Mice bearing Cdon-floxed allele (Cdonf/f) were obtained from EUCOMM and maintained as previously described17. For Cdon deletion in vascular muscle cells, Cdonf/f mice were crossed with SM22a-CreERT2 mice which express tamoxifen-inducible Cre recombinase under the transcriptional control of the SM22a (Tagln, smooth muscle protein 22-alpha) promoter. SM22a-CreERT2 mice were obtained from Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases (SIRIC, Yonsei University Health System). To induce the deletion of one of the exons of Cdon gene in VSMCs, around 8 weeks old mice were injected intraperitoneally with 100 mg/kg tamoxifen (tmx, Sigma-Aldrich) every two days for five times. To generate the model of vascular calcification in mice, 8 weeks old C57BL/6 male mice and vehicle or tamoxifen-injected mice were administrated by vitamin D3 (VD3, 5x105 IU/kg per day; Cayman Chemical). 14.575 mg VD3 in 70 ml of ethanol was mixed with 500 ml Cremophor for 15 min at room temperature, and this solution was then mixed with 6.2 ml sterilized water including 250 mg of dextrose for 15 min at room temperature. The mice were injected with a dose of VD3 (150 ml/25 g per day) subcutaneously for consecutive 3 days18.
The animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University School of Medicine (SUSM) and complied with the animal experiments guidelines of the SUSM Ethics Committee (the protocol number: SKKUIACUC 2020-04-14-1).
To measure the cardiac function and the pulse wave velocity (PWV), echocardiographic analysis was performed 1 day before sacrifice. Mice were anesthetized with 1-2 % (vol/vol) isoflurane and body temperature was maintained at 36-38 ºC with a heating lamp and a heating platform. Heart rates are monitored and generally maintained at 400-500 beats per minute. Echocardiography was carried out using a Vevo LAZR-X machine (the BIORP of Korea Basic Science Institute) with a 40-MHz probe (visual sonic). Analysis of M-mode images derived from the short-axis view of the left ventricle was carried out to measure the ejection fraction (EF) and the fractional shortening (FS)19. PWV was obtained from the B-mode and pulse-waved (PW) doppler mode of aortic arch view, calculated as PWV=Aortic arch distance/transit time (cm/s). The PW doppler mode sample volume was placed in the ascending aorta and the time (T1) from the onset of the QRS complex to the onset of the ascending aortic Doppler waveform was measured. On the same image plane, the PW doppler mode sample volume was placed as distal as possible in the descending aorta and the time (T2) from the onset of the QRS complex to the onset of the ascending aortic Doppler waveform was measured. Obtained values for T1 and T2 were averaged over 10 cardiac cycles. The aortic arch distance was measured between the 2 sample volume positions along the central axis of the aortic arch on the B-mode image, and the transit time was calculated by T2-T1 (ms)20.
A7R5 (ATCC, CRL-1444) cells were cultured in normal media containing DMEM, 10% FBS, and 1% P/S as previously described21. For inducing the vascular calcification in vitro, the cells incubated with normal media were switched to calcifying media (CM) for up to 3 days or 7 days. The media were replenished every 48 h and the first day of culture in CM was defined as day 0. CM was generated by adding 100nM dexamethasone, 1mM insulin, 50mg/ml ascorbic acid, 10mM b-glycerophosphate, and 8mM CaCl2 to normal media18. Transfection experiments were performed by utilizing 1mg/ml polyethylenimine (PEI, Sigma-Aldrich). To analyze the effects of Cdon on Wnt signaling, cells were treated with 20ng/ml Wnt3a (R&D systems) and 4mM XAV939 (Calbiochem). To deplete the expression of Cdon in A7R5, control shRNA (shcont) or Cdon shRNA (shCdon) were expressed by using lentiviral expression system as previously described17.
Aortas were isolated from PBS perfused mice and fixed with 4% PFA, embedded in paraffin, and sectioned with 5 mm thickness. For determination of calcium deposition in aortas, slide sections were stained with Von Kossa stain. Briefly, deparaffinized sections were incubated with 1% silver nitrate solution under ultraviolet for 2 h followed by the step removing unreacted silver with 5% sodium thiosulfate for 5 min. Sections were counterstained with nuclear fast red for 5 min before dehydration and mounting18. To quantify the amount of calcium deposition in aortas, the images obtained from TissueFAXS PLUS (TISSUEGNOSTICS) were analyzed in ImageJ software (NIH).
For the immunohistochemistry in aorta samples, deparaffinized samples were boiled in Tris-EDTA buffer (pH9.0, 0.05% Tween-20) for the antigen retrieval followed by the standard protocol. To analyze the cell death during vascular calcification, Click-iT TUNEL assay Alexa imaging assay kit (Invitrogen, C10246) was utilized on cryosections of aortas which were isolated from PBS perfused mice and sectioned with 5mm thickness. Fluorescence images were analyzed with an LSM-710 confocal microscope (Carl Zeiss) as previously described16.
Protein and RNA analysis
Immunoblot analysis was carried out as previously described16. In brief, cultured cells and homogenized tissues of aortas were lysed in RIPA buffer (protease inhibitor cocktail [Roche, 1183617001], pH8.0; 150mM NaCl; 1mM EDTA; 1% Triton X-100; 10mM Tris-HCl). The primary antibodies used in this study are listed in Supplementary table 1.
Quantitative RT-PCR and RNA sequencing analysis were performed as previously described16. Total RNAs from cells and homogenized tissues were extracted with easy-BLUE (iNtRON) reagent following the manufacturer’s instruction. cDNA samples were generated from 0.5 mg of RNAs by PrimeScript RT reagent (TaKaRa) according to the manuscript’s protocol. The primer sequences used in this study are listed in Supplementary table 2. High-throughput sequencing was performed as single-end 75 sequencings using Illumina NExtSEq 500 (ebiogen, Korea). The analysis for RNA sequencing data was perfomed by using ExDEGA v3.2 (ebiogen) and a heatmap was displayed utilizing Morpheous (http://software.broadinstitute.org/morpheus/). The global gene expression was assessed by the reactome with Gene Set Enrichment Analysis (GSEA) (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp) using MsigDB database v7.2 (>1.3 fold, |RC log2|>2, p<0.05).
The aortic transcriptomes of human from the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) database (GSE43292, GSE12644, and GSE83453) were computed for the analysis of gene expression and Pearson’s correlations with the Graphpad PRISM7. The single-cell transcriptome of human patients (GSE159677) was utilized for extracting each cell type in aortas including VSMCs, endothelial cells, macrophages, T-lymphocytes, B-lymphocytes, and NK-cells by marker genes. The genes of VSMCs were aligned and projected through Uniform manifold approximation and projection (UMAP) to explore the scRNA-seq data. Visualization of gene expression was carried out in Graphpad PRISM7.
Luciferase reporter assay was performed as previously described15. Briefly, A7R5 cells were transfected with control or Cdon overexpressing or siCdon plasmids, b-catenin-responsive Top-Flash luciferase construct plasmid. Cells were analyzed for luciferase activity with a microplate luminometer according to manufactures protocol 24 h after transfection (Promega).
Alizarin Red stain
Cells were washed with PBS and fixed with 4% PFA for 15 min at room temperature. Cells were then washed twice with deionized water and covered with 40mM Alizarin Red S (Sigma-Aldrich) at around pH4.2 for 2 h at room temperature with gentle shaking. To remove the unstained dye, cells were washed thrice with deionized water before images were obtained using Nikon CELIPS TE-2000U. To quantify the amount of calcification, Alizarin Red S was extracted with 10% acetic acid for 30 min at room temperature, scraped into a microcentrifuge tube, vortexed, and incubated at 85 ºC for 15 min. After chilling on ice for 5 min, the mixture was centrifuged at 20,000 g for 15 min at 4 ºC. The supernatant was transferred to a new tube and 10% ammonium hydroxide was added to the supernatant. Absorbance was read in triplicate at 405 nm using a 96-well plate spectrophotometer18.
HEK293T cells were transfected with recombinant proteins of Cdon fused with the Fc region of human IgG gamma listed as follows: Cdon-Fc; the entire ectodomain fused to Fc, Ig2-Fc; Fc fusion protein with Ig2 domain15. At 72 h after transfection, cells were pelleted by centrifugation at 2,000 g for 10 min. The supernatant was then filtered (0.45 mm), followed by a standard protocol for Protein A antibody purification22. In briefly, the supernatant was incubated with Protein A agarose (Millipore) for 1 h at room temperature under constant rotation. Bound proteins were eluted with 0.1M citric acid (pH 3.0) and immediately neutralized with 1M Tris-HCl (pH 8.0). Eluates were concentrated using Amicon Ultra-4 centrifugal filters (MWCO 3K, EMD Millipore) and dialysed against PBS. Protein concentrations were determined using A280 measurements. Protein purity and integrity was assessed by SDS-PAGE.
Values are means ±SEM or SD as noted. Statistical significance was calculated by paired or unpaired Student’s t-test or one-tailed Analysis of variance (ANOVA) test followed by Tukey’s test (GraphPad Prism software, v7). Differences were considered as significant at p<0.05.