The U937 human monocytic cell line (PCS-100-011) was procured from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), and incubated at 37 °C under a humidified atmosphere containing 5% CO₂. To differentiate U937 cells into foam cells, they were first stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 h, followed by treatment with 50 µg/mL oxidized low-density lipoprotein (ox-LDL; Sigma-Aldrich, St. Louis, MO, USA) for an additional 48 h.

Small interfering RNA targeting ASS1 (si-ASS1: 5^′-UCAAUGAACACCUUUUUGGCC-3^′), ASS1 overexpression plasmid (pCDNA3.0-ASS1), NLRP3 overexpression plasmid (pCDNA3.0-NLRP3), and their corresponding negative controls (scrambled siRNA [si-NC] for knockdown and empty vectors for overexpression) were commercially obtained from GenePharma (Shanghai, China). Foam cells were transfected with the indicated plasmids or siRNAs, either alone or in combination with their respective controls, using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). To further investigate the functional link between ASS1 and STAT3 signaling, we performed rescue experiments using STAT3-specific small interfering RNA (si-STAT3) and a constitutively active STAT3 mutant plasmid (STAT3-CA). The si-STAT3 (target sequence: 5^′-GAACCAUGUGGAUAUCAUA-3^′) and STAT3-CA overexpression plasmid (pCDNA3.1-STAT3-CA) were purchased from GenePharma (Shanghai, China). U937 foam cells were co-transfected with either si-STAT3 or STAT3-CA along with ASS1 overexpression or knockdown (si-ASS1) plasmids using Lipofectamine 2000 according to the manufacturer’s instructions. Cells were harvested 48 hours post-transfection for subsequent analyses.

Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent probe 2^′,7^′-dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich). Cells were incubated with 10 µM DCFH-DA for 30 min at 37 °C, washed thoroughly, and analyzed immediately on a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA). Data were processed with CellQuest software (version 4.1; BD Biosciences, Franklin Lakes, NJ, USA) and presented as fluorescence intensity histograms.

Total RNA was isolated from foam cells using Trizol reagent from Invitrogen (Carlsbad, CA, USA). Subsequently, cDNA synthesis was performed using the M-MLV reverse transcriptase kit from TaKaRa Co. (Dalian, Shandong, China). Quantitative real-time PCR amplification was conducted on an ABI 7500 Real-Time PCR System by Applied Biosystems. The amplification reaction was performed using the 1$\times$ FastStart Essential DNA Green Master from Roche (Mannheim, Germany), and the PCR protocol included an initial step at 50 °C for 2 minutes, followed by denaturation at 95 °C for 5 minutes, and 40 amplification cycles (4 seconds at 95 °C and 30 seconds at 60 °C). The relative quantity of target genes was determined using the 2^{-Δ⁢Δ⁢Ct} method, with normalization to GAPDH as the internal control. The primer sequences used were as follows:

ASS1 forward: 5^′-TGAAATTTGCTGAGCTGGTG-3^′ and ASS1 reverse: 5^′-ATGTACACCTGGCCCTTGAG-3^′; ST2L (membrane-bound isoform) forward: 5^′-CCTGGTGAAGTTCAACAGCA-3^′ and reverse: 5^′-TGTAGTAGGCGATGCCACCT-3^′;

sST2 (soluble isoform) forward: 5^′-GCTCCAGGAACTCCAACCTC-3^′ and reverse: 5^′-AGCTCCAGGTACATCGGTGT-3^′; GAPDH forward: 5^′-CAGCCTCAAGATCATCAGCA-3^′ and GAPDH reverse: 5^′-TGTGGTCATGAGTCCTTCCA-3^′.

Human umbilical vein endothelial cells (HUVECs) were obtained from the ATCC (Rockville, MD, USA), while the Human aortic vascular smooth muscle cells (HAVSMCs) were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). Both HUVECs and HAVSMCs were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT, USA), 1% penicillin and 1% streptomycin. To establish an indirect co-culture system, a Transwell chamber comprising a culture insert with a 10-µm thick porous membrane featuring 0.4-µm pores was used. In this system, foam cells (1 $\times$ 10⁵ cells/well) from various transfection groups or ox-LDL preprocessed groups were initially seeded in the upper layer of the Transwell, placed within the wells of a six-well plate, and co-cultured with either HUVECs or HAVSMCs positioned at the bottom of the Transwell chamber (Corning, NY, USA). For co-culture experiments, HUVECs were seeded at a density of 1 $\times$ 10⁵ cells per well and HAVSMCs at 5 $\times$ 10⁴ cells per well in the lower compartment. All cell lines were validated by STR profiling and tested negative for mycoplasma.

Apoptosis was quantified by flow cytometry using an Annexin V-FITC/PI Apoptosis Detection Kit (Cat# KGA108, KeyGen Biotech, Nanjing, Jiangsu, China) according to the manufacturer’s protocol. Briefly, cells were harvested with trypsin-EDTA, washed twice with PBS, and stained with FITC-labeled Annexin V (1 µg/mL) and propidium iodide (PI, 10 µg/mL) for 20 min in the dark. Samples were then analyzed on a FACScan flow cytometer (Beckman Coulter, Brea, CA, USA).

The expression of ASS1 and NLRP3 in foam cells, and of the endothelial marker CD34 in HUVECs, was examined by immunofluorescence. Cells were seeded in sterile Millicell® EZ-Slide eight-well glass plates (PEZGS0816; Millipore, MA, USA). After reaching appropriate confluency, they were rinsed with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 for 30 min. Non-specific binding was blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature. Cells were then incubated overnight at 4 °C with primary antibodies diluted as follows: anti-ASS1 (ab172730, 1:100; Abcam, Cambridge, UK) and anti-NLRP3 (ab270449, 1:100; Abcam) for foam cells, or anti-CD34 (ab81289, 1:200; Abcam) for HUVECs. After washing, samples were incubated for 2 h with a FITC-conjugated secondary antibody (BA1105, Boster, Wuhan, Hubei, China). Nuclei were counterstained with DAPI (10 µg/mL) for 5 min in the dark. Following final PBS washes, fluorescence images were acquired using a fluorescence microscope (Leica, Wetzlar, Germany).

Cell viability of HAVSMCs was assessed using the Cell Counting Kit-8 (HY-K0301, CCK-8; MedChem Express, Monmouth Junction, NJ, USA). Cells from different experimental groups were seeded in 96-well plates. After the respective treatments, 10 µL of CCK-8 solution was added to each well, followed by incubation at 37 °C for 2 h. Absorbance was then measured at 450 nm using a microplate reader.

Cell proliferation was evaluated using the Cell-Light EdU Apollo®567 In Vitro Imaging Kit (C10371-1, RiboBio, Guangzhou, Guangdong, China) according to the manufacturer’s instructions. HAVSMCs in logarithmic growth phase were seeded into 96-well plates at a density of 4 $\times$ 10³ cells per well and cultured for 24 h. Cells were then pulsed with 50 µM EdU for 2 h, followed by fixation with 4% paraformaldehyde in PBS for 30 min. After washing with PBS, samples were incubated with 1$\times$ Apollo staining solution for 30 min, then permeabilized with 0.5% Triton X-100 in PBS for 10 min. Nuclei were counterstained with 1$\times$ Hoechst 33342 for 30 min at room temperature. Following a final PBS wash, EdU-positive cells were visualized and quantified under a fluorescence microscope.

Cell migration and invasion assays were performed using Transwell® chambers (Corning Life Sciences, Corning, NY, USA). For migration, uncoated inserts were used; for invasion, inserts pre-coated with Matrigel® matrix were employed. HAVSMCs were suspended in 100 µL of serum-free RPMI-1640 medium and seeded into the upper chamber. The lower compartment was filled with 500 µL of complete medium containing 10% FBS to serve as a chemoattractant. After incubation for 24 h at 37 °C under 5% CO₂, cells that had migrated or invaded to the lower surface of the membrane were fixed with methanol, stained with 0.1% crystal violet for 35 min, and counted in five randomly selected fields per insert under a light microscope.

Total cellular protein was extracted using RIPA lysis buffer (Pierce, Thermo Fisher Scientific), and protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Protein samples (30 µg per lane) were separated on 12% SDS-PAGE gels and transferred onto PVDF membranes. After blocking with 5% non-fat milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with the following primary antibodies (all from Abcam, unless otherwise noted): anti-ASS1 (ab172730), anti-ICAM-1 (ab282575), anti-VCAM-1 (ab134047), anti-STAT3 (ab68153), anti-phospho-STAT3 (ab267373), anti-ST2 (total; ab194113), anti-sST2 (soluble isoform; ab272372), anti-SMHC (ab133567), anti-CNN1 (ab46794), anti-$\alpha$-SMA (ab7817), and anti-$\beta$-actin (ab8226). All Abcam antibodies were used at 1:1000 dilution, except $\beta$-actin (1:5000). To assess mitochondrial apoptosis in ASS1-treated HUVECs, additional blots were probed with anti-cleaved PARP (ab32561), anti-Bax (ab32503), and anti-Bcl-2 (ab32124). Following primary antibody incubation, membranes were washed and probed with a horseradish peroxidase-conjugated secondary antibody (sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific) and imaged with a chemiluminescence detection system.

IL-33 levels in foam cell supernatants and IL-1$\beta$ levels in mouse serum were quantified using commercially available ELISA kits (IL-33 ELISA Kit (Cat# SEA568Hu), IL-1$\beta$ ELISA Kit (Cat# SEA563Mu), Cloud-Clone Corp, Wuhan, Hubei, China) according to the manufacturer’s protocols. Briefly, samples and standards (100 µL each) were added to the wells of a 96-well plate and incubated at 37 °C for 90 min. After washing three times, 100 µL of biotinylated detection antibody was added and incubated for 60 min. Following another wash step, 100 µL of horseradish peroxidase (HRP)-conjugated streptavidin working solution was applied for 30 min. Color development was initiated by adding substrate solution, and the reaction was stopped before absorbance was measured at 450 nm and 630 nm using a microplate reader (Thermo Fisher Scientific).

Apoptotic endothelial cells were detected by TUNEL staining using a fluorescence FITC kit (12156792910, Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. HUVECs grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After washing, cells were incubated with the TUNEL reaction mixture for 1 h at 37 °C in the dark. Following three washes with PBS, nuclei were counterstained with DAPI (10 µg/mL; Solarbio, Beijing, China). Images were captured using a fluorescence microscope (Olympus BX51, Tokyo, Japan) at 200$\times$ magnification. TUNEL-positive cells, identified by green nuclear fluorescence, were quantified in at least five random fields per sample.

Male apolipoprotein E-deficient (ApoE^-⁣/-) mice were obtained from the Laboratory Animal Center of Fuwai Hospital, National Center for Cardiovascular Diseases. All animal procedures were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College (Approval No. 0105-1-6-ZX(X)-4). Mice were housed under specific pathogen-free conditions with a 12 h light/dark cycle and free access to food and water. Atherosclerosis was induced by feeding 8-week-old mice a high-fat diet (21% fat, 0.15% cholesterol, 3.2% cholate) for 12 weeks. Body weight and food intake were recorded weekly.

After the 12-week dietary intervention (high-fat diet #D12079B, Research Diets Inc., New Brunswick, NJ, USA), mice were euthanized in strict accordance with institutional animal care guidelines. Euthanasia was performed via intraperitoneal injection of sodium pentobarbital (Euthasol®) at a dose of 200 mg kg^-1 body weight. The stock solution (39% w/v) was diluted 1:10 with 0.9% saline to obtain a 3.9% w/v working solution. Prior to injection, mice were gently restrained to minimize stress. Depth of anesthesia was confirmed by loss of pedal reflex and cessation of spontaneous respiration. Following this confirmation, euthanasia was carried out as a result of the administered pentobarbital overdose, leading to cardiac arrest. Cardiac arrest was verified by thoracotomy within 5 min of respiratory cessation. Cervical dislocation was performed immediately after cardiac arrest as a secondary physical method to ensure death. All procedures were conducted by trained personnel between 08:00 and 11:00 to minimize circadian influences. Tissues were subsequently harvested for analysis.

To modulate mouse ASS1 (NCBI reference: NM_007494.3) expression in vivo, adeno-associated virus serotype 9 (AAV9) vectors were constructed for overexpression and knockdown. For overexpression, the full-length ASS1 coding sequence was subcloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) and then inserted into the AAV9 backbone plasmid pAV-F4/80 (Weizhen Biotechnology Co., Jinan, Shandong, China). For knockdown, potential shRNA target sites were screened by co-transfecting the pcDNA3.1-ASS1 plasmid and the pAV-CAG-GFP-mir30 shRNA library plasmid (Boshang Biotechnology Co., Jinan, Shandong, China) into HEK-293 cells. The most efficient shRNA sequence was subsequently cloned into the pAV-F4/80-GFP-mir30 shRNA vector (Wuyue Biotechnology Co., Jinan, Shandong, China) and packaged into AAV9 particles. Control vectors included AAV9 expressing a scrambled shRNA (for knockdown experiments) and empty pAV-F4/80 (for overexpression experiments).

Mice were randomly assigned to five groups: (1) wild-type sham, (2) atherosclerosis model, (3) negative control (empty vector), (4) ASS1 knockdown, and (5) ASS1 overexpression. After 12 weeks of a high-fat diet, mice received an intravenous injection of the respective AAV9 vector (1 $\times$ 10¹² vp/mL, 50 µL per mouse) via the tail vein without anesthesia to minimize stress and physiological disturbance. Four weeks post-injection, all mice were euthanized for tissue collection. Prior to euthanasia, mice were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (200 mg/kg). Adequate depth of anesthesia was confirmed by the loss of the pedal reflex. Following anesthesia, tissues were harvested for subsequent analyses. All animal procedures were performed in accordance with institutional animal care guidelines.

Plaque lipid content was evaluated by Oil Red O staining. Tissue samples from the transverse cardiac section (TCS) and aortic arch (AAC) were fixed in 10% neutral-buffered formalin for 24 h at room temperature, embedded in paraffin, and sectioned at 5 µm. Sections were deparaffinized in xylene, rehydrated through a graded ethanol series (100% $\to$ 95% $\to$ 80% $\to$ 70%), and rinsed in distilled water.

Sections were stained with Oil Red O working solution (prepared by dissolving Oil Red O powder [Sigma-Aldrich, Cat. No. O0625] in isopropanol and diluting with distilled water at a 3:2 ratio) for 15 min at room temperature. After rinsing with distilled water, nuclei were counterstained with hematoxylin (Merck, Cat. No. 109297) for 1 min, differentiated in 70% ethanol, and mounted with neutral mounting medium. Lipid droplets were visualized as red areas under a light microscope.

The Oil Red O-positive area was quantified as a percentage of total plaque area using image-analysis software. For each mouse, three non-consecutive sections were analyzed, and the mean value was used for statistical comparisons (n = 6 mice per group).

Data are presented as mean $\pm$ standard error of the mean (SD). Comparisons between two groups were performed using Student’s t-test. For multiple-group comparisons, one-way analysis of variance (ANOVA) with repeated measures was applied, followed by Tukey’s post-hoc test for pairwise comparisons. A p-value  0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA).

To examine the role of ASS1 in atherosclerosis, foam cells were generated by treating U937 human monocytes with phorbol 12-myristate 13-acetate (PMA) and oxidized low-density lipoprotein (ox-LDL). ELISA showed that PMA + ox-LDL treatment significantly increased IL-33 secretion (Fig. 1a), and flow cytometry confirmed elevated intracellular reactive oxygen species (ROS) levels (Fig. 1b), confirming successful foam cell differentiation.

Fig. 1.

ASS1 expression is upregulated in ox-LDL-induced U937 foam cells. U937 foam cells were generated through co-treatment with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 h and 50 µg/mL oxidized low-density lipoprotein (ox-LDL) for an additional 48 h. (a) IL-33 concentration measured by ELISA assay. (b) ROS generation analyzed by flow cytometry. (c) ASS1 expression assessed via quantitative real-time PCR. (d) Immunofluorescence assay and (e) Western blot showing ASS1 expression in foam cells. (f) Western blot analysis demonstrating NLRP3 expression in foam cells. The scale bar is 50 µm in (d). For all panels, n = 3 biologically independent experiments, each performed on separate days with 1 $\times$ 10⁶ cells per well; technical replicates: 3 wells per condition per biological replicate. Data are presented as mean $\pm$ standard deviation (SD), error bars indicate SD. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. ***p 0.001 versus the control group.

ASS1 expression was then evaluated in these cells. qPCR analysis revealed a marked upregulation of ASS1 mRNA in foam cells compared with untreated controls (Fig. 1c). Consistent with this, both immunofluorescence and Western blotting demonstrated increased ASS1 protein expression in foam cells (Fig. 1d,e). Interestingly, Western blot also indicated an upward trend in NLRP3 protein levels in foam cells (Fig. 1f).

To determine the functional role of ASS1 in foam cells, we modulated its expression by siRNA-mediated knockdown and plasmid-mediated overexpression. qPCR confirmed efficient reduction of ASS1 mRNA after si-ASS1 transfection and increased expression after ASS1 overexpression (Fig. 2a).

Fig. 2.

ASS1 modulates ROS production, inflammatory marker expression, and IL-33 secretion in foam cells. U937 foam cells were transfected with si-ASS1 or ASS1 overexpression plasmid for 48 h. (a) Quantitative real-time PCR analysis of ASS1 mRNA expression. (b,c) Flow cytometry analysis of ROS generation. (d) Immunofluorescence assay showing ASS1 protein levels. (e) Western blot analysis of ASS1, ICAM-1, VCAM-1, (f) STAT3, and p-STAT3 protein levels. (g) ELISA assay for IL-33 concentration. The scale bar is 50 µm in (d). For all panels, n = 3 biologically independent experiments (performed on different days with different cell passages, 1 $\times$ 10⁶ cells per well); technical replicates: 3 wells per condition in each biological replicate. Data are presented as mean $\pm$ standard deviation (SD), and error bars indicate SD. Statistical significance: ***p 0.001 versus the corresponding baseline by one-way ANOVA with Tukey’s post-hoc test.

Intracellular ROS levels, measured by DCFH-DA fluorescence, were elevated in ox-LDL-treated foam cells. ASS1 knockdown significantly attenuated this increase, whereas ASS1 overexpression further enhanced ROS production (Fig. 2b,c).

Western blotting verified the corresponding changes in ASS1 protein (Fig. 2d,e). Notably, ASS1 knockdown reduced the protein levels of the adhesion molecules ICAM-1 and VCAM-1, while ASS1 overexpression increased them (Fig. 2e). Phosphorylation of STAT3 (p-STAT3) was also diminished by ASS1 knockdown and augmented by ASS1 overexpression (Fig. 2f).

Consistent with these pro-inflammatory changes, IL-33 secretion—quantified by ELISA—was decreased upon ASS1 knockdown and elevated upon ASS1 overexpression (Fig. 2g). Together, these data indicate that ASS1 drives oxidative stress and inflammatory activation in ox-LDL-stimulated foam cells.

To determine whether ASS1 acts through STAT3 signaling, rescue experiments were conducted using STAT3-specific siRNA (si-STAT3) and a constitutively active STAT3 mutant (STAT3-CA) in U937 foam cells. Western blot analysis revealed that ASS1 overexpression upregulated NLRP3, ICAM-1, and VCAM-1, an effect that was largely abolished by co-transfection with si-STAT3 (Fig. 3a). Conversely, expression of STAT3-CA reversed the downregulation of these proteins caused by ASS1 knockdown (Fig. 3a).

Fig. 3.

ASS1 regulates NLRP3 inflammasome activation and IL-33 secretion through STAT3 phosphorylation in U937 foam cells. Foam cells were co-transfected with indicated plasmids/siRNAs and treated with ox-LDL (50 µg/mL). The experimental groups were as follows: ox-LDL + vector, ox-LDL + ASS1, ox-LDL + ASS1 + si-STAT3, ox-LDL + si-STAT3, ox-LDL + si-ASS1, and ox-LDL + si-ASS1 + STAT3-CA. (a) Western blot analysis of NLRP3, ICAM-1, and VCAM-1 expression. (b) STAT3 and p-STAT3 protein levels. (c,d) ROS production measured by DCFH-DA flow cytometry. (e) IL-33 concentration determined by ELISA. For all panels, n = 3 biologically independent experiments (performed on different days, 1 $\times$ 10⁶ cells per well); technical replicates: 3 wells per condition in each biological replicate. Data are presented as mean $\pm$ standard deviation (SD). Error bars represent SD. ns, not significant (p $\ge$ 0.05), *p 0.05, **p 0.01, ***p 0.001 by one-way ANOVA with Tukey post-hoc test relative to the corresponding baseline.

ASS1 overexpression increased STAT3 phosphorylation (p-STAT3), while ASS1 knockdown reduced it; these changes were respectively blocked by si-STAT3 or restored by STAT3-CA (Fig. 3b). Similarly, ASS1-induced elevation of ROS production and IL-33 secretion was suppressed by si-STAT3, whereas the decreases resulting from ASS1 knockdown were rescued by STAT3-CA (Fig. 3c–e).

Collectively, these data demonstrate that STAT3 activation is an essential downstream event through which ASS1 promotes NLRP3 inflammasome activity and IL-33 release in foam cells.

To further examine the role of ASS1 in ox-LDL-induced endothelial injury, we co-cultured HUVECs with U937-derived foam cells. Apoptosis was significantly increased in HUVECs exposed to ox-LDL-induced foam cells, as shown by flow cytometry (Fig. 4a). This effect was attenuated by ASS1 knockdown in foam cells and further exacerbated by ASS1 overexpression, which was confirmed by TUNEL staining (Fig. 4b).

Fig. 4.

ASS1 in foam cells promotes HUVEC apoptosis via the mitochondrial pathway without affecting CD34 or total ST2 expression. HUVECs were co-cultured with U937 foam cells from control, si-NC, si-ASS1, vector, and ASS1 groups. (a) Flow cytometry analysis of the apoptotic rate of endothelial cells. (b) TUNEL fluorescence FITC assay for apoptotic endothelial cells. (c) Immunofluorescence staining of the vascular marker CD34. (d) Western blot analysis of ST2 protein levels. The scale bar is 50 µm in (b,c). For all panels, n = 3 biologically independent experiments (performed on different days with different HUVEC donors and different U937 passages); each experiment used 1 $\times$ 10⁵ HUVECs per well (12-well plate) and 2 $\times$ 10⁵ U937 foam cells per insert, with 3 technical replicates per group. Data are presented as mean $\pm$ standard deviation (SD). Error bars represent SD. ***p 0.001, ns, no significance (one-way ANOVA with repeated measures and Tukey post-hoc test relative to corresponding baseline).

Western blot analysis of HUVECs revealed that ASS1 knockdown reduced the levels of cleaved PARP and Bax while increasing Bcl-2, whereas ASS1 overexpression had the opposite effect. The Bax/Bcl-2 ratio was approximately 3-fold higher in the ASS1-overexpression group than in the vector control, indicating activation of the mitochondrial apoptosis pathway (Supplementary Fig. 1).

Immunofluorescence staining showed that the endothelial marker CD34 was not altered by ASS1 manipulation in foam cells (Fig. 4c). Interestingly, while co-culture with ox-LDL-induced U937 foam cells significantly enhanced total ST2 expression in HUVECs, these levels were maintained independently of ASS1 manipulation (knockdown or overexpression) (Fig. 4d).

These data suggest that ASS1 in foam cells enhances ox-LDL-driven endothelial apoptosis through the mitochondrial pathway, without affecting endothelial marker expression or total ST2 levels.

Given the opposing roles of the soluble decoy receptor sST2 and the membrane-bound signaling receptor ST2L, we examined how ASS1 regulates their expression. In U937 foam cells, ASS1 overexpression significantly increased sST2 mRNA and decreased ST2L mRNA, raising the sST2/ST2L ratio. Conversely, ASS1 knockdown reduced sST2 and increased ST2L mRNA, lowering the sST2/ST2L ratio (Supplementary Fig. 2a,b).

A similar regulatory pattern was observed in HUVECs and HAVSMCs co-cultured with ASS1-modulated foam cells. Co-culture with ASS1-overexpressing foam cells elevated sST2 mRNA and reduced ST2L mRNA in both HUVECs (Supplementary Fig. 2c,d) and HAVSMCs (Supplementary Fig. 2e,f), again increasing the sST2/ST2L ratio. Western blot analysis confirmed these changes at the protein level: ASS1 overexpression in foam cells increased sST2 protein in co-cultured HUVECs (Supplementary Fig. 2g) and HAVSMCs (Supplementary Fig. 2h), whereas ASS1 knockdown produced the opposite effect.

Together, these findings indicate that ASS1 drives a shift in the ST2 isoform balance toward a pro-inflammatory state—characterized by elevated sST2 and reduced ST2L—thereby potentiating IL-33-mediated signaling activity.

Given the contribution of vascular smooth muscle cells (VSMCs) to atherosclerotic plaque progression, we examined whether ASS1 in foam cells influences HAVSMC behavior. HAVSMCs were co-cultured with ox-LDL-induced U937 foam cells in which ASS1 was either knocked down or overexpressed.

CCK-8 and EdU assays showed that ox-LDL-treated foam cells stimulated HAVSMC proliferation, an effect that was further enhanced by ASS1 overexpression and reduced by ASS1 knockdown (Fig. 5a,b).

Fig. 5.

ASS1 in foam cells promotes HAVSMC proliferation, migration, and dedifferentiation. HAVSMCs were co-cultured with U937 foam cells from control, si-NC, si-ASS1, vector, and ASS1 groups. (a) Cell viability was assessed by the CCK-8 assay. (b) Cell proliferation of HAVSMCs was evaluated using EdU staining. (c) Western blot analysis of SMHC, CNN1, and $\alpha$-SMA protein levels. (d) Western blot analysis of ST2 protein levels. (e) Transwell assay to determine cell migration and invasion in HAVSMCs. The scale bar is 50 µm in (b), and the scale bar is 100 µm in (e). For all panels, n = 3 biologically independent experiments (performed on different days with different HAVSMC lots and different U937 passages); each experiment used 1 $\times$ 10⁴ HAVSMCs per well (96-well plate for CCK-8, 24-well plate for EdU, 6-well plate for Western blot, and 24-well Transwell inserts for migration/invasion) with 3 technical replicates per group. Data presented as mean $\pm$ standard deviation (SD). Error bars represent SD. ns, not significant (p $\ge$ 0.05), **p 0.01, ***p 0.001 by one-way ANOVA with Tukey post-hoc test relative to the corresponding baseline.

Western blot analysis revealed that co-culture with ox-LDL-induced foam cells downregulated the VSMC differentiation markers SMHC, CNN1, and $\alpha$-SMA in HAVSMCs. This downregulation was partially reversed by ASS1 knockdown in foam cells and exacerbated by ASS1 overexpression (Fig. 5c), indicating that ASS1 promotes VSMC dedifferentiation.

Notably, total ST2 protein levels in HAVSMCs were elevated following co-culture with ox-LDL-treated foam cells, but were not significantly altered by modulating ASS1 expression in foam cells (Fig. 5d).

Finally, Transwell migration assays demonstrated that ASS1 knockdown in foam cells attenuated the enhanced migration of HAVSMCs induced by co-culture, whereas ASS1 overexpression further increased migratory capacity (Fig. 5e).

Together, these results indicate that ASS1 in foam cells drives HAVSMC proliferation, migration, and dedifferentiation—key processes in atheroma progression—without affecting total ST2 expression in co-cultured HAVSMCs.

To determine whether NLRP3 mediates the pro-inflammatory effects of ASS1, rescue experiments were performed in U937 foam cells. ELISA showed that ASS1 knockdown reduced IL-33 secretion, and this reduction was largely reversed by NLRP3 overexpression (Fig. 6a). Similarly, the decrease in ROS production caused by ASS1 knockdown was restored upon NLRP3 overexpression (Fig. 6b,c).

Fig. 6.

NLRP3 overexpression rescues the anti-inflammatory effects of ASS1 knockdown in foam cells. U937 foam cells were co-transfected with si-ASS1 and NLRP3 overexpression plasmid or transfected with si-ASS1 alone. (a) IL-33 concentration determined by ELISA assay. (b,c) Flow cytometry analysis of ROS generation. (d) Immunofluorescence assay showing NLRP3 protein expression in U937 foam cells. (e) Western blot analysis of ASS1 protein levels. (f) Western blot analysis of adhesion molecules ICAM-1 and VCAM-1, (g) inflammasome-associated proteins NLRP3 and PYCARD, and (h) STAT3 and p-STAT3 associated with the STAT3 signaling pathway. The scale bar is 50 µm in (d). For all panels, n = 3 biologically independent experiments (performed on different days with different cell passages); each experiment contained 3 technical replicates (wells) per condition, with 2 $\times$ 10⁵ U937 foam cells per well. Data are presented as mean $\pm$ standard deviation (SD). Error bars represent SD. ns, not significant (p $\ge$ 0.05), **p 0.01, ***p 0.001 by one-way ANOVA with Tukey post-hoc test relative to the corresponding baseline.

Immunofluorescence confirmed successful NLRP3 overexpression (Fig. 6d). ASS1 protein downregulation by siRNA was not altered by NLRP3 overexpression (Fig. 6e), indicating that NLRP3 acts downstream of ASS1.

Western blot analysis revealed that the downregulation of adhesion molecules (ICAM-1 and VCAM-1) induced by ASS1 knockdown was abolished by NLRP3 overexpression (Fig. 6f). Likewise, the reduced expression of NLRP3 itself and its adaptor protein PYCARD after ASS1 knockdown was restored by NLRP3 overexpression (Fig. 6g). In contrast, NLRP3 overexpression did not reverse the decrease in STAT3 phosphorylation (p-STAT3) caused by ASS1 knockdown (Fig. 6h).

These results demonstrate that NLRP3 activation is a key downstream event responsible for ASS1-driven inflammatory signaling and oxidative stress in foam cells, independent of changes in ASS1 expression or STAT3 phosphorylation.

To determine whether NLRP3 activation itself is sufficient to induce ASS1-like inflammatory responses, we overexpressed NLRP3 alone in ox-LDL-treated U937 foam cells without altering ASS1 expression. Western blot analysis showed that NLRP3 overexpression significantly increased the protein levels of adhesion molecules ICAM-1 and VCAM-1 (Fig. 7a), as well as NLRP3 and its adaptor protein PYCARD (Fig. 7b), to a degree comparable to ASS1 overexpression.

Fig. 7.

NLRP3 overexpression increased downstream factors to levels comparable to ASS1 overexpression, and reversed the effects of ASS1 knockdown. U937 foam cells were treated as follows: Ctrl (untreated), ox-LDL + vector, ox-LDL + ASS1, ox-LDL + NLRP3, ox-LDL + si-NC, ox-LDL + si-ASS1, or ox-LDL + si-ASS1 + NLRP3. (a) Western blot analysis of adhesion molecules ICAM-1 and VCAM-1. (b) Inflammasome-associated proteins NLRP3 and PYCARD expression. (c,d) ROS production measured by DCFH-DA flow cytometry. (e) IL-33 concentration determined by ELISA assay. For all panels, n = 3 biologically independent experiments (performed on different days, 1 $\times$ 10⁶ cells per well); technical replicates: 3 wells per condition in each biological replicate. Data are presented as mean $\pm$ standard deviation. ns, not significant (p $\ge$ 0.05), **p 0.01, ***p 0.001 by one-way ANOVA with Tukey post-hoc test relative to the corresponding baseline.

Flow cytometry revealed that NLRP3 overexpression alone markedly elevated intracellular ROS production (Fig. 7c,d), similar to the effect of ASS1 overexpression. ELISA further demonstrated that IL-33 secretion was increased by approximately 70% in NLRP3-overexpressing cells, with no statistically significant difference from the ASS1-overexpression group (Fig. 7e).

Importantly, the anti-inflammatory effects of ASS1 knockdown—reduced ROS, lower adhesion molecule expression, and decreased IL-33 secretion—were largely reversed when NLRP3 was co-overexpressed (Fig. 7a–e).

Together, these results indicate that NLRP3 activation is sufficient to drive a pro-inflammatory phenotype in foam cells, and that the pro-atherogenic effects of ASS1 are mediated primarily through upregulation of NLRP3.

We next examined whether NLRP3 modulates the impact of ASS1 on vascular cells using a foam-cell co-culture model.

HUVECs: ASS1 knockdown in foam cells reduced HUVEC apoptosis, an effect that was reversed by NLRP3 overexpression in foam cells, as shown by flow cytometry and TUNEL staining (Fig. 8a,b). The endothelial marker CD34 was unaffected by either ASS1 or NLRP3 manipulation (Fig. 8c). Total ST2 expression in HUVECs was elevated after co-culture with ox-LDL-induced foam cells, but remained elevated upon ASS1 knockdown or NLRP3 overexpression (Fig. 8d).

Fig. 8.

NLRP3 overexpression in foam cells reverses the protective effects of ASS1 knockdown on HUVEC apoptosis. HUVECs were co-cultured with U937 foam cells from control, si-NC, si-ASS1, si-ASS1 + vector, and si-ASS1 + NLRP3 groups. (a) Flow cytometry assay to determine cell apoptotic rate. (b) TUNEL fluorescence FITC kit used to detect apoptotic endothelial cells. (c) Immunofluorescence staining of the vascular marker CD34. (d) Western blot analysis of ST2 protein levels. The scale bar is 50 µm in (b,c). For all panels, n = 3 biologically independent experiments (performed on different days with different HUVEC donors and different U937 passages); each experiment contained 3 technical replicates (wells) per group, with 1 $\times$ 10⁵ HUVECs per well. Data are presented as mean $\pm$ standard deviation (SD). Error bars represent SD. **p 0.01, ***p 0.001. ns, no significance (one-way ANOVA with repeated measures and Tukey post-hoc test relative to the corresponding baseline).

HAVSMCs: ASS1 knockdown in foam cells attenuated HAVSMC proliferation, which was restored by NLRP3 overexpression (CCK-8 and EdU assays; Fig. 9a,b). Western blotting showed that ASS1 knockdown increased the expression of smooth-muscle differentiation markers (SMHC, CNN1, $\alpha$-SMA), an effect that was blunted by NLRP3 overexpression (Fig. 9c). Total ST2 levels in HAVSMCs were also raised after co-culture, independent of ASS1 or NLRP3 modulation (Fig. 9d). Furthermore, Transwell migration assays confirmed that the decreased HAVSMC migration caused by ASS1 knockdown was rescued by NLRP3 overexpression (Fig. 9e).

Fig. 9.

NLRP3 overexpression in foam cells restores HAVSMC proliferation, migration, and dedifferentiation suppressed by ASS1 knockdown. HAVSMCs were co-cultured with U937 foam cells from control, si-NC, si-ASS1, si-ASS1 + vector, and si-ASS1 + NLRP3 groups. (a) Cell viability was assessed by the CCK-8 assay. (b) Cell proliferation of HAVSMCs was evaluated using EdU staining. (c) Western blot analysis of SMHC, CNN1, and $\alpha$-SMA protein levels. (d) Western blot analysis of ST2 protein levels. (e) Transwell assay to determine cell migration in HAVSMCs. The scale bar is 50 µm in (b), and the scale bar is 100 µm in (e). For all panels, n = 3 biologically independent experiments (performed on different days with different HAVSMC lots and different U937 passages); each experiment contained 3 technical replicates (wells) per group, with 1 $\times$ 10⁴ HAVSMCs per well for CCK-8, 2 $\times$ 10⁴ HAVSMCs per well for EdU, 6-well plates for Western blot, and 24-well Transwell inserts for migration. Data are presented as mean $\pm$ standard deviation (SD). Error bars represent SD. ns, not significant (p $\ge$ 0.05), **p 0.01, ***p 0.001 by one-way ANOVA with Tukey post-hoc test relative to the corresponding baseline.

These results indicate that NLRP3 activation in foam cells can counteract the regulatory effects of ASS1 on endothelial apoptosis and smooth muscle proliferation/migration, whereas total ST2 expression in vascular cells is influenced by ox-LDL-exposed foam cells but not directly by ASS1 or NLRP3.

To further evaluate the role of ASS1 in atherosclerosis, we performed histological and biochemical analyses in ApoE-deficient mice. Mice were fed a high-fat diet for 12 weeks to induce atherosclerosis, after which ASS1 expression was modulated via AAV9 vectors. Plaques from the transverse cardiac section (TCS) and aortic arch (AAC) were examined.

Oil Red O staining showed that lipid accumulation was markedly increased in atherosclerotic (AS) mice compared with sham-operated controls (Fig. 10a). Quantification confirmed the most severe lipid deposition in the ASS1-overexpression (ASS1^OE) group, intermediate levels in the AS and negative control (NC) groups, and significantly reduced lipid in the ASS1-knockdown (ASS1^kd) group (Fig. 10b,c). Body weight was also highest in ASS1^OE mice, while ASS1^kd mice maintained weights similar to the Sham group, suggesting a systemic metabolic influence of ASS1 (Fig. 10d).

Fig. 10.

ASS1 exacerbates atherosclerotic plaque progression, inflammation, and systemic metabolism in ApoE-deficient mice. Atherosclerosis was induced by feeding a high-fat diet for 12 weeks, followed by AAV9-mediated knockdown or overexpression of ASS1. Grouping: wild-type sham-operated group (Sham), atherosclerotic model group (AS), negative control group (NC, empty vector), ASS1 knockdown group (ASS1^kd), and ASS1 overexpression group (ASS1^OE). (a) Representative images of Oil Red O staining of the transverse cardiac section (TCS) and aortic arch cut (AAC). The black arrows in the figure indicate the severe areas of plaque lipid accumulation. (b,c) Quantification of lipid deposition of the TCS and AAC (Oil Red O-positive area percentage within plaque). (d) Body weight of mice measured at the study endpoint and comparative analysis between groups. (e) Western blot analysis (left) and quantitative summary (right) of ASS1 and NLRP3 protein expression in vascular tissues (n = 3 independent biological replicates). (f) Western blot analysis (left) and quantitative summary (right) of STAT3 and phosphorylated STAT3 (p-STAT3) protein levels in vascular tissues. (g,h) Serum IL-33 and serum IL-1$\beta$ concentration measured by ELISA. The scale bar is 100 µm for TCS and 200 µm for AAC in (a). For all panels: n = 6 mice per group (exact number); each mouse represents one biological replicate. Animals were 8-week-old male ApoE^-⁣/- mice (C57BL/6 background), single-caged, fed a high-fat diet for 12 weeks, and injected with AAV9 on three separate days (3 independent cohorts) to ensure reproducibility. Tissue samples were collected once per mouse. Data presented are as mean $\pm$ standard deviation (SD). Error bars represent SD. **p 0.01, ***p 0.001 compared to the control. Comparisons were made with 1-way ANOVA.

Western blot analysis of vascular tissues revealed that ASS1 overexpression increased NLRP3 protein levels, whereas ASS1 knockdown decreased them (Fig. 10e). Similarly, phosphorylated STAT3 (p-STAT3) was upregulated by ASS1 overexpression and downregulated by ASS1 knockdown (Fig. 10f), consistent with the in vitro findings.

ELISA of serum cytokines showed that IL-33 was elevated in AS mice, further increased by ASS1 overexpression, and attenuated by ASS1 knockdown (Fig. 10g). To determine whether NLRP3 activation also affects other inflammatory mediators, we measured IL-1$\beta$. Serum IL-1$\beta$ was significantly higher in AS mice than in sham controls. ASS1 overexpression further raised IL-1$\beta$ levels by ~1.7-fold relative to the NC group, while ASS1 knockdown reduced IL-1$\beta$ by 42% (p  0.001; Fig. 10h).

These in vivo results demonstrate that ASS1 exacerbates plaque lipid deposition, activates the NLRP3 inflammasome and STAT3 signaling, and enhances the secretion of pro-inflammatory cytokines IL-33 and IL-1$\beta$, thereby promoting atherosclerotic progression.