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Determination of PPi plasma content for the 4 samples. Means for all duplicates (shown individually in Supplementary Table S1) were determined, and blanks were subtracted. These mean values were plotted, and the linear gradient was calculated. Conversion of the formulae (including a 10× correction) yielded the PPi content (µM) of each sample. The gradient and intercept varied between patient samples, again suggesting that the plasma content may affect the luminescence assay readout. For instance, sample 12.1 had a higher PPi level at readout compared to the other samples, notwithstanding its lower luminescence output.
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Pseudoxanthoma elasticum (PXE) is a rare ectopic calcification disorder affecting soft connective tissues that is caused by biallelic ABCC6 mutations. While the underlying pathomechanisms are incompletely understood, reduced circulatory levels of inorganic pyrophosphate (PPi)—a potent mineralization inhibitor—have been reported in PXE patients and...
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Citations
... The ABCC6 gene involved in PXE encodes the ATP-binding cassette transporter ABCC6, the activity of which appears to be linked to the cellular outflow of ATP and the subsequent generation of inorganic pyrophosphate (PPi) [10]. Pyrophosphate is known as a physiological plasma anti-calcifying factor, and plasma PPi is chronically decreased in Abcc6 −/− animal models [10][11][12] and by 25 to 60% in PXE patients [12][13][14][15][16][17]. To date, in the absence of standardized measurement methods to determine PPi concentration in the plasma, the diagnostic value of PPi for PXE has never been tested, and its relevance remains to be established. ...
... A low plasma PPi has been consistently reported in PXE patients with a reduction rate ranging from 25 to 60% [12][13][14][15][16][17] despite significant differences in the absolute values: plasma PPi levels in PXE patients versus healthy controls were reported in a Belgian cohort (mean value 0.497 vs. 0.985 µM [17]), in a French cohort (median value 0.77 vs. 1.53 µM [15]), in a Dutch cohort (0.53 vs. 1.13 µM [14]) and in a Spanish cohort (1.11 vs. 1.43 µM [16]). Altogether, comparisons of plasma PPi concentration between all these studies are somewhat difficult since it depends on (1) the type of blood collection tube (CTAD, citrate, heparin, EDTA, . . ...
... A low plasma PPi has been consistently reported in PXE patients with a reduction rate ranging from 25 to 60% [12][13][14][15][16][17] despite significant differences in the absolute values: plasma PPi levels in PXE patients versus healthy controls were reported in a Belgian cohort (mean value 0.497 vs. 0.985 µM [17]), in a French cohort (median value 0.77 vs. 1.53 µM [15]), in a Dutch cohort (0.53 vs. 1.13 µM [14]) and in a Spanish cohort (1.11 vs. 1.43 µM [16]). Altogether, comparisons of plasma PPi concentration between all these studies are somewhat difficult since it depends on (1) the type of blood collection tube (CTAD, citrate, heparin, EDTA, . . ...
Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, p < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE.
... Moreover, it would also be a convenient diagnostic aid for HPP and disorders with decreased plasma PPi levels, such as generalized arterial calcification of infancy, autosomal recessive hypophosphatemic rickets 2, and pseudoxanthoma elasticum with ENPP1 and ABCC6 variants. [15,[19][20][21] Contrary to the result in our case, a previous report on a murine model of HPP (ALPL À/À mice) injected with adeno-associated virus (AAV) 8-TNSALP-deca-aspartate (D 10 ) did not present uPPi/Cre reduction after treatment with AAV8-TNSALP-D 10 , although plasma PPi significantly decreased. [22] AAV8-TNSALP-D 10 differs from AA in that the latter has a human IgG1 Fc domain for drug purification; however, they are qualitatively identical treatments. ...
Hypophosphatasia (HPP) is an inherited disease caused by variants of the ALPL gene encoding tissue‐nonspecific alkaline phosphatase. Adult‐onset HPP (adult HPP), known as a mild form of HPP, develops symptoms involving osteomalacia after the age of 18 years. Asfotase alfa (AA) is a modulated recombinant human alkaline phosphatase (ALP) that has been established as a first‐line therapy for severe forms of HPP, such as perinatal and infantile forms. We described a 64‐year‐old female who presented with pseudofractures in bilateral femur diaphyses and impaired mobility. Low serum ALP activity and a high concentration of urine phosphoethanolamine indicated the diagnosis of HPP, which was confirmed by the identification of a homozygous variant in the ALPL gene (c.319G > A; p.Val107Ile). An in vitro transfection experiment to measure the ALP activity of this novel variant protein was performed, resulting in 40% of the residual enzymatic activity compared with the wild type. AA was initiated to facilitate the union of pseudofracture and to improve mobility. After 6 months, radiographic images revealed the disappearance of fracture lines, and improvement of ambulatory ability was confirmed by the 6‐minute walk test (525 to 606 m). The EQ‐5D‐5L index was also improved (0.757 to 0.895). Within a follow‐up period, the levels of urine pyrophosphate corrected by urine creatinine (uPPi/Cre) declined in parallel with the level of plasma PPi (plasma PPi: 6.34 to 1.04 μM, uPPi/Cre: 226.8 to 75.4 nmol/mg). The beneficial effect of AA on pseudofracture healing in adult HPP was presented, although the application of AA should be restricted to patients exhibiting relatively severe manifestations. In addition, a novel pathogenic variant of the ALPL gene was identified with the supportive result of functional analysis. Furthermore, when monitoring patients with HPP treated with AA, uPPi/Cre might be a convenient substitute for plasma PPi, which requires immediate filtration after blood sampling. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
... The authors suggested it to be closely linked to increased osteoclastic activity, but, at the same time, these observations pose the question of the importance of PPi/Pi balance in bone homeostasis ( Figure 3) [111,116]. Moreover, the ABCC6 transporter facilitates ATP efflux, and PXE patients have lowered plasma levels of PPi compared to healthy controls and heterozygous carriers [49,[117][118][119][120]. The existence of a subset of adult GACI patients with pathogenic variants in ABCC6 or ENPP1 that have a complete lack of PPi in their plasma and suffer from hypophosphatemic rickets strengthens the belief that PPi/Pi balance may have an influence on bone, though there is currently little additional experimental evidence for this hypothesis [121]. ...
Zebrafish are increasingly becoming an important model organism for studying the pathophysiological mechanisms of human diseases and investigating how these mechanisms can be effectively targeted using compounds that may open avenues to novel treatments for patients. The zebrafish skeleton has been particularly instrumental in modeling bone diseases as—contrary to other model organisms—the lower load on the skeleton of an aquatic animal enables mutants to survive to early adulthood. In this respect, the axial skeletons of zebrafish have been a good read-out for congenital spinal deformities such as scoliosis and degenerative disorders such as osteoporosis and osteoarthritis, in which aberrant mineralization in humans is reflected in the respective zebrafish models. Interestingly, there have been several reports of hereditary multisystemic diseases that do not affect the vertebral column in human patients, while the corresponding zebrafish models systematically show anomalies in mineralization and morphology of the spine as their leading or, in some cases, only phenotype. In this review, we describe such examples, highlighting the underlying mechanisms, the already-used or potential power of these models to help us understand and amend the mineralization process, and the outstanding questions on how and why this specific axial type of aberrant mineralization occurs in these disease models.
The global population of individuals with cardiovascular disease is expanding, and a key risk factor for major adverse cardiovascular events is vascular calcification. The pathogenesis of cardiovascular calcification is complex and multifaceted, with external cues driving epigenetic, transcriptional, and metabolic changes that promote vascular calcification. This review provides an overview of some of the lesser understood molecular processes involved in vascular calcification and discusses the links between calcification pathogenesis and aspects of adenosine signaling and the methionine pathway; the latter of which salvages the essential amino acid methionine, but also provides the substrate critical for methylation, a modification that regulates the function and activity of DNA and proteins. We explore the complex and dynamic nature of osteogenic reprogramming underlying intimal atherosclerotic calcification and medial arterial calcification (MAC). Atherosclerotic calcification is more widely studied however emerging studies now show MAC is a significant pathology independent from atherosclerosis. Further, we emphasize metabolite and metabolic-modulating factors that influence vascular calcification pathogenesis. While the contribution of these mechanisms are more well-define in relation to atherosclerotic intimal calcification, understanding these pathways may provide crucial mechanistic insights into MAC and inform future therapeutic approaches. Herein we highlight the significance of adenosine and methyltransferase pathways as key regulators of vascular calcification pathogenesis.
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.
Pseudoxanthoma Elasticum (PXE) is an inherited disease characterized by elastic fiber calcification in the eyes, the skin and the cardiovascular system. PXE results from mutations in ABCC6 that encodes an ABC transporter primarily expressed in the liver and kidneys. It took nearly 15 years after identifying the gene to better understand the etiology of PXE. ABCC6 function facilitates the efflux of ATP, which is sequentially hydrolyzed by the ectonucleotidases ENPP1 and CD73 into pyrophosphate (PPi) and adenosine, both inhibitors of calcification. PXE, together with General Arterial Calcification of Infancy (GACI caused by ENPP1 mutations) as well as Calcification of Joints and Arteries (CALJA caused by NT5E/CD73 mutations), forms a disease continuum with overlapping phenotypes and shares steps of the same molecular pathway. The explanation of these phenotypes place ABCC6 as an upstream regulator of a purinergic pathway (ABCC6 → ENPP1 → CD73 → TNAP) that notably inhibits mineralization by maintaining a physiological Pi/PPi ratio in connective tissues. Based on a review of the literature and our recent experimental data, we suggest that PXE (and GACI/CALJA) be considered as an authentic “purinergic disease”. In this article, we recapitulate the pathobiology of PXE and review molecular and physiological data showing that, beyond PPi deficiency and ectopic calcification, PXE is associated with wide and complex alterations of purinergic systems. Finally, we speculate on the future prospects regarding purinergic signaling and other aspects of this disease.
Background and aims
– Pseudoxanthoma elasticum (PXE) is a rare genetic disease caused by pathogenic mutations in the ABCC6 gene, resulting in low values of inorganic pyrophosphate (PPi). While low PPi is thought to contribute to arterial calcification, it remains unclear whether this fully explains premature calcification in PXE. It has been hypothesized that the ABCC6 gene could be related to dyslipidemia, which could contribute to vascular calcification seen in PXE. The aim of this study is to evaluate the relation between PXE and plasma lipid concentrations in a large cohort of PXE patients compared with reference values for the general population and compared with non-PXE controls.
Methods
– The plasma concentrations of total cholesterol, HDL-cholesterol, tiglycerides, and LDL-cholesterol of 312 PXE patients were compared to age- and sex-matched modeled data of the general Dutch population. Differences in median lipid levels were compared with Mann-Whitney-U test. Secondly, plasma lipid concentrations of 44 PXE patients were compared to 44 not-genetically related relatives (spouses or friends), with linear models adjusted for age, sex and BMI.
Results
– Total cholesterol in PXE patients was 5.6 [IQR 4.6–6.4] mmol/L versus 5.3 [IQR 4.7–6.0] mmol/L (p < 0.01) in the general population; triglycerides were 1.1 [IQR 0.9–1.7] mmol/L versus 1.0 [0.7–1.4] mmol/L (p < 0.01); HDL-c was 1.4 [IQR 1.2–1.7] mmol/L versus 1.5 [IQR 1.2–1.8] mmol/L (p = 0.03) and LDL-c was 3.3 [IQR 2.7–4.1] mmol/L versus 3.2 [IQR 2.7–3.8] mmol/L (p = 0.01). In the patient control analysis with 44 pairs and age, sex and BMI adjusted, comparison with the non-PXE controls only triglycerides were significantly different (mean difference: 0.38 (0.13–0.63)).
Conclusion
–The lipid profiles of PXE patients are marginally different from the general population or compared to a matched control group, but the differences are unlikely to be clinically relevant. It is therefore unlikely that plasma lipids contribute to the premature vascular calcifications in PXE patients.
