Animals

Male Sprague‒Dawley rats (8–9 weeks old) were obtained from Charles River Laboratories (France). To induce diabetes, a single intraperitoneal injection of streptozotocin (55 mg/kg body weight, Sigma‒Aldrich) was administered in a 0.9% NaCl solution. The control rats received an equivalent volume of isotonic saline solution. The protocol was approved by the ethics committee (PROEX 427/15) and conformed to directive 2010/63EU and recommendation 2007/526/EC on the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under RD1201/2005.

ASMC isolation and culture

Rat aortic smooth muscle cells (ASMCs) were isolated using a double digestion protocol with collagenase [32]. The ASMCs were cultured in Minimum Essential Medium Eagle (MEM) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. All cell culture reagents were obtained from Invitrogen (Paisley, UK). The control group was maintained in MEM with a glucose concentration of 1 g/L (control medium). To prepare glucose-enriched medium, MEM supplemented with glucose (Sigma‒Aldrich) was filtered through a 0.22 μm filter (PES033S0221, Scharlab, Barcelona, Spain), resulting in a final concentration of 4.5 g/L. After initial trypsinization (passage 1), the cells were incubated in either 1 g/L or 4.5 g/L glucose MEM and passaged to passage 12. The cells were maintained at a 1:3 split ratio during each trypsinization step. The medium was changed every 2–3 days.

Human diabetic and non-diabetic aortic smooth muscle cells (Lonza, Walkersville, USA) were cultured in SmGM-2 medium (Lonza) according to the manufacturer’s instructions. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. A 1:3 split ratio was used during each trypsinization step to maintain the cultures.

Cell proliferation

Cell counting was performed regularly over a 6-week period to assess the division rate of rat aortic smooth muscle cells cultured under both experimental conditions. A Neubauer counting chamber (717805, Brand™, Arnedo, Spain), also known as a hemocytometer, was used for accurate cell counting. Cell passages were carried out upon reaching 50% confluence, using trypsin (Thermo Fisher).

Cell proliferation was evaluated by measuring the incorporation of 5-bromodeoxyuridine (BrdU) via the BrdU Cell Proliferation ELISA Kit (Abcam, ab126556) following the manufacturer’s protocol. The absorbance was read at dual wavelengths of 450/550 nm using a Varioskan™ LUX multimode microplate reader, and the results are expressed as the optical density (OD) per cell.

Cell viability

The viability of aortic smooth muscle cells was evaluated via the PrestoBlue™ Cell Viability Reagent (A13261; Invitrogen) according to the manufacturer’s protocol. This assay utilizes resazurin as a redox indicator to measure cellular viability. Upon incubation with viable cells, resazurin, a cell-permeable, nonfluorescent blue dye, is reduced by mitochondrial oxidoreductase enzymes in metabolically active cells to resorufin, a pink, fluorescent compound.

Briefly, subconfluent aortic smooth muscle cells were dissociated using trypsin (200 U/ml, Thermo Fisher) and counted. Five serial dilutions of the cells were prepared in the appropriate culture medium for each experimental group. Following the manufacturer’s instructions, 90 µL of the cell suspensions and 10 µL of PrestoBlue reagent were added to each well of a 96-well plate, resulting in a final volume of 100 µL per well. The plate was incubated in the dark at 37 °C, and the absorbance was measured at 570 nm (to detect resorufin) and 600 nm (background) at 30-minute intervals over a 2-hour period using a Varioskan™ LUX multimode microplate reader.

ATP and pyrophosphate quantification

The intracellular and extracellular ATP levels were measured using a coupled luciferin/luciferase reaction with an ATP Determination Kit (Invitrogen) following the manufacturer’s instructions and previous studies [14, 27]. For intracellular ATP quantification, aortic smooth muscle cells were lysed in lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, and 0.1% Triton X-100 (pH 7.4). Intracellular ATP measurements were performed on cell lysates alongside ATP standards for calibration. Extracellular pyrophosphate was measured with an enzyme-linked bioluminescence assay as described previously [14]. ATP and pyrophosphate levels were normalized to the cellular protein content, which was quantified using the Pierce™ BCA Protein Assay Kit according to the manufacturer’s instructions.

Mitochondrial ATP synthesis in digitonin-permeabilized aortic smooth muscle cells (2 × 106 cells) was evaluated via a kinetic luminescence assay based on the previous studies [14, 27].

Extracellular pyrophosphate metabolism

Aortic smooth muscle cells (ASMCs) or aortic rings were incubated in vitro or ex vivo, respectively, in Hank’s balanced salt solution (HBSS, BE10-527 F, Lonza) containing specified concentrations of pyrophosphate (Sigma‒Aldrich) and pyrophosphate-32 (Perkin-Elmer) or ATP (Sigma‒Aldrich) and [γ-32P]ATP (Perkin-Elmer). After the designated incubation periods, ATP and pyrophosphate were separated from orthophosphate as described in previous protocols [24, 27].

Briefly, 20 µL of the sample was mixed with 400 µL of ammonium molybdate (Sigma‒Aldrich) to bind orthophosphate, followed by the addition of 0.75 mol/L sulfuric acid (Sigma‒Aldrich). The solution was then extracted with 800 µL of an isobutanol/petroleum ether mixture (4:1; Sigma‒Aldrich) to separate phosphomolybdate from pyrophosphate and ATP. A 400 µL aliquot of the organic phase containing phosphomolybdate was collected, and its radioactivity was measured.

The same aortic rings were used for both the ATP and pyrophosphate hydrolysis assays. Following the ATP hydrolysis assays, the rings were washed five times with HBSS before the pyrophosphate hydrolysis assays were conducted. Finally, the aortic rings were dried and weighed.

To analyze the products released during ATP hydrolysis, ASMCs or aortic rings were incubated in HBSS containing ATP and [γ-32P]ATP at final concentrations of 1 µmol/L and 10 µCi/mL, respectively. After the designated incubation periods, the production of phosphate-32 (32Pi) and pyrophosphate-32 (32PPi) was determined by chromatography using PEI-cellulose plates (50488-25EA-F, Sigma‒Aldrich). The plates were developed with 650 mmol/L K2HPO4 (Sigma‒Aldrich) at pH 3.0, following previous studies. The resulting spots were excised and analyzed via liquid scintillation counting via UltraGold (6013329, Perkin-Elmer).

Recombinant enzymes and inhibitors

The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA). The eNTPD1 inhibitor PSB609 [33, 34] was obtained from Tocris Bioscience (Minneapolis, MN, USA; catalog number 2573). The TNAP inhibitor SBI425 [35, 36] was sourced from Sigma‒Aldrich (catalog number SML2935).

Real-time polymerase chain reaction

Total RNA was isolated using TRIzol reagent (Invitrogen), and cDNA synthesis was performed with the Superscript III cDNA Synthesis System (Invitrogen) following the manufacturer’s instructions. Relative quantification of the expression of the rat genes BMP2, SM22α, TNAP, eNTPD1, and eNPP1 was conducted via real-time PCR with SYBR Green according to the manufacturer’s protocol. The sequences of primers used for amplification were as follows [25]: BMP2: 5′-GTTCTGTCCCTACTGATGAG-3′ (forward) and 5′-ATTCGGTGCTGGAAACTAC-3′ (reverse); SM22α: 5′-CAGACTGTTGACCTCTTTGAAG-3′ (forward) and 5′-TCTTATGCTCCTGGGCTTTC-3′ (reverse); TNAP: 5′-TGAATCGGAACAACCTGACTG-3′ (forward) and 5′-GCCTCCTTCCACTAGCAAGAA-3′ (reverse); eNTPD1: 5′-CAGGTTTCAAGTGGTGGGATT-3′ (forward) and 5′-GAAGGCACACTGGGAGTAAGG-3′ (reverse); eNPP1: 5′-AAGGTATGCCCAAGAAAGGAA-3′ (forward) and 5′-TTCTTGACTGCGGATGACTCT-3′ (reverse).

The comparative ΔΔCT method was used for quantification, with acidic ribosomal phosphoprotein (ARP) RNA serving as the endogenous reference. The primers used for rat ARP amplification were 5′-CACCTTCCCACTGGCTGAA-3′ (forward) and 5′-CACCTTCCCACTGGCTGAA-3′ (reverse).

ASMC calcification assay

Rat aortic smooth muscle cells (ASMCs) were cultured to confluence and subjected to a quiescence step by incubation overnight in culture medium containing 0.1% fetal bovine serum. Calcification assays were then performed by incubating the cells for 7 days in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.1% fetal bovine serum, and 2 mmol/L phosphate (phosphate-calcifying medium), as previously described [32, 37]. The phosphate-calcifying medium and control medium were replaced daily. To quantify the calcium content in rat ASMCs, the wells were treated with 0.6 M HCl overnight at 4 °C, and the calcium levels were analyzed via a QuantiChrom Calcium Assay Kit (BioAssay Systems, Hayward, CA) via a colorimetric method. Cell fixation was carried out according to previous studies [37, 38].

Human aortic smooth muscle cells (ASMCs) were cultured to confluence in SmGM-2 medium and subjected to a quiescence step by incubating overnight in MEM containing 0.1% fetal bovine serum. Calcification assays were performed by incubating the cells for 4 days in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.1% fetal bovine serum, and 10 µCi/mL calcium-45 as a radiotracer.

To quantify the calcium content in ASMCs, the wells were washed five times with 9 g/L NaCl, treated with 200 µL of 0.6 M HCl and incubated overnight at 4 °C to release calcium. Following incubation, 50 µL of the calcium-containing HCl solution was transferred to a liquid scintillation fluid (UltimaGold™, 6013329; Perkin Elmer), and radioactivity was measured via a Tri-Carb 2810TR liquid scintillation analyzer (Perkin Elmer).

Aorta isolation and calcification assay

The rats were euthanized via carbon dioxide inhalation, and the thoracic aorta tissue was perfused with saline and removed following previously published protocols [25, 32]. For calcification assays, aortic rings were cultured ex vivo at 37 °C with 5% CO2 in minimum essential medium (MEM) supplemented with calcium-45 (45Ca) as a radiotracer (Perkin Elmer, Boston), 2 mmol/L L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 0.1% fetal bovine serum. Aortic rings were further cultured in MEM supplemented with 2 mmol/L phosphate (KH2PO4/K2HPO4, pH 7.4) for calcification assays, whereas the control groups received 1 mmol/L phosphate. After 7 days of incubation, the aortic rings were dried, and the radioactivity was measured using liquid scintillation counting (Perkin Elmer Tri-Carb 2810TR).

Aortic staining

Rat aortas were embedded in optimal cutting temperature compound (Sakura, Alphen aan den Rijn, The Netherlands), and 5-µm cross-sections were prepared using a cryostat (Leica CM1940). The calcification of the aortic tissue was assessed through Alizarin Red and Von Kossa staining, while the cell content and tissue architecture were visualized via hematoxylin and eosin (H&E) staining.

Analytical parameters

For the analytical parameters shown in Fig. 1, blood samples were collected from euthanized rats into serum or heparinized tubes and centrifuged to separate the serum or plasma, respectively. Liver glycogen, as well as plasma glucose, insulin, AGEs, GSP, and TNFα, were quantified using the following kits according to the manufacturer’s protocols: a glycogen assay kit (Abcam, ab65620), a glucose assay kit (Abcam, ab65333), a rat insulin ELISA kit (Thermo Fisher Scientific), a rat advanced glycation end products (AGEs) ELISA kit (RTEB0188), a glycated serum protein ELISA kit (ABIN771898), and a rat TNFα ELISA kit (Thermo Fisher Scientific).

Fig. 1

Impairment of glucose homeostasis in STZ-treated rats.A Body weight, B blood glucose levels, C blood insulin levels, D advanced glycation end products (AGEs), E liver glycogen content, F glycated serum protein (GSP), and G tumor necrosis factor-alpha (TNFα) levels in plasma from STZ-treated (left, green) and control rats (right, red). The data are shown as the mean ± SEM (n = 12). Statistical significance was assessed via Student’s t test. Asterisks denote significant differences, with ***P < 0.001

Immunoblotting and ELISA

Immunoblot assays were conducted via a chemiluminescent detection method with a Millipore kit, as previously described [14]. Primary antibodies against TNAP (LS-C171640), eNPP1 (LS-C780091), and eNTPD1 (LS-C387669) were obtained from LSBio (Shirley, MA, USA) and used following the manufacturer’s protocols. For the sandwich ELISAs, commercial kits for eNPP1 (LS-F33284), eNTPD1 (LS-F20306), TNAP (LS-F4993), MGP (LS-F6344), OPN (LS-F2199), BMP2 (LS-F2407), SM22α (LS-F7231), and Cbfa1/Runx2 (LS-F53973) were also purchased from LSBio and used according to the manufacturer’s instructions.

Statistical analysis

The Kolmogorov‒Smirnov test was used to assess the normality of the data. Student’s t test or one-way ANOVA and Tukey’s multiple comparison post hoc test were used for statistical analyses (according to the figure legends). Statistical significance was determined via GraphPad Prism 5 software.