Fig 2: Visfatin promotes vascular calcification both in vivo and in vitro. (A) Experimental workflow for assessing adipose tissue-specific visfatin overexpression in a mice vascular calcification model. (B) The serum visfatin levels in mice with epididymal adipose tissue-specific overexpression of visfatin were measured using ELISA (n = 6). (C) Representative gross alizarin red staining of murine aortas (n = 6). (D) Immunohistochemical staining of RUNX2 in the aortas of mice in the calcified empty vector group and in specific epididymal adipose tissue from visfatin-overexpressing mice (n = 6). Scale bar: 50 μm. (E) Assessment of matrix mineral deposition in aortic tissue through von Kossa staining (n = 6). Scale bar: 200 μm. (F) Experimental workflow for visfatin treatment in in vitro aortic ring and VSMC calcification assays. (G) The deposition of mineral matrix in aortic tissue was analyzed using alizarin red S and von Kossa staining of murine aortic rings (n = 5 per group). Scale bar: 100 μm. H and J. Representative alizarin red staining of VSMCs (n = 4). I and K. Western blot bands and semiquantitative analysis of the representative markers OPN, RUNX2, BMP2, and calponin in VSMCs after treatment with calcified culture medium and visfatin (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: Visfatin accelerates vascular calcification via TLR4 and inflammatory pathways. (A) Western blot bands and semiquantitative analysis of inflammatory markers (NLRP3, TNF-α, and IL-1β), osteogenic markers (RUNX2), and contractile markers (calponin) in VSMCs treated with calcification medium and LPS (n = 5). (B) Western blot bands and semiquantitative analysis of inflammatory markers (NLRP3, TNF-α, IL-1β) in VSMCs treated with calcification medium in the presence or absence of visfatin (n = 5). (C) Western blot bands and semiquantitative analysis of inflammatory markers (NLRP3, TNF-α, and IL-1β) in VSMCs from TLR4−/− mice treated with calcification medium and visfatin (n = 5). (D) VSMC lysates were subjected to immunoprecipitation with either IgG or a TLR4 antibody, and the outcomes were analyzed by Western blotting with an anti-visfatin antibody. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: Empagliflozin suppresses the production and release of visfatin in adipose tissue and adipocytes. (A) Serum visfatin levels measured by ELISA (n = 6). (B) Western blot analysis of visfatin levels in the serum of mice with vascular calcification and vascular calcification + empagliflozin gavage (n = 6). (C) RT‒qPCR was used to assess the mRNA levels of visfatin in visceral adipose tissue (n = 6). (D) Immunohistochemical staining of visfatin in the epididymal adipose tissue of mice with vascular calcification and vascular calcification + empagliflozin gavage (n = 6). Scale bar: 50 μm. (E) Visfatin levels in the culture supernatant were measured by ELISA (n = 6). F‒G. The protein and mRNA levels of visfatin in differentiated 3T3-L1 adipocytes (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001