequence so far. In silico characterization of this unconsidered fragment depicted an evolutionary conservation between sequences of Homo sapiens and Pan troglodytes (chimpanzee) or Mus musculus (mouse), respectively. Promoter activity studies indicated that XYLT1238* harbors various transcription factor binding sites affecting basal XYLT1 expression and inducibility by transforming growth factor-β1, the key fibrotic mediator. A microsatellite and two single nucleotide variants (SNV), c.-403C>T and c.-1088C>A, were identified and genotyped in 100 healthy blood donors. Construct associated changes in XYLT1 promoter activity were detected for several sequence variants, whereas serum XT activity was only marginally affected. Conclusions Our findings describe for the first time the entire XYLT1 promoter sequence and provide new insights into transcriptional regulation of XT-I. Future studies should analyze the impact of regulatory XYLT1 promoter variations on XT-associated diseases.

Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization.

Background Dysregulations in cholesterol and lipid metabolism have been linked to human diseases like hypercholesterolemia, atherosclerosis or the metabolic syndrome. Many ABC transporters are involved in trafficking of metabolites derived from these pathways. Pseudoxanthoma elasticum (PXE), an autosomal-recessive disease caused by ABCC6 mutations, is characterized by atherogenesis and soft tissue calcification. Methods In this study we investigated the regulation of cholesterol biosynthesis in human dermal fibroblasts from PXE patients and healthy controls. Results Gene expression analysis of 84 targets indicated dysregulations in cholesterol metabolism in PXE fibroblasts. Transcript levels of ABCC6 were strongly increased in lipoprotein-deficient serum (LPDS) and under serum starvation in healthy controls. For the first time, increased HMG CoA reductase activities were found in PXE fibroblasts. We further observed strongly elevated transcript and protein levels for the proprotein convertase subtilisin/kexin type 9 (PCSK9), as well as a significant reduction in APOE mRNA expression in PXE. Conclusion Increased cholesterol biosynthesis, elevated PCSK9 levels and reduced APOE mRNA expression newly found in PXE fibroblasts could enforce atherogenesis and cardiovascular risk in PXE patients. Moreover, the increase in ABCC6 expression accompanied by the induction of cholesterol biosynthesis supposes a functional role for ABCC6 in human lipoprotein and cholesterol homeostasis.

Hydroxymethylglutaryl coenzyme A reductase (HMGCR) catalyzes the rate limiting step in cholesterol biosynthesis converting HMG-CoA into mevalonic acid (MVA), which equilibrates with mevalonic acid lactone (MVL) under neutral pH conditions. We developed a fast, sensitive, and efficient method to determine HMGCR activity in human cell lines measuring MVL levels by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Convenient prepared samples containing MVL-D7 as an internal standard were injected, separated, and eluted from an ACQUITY HSS PFP column. Measurement of MVL was performed by electrospray ionization mass spectrometry with multiple reaction monitoring. Calibration curves were linear and reproducible in the range of 0.15-165 μg/l (r>0.99). Lower limit of quantification was 0.12 μg/l. Intra- and interassay imprecision were

We developed a stable isotope dilution ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay to measure nicotine and cotinine, the major oxidative and pharmacologically less active metabolite of nicotine, in human urine. A simple dilution step was used as sample preparation and the measurement of nicotine and cotinine was performed during a 1.5-min run-time using nicotine-D₄ and cotinine-D₄ as internal standards. Multiple calibration curves for the analysis of both nicotine and cotinine exhibited a consistent excellent linearity and reproducibility in the range of 5-35,000 μg/L (r>0.999). Limits of Detection were 0.7 μg/L for nicotine and 0.4 μg/L for cotinine, and Lower Limits of Quantification were 1.7 μg/L for nicotine and 1.1 μg/L for cotinine. The intraassay coefficients of variation (CVs) for nicotine and cotinine were

Purpose To investigate the Bruch's membrane–retinal pigment epithelium (RPE) complex in pseudoxanthoma elasticum (PXE) by imaging techniques capable of visualizing deep retinal structures with high contrast. Design Prospective cross-sectional study. Participants Patients with PXE, confirmed by mutation analysis, skin histopathologic examination, or both. Methods Sixteen patients were investigated by indocyanine green (ICG) and fluorescein angiography, confocal near-infrared (NIR) reflectance, and fundus autofluorescence imaging using a confocal scanning laser ophthalmoscope (Spectralis HRA-OCT; Heidelberg Engineering, Heidelberg, Germany). Composite digital fundus photography also was performed. Main Outcome Measures Characterization and topographic distribution of abnormalities detected by retinal imaging. Results On late-phase ICG angiography, a central area of decreased fluorescence centered on the posterior pole was a characteristic finding in all patients. A second area characterized by increased fundus reflectivity on NIR reflectance imaging extended further into the periphery. A third and most eccentric area showed neither decreased ICG fluorescence nor increased fundus reflectivity. These 3 areas were separated by 2 transition zones, the second being the equivalent of peau d'orange. Angioid streaks did not extend into the third area. Conclusions The abnormalities detected by this multimodal imaging approach suggest a centrifugal spread of the retinal pathologic features of the Bruch's membrane–RPE complex in PXE. Decreased fluorescence on late-phase ICG angiography is a consistent sign of retinal pathologic features in PXE. Bruch's membrane calcification may be the underlying cause for the increased reflectivity observed on NIR reflectance imaging. Financial Disclosure(s) Proprietary or commercial disclosure may be found after the references.

Pseudoxanthoma elasticum (PXE) is a rare 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. In the present study, we investigated matrix metalloproteinases MMP-2 and MMP-9 in PXE patients compared to healthy controls. We analyzed the serum concentrations of MMP-2 and MMP-9 in a cohort of 69 German PXE patients and in 69 healthy, age-, and sex-matched control subjects using commercially available ELISA assays. We found elevated concentrations of both MMPs in the sera of PXE patients. MMP-2 levels were significantly higher in patients than controls (231 +/- 5.89 vs 202 +/- 5.17 ng/ml, p = 0.0002), as were MMP-9 levels (841 +/- 65.9 vs 350 +/- 30.8 ng/ml, p0.0001). Our findings point to an involvement of matrix metalloproteinases in PXE pathology. ECM remodeling in PXE is reflected by elevated levels of circulating MMP-2 and MMP-9. Those MMPs might, therefore, be applicable as serum markers for the matrix-degradative process in PXE.

Mutations in the ABCC6 gene, encoding the multidrug resistance-associated protein 6 (MRP6), cause pseudoxanthoma elasticum (PXE). This heritable disorder leads to pathological alterations in connective tissues. The implication of MRP6 deficiency in PXE is still unknown. Moreover, nothing is known about a possible compensatory expression of other ATP binding-cassette (ABC) transporter proteins in MRP6-deficient cells. We investigated the gene expression profile of 47 ABC transporters in human dermal fibroblasts of healthy controls (n=2) and PXE patients (n=4) by TaqMan low-density array. The analysis revealed the expression of 37 ABC transporter genes in dermal fibroblasts. ABCC6 gene expression was not quantifiable in fibroblasts derived from PXE patients. Seven genes (ABCA6, ABCA9, ABCA10, ABCB5, ABCC2, ABCC9 and ABCD2) were induced, whereas the gene expression of one gene (ABCA3) was decreased, comparing controls and PXE patients (with at least twofold changes). We reanalyzed the gene expression of selected ABC transporters in a larger set of dermal fibroblasts from controls and PXE patients (n=6, each). Reanalysis showed high interindividual variability between samples, but confirmed the results obtained in the array analysis. The gene expression of ABC transporter genes, as well as lineage markers of PXE, was further examined after inhibition of ABCC6 gene expression by using specific small-interfering RNA. These experiments corroborated the observed gene expression alterations, most notably in the ABCA subclass (up to fourfold, P

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.

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.1277TC (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.1277TC (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.1277TC (p.Y402H) is not a genetic risk factor for PXE.

Background Proteoglycans are major components of the glomerular basement membrane, being responsible for their permeability properties. Type 1 diabetic patients have an altered proteoglycan metabolism, which contributes to microvascular complications like diabetic nephropathy. Xylosyltransferase II (XT-II) is a chain-initiating enzyme in the biosynthesis of basement membrane proteoglycans and catalyzes the transfer of xylose to selected serine residues in the core protein. Thus, genetic variations in the XT-II coding gene XYLT2 might be implicated in the initiation and progression of late diabetic complications. Methods Genotyping of 6 genetic variations in the XYLT2 gene and haplotype analysis was performed in 697 type 1 diabetic patients (358 with and 338 without diabetic nephropathy). Results The haplotype analysis of 6 XYLT2 polymorphisms revealed one haplotype (GATTCG) to be significantly less frequent among type 1 patients with diabetic nephropathy (p = 0.002, OR = 0.13, 95% CI = 0.03–0.59). The haplotype GATTCG consist of the XYLT2 variations c.166G>A, c.177A>G, c.342T>C, IVS6-9T>C, c.1569C>T and c.2402C>G. No genotype–phenotype interactions were revealed. Conclusions Our data show that a XYLT2 haplotype is associated with nephropathy in type 1 diabetic patients.

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.

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 ∼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_156insG, and c.244_245insTG) were significantly more frequent in PXE patients than in 93 age- and sex-matched healthy controls (Pcorrected < 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 (Pcorrected = 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.