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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.

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]

Aortic stenosis (AS) affects more than 10% of the population over 80 years of age and constitutes a major risk factor for heart failure, thromboembolic stroke, and death. A better understanding of the disease, including its interaction with the haemostatic system, is a prerequisite to develop prophylactic treatments. AS pathogenesis is a dynamic process involving endothelial dysfunction, inflammation, fibrosis, and calcification. Several studies support the interplay between the components of the haemostatic system such as platelets, the coagulation system, von Willebrand factor, and extracellular micro-particles at each pathophysiological stage of AS. Previous reports have evidenced persistent biological activity of the native valve after transcatheter aortic valve replacement and the subsequent development of microthrombosis that may impact the function of the newly implanted valve. Here, we review the current evidence on the interplay between AS and prothrombotic activity, and we emphasize the clinical consequences of these interactions after aortic valve replacement. [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]

Extracellular nucleotides and adenosine regulate endocrine pancreatic functions such as insulin secretion by Langerhans islet B-cells via the activation of specific P2 and P1 receptors. Membrane-bound ectonucleotidases regulate the local concentration of these ligands and consequently control the activation of their receptors. The objective of this study was to identify and localize the major ectonucleotidases, namely NTPDases and ecto-5'-nucleotidase, present in the endocrine pancreas. In addition, the potential implication of ecto-ATPase activity on insulin secretion was investigated in the rat [beta]-cell line INS-1 (832/13). The localization of ectonucleotidase activity and protein was carried out in situ by enzyme histochemistry and immunolocalization in mouse, rat, and human pancreas sections. NTPDasel was localized in all blood vessels and acini, and NTPDase2 was localized in capillaries of Langerhans islets and in peripheral conjunctive tissue, whereas NTPDase3 was detected in all Langerhans islet cell types. Interestingly, among the mammalian species tested, ecto-5'-nucleotidase was present only in rat Langerhans islet cells, where it was coexpressed with NTPDase3. Notably, the inhibition of NTPDase3 activity by BG0136 and NF279 facilitated insulin release from INS-1 (832/13) cells under conditions of low glycemia, probably by affecting P2 receptor activation. NTPDase3 activity also regulated the inhibitory effect of exogenous ATP in the presence of a high glucose concentration most likely by controlling adenosine production. In conclusion, all pancreatic endocrine ceils express NTPDase3 that was shown to modulate insulin secretion in rat INS-1 (832/13) [beta]-cells. Ecto-5'-nucleotidase is expressed in rat Langerhans islet cells but absent in human and mouse endocrine cells. nucleoside triphosphate diphosphohydrolase; ecto-5'-nucleotidase/ CD73; P2 receptors; purinergic signaling; INS-1 (832/13) doi: 10.1152/ajpendo.00126.2010.

Keywords: ABCC6; ageing; bone loss; PXE; pyrophosphate EN ABCC6 ageing bone loss PXE pyrophosphate 1635 1637 3 10/06/22 20221001 NES 221001 ABCC6 transporter deficiency causes pseudoxanthoma elasticum (PXE), a rare disease associated with progressive calcification in the eyes, skin and arteries. This phenotype is essentially characterized by trabecular bone loss and likely linked to an increased osteoclastic activity.3 Our group, using a similar CT-scan approach4 in 2-year-old I Abcc6-/- i mice,5 evidenced a similar bone loss of the tibiae characterized by decreased trabecular bone percentage, trabecular number and increased inter trabecular spacing (see Figure 1). [Extracted from the article]

Background: Antiphospholipid syndrome is associated with endothelial dysfunction, which leads to thrombosis and early atheroma. Given that hydroxychloroquine has anti-thrombotic properties in lupus, we hypothesized that it could reduce endothelial dysfunction in an animal model of antiphospholipid syndrome. We evaluated the effect of hydroxychloroquine in preventing endothelial dysfunction in a mouse model of antiphospholipid syndrome. Methods: Antiphospholipid syndrome was induced by an injection of monoclonal anti-beta-2-GPI antibodies. Vascular reactivity was evaluated in mesenteric resistance arteries isolated from mice 3 weeks (APL3W) after receiving a single injection of anti-beta-2-GPI antibodies and after 3 weeks of daily oral hydroxychloroquine treatment (HCQ3W) compared to control mice (CT3W). We evaluated endothelial dysfunction by measuring acetylcholine-mediated vasodilation. A pharmacological approach was used to evaluate NO synthase uncoupling (tetrahydrobiopterin) and the generation of reactive oxygen species (Tempol). Results: Impaired acetylcholine-mediated dilation was evidenced in mice 3 weeks after anti-beta-2-GPI antibodies injection compared to CT3W, by reduced maximal dilation (p<0.0001) and sensitivity (pKd) (p = 0.01) to acetylcholine. Hydroxychloroquine improved acetylcholine-dependent dilation, on pKd (p = 0.02) but not maximal capacity compared to untreated mice. The addition of tetrahydrobiopterin (p = 0.02) and/or Tempol (p = 0.0008) improved acetylcholine-mediated dilation in APL3W but not in HCQ3W. Conclusions: We demonstrated that endothelial dysfunction in mouse resistance arteries persisted at 3 weeks after a single injection of monoclonal anti-beta-2-GPI antibodies, and that hydroxychloroquine improved endothelium-dependent dilation at 3 weeks, through improvement of NO synthase coupling and oxidative stress reduction. [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]

Background and aims: The contribution of arterial calcification (AC) in peripheral arterial disease (PAD) and arterial wall compressibility is a matter of debate. Pseudoxanthoma elasticum (PXE), an inherited metabolic disease due to ABCC6 gene mutations, combines elastic fiber fragmentation and calcification in various soft tissues including the arterial wall. Since AC is associated with PAD, a frequent complication of PXE, we sought to determine the role of AC in PAD and arterial wall compressibility in this group of patients. Methods and Results: Arterial compressibility and patency were determined by ankle-brachial pressure index (ABI) in a cohort of 71 PXE patients (mean age 48±SD 14 yrs, 45 women) and compared to 30 controls without PAD. Lower limb arterial calcification (LLAC) was determined by non-contrast enhanced helicoidal CT-scan. A calcification score (Ca-score) was computed for the femoral, popliteal and sub-popliteal artery segments of both legs. Forty patients with PXE had an ABI<0.90 and none had an ABI>1.40. LLAC increased with age, significantly more in PXE subjects than controls. A negative association was found between LLAC and ABI (r = −0.363, p = 0.002). The LLAC was independently associated with PXE and age, and ABI was not linked to cardiovascular risk factors. Conclusions: The presence of AC was associated with PAD and PXE without affecting arterial compressibility. PAD in PXE patients is probably due to proximal obstructive lesions developing independently from cardiovascular risk factors. [ABSTRACT FROM AUTHOR]

Hypertension is a major risk factor for cardiovascular disorders. As flow-mediated outward remodeling has a key role in postischemic revascularization, we investigated this remodeling in mesenteric resistance arteries of normotensive (WKY) and spontaneously hypertensive rats (SHRs) aged 3 to 9 months. Sequential ligation of mesenteric resistance arteries allowed modifying blood flow in vivo, thus exposing arteries to low, normal, or high flow. After 1, 3, 8, or 24 weeks, arteries were isolated for in vitro study. High flow (HF) induced outward hypertrophic remodeling in WKY rats after 1 week and persisted until 24 weeks without change in wall to lumen ratio. In SHRs, diameter increase was delayed, occurring only after 3 weeks. Nevertheless, it was reduced at 8 weeks and no longer significant after 24 weeks. In parallel, media cross-section area increased more with time in SHRs than in WKY rats and this was associated with increased contractility and oxidative stress with decreased NO-dependent relaxation. Low flow induced progressive inward remodeling until 24 weeks in both strains with excessive hypertrophy in SHRs. Thus, a chronic increase in flow induced transitory diameter expansion and long-lasting hypertrophy in SHRs. This could contribute to the higher susceptibility of hypertensive subjects to ischemic diseases. [ABSTRACT FROM AUTHOR]

Background: Pseudoxanthoma elasticum (PXE), caused by mutations in the ABCC6 gene, is a rare multiorgan disease characterized by the mineralization and fragmentation of elastic fibers in connective tissue. Cardiac complications reportedly associated with PXE are mainly based on case reports. Methods: A cohort of 67 PXE patients was prospectively assessed. Patients underwent physical examination, electrocardiogram, transthoracic echocardiography, cardiac magnetic resonance imaging (CMR), treadmill testing, and perfusion myocardial scintigraphy (SPECT). Additionally, the hearts of a PXE mouse models (Abcc6−/−) and wild-type controls (WT) were analyzed. Results: Three patients had a history of proven coronary artery disease. In total, 40 patients underwent exercise treadmill tests, and 28 SPECT. The treadmill tests were all negative. SPECT showed mild perfusion abnormalities in two patients. Mean left ventricular (LV) dimension and function values were within the normal range. LV hypertrophy was found in 7 (10.4%) patients, though the hypertrophy etiology was unknown for 3 of those patients. Echocardiography revealed frequent but insignificant mitral and tricuspid valvulopathies. Mitral valve prolapse was present in 3 patients (4.5%). Two patients exhibited significant aortic stenosis (3.0%). While none of the functional and histological parameters diverged significantly between the Abcc6−/− and WT mice groups at age of 6 and 12 months, the 24-month-old Abcc6−/− mice developed cardiac hypertrophy without contractile dysfunction. Conclusions: Despite sporadic cases, PXE does not appear to be associated with frequent cardiac complications. However, the development of cardiac hypertrophy in the 24-month-old Abcc6−/− mice suggests that old PXE patients might be prone to developing late cardiopathy. [ABSTRACT FROM AUTHOR]

Ectonucleotidases modulate purinergic signaling by hydrolyzing ATP to adenosine. Here we characterized the impact of the cellular distribution of hepatic ectonucleotidases, namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39, NTPDase2/CD39L1, NTPDase8, and ecto-5'-nucleotidase/CD73, and of their specific biochemical properties, on the levels of P1 and P2 receptor agonists, with an emphasis on adenosine-producing CD73. Immunostaining and enzyme histochemistry showed that the distribution of CD73 (protein and AMPase activity) overlaps partially with those of NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activities) in normal rat liver. CD73 is expressed in fibroblastic cells located underneath vascular endothelial cells and smooth muscle cells, which both express NTPDase1, in portal spaces in a distinct fibroblast population next to NTPDase2-positive portal fibroblasts, and in bile canaliculi, together with NTPDase8. In fibrotic rat livers, CD73 protein expression and activity are redistributed but still overlap with the NTPDases mentioned. The ability of the observed combinations of ectonucleotidases to generate adenosine over time was evaluated by reverse-phase HPLC with the recombinant rat enzymes at high "inflammatory" (500 μM) and low "physiological" (1 μM) ATP concentrations. Overall, ATP was rapidly converted to adenosine by the NTPDase1+CD73 combination, but not by the NTPDase2+CD73 combination. In the presence of NTPDase8 and CD73, ATP was sequentially dephosphorylated to the CD73 inhibitor ADP, and then to AMP, thus resulting in a delayed formation of adenosine. In conclusion, the specific cellular cocompartmentalization of CD73 with hepatic NTPDases is not redundant and may lead to the differential activation of P1 and P2 receptors, under normal and fibrotic conditions. [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]

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]

Tumor-associated macrophages (TAM) are immunosuppressive cells that can massively accumulate in the tumor microenvironment. In patients with ovarian cancer, their density is correlated with poor prognosis. Targeting mediators that control the generation or the differentiation of immunoregulatory macrophages represents a therapeutic challenge to overcome tumor-associated immunosuppression. The ectonucleotidase CD39 hydrolyzes ATP into extracellular adenosine that exhibits potent immunosuppressive properties when signaling through the A2A adenosine receptor. We report here that CD14+CD163+TAM isolated from ovarian cancer patients and macrophages generatedin vitrowith M-CSF, express high levels of the membrane ectonucleotidase CD39 compared to classically activated macrophages. The CD39 inhibitor POM-1 and adenosine deaminase (ADA) diminished some of the immunosuppressive functions of CD14highCD163highCD39highmacrophages, such as IL-10 secretion. We identified the cytokine IL-27, secreted by tumor-infiltrating neutrophils, located close to infiltrating CD163+macrophages, as a major rheostat of CD39 expression and consequently, on the acquisition of immunoregulatory properties by macrophages. Accordingly, the depletion of IL-27 downregulated CD39 and PD-L1 expression as well as IL-10 secretion by M-CSF-macrophages. Collectively, these data suggest that CD39, drived by IL-27 and CD115 ligands in ovarian cancer, maintains the immunosuppressive phenotype of TAM. This work brings new information on the acquisition of immunosuppressive properties by tumor-infiltrating macrophages. [ABSTRACT FROM AUTHOR]

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (P110β and p110γ) have been implicated in Gi-dependent integrin αIIbβ3 regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110β is primarily responsible for G1-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin αIIbβ3 activation in p110γ-/- platelets was not associated with alterations in the levels of PI(3,4)P2 or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that P110γ principally regulated integrin αIIbβ3 activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110γ, led to a major reduction in integrin αIIbβ3 activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110γ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110β and p110γ is critical for P2Y12/Gi-dependent integrin αIIbβ3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways. [ABSTRACT FROM AUTHOR]

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease. [Display omitted] • Sepsis induces expansion of an immune-suppressive CD39-expressing B cell population • Septic B cells have metabolic reprogramming supporting their suppressive activity • Adenosine derived from CD39+ B cells impairs macrophage bacterial killing • Adenosine signaling via A2aR promotes macrophage production of suppressive IL-10 Sepsis causes immunosuppression and increased susceptibility to infection by unknown means. Nascimento et al. show sepsis-induced expansion of a CD39hi plasmablast population that elevated circulating adenosine. Adenosine signaling in macrophages suppressed microbial killing and promoted IL-10 production. This work highlights CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression. [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.

Background: Pseudoxanthoma elasticum (PXE) is a genetic disease caused by mutations in the ABCC6 gene that result in low pyrophosphate levels and subsequent progressive soft tissue calcifications. PXE mainly affects the skin, retina, and arteries. However, many patients with PXE experience kidney stones. We determined the prevalence of this pathology in patients with PXE and examined the possible underlying mechanisms in murine models., Methods: We conducted a retrospective study in a large cohort of patients with PXE and analyzed urine samples and kidneys from Abcc6 -/- mice at various ages. We used Yasue staining, scanning electron microscopy, electron microscopy coupled to electron energy loss spectroscopy, and Fourier transform infrared microspectroscopy to characterize kidney calcifications., Results: Among 113 patients with PXE, 45 (40%) had a past medical history of kidney stones. Five of six computed tomography scans performed showed evidence of massive papillary calcifications (Randall plaques). Abcc6 -/- mice spontaneously developed kidney interstitial apatite calcifications with aging. These calcifications appeared specifically at the tip of the papilla and formed Randall plaques similar to those observed in human kidneys. Compared with controls, Abcc6 -/- mice had low urinary excretion of pyrophosphate., Conclusions: The frequency of kidney stones and probably, Randall plaque is extremely high in patients with PXE, and Abcc6 -/- mice provide a new and useful model in which to study Randall plaque formation. Our findings also suggest that pyrophosphate administration should be evaluated for the prevention of Randall plaque and kidney stones., (Copyright © 2018 by the American Society of Nephrology.)

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

Objective: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT., Approach and Results: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries., Conclusions: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases., (© 2016 American Heart Association, Inc.)