Influence of pseudoxanthoma elasticum on the lipid profile and prognostic implications.

Background: Pseudoxanthoma elasticum (PXE) is a rare, inherited disease characterised by specific skin lesions, progressive loss of vision and early onset atherosclerosis. Atherosclerosis in PXE leads to an increased rate of vascular occlusion and severe intermittent claudication. Although genetically determined, the individual course of PXE is highly variable. Up to now, there is no sufficient parameter to identify individuals at risk of rapid disease progression. This present study focused the lipid profile of patients with PXE and its possible influence on the clinical severity of peripheral artery disease (PAD). Patients and methods: 112 patients with PXE were retrospectively screened. Patients without a complete lipid profile consisting of total cholesterol (TC), triglycerides (TGC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and Lipoprotein(a) (Lp[a]) where excluded as well as patients with already initiated lipid-lowering therapy. 52 patients met the inclusion criteria. An age-adjusted ordinal regression model was applied to determine the association of each lipid fraction with the severity of PAD assessed as Fontaine classification. Results: The lipid profile of patients with PXE was unremarkable (TGC: 135.8±105.8 mg/dl; TC: 172.5±44.4 mg/dl; HDL: 63.0±18.2 mg/dl; Lp[a]: 64.7±93.5 nmol/l). Ordinal regression showed a significant association of Lp(a) with the severity of PAD with an odds ratio of 1.01 (1.00-1.02; p = 0.004), whereas the other fractions of the lipid profile had no significant influence. Conclusions: This study provides the largest evaluation of blood lipids up to now and the first characterization of Lp(a) levels in patients with PXE. We were able to provide first evidence of a correlation between elevated levels of Lp(a) and the severity of PAD. The present results suggest that determination of Lp(a) in early stages of PXE could help to identify patients at risk of rapid disease progression and with the need of intensified walking exercise training.

Targeting ABCC6 in Mesenchymal Stem Cells: Impairment of Mature Adipocyte Lipid Homeostasis.

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.

Cytokine-mediated induction of human xylosyltransferase-I in systemic sclerosis skin fibroblasts.

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-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß). 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-ß signaling. Upon IL-1ß treatment, SScF showed an increased mRNA expression level of XT-I and TGF-ß receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1ß in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-ß1, we showed that the pro-fibrotic effect of IL-1ß may be mediated by a miRNA-21/TGF-ß receptor II axis, enhancing the autocrine TGF-ß signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1ß-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.

Xylosyltransferase-deficient human HEK293 cells show a strongly reduced proliferation capacity and viability.

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.

microRNA-145 mediates xylosyltransferase-I induction in myofibroblasts via suppression of transcription factor KLF4.

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 ß1 (TGFß1). 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 TGFß1-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 TGFß1-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.

IMPAIRED DARK ADAPTATION ASSOCIATED WITH A DISEASED BRUCH MEMBRANE IN PSEUDOXANTHOMA ELASTICUM.

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.

Linking ABCC6 Deficiency in Primary Human Dermal Fibroblasts of PXE Patients to p21-Mediated Premature Cellular Senescence and the Development of a Proinflammatory Secretory Phenotype.

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-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 ß-galactosidase (SA-ß-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.

First description of a compensatory xylosyltransferase I induction observed after an antifibrotic UDP-treatment of normal human dermal fibroblasts.

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-ß1 (TGF-ß1), 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.

Abcc6 deficiency in mice leads to altered ABC transporter gene expression in metabolic active tissues.

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in a huge range of physiological processes. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum, a metabolic disease with progressive soft tissue calcification. METHODS: The aim of the present study was to analyze gene expression levels of selected ABC transporters associated with cholesterol homeostasis in metabolic active tissues, such as the liver, kidney and white adipose tissue (WAT) of Abcc6-/- mice from an early and late disease stage (six-month-old and 12-month-old mice). RESULTS: The strongest regulation of ABC transporter genes was observed in the liver tissue of six-month-old Abcc6-/- mice. Here, we found a significant increase of mRNA expression levels of phospholipid, bile salt and cholesterol/sterol transporters Abcb1b, Abcb11, Abcg1, Abcg5 and Abcg8. Abcd2 mRNA expression was increased by 3.2-fold in the liver tissue. We observed strong upregulation of Abca3 and Abca1 mRNA expression up to 3.3-fold in kidney and WAT, and a 2-fold increase of Abca9 mRNA in the WAT of six-month-old Abcc6 knockout mice. Gene expression levels of Abcb1b and Abcg1 remained increased in the liver tissue after an age-related disease progression, while we observed lower mRNA expression of Abca3 and Abca9 in the kidney and WAT of 12-month-old Abcc6-/- mice. CONCLUSIONS: These data support previous findings that Abcc6 deficiency leads to an altered gene expression of other ABC transporters depending on the status of disease progression. The increased expression of fatty acid, bile salt and cholesterol/sterol transporters may be linked to an altered cholesterol and lipoprotein metabolism due to a loss of Abcc6 function.

Retinal imaging including optical coherence tomography angiography for detecting active choroidal neovascularization in pseudoxanthoma elasticum.

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.

Characterization of dermal myofibroblast differentiation in pseudoxanthoma elasticum.

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-ß1 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.

Increased vascular occlusion in patients with pseudoxanthoma elasticum.

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.
.

Identification and characterization of human xylosyltransferase II promoter single nucleotide variants.

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.

Xylosyltransferase II is the predominant isoenzyme which is responsible for the steady-state level of xylosyltransferase activity in human serum.

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.

Measurement of HMG CoA reductase activity in different human cell lines by ultra-performance liquid chromatography tandem mass spectrometry.

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 <1.3% and <2.9%, respectively. HMGCR enzymatic activity measurements of cells cultivated under different cell culture conditions (with 10% FCS, with 10% lipoprotein-deficient serum and under serum starvation) revealed the applicability of this test system for various experimental settings. This efficient UPLC-MS/MS assay permits rapid and high sensitive determination of HMGCR enzyme activity, tracing potential alterations in cholesterol biosynthesis.

First description of the complete human xylosyltransferase-I promoter region.

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 XYLT1 238*, 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 XYLT1 238* harbors various transcription factor binding sites affecting basal XYLT1 expression and inducibility by transforming growth factor-ß1, 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.

ABCC6- a new player in cellular cholesterol and lipoprotein metabolism?

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.

Optic Disc Drusen in Pseudoxanthoma Elasticum Are Associated with the Extent of Bruch's Membrane Calcification.

Background/Objectives: To assess the frequency, extent, localization and potential progression of optic disc drusen (ODD) and the correlation with the angioid streak (AS) length and retinal atrophy in patients with pseudoxanthoma elasticum (PXE). Methods: This retrospective study included patient data from a dedicated PXE clinic at the Department of Ophthalmology, University of Bonn, Germany (observation period from February 2008 to July 2023). Two readers evaluated the presence, localization, and the extent of the ODD on fundus autofluorescence (FAF) imaging at baseline and the follow-up assessments. Additionally, we measured the length of the longest AS visible at baseline and follow-up and the area of atrophy at baseline, both on FAF. Results: A total of 150 eyes of 75 PXE patients (median age at baseline 51.8 years, IRQ 46.3; 57.5 years, 49 female) underwent retrospective analysis. At baseline, 23 of 75 patients exhibited ODD in a minimum of one eye, resulting in an ODD prevalence of 30.7% in our cohort of PXE patients. Among these, 14 patients showed monocular and 9 binocular ODD that were localized predominantly nasally (46.9%). During the observational period (mean 97.5 ± 44.7 months), only one patient developed de novo ODD in one eye and one other patient showed a progression in the size of the existing ODD. The group of patients with ODD had significantly longer ASs (median 7020 µm, IQR 4604; 9183, vs. AS length without ODD: median 4404 µm, IQR 3512; 5965, p < 0.001). No association with the size of the atrophy was found at baseline (p = 0.27). Conclusions: This study demonstrates a prevalence of ODD of 30.7%. ODD presence is associated with longer ASs (an indicator of the severity and extent of ocular Bruch's membrane calcification), suggesting that ODD formation is tightly related to ectopic calcification-possibly secondary to calcification of the lamina cribrosa. Prospective studies investigating the impact of ODD (in conjunction with intraocular pressure) on visual function in PXE warrant consideration.

Matrix Metalloproteinases Contribute to the Calcification Phenotype in Pseudoxanthoma Elasticum.

Ectopic calcification and dysregulated extracellular matrix remodeling are prominent hallmarks of the complex heterogenous pathobiochemistry of pseudoxanthoma elasticum (PXE). The disease arises from mutations in ABCC6, an ATP-binding cassette transporter expressed predominantly in the liver. Neither its substrate nor the mechanisms by which it contributes to PXE are completely understood. The fibroblasts isolated from PXE patients and Abcc6-/- mice were subjected to RNA sequencing. A group of matrix metalloproteinases (MMPs) clustering on human chromosome 11q21-23, respectively, murine chromosome 9, was found to be overexpressed. A real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunofluorescent staining confirmed these findings. The induction of calcification by CaCl2 resulted in the elevated expression of selected MMPs. On this basis, the influence of the MMP inhibitor Marimastat (BB-2516) on calcification was assessed. PXE fibroblasts (PXEFs) exhibited a pro-calcification phenotype basally. PXEF and normal human dermal fibroblasts responded with calcium deposit accumulation and the induced expression of osteopontin to the addition of Marimastat to the calcifying medium. The raised MMP expression in PXEFs and during cultivation with calcium indicates a correlation of ECM remodeling and ectopic calcification in PXE pathobiochemistry. We assume that MMPs make elastic fibers accessible to controlled, potentially osteopontin-dependent calcium deposition under calcifying conditions.

The Activation of JAK/STAT3 Signaling and the Complement System Modulate Inflammation in the Primary Human Dermal Fibroblasts of PXE Patients.

Previous studies revealed a link between inflammation and overactivation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling in syndromes associated with aging. Pseudoxanthoma elasticum (PXE), a rare autosomal-recessive disorder, arises from mutations in ATP-binding cassette subfamily C member 6 (ABCC6). On a molecular level, PXE shares similarities with Hutchinson-Gilford progeria syndrome, such as increased activity of senescence-associated- beta-galactosidase or high expression of inflammatory factors. Thus, this study's aim was the evaluation of activated STAT3 and the influence of JAK1/2-inhibitor baricitinib (BA) on inflammatory processes such as the complement system in PXE. Analysis of activation of STAT3 was performed by immunofluorescence and Western blot, while inflammatory processes and complement system factors were determined based on mRNA expression and protein level. Our results assume overactivation of JAK/STAT3 signaling, increased expression levels of several complement factors and high C3 protein concentration in the sera of PXE patients. Supplementation with BA reduces JAK/STAT3 activation and partly reduces inflammation as well as the gene expression of complement factors belonging to the C1 complex and C3 convertase in PXE fibroblasts. Our results indicate a link between JAK/STAT3 signaling and complement activation contributing to the proinflammatory phenotype in PXE fibroblasts.