Vascular Smooth Muscle–Specific NLRP3 Hyperactivation Drives Arterial Intimal Hyperplasia in Mice
Yun-Ting Wang, Alexandra K Moura, Rui Zuo, Wei Gao, Bradley K McConnell, Guangbi Li, Pin-Lan Li, Xiang Li, Yang Zhang
TL;DR
Hyperactivation of the NLRP3 inflammasome in vascular smooth muscle cells worsens arterial thickening and inflammation in mice, suggesting a new therapeutic target for vascular diseases.
Contribution
First direct evidence that VSMC-specific NLRP3 hyperactivation drives intimal hyperplasia and foam cell-like transition in vivo.
Findings
VSMC NLRP3 gain-of-function increases vascular inflammation and cell death.
NLRP3 hyperactivation worsens neointimal lesion growth and lipid accumulation.
Impaired TFEB-dependent lysosome-autophagy homeostasis contributes to VSMC dysfunction.
Abstract
Intimal hyperplasia is a major contributor to restenosis after vascular interventions and to atherosclerotic lesion progression, driven largely by vascular smooth muscle cell (VSMC) inflammatory activation, phenotypic switching, and maladaptive remodeling. While NOD-like receptor pyrin domain 3 (NLRP3) inflammasome activity has been linked to vascular diseases, direct evidence that VSMC-intrinsic NLRP3 hyperactivation drives VSMC dysfunction and intimal hyperplasia in vivo has been lacking. Here, we generated a VSMC-specific Nlrp3 knock-in mouse (Nlrp3 L351P/+/Myh11−Cre, “Nlrp3SMKI”) and subjected it to carotid partial ligation under hypercholesterolemic conditions. VSMC Nlrp3 gain-of-function knock-in induced robust caspase-1 activation in vivo, including in unligated arteries, and markedly amplified injury-triggered inflammasome activation. Nlrp3SMKI arteries exhibited heightened…
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Taxonomy
TopicsInflammasome and immune disorders · Curcumin's Biomedical Applications · Phagocytosis and Immune Regulation
Introduction
Intimal hyperplasia is a major pathological driver of restenosis after vascular interventions and contributes to lumen narrowing during atherosclerotic disease progression^1,2^. Despite improvements in stent design and adjunct pharmacotherapy, neointimal lesion formation remains a common and clinically significant obstacle^3^. At the tissue level, intimal hyperplasia reflects a coordinated response to vascular injury and disturbed flow, involving inflammatory activation of the vessel wall, recruitment of leukocytes, and remodeling of extracellular matrix that collectively promote neointimal growth^4^. Defining the cell-intrinsic mechanisms that amplify this maladaptive remodeling program is essential for developing more precise, vessel wall–directed therapies.
Vascular smooth muscle cells (VSMCs) are central effectors of intimal hyperplasia. Following injury, VSMCs undergo phenotypic switching from a contractile to a synthetic state, characterized by enhanced proliferation, migration into the intima, and secretion of remodeling factors, such as matrix metalloproteinases^1,5,6^. This plasticity is strongly influenced by inflammatory cues and cell stress, which can create feed-forward loops that perpetuate VSMC dysfunction and accelerate lesion progression^7^. Beyond classical neointimal expansion, growing evidence from lineage-tracing and single-cell studies indicates that VSMCs can adopt macrophage-like gene programs and contribute substantially to foam cell populations in mouse and human atherosclerotic plaques^8,9^. Thus, VSMC maladaptation encompasses not only proliferative/migratory remodeling but also lipid-driven phenotypic transitions that can worsen lesion burden and complexity.
Inflammasomes are innate immune signaling platforms that couple cellular stress sensing to inflammatory effector outputs^10^. Among them, the NLRP3 inflammasome is activated by diverse vascular disease–relevant stimuli, including oxidative stress, and lipid-associated danger signals^11–13^. Upon activation, NLRP3 promotes caspase-1 activation, which drives the maturation of IL-1β/IL-18 and the cleavage of GSDMD, leading to pore formation, the release of inflammatory mediators, and pyroptosis-associated cell injury^14,15^. Consistent with these functions, NLRP3 pathway activation has been implicated in atherosclerosis and post-injury vascular remodeling^16–18^. However, the vascular wall contains multiple potential cellular sources of NLRP3 activity, including infiltrating immune cells, endothelial cells, and VSMCs, making it difficult to determine whether NLRP3 signaling in VSMCs is sufficient to initiate and sustain the remodeling cascade culminating in intimal hyperplasia and lipid-laden VSMC phenotypes.
Our recent work showed that carotid partial ligation injury under hypercholesterolemic conditions activates the NLRP3 inflammasome in VSMCs and that global Nlrp3 deficiency attenuates injury-induced inflammasome activation and intimal hyperplasia^18^. While these findings support a causal role for NLRP3 signaling in vascular remodeling, global loss-of-function approaches cannot distinguish the relative contributions of hematopoietic versus vessel wall–resident compartments, nor can they directly establish the sufficiency of VSMC-intrinsic NLRP3 activation in driving VSMC dysfunction in vivo^18^. This knowledge gap is highly relevant to translational strategy: if VSMC-intrinsic NLRP3 hyperactivation is sufficient to amplify inflammation, pyroptosis-associated injury, and proliferative/migratory remodeling, then targeting NLRP3 signaling specifically within VSMCs could represent a focused therapeutic avenue to limit neointimal growth while reducing systemic immune perturbation.
Here, we directly test the VSMC-intrinsic contribution of NLRP3 by generating a VSMC-specific Nlrp3 gain-of-function model. We crossed Nlrp3 ^L351PneoR^ mice with Myh11-Cre to create Nlrp3 ^L351P/+/Myh11−Cre^ mice (Nlrp3 ^SMKI^) and littermate controls, leveraging a gain-of-function NLRP3 substitution analogous to a human cryopyrin-associated periodic syndromes (CAPS)-associated mutation that enhances inflammasome activation^19^. Using carotid partial ligation combined with a Paigen diet to induce hypercholesterolemia-associated remodeling^20^, we assessed inflammasome activation, vascular inflammation, GSDMD-linked cell injury, VSMC proliferation and migration, neointimal lesion growth, and lipid accumulation associated with VSMC-to–foam cell–like transition in vivo. This approach provides direct mechanistic evidence for how VSMC-intrinsic NLRP3 hyperactivation programs maladaptive vascular remodeling and identifies VSMC NLRP3 signaling as a translationally actionable target to treat intimal hyperplasia and VSMC-driven foam cell transition.
Materials and Methods
Antibodies and reagents
Primary antibodies: NLRP3 (Biotechne MAB7578), cleaved-caspase1 (Affinity AF4005), FITC-α-SMA (Sigma F3777), Cy3-αSMA (Sigma C6198), VCAM1 (Abcam ab134047), F4/80 (BD 565411), CD206 (CST 24595), GSDMD (Sigma G7422), Ki67 (Abcam ab16667), MMP2 (Abcam ab92536), PLIN2 (Proteintech 15294–1-AP), TFEB (Bethyl Laboratories A303–673A), LAMP2A (Abcam ab18528), LAMP1 (BD 553792), LC3 (CST 12741S).
Secondary antibody for immunofluorescence (1:500 dilution): Donkey anti-Mice IgG (H + L) Secondary Antibody, Alexa Fluor^®^ 488 conjugate (Thermo Fisher A-21202); Donkey anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor^®^ 488 conjugate (Thermo Fisher A21206); Donkey anti-Mice IgG (H + L) Secondary Antibody, Alexa Fluor^®^ 555 conjugate (Thermo Fisher A-31570); Donkey anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor^®^ 555 conjugate (Thermo Fisher A-31572); Donkey anti-Rat IgG (H + L) Highly Cross-Adsorbed, Alexa Fluor 594 (Thermo Fisher A21209); Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor^™^ Plus 647 (Thermo Fisher A32795TR); Donkey anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor^™^ 647 (Thermo Fisher A78947).
Reagents: Green FLICA Caspase-1 Assay Kit (Immunochemistry Technologies #98), In Situ Cell Death Detection Kit (Sigma 11684795910), Sirius Red (Sigma 365548–5g), Fast green (Sigma F7252–5g), Hematoxylin and Eosin staining kit (Teomics HAE- 1), Oil red O (VWR 135140–100G), Bodipy (Thermo Fisher D3922).
Mice
All procedures involving animals were conducted in accordance with protocols approved by the University of Houston Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC). Mice were maintained in a specific pathogen-free, temperature-regulated animal facility on a 12 h light/12 h dark schedule, with free access to water and a standard chow diet (PicoLab^®^ Rodent Diet 20; 5053) unless noted otherwise.
Generation of VSMC-specific Nlrp3 Knock-in mice
Nlrp3 ^L351PneoR^ mice (JAX:017970) harbor a Cre-conditional Nlrp3 ^L351P^ allele in the absence of Cre; the neoR cassette disrupts Nlrp3 expression, whereas Cre-mediated recombination restores expression of an Nlrp3 transcript carrying the L351P mutation in Cre-specific tissues. Myh11-Cre transgenic mice (JAX:007742) were obtained from The Jackson Laboratory. To generate a VSMC-specific gain-offunction (GOF) model, Nlrp3 ^L351PneoR^ mice were crossed with Myh11-Cre mice to produce Nlrp3 ^L351P/+/Myh11−Cre^ (Nlrp3 ^SMKI^) mice and Cre-negative littermate Nlrp3 ^L351P/+/WT^ (Nlrp3 ^ctrl^) controls. Both Nlrp3 ^SMKI^ and littermate control Nlrp3 ^ctrl^ mice were heterozygous for the Nlrp3 ^L351P/+^ allele. Genotypes were confirmed by PCR using primers and conditions provided by the vendor.
Partial carotid ligation surgery
Partial carotid ligation was performed in 6-month-old male Nlrp3 ^SMKI^ mice and Nlrp3 ^ctrl^ littermates. The left carotid artery was subjected to partial ligation, while the right side remained unligated and was used as the control vessel^18,20,21^. Following surgery, mice were maintained on a Paigen’s diet (Research Diets, D12336) containing 35% kcal from fat and 1.25% cholesterol for 4 weeks. Mice were anesthetized and euthanized by cervical dislocation, and bilateral carotid arteries were excised for immunohistochemistry and immunofluorescence. Tissues were embedded in Tissue-Tek O.C.T., snap-frozen in liquid nitrogen, stored at − 80°C, and cryosectioned at 8 μm.
Immunofluorescence staining
Frozen sections were briefly equilibrated at room temperature and air-dried for no longer than 15 min. Sections were then fixed in freshly prepared 4% paraformaldehyde for 15 min at room temperature, followed by rinsing in PBS. For simultaneous permeabilization and blocking, sections were incubated for 1 h at room temperature in PBS containing 5% BSA and 0.3% Triton X-100. Primary antibodies (as indicated) were applied and incubated overnight at 4°C. After washing, sections were incubated with species-matched Alexa Fluor–conjugated secondary antibodies (488/555/647) for 1 h at room temperature. Nuclei were counterstained with DAPI for 15 min and slides were mounted using an antifade mounting medium.
Fluorescence images were acquired using an Olympus IX73 imaging system under identical exposure and acquisition parameters across groups. Quantification was performed in Image-Pro Plus 6.0 by measuring mean fluorescence density (as applicable) or calculating Pearson’s correlation coefficient (PCC) for protein colocalization within the carotid intima and media. For each carotid artery, three consecutive serial sections were stained and analyzed for each marker, and values from the three sections were averaged to generate a single measurement per animal.
For regional analysis, unligated arteries were quantified in the medial layer only, identified as the continuous α-SMA–positive band. In ligated arteries, α-SMA–positive VSMCs in both the neointima (migrated VSMCs) and the media were included. Specificity was assessed using adjacent sections incubated with matched isotype IgG at the same concentration in place of primary antibodies; no appreciable nonspecific background signal was detected. Potential elastin autofluorescence in the AF488 channel under low-signal conditions was minimized by imaging α-SMA with strong FITC signals (≤ 50 ms exposure), while most targets were collected in the AF555 or AF647 channels, thereby avoiding interference from elastin-derived fluorescence^18^.
FLICA analysis of caspase-1 activation
Frozen sections were equilibrated to room temperature and air-dried for ≤ 15 min. Without fixation, sections were incubated with the FLICA reagent together with Hoechst 33342 (FLICA Assay Kit) for 1.5 h at 37°C. Slides were then washed three times in pre-warmed assay buffer (10 min each, 37°C). The FLICA-derived green fluorescence was captured using an Olympus IX73 imaging system^18,22^.
TUNEL staining
Frozen tissue sections were fixed with freshly prepared 4% paraformaldehyde for 20 min at room temperature, followed by washing in PBS for 30 min. Sections were then permeabilized in 0.1% Triton X-100/0.1% sodium citrate for 2 min on ice. TUNEL reaction mixture was freshly prepared and 50 μL was applied to each section, followed by incubation for 60 min at 37°C in a humidified chamber protected from light; negative controls were incubated with Label Solution lacking enzyme. After incubation, slides were rinsed three times with PBS and imaged by fluorescence microscopy using FITC settings (Ex 450–500 nm; Em 515–565 nm)^18^.
Sirius red and fast green staining
Frozen sections were fixed in Kahle’s fixative for 15 min at room temperature, rinsed with PBS, and stained for 60 min at room temperature in a picric acid–based Sirius Red/Fast Green solution (0.1% Sirius Red and 0.1% Fast Green in saturated aqueous picric acid). Slides were then rinsed twice in acidified water (0.1 N HCl in ddH_2_O; 1 min each). Sections were dehydrated through two changes of 100% ethanol and cleared in xylene prior to coverslipping with mounting medium. Images were acquired immediately using an Olympus IX73 imaging system^23^.
H&E staining
Hematoxylin and eosin staining was performed using a commercial kit (Teomics, HAE-1). Briefly, frozen sections were fixed in 4% paraformaldehyde for 15 min at room temperature and rinsed with distilled water. Sections were stained with Mayer’s hematoxylin for 5 min, washed twice in distilled water, and briefly blued using bluing reagent (10–15 s) followed by two additional water rinses. Slides were then dipped in absolute ethanol to remove residual water, counterstained with Eosin Y for 2–3 min and rinsed in absolute ethanol. After clearing, sections were mounted with synthetic resin and imaged on an Olympus IX73 microscope^18,23^.
Oil red O staining
Frozen sections (8 μm) were allowed to air-dry and fixed in 4% PFA for 15 min. Slides were then rinsed in distilled water (three changes) and immersed in distilled water for 20 min. To minimize aqueous carryover, sections briefly dipped in 60% isopropanol for 1 min prior to staining. Sections were incubated with freshly prepared Oil Red O working solution for 30 min, rinsed in 60% isopropanol for 2 min, and washed twice with distilled water. Slides were mounted with mounting medium and imaged immediately using an Olympus IX73 imaging system^22,23^.
BODIPY 493/503 staining
Neutral lipid droplets were visualized using BODIPY 493/503 (Invitrogen, D3922). Frozen sections were fixed in 4% paraformaldehyde for 10 min and rinsed with PBS-T. Sections were then incubated for 1 h at room temperature with BODIPY 493/503 (40 μg/mL) together with DAPI diluted in PBS-T. After washing in PBS-T, slides were mounted with Mowiol and imaged immediately using an Olympus IX73 imaging system^24,25^.
Statistical analysis
All quantitative results are reported as mean ± SEM. Given the small cohort sizes (N < 10) and the lack of normality in our datasets, nonparametric tests were applied throughout. Comparisons among three or more groups were performed using the Kruskal–Wallis test with Dunn’s post hoc multiple-comparisons correction. For two-group comparisons, the Mann–Whitney U test was used. Analyses were conducted in GraphPad Prism (version 9.0; GraphPad Software, USA). A two-sided P value < 0.05 was considered statistically significant. Sample sizes were selected based on prior publications and pilot data to provide adequate sensitivity for detecting biologically relevant effects. Data was excluded only for pre-specified reasons, including sample contamination, technical failure, or clear measurement artifacts; outliers were handled according to predefined criteria. Investigators were blinded to group assignment during experimental procedures and outcome quantification to reduce bias and ensure objective interpretation.
Results
VSMC-specific Nlrp3 gain-of-function mutation activates the NLRP3 inflammasome in vivo.
In our recent work, we showed that carotid partial ligation injury under hypercholesterolemic conditions activates the NLRP3 inflammasome in VSMCs, and that global Nlrp3 deficiency attenuates injury-induced inflammasome activation and intimal hyperplasia^18^. To directly define the contribution of VSMC-intrinsic NLRP3 to pathological vascular remodeling, we generated a VSMC-specific Nlrp3 knock-in model by crossing Nlrp3 ^L351PneoR^ mice with Myh11-Cre mice, yielding Nlrp3 ^L351P/+/Myh11−Cre^ (Nlrp3 ^SMKI^) mice and littermate Nlrp3 ^L351P/+/WT^ (Nlrp3 ^ctrl^) controls (Fig. 1A). The L351P substitution (from leucine to proline at residue 351) corresponds to the human L353P mutation associated with cryopyrin-associated periodic syndromes (CAPS) and is predicted to enhance NLRP3 inflammasome assembly/activation through altered interdomain or protein–protein interactions^19^.
We subjected Nlrp3 ^SMKI^ and Nlrp3 ^ctrl^ mice to partial carotid ligation and Paigen’s diet to induce intimal hyperplasia^18,20,21^, then assessed inflammasome activation in ligated and contralateral unligated arteries. NLRP3 immunostaining was increased after ligation in both genotypes, and this injury-induced upregulation was significantly greater in Nlrp3 ^SMKI^ arteries; by contrast, NLRP3 levels were comparable between genotypes in unligated arteries (Fig. 1B–C). Importantly, FLICA labeling (Fig. 1D–E) and cleaved caspase-1 staining (Fig. 1F–G) demonstrated that VSMC Nlrp3 GOF was sufficient to induce caspase-1 activation even in unligated arteries and further amplified ligation-induced caspase-1 activation in injured arteries. Together, these data establish a VSMC-intrinsic model of enhanced NLRP3–caspase-1 activity in the injured artery. This heightened inflammasome activation provides a mechanistic basis for downstream inflammatory signaling and vascular wall immune cell recruitment during remodeling.
VSMC Nlrp3 gain-of-function exacerbates vascular inflammation after injury
Vascular inflammation is a hallmark of intimal hyperplasia and is characterized by increased adhesion molecule expression and recruitment of inflammatory cells^26^. We therefore examined whether VSMC Nlrp3 GOF enhances inflammatory activation after injury. Consistent with our prior observations^18^, partial ligation markedly increased VCAM-1 expression in VSMC of the vessel wall, and this response was significantly greater in Nlrp3 ^SMKI^ mice, with the most prominent signal localized to the neointimal region (Fig. 2A–B). In parallel, immunostaining for the pan-macrophage marker F4/80 (Fig. 2C–D) revealed increased macrophage accumulation in ligated arteries, which was further amplified in Nlrp3 ^SMKI^ mice. Staining for M2-polarized macrophage marker CD206 (Fig. 2E–F) also increased after injury and was elevated to a greater extent in ligated arteries from Nlrp3 ^SMKI^ mice. Together, these results support that VSMC-intrinsic NLRP3 hyperactivation potentiates injury-induced VCAM-1 upregulation and promotes macrophage accumulation within the remodeling vessel wall, thereby exacerbating vascular inflammation. Thus, VSMC NLRP3 hyperactivation amplifies endothelial/vascular inflammatory activation and creates a permissive environment for leukocyte adhesion and accumulation in the lesion.
VSMC Nlrp3 gain-of-function enhances GSDMD upregulation and cell death after vascular injury
Given the canonical linkage between NLRP3–caspase-1 activation and GSDMD-mediated pyroptotic signaling, we next assessed GSDMD and cell death in injured arteries. Consistent with our previous study^18^, partial ligation induced VSMC GSDMD upregulation in the vessel wall; notably, this increase was significantly greater in Nlrp3 ^SMKI^ mice (Fig. 3A–B). Moreover, TUNEL staining showed that partial ligation-induced cell death was further increased by VSMC Nlrp3 GOF (Fig. 3C–D). These findings are consistent with activation of the NLRP3–caspase-1–GSDMD axis in vivo. These findings link VSMC NLRP3 hyperactivation to activation of the caspase-1–GSDMD effector arm and increased cell death within the remodeling vessel wall.
VSMC Nlrp3 gain-of-function promotes VSMC proliferation/migration and aggravates intimal hyperplasia
Excessive VSMC proliferation and migration into the intima are central drivers of intimal hyperplasia during vascular remodeling^1,27^. Ki-67 is expressed in all active phases of the cell cycle and is widely used to quantify proliferating VSMCs in vascular lesions^28^. We observed that partial ligation increased the number of Ki-67-positive VSMCs in the vessel wall, and this proliferative response was significantly enhanced in Nlrp3 ^SMKI^ mice (Fig. 4A–B). We next assessed VSMC migratory remodeling by examining MMP-2, a key mediator of extracellular matrix degradation and VSMC migration^29^. Strikingly, VSMC Nlrp3 GOF increased MMP-2 expression even in unligated arteries and further amplified ligation-induced MMP-2 upregulation in injured arteries (Fig. 4C–D).
We then evaluated structural remodeling endpoints. Sirius red staining demonstrated robust collagen deposition after ligation; however, total collagen accumulation was not further increased by VSMC Nlrp3 GOF (Fig. 5A, 5C). In contrast, H&E staining and morphometric analysis showed that ligation induced substantial intimal hyperplasia in Nlrp3 ^ctrl^ mice, and this response was significantly exacerbated in Nlrp3 ^SMKI^ mice, as reflected by increased intimal area and elevated intima-to-media ratio (Fig. 5B, 5D–E). Together, these data indicate that VSMC NLRP3 hyperactivation enhances proliferative/migratory remodeling and markedly worsens injury-induced intimal hyperplasia.
VSMC Nlrp3 gain-of-function accelerates VSMC-to-foam cell transition
VSMC-to-foam cell transition is a lipid-driven phenotypic switch involving metabolic reprogramming, and lineage-tracing/single-cell studies indicate that a substantial fraction of foam cells within mouse and human plaques can originate from VSMCs^8,9^. We therefore tested whether VSMC Nlrp3 GOF affects lipid accumulation within the injured vessel wall. Compared with controls, Nlrp3 ^SMKI^ arteries displayed significantly greater lipid deposition after injury, as demonstrated by Oil Red O staining (Fig. 6A–B) and BODIPY staining (Fig. 6C–D). Consistently, the lipid droplet coat protein PLIN2 was increased to a greater extent in Nlrp3 ^SMKI^ lesions (Fig. 6E–F). These results demonstrate that VSMC NLRP3 hyperactivation promotes VSMC lipid loading and foam cell–like phenotypic transition in vivo. The enhanced lipid accumulation and PLIN2 induction suggest that NLRP3 hyperactivation promotes a metabolic shift in VSMCs toward lipid storage and foam cell–like phenotype.
VSMC Nlrp3 gain-of-function impairs TFEB-mediated autophagy–lysosome signaling
TFEB is a master transcriptional regulator of autophagy–lysosome pathways, coordinating lysosomal biogenesis, autophagosome formation, and cargo degradation^30–32^. Our prior study showed that TFEB signaling is suppressed in ligated carotid arteries^21^. Consistent with this, ligation reduced TFEB expression in VSMCs, and this decrease was significantly more pronounced in Nlrp3 ^SMKI^ arteries (Fig. 7A–B), indicating that VSMC NLRP3 hyperactivation further suppresses TFEB during vascular remodeling. We next assessed lysosomal and autophagy markers. LAMP2A, a key receptor required for chaperone-mediated autophagy^33^, was increased by ligation in this dataset (in contrast to our previous observation^21^). Notably, VSMC Nlrp3 gain-of-function counteracted this ligation-induced LAMP2A increase and reduced LAMP2A levels in VSMCs (Fig. 7C–D). In addition, Nlrp3 ^SMKI^ arteries exhibited greater impairment of lysosome/autophagy markers, including reduced LAMP1 (Fig. 7E–F) and decreased LC3 signal (Fig. 7G–H) compared with Nlrp3 ^ctrl^ arteries after injury. Collectively, these data support that VSMC NLRP3 gain-of-function disrupts TFEB-associated autophagy–lysosome homeostasis during injury-induced vascular remodeling.
Discussion
In this study, we provide direct in vivo evidence that VSMC-intrinsic NLRP3 hyperactivation is sufficient to drive maladaptive vascular remodeling and markedly worsen intimal hyperplasia under hypercholesterolemic injury conditions. Using a VSMC-specific Nlrp3 gain-of-function knock-in model, we bypass the long-standing ambiguity inherent to systemic loss-of-function approaches and directly assign causality to VSMCs as the initiating and amplifying compartment in inflammasome-dependent lesion progression. Our data support a mechanistic cascade in which enhanced NLRP3–caspase-1 signaling in VSMCs augments vascular inflammation, engages the caspase-1–GSDMD effector arm, leading to increased cell death, promotes proliferative and migratory remodeling, and accelerates lipid loading and a VSMC-to–foam cell–like phenotypic transition, culminating in exacerbated neointimal growth.
Recent studies have leveraged gain-of-function NLRP3 knock-in (KI) mice to model cryopyrin-associated periodic syndromes (CAPS) and to dissect how inflammasome hyperactivation drives tissue pathology. Seminal work by Hoffman and colleagues established KI strains carrying CAPS-linked mutations (e.g., A352V and L353P) and showed that these variants can trigger systemic inflammation and early mortality when expressed in myeloid lineages^19^. Since then, additional CAPS-relevant NLRP3 KI models—including D301N, R258W, and others—have been applied across diverse organ systems and cell types to reveal how cell-intrinsic NLRP3 hyperactivation shapes inflammation and disease phenotypes^34,35^. Although these models have been highly informative and increasingly adopted beyond classic autoinflammatory contexts, the potential contribution of cardiovascular-resident cells, particularly endothelial cells and vascular smooth muscle cells (VSMCs), to NLRP3-driven CAPS-like pathology and vascular remodeling remains poorly defined. Addressing this gap, we provide the first direct evidence that smooth muscle–restricted NLRP3 gain-of-function (L351P KI) is sufficient to activate the inflammasome program in vivo and to exacerbate injury-driven vascular remodeling. Notably, VSMC Nlrp3 gain-of-function increased inflammasome activation not only after partial carotid ligation under hypercholesterolemia, but also detectably in unligated arteries, as indicated by elevated FLICA labeling and cleaved caspase-1 staining. This suggests that VSMC inflammasome hyperactivation can establish a “primed” pro-inflammatory state that lowers the activation threshold of the vessel wall, thereby sensitizing arteries to subsequent hemodynamic or metabolic stress. Following injury, this primed state translated into a markedly amplified caspase-1 response, enhanced inflammatory activation and macrophage accumulation, and accelerated neointimal growth. Together, these findings support a model in which VSMC-intrinsic NLRP3 hyperactivation is an upstream driver, rather than a passive byproduct, of the inflammatory microenvironment that fuels pathological vascular remodeling. Future work should determine whether other CAPS-linked NLRP3 variants similarly reprogram VSMC function, define the downstream mediators coupling VSMC inflammasome activation to lesion progression, and test whether targeting VSMC NLRP3 signaling can mitigate vascular pathology in relevant disease settings.
Consistent with this, VSMC NLRP3 hyperactivation significantly intensified vascular inflammatory activation and immune cell accumulation. VCAM-1 upregulation within the vessel wall was increased in Nlrp3 ^SMKI^ arteries, particularly within the neointimal region, accompanied by greater macrophage accumulation (F4/80^+^) and increased CD206^+^ macrophage staining after injury. Increased VCAM-1 provides a plausible molecular link between VSMC inflammasome activation and leukocyte adhesion and retention within lesions, thereby amplifying local inflammatory signaling. Elevated macrophage accumulation (F4/80+) indicates significant infiltration within the lesion, while increased CD206 + expression reflects a polarization toward a ‘repair-associated’ phenotype. These cells support lesion remodeling through growth factor production, matrix remodeling, and complex effects on lipid handling depending on context^36,37^. Together, these findings place VSMC NLRP3 activity upstream of a permissive inflammatory niche, poised to accelerate lesion development through both immune-dependent and immune-independent mechanisms.
We further demonstrate that VSMC NLRP3 hyperactivation activates the caspase-1–GSDMD effector axis and is associated with increased cell death within the remodeling vessel wall. In Nlrp3 ^SMKI^ arteries, ligation elicited greater GSDMD upregulation accompanied by increased TUNEL positivity, consistent with enhanced inflammasome-linked cellular injury. Importantly, in the setting of intimal hyperplasia, cell death can be pro-remodeling rather than protective: pyroptosis-associated membrane permeabilization and release of inflammatory mediators and danger signals can amplify paracrine activation of neighboring vascular cells, promote chemokine/adhesion molecule expression, and trigger compensatory VSMC proliferation and migration. In parallel, inflammasome-derived IL-1 family cytokines directly support VSMC activation states that favor migratory and proliferative remodeling^38–40^. Notably, our recent work further indicates that GSDMD can promote platelet-derived growth factor-BB (PDGF-BB)–induced VSMC proliferation, migration, and inflammation independent of overt cell death^18^, highlighting a non-lytic remodeling function for this pathway. Together, these findings support a model in which NLRP3–caspase-1–GSDMD signaling accelerates neointimal growth through both inflammatory injury–driven feed-forward loops and cell-intrinsic, pro-remodeling effects on VSMC behavior.
In line with this model, we observed that VSMC Nlrp3 gain-of-function robustly enhanced the proliferative and migratory programs that drive neointimal expansion. Nlrp3 ^SMKI^ arteries exhibited increased Ki-67–positive VSMCs after ligation and elevated MMP-2 expression, with a notable increase even in unligated arteries. These findings are consistent with the established paradigm that injury-induced phenotypic switching enables VSMCs to proliferate, degrade extracellular matrix via MMPs, and migrate into the intima to form neointimal lesions^41^. Importantly, these molecular and cellular changes translated into clear structural consequences: Nlrp3 ^SMKI^ mice developed significantly greater intimal hyperplasia, reflected by increased intimal area and a higher intima-to-media ratio—standard morphometric endpoints tightly linked to restenosis severity^42,43^. Although collagen deposition increased after injury, VSMC Nlrp3 gain-of-function did not further augment total collagen accumulation, suggesting that the predominant impact of VSMC inflammasome hyperactivation in this setting is cellular expansion and lesion architecture, rather than generalized fibrosis. This distinction is clinically relevant because VSMC accumulation and neointimal cellularity, more than collagen content per se, are major determinants of luminal narrowing and restenosis risk.
Beyond lesion size, our findings extend VSMC inflammasome biology into the domain of lipid-driven phenotypic switching. We show that VSMC Nlrp3 gain-of-function significantly increased vascular lipid deposition (Oil Red O and BODIPY staining) and elevated PLIN2 expression in injured arteries, supporting accelerated VSMC lipid loading and foam cell–like transition. This observation is particularly important given accumulating evidence that VSMCs contribute substantially to foam cell populations in atherosclerotic plaques and can acquire macrophage-like transcriptional programs that complicate lesion biology and therapeutic targeting^8,9^. Functionally, VSMC-to-foam cell transition is not a benign lipid-storage state: it is associated with loss of canonical contractile identity, impaired matrix homeostasis, and reduced plaque-stabilizing functions of VSMCs, while promoting a pro-inflammatory, protease-rich, and stress-vulnerable phenotype that can increase necrotic core formation and lesion complexity^44^. In addition, lipid-laden VSMCs may be more prone to cell death and defective efferocytosis, thereby amplifying inflammatory signaling and contributing to the expansion of acellular/necrotic regions within lesions^45^. Consistent with this concept, an in vitro/in vivo study demonstrated that NLRP3 inflammasome activation promotes foam cell formation in human VSMCs and atherogenesis via HMGB1^46^. Thus, our data suggest that inflammasome hyperactivation within VSMCs is sufficient to bias VSMCs toward a lipid-storing, foam-like phenotype in vivo, providing a mechanistic link between inflammatory danger sensing and metabolic remodeling that can accelerate plaque progression and destabilizing features, beyond simply increasing lesion burden.
Mechanistically, we observed that VSMC NLRP3 hyperactivation was accompanied by suppression of TFEB and broad impairment of autophagy–lysosome markers (LAMP1, LAMP2A, LC3) during vascular remodeling. These results suggest that lysosomal quality control is not simply a downstream bystander of injury, but a vulnerable node that is further destabilized by excessive VSMC inflammasome activity. Because lysosome–autophagy pathways are central to lipid droplet turnover and cellular stress resolution, their impairment provides a plausible explanation for the increased lipid accumulation and PLIN2 induction observed in Nlrp3 ^SMKI^ lesions. Moreover, lysosomal dysfunction can reinforce inflammation by promoting the accumulation of damaged organelles and endogenous danger signals, thereby creating a feed-forward loop that sustains, and can further amplify inflammasome signaling^47^.
Importantly, TFEB suppression provides a unifying mechanism that extends beyond lipid handling. In our previous study, TFEB activation/overexpression restrained VSMC proliferation and migration, whereas TFEB silencing promoted these pro-remodeling behaviors^21,48,49^. Thus, NLRP3 hyperactivation–induced TFEB downregulation may contribute not only to VSMC foam cell–like transition, but also to the heightened proliferative and migratory remodeling that drives neointimal expansion. In addition, TFEB overexpression has been linked to enhanced antioxidant defenses and anti-inflammatory effects in endothelial cells^50^, offering a plausible pathway by which TFEB loss could further amplify vascular inflammation and injury responses in the lesion microenvironment.
One unexpected observation was that LAMP2A increased in Nlrp3 ^ctrl^ arteries after ligation in this dataset, which differs from our prior report showing reduced LAMP2A in ligated arteries from wild type mice^21^. We interpret this injury-induced upregulation of LAMP2A as a potential compensatory response aimed at enhancing chaperone-mediated autophagy and restoring proteostasis under vascular stress. In contrast, LAMP2A was reduced in ligated arteries from Nlrp3 ^SMKI^ mice, indicating that VSMC Nlrp3 gain-of-function not only prevents the normal injury-induced LAMP2A induction, but shifts the response toward an overall loss of CMA capacity during remodeling. One possible contributor to the discrepancy across datasets is the Nlrp3 heterozygous background, which may alter baseline stress signaling and the magnitude/timing of compensatory programs. Alternatively, LAMP2A regulation may be stage-dependent, with an early adaptive induction in controls that becomes blunted, reversed, or insufficient when inflammasome activity is chronically heightened in VSMCs. Importantly, the broader marker pattern is internally consistent: TFEB was reduced, and lysosome/autophagy markers were further diminished in Nlrp3 ^SMKI^ arteries, supporting a net impairment of autophagy–lysosome homeostasis during remodeling. Collectively, these findings support an integrated model in which VSMC NLRP3 hyperactivation couples inflammatory/pyroptotic signaling with suppression of TFEB-linked stress-adaptive clearance pathways, thereby disabling compensatory proteostasis responses, promoting VSMC dysfunction, and accelerating lesion progression and foam cell–like phenotypic switching.
From a translational perspective, these results elevate VSMC NLRP3 signaling as a therapeutic target to limit both restenosis-like neointimal hyperplasia and lipid-driven VSMC phenotypic switching. Systemic inflammasome or IL-1 pathway inhibition has shown promise in inflammatory cardiovascular contexts^51^, but broad immunosuppression can increase infection risk and may not optimally suppress vessel wall–localized remodeling programs. Our findings, therefore, motivate an alternative strategy: cell- or lesion-targeted inhibition of the NLRP3–caspase-1–GSDMD axis within the vascular wall, potentially using local delivery approaches (e.g., targeted nanoparticles) to reduce off-target effects while maximizing efficacy at the remodeling site. In addition, the tight coupling between inflammasome hyperactivation and foam-like lipid accumulation suggests that targeting VSMC inflammasome signaling may provide dual benefit—restraining neointimal growth while reducing lesion lipid burden and VSMC-derived foam cell formation. Complementing inflammasome-directed approaches, our data also suggest that restoring VSMC stress-adaptive programs downstream of NLRP3—particularly TFEB-mediated lysosome–autophagy capacity—may represent a second translational direction for vascular disease in CAPS-associated settings and cardiovascular diseases characterized by intimal hyperplasia. Given our prior evidence that TFEB activation suppresses VSMC proliferation and migration^21,48,49^, and the established role of lysosome–autophagy pathways in lipid clearance and inflammation control^50,52^, pharmacologic or gene-based activation of TFEB in VSMCs—potentially using the same lesion-targeted delivery platforms—could mitigate both structural remodeling and lipid-driven phenotypic switching. Thus, combined strategies that inhibit VSMC NLRP3 effector signaling and/or boost VSMC TFEB-driven protective clearance pathways may offer a rational, vessel wall–focused therapeutic framework to prevent restenosis and limit pathological VSMC plasticity.
This study has limitations. First, our conclusions are based on a single injury paradigm (partial carotid ligation) under a hypercholesterolemic diet, and additional models, such as wire injury, will be needed to establish generalizability across clinically relevant forms of intimal hyperplasia. Second, although our data support a causal role for VSMC NLRP3 hyperactivation, the molecular intermediates linking VSMC inflammasome signaling to the suppression of TFEB and lysosome–autophagy programs remain to be defined. Third, future studies should determine whether pharmacologic inhibition of NLRP3/GSDMD (or VSMC-targeted inhibition) can rescue lesion phenotypes in Nlrp3 ^SMKI^ mice, and whether restoring TFEB-mediated lysosomal/autophagic capacity can blunt VSMC foam transition and lesion growth.
In summary, our work establishes that VSMC-intrinsic NLRP3 hyperactivation is sufficient to drive VSMC dysfunction and accelerate intimal hyperplasia, while also promoting lipid accumulation and VSMC-to–foam cell–like phenotypic transition. Mechanistically, these pathological responses are accompanied by suppression of TFEB and impairment of TFEB-regulated lysosome–autophagy programs, identifying defective cellular clearance and stress adaptation as an additional axis downstream of inflammasome hyperactivation. Together, these findings resolve a key mechanistic gap by directly implicating VSMCs as a causative inflammasome-active compartment during vascular remodeling and suggest that the NLRP3–TFEB/lysosome pathway represents a translationally actionable target for preventing restenosis, limiting plaque progression, and restraining maladaptive VSMC phenotypic switching.
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