Visfatin from Adipocyte Accelerates Vascular Calcification via TLR4

Visfatin from Adipocyte Accelerates Vascular Calcification via TLR4 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Visfatin from Adipocyte Accelerates Vascular Calcification via TLR4 Yue Zhang, Yarui Zhao, Yijie Luo, Lei Wang, Jing Liao, Weijie Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7284693/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Vascular calcification is a common complication in metabolic diseases, such as diabetes and obesity, contributing to cardiovascular morbidity. Visfatin, an adipokine secreted by visceral fat, has been confirmed to be closely associated with metabolic dysfunctions. Methods: Vascular calcification models were established in mice through intraperitoneal injection of vitamin D (VitD). Serum visfatin levels were measured in both patients with coronary artery calcification and calcified mice. Visfatin expression in visceral fat was assessed by molecular analyses. To examine the functional role of visfatin, mice with adipose-specific visfatin overexpression were generated. Primary vascular smooth muscle cells (VSMCs) were treated with calcification medium and recombinant visfatin to assess osteogenic differentiation. The interaction between visfatin and Toll-like receptor 4 (TLR4) was investigated, and Tlr4 knockout models were used to verify its role. Empagliflozin was administered to evaluate its effects on vascular calcification and visfatin expression, along with related signaling pathway analyses. Results: Serum visfatin levels were significantly elevated in patients with high coronary artery calcification scores and in mice with vascular calcification compared to controls. Visfatin expression was also markedly increased in the visceral adipose tissue of calcified mice. Mice with adipose-specific visfatin overexpression showed aggravated vascular calcification following vitamin D treatment. In vitro, visfatin enhanced the osteogenic differentiation of VSMCs in response to calcification medium. Mechanistically, visfatin directly bound to TLR4 and promoted the osteogenic transformation of VSMCs. Tlr4 deletion significantly attenuated aortic calcification induced by visfatin both in vivo and in vitro. Empagliflozin treatment significantly reduced vascular calcification and lowered circulating visfatin levels. Furthermore, empagliflozin inhibited the activation of the p38/NF-κB signaling pathway in adipose tissue, reduced nuclear translocation of NF-κB, suppressed its binding to the visfatin promoter, and thereby downregulated visfatin expression in adipocytes. Conclusions: that visfatin secreted from visceral fat accelerates VSMCs osteogenic differentiation and vascular calcification via TLR4. Empagliflozin inhibits the expression of visfatin in adipocytes through the p38/NF-κB signaling pathway, thereby suppressing vascular calcification. The results suggest that visfatin may represent a novel therapeutic strategy for preventing or treating vascular calcification and related cardiovascular diseases. visfatin vascular calcification TLR4 empagliflozin adipocyte p38/NF-κB pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Vascular calcification is a key pathological process in arteriosclerosis, hypertension, and chronic kidney disease, with its incidence increasing significantly with age and being more prevalent in patients with diabetes and chronic kidney disease 1-5 . It is classified into intimal and medial calcification, with medial calcification commonly seen in hypertension and chronic kidney disease, driven by osteogenic differentiation of VSMCs due to calcium phosphate deposition 6 7 . Research indicates that inflammation plays a crucial role in vascular calcification, as cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), along with inflammasomes, promote calcium deposition and VSMCs phenotypic transformation 8-10 . The inhibition of inflammation and VSMCs osteogenic transformation has become a strategy for preventing vascular calcification. TLR4 is an important member of the TLR family and plays a central role in innate immunity 11 . As a transmembrane receptor, it binds to both extracellular and intracellular ligands, initiating complex signaling pathways 12 . TLR4 can be activated by lipopolysaccharides (LPS), well-known pathogen-associated molecular patterns (PAMPs), as well as endogenous molecules such as peptides and phospholipids 13-15 . Recent studies have highlighted the role of TLR4 in vascular calcification, where it mediates inflammatory signaling, induces VSMC osteogenic differentiation, and promotes mineral deposition 16 . TLR4 has emerged as a potential target for therapeutic strategies aimed at preventing or mitigating vascular calcification. However, its regulation of cell expression is still poorly understood. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), is an adipokine highly expressed in visceral adipose tissue 17 . It exists both intracellularly and extracellularly, playing distinct roles in various physiological and pathological processes 18 . Intracellular NAMPT (iNAMPT) regulates nicotinamide adenine dinucleotide (NAD + ) biosynthesis, influencing immune function, aging, and cancer through NAD-dependent proteins such as sirtuins and poly (ADP-ribose) polymerases (PARPs) 19 . Extracellular NAMPT (eNAMPT) participates in hormone-like signaling and activates NF-κB and mitogen-activated protein kinase (MAPK) pathways, which regulate inflammation, cell growth, and metabolism 19-21 . Studies show that visfatin levels are elevated in diseases like rheumatoid arthritis, cancer, and diabetes, and are linked to vascular remodeling, inflammation, and atherosclerosis, increasing cardiovascular risk 22-24 . However, research on the role of visfatin in vascular calcification is rarely seen. Visfatin is a pro-inflammatory adipokine. Studies suggest that exogenous visfatin promotes inflammatory injury in pulmonary endothelial cells via TLR4/NF-κB, exacerbating endothelial dysfunction 25 . We speculate that visfatin may enhance inflammation through TLR4, induce phenotypic changes in vascular smooth muscle cells, promote calcium deposition, and drive vascular calcification. Thus, the visfatin-TLR4 axis may be a key regulator of vascular calcification, offering new insights into its pathophysiology and potential treatments. eNAMPT is secreted via a non-classical pathway rather than the traditional Golgi-endoplasmic reticulum system 26 . The mechanisms regulating visfatin expression and secretion remain unclear but may involve acetylation status, metabolic state, inflammation, and hormone levels 27 . Clinical studies show that sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce serum visfatin levels in type 2 diabetes patients, suggesting their role in metabolic regulation 28 . Empagliflozin, a widely studied SGLT2i, lowers blood glucose by inhibiting SGLT2 in renal proximal tubules, reducing glucose reabsorption and promoting urinary glucose excretion. Beyond glycemic control, it also improves cardiovascular outcomes 29 . The EMPA-REG OUTCOME trial demonstrated that empagliflozin reduces major cardiovascular events, heart failure hospitalizations, and cardiovascular-related mortality in type 2 diabetes patients with cardiovascular disease 30-32 . However, the role of Empagliflozin in vascular calcification remains unknown. This study aims to investigate the role and mechanisms of visfatin in vascular smooth muscle cell osteogenic differentiation and vascular calcification, as well as evaluate its potential for pharmacological intervention. 2. MATERIALS AND METHODS 2.1 Reagents Foetal bovine serum (FBS; 10099141), newborn calf serum (NCS; 16010159) and Dulbecco's Modified Eagle's medium (DMEM; 11965092) were obtained from Gibco (Grand Island, NY, USA). β‐Glycerophosphate (β‐GP; G5422), calcium dichloride (C5670), dexamethasone(D1756) and IBMX(I7018) were purchased from Sigma‐Aldrich (Louis, Missouri, USA). SP600125 (S1460), PD98059 (S1177), and SB203580 (S1076) were obtained from Selleck Chemicals (Houston, TX, United States). Empagliflozin (HY-15409), BAY 11-7085 (HY-10257), insulin (HY-P0035), rosiglitazone (HY-17386), and visfatin (HY-P701314) were purchased from MedChemExpress (New Jersey, USA). Antibodies against OPN (22952-1-AP), NF-κB (10745-1-AP), Transferrin (66171-1-Ig), GAPDH (10494-1-AP), visfatin (11776-1-AP), SGLT2 (24654-1-AP), NLRP3 (30109-1-AP), TNF-α (26162-1-AP), IL-1β (16806-1-AP), and TLR4 (19811-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies against JNK (#9252), p-p44/42 MAPK (Erk1/2) (#4370), p44/42 MAPK (Erk1/2) (#4695), p-p38 MAPK (#4511), p38 MAPK (#8690), β-actin (#4970), and Calponin (#17819) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Antibodies against RUNX2 (ab236639), BMP2 (ab214821), and p-JNK (ab76572) were purchased from Abcam (Cambridge, UK). An adeno-associated virus delivering adipose-specific FABP4 promoter-driven control shRNA (pAAV-FABP4p-scramble, AAV- NC ) and shRNA against murine Visfatin (pAAV-FABP4p- Visfatin , AAV- Visfatin ) were obtained from Shanghai Genechem Co. Ltd. The oligonucleotide primer sequences were as follows: for Visfatin , 5′-TTCAAGGAGATGGCGTGGAT-3′ and 5′-CCACCAGAACCGAAGGAGAC-3′; for SGLT2, 5′- GTTCCGACCATAAACGATGCC-3′ and 5′-TGGTGGTTGCCCTTCCGTCAAT-3′; fragment1 of Visfatin promoter, 5′- AGGATCGGAATCCACAAGACG-3′ and 5′-TTACCTTTGTCTCCCGCTTGG-3′; fragment2 of Visfatin promoter, 5′-CATATAGCCCTTTGCGGGAGA-3′ and 5′-TACTGGCTTAATCCCACAGCA-3′; fragment3 of Visfatin promoter, 5′-ATGAGCCAAATAGATGTTTCCTCT-3′ and 5′-TTGCCCCATCTGACTTGCTATT-3′; fragment4 of Visfatin promoter, 5′-CCGGGGTACTGCTTAGTTCAT-3′ and 5′-ACACAGAAGTGGATGCTCACA-3′; Gapdh: 5′-TGTCTCCTGCGACTTCAACA-3′ and 5′-GGTGGTCCAGGGTTTCTTACT-3′. 2.2 Human samples Venous blood samples were collected from 78 patients who had undergone Coronary Computed Tomography Angiography and had a confirmed coronary artery calcification score. The coronary artery calcification score was calculated using the Agatston method 33 . Patients were divided into two groups based on a score threshold of 100. All participants had normal liver and kidney function. Serum was obtained by centrifugation at 3000 rpm for 15 minutes and subsequently used for ELISA analysis. The use of blood samples was approved by the Medical Institutional Ethics Committee of Qilu Hospital, Shandong University, China (Approval No. KYLL-2020(KS)-537), and all donors provided informed consent. 2.3 Animals Eight-week-old male C57BL/6J mice were obtained from ViewSolid Biotech (Beijing, China). TLR4 knockout mice ( TLR4 -/- ; JAX:003752, Jackson Laboratory) were purchased from the Jackson laboratory. Eight-week-old male mice were subcutaneously injected with VitD at a dose of 500,000 IU/kg/day for 4 days, while mice in the control group were injected with the same volume of 5% ethanol (the solvent for VitD). Mice were fed a normal diet for 10 days before sacrificed. Eight-week-old male mice were administered empagliflozin via gavage at a dose of 10 mg/kg/day for 10 days, while mice in the control group received the same volume of normal saline. Mice were sacrificed 10 days after the administration. Eight-week-old male C57BL/6J mice were anesthetized via an intraperitoneal injection of tribromoethanol (10 mL/kg body weight, M2910, Nanjing Aibei Biotechnology, Nanjing, China). Epididymal fat tissues were exposed, and multiple-site injections of either AAV- CTR or AAV- Visfatin were performed bilaterally. A total of 10 μl of the viral solution (5 μl per side) was injected, with a viral titer of 5×10¹² virus genomes/ml. After a 4-week, mice were subcutaneously injected with vitamin D or 5% ethanol to induce vascular calcification. All animals were maintained under a 12-hour light/dark cycle at a temperature of 25 °C. All procedures adhered to protocols approved by the Animal Care and Use Committee of Shandong University. 2.4 Von Kossa staining After routine deparaffinization and rehydration, 1% silver nitrate (G5491, Solarbio, Beijing, China) was added to the aortic sections, completely covering tissues. The sections were then exposed to ultraviolet light for 20 minutes, rinsed with distilled water, and incubated in 5% sodium thiosulfate for 2 minutes at room temperature. The sections were dehydrated and mounted. Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan). 2.5 mice aortic ring culture Mice thoracic aortas were immediately isolated and transferred to a sterile Petri dish containing PBS ice after anesthetized. After removing the outer membrane, the aortas were cut into rings with width of 3-5mm, which were then placed in a culture medium containing 15% FBS, 100 μg/mL penicillin-streptomycin. The aortic rings were incubated at 37°C in 5% CO2. The culture medium was replaced every other day. To induce calcification, aortic rings were cultured in DMEM with or without calcified medium (3mM calcium chloride and 10mM β-glycerophosphate) for 6 days, with the medium changed every other day. These aortic rings were co-cultured with approximately 5 cubic millimeters of epididymal adipose tissue. 2.6 Cell culture Eight-week-old C57BL/6J and TLR4⁻/⁻ mice were euthanized via CO₂ asphyxiation. The skin and muscles were dissected, and the aortas were quickly removed and placed in a sterile Petri dish containing phosphate-buffered saline (PBS). After removing the outer membrane, the aortas were cut into small pieces and evenly distributed at the bottom of a culture flask. The tissues were incubated at 37°C for 2 hours, followed by the addition of DMEM containing 15% FBS and 100 μg/mL penicillin-streptomycin, and maintained at 37°C in 5% CO2 for a minimum of 5 days. VSMCs were maintained in DMEM supplemented with 10% FBS, 100 μg/mL penicillin- streptomycin. To induce calcification, cells were cultured in DMEM containing calcified medium for 6 days. The culture medium was replaced every other day. 3T3-L1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. 3T3-L1 preadipocytes were cultured in complete culture medium until 70-80% confluent. Differentiation of 3T3-L1 was induced by addition of 0.25 μM dexamethasone, 0.5 mM IBMX, 1 μg/ml insulin and 2 μM rosiglitazone for 4 days, at which time the medium was replaced with growth medium containing 2 μM insulin for another 4 days. Under high magnification microscopy, bright circular lipid droplets were observed inside the cells, indicating adipocyte differentiation and maturation. 2.7 Oil red O staining Dilute the saturated Oil Red O solution (G1015, Servicebio, Wuhan, China) with double-distilled water in a 3:2 ratio and heat at 65°C for 1 hour. Allow the solution to cool to room temperature and then filter it. For induced mature adipocytes, gently rinse with PBS and fix with 4% paraformaldehyde at room temperature for 30 minutes. After removing the fixative, rinse cells again with PBS. Subsequently, stain with the Oil Red O working solution at room temperature for 10 minutes avoiding light. Finally, rinse off unbound dye with PBS. The sections were dehydrated and mounted. Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan). 2.8 Co-Culture of 3T3-L1 and VSMCs 3T3-L1 cells were seeded in the lower chamber of a trans-well 12-well plate and incubated at 37 °C in 5% CO₂ for 24 hours, followed by induction into adipocytes. VSMCs were seeded in the upper chamber of the trans-well 12-well plate and incubated at 37℃ in a 5% CO₂ for 24 hours. The adherent VSMCs in the upper chamber were then transferred to 12-well plate with or without mature adipocytes in the lower chamber to establish a VSMC-adipocyte co-culture system. After the co-culture system was established, the cells were serum-starved in low-serum medium (2% FBS-H-DMEM) for 1 day. Then, the medium was replaced with calcification medium to induce calcification. 2.9 Alizarin Red S staining VSMCs were washed with PBS and fixed with paraformaldehyde for 30 minutes. After fixation, the cells were washed with PBS and stained with 1% alizarin red S staining solution (G1452, Solarbio, Beijing, China) in the dark for 10 minutes. Following washing, images were captured using a digital microscope. Tissues and vascular rings were fixed in 10% paraformaldehyde, embedded in paraffin, and sectioned into 5 µm thick slices. After routine deparaffinization and rehydration, the sections were stained with 1% alizarin red S solution in the dark for 5 minutes, followed by three washes with PBS. The sections were then treated with xylene and mounted using neutral resin. The sections were dehydrated and mounted. Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan). 2.10 Aortic gross staining with alizarin red Mice aortas were fixed in 10% formalin overnight, followed by dehydration in 95% ethanol for 24 hours. The aortas were then stained with 0.003% alizarin red solution in 1% potassium hydroxide for 24 hours and rinsed with 2% potassium hydroxide to eliminate any excess stain. Images were captured for analysis. 2.11 Western blot analysis Proteins were extracted from cells or tissues using RIPA lysis buffer (R0010, Solarbio, Beijing, China), separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (ISEQ00010, Millipore, Boston, Massachusetts, USA). The membrane was blocked with 5% skim milk in Tween 20-Tris-buffered saline (TBST) for 1 hour, followed by incubation with the appropriate primary antibody at 4 °C overnight. The membrane was washed 3 times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. After 3 additional washes with TBST, the protein signals were detected using enhanced chemiluminescence (WBKLS0500, Millipore, Boston, Massachusetts, USA). The results were analyzed with ImageJ software. All experiments were performed at least 3 times. 2.12 Quantitative real-time PCR (qPCR) Total RNA was isolated from cell and mouse tissues using Tri reagent (Ambion, Austin, TX, USA) following the manufacturer's protocol. 1 μg of RNA was then reverse-transcribed into complementary DNA with the iScriptcDNA synthesis kit (Bio-Rad, Hercules, CA, USA). PCR amplification was performed using the SYBR PCR mix (Bio-Rad, Hercules, CA, USA). 2.13 Immunoprecipitation (IP) analysis VSMCs were disrupted using a specialized immunoprecipitation lysis solution composed of 150 mM saline, 50 mM Tris–HCl and 1% NP-40 (88805, Thermo Fisher Scientific, Waltham, MA, USA) at pH 7.8, supplemented with protease inhibitor cocktail. Following centrifugation and supernatant collection, the lysates were mixed with primary antibodies pre-conjugated to 30 µl of pre-washed protein A/G magnetic beads (HY-K0202, MedChemExpress, New Jersey, USA) and incubated at 4 °C overnight. The resulting immunoprecipitated complexes were then rinsed and heated in SDS loading buffer at 95°C using a metal bath, and subsequently analyzed via western blotting. 2.14 Serum and Culture medium supernatant Visfatin analysis Serum and Culture medium supernatant visfatin levels were measured by an ELISA assay. The eNAMPT ELISA kit (DY4335-05 and NBP3-43467) was purchased from R&D Systems (Minneapolis, MN, USA). Mix serum or culture supernatant with acetone at a 1:4 ratio, incubate overnight at -80°C, then centrifuge and dry the precipitate. Dissolve the precipitate in RIPA lysis buffer and adjust the concentration for subsequent experiments. 2.15 Immunohistochemical analysis After deparaffinization, rehydration, antigen retrieval, and blocking of non-specific binding, the sections were incubated with the appropriate primary antibody at 4°C overnight. The sections were washed 3 times with PBS and incubated with the secondary antibody at 37°C for 30 minutes. The bound secondary antibody was detected using DAB solution (ZLI-9018, Zhong Shan Golden Bridge Biological Technology, Beijing, China). Haematoxylin was applied to counterstain the nucleus. Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan). 2.16 Immunofluorescence (IF) analysis After deparaffinization, rehydration, antigen retrieval, sections were permeabilized with 0.3% Triton X-100 (T8200, Solarbio, Beijing, China) for 15 minutes. After washing with PBS, the slides were blocked. The sections were incubated with the appropriate primary antibody at 4°C overnight. After washing with PBS, the sections were incubated with the secondary antibody at 37 °C for 1 hour. The slides were covered by a drop of Fluoroshield Mounting Medium containing 40,6‐diamidino‐2‐phenylindole (DAPI; ab104139, Abcam, Cambridge, UK) before being observed with laser scanning confocal microscopy (LSM710, Zeiss, Oberkochen, Germany). Cell slides with mature adipocytes were fixed with immunostaining fixative (P0098, Beyotime Biotechnology, Shanghai, China) at room temperature for 1 hour. The slides were blocked, probed with antibodies, stained with DAPI, and observed under laser confocal microscopy (LSM710, Zeiss, Oberkochen, Germany). 2.17 Dual-luciferase reporter assay The promoter region of mice Visfatin was amplified using PCR and subsequently inserted into the pGL3-Basic vector (E1751, Promega, Madison, WI, USA) to generate the Visfatin promoter-driven luciferase reporter construct (designated as Mus-Nampt). The Mus-Nampt plasmid was then co-transfected with an NF-κB overexpression plasmid into HEK293 cells. Following a 48-hour transfection period, the activities of Firefly and Renilla luciferase were measured utilizing the Dual-Luciferase Reporter Assay System (E1751, Promega, Madison, Wisconsin, USA). 2.18 Chromatin immunoprecipitation (ChIP) assay Chromatin immunoprecipitation (ChIP) was performed following the manufacturer’s protocol using the Enzymatic Chromatin IP Kit (9003; Cell Signaling Technology, Danvers, Massachusetts, USA). Briefly, 3T3-L1-induced adipocytes were cross-linked with 1% formaldehyde for 10 minutes, followed by sonication to shear the DNA into fragments. The lysates were then incubated with either anti-IgG or anti-NF-κB antibody in the presence of magnetic beads. After collection and purification, the immunoprecipitated DNA samples were analyzed by RT-qPCR. After performing the ChIP experiment, the qPCR products were processed on a 2% agarose gel and visualized using a UV transilluminator. 2.19 Statistical analysis All statistical analysis were carried out using GraphPad Prism 6.0, with results expressed as mean ± SEM. Differences between two groups were assessed using the Student's t-test, while comparisons across multiple groups were analyzed through one-way ANOVA. A p-value of less than 0.05 was deemed to indicate statistical significance. 3. Results 3.1 Adipose promotes calcified medium-induced calcification of VSMCs and aortic vascular rings To study the effect of adipose tissue on vascular calcification. We harvested mouse aortic rings and induced calcification using a calcification medium, co-culturing them with approximately 5 cubic millimeters of epididymal adipose tissue during this process. Compared to aortic rings cultured alone and induced to calcify, the co-culture group exhibited a significantly increased deposition of mineralized matrix, as assessed by Alizarin Red and Von-Kossa staining (Fig.1A). Subsequently, 3T3-L1 preadipocytes were induced to differentiate into adipocytes over an 8-day period for in vitro experiments. Oil Red O staining was used to assess the state of the differentiated cells (Fig.1B). To further validate the effect of adipocytes on VSMC calcification, we co-cultured 3T3-L1-induced differentiated adipocytes with primary mouse VSMCs and incubated them in calcification medium for 6 days. Compared to VSMCs cultured alone and induced to calcify, the expression levels of calcification markers RUNX2, OPN, and BMP2 in VSMCs from the co-culture group were significantly upregulated (Fig.1C and D). The results indicate that adipose tissue or adipocytes significantly promote the calcification process of vascular smooth muscle cells through co-culture, further supporting the regulatory role of adipocytes in vascular calcification. 3.2 The disorder of calcium and phosphorus promotes the expressed and secretion of visfatin in vivo and in vitro After confirming the pro-calcification effect of adipocytes on blood vessels, we first investigated whether calcium-phosphorus imbalance affects the expression and secretion of visfatin in both adipose tissue and adipocytes to further explore its role in vascular calcification. Patients with moderate to severe coronary artery calcification (CAC score >100) exhibit significantly higher serum visfatin levels compared to those with mild or no coronary calcification (CAC score ≤100) (Fig.2A). Similarly, after vitamin D treatment, serum visfatin protein levels were significantly elevated in calcified mice compared to control mice (Fig.2B and C). In mice with vascular calcification, both the protein and mRNA levels of visfatin are significantly higher in visceral adipose tissue (VAT) than in subcutaneous adipose tissue (SAT) (Fig.2D and E), suggesting that visfatin is primarily produced and secreted by VAT. Therefore, we focused our investigation on visceral adipose tissue. Western blot and qPCR demonstrated that visfatin protein and mRNA levels were elevated in the epididymal adipose tissue of calcified mice (Fig.2F and G). Additionally, immunohistochemical analysis indicated that visfatin expression was markedly upregulated in the epididymal adipose tissue of calcified mice compared to the control group (Fig.2H). We simulated calcium-phosphorus imbalance in vivo using calcification medium prepared with β-GP and CaCl 2 . After stimulating 3T3-L1 differentiated adipocytes with the calcification medium for 3 days, ELISA results showed that the level of visfatin in the supernatant of the adipocyte culture medium was increased (Fig.2I). Further analysis by Western blot and RT-qPCR revealed that both the protein and mRNA levels of visfatin in the adipocytes treated with the calcification medium were significantly elevated compared to the control group (Fig.2J and K). Thus, both in vivo and in vitro disturbances in calcium and phosphorus balance can promote the expression and secretion of visfatin in adipocytes. 3.3 Visfatin promotes VSMC phenotype switching in vivo and in vitro Vascular calcification has been shown to result from multifactorial vascular changes, not merely calcium deposition, with osteogenic transformation of VSMCs being one of the key mechanisms in vascular calcification. To further investigate the role of visfatin in vascular calcification, we examined whether visfatin could induce phenotypic transition in vascular smooth muscle cells and promote vascular calcification. We constructed a mouse model with epididymal adipose tissue-specific overexpression of visfatin by locally injecting AAV carrying the FABP4 promoter into the bilateral epididymal adipose tissue of mice. After inducing calcification in mice with vitamin D, ELISA analysis demonstrated that the serum visfatin levels in mice injected with AAV- Visfatin were significantly higher compared to those injected with AAV- NC (Fig.3A). The mouse aorta was then collected for whole-mount Alizarin Red staining, which revealed that visfatin overexpression in epididymal adipose tissue significantly promoted aortic calcium deposition (Fig.3B). Immunohistochemistry showed that RUNX2 expression was significantly increased in the aortas of mice with overexpression of visfatin in epididymal adipose tissue (Fig.3C). Likewise, Von Kossa staining revealed an increase in calcium deposition in the aortas of mice with visfatin overexpression (Fig.3D). To investigate the role of visfatin in vascular calcification, mouse aortic rings were isolated and induced with calcification medium. The aortic rings were divided into two groups: one group was treated with visfatin (100 ng/mL), while the other group received an equal volume of PBS as a control. Alizarin Red staining and Von-Kossa staining results demonstrated that visfatin significantly enhanced matrix mineralization deposition in the mouse aortic rings (Fig.3E). Subsequently, VSMC treated with calcification medium were exposed to visfatin (100 ng/mL) for 6 days. The results showed that visfatin significantly enhanced calcium deposition in VSMCs (Fig.3F and H). Consistently, visfatin upregulated the expression of calcification markers, including Runx2, OPN, and BMP2, while downregulating the expression of the contractile marker calponin (Fig.3G and I). 3.4 TLR4 Knockout Attenuates Visfatin-Induced Vascular Calcification Previous studies have suggested that the effects of visfatin may be associated with TLR4. To gain a deeper understanding of the mechanism by which visfatin promotes vascular calcification, we investigated the involvement of the TLR4 signaling pathway in this process. After vitamin D-induced calcification, whole-mount Alizarin Red staining of the aortic tissue showed that matrix mineral deposition was significantly reduced in TLR4 -/- mice compared to wild-type mice (Fig.4A). Sectional Von Kossa staining further confirmed that TLR4 knockout significantly reduced calcium deposition in the aortic tissue (Fig.4B). Immunohistochemistry showed that RUNX2 expression was significantly increased in the aortas of TLR4 -/- mice (Fig.4C and D). To specifically assess the role of TLR4 in visfatin-mediated vascular calcification, aortic rings from TLR4 -/- mice were treated with both visfatin and calcification medium. Alizarin Red and Von Kossa staining revealed that TLR4 knockout significantly reduced calcium deposition in the aortic rings compared to the control group (Fig.4E). Primary VSMCs from control and TLR4 -/- mice were cultured in calcification medium with visfatin for 6 days. The results showed that compared to the control group, VSMCs from TLR4 -/- mice exhibited higher expression of contractile phenotype markers and lower expression of osteogenic phenotype markers (Fig.4F). Together, these results demonstrate that TLR4 deletion not only mitigates baseline vascular calcification but also significantly inhibits the pro-calcific actions of visfatin. These findings underscore the pivotal role of TLR4 in mediating visfatin-driven osteogenic transition and mineral deposition in vascular tissues. 3.5 TLR4 Knockout Attenuates Visfatin-Induced Upregulation of Inflammatory Mediator-Related Proteins Inflammatory responses play a critical role in the initiation and progression of vascular calcification. As a classical innate immune receptor, TLR4 is capable of sensing both exogenous and endogenous pro-inflammatory stimuli and activating downstream signaling pathways, thereby promoting the expression of various inflammatory mediators and accelerating the calcification process. In this study, we found that TLR4 plays an essential regulatory role in visfatin-induced vascular calcification. To further elucidate the underlying mechanism, we specifically investigated the involvement of inflammation in this process. To begin with, VSMCs cultured in calcification medium were treated with LPS to activate TLR4. Compared with cells cultured in calcification medium alone, LPS stimulation significantly increased the expression of inflammatory markers such as NLRP3, TNF-α, and IL-1β, along with an enhanced phenotypic switch of VSMCs from a contractile to an osteogenic state (Fig.5A). Similarly, treatment of VSMCs cultured in calcification medium with visfatin also led to a pronounced elevation in the levels of inflammatory mediators, including NLRP3, TNF-α, and IL-1β, when compared to cells exposed to calcification medium alone (Fig.5B). These findings indicate that visfatin, like LPS, can amplify inflammatory signaling within VSMCs under pro-calcific conditions. To further validate the role of TLR4 in visfatin-induced inflammation, primary VSMCs were isolated from TLR4 knockout mice and cultured in calcification medium supplemented with visfatin. Compared with VSMCs derived from WT mice, TLR4 -/- VSMCs exhibited significantly lower expression levels of inflammatory markers (Fig.5C). Immunoprecipitation demonstrated that visfatin can bind to TLR4 (Fig.5D). Collectively, these findings indicate that visfatin promotes inflammatory responses in VSMCs at least in part through direct interaction with TLR4, thereby contributing to vascular calcification. 3.6 Empagliflozin inhibits the expression and secretion of visfatin in both adipose tissue and adipocytes Currently, studies investigating the regulation of visfatin expression and secretion are limited. Previous reports have shown that SGLT2 inhibitors can reduce serum visfatin levels in patients with type 2 diabetes 28 . Therefore, we next explored the effects of the SGLT2 inhibitor empagliflozin on the regulation of visfatin expression and secretion in adipose tissue. Mice were administered empagliflozin by gavage at a dose of 3.8 mg/kg/d, while the control group received an equivalent volume of double-distilled water. After vitamin D-induced calcification, the serum visfatin levels were significantly reduced in the empagliflozin gavage group of mice (Fig.6A and B). The mRNA level of visfatin in the visceral adipose tissue of empagliflozin-treated mice with vascular calcification was decreased (Fig.6C). Consistently, Immunohistochemical analysis revealed that the expression of visfatin in the epididymal adipose tissue of mice with vascular calcification treated with empagliflozin was significantly reduced compared to the vascular calcification group without treatment (Fig.6D). After differentiating 3T3-L1 cells into adipocytes, they were treated with either calcification medium alone or calcification medium combined with empagliflozin. ELISA analysis showed that visfatin levels in the culture supernatant were significantly reduced in the empagliflozin-treated group compared to the group treated with calcification medium alone (Fig.6E), and both protein and mRNA expression levels of visfatin in adipocytes were also markedly decreased (Fig.6F and G). In summary, our findings demonstrate that empagliflozin significantly suppresses the expression and secretion of visfatin in adipose tissue, which may represent a potential mechanism underlying its ability to ameliorate vascular calcification. These results suggest that empagliflozin, by modulating visfatin-mediated signaling pathways, may offer a novel therapeutic strategy for the treatment of vascular calcification. 3.7 Empagliflozin can improve vascular calcification Given that empagliflozin suppresses the expression and secretion of visfatin in adipocytes, we next explored whether empagliflozin could attenuate vascular calcification through this mechanism. After inducing vascular calcification in mice with vitamin D, one group of mice was treated with empagliflozin gavage, while the other group received an equal volume of distilled water as a control. Whole-mount Alizarin Red staining of the aorta revealed that empagliflozin significantly improved vascular calcification and reduced calcium deposition (Fig.7A). Meanwhile, Von Kossa staining of aortic sections further confirmed this result (Fig.7B). Immunohistochemical staining of aortic sections showed a decreased expression of RUNX2 protein in the empagliflozin group (Fig.7C and D). In vitro experiments revealed that primary VSMCs were divided into two groups: one group was induced for calcification, and the other group was induced for calcification with the addition of empagliflozin (10 µM). Western blot analysis showed no significant differences in the expression levels of osteogenic and contractile markers between the two groups (Fig.7E). However, in a co-culture system of 3T3-L1 adipocytes and VSMCs under calcification induction with calcification medium, the addition of empagliflozin (10 µM) significantly reduced the expression of osteogenic markers OPN, RUNX2, and BMP2, while increasing the expression of the contractile marker calponin compared to the control group (Fig.7F). To further validate these findings, a co-culture system of mouse aortic rings and epididymal adipose tissue was established. In the presence of calcification medium, which promotes calcification, the group treated with empagliflozin exhibited a significant reduction in calcium deposition compared to the untreated control group (Fig.7G). Both the protein and mRNA expression levels of SGLT2 are higher in adipocytes than in VSMCs (Fig.7H). In summary, the results of this section indicate that the SGLT2 inhibitor empagliflozin reduces calcium deposition in the aortas of calcified mice both in vivo and in vitro. This phenomenon may be attributed to empagliflozin's inhibition of the pro-calcification effect of adipocytes on VSMCs. 3.8 Empagliflozin reduces the expression and secretion of visfatin in adipocytes by inhibiting the p38 MAPK pathway. Some potential signaling pathways targeted by SGLT2 inhibitors have been reported, including ERK, JNK, and p38 MAPK 34 . The p38 signaling pathway plays a crucial role in regulating inflammatory and metabolic responses in adipocytes. To investigate whether empagliflozin modulates visfatin expression through this pathway, we examined the phosphorylation status of p38 in adipose tissue following empagliflozin treatment. When 3T3-L1 induced adipocytes were incubated with the calcification medium for 3 days, there was a significant increase in the phosphorylation of JNK, ERK, and p38 compared to the control group (Fig.8A). The application of specific inhibitors for JNK (SP600125), ERK (PD98059), and p38 (SB203580) revealed that only the p38 inhibitor significantly reduced the protein levels of visfatin in adipocytes (Fig.8B). Co-treatment of 3T3-L1 induced adipocytes with calcification medium and the p38 inhibitor resulted in a significant decrease in both the protein and mRNA levels of visfatin, compared to treatment with calcification medium alone (Fig.8C and D). Furthermore, empagliflozin markedly suppressed the phosphorylation of p38 in adipocytes stimulated by calcification medium (Fig.8E). In summary, empagliflozin inhibits the upregulation of visfatin expression in adipocytes induced by calcification medium by suppressing the p38 phosphorylation pathway. 3.9 Empagliflozin inhibits the endogenous binding of NF-κB to the visfatin promoter. Building on the finding that empagliflozin and p38 regulate visfatin expression, we next investigated whether NF-κB nuclear translocation serves as a downstream mechanism controlling visfatin transcription. We found that calcification medium promotes the nuclear translocation of NF-κB in 3T3-L1 induced adipocytes (Fig.9A). The use of a specific NF-κB inhibitor (BAY 11-7085) significantly reduced both the protein and mRNA levels of visfatin in adipocytes (Fig.9B). Subsequently, we examined the effects of empagliflozin and the p38 inhibitor on NF-κB in adipocytes. In 3T3-L1-induced adipocytes treated with calcification medium, both empagliflozin and the p38 inhibitor were able to suppress the nuclear translocation of NF-κB (Fig.9C). Based on previous results, we investigated the effect of NF-κB on the transcriptional regulation of visfatin. We synthesized luciferase reporter plasmids targeting the promoter region of visfatin and transfected HEK293T cells with these plasmids. Subsequent luciferase activity assays demonstrated that NF-κB enhances the transcriptional activity of visfatin (Fig.9D). The results of binding site prediction for the visfatin promoter using JASPAR revealed that NF-κB binds to the visfatin promoter at the sequences ggtatttccc (-1989 bp to -1980 bp) or ggtaatttct (-1696 bp to -1687 bp) (Fig.9E). The visfatin promoter was divided into four fragments of approximately 200 bp in length, including two random fragments without predicted binding sites (fragment1: -1526bp to -1433bp and fragment2: -438bp to -369bp) and two fragments containing predicted binding sites (fragment3: -2032bp to -1924bp and fragment4: -1761bp to -1622bp). Primers targeting these four regions were synthesized, and ChIP experiments were conducted for analysis. The results of the ChIP assay and agarose gel electrophoresis demonstrated that the region where NF-κB directly binds to the visfatin promoter is fragment4 (Fig.9F and G). In summary, our findings demonstrate that NF-κB directly binds to the visfatin promoter at specific sites, thereby enhancing its transcriptional activity. This regulatory mechanism is modulated by empagliflozin and p38 inhibition, which suppress NF-κB nuclear translocation and subsequently reduce visfatin expression. 4. Discussion In this study, we experimentally discovered that serum levels of visfatin were significantly elevated in mice with vascular calcification, and the expression and secretion of visfatin in visceral fat tissue were also notably increased. This suggests that visfatin, as an adipokine secreted by visceral fat, may play an important role in the occurrence and progression of vascular calcification. Further investigation revealed that visfatin directly binds to the TLR4 receptor on vascular smooth muscle cells, promoting the increased expression of osteogenic markers and the decreased expression of contractile markers, thereby facilitating the process of vascular calcification. This finding provides new insights into the link between visceral fat and vascular diseases. Additionally, we found that empagliflozin, an SGLT2 inhibitor, can inhibit the activation of p38 in adipocytes, thereby preventing the nuclear translocation of NF-κB, which ultimately suppresses the binding of NF-κB to the visfatin promoter and reduces visfatin expression. This mechanism not only highlights the potential role of empagliflozin in ameliorating vascular calcification but also provides new theoretical insights into its pleiotropic effects in metabolic and cardiovascular diseases, while further elucidating the critical regulatory role of visfatin in vascular calcification. Visceral fat is closely associated with vascular calcification. In addition to serving as an energy storage tissue, visceral fat secretes various adipokines and inflammatory factors that promote the occurrence of vascular calcification 35 . Excessive accumulation of visceral fat leads to chronic low-grade inflammation 36 , activating signaling pathways such as NF-κB 37 , MAPK 38 , and TLR4 39 . These pathways not only promote immune responses but also affect the phenotypic transition of vascular smooth muscle cells. Upon stimulation by these signals, vascular smooth muscle cells shift from a contractile phenotype to an osteoblast-like phenotype, which promotes calcium salt deposition and leads to vascular wall calcification. Through this mechanism, visceral fat increases the risk of vascular calcification, especially in patients with metabolic diseases such as obesity and diabetes, where the occurrence of vascular calcification is closely linked to excessive visceral fat accumulation. Therefore, abnormalities in visceral fat metabolism not only affect overall metabolic function but may also exacerbate vascular calcification through inflammation and cellular transformation, further increasing the risk of cardiovascular diseases. The role of TLR4 in vascular calcification is primarily through its regulation of inflammatory responses and intracellular mineralization processes. VSMCs respond to inflammatory signals via TLR4, activating pathways like NF-κB and MAPK 16 40-42 . These pathways enhance inflammation and promote cell phenotypic transition, driving vascular calcification. VSMCs shift from a contractile phenotype to an osteoblast-like phenotype, expressing osteogenic markers such as osteocalcin and alkaline phosphatase 43 . TLR4 activates these pathways, accelerating vascular calcification. Visfatin, though known to play a role in inflammation and insulin resistance 44 45 , has not been fully explored in vascular calcification. Existing studies show that visfatin activates the TLR4 pathway in various cell types 46-49 , but its role in VSMCs is not well understood. Our research reveals that visfatin binds to TLR4 on VSMCs, initiating signal transduction, and emphasizes the importance of TLR4 in regulating vascular calcification. TLR4 activation enhances inflammation in VSMCs and may interact with osteogenic signaling pathways to promote vascular calcification. Studying the TLR4-visfatin interaction provides insights into the molecular mechanisms of vascular calcification and potential therapeutic strategies. In this study, we identified empagliflozin as a regulator of visfatin expression in adipocytes. Our results demonstrate that empagliflozin inhibits the activation of p38, preventing the nuclear translocation of NF-κB and its binding to the visfatin promoter, leading to reduced visfatin secretion. These findings suggest that empagliflozin, beyond its glucose-lowering effects, may exert protective effects against vascular calcification by modulating adipokine signaling. Previous studies have focused on SGLT2 inhibitors' role in improving glycemic control and reducing heart failure risk. However, our study is the first to show that empagliflozin can directly suppress visfatin expression in adipocytes via the p38/NF-κB pathway, providing a new cardiovascular benefit mechanism. Recent studies have also shown that empagliflozin modulates multiple signaling pathways, including AMPK activation and oxidative stress reduction, which may enhance its cardiovascular protective effects 50-54 . For example, empagliflozin has been reported to reduce inflammation through AMPK activation and improve vascular calcification through anti-inflammatory pathways 55 . In this study, the direct effect of SGLT2 inhibitors on calcification medium-induced vascular smooth muscle calcification was not prominent, potentially due to the short stimulation time of the calcification medium, their cell type-specific action, and their primary reliance on regulating visfatin expression in adipocytes. The calcification medium may activate pathways less responsive to SGLT2 inhibition, and its complex mechanism compared to high-phosphate conditions could contribute to the limited direct impact. Future studies should further investigate SGLT2 inhibitor targets in smooth muscle cells and their indirect mechanisms in vivo. Given the elevated levels of visfatin in patients with metabolic disorders such as obesity and diabetes 56 57 , our findings suggest that empagliflozin could be particularly beneficial in these populations. This study is the first to show that visceral adipose-derived visfatin binds to TLR4 on vascular smooth muscle cells, promoting vascular calcification by upregulating osteogenic markers and downregulating contractile markers. While visfatin's role in inflammation and insulin resistance is well-established, its direct involvement in vascular calcification has remained unclear. We also discovered that empagliflozin, an SGLT2 inhibitor, suppresses visfatin expression in adipocytes via the p38/NF-κB pathway, expanding the understanding of SGLT2 inhibitors' pleiotropic effects beyond glucose-lowering. By reducing visfatin expression, empagliflozin may help mitigate the risk of vascular calcification and its associated complications, such as atherosclerosis and cardiovascular events. Future studies should investigate whether other SGLT2 inhibitors share empagliflozin's ability to suppress visfatin expression and whether this effect is consistent across different patient populations. Additionally, long-term clinical trials are needed to evaluate empagliflozin's effects on vascular calcification in patients with metabolic disorders. In conclusion, our study highlights empagliflozin's multifaceted role in metabolic and cardiovascular health, demonstrating its potential as a comprehensive therapeutic agent for preventing vascular calcification in patients with metabolic disorders. This study has several limitations. First, the use of a single calcification model may not fully capture the complexity of vascular calcification. Future studies should consider employing multiple models, such as high-phosphate diet feeding or renal resection surgery-induced calcification models, to verify the effects of empagliflozin and assess its mechanisms across different conditions. Second, while we demonstrated that visfatin promotes vascular calcification through TLR4, the detailed mechanisms behind this interaction remain unclear. Future research should further explore the downstream signaling pathways activated by TLR4 and the role of visfatin in this process. Additionally, this study focused on the short-term effects of empagliflozin, and the long-term impact on vascular calcification needs further investigation. Finally, while the study concentrated on the visfatin-p38/NF-κB pathway, other factors, such as oxidative stress and inflammation, may also contribute to vascular calcification. Future studies should investigate the involvement of these factors and evaluate empagliflozin's potential to modulate them. Conclusion This study reveals that visceral adipose-derived visfatin promotes vascular calcification by binding to TLR4 on vascular smooth muscle cells. Additionally, we found that empagliflozin improves vascular calcification by inhibiting the activation of the p38/NF-κB pathway in adipocytes, thereby reducing visfatin expression. These findings provide new theoretical support for the use of SGLT2 inhibitors in preventing and treating vascular calcification in patients with metabolic disorders. Future studies should explore whether other SGLT2 inhibitors exhibit similar effects and validate these mechanisms in clinical applications. Further investigation into the role of visfatin in vascular calcification and the effects of empagliflozin under different pathological conditions will help develop more effective therapeutic strategies to reduce cardiovascular risk in patients with metabolic disorders. Abbreviations β‐GP: β‐Glycerophosphate; CAC: coronary artery calcification; CHIP: Chromatin immunoprecipitation; DMEM: Dulbecco's Modified Eagle's medium; eNAMPT: Extracellular NAMPT; FBS: foetal bovine serum; IF: Immunofluorescence; IL-6: Interleukin-6; iNAMPT: Intracellular NAMPT; IP: Immunoprecipitation; LPS: lipopolysaccharides; MAPK: Mitogen-activated protein kinase; NF-κB: nuclear factor-kappa B; NAMPT: nicotinamide phosphoribosyltransferase; NAD+: nicotinamide adenine dinucleotide; NCS: newborn calf serum; PAMPs: pathogen-associated molecular patterns; PARPs: poly (ADP-ribose) polymerases; PBS: phosphate-buffered saline; qPCR: Quantitative real-time PCR; SAT: subcutaneous adipose tissue; SGLT2i: sodium-glucose cotransporter 2 inhibitors ; TBST: Tween 20-Tris-buffered saline (TBST); TLR4: Toll-like receptor 4; TNF-α: Tumour necrosis factor alpha; VAT: visceral adipose tissue; VitD: vitamin D; VSMCs: vascular smooth muscle cells Declarations Acknowledgements We would like to express our deepest appreciation to the patients who consented to participate and donated blood samples, making this study possible. Funding statement This work was supported by the National Natural Science Foundation of China (grant nos. 81873516, 82170463, 81900444), the National Key Research and Development Program of China (grant nos. 2021YFF0501403, 2021YFF0501404, 2017YFC1308303), the Natural Science Foundation of Shandong Province (grant nos. ZR2023QH398, ZR2024MH019, ZR2019BH052, ZR2020QH007, and ZR2019PH030), China International Medical Foundation (grant nos. Z-2016-23-2001-01). Ethics approval and consent to participate The use of blood samples was approved by the Medical Institutional Ethics Committee of Qilu Hospital, Shandong University, China (Approval No. KYLL-2020(KS)-537), and all donors provided informed consent. All procedures adhered to protocols approved by the Animal Care and Use Committee of Shandong University (Approval No. DWLL-2021-143). Author Contribution X.J., X.Z. and Y.Z. designed and conducted this research, wrote the manuscript. Y.Z. and Y.L. prepared the figure. 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Cardiovasc Res 2023;119(5):1175-89. doi: 10.1093/cvr/cvad009 [published Online First: 2023/01/11] Zhou H, Wang S, Zhu P, et al. Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission. Redox Biol 2018;15:335-46. doi: 10.1016/j.redox.2017.12.019 [published Online First: 2018/01/08] Cai W, Chong K, Huang Y, et al. Empagliflozin improves mitochondrial dysfunction in diabetic cardiomyopathy by modulating ketone body metabolism and oxidative stress. Redox Biol 2024;69:103010. doi: 10.1016/j.redox.2023.103010 [published Online First: 2024/01/02] Kolijn D, Pabel S, Tian Y, et al. Empagliflozin improves endothelial and cardiomyocyte function in human heart failure with preserved ejection fraction via reduced pro-inflammatory-oxidative pathways and protein kinase Galpha oxidation. Cardiovasc Res 2021;117(2):495-507. doi: 10.1093/cvr/cvaa123 [published Online First: 2020/05/13] Li C, Zhang J, Xue M, et al. SGLT2 inhibition with empagliflozin attenuates myocardial oxidative stress and fibrosis in diabetic mice heart. Cardiovasc Diabetol 2019;18(1):15. doi: 10.1186/s12933-019-0816-2 [published Online First: 2019/02/04] Lu CW, Lee CJ, Hsieh YJ, et al. Empagliflozin Attenuates Vascular Calcification in Mice with Chronic Kidney Disease by Regulating the NFR2/HO-1 Anti-Inflammatory Pathway through AMPK Activation. Int J Mol Sci 2023;24(12) doi: 10.3390/ijms241210016 [published Online First: 2023/06/28] Retnakaran R, Youn BS, Liu Y, et al. Correlation of circulating full-length visfatin (PBEF/NAMPT) with metabolic parameters in subjects with and without diabetes: a cross-sectional study. Clin Endocrinol (Oxf) 2008;69(6):885-93. doi: 10.1111/j.1365-2265.2008.03264.x [published Online First: 2008/04/16] Sandeep S, Velmurugan K, Deepa R, et al. Serum visfatin in relation to visceral fat, obesity, and type 2 diabetes mellitus in Asian Indians. Metabolism 2007;56(4):565-70. doi: 10.1016/j.metabol.2006.12.005 [published Online First: 2007/03/24] Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.tiff Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7284693","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496420843,"identity":"360b1d5d-1571-44f1-9756-710e2f2c942a","order_by":0,"name":"Yue Zhang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhang","suffix":""},{"id":496420844,"identity":"8ebd9eb0-f4d8-454d-be29-9c293f1646b6","order_by":1,"name":"Yarui Zhao","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yarui","middleName":"","lastName":"Zhao","suffix":""},{"id":496420845,"identity":"0ab88b11-39f8-4482-b80b-94cdb57ed42f","order_by":2,"name":"Yijie Luo","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yijie","middleName":"","lastName":"Luo","suffix":""},{"id":496420846,"identity":"bd5c8c1a-ef28-4770-86f2-ba650700420a","order_by":3,"name":"Lei Wang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wang","suffix":""},{"id":496420847,"identity":"994aac00-a0ec-4803-8daa-1cc61f05fdc7","order_by":4,"name":"Jing Liao","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liao","suffix":""},{"id":496420848,"identity":"bb852dde-94ea-44c6-bc47-e34433833663","order_by":5,"name":"Weijie Wu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Weijie","middleName":"","lastName":"Wu","suffix":""},{"id":496420849,"identity":"d5044767-413a-4d3e-8f66-ba6532d8ad8f","order_by":6,"name":"Huixia Lu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Huixia","middleName":"","lastName":"Lu","suffix":""},{"id":496420850,"identity":"ef30eb67-f0bc-4d81-a282-359fdf54368d","order_by":7,"name":"Xinyu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDACCTjJfICBB8Q+QLwWtgSStIAAjwFxWuRnNz97+LXNIs/gRs7nD2/bGOT4biQwfi7Ao4VxzjFzY5kzEsUGN3K3Sc5tYzCWvJHALD0DjxZmiQQzaYkKicQNQC3MvG0MQEYCGzMPHi1sEunfpCUMQFpyHn8GaqknqIVHIsdM8gPYlhwGaaCWBANCWiQkcsqkGc5IJM4888xMcs45CcOZZx42S+PTIj8jfZvkz7a6xL7jyY8/vCmzkec7nnzwMz4tIAB2hsIBiK1AzNhAQANQyQ+QdYTVjYJRMApGwUgFAD6/SxfShrOtAAAAAElFTkSuQmCC","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Zhang","suffix":""},{"id":496420851,"identity":"07e9e65b-d750-4b94-8b72-b7dae81542f4","order_by":8,"name":"Xiaoping Ji","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2025-08-03 17:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7284693/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7284693/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88836982,"identity":"05367b22-a7f8-45f3-9908-8fda2b20e31f","added_by":"auto","created_at":"2025-08-12 01:20:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2344814,"visible":true,"origin":"","legend":"\u003cp\u003eAdipose promotes calcified medium-induced calcification. A. Alizarin Red S and Von Kossa staining of mouse aortic rings to evaluate matrix mineral deposition in aortic tissue (n = 5). Scale bar: 100 μm. B. 3T3-L1 preadipocytes were induced to differentiate into adipocytes, and the differentiation status was evaluated by Oil Red O staining. C-D. Representative Western blot bands and semiquantitative analysis of OPN, RUNX2, and BMP2 in co-cultured VSMCs and VSMCs induced to calcify with calcified medium (n = 5 per group). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/188b4349a9420e5339498327.jpg"},{"id":88836162,"identity":"af26a93a-f9e3-4306-89a4-eade3ca65af0","added_by":"auto","created_at":"2025-08-12 01:12:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2435810,"visible":true,"origin":"","legend":"\u003cp\u003eCalcium-phosphorus imbalance promotes the expression and secretion of visfatin in both adipocytes and adipose tissue. A. Serum visfatin levels in human blood samples were measured using ELISA. B. The level of serum visfatin in mice was measured using ELISA (n = 6). C. Western blot analysis of visfatin levels in the serum of control and vascular calcification mice (n = 5). D. RT-qPCR analysis of visfatin mRNA levels in SAT and VAT (n = 5). E. Western blot analysis was performed to measure visfatin protein levels in SAT and VAT (n = 5). F. RT-qPCR was used to detect the mRNA levels of visfatin in visceral adipose (n = 6). G. Western blot analysis was used to detect the protein levels of visfatin in epididymal adipose tissue (n = 5). H. Immunohistochemical staining of visfatin in the epididymal adipose tissue of control and vascular calcification mice (n = 6). Scale bar: 50 μm. I. Culture supernatant visfatin measured by ELISA (n = 6). J-K. Visfatin protein and mRNA levels in adipocytes induced from 3T3-L1 preadipocytes in the control group and calcification medium induced group (n = 6). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/8ea0f9b7479664e064e349c3.jpg"},{"id":88836195,"identity":"f4c08443-2ba8-4253-8e81-cf1db36c6d34","added_by":"auto","created_at":"2025-08-12 01:12:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5396891,"visible":true,"origin":"","legend":"\u003cp\u003eVisfatin promotes vascular calcification both in vivo and in vitro. A. The serum visfatin levels in mice with epididymal adipose tissue-specific overexpression of visfatin were measured using ELISA (n = 6). B. Representative gross alizarin red staining in mouse aorta (n = 6). C. Immunohistochemistry of RUNX2 in the aorta of calcified empty vector group and specific epididymal adipose tissue visfatin overexpression mice (n = 6). Scale bar: 50 μm. D. Assessment of matrix mineral deposition in aortic tissue through Von Kossa staining (n=6). Scale bar: 100 μm. E. The deposition of mineral matrix in aortic tissue was analyzed using Alizarin Red S and Von Kossa staining on mouse aortic rings. (n = 5 per group). Scale bar: 100 μm. F and H. Representative alizarin red staining in VSMCs (n = 4). G and I. Western blot bands and semi-quantitative analysis of representative markers OPN, RUNX2, BMP2, and calponin in VSMCs after treatment with calcified culture medium and visfatin (n = 6). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/17056f5d3140a5dbdeef6b48.jpg"},{"id":88836167,"identity":"629c1478-5724-40b3-8b21-87317819ef41","added_by":"auto","created_at":"2025-08-12 01:12:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3602489,"visible":true,"origin":"","legend":"\u003cp\u003eTLR4 Mediates vsfatin-induced vascular calcification. A. Representative gross alizarin red staining in mouse aorta (n = 6). B. Assessment of matrix mineral deposition in aortic tissue through Von Kossa staining (n = 6). Scale bar: 200 μm. C-D. Immunohistochemistry of RUNX2 in the aorta of the WT group and \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e group. (n = 5). Scale bar: 50 μm. E. Alizarin Red S and Von Kossa staining of mouse aortic rings to evaluate matrix mineral deposition in aortic tissue (n = 5). Scale bar: 100 μm. F. Western blot bands and semi-quantitative analysis of representative markers OPN, RUNX2, and calponin in VSMCs after treatment with calcified culture medium and visfatin (n=5). *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001.\u003c/p\u003e","description":"","filename":"figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/db24e0bd188fb32d12bcef4d.jpg"},{"id":88836159,"identity":"8a596128-41e7-4d08-8f65-f8830b09f600","added_by":"auto","created_at":"2025-08-12 01:12:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2942128,"visible":true,"origin":"","legend":"\u003cp\u003eVisfatin Accelerates Vascular Calcification via TLR4 and Inflammatory Pathways. A. Western blot bands and semi-quantitative analysis of inflammatory markers (NLRP3, TNF-α, IL-1β), osteogenic marker (RUNX2), and contractile marker (calponin) in VSMCs treated with calcification medium and LPS (n = 5). B. Western blot bands and semi-quantitative 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 semi-quantitative analysis of inflammatory markers (NLRP3, TNF-α, IL-1β) in VSMCs from \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice treated with calcification medium and visfatin (n = 5). D. VSMC lysates were subjected to immunoprecipitation with either IgG or TLR4 antibody, and the outcomes were analyzed using Western blot with anti-visfatin antibody. *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001.\u003c/p\u003e","description":"","filename":"figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/025afe2500d003b3aa519805.jpg"},{"id":88836166,"identity":"37d4add4-0d2c-4f67-a42a-ffbf0c1dd592","added_by":"auto","created_at":"2025-08-12 01:12:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2309286,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin suppresses the production and release of visfatin in adipose tissue and adipocytes. A. Serum visfatin 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 detect the mRNA levels of visfatin in visceral adipose (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. Culture supernatant visfatin measured by ELISA (n = 6). F-G. The protein and mRNA levels of visfatin in 3T3-L1 induced adipocytes (n = 6). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/bed84d81b336c298d4816103.jpg"},{"id":88836163,"identity":"0ec6a1b1-5b0a-4d11-9467-2f28c0c211ac","added_by":"auto","created_at":"2025-08-12 01:12:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5019109,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin can improve vascular calcification. A. Representative gross alizarin red staining of the aorta in mice from the vascular calcification group and the vascular calcification with empagliflozin gavage group (n = 6). B. Assessment of matrix mineral deposition in aortic tissue through Von Kossa staining (n = 6). Scale bar: 200 μm. C-D. Immunohistochemical analysis to assess RUNX2 levels in mouse aortic tissue (n = 6). Scale bar: 50 μm. E. Western blot analysis was performed to assess the protein levels of RUNX2, OPN, and BMP2 in VSMCs (n = 6). F.\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was performed to assess the protein levels of RUNX2, OPN, and BMP2 in VSMCs within the 3T3-L1 adipocyte and VSMC co-culture system (n = 5). G. Alizarin Red S and Von Kossa staining of mouse aortic rings to evaluate matrix mineral deposition in aortic tissue (n = 5). Scale bar: 100 μm. H. Protein and mRNA expression levels of SGLT2 in adipocytes and VSMCs (n = 5). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/beb95501a1b4f477a34b1f3b.jpg"},{"id":88836165,"identity":"98fa34d1-77aa-457c-be3e-bc75b39f5287","added_by":"auto","created_at":"2025-08-12 01:12:41","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2246225,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of the p38 MAPK pathway on empagliflozin-mediated regulation of visfatin expression and secretion in adipocytes. A. Western blot analysis was used to evaluate the effect of calcification medium on the activation of JNK, ERK, and p38 MAPK in adipocytes. β-actin was used as a loading control (n=5). B. Adipocytes were treated with SP600125 (JNK inhibitor), PD098059 (ERK1/2 inhibitor), or SB203580 (p38 inhibitor) during exposure to calcification medium. Western blot analysis was used to measure the protein levels of visfatin (n=5). C. RT-qPCR was used to detect the mRNA levels of visfatin (n = 6). D. During exposure to calcification medium, adipocytes were treated with SB203580 (p38 inhibitor), and Western blot was used to measure the protein levels of visfatin in adipocytes (n=6). E. During the treatment with calcification medium, the effect of empagliflozin on the phosphorylation of p38 in adipocytes was assessed by Western blot analysis. β-actin was used as a loading control (n=5). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/83d59c14ee39b960d80b23bd.jpg"},{"id":88836168,"identity":"1cdd3e8d-822e-49b0-a5c4-df63b820d698","added_by":"auto","created_at":"2025-08-12 01:12:42","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4869924,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin inhibits the endogenous binding of NF-κB to the visfatin promoter. A. Confocal microscopy of immunofluorescence staining for NF-κB in VSMCs, both in the presence and absence of calcification medium. Scale bar: 20 μm. B. During exposure to calcification medium, adipocytes were treated with BAY 11-7085 (NF-κB inhibitor), and Western blot was used to measure the protein levels of visfatin in adipocytes (n=6). RT-qPCR was used to detect the mRNA levels of visfatin (n = 6). C. Confocal microscopy of immunofluorescence staining for NF-κB in VSMCs following treatment of adipocytes with empagliflozin and SB203580 (p38 inhibitor). Scale bar: 20 μm. D. NF-κB was overexpressed in HEK293T cells, followed by co-transfection with the visfatin promoter carrying luciferase activity and Renilla luciferase. Subsequently, luciferase and Renilla luciferase activity assays were performed (n = 3). E. The sequence labeled as 1 corresponds to the first exon of the mRNA, indicated in bold and enclosed in a box. Additionally, the sequences within the other two rectangles represent the putative NF-κB binding sites. F. RT-qPCR was used to quantitatively analyze the binding of NF-κB to the visfatin promoter region (n = 3). G. Following the ChIP assay, agarose gel electrophoresis detected that NF-κB can bind to the visfatin promoter region (n = 3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/ff49dbc83e3e98ef927082c5.jpg"},{"id":89619030,"identity":"2b014ce3-3a4d-4bca-a82c-5c987ca2e0a0","added_by":"auto","created_at":"2025-08-22 03:47:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32255789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/ed5fdf71-f157-4b40-a05a-3428a25b2da0.pdf"},{"id":88836161,"identity":"951fe0d0-7bb5-4e78-b72f-16e6690feaa5","added_by":"auto","created_at":"2025-08-12 01:12:41","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1984937,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7284693/v1/10fc9b1b13d6918382368c7e.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Visfatin from Adipocyte Accelerates Vascular Calcification via TLR4","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVascular calcification is a key pathological process in arteriosclerosis, hypertension, and chronic kidney disease, with its incidence increasing significantly with age and being more prevalent in patients with diabetes and chronic kidney disease\u003csup\u003e1-5\u003c/sup\u003e. It is classified into intimal and medial calcification, with medial calcification commonly seen in hypertension and chronic kidney disease, driven by osteogenic differentiation of VSMCs due to calcium phosphate deposition\u003csup\u003e6 7\u003c/sup\u003e. Research indicates that inflammation plays a crucial role in vascular calcification, as cytokines such as tumour necrosis factor alpha (TNF-\u0026alpha;) and interleukin-6 (IL-6), along with inflammasomes, promote calcium deposition and VSMCs phenotypic transformation\u003csup\u003e8-10\u003c/sup\u003e. The inhibition of inflammation and VSMCs osteogenic transformation has become a strategy for preventing vascular calcification.\u003c/p\u003e\n\u003cp\u003eTLR4 is an important member of the TLR family and plays a central role in innate immunity\u003csup\u003e11\u003c/sup\u003e. As a transmembrane receptor, it binds to both extracellular and intracellular ligands, initiating complex signaling pathways\u003csup\u003e12\u003c/sup\u003e. TLR4 can be activated by lipopolysaccharides (LPS), well-known pathogen-associated molecular patterns (PAMPs), as well as endogenous molecules such as peptides and phospholipids\u003csup\u003e13-15\u003c/sup\u003e. Recent studies have highlighted the role of TLR4 in vascular calcification, where it mediates inflammatory signaling, induces VSMC osteogenic differentiation, and promotes mineral deposition\u003csup\u003e16\u003c/sup\u003e. TLR4 has emerged as a potential target for therapeutic strategies aimed at preventing or mitigating vascular calcification. However, its regulation of cell expression is still poorly understood.\u003c/p\u003e\n\u003cp\u003eVisfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), is an adipokine highly expressed in visceral adipose tissue\u003csup\u003e17\u003c/sup\u003e. It exists both intracellularly and extracellularly, playing distinct roles in various physiological and pathological processes\u003csup\u003e18\u003c/sup\u003e. Intracellular NAMPT (iNAMPT) regulates nicotinamide adenine dinucleotide (NAD\u003csup\u003e+\u003c/sup\u003e) biosynthesis, influencing immune function, aging, and cancer through NAD-dependent proteins such as sirtuins and poly (ADP-ribose) polymerases (PARPs)\u003csup\u003e19\u003c/sup\u003e. Extracellular NAMPT (eNAMPT) participates in hormone-like signaling and activates NF-\u0026kappa;B and\u0026nbsp;mitogen-activated protein kinase (MAPK) pathways, which regulate inflammation, cell growth, and metabolism\u003csup\u003e19-21\u003c/sup\u003e. Studies show that visfatin levels are elevated in diseases like rheumatoid arthritis, cancer, and diabetes, and are linked to vascular remodeling, inflammation, and atherosclerosis, increasing cardiovascular risk\u003csup\u003e22-24\u003c/sup\u003e. However, research on the role of visfatin in vascular calcification is rarely seen.\u003c/p\u003e\n\u003cp\u003eVisfatin is a pro-inflammatory adipokine. Studies suggest that exogenous visfatin promotes inflammatory injury in pulmonary endothelial cells via TLR4/NF-\u0026kappa;B, exacerbating endothelial dysfunction\u003csup\u003e25\u003c/sup\u003e. We speculate that visfatin may enhance inflammation through TLR4, induce phenotypic changes in vascular smooth muscle cells, promote calcium deposition, and drive vascular calcification. Thus, the visfatin-TLR4 axis may be a key regulator of vascular calcification, offering new insights into its pathophysiology and potential treatments.\u003c/p\u003e\n\u003cp\u003eeNAMPT is secreted via a non-classical pathway rather than the traditional Golgi-endoplasmic reticulum system\u003csup\u003e26\u003c/sup\u003e. The mechanisms regulating visfatin expression and secretion remain unclear but may involve acetylation status, metabolic state, inflammation, and hormone levels\u003csup\u003e27\u003c/sup\u003e. Clinical studies show that sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce serum visfatin levels in type 2 diabetes patients, suggesting their role in metabolic regulation\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEmpagliflozin, a widely studied SGLT2i, lowers blood glucose by inhibiting SGLT2 in renal proximal tubules, reducing glucose reabsorption and promoting urinary glucose excretion. Beyond glycemic control, it also improves cardiovascular outcomes\u003csup\u003e29\u003c/sup\u003e. The EMPA-REG OUTCOME trial demonstrated that empagliflozin reduces major cardiovascular events, heart failure hospitalizations, and cardiovascular-related mortality in type 2 diabetes patients with cardiovascular disease\u003csup\u003e30-32\u003c/sup\u003e. However, the role of Empagliflozin in vascular calcification remains unknown.\u003c/p\u003e\n\u003cp\u003eThis study aims to investigate the role and mechanisms of visfatin in vascular smooth muscle cell osteogenic differentiation and vascular calcification, as well as evaluate its potential for pharmacological intervention.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1 Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFoetal bovine serum (FBS; 10099141), newborn calf serum (NCS; 16010159) and Dulbecco\u0026apos;s Modified Eagle\u0026apos;s medium (DMEM; 11965092) were obtained from Gibco (Grand Island, NY, USA). \u0026beta;‐Glycerophosphate (\u0026beta;‐GP; G5422), calcium dichloride (C5670), dexamethasone(D1756) and IBMX(I7018) were purchased from Sigma‐Aldrich (Louis, Missouri, USA).\u0026nbsp;SP600125 (S1460), PD98059 (S1177), and SB203580 (S1076) were obtained from Selleck Chemicals (Houston, TX, United States).\u0026nbsp;Empagliflozin (HY-15409), BAY 11-7085 (HY-10257), insulin (HY-P0035), rosiglitazone (HY-17386), and visfatin (HY-P701314) were purchased from MedChemExpress (New Jersey, USA).\u003c/p\u003e\n\u003cp\u003eAntibodies against OPN (22952-1-AP), NF-\u0026kappa;B (10745-1-AP), Transferrin (66171-1-Ig), GAPDH (10494-1-AP), visfatin (11776-1-AP), SGLT2 (24654-1-AP), NLRP3 (30109-1-AP), TNF-\u0026alpha;\u0026nbsp;(26162-1-AP), IL-1\u0026beta;\u0026nbsp;(16806-1-AP), and TLR4 (19811-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies against JNK (#9252), p-p44/42 MAPK (Erk1/2) (#4370), p44/42 MAPK (Erk1/2) (#4695), p-p38 MAPK (#4511), p38 MAPK (#8690), \u0026beta;-actin (#4970), and Calponin (#17819) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).\u0026nbsp;Antibodies against RUNX2 (ab236639), BMP2 (ab214821), and p-JNK (ab76572) were purchased from Abcam (Cambridge, UK).\u003c/p\u003e\n\u003cp\u003eAn adeno-associated virus delivering adipose-specific FABP4 promoter-driven control shRNA (pAAV-FABP4p-scramble, AAV-\u003cem\u003eNC\u003c/em\u003e) and shRNA against murine \u003cem\u003eVisfatin\u003c/em\u003e (pAAV-FABP4p-\u003cem\u003eVisfatin\u003c/em\u003e, AAV-\u003cem\u003eVisfatin\u003c/em\u003e) were obtained from Shanghai Genechem Co. Ltd.\u003c/p\u003e\n\u003cp\u003eThe oligonucleotide primer sequences were as follows: for \u003cem\u003eVisfatin\u003c/em\u003e, 5\u0026prime;-TTCAAGGAGATGGCGTGGAT-3\u0026prime; and 5\u0026prime;-CCACCAGAACCGAAGGAGAC-3\u0026prime;; for SGLT2, 5\u0026prime;- GTTCCGACCATAAACGATGCC-3\u0026prime; and 5\u0026prime;-TGGTGGTTGCCCTTCCGTCAAT-3\u0026prime;; fragment1 of \u003cem\u003eVisfatin\u003c/em\u003e promoter, 5\u0026prime;- AGGATCGGAATCCACAAGACG-3\u0026prime; and 5\u0026prime;-TTACCTTTGTCTCCCGCTTGG-3\u0026prime;; fragment2 of \u003cem\u003eVisfatin\u003c/em\u003e promoter, 5\u0026prime;-CATATAGCCCTTTGCGGGAGA-3\u0026prime; and 5\u0026prime;-TACTGGCTTAATCCCACAGCA-3\u0026prime;; fragment3 of \u003cem\u003eVisfatin\u003c/em\u003e promoter, 5\u0026prime;-ATGAGCCAAATAGATGTTTCCTCT-3\u0026prime; and 5\u0026prime;-TTGCCCCATCTGACTTGCTATT-3\u0026prime;; fragment4 of \u003cem\u003eVisfatin\u003c/em\u003e promoter, 5\u0026prime;-CCGGGGTACTGCTTAGTTCAT-3\u0026prime; and 5\u0026prime;-ACACAGAAGTGGATGCTCACA-3\u0026prime;; Gapdh: 5\u0026prime;-TGTCTCCTGCGACTTCAACA-3\u0026prime; and 5\u0026prime;-GGTGGTCCAGGGTTTCTTACT-3\u0026prime;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Human samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVenous blood samples were collected from 78 patients who had undergone Coronary Computed Tomography Angiography and had a confirmed coronary artery calcification score. The coronary artery calcification score was calculated using the Agatston method\u003csup\u003e33\u003c/sup\u003e. Patients were divided into two groups based on a score threshold of 100. All participants had normal liver and kidney function. Serum was obtained by centrifugation at 3000 rpm for 15 minutes and subsequently used for ELISA analysis. The use of blood samples was approved by the Medical Institutional Ethics Committee of Qilu Hospital, Shandong University, China (Approval No. KYLL-2020(KS)-537), and all donors provided informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old male C57BL/6J mice were obtained from ViewSolid Biotech (Beijing, China).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eknockout mice (\u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e; JAX:003752, Jackson Laboratory) were purchased from the Jackson laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEight-week-old male mice were subcutaneously injected with VitD at a dose of 500,000 IU/kg/day for 4 days, while mice in the control group were injected with the same volume of 5% ethanol (the solvent for VitD).\u0026nbsp;Mice were fed a normal diet for 10 days before sacrificed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEight-week-old male mice were administered empagliflozin via gavage at a dose of 10 mg/kg/day for 10 days, while mice in the control group received the same volume of normal saline. Mice were sacrificed 10 days after the administration.\u003c/p\u003e\n\u003cp\u003eEight-week-old male C57BL/6J mice were anesthetized via an intraperitoneal injection of tribromoethanol (10 mL/kg body weight, M2910, Nanjing Aibei Biotechnology, Nanjing, China). Epididymal fat tissues were exposed, and multiple-site injections of either AAV-\u003cem\u003eCTR\u003c/em\u003e or AAV-\u003cem\u003eVisfatin\u003c/em\u003e were performed bilaterally. A total of 10 \u0026mu;l of the viral solution (5 \u0026mu;l per side) was injected, with a viral titer of 5\u0026times;10\u0026sup1;\u0026sup2; virus genomes/ml. After a 4-week, mice were subcutaneously injected with vitamin D or 5% ethanol to induce vascular calcification.\u003c/p\u003e\n\u003cp\u003eAll animals were maintained under a 12-hour light/dark cycle at a temperature of 25 \u0026deg;C. All procedures adhered to protocols approved by the Animal Care and Use Committee of Shandong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Von Kossa staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter routine deparaffinization and rehydration, 1% silver nitrate (G5491, Solarbio, Beijing, China) was added to the aortic sections, completely covering tissues. The sections were then exposed to ultraviolet light for 20 minutes, rinsed with distilled water, and incubated in 5% sodium thiosulfate for 2 minutes at room temperature. The sections were dehydrated and mounted.\u0026nbsp;Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 mice aortic ring culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice thoracic aortas were immediately isolated and transferred to a sterile Petri dish containing PBS ice after anesthetized. After removing the outer membrane, the aortas were cut into rings with width of 3-5mm, which were then placed in a culture medium containing 15% FBS, 100 \u0026mu;g/mL penicillin-streptomycin. The aortic rings were incubated at 37\u0026deg;C in 5% CO2. The culture medium was replaced every other day. To induce calcification, aortic rings were cultured in DMEM with or without calcified medium (3mM calcium chloride and 10mM \u0026beta;-glycerophosphate) for 6 days, with the medium changed every other day. These aortic rings were co-cultured with approximately 5 cubic millimeters of epididymal adipose tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old C57BL/6J and \u003cem\u003eTLR4⁻/⁻\u003c/em\u003e mice were euthanized via CO₂ asphyxiation. The skin and muscles were dissected, and the aortas were quickly removed and placed in a sterile Petri dish containing phosphate-buffered saline (PBS). After removing the outer membrane, the aortas were cut into small pieces and evenly distributed at the bottom of a culture flask. The tissues were incubated at 37\u0026deg;C for 2 hours, followed by the addition of DMEM containing 15% FBS and 100 \u0026mu;g/mL penicillin-streptomycin, and maintained at 37\u0026deg;C in 5% CO2 for a minimum of 5 days. VSMCs were maintained in DMEM supplemented with 10% FBS, 100 \u0026mu;g/mL penicillin- streptomycin. To induce calcification, cells were cultured in DMEM containing calcified medium for 6 days. The culture medium was replaced every other day.\u003c/p\u003e\n\u003cp\u003e3T3-L1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. 3T3-L1 preadipocytes were cultured in complete culture medium until 70-80% confluent. Differentiation of 3T3-L1 was induced by addition of 0.25 \u0026mu;M dexamethasone, 0.5 mM IBMX, 1 \u0026mu;g/ml insulin and 2 \u0026mu;M rosiglitazone for 4 days, at which time the medium was replaced with growth medium containing 2 \u0026mu;M insulin for another 4 days. Under high magnification microscopy, bright circular lipid droplets were observed inside the cells, indicating adipocyte differentiation and maturation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Oil red O staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDilute the saturated Oil Red O solution (G1015, Servicebio, Wuhan, China) with double-distilled water in a 3:2 ratio and heat at 65\u0026deg;C for 1 hour. Allow the solution to cool to room temperature and then filter it. For induced mature adipocytes, gently rinse with PBS and fix with 4% paraformaldehyde at room temperature for 30 minutes. After removing the fixative, rinse cells again with PBS. Subsequently, stain with the Oil Red O working solution at room temperature for 10 minutes avoiding light. Finally, rinse off unbound dye with PBS. The sections were dehydrated and mounted.\u0026nbsp;Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Co-Culture of 3T3-L1 and VSMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3T3-L1 cells were seeded in the lower chamber of a trans-well 12-well plate and incubated at 37 \u0026deg;C in 5% CO₂ for 24 hours, followed by induction into adipocytes. VSMCs were seeded in the upper chamber of the trans-well 12-well plate and incubated at 37℃ in a 5% CO₂ for 24 hours. The adherent VSMCs in the upper chamber were then transferred to 12-well plate with or without mature adipocytes in the lower chamber to establish a VSMC-adipocyte co-culture system. After the co-culture system was established, the cells were serum-starved in low-serum medium (2% FBS-H-DMEM) for 1 day. Then, the medium was replaced with calcification medium to induce calcification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Alizarin Red S staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVSMCs were washed with PBS and fixed with paraformaldehyde for 30 minutes. After fixation, the cells were washed with PBS and stained with 1% alizarin red S staining solution (G1452, Solarbio, Beijing, China) in the dark for 10 minutes. Following washing, images were captured using a digital microscope.\u003c/p\u003e\n\u003cp\u003eTissues and vascular rings were fixed in 10% paraformaldehyde, embedded in paraffin, and sectioned into 5 \u0026micro;m thick slices. After routine deparaffinization and rehydration, the sections were stained with 1% alizarin red S solution in the dark for 5 minutes, followed by three washes with PBS. The sections were then treated with xylene and mounted using neutral resin. The sections were dehydrated and mounted.\u0026nbsp;Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Aortic gross staining with alizarin red\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice aortas were fixed in 10% formalin overnight, followed by dehydration in 95% ethanol for 24 hours. The aortas were then stained with 0.003% alizarin red solution in 1% potassium hydroxide for 24 hours and rinsed with 2% potassium hydroxide to eliminate any excess stain. Images were captured for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Western blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from cells or tissues using RIPA lysis buffer (R0010, Solarbio, Beijing, China), separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (ISEQ00010, Millipore, Boston, Massachusetts, USA). The membrane was blocked with 5% skim milk in Tween 20-Tris-buffered saline (TBST) for 1 hour, followed by incubation with the appropriate primary antibody at 4 \u0026deg;C overnight. The membrane was washed 3 times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. After 3 additional washes with TBST, the protein signals were detected using enhanced chemiluminescence (WBKLS0500, Millipore, Boston, Massachusetts, USA). The results were analyzed with ImageJ software. All experiments were performed at least 3 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Quantitative real-time PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from cell and mouse tissues using Tri reagent (Ambion, Austin, TX, USA) following the manufacturer\u0026apos;s protocol. 1 \u0026mu;g of RNA was then reverse-transcribed into complementary DNA with the iScriptcDNA synthesis kit (Bio-Rad, Hercules, CA, USA). PCR amplification was performed using the SYBR PCR mix (Bio-Rad, Hercules, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 Immunoprecipitation (IP) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVSMCs were disrupted using a specialized immunoprecipitation lysis solution composed of 150 mM saline, 50 mM Tris\u0026ndash;HCl and 1% NP-40 (88805, Thermo Fisher Scientific, Waltham, MA, USA) at pH 7.8, supplemented with protease inhibitor cocktail. Following centrifugation and supernatant collection, the lysates were mixed with primary antibodies pre-conjugated to 30 \u0026micro;l of pre-washed protein A/G magnetic beads (HY-K0202, MedChemExpress, New Jersey, USA) and incubated at 4 \u0026deg;C overnight. The resulting immunoprecipitated complexes were then rinsed and heated in SDS loading buffer at 95\u0026deg;C using a metal bath, and subsequently analyzed via western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 Serum and Culture medium supernatant Visfatin analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum and Culture medium supernatant visfatin levels were measured by an ELISA assay. The eNAMPT ELISA kit (DY4335-05 and NBP3-43467) was purchased from R\u0026amp;D Systems (Minneapolis, MN, USA). Mix serum or culture supernatant with acetone at a 1:4 ratio, incubate overnight at -80\u0026deg;C, then centrifuge and dry the precipitate. Dissolve the precipitate in RIPA lysis buffer and adjust the concentration for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15\u0026nbsp;Immunohistochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter deparaffinization, rehydration, antigen retrieval, and blocking of non-specific binding, the sections were incubated with the appropriate primary antibody at 4\u0026deg;C overnight. The sections were washed 3 times with PBS and incubated with the secondary antibody at 37\u0026deg;C for 30 minutes. The bound secondary antibody was detected using DAB solution (ZLI-9018, Zhong Shan Golden Bridge Biological Technology, Beijing, China).\u0026nbsp;Haematoxylin was applied to counterstain the nucleus. Images were captured using the Olympus DP72 digital imaging system (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.16 Immunofluorescence (IF) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter deparaffinization, rehydration, antigen retrieval, sections were permeabilized with 0.3% Triton X-100 (T8200, Solarbio, Beijing, China) for 15 minutes. After washing with PBS, the slides were blocked. The sections were incubated with the appropriate primary antibody at 4\u0026deg;C overnight. After washing with PBS, the sections were incubated with the secondary antibody at 37 \u0026deg;C for 1 hour. The slides were covered by a drop of Fluoroshield Mounting Medium containing 40,6‐diamidino‐2‐phenylindole (DAPI; ab104139, Abcam, Cambridge, UK) before being observed with laser scanning confocal microscopy (LSM710, Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003eCell slides with mature adipocytes were fixed with immunostaining fixative (P0098, Beyotime Biotechnology, Shanghai, China) at room temperature for 1 hour. The slides were blocked, probed with antibodies, stained with DAPI,\u0026nbsp;and observed under laser confocal microscopy\u0026nbsp;(LSM710, Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.17 Dual-luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe promoter region of mice \u003cem\u003eVisfatin\u003c/em\u003e was amplified using PCR and subsequently inserted into the pGL3-Basic vector (E1751, Promega, Madison, WI, USA) to generate the \u003cem\u003eVisfatin\u003c/em\u003e promoter-driven luciferase reporter construct (designated as Mus-Nampt). The Mus-Nampt plasmid was then co-transfected with an NF-\u0026kappa;B overexpression plasmid into HEK293 cells. Following a 48-hour transfection period, the activities of Firefly and Renilla luciferase were measured utilizing the Dual-Luciferase Reporter Assay System (E1751, Promega, Madison, Wisconsin, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.18 Chromatin immunoprecipitation (ChIP) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin immunoprecipitation (ChIP) was performed following the manufacturer\u0026rsquo;s protocol using the Enzymatic Chromatin IP Kit (9003; Cell Signaling Technology, Danvers, Massachusetts, USA). Briefly, 3T3-L1-induced adipocytes were cross-linked with 1% formaldehyde for 10 minutes, followed by sonication to shear the DNA into fragments. The lysates were then incubated with either anti-IgG or anti-NF-\u0026kappa;B antibody in the presence of magnetic beads. After collection and purification, the immunoprecipitated DNA samples were analyzed by RT-qPCR. After performing the ChIP experiment, the qPCR products were processed on a 2% agarose gel and visualized using a UV transilluminator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.19 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analysis were carried out using GraphPad Prism 6.0, with results expressed as mean \u0026plusmn; SEM. Differences between two groups were assessed using the Student\u0026apos;s t-test, while comparisons across multiple groups were analyzed through one-way ANOVA. A p-value of less than 0.05 was deemed to indicate statistical significance.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Adipose promotes calcified medium-induced calcification of VSMCs and aortic vascular rings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the effect of adipose tissue on vascular calcification. We harvested mouse aortic rings and induced calcification using a calcification medium, co-culturing them with approximately 5 cubic millimeters of epididymal adipose tissue during this process. Compared to aortic rings cultured alone and induced to calcify, the co-culture group exhibited a significantly increased deposition of mineralized matrix, as assessed by Alizarin Red and Von-Kossa staining (Fig.1A). Subsequently, 3T3-L1 preadipocytes were induced to differentiate into adipocytes over an 8-day period for in vitro experiments. Oil Red O staining was used to assess the state of the differentiated cells (Fig.1B).\u0026nbsp;To further validate the effect of adipocytes on VSMC calcification, we co-cultured 3T3-L1-induced differentiated adipocytes with primary mouse VSMCs and incubated them in calcification medium for 6 days. Compared to VSMCs cultured alone and induced to calcify, the expression levels of calcification markers RUNX2, OPN, and BMP2 in VSMCs from the co-culture group were significantly upregulated (Fig.1C and D). The results indicate that adipose tissue or adipocytes significantly promote the calcification process of vascular smooth muscle cells through co-culture, further supporting the regulatory role of adipocytes in vascular calcification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 The disorder of calcium and phosphorus promotes the expressed and secretion of visfatin in vivo and in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter confirming the pro-calcification effect of adipocytes on blood vessels, we first investigated whether calcium-phosphorus imbalance affects the expression and secretion of visfatin in both adipose tissue and adipocytes to further explore its role in vascular calcification. Patients with moderate to severe coronary artery calcification (CAC score \u0026gt;100) exhibit significantly higher serum visfatin levels compared to those with mild or no coronary calcification (CAC score \u0026le;100) (Fig.2A). Similarly, after vitamin D treatment, serum visfatin protein levels were significantly elevated in calcified mice compared to control mice (Fig.2B and C). In mice with vascular calcification, both the protein and mRNA levels of visfatin are significantly higher in visceral adipose tissue (VAT) than in subcutaneous adipose tissue (SAT) (Fig.2D and E), suggesting that visfatin is primarily produced and secreted by VAT. Therefore, we focused our investigation on visceral adipose tissue. Western blot and qPCR demonstrated that visfatin protein and mRNA levels were elevated in the epididymal adipose tissue of calcified mice (Fig.2F and G). Additionally, immunohistochemical analysis indicated that visfatin expression was markedly upregulated in the epididymal adipose tissue of calcified mice compared to the control group (Fig.2H). \u0026nbsp;We simulated calcium-phosphorus imbalance in vivo using calcification medium prepared with \u0026beta;-GP and CaCl\u003csub\u003e2\u003c/sub\u003e. After stimulating 3T3-L1 differentiated adipocytes with the calcification medium for 3 days, ELISA results showed that the level of visfatin in the supernatant of the adipocyte culture medium was increased (Fig.2I). Further analysis by Western blot and RT-qPCR revealed that both the protein and mRNA levels of visfatin in the adipocytes treated with the calcification medium were significantly elevated compared to the control group (Fig.2J and K). Thus, both in vivo and in vitro disturbances in calcium and phosphorus balance can promote the expression and secretion of visfatin in adipocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Visfatin promotes VSMC phenotype switching in vivo and in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVascular calcification has been shown to result from multifactorial vascular changes, not merely calcium deposition, with osteogenic transformation of VSMCs being one of the key mechanisms in vascular calcification. To further investigate the role of visfatin in vascular calcification, we examined whether visfatin could induce phenotypic transition in vascular smooth muscle cells and promote vascular calcification.\u0026nbsp;We constructed a mouse model with epididymal adipose tissue-specific overexpression of visfatin by locally injecting AAV carrying the FABP4 promoter into the bilateral epididymal adipose tissue of mice.\u0026nbsp;After inducing calcification in mice with vitamin D, ELISA analysis demonstrated that the serum visfatin levels in mice injected with AAV-\u003cem\u003eVisfatin\u003c/em\u003e were significantly higher compared to those injected with AAV-\u003cem\u003eNC\u003c/em\u003e (Fig.3A). The mouse aorta was then collected for whole-mount Alizarin Red staining, which revealed that visfatin overexpression in epididymal adipose tissue significantly promoted aortic calcium deposition (Fig.3B). Immunohistochemistry showed that RUNX2 expression was significantly increased in the aortas of mice with overexpression of visfatin in epididymal adipose tissue (Fig.3C). Likewise, Von Kossa staining revealed an increase in calcium deposition in the aortas of mice with visfatin overexpression (Fig.3D). To investigate the role of visfatin in vascular calcification, mouse aortic rings were isolated and induced with calcification medium. The aortic rings were divided into two groups: one group was treated with visfatin (100 ng/mL), while the other group received an equal volume of PBS as a control. Alizarin Red staining and Von-Kossa staining results demonstrated that visfatin significantly enhanced matrix mineralization deposition in the mouse aortic rings (Fig.3E). Subsequently, VSMC treated with calcification medium were exposed to visfatin (100 ng/mL) for 6 days. The results showed that visfatin significantly enhanced calcium deposition in VSMCs (Fig.3F and H). Consistently, visfatin upregulated the expression of calcification markers, including Runx2, OPN, and BMP2, while downregulating the expression of the contractile marker calponin (Fig.3G and I).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTLR4 Knockout Attenuates Visfatin-Induced Vascular Calcification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have suggested that the effects of visfatin may be associated with TLR4. To gain a deeper understanding of the mechanism by which visfatin promotes vascular calcification, we investigated the involvement of the TLR4 signaling pathway in this process. After vitamin D-induced calcification, whole-mount Alizarin Red staining of the aortic tissue showed that matrix mineral deposition was significantly reduced in \u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice compared to wild-type mice (Fig.4A).\u0026nbsp;Sectional Von Kossa staining further confirmed that TLR4 knockout significantly reduced calcium deposition in the aortic tissue (Fig.4B). Immunohistochemistry showed that RUNX2 expression was significantly increased in the aortas of \u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Fig.4C and D).\u003c/p\u003e\n\u003cp\u003eTo specifically assess the role of TLR4 in visfatin-mediated vascular calcification, aortic rings from \u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice were treated with both visfatin and calcification medium. Alizarin Red and Von Kossa staining revealed that TLR4 knockout significantly reduced calcium deposition in the aortic rings compared to the control group (Fig.4E). Primary VSMCs from control and \u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice were cultured in calcification medium with visfatin for 6 days. The results showed that compared to the control group, VSMCs from \u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice exhibited higher expression of contractile phenotype markers and lower expression of osteogenic phenotype markers (Fig.4F). Together, these results demonstrate that TLR4 deletion not only mitigates baseline vascular calcification but also significantly inhibits the pro-calcific actions of visfatin. These findings underscore the pivotal role of TLR4 in mediating visfatin-driven osteogenic transition and mineral deposition in vascular tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 TLR4 Knockout Attenuates Visfatin-Induced Upregulation of Inflammatory Mediator-Related Proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInflammatory responses play a critical role in the initiation and progression of vascular calcification. As a classical innate immune receptor, TLR4 is capable of sensing both exogenous and endogenous pro-inflammatory stimuli and activating downstream signaling pathways, thereby promoting the expression of various inflammatory mediators and accelerating the calcification process. In this study, we found that TLR4 plays an essential regulatory role in visfatin-induced vascular calcification. To further elucidate the underlying mechanism, we specifically investigated the involvement of inflammation in this process. To begin with, VSMCs cultured in calcification medium were treated with LPS to activate TLR4. Compared with cells cultured in calcification medium alone, LPS stimulation significantly increased the expression of inflammatory markers such as NLRP3, TNF-\u0026alpha;, and IL-1\u0026beta;, along with an enhanced phenotypic switch of VSMCs from a contractile to an osteogenic state (Fig.5A). Similarly, treatment of VSMCs cultured in calcification medium with visfatin also led to a pronounced elevation in the levels of inflammatory mediators, including NLRP3, TNF-\u0026alpha;, and IL-1\u0026beta;, when compared to cells exposed to calcification medium alone (Fig.5B). These findings indicate that visfatin, like LPS, can amplify inflammatory signaling within VSMCs under pro-calcific conditions. To further validate the role of TLR4 in visfatin-induced inflammation, primary VSMCs were isolated from TLR4 knockout mice and cultured in calcification medium supplemented with visfatin. Compared with VSMCs derived from WT mice, \u003cem\u003eTLR4\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e VSMCs exhibited significantly lower expression levels of inflammatory markers (Fig.5C). Immunoprecipitation demonstrated that visfatin can bind to TLR4 (Fig.5D). Collectively, these findings indicate that visfatin promotes inflammatory responses in VSMCs at least in part through direct interaction with TLR4, thereby contributing to vascular calcification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Empagliflozin inhibits the expression and secretion of visfatin in both adipose tissue and adipocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCurrently, studies investigating the regulation of visfatin expression and secretion are limited. Previous reports have shown that SGLT2 inhibitors can reduce serum visfatin levels in patients with type 2 diabetes\u003csup\u003e28\u003c/sup\u003e. Therefore, we next explored the effects of the SGLT2 inhibitor empagliflozin on the regulation of visfatin expression and secretion in adipose tissue.\u003c/p\u003e\n\u003cp\u003eMice were administered empagliflozin by gavage at a dose of 3.8 mg/kg/d, while the control group received an equivalent volume of double-distilled water. After vitamin D-induced calcification, the serum visfatin levels were significantly reduced in the empagliflozin gavage group of mice (Fig.6A and B). The mRNA level of visfatin in the visceral adipose tissue of empagliflozin-treated mice with vascular calcification was decreased (Fig.6C). Consistently, Immunohistochemical analysis revealed that the expression of visfatin in the epididymal adipose tissue of mice with vascular calcification treated with empagliflozin was significantly reduced compared to the vascular calcification group without treatment (Fig.6D). After differentiating 3T3-L1 cells into adipocytes, they were treated with either calcification medium alone or calcification medium combined with empagliflozin. ELISA analysis showed that visfatin levels in the culture supernatant were significantly reduced in the empagliflozin-treated group compared to the group treated with calcification medium alone (Fig.6E), and both protein and mRNA expression levels of visfatin in adipocytes were also markedly decreased (Fig.6F and G). In summary, our findings demonstrate that empagliflozin significantly suppresses the expression and secretion of visfatin in adipose tissue, which may represent a potential mechanism underlying its ability to ameliorate vascular calcification. These results suggest that empagliflozin, by modulating visfatin-mediated signaling pathways, may offer a novel therapeutic strategy for the treatment of vascular calcification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Empagliflozin can improve vascular calcification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that empagliflozin suppresses the expression and secretion of visfatin in adipocytes, we next explored whether empagliflozin could attenuate vascular calcification through this mechanism. After inducing vascular calcification in mice with vitamin D, one group of mice was treated with empagliflozin gavage, while the other group received an equal volume of distilled water as a control. Whole-mount Alizarin Red staining of the aorta revealed that empagliflozin significantly improved vascular calcification and reduced calcium deposition (Fig.7A). Meanwhile, Von Kossa staining of aortic sections further confirmed this result (Fig.7B). Immunohistochemical staining of aortic sections showed a decreased expression of RUNX2 protein in the empagliflozin group (Fig.7C and D). In vitro experiments revealed that primary VSMCs were divided into two groups: one group was induced for calcification, and the other group was induced for calcification with the addition of empagliflozin (10 \u0026micro;M). Western blot analysis showed no significant differences in the expression levels of osteogenic and contractile markers between the two groups (Fig.7E). However, in a co-culture system of 3T3-L1 adipocytes and VSMCs under calcification induction with calcification medium, the addition of empagliflozin (10 \u0026micro;M) significantly reduced the expression of osteogenic markers OPN, RUNX2, and BMP2, while increasing the expression of the contractile marker calponin compared to the control group (Fig.7F). To further validate these findings, a co-culture system of mouse aortic rings and epididymal adipose tissue was established. In the presence of calcification medium, which promotes calcification, the group treated with empagliflozin exhibited a significant reduction in calcium deposition compared to the untreated control group (Fig.7G). Both the protein and mRNA expression levels of SGLT2 are higher in adipocytes than in VSMCs (Fig.7H). In summary, the results of this section indicate that the SGLT2 inhibitor empagliflozin reduces calcium deposition in the aortas of calcified mice both in vivo and in vitro. This phenomenon may be attributed to empagliflozin\u0026apos;s inhibition of the pro-calcification effect of adipocytes on VSMCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Empagliflozin reduces the expression and secretion of visfatin in adipocytes by inhibiting the p38 MAPK pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSome potential signaling pathways targeted by SGLT2 inhibitors have been reported, including ERK, JNK, and p38 MAPK\u003csup\u003e34\u003c/sup\u003e. The p38 signaling pathway plays a crucial role in regulating inflammatory and metabolic responses in adipocytes. To investigate whether empagliflozin modulates visfatin expression through this pathway, we examined the phosphorylation status of p38 in adipose tissue following empagliflozin treatment. When 3T3-L1 induced adipocytes were incubated with the calcification medium for 3 days, there was a significant increase in the phosphorylation of JNK, ERK, and p38 compared to the control group (Fig.8A). The application of specific inhibitors for JNK (SP600125), ERK (PD98059), and p38 (SB203580) revealed that only the p38 inhibitor significantly reduced the protein levels of visfatin in adipocytes (Fig.8B). Co-treatment of 3T3-L1 induced adipocytes with calcification medium and the p38 inhibitor resulted in a significant decrease in both the protein and mRNA levels of visfatin, compared to treatment with calcification medium alone (Fig.8C and D). Furthermore, empagliflozin markedly suppressed the phosphorylation of p38 in adipocytes stimulated by calcification medium (Fig.8E). In summary, empagliflozin inhibits the upregulation of visfatin expression in adipocytes induced by calcification medium by suppressing the p38 phosphorylation pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9 Empagliflozin inhibits the endogenous binding of NF-\u0026kappa;B to the visfatin promoter.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuilding on the finding that empagliflozin and p38 regulate visfatin expression, we next investigated whether NF-\u0026kappa;B nuclear translocation serves as a downstream mechanism controlling visfatin transcription. We found that calcification medium promotes the nuclear translocation of NF-\u0026kappa;B in 3T3-L1 induced adipocytes (Fig.9A). The use of a specific NF-\u0026kappa;B inhibitor (BAY 11-7085) significantly reduced both the protein and mRNA levels of visfatin in adipocytes (Fig.9B). Subsequently, we examined the effects of empagliflozin and the p38 inhibitor on NF-\u0026kappa;B in adipocytes. In 3T3-L1-induced adipocytes treated with calcification medium, both empagliflozin and the p38 inhibitor were able to suppress the nuclear translocation of NF-\u0026kappa;B (Fig.9C). Based on previous results, we investigated the effect of NF-\u0026kappa;B on the transcriptional regulation of visfatin. We synthesized luciferase reporter plasmids targeting the promoter region of visfatin and transfected HEK293T cells with these plasmids. Subsequent luciferase activity assays demonstrated that NF-\u0026kappa;B enhances the transcriptional activity of visfatin (Fig.9D). The results of binding site prediction for the visfatin promoter using JASPAR revealed that NF-\u0026kappa;B binds to the visfatin promoter at the sequences ggtatttccc (-1989 bp to -1980 bp) or ggtaatttct (-1696 bp to -1687 bp) (Fig.9E). The visfatin promoter was divided into four fragments of approximately 200 bp in length, including two random fragments without predicted binding sites (fragment1: -1526bp to -1433bp and fragment2: -438bp to -369bp) and two fragments containing predicted binding sites (fragment3: -2032bp to -1924bp and fragment4: -1761bp to -1622bp). Primers targeting these four regions were synthesized, and ChIP experiments were conducted for analysis. The results of the ChIP assay and agarose gel electrophoresis demonstrated that the region where NF-\u0026kappa;B directly binds to the visfatin promoter is fragment4 (Fig.9F and G). In summary, our findings demonstrate that NF-\u0026kappa;B directly binds to the visfatin promoter at specific sites, thereby enhancing its transcriptional activity. This regulatory mechanism is modulated by empagliflozin and p38 inhibition, which suppress NF-\u0026kappa;B nuclear translocation and subsequently reduce visfatin expression.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we experimentally discovered that serum levels of visfatin were significantly elevated in mice with vascular calcification, and the expression and secretion of visfatin in visceral fat tissue were also notably increased. This suggests that visfatin, as an adipokine secreted by visceral fat, may play an important role in the occurrence and progression of vascular calcification. Further investigation revealed that visfatin directly binds to the TLR4 receptor on vascular smooth muscle cells, promoting the increased expression of osteogenic markers and the decreased expression of contractile markers, thereby facilitating the process of vascular calcification. This finding provides new insights into the link between visceral fat and vascular diseases. Additionally, we found that empagliflozin, an SGLT2 inhibitor, can inhibit the activation of p38 in adipocytes, thereby preventing the nuclear translocation of NF-\u0026kappa;B, which ultimately suppresses the binding of NF-\u0026kappa;B to the visfatin promoter and reduces visfatin expression. This mechanism not only highlights the potential role of empagliflozin in ameliorating vascular calcification but also provides new theoretical insights into its pleiotropic effects in metabolic and cardiovascular diseases, while further elucidating the critical regulatory role of visfatin in vascular calcification.\u003c/p\u003e\n\u003cp\u003eVisceral fat is closely associated with vascular calcification. In addition to serving as an energy storage tissue, visceral fat secretes various adipokines and inflammatory factors that promote the occurrence of vascular calcification\u003csup\u003e35\u003c/sup\u003e. Excessive accumulation of visceral fat leads to chronic low-grade inflammation\u003csup\u003e36\u003c/sup\u003e, activating signaling pathways such as NF-\u0026kappa;B\u003csup\u003e37\u003c/sup\u003e, MAPK\u003csup\u003e38\u003c/sup\u003e, and TLR4\u003csup\u003e39\u003c/sup\u003e. These pathways not only promote immune responses but also affect the phenotypic transition of vascular smooth muscle cells. Upon stimulation by these signals, vascular smooth muscle cells shift from a contractile phenotype to an osteoblast-like phenotype, which promotes calcium salt deposition and leads to vascular wall calcification. Through this mechanism, visceral fat increases the risk of vascular calcification, especially in patients with metabolic diseases such as obesity and diabetes, where the occurrence of vascular calcification is closely linked to excessive visceral fat accumulation. Therefore, abnormalities in visceral fat metabolism not only affect overall metabolic function but may also exacerbate vascular calcification through inflammation and cellular transformation, further increasing the risk of cardiovascular diseases.\u003c/p\u003e\n\u003cp\u003eThe role of TLR4 in vascular calcification is primarily through its regulation of inflammatory responses and intracellular mineralization processes. VSMCs respond to inflammatory signals via TLR4, activating pathways like NF-\u0026kappa;B and MAPK\u003csup\u003e16 40-42\u003c/sup\u003e. These pathways enhance inflammation and promote cell phenotypic transition, driving vascular calcification. VSMCs shift from a contractile phenotype to an osteoblast-like phenotype, expressing osteogenic markers such as osteocalcin and alkaline phosphatase\u003csup\u003e43\u003c/sup\u003e. TLR4 activates these pathways, accelerating vascular calcification. Visfatin, though known to play a role in inflammation and insulin resistance\u003csup\u003e44 45\u003c/sup\u003e, has not been fully explored in vascular calcification. Existing studies show that visfatin activates the TLR4 pathway in various cell types\u003csup\u003e46-49\u003c/sup\u003e, but its role in VSMCs is not well understood. Our research reveals that visfatin binds to TLR4 on VSMCs, initiating signal transduction, and emphasizes the importance of TLR4 in regulating vascular calcification. TLR4 activation enhances inflammation in VSMCs and may interact with osteogenic signaling pathways to promote vascular calcification. Studying the TLR4-visfatin interaction provides insights into the molecular mechanisms of vascular calcification and potential therapeutic strategies.\u003c/p\u003e\n\u003cp\u003eIn this study, we identified empagliflozin as a regulator of visfatin expression in adipocytes. Our results demonstrate that empagliflozin inhibits the activation of p38, preventing the nuclear translocation of NF-\u0026kappa;B and its binding to the visfatin promoter, leading to reduced visfatin secretion. These findings suggest that empagliflozin, beyond its glucose-lowering effects, may exert protective effects against vascular calcification by modulating adipokine signaling. Previous studies have focused on SGLT2 inhibitors\u0026apos; role in improving glycemic control and reducing heart failure risk. However, our study is the first to show that empagliflozin can directly suppress visfatin expression in adipocytes via the p38/NF-\u0026kappa;B pathway, providing a new cardiovascular benefit mechanism. Recent studies have also shown that empagliflozin modulates multiple signaling pathways, including AMPK activation and oxidative stress reduction, which may enhance its cardiovascular protective effects\u003csup\u003e50-54\u003c/sup\u003e. For example, empagliflozin has been reported to reduce inflammation through AMPK activation and improve vascular calcification through anti-inflammatory pathways\u003csup\u003e55\u003c/sup\u003e. In this study, the direct effect of SGLT2 inhibitors on calcification medium-induced vascular smooth muscle calcification was not prominent, potentially due to the short stimulation time of the calcification medium, their cell type-specific action, and their primary reliance on regulating visfatin expression in adipocytes. The calcification medium may activate pathways less responsive to SGLT2 inhibition, and its complex mechanism compared to high-phosphate conditions could contribute to the limited direct impact. Future studies should further investigate SGLT2 inhibitor targets in smooth muscle cells and their indirect mechanisms in vivo.\u003c/p\u003e\n\u003cp\u003eGiven the elevated levels of visfatin in patients with metabolic disorders such as obesity and diabetes\u003csup\u003e56 57\u003c/sup\u003e, our findings suggest that empagliflozin could be particularly beneficial in these populations. This study is the first to show that visceral adipose-derived visfatin binds to TLR4 on vascular smooth muscle cells, promoting vascular calcification by upregulating osteogenic markers and downregulating contractile markers. While visfatin\u0026apos;s role in inflammation and insulin resistance is well-established, its direct involvement in vascular calcification has remained unclear. We also discovered that empagliflozin, an SGLT2 inhibitor, suppresses visfatin expression in adipocytes via the p38/NF-\u0026kappa;B pathway, expanding the understanding of SGLT2 inhibitors\u0026apos; pleiotropic effects beyond glucose-lowering. By reducing visfatin expression, empagliflozin may help mitigate the risk of vascular calcification and its associated complications, such as atherosclerosis and cardiovascular events. Future studies should investigate whether other SGLT2 inhibitors share empagliflozin\u0026apos;s ability to suppress visfatin expression and whether this effect is consistent across different patient populations. Additionally, long-term clinical trials are needed to evaluate empagliflozin\u0026apos;s effects on vascular calcification in patients with metabolic disorders. In conclusion, our study highlights empagliflozin\u0026apos;s multifaceted role in metabolic and cardiovascular health, demonstrating its potential as a comprehensive therapeutic agent for preventing vascular calcification in patients with metabolic disorders.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, the use of a single calcification model may not fully capture the complexity of vascular calcification. Future studies should consider employing multiple models, such as high-phosphate diet feeding or renal resection surgery-induced calcification models, to verify the effects of empagliflozin and assess its mechanisms across different conditions. Second, while we demonstrated that visfatin promotes vascular calcification through TLR4, the detailed mechanisms behind this interaction remain unclear. Future research should further explore the downstream signaling pathways activated by TLR4 and the role of visfatin in this process. Additionally, this study focused on the short-term effects of empagliflozin, and the long-term impact on vascular calcification needs further investigation. Finally, while the study concentrated on the visfatin-p38/NF-\u0026kappa;B pathway, other factors, such as oxidative stress and inflammation, may also contribute to vascular calcification. Future studies should investigate the involvement of these factors and evaluate empagliflozin\u0026apos;s potential to modulate them.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study reveals that visceral adipose-derived visfatin promotes vascular calcification by binding to TLR4 on vascular smooth muscle cells. Additionally, we found that empagliflozin improves vascular calcification by inhibiting the activation of the p38/NF-\u0026kappa;B pathway in adipocytes, thereby reducing visfatin expression. These findings provide new theoretical support for the use of SGLT2 inhibitors in preventing and treating vascular calcification in patients with metabolic disorders. Future studies should explore whether other SGLT2 inhibitors exhibit similar effects and validate these mechanisms in clinical applications. Further investigation into the role of visfatin in vascular calcification and the effects of empagliflozin under different pathological conditions will help develop more effective therapeutic strategies to reduce cardiovascular risk in patients with metabolic disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026beta;‐GP: \u0026beta;‐Glycerophosphate; CAC: coronary artery calcification; CHIP: Chromatin immunoprecipitation; DMEM: Dulbecco\u0026apos;s Modified Eagle\u0026apos;s medium; eNAMPT: Extracellular NAMPT; FBS: foetal bovine serum; IF: Immunofluorescence; IL-6: Interleukin-6; iNAMPT: Intracellular NAMPT; IP: Immunoprecipitation; LPS: lipopolysaccharides; MAPK: Mitogen-activated protein kinase; NF-\u0026kappa;B: nuclear factor-kappa B; NAMPT: nicotinamide phosphoribosyltransferase; NAD+: nicotinamide adenine dinucleotide; NCS: newborn calf serum; PAMPs: pathogen-associated molecular patterns; PARPs: poly (ADP-ribose) polymerases; PBS: phosphate-buffered saline; qPCR: Quantitative real-time PCR; SAT: subcutaneous adipose tissue; SGLT2i: sodium-glucose cotransporter 2 inhibitors ; TBST: Tween 20-Tris-buffered saline (TBST); TLR4: Toll-like receptor 4; TNF-\u0026alpha;: Tumour necrosis factor alpha; VAT: visceral adipose tissue; VitD: vitamin D; VSMCs: vascular smooth muscle cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe would like to express our deepest appreciation to the patients who consented to participate and donated blood samples, making this study possible.\u003c/p\u003e\n\u003cp\u003eFunding statement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant nos. 81873516, 82170463, 81900444), the National Key Research and Development Program of China (grant nos. 2021YFF0501403, 2021YFF0501404, 2017YFC1308303), the Natural Science Foundation of Shandong Province (grant nos. ZR2023QH398, ZR2024MH019, ZR2019BH052, ZR2020QH007, and ZR2019PH030), China International Medical Foundation (grant nos. Z-2016-23-2001-01).\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe use of blood samples was approved by the Medical Institutional Ethics Committee of Qilu Hospital, Shandong University, China (Approval No. KYLL-2020(KS)-537), and all donors provided informed consent.\u0026nbsp;All procedures adhered to protocols approved by the Animal Care and Use Committee of Shandong University (Approval No. DWLL-2021-143).\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eX.J., X.Z. and Y.Z. designed and conducted this research, wrote the manuscript. Y.Z. and Y.L. prepared the figure. L.W., J.L., W.W. and H.L. were responsible for resources acquisition and data collection. Y.Z., Y.Z., Y.L. and J.L. analyzed and interpreted the data. All the authors read and approved the submitted version.\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShanahan CM, Crouthamel MH, Kapustin A, et al. Arterial calcification in chronic kidney disease: key roles for calcium and phosphate. \u003cem\u003eCirc Res\u003c/em\u003e 2011;109(6):697-711. doi: 10.1161/CIRCRESAHA.110.234914 [published Online First: 2011/09/03]\u003c/li\u003e\n\u003cli\u003eLiu W, Zhang Y, Yu CM, et al. Current understanding of coronary artery calcification. \u003cem\u003eJ Geriatr Cardiol\u003c/em\u003e 2015;12(6):668-75. doi: 10.11909/j.issn.1671-5411.2015.06.012 [published Online First: 2016/01/21]\u003c/li\u003e\n\u003cli\u003eBostrom KI. 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Serum visfatin in relation to visceral fat, obesity, and type 2 diabetes mellitus in Asian Indians. \u003cem\u003eMetabolism\u003c/em\u003e 2007;56(4):565-70. doi: 10.1016/j.metabol.2006.12.005 [published Online First: 2007/03/24]\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"visfatin, vascular calcification, TLR4, empagliflozin, adipocyte, p38/NF-κB pathway","lastPublishedDoi":"10.21203/rs.3.rs-7284693/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7284693/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Vascular calcification is a common complication in metabolic diseases, such as diabetes and obesity, contributing to cardiovascular morbidity. Visfatin, an adipokine secreted by visceral fat, has been confirmed to be closely associated with metabolic dysfunctions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eVascular calcification models were established in mice through intraperitoneal injection of vitamin D (VitD). Serum visfatin levels were measured in both patients with coronary artery calcification and calcified mice. Visfatin expression in visceral fat was assessed by molecular analyses. To examine the functional role of visfatin, mice with adipose-specific visfatin overexpression were generated. Primary vascular smooth muscle cells (VSMCs) were treated with calcification medium and recombinant visfatin to assess osteogenic differentiation. The interaction between visfatin and Toll-like receptor 4 (TLR4) was investigated, and Tlr4 knockout models were used to verify its role. Empagliflozin was administered to evaluate its effects on vascular calcification and visfatin expression, along with related signaling pathway analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Serum visfatin levels were significantly elevated in patients with high coronary artery calcification scores and in mice with vascular calcification compared to controls. Visfatin expression was also markedly increased in the visceral adipose tissue of calcified mice. Mice with adipose-specific visfatin overexpression showed aggravated vascular calcification following vitamin D treatment. In vitro, visfatin enhanced the osteogenic differentiation of VSMCs in response to calcification medium. Mechanistically, visfatin directly bound to TLR4 and promoted the osteogenic transformation of VSMCs. Tlr4 deletion significantly attenuated aortic calcification induced by visfatin both in vivo and in vitro. Empagliflozin treatment significantly reduced vascular calcification and lowered circulating visfatin levels. Furthermore, empagliflozin inhibited the activation of the p38/NF-κB signaling pathway in adipose tissue, reduced nuclear translocation of NF-κB, suppressed its binding to the visfatin promoter, and thereby downregulated visfatin expression in adipocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e that visfatin secreted from visceral fat accelerates VSMCs osteogenic differentiation and vascular calcification via TLR4. Empagliflozin inhibits the expression of visfatin in adipocytes through the p38/NF-κB signaling pathway, thereby suppressing vascular calcification. The results suggest that visfatin may represent a novel therapeutic strategy for preventing or treating vascular calcification and related cardiovascular diseases.\u003c/p\u003e","manuscriptTitle":"Visfatin from Adipocyte Accelerates Vascular Calcification via TLR4","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 01:12:37","doi":"10.21203/rs.3.rs-7284693/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9aa04e71-4fc4-4185-8d38-a9f31f282f9a","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-22T03:38:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 01:12:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7284693","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7284693","identity":"rs-7284693","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}