apyrase and Calcinosis

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Antigens, CD; Aortic Diseases; Apyrase; Calcinosis; Diphosphates; Disease Models, Animal; Gene Knock-In Techniques; Humans; Lamin Type A; Levamisole; Longevity; Male; Mice; Mice, Transgenic; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; Progeria; Pyrophosphatases; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.

Topics: 5'-Nucleotidase; Adult; Aged; Aortic Valve; Aortic Valve Stenosis; Apyrase; Biomarkers; Calcinosis; Case-Control Studies; Chromatography, Liquid; Female; Filtration; Gene Expression; GPI-Linked Proteins; Heme Oxygenase-1; Humans; Male; Middle Aged; Proteolysis; Proteomics; Specimen Handling; Tandem Mass Spectrometry

Calcific aortic valve disease is an active disease process with lipoprotein deposition, chronic inflammation, and progressive leaflet degeneration. Expression of ectonucleotidases, a group of membrane-bound enzymes that regulate the metabolism of ATP and its metabolites, may coregulate the degeneration process of valvular interstitial cells (VICs). The aim of this study was to investigate the role of the enzymes of the purinergic system in the degeneration process of VICs. Ovine VICs were cultivated in vitro under different prodegenerative conditions and treated with inhibitors of ectonucleoside triphosphate diphosphohydrolase 1 (CD39)/ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and 5'-nucleotidase (CD73), as well as with adenosine and adenosine receptor agonists. Experiments were performed both in 2-dimensional (2-D) and 3-dimensional (3-D) cell-culture models. Our main findings were that VICs continuously release ATP. Inhibition of ATP hydrolyzing enzymes (CD39 and ENPP1) resulted in profound prodegenerative effects with a vigorous up-regulation of CD39, ENPP1, and CD73, as well as TGF-β1 and osteopontin at the gene level. In our 3-D model, the effect was more pronounced than in 2-D monolayers. Increasing adenosine levels, as well as stimulating the adenosine receptors A

Topics: 5'-Nucleotidase; Adenosine Triphosphate; Animals; Antigens, CD; Aortic Valve; Aortic Valve Stenosis; Apyrase; Bicuspid Aortic Valve Disease; Calcinosis; Cell Culture Techniques; Cellular Microenvironment; Heart Defects, Congenital; Heart Valve Diseases; Phosphoric Diester Hydrolases; Purinergic Agents; Pyrophosphatases; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Sheep; Signal Transduction; Up-Regulation

Topics: 5'-Nucleotidase; Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Antigens, CD; Apyrase; Atherosclerosis; Calcinosis; Fibroblasts; Gene Deletion; Humans; Hydrolysis; Mutation; Phosphoric Diester Hydrolases; Pyrophosphatases

Calcific aortic valve disease is a common condition and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes, but the cellular mechanisms are unclear. Recent studies identified extracellular ATP and P2Y receptor cascade as important regulators of bone remodeling, whereas its breakdown product, adenosine, is known to have anti-inflammatory properties. We hypothesize that extracellular ATP and adenosine have important roles in regulating osteoblast differentiation in human valve interstitial cells, and that this can be a potential target for therapy. Method and Results- Primary cultures of human valve interstitial cells (ICs) treated for 21 days with osteogenic media, ATP, and ATP-gamma-S (a stable agonist of the P2Y receptor) revealed a significant increase in alkaline phosphatase (ALP) (an osteoblast marker) activity and expression as measured using spectrophotometric assay and immunocytochemistry staining. Valve ICs treated with adenosine alone did not cause an increase in ALP activity; however, adenosine treatment decreased the ALP activity and expression induced by osteogenic media after 21 days of incubation. In addition, atorvastatin inhibited the activity of ALP induced by ATP in human valve ICs, and enzyme studies revealed that atorvastatin upregulated the breakdown of extracellular ATP into adenosine in human valve ICs after 24-hour treatment.. These findings identify a novel role for extracellular nucleotides in inducing osteoblast differentiation in human valve ICs in vitro and provide a potential therapeutic target for preventing the disease progression.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphatases; Adenosine Triphosphate; Aged; Alkaline Phosphatase; Aortic Valve; Aortic Valve Stenosis; Apyrase; Atorvastatin; Biomarkers; Calcinosis; Cell Differentiation; Cells, Cultured; Drug Evaluation, Preclinical; Heptanoic Acids; Humans; Middle Aged; Osteoblasts; Pyrroles; Receptors, Purinergic P2; Second Messenger Systems

Aortic medial calcification was investigated in rats in which the progeria-like syndrome (PLS) was evoked by administering dihydrotachysterol. In 35 experimental rats and 15 controls, calcification was studied morphologically by light and electron microscopy, and by enzyme histochemistry. Body weight, food intake and serum calcium levels were also determined. Calcification occurred along and on the elastic lamellae in association with the accumulation of ground substance. In the smooth-muscle cells surrounding the calcified foci, the activities of various lysosomal enzymes increased concomitantly with a tendency toward transformation of smooth-muscle cells to a modified form. From these observations, the role of ground-substance formation by smooth-muscle cells is postulated, and participation in the catabolism of ground substance by the lysosomal enzymes of these cells is suggested. It appears the increased activity of adenosine monophosphatase should be linked to the calcification. The etiology of weight loss, skin manifestations and aortic calcification in PLS rats seems to be different from that in human progeric diseases. Therefore, the PLS rat should not be readily accepted as an animal model for the study of progeric diseases.

Topics: Acid Phosphatase; Adenosine Triphosphatases; Animals; Aorta; Aortic Diseases; Apyrase; Body Weight; Calcinosis; Calcium; Dihydrotachysterol; Female; Glucuronidase; Hexosaminidases; Histocytochemistry; Phosphoric Monoester Hydrolases; Rats; Werner Syndrome