GRAPHICAL ABSTRACT

Highlights

• SLC25A33 is upregulated in PDGF-treated VSMCs and ligation-induced neointimal lesions.

• Silencing SLC25A33 inhibits PDGF-induced VSMC proliferation and migration.

• SLC25A33 knockdown decreases mtDNA synthesis and cytosolic release in VSMCs.

• Reduced cytosolic mtDNA attenuates cGAS–STING–TBK1–NF-κB signaling activation.

• Targeting SLC25A33 mitigates neointima formation in occlusive vascular diseases.

INTRODUCTION

Vascular occlusions, including atherosclerosis, stenosis, and restenosis, remain a leading cause of death in patients with diabetes [1]. To address these pathologies, procedures such as vascular angioplasty, coronary artery bypass grafting, and vascular transplantation are often performed. Unfortunately, these interventions are frequently associated with unfavorable outcomes [2-4]. The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) represent a key pathological process that significantly contributes to the development and progression of vascular occlusion [5].

Mitochondria are found in all eukaryotic cells and play essential roles in energy production, calcium regulation, and cell signaling [6]. In VSMCs, mitochondrial metabolism is increasingly recognized as a vital regulator of cell growth, proliferation, and migration [7-11]. Recent studies investigating the link between mitochondria and vascular diseases have revealed that mitochondrial alterations—such as oxidative stress resulting from changes in reactive oxygen species (ROS) production and dysfunction-induced vascular disorders—are implicated as underlying causes of vascular pathologies [7,12]. However, research on how aberrant mitochondrial DNA (mtDNA) synthesis in VSMCs leads to proliferation, migration, and subsequent neointima formation remains limited.

Mitochondrial carrier family proteins (solute carrier family 25 [SLC25]), mainly localized within the inner mitochondrial membrane, are crucial for regulating cellular metabolism and sustaining energy homeostasis through the transport of amino acids, fatty acids, carboxylic acids, coenzymes, inorganic ions, and nucleotides [13]. Among these, mitochondrial pyrimidine nucleotide carriers mediate the movement of pyrimidine nucleotides across the mitochondrial membrane, a critical process for mtDNA homeostasis [14]. Dysregulation of these transporters has been implicated in several disorders, including cardiac dysfunction, hypothyroidism, hyperinsulinism, and hyperammonemia [15,16]. In particular, solute carrier family 25 member 33 (SLC25A33) facilitates the transport of pyrimidine-derived (deoxy)nucleoside di- and triphosphates, underscoring its essential role in mtDNA maintenance [14]. Recent findings indicate that overexpression of SLC25A33 disrupts pyrimidine pools, leading to mtDNA release and inflammation in mouse embryonic fibroblasts [17]. Other studies have shown that mtDNA release into the cytosol activates the cyclic GMP-AMP synthesis (cGAS)-stimulator of interferon genes (STING) pathway, enhancing type I interferon and nuclear factor kappa B (NF-κB) responses in VSMCs associated with chronic kidney disease [18]. Although cytosolic mtDNA is known to trigger inflammatory responses, the precise role of SLC25A33 in regulating VSMC proliferation, migration, and neointima formation remains unclear. Given the specialized function of SLC25A33 in mitochondrial pyrimidine nucleotide homeostasis and mtDNA replication, its dysregulation may contribute to the release of mtDNA into the cytosol and subsequent activation of the cGAS-STING pathway in VSMCs. Here, we investigated whether downregulation of SLC25A33 suppresses the release of mtDNA into the cytosol of VSMCs, thereby reducing VSMC proliferation and migration and ultimately inhibiting neointima formation in a murine carotid artery ligation model.

METHODS

Ethical statement

All animal procedures were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (KNU2024-0379).

Isolation of primary VSMCs

Aortic smooth muscle cells were isolated from the aortas of 4-week-old male Sprague-Dawley rats (90 to 100 g), as previously described [19]. Briefly, trimmed aortic tissues were rinsed with sterilized cold phosphate-buffered saline (PBS) and then cut into small pieces. These tissue fragments were incubated on dishes containing low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 20% fetal bovine serum (FBS) (HyClone) and 1% antibiotic–antimycotic (Gibco, Grand Island, NY, USA) at 37°C in 5% CO2. The medium was replaced daily. VSMCs from passages four to nine were used in the experiments.

Cell culture and treatments

Primary VSMCs were maintained in low-glucose DMEM (Hy-Clone) with 10% FBS (HyClone) and 1% penicillin–streptomycin (Gibco). For treatments, primary VSMCs were cultured for 24 hours under serum-starved conditions. The cells were then treated with platelet-derived growth factor (PDGF; 10 ng/mL), in the presence or absence of voltage-dependent anion channel 1 (VDAC1)-based inhibitory tool 4 (VBIT-4) (10 μM; Selleckchem, Houston, TX, USA) for 24 hours.

Cell counting

Primary VSMCs were detached using trypsin/ethylenediaminetetraacetic acid (EDTA) solution and counted using a hemocytometer and trypan blue staining.

Small interfering RNA transfection and lentiviral transduction

Cells were transfected with scramble small interfering RNA (siRNA), siSLC25A33, small interfering solute carrier family 25 member 36 (siSLC25A36), sip65, siPDHA1, or si-thiamin pyrophosphokinase 1 (siTPK1; Bioneer, Seoul, Korea) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Lentiviral shSLC25A33 and a control short hairpin RNA (shRNA) plasmid vector (Ori-Gene, Rockville, MD, USA) were transduced into HEK293T cells using the Lenti-X Packaging Single Shot system (Takara Co., Tokyo, Japan) for 48 hours, after which the supernatants were collected.

Quantitative polymerase chain reaction and assessments of mtDNA copy number

Total RNA was isolated using the AccuPrep Universal RNA Extraction Kit (Bioneer). Complementary DNA (cDNA) was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Gene expression analyses were performed using QuantStudio 5 (Applied Biosystems, Foster City, CA, USA) with SYBR Green polymerase chain reaction Master Mix (Applied Biosystems), and expression levels were normalized to those of 36B4. The primer sequences used were as follows: 36B4 forward, TTC-CCA-CTG-GCT-GAA-AAGGT; 36B4 reverse, GCC-GCA-GCC-GCA-AA; SLC25A33 forward, AGA-CTA-GCT-CTT-CGG-ACG-GT; SLC25A33 reverse, TTT-GGA-GTA-GCA-CGC-GAA-GT; SLC25A36 forward, GGC-AGG-TTT-TAC-TGC-GAT-CAC; SLC25A36 reverse, CAA-GCT-GCA-GCC-GAG-TCT-TT; TPK1 forward, CTC-CTC-CAA-CCA-GGA-AAG-CA; TPK1 reverse, ACACCA-GCT-GCC-TTC-CAT-TC; PDHA1 forward, GCCTGG-AGG-CTG-GCA-TAA-A; and PDHA1 reverse, ATGGGC-TCG-ATA-GGC-AGT-GA. For total mtDNA measurement, total DNA was extracted using an AccuPrep Genomic DNA Extraction Kit (Bioneer), and mtDNA levels were normalized to β-actin. The primer sequences were as follows: β-actin forward, GGG-ATG-TTT-GCT-CCA-ACC-AA; β-actin reverse, GCG-CTT-TTG-ACT-CAG-GAT-TTA-A; NADH dehydrogenase subunit 3 (ND3) forward, ACT-CCGAAA-AAG-CAA-ACC-CAT; and ND3 reverse, GAG-GGGGAG-TAG-TAA-GGC-GAT.

Western blot analysis

Cell lysates were separated on tris-glycine gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies against SLC25A33 (OriGene), SLC25A36 (Proteintech, Rosemont, IL, USA), TBK-1, p-TBK1 (S127) (ABclonal, Woburn, MA, USA), retinoblastoma protein (Rb), p-Rb (S780), interferon regulatory factor 3 (IRF3), p-IRF3 (S396), p65, p-p65 (S536), and Actin (Cell Signaling, Danvers, MA, USA). Membranes were then washed in tris-buffered saline with tween 20 (TBST) and visualized using a goat anti-immunoglobulin G secondary antibody (GeneTex, Irvine, CA, USA).

Quantification of uridine 5´-triphosphate

To quantify uridine 5´-triphosphate (UTP) levels in the cytosol and mitochondria, cell fractionation was carried out on SLC25A33-knockdown VSMCs treated with PDGF using the mitochondria/cytosol fraction kit (Abcam, Cambridge, UK). UTP measurements were performed in cytosolic and mitochondrial samples following the manufacturer’s protocol for the general uridine triphosphate enzyme-linked immunosorbent assay (ELISA) Kit (MyBioSource, San Diego, CA, USA).

Pyruvate dehydrogenase activity assay

Pyruvate dehydrogenase (PDH) activity was assessed using the PDH Activity Assay Kit (Elabscience, Houston, TX, USA) according to the manufacturer’s instructions. Briefly, cells were washed with PBS, homogenized in extraction solution, and the supernatant was collected. Standards and samples were incubated with the working solution in a 96-well microplate, and absorbance at 450 nm was measured using a microplate reader (Tecan, Mannedorf, Zurich, Switzerland).

Flow cytometric analysis

For cell cycle analysis, VSMCs were incubated in serum-free medium for 24 hours to synchronize them at the G1 phase, followed by an additional 24 hours incubation with or without PDGF. The cells were then harvested and washed with cold PBS containing 0.01 g/mL bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) and 1 mM EDTA (Sigma). Next, they were fixed in 70% ethanol for 30 minutes and centrifuged at 2,000 rpm for 5 minutes. After removing the supernatant, the cells were washed again with cold PBS and recentrifuged at 2,000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in propidium iodide (PI) staining solution (1×PBS containing 1 mg/mL RNase A, 33 μg/mL PI, and 0.2% Triton X-100) in the dark. Fluorescence from PIDNA complexes was then measured using an Accuri C6 flow cytometer (BD Biosciences, San Diego, CA, USA).

Migration assay

For the wound healing assay, VSMCs were plated at 2.5×10⁵ cells per 35-mm dish and serum-starved for 24 hours. A scratch was generated with a pipette tip, and the cells were washed twice in PBS (WELGENE, Gyeongsan, Korea). They were then incubated with or without PDGF (10 ng/mL) and VBIT-4 (10 μM). When the wound in the positive control had recovered, cells were imaged via microscopy, and the wound area (%) was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). The final area was divided by the initial area and expressed as a percentage.

For the Transwell migration assay, VSMCs (8×10⁴ cells) were first plated in a 60-mm dish and serum-starved for 24 hours. They were then transferred into a Transwell (Corning Incorporated, Kennebunk, ME, USA). VSMCs (1×10⁴ cells in 300 μL per well) were seeded onto the 8.0 μm microporous membrane in the upper chamber (in a medium containing 0.5% FBS and 1% penicillin–streptomycin). Meanwhile, 500 μL of medium with or without PDGF was added to the lower chamber. After 24 hours, cells that had migrated to the lower chamber were fixed with 4% paraformaldehyde for 30 minutes at room temperature and washed twice. They were then stained with 0.05% crystal violet. Microscopic images were acquired, and the number of migrated cells was quantified using ImageJ.

Immunofluorescence

VSMCs were stained with MitoTracker Red CMXRos (Invitrogen, Waltham, MA, USA) and fixed in 4% paraformaldehyde for 15 minutes at room temperature. The cells were then permeabilized with 0.3% Triton X-100 for 10 minutes and blocked with 1% BSA for 30 minutes. VSMCs were incubated overnight at 4°C with an anti-double-stranded DNA (dsDNA) antibody (Abcam). For vascular tissue staining, paraffin-embedded tissue sections were subjected to antigen retrieval, permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature, and blocked with 5% BSA in PBS for 30 minutes. Primary antibodies against cytochrome c oxidase (COX) IV (Cell Signaling) and 8-hydroxy-2´-deoxyguanosine (8-OHdG) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for immunostaining. After washing, cells or tissue sections were incubated with Alexa Fluor 568 or 488-conjugated secondary antibodies (Invitrogen) for 1 hour at room temperature. To evaluate mtDNA replication in VSMCs, the cells were incubated with or without PDGF in the presence of 6-O-propynyl-dG (PdG; Jena Bioscience, Jena, Germany) and analyzed using a CuAAC Cell Reaction Buffer Kit (BTTAA-based) (Jena Bioscience). Image quantification was performed in ImageJ.

Protein oligomerization assay

To examine voltage-dependent anion-selective channel 1 (VDAC1) oligomerization, VSMCs were treated with PDGF (10 ng/mL), with or without siSLC25A33 knockdown, and then washed with Dulbecco’s PBS. The cells were collected and crosslinked using 500 μM ethylene glycol bis (succinimidyl succinate) (EGS, Sigma) in PBS (pH 7.8) for 20 minutes at 30°C. To quench the excess crosslinker, 1.5 M Tris-HCl (pH 7.8) was added to a final concentration of 20 mM, followed by incubation for 5 minutes at room temperature. The processed samples were separated on a NuPAGE® 4%–12% gradient gel (Thermo Fisher Scientific) and analyzed by immunoblotting with an anti-VDAC1 antibody (Cell Signaling).

Mitochondrial reactive oxygen species

Mitochondrial ROS (mtROS) levels were measured using the MitoSOX Red mitochondrial superoxide indicator (Invitrogen). VSMCs were treated with 4 μM MitoSOX reagent working solution and incubated in the dark for 20 minutes at 37°C with 5% CO2. After incubation, the cells were washed with Hank’s balanced salt solution buffer and counterstained with NucBlue Live Cell Stain Ready Probes (Invitrogen). The fluorescence intensity of MitoSOX was quantified using ImageJ.

Immunohistochemistry

Arterial tissue specimens were fixed in 4% paraformaldehyde and embedded in paraffin. After deparaffinization, antigen retrieval was performed using the IHC-Tek Epitope Retrieval Steamer Set (IHC World, Ellicott, MD, USA). Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide for 10 minutes. The slides were then incubated overnight at 4°C with primary antibodies against mouse anti-SLC25A33, anti-p-p65 (S536), and anti-p-TBK1 (S127). Detection was conducted using either the UltraVision LP Detection System (Thermo Fisher Scientific) or horseradish peroxidase-conjugated secondary antibodies (Abcam), with 3,3’-diaminobenzidine as the chromogen. Nuclei were counterstained using Dako hematoxylin (Agilent, Santa Clara, CA, USA).

Murine model of carotid artery ligation

Four-week-old male C57BL/6J mice were subjected to carotid artery ligation-induced neointimal hyperplasia, as described previously [20]. Briefly, neointimal hyperplasia was induced at 4 weeks post-surgery by ligating the unilateral carotid artery near the distal bifurcation with a 5.0 suture tie to block blood flow. Immediately after ligation, Pluronic F-127 gel (Sigma) containing the supernatant of Lenti-shSLC25A33 was applied near the ligation site.

Statistical analysis

Data are presented as the mean±standard error of the mean from at least three independent experiments. Differences between two groups were evaluated for statistical significance using Student’s t-test. For multiple comparisons, one-way analysis of variance (ANOVA) was performed, and individual differences were evaluated using Tukey’s post hoc in GraphPad Prism version 8. 4. 3. (GraphPad Software, Boston, MA, USA). Differences with P<0.05 were considered statistically significant.

RESULTS

SLC25A33 is upregulated in carotid artery ligation-induced neointima of mice and in PDGF-treated VSMCs

To explore the relevance of SLC25A33 in VSMC proliferation and migration, we first examined the protein levels of SLC25A33 in carotid artery ligation-induced neointima and in PDGF-stimulated VSMCs. Histological analysis (hematoxylin and eosin [H&E] staining) of the artery revealed significant neointima formation, with a marked elevation in the intima-to-media ratio following carotid artery ligation compared to sham-operated control mice (Fig. 1A). Immunohistochemical analysis further showed that SLC25A33 was more abundant in the neointimal region (Fig. 1A). To determine whether this phenomenon also occurs in primary cells, we employed rat VSMCs isolated from the aortas of 4-week-old male Sprague-Dawley rats. The rat aorta offers an advantage for in vitro cell isolation and culture because of its greater thickness and more robust smooth muscle layers compared to the mouse aorta [21]. Upon stimulation with PDGF, both mRNA and protein levels of SLC25A33 in rat VSMCs were significantly increased (Fig. 1B and C). However, the expression levels of another mitochondrial pyrimidine nucleotide transporter, SLC25A36, showed no change under these conditions (Fig. 1A, B, and D). These findings indicate a specific role for SLC25A33 in neointima formation.

Knockdown of SLC25A33 inhibits PDGF-stimulated VSMC proliferation and migration

Given that VSMC proliferation and migration are critical processes in neointima formation, we investigated whether SLC25A33 contributes to these processes using siRNA-mediated knockdown. Silencing SLC25A33 significantly suppressed PDGF-induced VSMC proliferation (Fig. 2A). Furthermore, flow cytometric cell cycle analysis revealed that SLC25A33 knockdown reduced the progression from G1 to S phase in response to PDGF (Fig. 2B). Consistent with these results, SLC25A33 knockdown decreased the phosphorylation of Rb, a well-known proliferation marker (Fig. 2C). Additionally, both Transwell migration and wound healing assays demonstrated that PDGF-induced VSMC migration was diminished following SLC25A33 knockdown (Fig. 2D). Collectively, these results suggest that SLC25A33 is essential for VSMC proliferation and migration.

Knockdown of SLC25A33 decreases PDGF-stimulated mtROS and mtDNA production and attenuates the cytosolic release of mtDNA

A recent study showed that PDGF stimulation elevates mtROS levels in VSMCs, promoting proliferation and migration [22]. We confirmed that PDGF increases mtROS in VSMCs (Fig. 3A). Notably, SLC25A33 knockdown mitigated this PDGF-stimulated mtROS generation (Fig. 3A). Given that SLC25A33 is required for mtDNA synthesis as a pyrimidine transporter [14], we next examined whether SLC25A33 knockdown downregulates both uridine 5´-triphosphate (UTP) and mtDNA in PDGF-treated VSMCs. Indeed, PDGF-treated VSMCs had increased mitochondrial UTP levels and mtDNA, both of which were reversed by SLC25A33 knockdown (Fig. 3B and C). Considering that damaged mtDNA—often induced by stress factors such as mtROS—can be released into the cytosol [23], we investigated whether SLC25A33 modulates mtDNA synthesis and cytosolic mtDNA release in PDGF-treated VSMCs. Following PDGF treatment, newly synthesized mtDNA levels were increased, and SLC25A33 knockdown reversed this effect (Fig. 3D). Additionally, PDGF-stimulated VSMCs exhibited a pronounced rise in cytosolic mtDNA relative to untreated VSMCs, and this phenomenon was completely suppressed by SLC25A33 knockdown (Fig. 3E). Since a recent study showed that UTP sustains pyruvate oxidation by activating PDH [24], we assessed whether the alterations in mitochondrial UTP mediated by the inhibition of SLC25A33 also affect cytosolic UTP availability or PDH activity in VSMCs. However, silencing SLC25A33 did not induce notable changes in cytosolic UTP levels or PDH activity (Fig. 3B, Supplementary Fig. 1A). Moreover, neither TPK1 inhibition nor pyruvate dehydrogenase E1 component subunit alpha 1 (PDHA1) inhibition influenced total mtDNA levels or its cytosolic release (Supplementary Fig. 1B-E). Collectively, these data suggest that PDGF-treated VSMCs exhibit enhanced mtROS and upregulated SLC25A33-mediated mtDNA synthesis, culminating in increased cytosolic mtDNA.

SLC25A33 knockdown and VBIT-4 treatment inhibit VSMC proliferation and migration by reducing cytosolic mtDNA levels, thereby disrupting the cGAS-STING-TBK1-NF-κB pathway

Given that cytosolic mtDNA activates the cGAS-STING-TBK1 pathway, leading to phosphorylation of IRF3 and NF-κB [25], we asked whether SLC25A33 inhibition blocks the PDGF-induced cGAS-STING-TBK1 cascade by diminishing cytosolic mtDNA. PDGF-treated VSMCs showed elevated phosphorylation of TBK1, p65, and IRF3, which was reversed by SLC25A33 knockdown; in contrast, SLC25A36 inhibition had no significant effects (Fig. 4A and B). Previous reports have demonstrated that mtDNA associates with VDAC1 to promote VDAC1 oligomerization, thereby facilitating mtDNA release into the cytosol [23]. Consistent with this mechanism, either knocking down SLC25A33 or treating cells with 2’,3’-dideoxycytidine (ddC), which lowers mtDNA, counteracted the PDGF-induced increase in VDAC1 oligomerization (Fig. 4C and D). Furthermore, VBIT-4, a specific VDAC1 oligomerization inhibitor [26], attenuated the PDGF-stimulated cGAS-STING-TBK1 pathway (Fig. 4E) and reduced PDGF-induced proliferation and migration in VSMCs (Fig. 4F and G). To clarify whether cytosolic mtDNA drives the cGAS-STING-TBK1-mediated NF-κB activation that ultimately promotes VSMC proliferation and migration, we knocked down p65 in VSMCs using siRNA (Fig. 4H) and examined its impact on PDGF-stimulated proliferation and migration. As expected, p65 knockdown attenuated PDGF-induced proliferation and migration at levels similar to VBIT-4 treatment (Fig. 4I and J). It is likely that SLC25A33 regulates the cGAS-STING-TBK1-NF-κB pathway by modulating cytosolic mtDNA levels, influencing VSMC proliferation and migration.

These findings suggest that SLC25A33 influences VSMC proliferation and migration by regulating the cGAS-STING-TBK1-NF-κB pathway via modulation of cytosolic mtDNA.

Knockdown of SLC25A33 in VSMCs suppresses carotid artery ligation-induced neointimal hyperplasia

Finally, to determine whether SLC25A33 inhibition curbs neointima formation in an occlusive vascular disease model, we ligated the carotid arteries of mice and locally introduced shSLC25A33 lentivirus near the ligation site. Histological analysis (H&E staining) indicated that arteries treated with Lenti-shSLC25A33 exhibited markedly decreased neointima formation and intima-to-media ratio compared to arteries treated with a control shRNA plasmid vector (Fig. 5A). Immunohistochemistry also revealed that artery ligation led to enhanced expression of SLC25A33 in the neointimal region, accompanied by elevated p-p65 and p-TBK1 (Fig. 5B and C). Additionally, we observed that ligation substantially increased the cytosolic localization of oxidized DNA, detected by 8-OHdG, a marker of oxidative DNA damage (Fig. 5D and E) [27]. Conversely, arteries treated with Lenti-shSLC25A33 showed a significant reduction in these effects (Fig. 5D and E).

DISCUSSION

In this study, we found that SLC25A33 expression was upregulated in PDGF-stimulated VSMCs and in carotid artery ligation-induced neointima lesions in mice. The knockdown of SLC25A33 reduced mtDNA synthesis and mtROS production, leading to decreased cytosolic mtDNA levels. This suppression, in turn, inhibited VSMC proliferation and migration, which rely on the cGAS-STING-TBK1 pathway, and significantly attenuated neointima formation induced by arterial ligation in mice.

Several studies have demonstrated the therapeutic potential and research significance of mitochondrial transporters in obstructive vascular disease [28,29]. SLC25A3, which mediates phosphate uptake in the mitochondria of VSMCs, is associated with superoxide production and calcification [28]. In contrast, the mitochondrial calcium uniporter (MCU) increases mitochondrial calcium influx and mitophagy, thereby limiting excessive VSMC proliferation [29]. Additionally, accumulating evidence points to the importance of SLC25A33 in various cellular processes and diseases. Notably, pyrimidine transport via SLC25A33 is essential for maintaining cellular function, and its imbalance plays a critical role in mtROS generation [17,30]. Insulin-like growth factor 1-induced SLC25A33 expression promotes cell growth and protects breast and cervical cancer cells from differentiation driven by mitochondrial dysfunction-related ROS [30,31]. Additionally, YME1 like 1 ATPase (YME1L) knockout disrupts the regulation of SLC25A33, contributing to pyrimidine imbalance and triggering inflammatory responses in the retina [17]. While these findings highlight the involvement of SLC25A33 in cancer cells and innate immunity [17,30,31], its role in vascular diseases has been relatively unexplored. In the present study, we demonstrated that upregulated SLC25A33 increased UTP levels and mtDNA synthesis in PDGF-treated VSMCs, thereby promoting VSMC proliferation and migration. This observation is consistent with a recent study investigating coronary artery disease-associated genetic variants in human VSMCs, which showed that targeting pyrimidine transporters induces VSMC apoptosis [32]. Collectively, these findings suggest that SLC25A33 could be a promising therapeutic target for occlusive vascular diseases with excessive growth of VSMCs. Nonetheless, further investigations are needed to elucidate the role of SLC25A33 in occlusive vascular diseases and to determine its translational potential.

mtDNA is essential for cellular function, encoding oxidative phosphorylation complexes that are critical for respiration and adenosine triphosphate production [33]. Recent work has shown that mtDNA released into the cytoplasm can contribute to the onset of arthritis and promote cancer progression [34, 35]. Previous studies on harmful effects involving mtDNA mainly focus on the damage and mutations induced by ROS. Several reports indicate that ROS is involved in atherosclerotic plaque instability and VSMC migration, thereby driving vascular remodeling [36,37]. While ROS has been pursued as a therapeutic target for vascular diseases, the vicious cycle between ROS and mtDNA remains a major obstacle [38]. It has been reported that mtDNA stabilizes the N-terminus of VDAC1, facilitating VDAC1 oligomerization and increasing mtROS [23]. Our results show that inhibiting SLC25A33 not only decreased PDGF-induced VDAC1 oligomerization but also reduced mtROS generation. VDAC1 oligomerization is known to be key for the cytosolic release of mtDNA, which activates IRF3 and NF-κB via the cGAS-STING-TBK1 pathway; this, in turn, enhances VSMC proliferation and migration [23,25,39]. Consistent with these observations, our data revealed that VBIT-4 treatment suppressed the cGAS-STING-TBK1-NF-κB pathway, leading to substantial reductions in VSMC proliferation and migration. These effects closely mirrored those following SLC25A33 knockdown.

This study had some limitations. Although our in vitro findings demonstrated cytosolic mtDNA release in PDGF-stimulated VSMCs, and we confirmed the cytosolic accumulation of oxidized DNA using 8-OHdG in a carotid artery ligation model, 8-OHdG is an indirect marker and does not directly demonstrate cytosolic mtDNA release. Therefore, further in vivo experiments are required to validate our findings. Despite this limitation, these findings underscore the necessity for more fundamental strategies to tackle occlusive vascular diseases arising from the interplay of ROS and mtDNA.

In conclusion, our study demonstrated that targeting SLC25A33 inhibits VSMC proliferation and migration by reducing cytosolic mtDNA levels, thereby suppressing the cGAS-STING-TBK1-NF-κB pathway. These results indicate that SLC25A33 could serve as a potential therapeutic target for occlusive vascular diseases driven by the vicious cycle between mtDNA damage and ROS generation.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: Y.K.C., Y.M.L., K.G.P., H.S.K., J.K.B.

Acquisition, analysis, or interpretation of data: D.K., J.S.

Drafting the work or revising: D.K., K.G.P., H.S.K., J.K.B.

Final approval of the manuscript: K.G.P., H.S.K., J.K.B.

FUNDING

This work was supported by National Research Foundation of Korea (NRF) grants RS-2021-NR059744, RS-2020-NR049556, and RS-2021-NR061783, funded by the Ministry of Science and Information and Communication Technology (ICT).

ACKNOWLEDGMENTS

None

Fig. 1.

Solute carrier family 25 member 33 (SLC25A33) expression is increased by arterial ligation and platelet-derived growth factor (PDGF) treatment in vascular smooth muscle cells (VSMCs). (A) Representative images of hematoxylin and eosin (H&E) staining (upper panel; scale bar=50 μm) and immunohistochemical staining of SLC25A33 and solute carrier family 25 member 36 (SLC25A36) (lower panel; scale bar=10 μm) in the Sham and Ligation groups (left panel). The intimal area, medial area, intima/media ratio, SLC25A33-stained area, and SLC25A36-stained area are shown on the right. (B) Effect of PDGF on mRNA levels of SLC25A33 and SLC25A36. (C, D) Effect of PDGF on protein levels of SLC25A33 (C) and SLC25A36 (D) in VSMCs. Data are expressed as the mean±standard error of the mean of three independent measurements. NS, not significant; Ctrl, control. ^aP<0.01, ^bP<0.001.

Fig. 2.

Solute carrier family 25 member 33 (SLC25A33) regulates the proliferation and migration of vascular smooth muscle cells (VSMCs). (A) Representative images (left panel) and corresponding cell counts (right panel) in platelet-derived growth factor (PDGF)-treated VSMCs transfected with or without SLC25A33-targeting small interfering RNA (siRNA). (B) Histogram depicting flow cytometric analysis (left panel) and cell cycle distribution (right panel) in PDGF-treated VSMCs with or without SLC25A33-targeting siRNA. (C) Effect of SLC25A33 knockdown on protein levels of phosphorylated retinoblastoma protein (p-Rb), Rb, and SLC25A33 in PDGF-treated VSMCs. (D) Representative images illustrating the effects of SLC25A33-targeting siRNA on VSMC migration (left panel), with quantification of migration and wound area (right panel). siSLC25A33 is referred to as siA33. Data in the bar graphs represent the mean±standard error of the mean of three independent measurements. Scale bar=40 μm. Ctrl, control; NS, not significant; FL2-A, fluorescence channel 2-area; G1, gap 1 phase; M, mitosis phase; S, synthesis phase; sc, scrambled. ^aP<0.01, ^bP<0.001.

Fig. 3.

Solute carrier family 25 member 33 (SLC25A33) regulates mitochondrial ROS (mtROS) and mitochondrial DNA (mtDNA) production in response to platelet-derived growth factor (PDGF), thereby promoting mtDNA release into the cytosol. (A) Representative immunofluorescence images of MitoSOX Red (left panel; scale bar=10 μm) and quantification of fluorescence intensity (right panel) in PDGF-treated vascular smooth muscle cells (VSMCs) with or without SLC25A33-targeting small interfering RNA (siRNA). (B) Effect of SLC25A33 knockdown on mitochondrial and cytosolic uridine 5’-triphosphate (UTP) levels in PDGF-treated VSMCs. (C) Effect of SLC25A33 knockdown on total mtDNA levels in PDGF-treated VSMCs. (D) Representative immunofluorescence images acquired using 6-O-propynyl-dG (PdG) and click chemistry (left panel; scale bar=5 μm) and quantification of PdG (right panel) in PDGF-treated VSMCs with or without SLC25A33-targeting siRNA. (E) Representative immunofluorescence images of double-stranded DNA (dsDNA) and mitochondria (left panel; scale bar=10 μm) and quantification of cytosolic dsDNA foci (right panel) in PDGF-treated VSMCs with or without SLC25A33-targeting siRNA. siSLC25A33 is referred to as siA33. Data in the bar graphs represent the mean±standard error of the mean of three independent measurements. Ctrl, control; NS, not significant; DAPI, 4’,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 4.

Solute carrier family 25 member 33 (SLC25A33) knockdown and VDAC1-based inhibitory tool 4 (VBIT-4) treatment inhibit vascular smooth muscle cell (VSMC) proliferation and migration by suppressing the cytosolic mitochondrial DNA (mtDNA)-mediated cyclic GMP-AMP synthesis (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1)-nuclear factor kappa B (NF-κB) pathway. (A, B) Effect of SLC25A33 (A) or solute carrier family 25 member 36 (SLC25A36) (B) knockdown on the phosphorylation of TANK-binding kinase 1 (TBK1), p65, and interferon regulatory factor 3 (IRF3) in platelet-derived growth factor (PDGF)-treated VSMCs. (C, D) Effect of SLC25A33 knockdown (C) or 2’,3’-dideoxycytidine (ddC) treatment (D) on voltage-dependent anion-selective channel 1 (VDAC1) oligomerization in PDGF-treated VSMCs. (E) Effect of VBIT-4 on the phosphorylation of TBK1, p65, and IRF3 in PDGF-treated VSMCs. (F, G) Representative images showing the effects of VBIT-4 on VSMC proliferation (F) and migration (G) (upper panel), with quantification of cell number, migration, and wound area (lower panel). (H) Effect of p65 knockdown on p65 protein expression in PDGF-treated VSMCs. (I, J) Representative images illustrating the effects of p65-targeting small interfering RNA (siRNA) on VSMC proliferation (I) and migration (J) (upper panel), with quantification of cell number, migration, and wound area (lower panel). Asterisks indicate nonspecific bands. siSLC25A33 and siSLC25A36 are referred to as siA33 and siA36, respectively. Data in the bar graphs represent the mean±standard error of the mean of three independent measurements. Scale bar=40 μm. EGS, ethylene glycol bis; sc, scrambled. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 5.

Solute carrier family 25 member 33 (SLC25A33) knockdown in vascular smooth muscle cells (VSMCs) reduces neointima formation in a carotid artery ligation model. (A) Representative images of hematoxylin and eosin (H&E) staining (upper panel; scale bar=50 μm), indicating the intimal and medial layers, as well as the intima/media ratio (lower panel). (B, C) Immunohistochemical staining with antibodies against SLC25A33, phosphorylated p65 (p-p65), and phosphorylated TANK-binding kinase 1 (p-TBK1) in mouse carotid artery tissue sections (B) and quantification of positive staining for SLC25A33, p-p65, and p-TBK1 (C). (D, E) Representative immunofluorescence images of cytochrome c oxidase (COX) IV and 8-hydroxy-2’-deoxyguanosine (8-OHdG) (D) and quantification of cytosolic 8-OHdG in mouse carotid artery tissue sections (E). COX IV protein is used as a mitochondrial marker. shSLC25A33 is referred to as shA33. Data in the bar graphs represent the mean±standard error of the mean of six independent animals. Scale bar=10 μm. NS, not significant; DAPI, 4’,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.01, ^cP<0.001.

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About this article

Kim D, Shin J, Choi YK, Lee YM, Park KG, Kim HS, Byun JK. Targeting SLC25A33 Suppresses Vascular Smooth Muscle Cell Proliferation and Migration by Reducing Cytosolic mtDNA Levels: Implications for Occlusive Vascular Diseases. Diabetes Metab J. 2026;50(1):139-152.

Received: Oct 14, 2024; Accepted: Apr 08, 2025

DOI: https://doi.org/10.4093/dmj.2024.0632.

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