Animal experiments

All animal experiments were approved by the Institutional Animal Care Committee at Beijing Chaoyang Hospital, Capital Medical University, Beijing, China and performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. Vitamin D3 (VitD3, MedChemExpress, 5.5 × 105 IU/kg subcutaneous injection) was injected once a day for three times to induce calcification models in young (8-week-old) and aged (72-week-old) SD rats, as well as C57BL/6J mice (8-week-old) as previous described(Chen et al. 2023a, b, c, d; Wang et al. 2022a, b). In some experiments, VitD3-overloaded mice were received with Elabela (1 mg/kg/d, intraperitoneal injection, MedChemExpress), PPAR-γ activator Rosiglitazone (5 mg/kg/d, intraperitoneal injection, MedChemExpress) or Elesclomol (20 mg/kg/d, subcutaneous injection, Topscience), respectively. All animals were kept under standard conditions, including temperature range of 22–25 °C and12-hour light/dark cycle. After treatment for 7 days, the mice were euthanized, then the aortas were collected for further analysis.

Arterial ring organ culture

As previous described(Chen et al. 2023a, b, c, d), two-month-old male SD rats were euthanized, and thoracic aortas were dissected under sterile conditions. The arteries were then cut into 5–6 mm rings for consistency. Subsequently, the vascular rings were subjected to treatment with growth medium (GM, DMEM/F12 medium) or calcifying medium (CM, DMEM/F12 medium containing 10 mM β-GP and 3 mM CaCl2), with or without Elabela (1 µM), Elesclomol (50 nM), or GW9662 (5 µmol) in the presence of CM in a 37 ℃ incubator with 5% CO2. To induce vascular calcification, the arterial rings were immersed in CM supplemented with 10 mM of β-glycerophosphate (β-GP, Sigma-Aldrich, St. Louis, MO, USA) and 3 mM CaCl2 (Sigma-Aldrich, St. Louis, MO, USA) for a duration of 7 days.

Cell culture and treatment

Primary rat VSMCs were isolated from aortas by using the explant method(Chen et al. 2023a, b, c, d). Following euthanasia, the thoracic aortas were isolated; then the adventitia and intima were removed. The aortas were then cut into small pieces to create aortic explants, which were cultured in DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pricella, Wuhan, China). VSMCs were passaged when they reached approximately 80% confluence. To induce osteogenic differentiation and cell calcification, VSMCs were treated with CM (containing 10 mM β-GP and 3 mM CaCl2) for 7 days. Elabela was administered at a concentration of 1 µM in the presence of CM. The GM and CM medium were refreshed every 2 days. In some experiments, Elesclomol (50 nM), GW9662 (5 µmol) and H2O2 (100 µM) were used to treat rat VSMCs.

Small interfering RNA (siRNA) transfection

The siRNA used in this study was obtained from GenePharma Co. Ltd. (Shanghai, China). When VSMCs reached 80% confluence, transfection was carried out with 100nM ATP7a siRNA or scrambled siRNA (si-NC) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Following 24 h of transfection, the medium was changed, and additional experiments were conducted. The protein level of ATP7a in VSMCs was evaluated through Western Blot analysis.

Western blot analysis

Total protein was extracted from VSMCs or arterial tissues using radio immunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) containing proteinase (Roche, Mannheim, BW, Germany). The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Next, protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). After blocking with 5% milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the specified primary antibodies as detailed in the supplementary materials. Subsequently, the membranes were washed three times with TBST and incubated with secondary anti-rabbit or anti-mouse HRP-conjugated antibodies for 2 h at room temperature. Antibody binding was visualized using ECL detection reagent (Bio-Rad, Hercules, CA, USA). Protein bands were analyzed with Image J software and normalized to GAPDH or Tubulin expression.

Quantitative real-time polymerase chain reaction (qRt-PCR)

Total RNA was extracted from tissues and VSMCs by using TRIzol reagent (Invitrogen, Waltham, MA, USA). Subsequently, the RNA was quantified and reverse-transcribed using a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) following the manufacturer’s instructions. Gene expression levels were measured via quantitative PCR using Synergy Brands (SYBR) Green mixture in a 7500 FAST Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers utilized in this study were synthesized by Sangon Biotech (Shanghai, China), and the sequences can be found in the supplementary materials (Supplementary Table 1).

Immunofluorescence and immunohistochemistry staining

Aortic tissues and cultured VSMCs were collected at specified time points. For tissue staining, arteries were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) overnight and then embedded in paraffin. Sections of 4–6 μm thickness were prepared for further staining. The paraffin-embedded sections were deparaffinized, and antigen retrieval was carried out using EDTA Antigen Retrieval Solution. After blocking with 5% bovine serum albumin (BSA, Solarbio, Beijing, China) for 30 min at room temperature, the sections were incubated with primary antibodies diluted in immunostaining primary antibody dilution solution overnight at 4 °C. Subsequently, FITC-labeled or Alexa Fluor 647 labeled secondary antibodies were applied for 1 h at room temperature. The coverslips were mounted on glass slides using a mounting medium (Beyotime, Shanghai, China) containing DAPI for counterstaining of the nuclei. Imaging was performed using an Olympus IX73 fluorescence microscope. For immunohistochemistry, the endogenous peroxidase activity in tissues was blocked with 3% H2O2 and then blocked with 5% BSA. The samples were incubated with primary antibodies overnight, followed by staining with an HRP-labeled secondary antibody at room temperature for 45 min, and visualization with DAB. Finally, the sections were counterstained with hematoxylin solution and observed using a microscope (Leica, Wetzlar, Germany).

For VSMCs, the cells were washed with 1× PBS, fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton-X for 20 min at room temperature, blocked with 5% BSA for 30 min, and then incubated with the primary antibody overnight at 4 °C. After washing, the VSMCs were incubated with Alexa Fluor secondary antibodies for 1 h at room temperature in the dark. The cell nuclei were counterstained with DAPI, and images were captured using a fluorescence microscope.

Alizarin red staining

As previously described in the literature(Li et al. 2024; Chen et al. 2023a, b, c, d), alizarin red staining was utilized to evaluate the calcification of VSMCs. Briefly, VSMCs were fixed in 4% paraformaldehyde for 10 min and then stained with a 0.2% alizarin red solution (Solarbio, Beijing, China) for 3 min. As for vascular tissues, sections were cut and mounted on glass slides. The sections were then incubated with 2% Alizarin Red S solution for 30 min at room temperature, allowing the dye to bind specifically to calcium deposits. After thorough washing with distilled water, the slides were counterstained with hematoxylin. Finally, the stained sections were examined under a light microscope, and images were captured for quantitative analysis of calcification.

Von-Kossa staining

Arterial samples were fixed in 10% formalin solution, embedded in paraffin, cut into sections, and then stained with 5% silver nitrate (Servicebio, Wuhan, China) followed by exposure to ultraviolet light. The stained sections were examined under a light microscope, and images were captured for quantitative analysis.

Calcium content assay

The measurement of calcium levels in VSMCs and arterial tissues was conducted using Calcium Test Kits (Nanjing Jiancheng Bioengineering, Nanjing, China) following the guidelines outlined by the manufacturer. Initially, the cells or tissues underwent homogenization and centrifugation for the collection of the supernatants. These supernatants were then treated with methyl thymol blue solution for a duration of 5 min at ambient temperature, with subsequent measurement of optical density at 610 nm. The quantification of protein concentration was achieved through the utilization of a BCA assay kit. Normalization of calcium content was performed in relation to protein concentrations and reported as µg/mg of protein.

Alkaline phosphatase (ALP) activity assay

The assessment of ALP activity was carried out using an ALP assay kit (Nanjing Jiancheng Bioengineering, Nanjing, China) as per the instructions. Arteries were homogenized, resulting in the isolation of supernatants through the process of centrifugation. The samples were then added p-nitrophenyl phosphate (p-NPP) substrate and were incubated for a period of 10 min at 37 ℃. The reaction was halted with 3 mol/l NaOH, following which the absorbance was determined at 405 nm. The quantification of protein concentration was ascertained utilizing a BCA assay kit. Subsequently, the outcomes were adjusted based on the protein levels, with the ALP activity being calculated in units per milligram of protein.

Copper content detection

Copper Microplate Assay Kit (Absin, Shanghai, China) was used to measure the copper content in vascular tissues and VSMCs. In brief, the supernatant of sample homogenate was mixed with the assay reagent and incubated at room temperature for 15 min. The absorbance of the samples at 605 nm was then measured using an Enzyme-linked immunosorbent assay reader. The quantification of protein concentration was ascertained utilizing a BCA assay kit. Subsequently, normalization of copper content was performed in relation to protein concentrations.

Coppersensor-1 staining

To assess intracellular copper levels, VSMCs were cultured in different medium supplemented with 1 µM copper. After 24 h, cells were incubated with 5 µM Coppersensor-1, a synthetic fluorophore for cell copper imaging, for 30 min at 37 °C. Following incubation, cells were washed and analyzed using fluorescence microscopy.

Detection of mitochondrial function

Mitochondrial damage in VSMCs were assessed by measuring the mitochondrial membrane potential using the JC-1 staining assay kit (Beyotime, Shanghai, China). VSMCs were treated with the JC-1 working solution for 30 min as per the manufacturer’s instructions. Fluorescence microscopy was utilized to capture images, and the Red/Green fluorescence intensity ratio was analyzed to assess potential mitochondrial damage. Furthermore, mitochondrial permeability transition pore opening was measured using the Mitochondrial Permeability Transition Pore (MPTP) Assay Kit (Beyotime, Shanghai, China). The assay was conducted according to the manufacturer’s guidelines. After treatment with stimuli, VSMCs were stained with a fluorescence probe (Calcein AM) provided in the kit, and images were captured using a fluorescence microscope. MitoSOX Red mitochondrial superoxide indicator (Beyotime, Shanghai, China) was used to detect the levels of superoxide in mitochondria. MitoTracker Deep Red FM (Beyotime, Shanghai, China) staining was utilized to investigate morphological alteration of mitochondria.

Measurement of ATP levels

According to the manufacturer’s protocol, an ATP Assay Kit (Nanjing Jiancheng Bioengineering, Nanjing, China) was used to measure the ATP level in cells. VSMCs were harvested and lysed in a buffer to release intracellular ATP. Then, the lysates were then mixed with working reagents, following which the absorbance was determined at 636 nm. Subsequently, absorbance was measured using enzyme labelling equipment and ATP concentration was calculated based on a standard curve generated with known concentrations of ATP.

Assessment of β-galactosidase (β-gal) activity

To analyze senescence, the β-gal assay (Beyotime, Shanghai, China) was employed. VSMCs were cultured in 6-well plates and subjected to H2O2 treatment to induce senescence. After rinsing with PBS, the VSMCs were fixed at room temperature for 30 min and then incubated wit β-gal staining solution overnight at 37 °C. The percentage of senescent cells was determined by the ratio of blue-stained cells to the total cell count.

Measurement of NAD+ levels

The NAD+ levels was determined by using an assay kit (Beyotime, Shanghai, China). Cellular samples were lysed with the provided extraction buffer, mixed with ethanol dehydrogenase working solution, and incubated at 37 °C for 10 min. The enzymatic reaction was stopped, and the absorbance was read at 450 nm. Subsequently, NAD+ levels were normalized to the total protein content.

Statistical analysis

Statistical analyses were conducted using Graphpad Prism 9. Data was presented as mean ± SD. Group differences were compared using Student’s t-test for two-group comparisons and one-way analysis of variance (ANOVA) for multiple group comparisons. Statistical significance was considered at a P value below 0.05.