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Fibroblast growth factor 23 inhibits osteoblastic gene expression and induces osteoprotegerin in vascular smooth muscle cellsTakehiro Nakahara M.D., Ph.D 1 *, Keiko Kawai-Kowase M.D., Ph.D 1,2 *, Hiroki Matsui, Ph.D 1, Hiroaki Sunaga, Ph.D 1, Toshihiro Utsugi M.D., Ph.D 1, Tatsuya Iso M.D., Ph.D 1, Masashi Arai M.D., Ph.D 1, Shouichi Tomono M.D., Ph.D 1, Masahiko Kurabayashi M.D., Ph.D 1 (1)Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan (2)Department of General Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan * These authors contributed equally to this work. Corresponding author and contact details; Keiko Kawai-Kowase, MD, PhDDepartment of General Medicine, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, JapanTel: (+81)27-220-8666, Fax: (+81)27-220-8666E-mail: kowasek@gunma-u.ac.jpSupplementary appendixes2. Supplementary Material and Methods2.1 RNA extraction and quantitative polymerase chain reaction (qPCR)Total RNA was prepared from cultured human aortic smooth muscle cells (HASMCs) using RNAiso Plus (Takara Otsu, Japan) according to the manufacturer’s protocol. One microgram of RNA was used for reverse transcription with an Advantage2 Polymerase Mix kit (Takara, Otsu, Japan). For semi-quantitative RT-PCR analysis, TaKaRa PCR Thermal Cycler (Takara, Otsu, Japan) and Advantage cDNA PCR Kit (Takara, Otsu, Japan) were used. For qPCR, Stratagene Mx3000P QPCR system (Stratagene, Amsterdam, Netherlands) and 2x THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) were used. Specificity of the THUNDERBIRD SYBR amplicons was confirmed by melting point analysis and gel electrophoresis in the presence of ethidium bromide (Nippon Gene, Tokyo, Japan). Expression of the housekeeping gene ribosomal 18S was used for normalization. Specific oligonucleotide primer for human FGFR1, FGFR2, FGFR3, FGFR4 and KLOTHO were as previous investigators.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Eb25hdGUtQ29ycmVhPC9BdXRob3I+PFllYXI+MjAxMzwv

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ADDIN EN.CITE.DATA 1 Specific primers for human BMP2, BMP4, MSX2, RUNX2, ALP and OPG were described in Supplemental Table 1. 2.2 Generation of Recombinant AdenovirusAdenoviruses expressing LacZ (ad-LacZ), human FGF23 (ad-FGF23), and human KLOTHO (ad-KLOTHO) were generated as previously described2. Their protein expressions were confirmed by Western blotting (data not shown). Recombinant human FGF23 (R&D Systems, Minneapolis, MN) and the supernatant medium from HASMCs infected with adenovirus were used. Recombinant soluble RANKL was purchaced from PeproTech (Rocky Hill, NJ).2.3 ALP, BCIP/NBT, Scratch-wound and MTS assaysALP activity of cells was measured using LabAssay ALP (Wako, Japan), according to the manufacturer’s protocol. ALP activity was normalized to total protein determined with a Coomassie Plus (Bradford) Assay Reagent (Thermo scientific). BCIP/NBT was stained BCIP/NBT kit (Takara) according to the manufacturer’s protocol. HASMC migration was assessed by scratch-wound assay as described previously3. MTS assays were measured by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacture’s protocol and measured 490nm OD by using Wallac ARVO SX 1420 multilabel counter (PerkinElmer). 2.4. Alizarin Red S stainingHASMCs were fixed with 4% formaldehyde and incubated with Alizarin Red S solution whose pH was adjusted to 4.1-4.3 using 0.5% ammonium hydroxide. To induce mineralization in the HASMC cultures, culture media were supplemented with Pi for final Pi concentration of 2.0-4.0 mmol/L.2.5. Coronary calcium score (CCS)The institutional review board (IRB) approved this study and written informed consent was obtained from all patients. Patients underwent cardiac CT (VCT: GE Healthcare) and CCS were measured on a workstation (Advantage Workstation, GE Healthcare) as the clinical guideline4-5.Supplementary Figure legendsSupplementary Figure 1. FGF23 does not affect SMC migration, proliferation, and SMC-specific marker gene expressions in HASMCs. (A) HASMCs was infected with adenovirus containing either human FGF23 (ad-FGF23) or LacZ (ad-LacZ) at a multiplicity of infection (MOI) of 20 for two days, and then scratched after changes the medium without serum. Migration images showing HASMC cultures after 0h (upper) and 22h (lower). Each photograph is representative of the results from five independent experiments. (B) HASMCs were infected with an MOI of 20 of indicated adenoviruses for two days and changed the medium. At three days after the medium change (day3), MTS assay were performed. (C-E) HASMCs infected with an MOI of 20 of indicated adenoviruses. At tow days, HASMCs were changed media and defined as “day0”. Total RNA was extracted at day 3, 5 and 7, and SM -ACTIN (C), SM 22 (D) and MMP2 (E) mRNA were measured by real-time PCR. An arbitrary value of 1.0 was assigned to the cells infected with LacZ at day3. All experiments were performed in technical duplicates and were repeated three times. Values represent means standard deviation of mean. Supplementary Figure 2. FGF2 does not affect OPG mRNA expression. HASMC were treated with recombinant FGF2 and vehicle for 3, 5, and 7 days, and mRNA level of OPG was measured by qPCR. An arbitrary value of 1.0 was assigned to the cells treated with vehicle at day3. All experiments were performed in technical duplicates and were repeated three times. Values represent means standard deviation of mean. Supplementary Figure 3. FGF23 does not affect RANKL expression. HASMC were infected with an MOI of 20 of indicated adenoviruses for two days and changed the medium. The supernatant were harvested at day 6 and 12 after infection of the indicated adenovirus and measured protein levels RANKL by ELISA. All experiments were performed in technical duplicates and were repeated three times. Values represent means standard deviation of mean..Reference ADDIN EN.REFLIST [1]Donate-Correa, J, Mora-Fernandez, C, Martinez-Sanz, R, et al., Expression of FGF23/KLOTHO system in human vascular tissue, International journal of cardiology, 2013;165:179-183.[2] He, TC, Zhou, S, da Costa, LT, et al., A simplified system for generating recombinant adenoviruses, ?Proc Natl Acad Sci U S A 95:2509-2514, 1998. [3]Liang, CC, Park, AY and Guan, JL, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro, Nature protocols, 2007;2:329-333.[4]Taylor, AJ, Cerqueira, M, Hodgson, JM, et al., ACCF/SCCT/ACR/AHA/ASE/ASNC/NASCI/SCAI/SCMR 2010 Appropriate Use Criteria for Cardiac Computed Tomography. A Report of the American College of [5]Abbara, S, Arbab-Zadeh, A, Callister, TQ, et al. SCCT guidelines for performance of coronary computed tomographic angiography: a report of the Society of Cardiovascular Computed Tomography Guidelines Committee. J Cardiovasc Comput Tomogr 3:190-204, 2009. ................
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