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Simple Summary: Pseudoxanthoma Elasticum (PXE) is an inherited disease that manifests in abnormal elastic fiber calcification in the eyes, the skin and the blood vessels. The gene responsible, called ABCC6, was identified in 2000 but it took nearly 15 years to understand how it works and how this gene's function relates to elastic fiber calcification. In essence, ABCC6 functions with at least two other enzymes (ENPP1 and CD73) to produce two inhibitors of calcification called pyrophosphate (PPi) and adenosine. In the absence of ABCC6, there is less PPi and adenosine produced, which leads to abnormal calcification in PXE patients. Remarkably, when ENPP1 or CD73 are mutated and non-functional this leads to other inherited diseases called General Arterial Calcification of Infancy (GACI) and Calcification of Joints and Arteries (CALJA), with overlapping symptoms. The three genes (ABCC6 → ENPP1 → CD73) normally work together to not only produce PPi, to prevent abnormal calcification, but also adenosine, which possesses numerous biological functions via a process called purinergic signaling. Our own work and a review of the scientific literature now indicate that PXE (and also GACI and CALJA) is an authentic "purinergic disease". In this article, we summarize the manifestations of PXE and review molecular and physiological data showing that PXE is indeed associated with a wide range of purinergic systems. Finally, we speculate on the future prospects regarding purinergic signaling and other aspects of this disease. 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. [ABSTRACT FROM AUTHOR]

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1−/− male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1−/− mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1−/− mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1−/− mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1−/− mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications. [ABSTRACT FROM AUTHOR]

Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease with autosomal recessive inheritance caused by mutations in the ABCC6 gene. Since the first description of the disease in 1896, alleging a disease involving the elastic fibers, the concept evolved with the further discoveries of the pivotal role of ectopic mineralization that is preponderant in the elastin-rich tissues of the skin, eyes and blood vessel walls. After discovery of the causative gene of the disease in 2000, the function of the ABCC6 protein remains elusive. More than 300 mutations have been now reported and the concept of a dermal disease has progressively evolved toward a metabolic disorder resulting from the remote effects caused by lack of a circulating anti-mineralization factor. Very recently, evidence has accumulated that this anti-mineralizing factor is inorganic pyrophosphate (PPi). This leads to decreased PPi/Pi (inorganic phosphate) ratio that results from the lack of extracellular ATP release by hepatocytes and probably renal cells harboring the mutant ABCC6 protein. However, the mechanism by which ABCC6 dysfunction causes diminished ATP release remains an enigma. Studies of other ABC transporters, such as ABCC7 or ABCC1 could help our understanding of what ABCC6 exact function is. Data and a hypothesis on the possible roles of ABCC6 in acquired metabolic diseases are also discussed. [ABSTRACT FROM AUTHOR]

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders. [ABSTRACT FROM AUTHOR]

Background: Pseudoxanthoma elasticum (PXE), due to rare sequence variants in the ABCC6 gene, is characterized by calcification of elastic fibers in several tissues/organs; however, the pathomechanisms have not been completely clarified. Although it is a systemic disorder on a genetic basis, it is not known why not all elastic fibers are calcified in the same patient and even in the same tissue. At present, data on soft connective tissue mineralization derive from studies performed on vascular tissues and/or on clinically affected skin, but there is no information on patients' clinically unaffected skin. Methods: Skin biopsies from clinically unaffected and affected areas of the same PXE patient (n = 6) and from healthy subjects were investigated by electron microscopy. Immunohistochemistry was performed to evaluate p-SMAD 1/5/8 and p-SMAD 2/3 expression and localization. Results: In clinically unaffected skin, fragmented elastic fibers were prevalent, whereas calcified fibers were only rarely observed at the ultrastructural level. p-SMAD1/5/8 and p-SMAD2/3 were activated in both affected and unaffected skin. Conclusion: These findings further support the concept that fragmentation/degradation is necessary but not sufficient to cause calcification of elastic fibers and that additional local factors (e.g., matrix composition, mechanical forces and mesenchymal cells) contribute to create the pro-osteogenic environment. [ABSTRACT FROM AUTHOR]

Background: Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease characterized by elastic fiber fragmentation and ectopic calcification. There is growing evidence that vascular calcification is associated with inflammatory status and is enhanced by inflammatory cytokines. Since PXE has never been considered as an inflammatory condition, no incidence of chronic inflammation leading to calcification in PXE has been reported and should be investigated. In atherosclerosis and aortic stenosis, positron emission tomography combined with computed tomographic (PET-CT) imaging has demonstrated a correlation between inflammation and calcification. The purpose of this study was to assess skin/artery inflammation and calcification in PXE patients. Methods: 18F-FluroDeoxyGlucose (18F-FDG) and 18F-Sodium Fluoride (18F-NaF) PET-CT, CT-imaging and Pulse wave velocity (PWV) were used to determine skin/vascular inflammation, tissue calcification, arterial calcium score (CS) and stiffness, respectively. In addition, inorganic pyrophosphate, high-sensitive C-reactive protein and cytokines plasma levels were monitored. Results: In 23 PXE patients, assessment of inflammation revealed significant 18F-FDG uptake in diseased skin areas contrary to normal regions, and exclusively in the proximal aorta contrary to the popliteal arteries. There was no correlation between 18F-FDG uptake and PWV in the aortic wall. Assessment of calcification demonstrated significant 18F-NaF uptake in diseased skin regions and in the proximal aorta and femoral arteries. 18F-NaF wall uptake correlated with CS in the femoral arteries, and aortic wall PWV. Multivariate analysis indicated that aortic wall 18F-NaF uptake is associated with diastolic blood pressure. There was no significant correlation between 18F-FDG and 18F-NaF uptake in any of the artery walls. Conclusion: In the present cross-sectional study, inflammation and calcification were not correlated. PXE would appear to more closely resemble a chronic disease model of ectopic calcification than an inflammatory condition. To assess early ectopic calcification in PXE patients, 18F-NaF-PET-CT may be more relevant than CT imaging. It potentially constitutes a biomarker for disease-modifying anti-calcifying drug assessment in PXE. [ABSTRACT FROM AUTHOR]

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids. [ABSTRACT FROM AUTHOR]

Background: Whilst the link between aging and thrombogenicity in atrial fibrillation (AF) is well established, the cellular underlying mechanisms are unknown. In AF, the role of senescence in tissue remodeling and prothrombotic state remains unclear. Aims: We investigated the link between AF and senescence by comparing the expression of senescence markers (p53 and p16), with prothrombotic and inflammatory proteins in right atrial appendages from patients in AF and sinus rhythm (SR). Methods: The right atrial appendages of 147 patients undergoing open-heart surgery were harvested. Twenty-one non-valvular AF patients, including paroxysmal (PAF) or permanent AF (PmAF), were matched with 21 SR patients according to CHA2DS2-VASc score and treatment. Protein expression was assessed by tissue lysates Western blot analysis. Results: The expression of p53, p16, and tissue factor (TF) was significantly increased in AF compared to SR (0.91 ± 0.31 vs. 0.58 ± 0.31, p = 0.001; 0.76 ± 0.32 vs. 0.35 ± 0.18, p = 0.0001; 0.88 ± 0.32 vs. 0.68 ± 0.29, p = 0.045, respectively). Expression of endothelial NO synthase (eNOS) was lower in AF (0.25 ± 0.15 vs. 0.35 ± 0.12, p = 0.023). There was a stepwise increase of p53, p16, TF, matrix metalloproteinase-9, and an eNOS progressive decrease between SR, PAF, and PmAF. AF was the only predictive factor of p53 and p16 elevation in multivariate analysis. Conclusions: The study brought new evidence indicating that AF progression is strongly related to human atrial senescence burden and points at a link between senescence, thrombogenicity, endothelial dysfunction and atrial remodeling. [ABSTRACT FROM AUTHOR]

Background: Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence. Methods and Results: Primary ECs were isolated from porcine atrial tissue. Endothelial senescence was assessed by measuring beta-galactosidase (SA-β-gal) activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, protein level by Western blot, and matrix metalloproteinases (MMPs) activity using zymography. Atrial endothelial senescence was induced by thrombin at clinically relevant concentrations. Thrombin induced the up-regulation of p53, a key regulator in cellular senescence and of p21 and p16, two cyclin-dependent kinase inhibitors. Nicotinamide adenine dinucleotide phosphate NADPH oxidase, cyclooxygenases and the mitochondrial respiration complex contributed to oxidative stress and senescence. Enhanced expression levels of vascular cell adhesion molecule (VCAM)-1, tissue factor, transforming growth factor (TGF)-β and MMP-2 and 9 characterized the senescence-associated secretory phenotype of atrial ECs. In addition, the pro-senescence endothelial response to thrombin was associated with an overexpression of both angiotensin converting enzyme and AT1 receptors and was inhibited by perindoprilat and losartan. Conclusions: Thrombin promotes premature ageing and senescence of atrial ECs and may pave the way to deleterious remodeling of atrial tissue by a local up-regulation of the angiotensin system and by promoting pro-inflammatory, pro-thrombotic, pro-fibrotic and pro-remodeling responses. Hence, targeting thrombin and/or angiotensin systems may efficiently prevent atrial endothelial senescence. [ABSTRACT FROM AUTHOR]