Dapagliflozin (#11574) was obtained from Cayman Chemical (MI, USA). Thrombin derived from bovine plasma (#T4648) and DMSO (#D8418) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Cell culture reagents, including Dulbecco’s Modified Eagle Medium (DMEM + GlutaMAX, #10569-010), fetal bovine serum (FBS, #10437-028), penicillin–streptomycin (#15140-122), non-essential amino acids (NEAA, #11140-035), and TrypLE (#12604-021), were supplied by Thermo Fisher Scientific (Waltham, MA, USA). The phospho–c-Jun primary antibody (RRID: AB_726900, #ab32385, 1:1000) and anti-c-Jun antibody (RRID: AB_731602, #ab40766, 1:1000) were obtained from Abcam (Cambridge, UK). Antibodies targeting phospho-JNK (RRID: AB_331659, #9251, 1:1000), total JNK (RRID: AB_2250373, #9252, 1:1000), phospho-ERK1/2 (RRID: AB_390779, #4695, 1:1000), total ERK1/2 (RRID: AB_2315112, #4370, 1:1000), and GAPDH (RRID: AB_561053, #2118, 1:3000) were purchased from Cell Signaling Technology (Danvers, MA, USA). The CTGF antibody (RRID: AB_10917259, sc-365970, 1:1000) was acquired from Santa Cruz Biotechnology (Dallas, TX, USA). The cleaved PAR-1 (Ser42) antibody (RRID: AB_10744166, #SAB4500823, 1:1000) was obtained from Sigma–Aldrich. The ADAM17 antibody (#GTX31632, 1:1000) was purchased from GeneTex (Irvine, CA, USA). The phospho-ADAM17 (Thr735) antibody (RRID: AB_2816411, #PA5-104938, 1:1000) and total PAR-1 antibody (RRID: AB_2900674, #PA5-116040, 1:1000) were sourced from Thermo Fisher Scientific. Dapagliflozin was dissolved in DMSO and kept at -20 °C until use.

Male Wistar rats (20–25 weeks old, 450–550 g) were obtained from BioLASCO (Taipei, Taiwan). All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Cathay General Hospital (CGH-IACUC-113-019). Experiments were performed following the 3Rs principles, the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th edition), and ARRIVE recommendations to ensure high ethical standards. Animals were examined before enrollment, and those presenting infection, inflammation, or neurological abnormalities were excluded. Surgical interventions were conducted under 2.5% isoflurane anesthesia, and analgesics were administered post-operatively to reduce discomfort. Throughout the study, animals were closely observed for unusual symptoms, and predefined humane endpoints were applied when necessary. Rats were randomly allocated to three groups: a sham-operated control (n = 6), a vehicle group receiving DMSO (n = 6), and a dapagliflozin-treated group (1.4 mg/kg/day for 7 days; n = 6). At the end of the 14-day protocol, animals were euthanized using CO₂ at a displacement rate of 50% of chamber volume per minute, consistent with NIH ARAC recommendations for rodent euthanasia. Dapagliflozin was administered using an implanted osmotic minipump at the time of balloon injury. The pump was loaded with a predefined amount of dapagliflozin sufficient to provide continuous drug release for 7 days and was not removed thereafter. This dosing strategy was designed to target the acute post-injury phase characterized by inflammatory activation and vascular smooth muscle cell proliferation. Neointimal formation was evaluated on day 14 following injury.

The A10 vascular smooth muscle (VSM) cell line (RRID: CVCL_0130), originally established from the thoracic aorta of embryonic rats, was sourced from the Bioresource Collection and Research Center (BCRC), Taiwan. Cells were cultured in DMEM supplemented with GlutaMAX, 10% fetal bovine serum, non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), and a standard antibiotic mixture (100 U/mL penicillin G and 1 mg/mL streptomycin). Cultures were maintained at 37 °C in a humidified incubator containing 5% CO₂. Depending on the experimental application, cells were seeded into 6-cm dishes for protein expression studies by Western blotting and RT-PCR.

For cell-based experiments, A10 vascular smooth muscle cells were subjected to a defined drug treatment protocol prior to analysis by real-time RT-PCR and immunoblotting. Cells were pretreated with dapagliflozin (1–10 µM) or vehicle control (DMSO) for 90 min. When pharmacological inhibitors were used, cells were pretreated with the ADAM17 inhibitor TAPI-1 or the ERK inhibitor U0126 for 60 min before thrombin stimulation. Following pretreatment, cells were stimulated with thrombin (1 U/mL) to induce downstream signaling responses.

Cells were harvested at specific time points after thrombin stimulation according to the signaling events being assessed. At 3 min, cells were collected to evaluate ADAM17 phosphorylation (p-ADAM17) and cleaved PAR-1 expression. At 5 min, cells were harvested to assess phosphorylation of JNK and ERK1/2. At 10 min, p-c-Jun expression was examined. For assessment of CTGF responses, cells were collected 2 h after thrombin stimulation to analyze CTGF protein and mRNA expression levels. All treatment durations and sampling time points were kept consistent across experiments to ensure comparability.

A10 cells (2.5 × 10⁴) were plated in 6 cm culture dishes and exposed to either the vehicle control (0.05% DMSO) or dapagliflozin at concentration of 10 µM. and RNA extracted using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany, #740955.5) and stored at -80 ℃. One µg of total RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Thermo Fisher Scientific, #4368814). Samples were quantified using QuantiNova PCR Kits (Qiagen, Hilden, #208252) by using the Step One Plus™ Real-Time PCR

System (Applied Biosystems™, Thermo Fisher Scientific, ). The nucleotide sequences of the primers used for amplification were as follows: for CTGF, sense 5’-CAAgCTgCCCgggAAAT-3’ and antisense 5’-CggTCCTTgggCTCATCA-3’; for GAPDH (as internal control), sense 5’-AgACAgCCgCATCTTCTTgT-3’ and antisense

5’-CTTgCCgTgggTAgAgTCAT-3’ ; for SGLT2, sense 5’-TgAgTggAATgCgCTCTTTg-3’ and antisense 5’-gAggCATggTAATCACTCCg-3’.

A10 cells (2.5 × 10⁴) were seeded into 6-cm plates and treated with either vehicle (0.05% DMSO) or dapagliflozin at 1, 5, or 10 µM. Following incubation, cells were harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, MA, USA). Protein concentrations were determined, and equivalent protein amounts from each sample were separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking, membranes were incubated overnight at 4 °C with the appropriate primary antibodies. Protein detection was achieved using enhanced chemiluminescence (ECL), and images were captured with a UVP ChemStudio imaging platform (Analytik Jena). Densitometric analysis of the immunoblots was performed using Image-Pro Plus 4.5 (Media Cybernetics, MD, USA), and quantified values were plotted to compare relative protein expression among groups.

A10 cells (1 × 10⁴ per insert) were seeded into Ibidi® culture inserts (#80366, Regensburg, Germany) and allowed to grow at 37 °C until reaching full confluence. To create a wound area [28], the inserts were gently lifted with sterile forceps to generate a defined cell-free gap. Cells were then treated for 24 h with either vehicle (DMSO), thrombin (1 U/mL), or thrombin in combination with dapagliflozin (10 µM). Phase-contrast images of the wound area were obtained immediately after insert removal (0 h) and again after 24 h to evaluate cell migration. The extent of gap closure was measured, and wound width was quantified using NIS-Elements image analysis software.

Balloon injury of the right common carotid artery in male Wistar rats was performed following established methodologies [28, 29]. In brief, animals were anesthetized with 2.5% isoflurane, and a 2 F Fogarty embolectomy catheter (#120602F, Edwards Lifesciences, Irvine, CA, USA) was inserted through the external carotid artery and advanced into the common carotid artery. The balloon was inflated to distend the vessel and then withdrawn toward the entry site; this maneuver was carried out three times before catheter removal. Immediately post-procedure, rats received an intraperitoneal injection of vehicle (DMSO) or dapagliflozin (0.7 mg), followed by continuous delivery of dapagliflozin for 7 days (1.4 mg/kg/day) using an ALZET® osmotic pump (Cupertino, CA, USA). 14 days after angioplasty, animals were euthanized with CO₂ and perfused transcardially with PBS. The injured carotid segments were excised, embedded, and sectioned at 5 μm thickness for hematoxylin and eosin staining. Images were acquired using an Echo Revolve microscope (Echo, San Diego, CA, USA), and morphometric parameters-including external and internal elastic laminae, lumen area, and neointimal thickness-were quantified with ImageJ 1.53a (NIH, Bethesda, MD, USA). The intima-to-media (I/M) area ratio was calculated using the formula: (IEL area - lumen area)/(EEL area - IEL area) [29]. Dapagliflozin was administered during the acute post-injury phase (days 0–7) to modulate early inflammatory and cellular responses, followed by a drug-free recovery period (days 7–14) prior to tissue collection, consistent with our previous experimental design [28].

Quantitative data are presented as the mean accompanied by the standard deviation (SD), with the number of experimental replicates specified for each dataset. To examine differences across multiple experimental conditions, one-way analysis of variance (ANOVA) was applied. In cases where ANOVA identified significant discrepancies among groups, Tukey’s post hoc analysis was utilized to perform pairwise comparisons. Statistical significance was defined as a p-value below 0.05. All statistical evaluations were conducted using the GraphPad Prism software, version 6 (GraphPad, San Diego, California, USA).