An expanded methods section is available in the Supplementary material online.
Patients and tissue collection
The study was performed based on the standards of the Declaration of Helsinki and it was approved by the local ethical committee. Informed consent has been obtained from the patients. Three leaflet aortic valves were collected during valve replacement for CAVD and named as stenotic aortic valves (total number of used valves: n = 71, mean age: 60, median age 62; 43 males; 28 females; age range: 36–74), while control aortic valves were obtained during heart transplantation or Bentall procedures and named as non-stenotic aortic valves (total number of used valves: n = 34, mean age: 53, median age: 53; 22 males; 13 females; age range: 28–75). The exact number of valves used for each experiment has been indicated in the figure legend. Dissected human aortic valve leaflets were immediately placed into ice-cold physiologic salt solution and transported to the laboratory on ice within 30 min of harvest.
Determination of aortic valve surface ecto-enzymes activities
For the determination of ecto-enzymes activities, valve leaflets were weighed and washed in Hanks Balanced Salt Solution (HBSS). Then, aortic valve leaflets were divided into 0.2 cm2 sections and in this intact condition directly placed into incubation solution. The modified assay system based on exposition into incubation medium by fibrosa and ventricularis surfaces separately. An intact valve leaflet fragment was fixed under the 0.5 cm diameter hole drilled in the bottom of one well of 24-well plate. It was supported by a plastic plate and the pressure was adjusted to ensure an effective seal. The leaflet fragment, clamped between two plastic plates, fully sealed the area exposed to the incubation medium . Next, each well has been washed twice with HBSS and 1 mL of HBBS with 50 µM adenosine, ATP or AMP was sequentially added with medium exchange after each substrate. 5 μm erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an inhibitor of adenosine deaminase, was present during incubation with ATP and AMP to block the conversion of adenosine to inosine . Although, nucleotides and nucleosides are maintained in extracellular space at nanomolar level, we adjusted the substrate concentration to micromolar as these compounds operate on the cell surface in the “pericellular halo” . To ensure that evaluated activities originate exclusively from the action of extracellular enzymes, part of experiments were conducted with the nucleoside transport inhibitor: 5 μm S-(4-Nitrobenzyl)-6-thioinosine (NBTI) . After 0, 5, 15 and 30 min of incubation at 37 °C samples were collected and concentrations of nucleotides and nucleosides were measured by reversed-phase HPLC according to the method described earlier . Enzyme activities were calculated from linear phase of the reaction and in the main experiment, the rates were normalized to the surface area. Final results for each patient based on the average activity obtained from 3 valve leaflets. After the experiment, valve leaflet fragments were washed in HBSS, dried and frozen at − 80 °C for later use.
Determination of valve deposits compounds concentrations
Sections of aortic valve leaflets, previously used for the estimation of ecto-enzymes activities, were quickly thawed and dissolved separately in 6 m HCl at 95 °C for 24 h followed by centrifugation at 2000×g during 30 min. The supernatant was collected and diluted with deionized water and used for the determination of calcium and magnesium or diluted with 0.6 m H2SO4 for phosphate determination as described in Supplementary material online.
Pre-operative echocardiography, blood pressure measurement and biochemical blood analyzes
All patients underwent a Doppler pre-operative echocardiographic examinations of aortic jet velocity (Vmax) and mean transvalvular gradient using the Vivid Q Portable Ultrasound (GE Healthcare, USA). Blood pressure measurement was taken with a validated electronic device, and it was the mean of two readings. For biochemical analysis, blood samples were collected from all subjects in a fasting state and serum or citrate plasma were obtained by centrifugation (1700×g, 10 min, 21 °C). Parameters of the coagulation system (prothrombin time, international normalized ratio), lipid profile and carbohydrate metabolism parameters (glycemia and glycated hemoglobin HbA1c) were measured in patients using standard methods.
All experiments were conducted in accordance with the guide for the care and use of laboratory animals published by the European Parliament, Directive 2010/63/EU and were approved by the local Bioethical Committee.
Male C57BL/6 J mice (wild type, WT) and double knock-out for apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) on the C57BL/6 J background (ApoE−/−LDLR−/−) , originally obtained from Jackson Lab (USA) were bred in house and used for the experiments at the age of 3, 6 or 10 months. Animals had an unlimited access to water and standard chow diet. Blood samples were collected by tail vein puncture. Serum was obtained after centrifugation (1700×g, 10 min, 21 °C). Mice were sacrificed under anaesthesia by a intraperitoneal injection of ketamine and xylazine (100 mg/kg/10 mg/kg). Isolated fragments of aortic roots were cleaved from a perivascular adventitia in cold HBSS and used for the measurements of nucleotides and adenosine conversion rates. For histological and immunofluorescence analyses, aortic roots were collected, placed into an Optimal Cutting Temperature (OCT) compound, and snap-frozen at − 80 °C or preserved in 4% buffered formalin solution and embedded in paraffin.
Treatment of ApoE−/−LDLR−/− mice
4-month-old ApoE−/−LDLR−/− mice were treated with adenosine deaminase inhibitor, deoxycoformycin (pentostatin, dCF) as shown before . Saline-treated ApoE−/−LDLR−/− mice were used as controls. Isolated fragments of aortic roots were cleaned as described above and used for the measurement of ALP activity. For histological analysis of aortic valve thickness, aortic roots were placed into an OCT compound and snap-frozen at − 80 °C. Serum samples were obtained as described above and used for the measurement of ALP activity, calcium, magnesium and phosphate concentration by standard methods using automated chemistry analyzer XL180 analyzer (ERBA Mannheim, Germany) Biochemica. The efficiency of dCF in blocking ADA and the effects of dCF in this animal model on endothelial function, blood nucleotide and adenosine concentration, biochemical parameters and morphology were investigated earlier .
Representative non-stenotic and stenotic aortic valve leaflets or mice aortic roots were fixed in 4% buffered formaldehyde, decalcified (if necessary) and embedded in paraffin. Then, the paraffin-embedded aortic valve leaflets or aortic roots were cut into 5 μm-thick cross-sections using a histological microtome, placed on microscopic slides and deparaffinized prior to staining. Aortic valve sections were stained with hematoxylin and eosin (HE) for general morphology. For the assessment of specific aortic valve morphology, adjacent sections were stained according to Masson’s Trichrome (TR) standard protocol and Orcein Martius Scarlet Blue (OMSB) protocol . These stainings allowed to characterize non-stenotic and stenotic valve composition, including cellular components as well as extracellular matrix fibers (loose connective tissue), collagen fibers (dense connective tissue), calcium nodules and myofibroblast-like cells, which far exceeds the capabilities of standard staining for calcium deposits. Mice aortic roots were stained according to OMSB and Oil Red O protocols [34, 35]. Human valvular interstitial cells were stained on 24-well-plate using von Kossa staining protocol . The acquisition and processing of stained section images were described in the Supplementary material online.
Adjacent aortic valve sections to sections used for histological stainings were used to immunofluorescence analysis (IF). 5 μm-thick paraffin-embedded aortic valve cross-sections were collected on polylysine-covered microscopic slides and deparaffinized using a standard protocol. Next, sections were pretreated according to the citrate-base HIER (Sigma) protocol to unmask the antigens and epitopes in formalin-fixed and paraffin-embedded sections. The OCT-frozen mice aortic roots originated from 6-month-old male WT and ApoE−/−LDLR mice were cut into 10 μm-thick cross sections (using Leica CM1920 cryotome), using a standardized protocol. Aortic root cross-sections were collected on polilisine-covered microscopic slides and fixed in acetone for 10 min. Human primary aortic valve endothelial (hVEC) and interstitial (hVIC) cells intended to IF were seeded on 96-well optical-bottom plate (Nunc ThermoFisher, USA) at a density 1 × 104 cells/well in a total volume of 200 μL cell culture medium. 24 h after seeding of hVEC and 72 h after seeding of hVIC, cell culture medium was removed and rinsed 3 times with PBS. Cells were fixed using 100 μL 4% paraformaldehyde (pH 7.4) for 10 min at 37 °C. Paraformaldehyde was removed and washed three times with PBS. To reduce non-specific antibody binding, slices or cells were preincubated with a PAD solution (5% of normal goat serum and 2% of filtered dry milk) (Sigma). The antibodies used and their origin were described in the Supplementary material online. Primary antibodies were used at 1:100 final dilution (1 h incubation), while secondary antibodies at 1:600 (30 min incubation). Negative controls omitted the primary antibodies (data not shown). Cell nuclei were counterstained with Hoechst 33258 (Sigma) (1:1500 final dilution, 5 min incubation). Images were recorded with an AxioCam MRc5 camera and an AxioObserved.D1 inverted fluorescent microscope (Zeiss) with appropriate filter cubes to show Cy3 (red), Alexa Fluor 488 (green) and Hoechst 33258 (blue) fluorescence, stored as tiff files and analyzed automatically using the Columbus Image Data Storage and Analysis System (Perkin Elmer). Total human CD39, CD73, eNPP1, ALP, ADA, A1R, A2aR, A2bR and A3R positive area in aortic valves were measured in each slide and the percentage of total aortic valve cross-sectional area covered by red signal was calculated from six sections.
Human non-stenotic and stenotic aortic valves were directly lysed with QIAzol® Lysis Reagent (Qiagen, Hilden, Germany) by shaking (5 min) in the presence of 3 mm diameter solid glass beads (Sigma, USA). Total RNA was isolated with RNeasy mini kit (Qiagen) according to the manufacturer’s instruction. To prevent DNA contamination, samples were pretreated with RNase-free DNase (Qiagen). The concentration of RNA was calculated based on the absorbance at 260 nm. RNA samples were stored at − 70 °C until use. For the measurement of CD39, CD73, ADA, ADORA1, ADORA2a, ADORA2b and ADORA3 mRNA expression, TaqManOne-Step RT-PCR Master MixReagents (Applied Biosystems, USA) were used as described previously [37, 38] according to the manufacturer’s protocol. The relative expressions were calculated using the comparative relative standard curve method . We used housekeeping gene, TATA-binding protein (TBP), as the relative control. TaqMan probes ids were given in the Supplementary material online.
Non-stenotic aortic valve cells isolation and culture
Aortic valve endothelial (hVEC) and interstitial (hVIC) cells were isolated from non-stenotic human aortic valves as was shown in Fig. 4a. The valve was digested with 5 mL collagenase A (0.15% w/v) for 10 min at 37 °C to obtain hVEC. 5 mL of EBM-2 Medium (Lonza, USA) was added to stop the action of collagenase. To isolate hVIC, the valve was minced and further digested with 5 mL collagenase A (0.15% w/v) for additional 45 min at 37 °C. 5 mL of DMEM (Sigma, USA) supplemented with 1 mmol/L l-glutamine, 10% FBS and 1% penicillin/streptomycin (v/v) (Sigma, USA) was added to neutralize the collagenase. Each of the suspensions, hVEC and hVIC, was purified using mesh filters 100 μm, 70 μm, 40 μm and centrifuged (150×g, 4 min). After centrifugation, hVEC pellet was resuspended in EBM-2 Medium (Lonza, USA), while hVIC pellet in a standard Dulbecco’s Modified Eagle’s medium (DMEM, Sigma, USA) supplemented with 1 mmol/L l-glutamine, 10% FBS and 1% penicillin/streptomycin (v/v) (Sigma, USA). Cells were cultured at 37 °C, in 5% CO2 atmosphere and used for experiments at passage 4.
Stenotic aortic valve cells isolation
Aortic valve endothelial and interstitial cells, as well as immune infiltrate, were also isolated from stenotic human aortic valves as was shown in Fig. S4a. hVEC and immune cells located in the upper layers of the valve were isolated after 10 min incubation with agitation in 5 mL of collagenase A (0.15% w/v) at 37 °C. 5 mL of EBM-2 Medium (Lonza, USA) was added to stop the action of collagenase. Aortic valve transport medium and suspension obtained after the first step of isolation were purified using mesh filters 100 μm, 70 μm, 40 μm. After centrifugation (150xg, 4 min), pellets were resuspended in MACS buffer, pooled and used for FACS analysis. hVIC and immune cells derived from the deeper layers of the valve were isolated after mincing of the valve and additional digestion for 45 min in 5 mL of collagenase A (0.15% w/v) at 37 °C. 5 mL of DMEM (Sigma, USA) supplemented with 1 mmol/L l-glutamine, 10% FBS and 1% penicillin/streptomycin (v/v) (Sigma, USA) was added to neutralize the collagenase. Petri place used for mincing of the tissue was washed using PBS and collected solution and suspension obtained after the second step of isolation were purified using mesh filters 100 μm, 70 μm, 40 μm. After centrifugation (150xg, 4 min), pellets were resuspended in MACS buffer, pooled and used for FACS analysis.
Peripheral blood mononuclear cells isolation
Peripheral blood mononuclear cells (PBMC) were isolated from healthy adult donors using a Histopaque procedure as described in the Supplementary material online.
Determination of specific ecto-enzyme activities on the surface of human aortic valve cells, immune cells and mice aortic roots
hVEC and hVIC isolated from non-stenotic aortic valves were used at passage 4 and seeded on 24-well plates at a density of 0.05 × 106 cells/well. hVIC treated with osteogenic medium was cultured for 19 days in cell culture medium supplemented with 3 mm phosphate with medium exchange every 2 days. Cells were used for the experiments at 90–100% confluency and washed with HBSS. Isolated human peripheral blood mononuclear cells and monocyte/macrophage cells (SC line, ATCC, cat. CRL-9855) that were used at passage 4, were plated in 24-well cell culture plate at a density 0.2 × 106 per well in a total volume of 1 mL HBSS. Mice aortic roots were cleaned of surrounding tissues as described above and used for experiment. Cells or mice aortic roots were pre-incubated in HBSS for 15 min at 37 °C with specific ecto-enzyme inhibitors, including 5 μm erythro-9-(2-hydroxy-3-nonyl) adenine for ADA1, 150 μm adenosine 5′-(α,β-methylene)diphosphate (AOPCP) for CD73 , 500 μm levamisole hydrochloride for ALP , 150 μm 6-N,N-Diethyl-β-γ-dibromomethylene-d-adenosine-5′-triphosphate trisodium salt hydrate (ARL67156) for ecto-ATPases, mainly NTPDases (including CD39) [42, 43] and 50 μm pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS) for ENPPs . After pre-incubation ecto-enzyme substrates were added (50 μm adenosine, AMP or ATP) and cells were incubated at 37 °C for 30 min. Samples of the incubation medium were collected in 0, 5, 15 and 30 min time points and analyzed for the concentration of nucleotides and their catabolites using HPLC as described above. Enzyme activities were calculated from linear phase of the reaction and normalized per cell protein concentration or aortic root surface area.
Determination of alkaline phosphatase activity on the surface of mice aortic roots
ALP activity on aortic roots obtained from ApoE−/−LDLR−/− mice treated in vivo with dCF and from WT mice that were treated ex vivo with osteogenic medium (DMEM supplemented with 1 mmol/L l-glutamine, 10% FBS and 1% penicillin/streptomycin (v/v) and 3 mm phosphate), adenosine (50 μm) and adenosine receptor antagonists (50 μm) in the presence of 150 μm AOPCP, 5 μm dCF and 5 μm NBTI has been measured as described in the Supplementary material online.
Flow cytometry analysis
Cells were resuspended in MACS buffer, preincubated with FcR Blocking Reagent (Miltenyi Biotech) and stained with specific antibodies that origin was described in the Supplementary material online.
To identify aortic valve endothelial cells and individual subsets of aortic valve interstitial cells and immune cells we used a panel of antibodies against different cell-specific markers, including markers for endothelial cells (CD45−, CD31high), activated VIC (CD45−, Vim+, Sial−, αSMAhigh), activated/osteoblast-like VIC (CD45−, Vim+, Sial+, αSMAint), osteoblast-like VIC (CD45−, Vim+, Sial+, αSMA−), T helper cells (CD45+, CD8+), T cytotoxic cells (CD45+,CD4+), B cells (CD45+, CD19+), monocytes/macrophages (CD45+, CD11b+, CD14+) and granulocytes (CD45+, CD11bint, CD14−). After 5 min of the incubation at room temperature cells were washed and resuspended in 200 µL MACS buffer for flow cytometry. Cell measurements were performed and analyzed as described in the Supplementary material online. The different cells subsets were enumerated and the percentage of CD39, CD73 and CD26 (adenosine deaminase binding-protein) and corresponding expression levels as measured by mean fluorescence intensity (MFI) was assessed.
Statistical analysis was performed using InStat software (GraphPad, San Diego, CA). Comparisons of mean values between groups were evaluated by one-way analysis of variance (ANOVA) followed by Holm–Sidak, or Sidak post hoc tests, two-way ANOVA followed by Sidak post hoc test, unpaired Student’s t test, or Mann–Whitney U test, as appropriate. Normality was assessed using the Kolmogorov–Smirnov test (when n = 5), Shapiro–Wilk test (when n = 7), and the D’Agostino and Pearson Omnibus (when n ≥ 8) normality tests. The exact value of n was provided for each type of experiments. Statistical significance was assumed at p < 0.05. Error bars indicated the standard error of the mean (SEM) unless otherwise described in the figure legend.