Highlights • A novel SPP1+ macrophage emerges early in pressure overload-driven cardiac remodeling. • Myeloid-specific Spp1 deletion alleviates pressure overload-induced cardiac inflammation, remodeling and dysfunction. • The HMGB1-NLRP3-NF-κB axis in macrophages drives pathogenic SPP1 production. • Arglabin, a selective NLRP3 inhibitor, suppresses SPP1 and thereby confers cardioprotection under pressure overload. Summary Cardiac macrophages display remarkable functional plasticity via heterogeneous subpopulations and a dynamic secretome, orchestrating key regulatory events during pressure overload–induced myocardial remodeling. However, the specific pathogenic subsets and their core regulatory mechanisms remain insufficiently defined. In this study, we identified a pathogenic secreted phosphoprotein 1 (SPP1)–expressing macrophage subset that emerges early after transverse aortic constriction. Myeloid-specific deletion of Spp1 potently attenuated transverse aortic constriction–induced cardiac inflammation, pathological remodeling, and dysfunction. Mechanistically, stressed cardiomyocytes release high-mobility group box 1 (HMGB1), which triggers NLRP3 inflammasome activation in macrophages and subsequent SPP1 production via NF-κB p65. Targeting this HMGB1-NLRP3-SPP1 axis with arglabin, an emerging NLRP3 inhibitor, effectively suppressed myocardial SPP1 expression, restricted proinflammatory immune cell infiltration and cardiac fibrosis, and thereby alleviated adverse myocardial remodeling induced by pressure overload. Our findings delineate a critical HMGB1-NLRP3-NF-κB-SPP1 axis in macrophages that drives pressure overload–induced cardiac pathogenesis. Targeting this pathway, exemplified by the natural compound arglabin, represents a promising therapeutic strategy against pathological cardiac remodeling. According to the Global Burden of Disease 2021 estimates, approximately 55.5 million individuals worldwide are affected by heart failure (HF). In the United States alone, 6.7 million people (2.3% of the population) lived with HF between 2017 and 2020.1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib1) Despite the high prevalence of HF, there is a dearth of efficacious therapeutic interventions. Hypertension is a major risk factor for HF.1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib1),2 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib2) In response to pathological pressure overload (PO), postnatal cardiomyocytes undergo hypertrophic growth. Although this initial hypertrophy serves as a compensatory mechanism to sustain cardiac output, persistent stress drives a transition from adaptive to maladaptive remodeling, ultimately culminating in HF.3 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib3),4 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib4) Previous mechanistic studies of pathological cardiac hypertrophy have largely focused on cardiomyocyte-intrinsic signaling pathways. However, the heart is a complex multicellular organ in which diverse non-myocyte cell populations, including fibroblasts, endothelial cells, and immune cells, also serve critical roles in coordinating cardiac homeostasis and pathological responses.5 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib5),6 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib6) A crucial but underappreciated aspect is the dynamic role of the cardiac immune landscape across different stages of disease progression. Recent evidence highlights that activation of macrophage inflammatory function marks a critical period of functional decline following PO induced by transverse aortic constriction (TAC).7 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib7) Additionally, monocyte-derived C-C chemokine receptor 2 (CCR2)+ macrophages infiltrate the myocardium early during TAC-induced PO. Therapeutic blockade of this CCR2-dependent macrophage infiltration has been shown to mitigate adverse left ventricular (LV) remodeling, dysfunction, T-cell expansion, and fibrosis.8 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib8) Consistent with this, by 1 week after TAC, mice exhibit a marked increase of cardiac CD45+ immune cells, with macrophages constituting the predominant population, followed by monocytes, dendritic cells, and other leukocytes.9 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib9) Recent single-cell RNA sequencing (scRNA-seq) analyses of cardiac CD45+ immune cells have provided additional support for this finding, confirming that monocytes/macrophages constitute the most abundant immune populations under PO.10 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib10) These cells can be broadly categorized by their origin: CCR2+ macrophages represent a proinflammatory subset derived from Ly6C hi monocytes.8 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib8),11 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib11),12 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib12) Upon cardiac injury, Ly6C hi monocytes are recruited via the CCL2-CCR2 chemotactic axis and infiltrate the heart, where they differentiate into CCR2+ macrophages that drive the development of cardiac hypertrophy and fibrosis.8 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib8),13 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib13) This pathogenic effect is potentially mediated through paracrine actions of factors such as cytokines, transforming growth factor-β, and platelet-derived growth factor on cardiomyocytes and fibroblasts.14 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib14) In contrast, CCR2– resident macrophages maintain tissue homeostasis under steady-state conditions, primarily through in situ proliferation.9 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib9),11 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib11),15 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib15) During pathological stress, such as angiotensin II (Ang II) infusion, this population expands through both enhanced local proliferation and monocyte recruitment.16 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib16) Functionally, these cells exert a cardioprotective role by promoting angiogenesis and attenuating early cardiac fibrosis following PO, thereby mitigating the progression toward HF.9 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib9) Furthermore, a distinct subset of CCR2+ resident macrophages has recently been identified. This subset contributes to postinjury monocyte recruitment, a finding that further underscores the considerable phenotypic and functional heterogeneity within the cardiac macrophage compartment.17 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib17) Intriguingly, we identified a novel subset of macrophages that specifically express secreted phosphoprotein 1 (SPP1, also known as osteopontin, OPN) in mouse hearts during the progression of PO-induced cardiac hypertrophy. The SPP1+ macrophage population peaked at 1 week after TAC. Thereafter, the density gradually declined by 5 weeks after TAC. Integrating the scRNA-seq of CD45+ immune cells from the PO-induced cardiac hypertrophy model (GSE137167) further revealed that SPP1+ macrophages are characterized by high expression of Trem2 and relatively elevated levels of Nlrp3. SPP1 is a multifunctional protein that acts both as an extracellular matrix (ECM) component and a proinflammatory cytokine.18-21 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib18) It is produced by various cardiac cells, including cardiomyocytes,22 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib22) fibroblasts,23 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib23) endothelial cells,24 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib24) and monocytes or macrophages,6 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib6),25 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib25) in response to stress. Its expression is markedly upregulated both in experimental models of LV hypertrophy and in human myocardial hypertrophy associated with ischemic or idiopathic cardiomyopathy,22 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib22) as well as in patients with dilated cardiomyopathy (DCM).26 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib26) Notably, excessive myocardial SPP1 accumulation is strongly associated with increased ventricular stiffness and systolic dysfunction in patients with hypertensive heart disease and HF, primarily by driving the formation of insoluble, degradation-resistant collagen and subsequent impairment of LV mechanical properties and function.27 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib27) Despite the growing recognition of the pathophysiological importance of SPP1 and the widespread use of Spp1-knockout mice in cardiovascular disease research, the cell type–specific functions of SPP1, particularly in defined immune subsets, remain poorly understood. In this study, we aimed to characterize the specific role of SPP1+ macrophages in the pathogenesis of PO-induced cardiac hypertrophy and remodeling and to dissect the mechanisms underlying SPP1 activation in macrophages. Furthermore, against this newly identified target, we sought to identify a potential small-molecule pharmacological inhibitor to facilitate clinical translation in the treatment of pathological cardiac hypertrophy and HF. Methods Animals LoxP-flanked Spp1 (Spp1 f/f) mice in a C57BL/6 background (NM-CKO-210205) were generated by flanking exon 5 to 8 of the mouse Spp1 gene with LoxP sites at Shanghai Model Organisms Center Inc. Spp1 f/f mice were crossed with mice expressing Cre recombinase under the control of the lysozyme M promoter28 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib28) (LysM-Cre line, JAX, 004781) and resulting myeloid-specific Spp1-knockout (Spp1 MyeKO) mice, and the Spp1 f/f littermates without Cre recombinase were used as control of Spp1 MyeKO mice. Wild-type C57BL/6 mice were purchased from Charles River Laboratories. Male mice were randomly assigned to treatment groups. All animal procedures and experiments were approved by the Animal Care and Use Committee of Wenzhou Medical University and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Statistical analysis Statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad Software). Data are presented as mean ± SEM. Normality was assessed with the Shapiro-Wilk test. For comparisons between 2 groups, normally distributed data were analyzed by unpaired 2-tailed Student's t test; if normality was met but homoscedasticity was violated, Welch's correction was applied. Non-normally distributed data were evaluated with the Mann-Whitney U test. For comparisons among more than 2 groups, 1-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for normally distributed variables; when heteroscedasticity was present, Brown-Forsythe or Welch ANOVA was conducted followed by Dunnett's T3 post hoc test. Non-normally distributed data were analyzed with the Kruskal-Wallis test and Dunn's post hoc test. For comparisons involving 2 independent factors, 2-way ANOVA with Sidak's post hoc test was performed. A P value <0.05 was defined as statistically significant. Results SPP1+ macrophages accumulate in heart tissues in response to PO To elucidate distinct macrophage subtypes and their functional roles during myocardial remodeling, we reanalyzed a publicly available scRNA-seq data set of CD45+ immune cells isolated from hearts of mice subjected to sham operation or TAC-induced HF (GSE137167).29 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib29) After integrating cellular data from both sham- and TAC-operated hearts, we performed clustering analysis and annotated the major cell types based on established markers. Monocytes/macrophages constituted the predominant population (Figures 1A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 1B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)). To further explore the heterogeneity of these monocytes/macrophages, we reclustered them into 6 distinct subtypes (Figure 1C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)), among which a subtype of SPP1+ macrophages (cluster 4) showed an increasing trend following TAC (Figures 1D to 1F). This subcluster was also characterized by high expression of Trem2 and relatively elevated levels of Nlrp3 (Figures 1D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 1E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)). To delineate the expression profile of SPP1 in human heart samples, we analyzed publicly available scRNA-seq data (GSE145154)30 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib30) from human hearts with DCM (Supplemental Figures 1A to 1D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Our analysis confirmed the presence of SPP1-expressing monocytes/macrophages in the LV tissues of patients with DCM. Although these cells did not form a discrete cluster (Supplemental Figures 1C to 1E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)), likely because of the limited sample size, their identification provides critical preliminary evidence for the existence of this cell population in human pathological myocardium. Figure 1 SPP1+ Macrophages Accumulate in Heart Tissues in Response to Pressure Overload (A) t-distributed Stochastic Neighbor Embedding (tSNE) plot of single-cell RNA-sequencing data (GSE137167) from CD45+ immune cells isolated from the hearts of sham- and transverse aortic constriction (TAC)–operated mice. (B) Dot plot showing the expression of signature genes in each major cell type. (C) Uniform Manifold Approximation and Projection (UMAP) plot displaying 6 distinct subpopulations obtained by further subclustering the total Mono_Mac population shown in (A). (D) UMAP visualization of the expression of genes Spp1, Nlrp3, and Trem2, (E) accompanied by violin plots illustrating their expression levels across different cell clusters. (F) The percentage of Spp1+ Mac (cluster 4) relative to the total Mono_Mac populations. (G) Quantitative analysis of total CD45+ immune cells in the hearts from sham- and TAC-operated mice at 1 week and 5 weeks after surgery, detected by flow cytometry (n = 6). (H) Representative immunofluorescence staining for SPP1 (red) and CD68 (green) in heart tissues from sham- and TAC-operated mice at 1 week and 5 weeks after surgery. Nuclei were labeled with DAPI (blue). Arrowheads indicate SPP1+CD68+ double-positive cells. Scale bars: 50 μm. (I) Quantitative analysis of SPP1+CD68+ cells (n = 6). (J) SPP1 protein levels in heart tissues from sham- and TAC-operated mice at 1 week and 5 weeks after surgery, as measured by enzyme-linked immunosorbent assay (n = 6). Statistical analyses were performed using 2-way analysis of variance followed by Sidak's post hoc test (G, I, and J). Values are mean ± standard error of the mean. ∗P< 0.05; ∗∗∗P< 0.001. cDC = conventional dendritic cell; Mono = monocyte; Mac = macrophage; NK = natural killer. • Download figure (https://www.jacc.org/cms/asset/5e18060c-6f2f-41b1-9c1a-82af43846c3e/gr1.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig1) The presence of SPP1+ macrophages in TAC-induced hearts was further confirmed through immunostaining with an anti-SPP1 antibody, along with costaining for the macrophage-specific marker CD68. Consistent with the overall trend of cardiac CD45+ immune cell dynamics, which flow cytometry revealed as a substantial increase at 1 week after TAC and then returned to levels slightly higher but not significantly different from sham controls (Figure 1G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1), Supplemental Figure 1F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)), the density of SPP1+ macrophages peaked at 1 week after TAC, as evidenced by significant colocalization of SPP1 and CD68 signals (Figures 1H (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 1I (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)). Subsequently, the density showed a clear reduction by 5 weeks after TAC (Figures 1H (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 1I (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)). However, the overall expression of SPP1 in the myocardial tissue continued to rise throughout this period (Figure 1J (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)). Myeloid-specific SPP1 deletion attenuates cardiac remodeling, inflammation, and dysfunction after TAC We next sought to elucidate the specific role of SPP1+ macrophages in TAC-induced cardiac remodeling. To this end, we crossed Spp1 f/f mice with mice expressing Cre recombinase under the control of the lysozyme M promoter (LysM-Cre) to generate Spp1 MyeKO mice (Figure 2A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2), Supplemental Figures 2A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1) and 2B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Enrichment of cardiac macrophages from TAC-operated hearts using anti-F4/80 MicroBeads followed by Western blot analysis confirmed efficient deletion of SPP1 in macrophages from Spp1 MyeKO hearts (Figure 2B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Moreover, macrophage-specific Spp1 knockout partially reduced SPP1 content in whole heart tissues after TAC (Figure 2C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Figure 2 Myeloid-Specific Spp1 Deletion Attenuates Cardiac Remodeling, Inflammation, and Dysfunction After Transverse Aortic Constriction (TAC) (A) Schematic representation of the experimental design. Myeloid-specific Spp1-knockout (Spp1 MyeKO) mice and their control littermates (Spp1 f/f) were subjected to sham or TAC surgery, with subsequent analyses conducted at the indicated time points. (B) Representative immunoblots and quantitative analysis of SPP1 protein expression in F4/80+ macrophages (Mφs) enriched by anti-F4/80 MicroBeads, isolated from Spp1 f/f and Spp1 MyeKO hearts after TAC. GAPDH was used as an internal control (n = 4). (C) Protein levels of SPP1 in heart tissues from Spp1 f/f and Spp1 MyeKO mice determined by ELISA (n = 6). (D) Representative M-mode echocardiographic (left), pulse-wave Doppler (middle), and tissue Doppler (right) images from Spp1 f/f and Spp1 MyeKO mouse hearts at 2 weeks after surgery. The left ventricular posterior wall thickness at end-diastole (LVPWd) was evaluated using M-mode echocardiography, whereas the ratio of peak early-diastolic mitral inflow velocity to peak early-diastolic mitral annular velocity (E/e′) was calculated according to the pulsed-wave Doppler and tissue Doppler imaging (n = 6). (E) Representative M-mode echocardiographic images of hearts from Spp1 f/f and Spp1 MyeKO mice at 5 weeks after surgery. Left ventricular ejection fraction (EF), fractional shortening (FS), and left ventricular internal dimension at end-systole (LVIDs) were determined by echocardiography (n = 6-9). (F-H) Representative images and quantitative analysis of wheat germ agglutinin (WGA) staining (scale bar: 50 μm, n = 6) (F), hematoxylin and eosin (H&E) staining (scale bar: 1 mm) (G), and Masson's trichrome staining (scale bar: 50 μm, n = 6) (H) in hearts from Spp1 f/f and Spp1 MyeKO mice at 5 weeks after surgery. (I) Representative immunofluorescence staining for vimentin (red) in heart tissues from Spp1 f/f and Spp1 MyeKO mice at 5 weeks after surgery. Nuclei were labeled with DAPI (blue). Scale bar: 50 μm. (J) Heart weight-to-body weight ratio (HW/BW, mg/g) in Spp1 f/f and Spp1 MyeKO mice at 5 weeks after surgery (n = 6-9). (K) Representative flow cytometry contour plots and statistical analysis of CD11b+Ly6C hi monocytes in the hearts of Spp1 f/f and Spp1 MyeKO mice at 1 week after surgery, pregated on CD45+ cells (n = 6). Statistical analyses were performed using the unpaired 2-tailed Student's t-test (B) and 2-way ANOVA followed by Sidak's post hoc test (C to K). Values are mean ± SEM. ∗P< 0.05; ∗∗P< 0.01; ∗∗∗P< 0.001. EF = ejection fraction; FS = fractional shortening; H&E = hematoxylin and eosin; HW/BW = heart weight -to -body weight ratio; LVIDs = left ventricular internal dimension at end-systole; WGA = wheat germ agglutinin; other abbreviations as in Figure 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1). • Download figure (https://www.jacc.org/cms/asset/13bbe6d4-952f-4b36-993d-6f1210191611/gr2.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig2) We then assessed cardiac function by echocardiography. Transaortic pressure gradients were comparable between groups (>50 mm Hg) (Supplemental Figure 2C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). During the compensatory hypertrophy phase (2 weeks after TAC), we observed pronounced ventricular wall thickening, reflected by an increased LV posterior wall thickness at end-diastole in Spp1 f/f mouse hearts (Figure 2D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). This increase was attenuated in Spp1 MyeKO mice (Figure 2D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Meanwhile, the ratio of peak early-diastolic mitral inflow velocity to peak early-diastolic mitral annular velocity (E/e′), a key indicator of diastolic function, was significantly elevated in Spp1 f/f mice after TAC but remained relatively normal in Spp1 MyeKO mice (Figure 2D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)), indicating preserved diastolic function. By 5 weeks after TAC, Spp1 f/f mice displayed pronounced systolic dysfunction and overt LV dilatation (Figure 2E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Conversely, Spp1 MyeKO mice exhibited reduced left ventricular internal dimension at end-systole (LVIDs), as well as preserved ejection fraction (EF) and fractional shortening (FS), compared with Spp1 f/f controls (Figure 2E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Furthermore, macrophage-specific deletion of Spp1 significantly reduced cardiomyocyte size at 5 weeks after TAC while exerting minimal effects in sham-operated control mice (Figures 2F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2) and 2G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Masson's trichrome staining further demonstrated attenuated interstitial and perivascular fibrosis in Spp1 MyeKO hearts relative to Spp1 f/f controls after TAC (Figure 2H (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)), accompanied by reduced accumulation of vimentin+ myofibroblasts in perivascular regions (Figure 2I (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). The heart weight-to-body weight ratio (HW/BW) was also reduced in Spp1 MyeKO mice (Figure 2J (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Correspondingly, expression of profibrotic genes, including Col1a1, Mmp2, Postn, Fn1, and Tgfb1, was also reduced in Spp1 MyeKO hearts (Supplemental Figure 2D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). To investigate whether SPP1+ macrophages activate fibroblasts, we used a coculture system. Macrophages isolated from TAC-operated hearts increased collagen I fluorescence intensity in cocultured adult mouse cardiac fibroblasts (AMCFs) (Supplemental Figure 2E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). This profibrotic effect was abolished when SPP1 was neutralized with a specific antibody (Supplemental Figure 2E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). In addition, treatment of cultured AMCFs with recombinant SPP1 exerted direct profibrotic effects, as evidenced by significantly elevated expression of profibrotic genes (except for Tgfb1) (Supplemental Figure 2F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)) and enhanced immunofluorescence intensity of collagen I (Supplemental Figure 2G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Beyond its function as an ECM protein, SPP1 also serves as a potent chemotactic cytokine. Consistent with this, flow cytometry revealed marked infiltration of Ly6C hi monocytes into Spp1 f/f hearts at 1 week after TAC (Figure 2K (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2)). Ly6C hi monocytes, characterized by high CCR2 expression, vigorously infiltrate inflamed tissues and give rise to CCR2+ macrophages that contribute to cardiac inflammation.8 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib8),12 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib12) Accordingly, myeloid-specific Spp1 deletion markedly attenuated the infiltration of Ly6C hi monocytes into the myocardium following TAC. Together, these results indicate that macrophage-derived SPP1 plays a critical role in post-TAC inflammatory responses and fibroblast activation. Myeloid-specific deletion of Spp1 confers substantial protection throughout the progression from compensated hypertrophy to overt HF. NLRP3 mediates SPP1 upregulation in macrophages in response to PO Next, we sought to explore how SPP1 is upregulated in macrophages in response to PO. Given that SPP1+ macrophages also exhibited relatively elevated Nlrp3 levels (Figure 1E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1)), we hypothesized that inflammasome-related mechanisms might be involved. To determine the role of NLRP3 in SPP1 activation, we isolated macrophages from sham- and TAC-operated mouse hearts. NLRP3 expression was significantly increased in macrophages from TAC hearts, along with activation of the inflammasome pathway, as indicated by elevated levels of cleaved caspase-1 and mature interleukin-1 beta (IL-1β) (Figure 3A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)). Meanwhile, SPP1 protein levels were also markedly elevated in these macrophages (Figure 3B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)). Conversely, Nlrp3 knockdown not only suppressed inflammasome signaling but also significantly reduced SPP1 expression (Figures 3A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3) and 3B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)), suggesting a potential mechanistic link between NLRP3 and SPP1 upregulation in macrophages under PO. Figure 3 NLRP3 Mediates SPP1 Upregulation in Macrophages in Response to Pressure Overload (A and B) Representative immunoblots and quantitative analysis of NLRP3, cleaved caspase-1, and mature interleukin (IL)-1β protein levels (A), as well as SPP1 protein levels (B), in enriched F4/80+ macrophages (Mφs) isolated from sham- or TAC-operated mouse hearts after transfection with small interfering RNA (siRNA) targeting Nlrp3 (si Nlrp3) or control siRNA (si Scr). GAPDH was used as an internal control (n = 4). (C) Representative immunoblots of nuclear and cytoplasmic p65 in enriched F4/80+ Mφs isolated from sham- or TAC-operated mouse hearts after transfection with si Nlrp3 or si Scr. GAPDH and histone H3 were used as cytoplasmic and nuclear loading controls, respectively. (D) Representative immunoblots and quantitative analysis of SPP1 protein expression in enriched F4/80+ Mφs treated as in (A), with additional transfection with siRNA targeting p65 (si p65) (n = 4). (E and F) Adult mouse cardiac fibroblasts (AMCFs) were cocultured in a transwell system with F4/80+ Mφs isolated from sham- or TAC-operated mouse hearts and pretransfected with si Nlrp3, si p65, or si Scr. Real-time quantitative polymerase chain reaction analysis for profibrotic genes including Col1a1, Mmp2, Postn, Fn1, and Tgfb1 in cocultured AMCFs (n = 6). Gapdh was used as a reference gene (E). Representative immunofluorescence staining for collagen I (red) in cocultured AMCFs. F-actin was labeled with phalloidin (green), and nuclei were labeled with DAPI (blue). Scale bar: 50 μm (F). Statistical analyses were performed using 1-way ANOVA followed by Tukey's post hoc test in (A) (NLRP3 and cleaved caspase-1), (B, D, E) (Mmp2 and Tgfb1), Brown-Forsythe or Welch ANOVA followed by Dunnett's T3 post hoc test in (A) (mature IL-1β) and (E) (Col1a1, Fn1, and Postn). Values are mean ± SEM. ∗P< 0.05; ∗∗P< 0.01; ∗∗∗P< 0.001. Abbreviations as in Figure 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1). • Download figure (https://www.jacc.org/cms/asset/d47098e7-b7e3-43c7-800b-c0c2588db8c9/gr3.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig3) Interestingly, we found that NLRP3 activation in macrophages from TAC hearts was accompanied by concurrent activation of the NF-κB/p65 pathway, indicated by an increase in p65 nuclear translocation (Figure 3C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)). Notably, this p65 translocation was substantially attenuated upon Nlrp3 knockdown (Figure 3C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)), suggesting that NF-κB/p65 may function downstream of NLRP3. Knockdown of either Nlrp3 or p65 in macrophages isolated from TAC-operated hearts suppressed SPP1 expression (Figure 3D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)) and subsequently reduced the levels of profibrotic genes (Figure 3E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)) as well as collagen I deposition in cocultured AMCFs (Figure 3F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig3)). Macrophage-specific NLRP3 knockdown suppresses SPP1 expression and attenuates cardiac remodeling following TAC To further delineate the role of NLRP3 in regulating SPP1 activation in macrophages, we used an adeno-associated virus (AAV)-DIO system in combination with LysM-Cre transgenic mice. The pAAV-Ef1α-DIO-miR30shRNA-WPRE vector, featuring lox sites flanking an inverted target gene sequence, represents an advanced tool enabling specific target gene expression in Cre-expressing cells.31 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib31) Using this system, short hairpin RNA (shRNA) targeting Nlrp3 could be successfully delivered to macrophages, achieving efficient gene knockdown in macrophages isolated from TAC-operated hearts. Notably, Nlrp3 knockdown significantly suppressed SPP1 activation in enriched macrophages from TAC-operated hearts (Figure 4A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4), Supplemental Figure 3A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)) and partially diminished SPP1 levels in whole heart tissues (Figure 4B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4)). Furthermore, compared with mice transduced with AAV-DIO-NC, mice receiving AAV-DIO-sh Nlrp3 showed significant attenuation of TAC-induced cardiomyocyte hypertrophy (Figures 4C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4) and 4D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4)) and cardiac fibrosis (Figure 4F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4)), as well as marked reduction in the HW/BW ratio at 5 weeks after TAC (Figure 4E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4)). Moreover, macrophage-specific Nlrp3 knockdown partially reversed TAC-induced declines in EF and FS while concurrently attenuating TAC-evoked increases in LVIDs (Figure 4G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig4), Supplemental Figure 3B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Figure 4 Macrophage-Specific Nlrp3 Knockdown Suppresses SPP1 Expression and Cardiac Remodeling After TAC Adeno-associated virus vectors pAAV-Ef1a-DIO-miR30shRNA (Nlrp3)-WPRE (AAV-DIO-sh Nlrp3) and the control pAAV-Ef1a-DIO-miR30shRNA (NC)-WPRE (AAV-DIO-NC) were administered to LysM-Cre mice, which were then subjected to TAC or sham surgery. (A) Representative immunoblots and quantitative analysis of SPP1 protein expression in enriched F4/80+ Mφs isolated from mouse hearts at 1 week after surgery. GAPDH was used as an internal control (n = 6). (B) Protein levels of SPP1 in mouse heart tissues determined by ELISA (n = 6). (C) Representative images of H&E staining in mouse hearts at 5 weeks after surgery. Scale bar: 1 mm. (D) Representative images and quantitative analysis of WGA staining in mouse hearts at 5 weeks after surgery (n = 6). Scale bar: 50 μm. (E) HW/BW ratio (mg/g) in mice at 5 weeks after surgery (n = 6-8). (F) Representative images and quantitative analysis of Masson's trichrome staining in mouse hearts at 5 weeks after surgery (n = 6). Scale bar = 50 μm. (G) Representative M-mode echocardiographic images of mouse hearts at 5 weeks after surgery. The EF, FS, and LVIDs were assessed by echocardiography (n = 6-8). Statistical analyses were performed using unpaired 2-tailed Student's t-test (A) and 2-way ANOVA followed by Sidak's post hoc test (B, D to F, and G). Values are mean ± SEM. ∗P< 0.05; ∗∗P< 0.01; ∗∗∗P< 0.001. shRNA = short hairpin RNA; NC = negative control; other abbreviations as in Figures 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 2 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2). • Download figure (https://www.jacc.org/cms/asset/db9c60de-4c68-4062-bd44-4ab7f4e39de8/gr4.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig4) Extracellular HMGB1 secreted by cardiomyocytes contributes to NLRP3-SPP1 activation in macrophages under PO Next, we further investigated the molecular mechanisms responsible for NLRP3 activation in cardiac macrophages under PO. Notably, in mice subjected to TAC, we observed a marked increase in serum HMGB1 levels at 1 week after surgery (Figure 5A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). HMGB1 is a well-established potent activator of the NLRP3 inflammasome. Accordingly, we sought to identify the source of elevated HMGB1 in response to PO. Our results revealed a significant increase in HMGB1 concentration in the supernatant of neonatal mouse cardiomyocytes (NMCMs) treated with Ang II, peaking at 1 hour and remaining elevated over the entire experimental period (Figure 5B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). These findings suggest that cardiomyocytes may act as a potential source of HMGB1 under stress conditions. To determine whether extracellular HMGB1 secreted by cardiomyocytes contributes to NLRP3 inflammasome activation in macrophages, we cocultured mouse peritoneal macrophages (PMs) with Ang II–stimulated NMCMs (Figure 5C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). Preactivation of NMCMs with Ang II significantly activated the NLRP3 inflammasome pathway in cocultured PMs, and this activation was markedly abrogated by neutralization of extracellular HMGB1 with a specific antibody (Figures 5D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5) and 5E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). Concurrently, nuclear translocation of p65 was observed in PMs cocultured with Ang II–preactivated NMCMs (Figure 5F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)), accompanied by substantial secretion of SPP1 into the culture medium (Figure 5G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). However, these phenomena were effectively abrogated following treatment with the HMGB1-neutralizing antibody (Figures 5F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5) and 5G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). Figure 5 Extracellular HMGB1 Secreted by Cardiomyocytes Contributes to NLRP3-SPP1 Activation in Macrophages Under Pressure Overload (A) Serum HMGB1 protein levels in sham- and TAC-operated mice at 1 week after surgery, determined by ELISA (n = 6). (B) Relative HMGB1 protein levels in the supernatant of neonatal mouse cardiomyocytes (NMCMs) at different time points after angiotensin II (Ang II) treatment, measured by ELISA (n = 9). (C) Schematic diagram of the transwell coculture system. NMCMs seeded in the upper chamber were first pretreated with 1 μM Ang II, followed by coculture with mouse peritoneal macrophages (PMs) in the presence of 10 μg/mL anti-HMGB1 neutralizing antibody (anti-HMGB1 nAb) or IgM isotype control. (D) Representative immunoblots and quantitative analysis of NLRP3, cleaved caspase-1, and mature IL-1β protein expression in cocultured PMs. GAPDH served as an internal control (n = 4). (E) Representative immunofluorescence staining and quantitative analysis for NLRP3 (red) in cocultured PMs (n = 6). Scale bar: 50 μm. (F) Representative immunofluorescence staining showing the cellular localization of p65 (red) in cocultured PMs. Scale bar: 50 μm. F-actin was label with phalloidin (green) and nuclei were labeled with DAPI (blue). (G) After completion of coculture, NMCMs in the upper chamber were removed, and macrophages in the lower chamber were left to culture alone. The release of SPP1 in the culture supernatant was detected by ELISA (n = 6). (H) RAW264.7 cells were treated with 400 ng/mL recombinant mouse HMGB1 in the presence of si Nlrp3 or si Scr. Chromatin immunoprecipitation (ChIP) assay was performed using a specific antibody against NF-κB p65 or control IgG. Input of sheared chromatin was prepared before immunoprecipitation. The immunoprecipitated DNA was quantified by real-time quantitative polymerase chain reaction analysis using 3 different primers (n = 3). Statistical analyses were performed using unpaired 2-tailed Student's t-test (A), Kruskal-Wallis test followed by the Dunn's post hoc test (B), 1-way ANOVA followed by Tukey's post hoc test (D, E, and H), and Brown-Forsythe or Welch ANOVA followed by Dunnett's T3 post hoc test (G). Values are mean ± SEM. ∗P< 0.05; ∗∗P< 0.01; ∗∗∗P< 0.001. Abbreviations as in Figures 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 2 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2). • Download figure (https://www.jacc.org/cms/asset/c96e6d88-342f-4f67-b786-250106cbc10e/gr5.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig5) We next elaborated on the mechanism by which HMGB1 regulates NLRP3-SPP1 activation in the RAW264.7 cell line. Recombinant HMGB1 protein upregulated the protein levels of NLRP3 and cleaved caspase-1, accompanied by increased SPP1 expression (Supplemental Figure 3C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). We subsequently knocked down the receptors of HMGB1, respectively, and confirmed that Tlr2 knockdown exerted an inhibitory effect on HMGB1-activated NLRP3-SPP1 signaling (Supplemental Figure 3C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Consistent with the HMGB1-induced p65 nuclear translocation observed in coculture experiments, recombinant HMGB1 protein upregulated phospho-IκB (Ser32) levels and promoted the degradation of total IκB (Supplemental Figure 3D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)), indicating activation of the NF-κB pathway. Treatment with an IL-1β–neutralizing antibody inhibited HMGB1-induced activation of NF-κB and upregulation of SPP1 (Supplemental Figure 3D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)); similarly, BAY 11-7085, a specific inhibitor of the NF-κB pathway, also suppressed HMGB1-mediated SPP1 upregulation (Supplemental Figure 3E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Furthermore, direct binding of NF-κB p65 to the Spp1 promoter upon HMGB1 stimulation was confirmed by chromatin immunoprecipitation assay, and this interaction was abolished upon Nlrp3 knockdown (Figure 5H (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig5)). These results suggest that HMGB1 activates NLRP3 to promote IL-1β release, which in turn activates NF-κB and promotes SPP1 upregulation. Endogenous HMGB1 neutralization inhibits the NLRP3-SPP1 axis in macrophages and alleviates cardiac remodeling Given that the interactions among various cell types in vivo are more complex than those observed in our in vitro experiments, we subsequently investigated the role of extracellular HMGB1 in TAC-operated mice. The anti-HMGB1 neutralizing antibody reduced the overall intensity of NLRP3 in the myocardium and decreased macrophage numbers (Figure 6A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)). Concurrently, NLRP3 intensity was diminished in CD68+ macrophages, indicative of suppressed NLRP3 expression in these cells (Figure 6A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)). Specifically, administration of the anti-HMGB1 neutralizing antibody in TAC-operated mice effectively inhibited the expression of NLRP3 inflammasome pathway proteins (Figure 6B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)) and concurrently suppressed SPP1 expression (Figure 6C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)) in macrophages subsequently isolated from the heart, resulting in a partial reduction of total cardiac SPP1 levels (Figure 6D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)). Furthermore, blockade of endogenous HMGB1 effectively reduced cardiomyocyte size (Figures 6E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6) and 6F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)), cardiac fibrosis (Figure 6G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)), and the HW/BW ratio (Figure 6H (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6)). Meanwhile, it decreased LVIDs and preserved cardiac function at 5 weeks after TAC (Figure 6I (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig6), Supplemental Figure 3F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Taken together, these results demonstrate that extracellular HMGB1 induces NLRP3-SPP1 activation in macrophages and at least partially contributes to cardiac remodeling under PO conditions. Figure 6 Endogenous HMGB1 Neutralization Inhibits NLRP3-SPP1 Axis in Macrophages and Alleviated Cardiac Remodeling Endogenous HMGB1 was blocked by injection of a neutralizing anti-HMGB1 antibody or an IgM isotype control at a dose of 100 μg in TAC-operated mice. (A) Representative immunofluorescence staining for NLRP3 (red) and CD68 (green) in mouse hearts at 1 week after TAC. Nuclei were labeled with DAPI (blue). Arrowheads indicate NLRP3+CD68+ double-positive cells. Scale bars: 50 μm. The NLRP3 fluorescence intensity in each CD68+ cell was also quantified (n = 6). (B and C) Representative immunoblots and quantitative analysis of NLRP3, cleaved caspase-1, and mature IL-1β (B), as well as SPP1 (C) protein expression in enriched F4/80+ Mφs isolated from mouse hearts at 1 week after TAC. GAPDH was used as an internal control (n = 6). (D) Protein levels of SPP1 in mouse heart tissues determined by ELISA (n = 6). (E-G) Representative images of WGA staining (scale bar: 50 μm, n = 6) (E), H&E staining (scale bar: 1 mm) (F), and Masson's trichrome staining (scale bar: 50 μm, n = 6) (G) in mouse hearts at 5 weeks after TAC. Also shown the quantitative analyses of WGA staining and Masson's trichrome staining. (H) HW/BW ratio (mg/g) in mice at 5 weeks after TAC (n = 6). (I) Representative M-mode echocardiographic images of mouse hearts at 5 weeks after TAC. The EF, FS, and LVIDs were assessed by echocardiography (n = 6). Statistical analyses were performed using unpaired 2-tailed Student's t-test (A, B, D, E, G, H, and I) and the Mann-Whitney U test (C). Values are mean ± SEM. ∗∗P< 0.01; ∗∗∗P< 0.001. Abbreviations as in Figures 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 2 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2). • Download figure (https://www.jacc.org/cms/asset/d344bd27-80c1-42ca-a1b8-f0674201b456/gr6.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig6) Pharmacological SPP1 inhibition by arglabin significantly attenuates PO-induced cardiac remodeling and dysfunction Given the critical role of the NLRP3 in driving SPP1 activation in macrophages and its pathological contribution to cardiac remodeling, we next sought to identify a therapeutic inhibitor targeting this axis to support clinical translation. Arglabin (ARG), a naturally occurring compound with potent anti-inflammatory properties that suppresses NLRP3 inflammasome activation in macrophages,32 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib32) was therefore selected for further investigation (Figure 7A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). We subsequently evaluated the therapeutic efficacy of ARG in mice subjected to TAC. ARG treatment markedly reduced cardiomyocyte size (Figures 7B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7) and 7C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)), cardiac fibrosis (Figure 7D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)), and the HW/BW ratio (Figure 7E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). In the assessment of cardiac remodeling and function, ARG significantly improved cardiac diastolic function, as evidenced by a reduced E/e′ ratio, and attenuated the LV posterior wall thickness at end-diastole during the compensatory hypertrophic phase (2 weeks after TAC) compared with untreated TAC mice (Supplemental Figure 4A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). At 5 weeks after TAC, administration of ARG ameliorated cardiac systolic dysfunction (as indicated by EF and FS) and suppressed the pathological increase in LVIDs (Figure 7F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). Figure 7 Pharmacological SPP1 Inhibition by Arglabin (ARG) Significantly Attenuates Pressure Overload–Induced Remodeling and Cardiac Dysfunction (A) Schematic representation of the experimental design. Mice were subjected to TAC or sham operation. Mice that underwent TAC were injected with or without arglabin (ARG) at a dose of 2.5 μg/kg, with subsequent analyses conducted at the indicated time points. (B to D) Representative images of WGA staining (scale bar: 50 μm, n = 6) (B), H&E staining (scale bar: 1 mm) (C), and Masson's trichrome staining (scale bar: 50 μm, n = 6) (D) in mouse hearts at 5 weeks after surgery. Also shown the quantitative analyses of WGA staining and Masson's trichrome staining. (E) HW/BW ratio (mg/g) in mice at 5 weeks after surgery (n = 6). (F) Representative M-mode echocardiographic images of mouse hearts at 5 weeks after surgery. The EF, FS, and LVIDs were assessed by echocardiography (n = 6). (G) Gene Ontology (GO) analysis of all differentially expressed genes (DEGs; Q value < 0.05 and |log 2 FC| ≥ 1) identified by RNA sequencing in heart tissues from the TAC + ARG group vs the TAC group (n = 4). Red bars indicate upregulated DEGs, while blue bars indicate downregulated DEGs. (H) Volcano plot showing the DEGs identified by RNA sequencing in heart tissues from the TAC + ARG group vs the TAC group (n = 4). Red dots represent upregulated DEGs, and blue dots represent downregulated DEGs. (I) Representative immunoblots and quantitative analysis of SPP1 protein expression in enriched F4/80+ Mφs isolated from mouse hearts at 1 week after surgery. GAPDH was used as an internal control (n = 6). (J) Protein levels of SPP1 in mouse heart tissues determined by ELISA (n = 6). (K) Representative flow cytometry contour plots and statistical analysis of CD11b+Ly6C hi monocytes in mouse hearts at 1 week after surgery, pregated on CD45+ cells (n = 6). (L) Representative immunofluorescence staining for SPP1 (red) and CD68 (green) in mouse hearts at 1 week after surgery. Nuclei were labeled with DAPI (blue). Arrowheads indicate SPP1+CD68+ double-positive cells. Scale bars: 50 μm. The SPP1+CD68+ cells were also quantified (n = 6). Statistical analyses were performed using Brown-Forsythe or Welch ANOVA followed by Dunnett's T3 post hoc test (B), 1-way ANOVA followed by Tukey's post hoc test in D to F (EF and FS), and (J)-(L), and Kruskal-Wallis test followed by the Dunn's post hoc test in F (LVIDs) and I. Values are mean ± SEM. ∗P< 0.05; ∗∗P< 0.01; ∗∗∗P< 0.001. FC = fold change; other abbreviations as in Figures 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 2 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2). • Download figure (https://www.jacc.org/cms/asset/c17dde95-a11d-4926-8dc3-ecc05c49a431/gr7.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig7) To further dissect the mechanism underlying ARG-mediated cardiac protection, RNA sequencing analysis identified differentially expressed genes (DEGs) in ARG-treated TAC hearts vs untreated TAC hearts, which were significantly enriched in Gene Ontology terms including cell adhesion (34 DEGs downregulated by ARG), ECM organization (23 DEGs downregulated by ARG), and collagen fibril organization (15 DEGs downregulated by ARG) (Figure 7G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7), Supplemental Figure 4B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). More specifically, the downregulated gene set in ARG-treated TAC hearts comprised key mediators of fibrosis and ECM structure, most notably multiple collagen isoforms, Fn1, ADAMTS family members, and Spp1 (Figure 7H (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). The significant inhibition of profibrotic gene expression was also verified in ARG-treated TAC hearts (Supplemental Figure 4C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). More importantly, in vivo ARG treatment in TAC mice significantly downregulated Spp1 gene expression in CD45+CD11b+ immune cells subsequently isolated from their hearts, compared with cells isolated from untreated TAC mice (Supplemental Figures 4D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1) and 4E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). In parallel, ARG-treated TAC mice exhibited marked inhibition of SPP1 protein expression in F4/80-enriched cardiac macrophages, as well as partial reduction in total myocardial SPP1 expression (Figures 7I (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7) and 7J (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). Consistent with its anti-inflammatory properties, ARG treatment also markedly attenuated the infiltration of Ly6C hi monocytes into the myocardium following TAC (Figure 7K (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). Concurrently, the number of SPP1+ macrophages was reduced in TAC hearts after ARG administration (Figure 7L (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig7)). Collectively, these results demonstrate that ARG acts as an effective inhibitor of SPP1 expression in macrophages and exerts cardioprotective effects against PO-induced cardiac remodeling. ARG downregulates macrophage SPP1 via inhibiting extracellular HMGB1-induced activation of NLRP3 signaling pathway We next sought to explore whether ARG's regulation of SPP1 is dependent on NLRP3 inhibition. In TAC-induced PO mice, ARG treatment reduced the abundance of myocardial macrophages and suppressed NLRP3 expression in these cells relative to untreated TAC controls (Figure 8A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8)). Furthermore, macrophages isolated from ARG-treated TAC hearts exhibited suppressed expression of NLRP3 inflammasome pathway proteins (Figure 8B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8)) and decreased SPP1 secretion in the supernatant (Figure 8C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8)). In macrophage-AMCF cocultures, macrophages isolated from ARG-treated TAC hearts decreased collagen I fluorescence intensity in cocultured AMCFs relative to those from untreated TAC controls (Supplemental Figure 5A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). However, reactivation of the ARG-inhibited NLRP3 inflammasome with nigericin abrogated ARG's suppressive effects on SPP1 expression and subsequent fibroblast activation (Figure 8C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8), Supplemental Figure 5A (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)). Figure 8 Arglabin (ARG) Downregulates Macrophage SPP1 via Inhibiting Extracellular HMGB1-Induced Activation of NLRP3 Signaling Pathway (A to C) Mice were subjected to TAC or sham operation. Mice that underwent TAC were injected with or without ARG at a dose of 2.5 μg/kg. Representative immunofluorescence staining for NLRP3 (red) and CD68 (green) in mouse hearts at 1 week after surgery. Nuclei were labeled with DAPI (blue). Arrowheads indicate NLRP3 expression in CD68+ cells. Scale bars: 50 μm (A). Representative immunoblots and quantitative analysis of NLRP3, cleaved caspase-1, and mature IL-1β protein expression in enriched F4/80+ Mφs isolated from mouse hearts at 1 week after surgery (n = 6). GAPDH was used as an internal control (B). (C) The release of SPP1 in the culture supernatant of enriched F4/80+ Mφs isolated from mouse hearts was detected by ELISA (n = 6). To reactivate the NLRP3 inflammasome, F4/80+ Mφs isolated from ARG-treated TAC hearts were further incubated with 10 μM nigericin (NIG). (D-F) Neonatal mouse cardiomyocytes (NMCMs) seeded in the upper chamber were first pretreated with 1 μM Ang II, followed by coculture with peritoneal macrophages (PMs) pretreated with ARG or not. To reactivate the NLRP3 inflammasome, PMs were further stimulated with 10 μM NIG after NMCMs were removed from the upper chamber. Representative immunoblots and quantitative analysis of NLRP3 protein expression in cocultured PMs (n = 4). GAPDH was used as an internal control (D). Representative immunofluorescence staining and quantitative analysis for NLRP3 (red) in cocultured PMs (n = 6). Scale bar: 50 μm (E). Representative immunofluorescence staining showing the cellular localization of p65 (red) in cocultured PMs. Scale bar: 50 μm (F). F-actin was labeled with phalloidin (green) and nuclei were labeled with DAPI (blue). (G) After completion of coculture, NMCMs in the upper chamber were removed, and macrophages in the lower chamber were left to culture alone. The release of SPP1 in the culture supernatant was detected by ELISA (n = 6). Statistical analyses were performed using 1-way ANOVA followed by Tukey's post hoc test (B to E, G). Values are mean ± SEM. ∗P< 0.05; ∗∗P< 0.01; ∗∗∗P< 0.001. Abbreviations as in Figures 1 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig1) and 2 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig2). • Download figure (https://www.jacc.org/cms/asset/8a65323e-9e92-4d79-911b-386a75512f63/gr8.jpg) • Download PowerPoint (https://www.jacc.org/action/downloadFigures?doi=10.1016%2Fj.jacbts.2026.101623&id=fig8) Given that cardiomyocyte-derived HMGB1 drives the NLRP3-SPP1 pathway in macrophages under PO, we used the coculture system of mouse PMs and Ang II–stimulated NMCMs for subsequent investigations. Ang II pretreatment of NMCMs markedly upregulated NLRP3 expression in cocultured macrophages, which was substantially reversed by ARG (Figures 8D (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8) and 8E (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8)). Concomitantly, Ang II–stimulated NMCMs induced p65 nuclear translocation in macrophages and increased SPP1 secretion, both of which were inhibited by ARG (Figures 8F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8) and 8G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8)). Notably, nigericin-mediated NLRP3 reactivation reversed ARG's effects on p65 nuclear translocation and SPP1 secretion (Figures 8F (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8) and 8G (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#fig8)). Consistently, the inhibitory effects of ARG on NLRP3 and NF-κB activation were confirmed in recombinant HMGB1-stimulated RAW264.7 cells. Exogenous IL-1β supplementation restored NF-κB activation despite ARG treatment (Supplemental Figure 5B (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)), confirming ARG modulates NF-κB indirectly via NLRP3 inhibition rather than direct targeting. Additionally, ARG also reduced HMGB1-induced Nlrp3 mRNA levels (Supplemental Figure 5C (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#mmc1)), indicating an additional transcriptional regulatory role. Collectively, our findings propose a positive feedback loop wherein HMGB1 activates the NLRP3/IL-1β axis to induce NF-κB activation, which in turn further amplifies Nlrp3 transcription. ARG disrupts this regulatory loop by inhibiting NLRP3 activation, consequently reducing NF-κB-driven SPP1 production in macrophages. Discussion SPP1 exerts dual roles in the pathogenesis of cardiovascular diseases. On the one hand, it is indispensable for postinjury cardiac repair: After myocardial infarction, SPP1 is predominantly secreted by galectin-3 hi CD206+ reparative macrophages in the infarct zone, whose polarization is governed by the IL-10-STAT3-galectin-3 axis.33 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib33) These SPP1-expressing macrophages facilitate tissue repair via enhancing fibrotic deposition and clearing apoptotic cells, supported by evidence that Spp1-knockout mice exhibited exacerbated LV dilatation with reduced collagen accumulation after myocardial infarction33 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib33),34 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib34)—highlighting SPP1's essential role in stabilizing infarcted myocardium through adequate interstitial collagen deposition. On the other hand, SPP1 is a key driver of pathological remodeling in multiple cardiovascular diseases: SPP1 is predictive of all-cause mortality and/or rehospitalizations in patients with heart failure with preserved ejection fraction (HFpEF)35 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib35); in a murine model of HFpEF, the disease is associated with accumulation of SPP1-expressing CCR2+ cardiac macrophages. SPP1 deletion or blockade of CCR2+ macrophage recruitment alleviated HFpEF phenotypes, as evidenced by improved diastolic function, attenuated cardiac hypertrophy, and reduced fibrosis.36 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib36) Similarly, single-cell transcriptomic analyses of atrial tissues from patients with atrial fibrillation identified an expansion of SPP1+ macrophages, which act as a pleiotropic amplifier of inflammation and fibrosis via cross talk with local immune and stromal cells.37 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib37) Complementing these observations, this macrophage population expands in response to platelet- and monocyte-derived CXCL4, thereby promoting tissue remodeling through fibroblast cross talk.38 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib38) Turning to a cardiac hypertrophy model more relevant to our work, in an Ang II–induced cardiac hypertrophy, Spp1 deficiency attenuated fibrosis but failed to repress hypertrophy; conversely, it promoted systolic dysfunction and exacerbated LV dilatation, implying that SPP1 contributes critically to compensatory adaptation during Ang II–driven cardiac remodeling.39 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib39) In contrast, our findings in a TAC-induced cardiac hypertrophy model demonstrate that myeloid-specific Spp1 knockout improves cardiac remodeling and dysfunction at both compensatory and decompensated stages. The opposing functional outcomes of Spp1 deficiency between our model and others may be attributed to several factors. First, the mechanisms underlying cardiac hypertrophy induced by PO-related biomechanical stress may differ from those driven by neurohumoral activation. More notably, it is the discrepancy brought about by myeloid-specific knockout compared with global knockout. SPP1 expression can be induced in multiple cardiac cell types, including cardiomyocytes,22 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib22) fibroblasts,23 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib23) monocytes/macrophages,6 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib6),25 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib25) and endothelial cells.24 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib24) Thus, specifically targeting macrophage-derived SPP1 may allow for more precise modulation of disease progression without compromising the overall reparative effects. Beyond promoting fibroblast activation, SPP1 also functions as a chemotactic cytokine that modulates immune cell adhesion, migration, and activation.20 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib20),21 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib21) Leukocyte Spp1 deficiency protected against Ang II–accelerated atherosclerosis and abdominal aortic aneurysm formation by inhibiting leukocyte recruitment, reducing cell viability, and suppressing the expression of proinflammatory cytokines and matrix metalloproteinases.21 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib21) Consistent with this, myeloid-specific Spp1 knockout markedly diminished cardiac infiltration of Ly6C hi proinflammatory monocytes following TAC. Recruited monocytes and monocyte-derived macrophages are established key mediators of inflammation and oxidative stress. Given compelling evidence that CCR2+ monocyte/macrophage blockade or depletion potently alleviates late pathological LV remodeling, dysfunction, and fibrosis in the TAC model,8 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib8) we conclude that the cardioprotective effect of myeloid-specific Spp1 deletion may, in large part, be attributed to suppressed infiltration of proinflammatory monocytes and macrophages. The NLRP3 inflammasome has been demonstrated to be activated in several cardiac hypertrophy models, and it is closely associated with disease progression.40-43 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib40) Functionally, NLRP3 inflammasome initiates caspase-1 activation in response to cellular damage and microbial infection.44 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib44) This action subsequently drives the production of proinflammatory cytokines IL-1β and IL-18 while triggering gasdermin D–mediated pyroptosis.45 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib45),46 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib46) Consistent with previous studies demonstrating that NLRP3 inhibition effectively mitigates myocardial hypertrophy and improves cardiac function under PO,47 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib47),48 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib48) we used an AAV-DIO system to selectively knock down Nlrp3 in macrophages. This targeted intervention resulted in alleviated cardiac fibrosis and restored cardiac function in the TAC model. Further mechanistic analysis revealed that macrophages isolated from TAC-operated hearts displayed a significantly activated NLRP3 inflammasome pathway compared to those from the sham group, accompanied by enhanced nuclear translocation of NF-κB p65 and upregulated SPP1 expression. Notably, Nlrp3 knockdown suppressed p65 nuclear translocation in macrophages, which was accompanied by a significant reduction in SPP1 expression. These observations align with previous research demonstrating that NF-κB p65 directly transcriptionally regulates SPP1 expression in ACKR1 hi endothelial cells, with SPP1 functioning as a key mediator underlying endothelial-macrophage cross talk.49 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib49) Collectively, our results establish that the NLRP3-p65 axis plays a central regulatory role in mediating SPP1 expression in macrophages under TAC-induced PO. Emerging experimental evidence highlights the pivotal role of HMGB1 in diverse cardiac pathologies, including myocardial ischemia-reperfusion, infarction, and cardiac hypertrophy.50-53 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib50) Beyond its canonical nuclear function in stabilizing nucleosomes and regulating transcription factors,54 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib54) HMGB1 can be actively secreted by injured cells, activated platelets, and stimulated immune cells.55-57 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib55) Notably, the biological function of HMGB1 is tightly dictated by its subcellular localization: Nuclear HMGB1 overexpression was shown to attenuate endothelin 1–induced cardiomyocyte hypertrophy and was associated with significantly alleviated DNA damage in response to TAC,53 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib53) whereas extracellular HMGB1 was demonstrated to promote cardiomyocyte hypertrophy.58 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib58) In line with previous reports,59 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib59) we detected elevated HMGB1 levels during the pathological progression of cardiac hypertrophy. Cardiomyocytes not only mediate cardiac contraction but also serve as a rich source of proinflammatory cytokines.60 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib60) Consistent with this, our data demonstrated that Ang II induced a prominent increase in extracellular HMGB1 secretion in isolated cardiomyocytes, implying that cardiomyocytes partially contribute to the upregulation of extracellular HMGB1 during stress. As a well-defined damage-associated molecular pattern, HMGB1 activates the NLRP3 inflammasome and NF-κB signaling in inflammatory cells.61 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib61) In the present study, coculture of macrophages with Ang II–stimulated cardiomyocytes markedly upregulated NLRP3 expression and p65 nuclear translocation in macrophages, effects that were significantly blunted by HMGB1-neutralizing antibody, accompanied by decreased SPP1 secretion in the culture supernatant. Furthermore, chromatin immunoprecipitation assay confirmed that HMGB1 stimulation promoted the direct binding of p65 to the SPP1 promoter, and this interaction was abolished by Nlrp3 knockdown. Consistently, in the TAC model, HMGB1 neutralization substantially suppressed macrophage NLRP3 expression, decreased SPP1 levels, attenuated myocardial fibrosis, and improved cardiac function. Collectively, these results demonstrate that extracellular HMGB1 functions as a critical upstream regulator of NLRP3/p65-SPP1 signaling activation in macrophages in response to PO. ARG, a natural sesquiterpene lactone isolated from plants including Artemisia glabella,62 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib62) exhibits potent anti-inflammatory activity.63 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib63) ARG suppressed NLRP3 expression, which abolished caspase-1 maturation and concomitantly reduced the secretion of active IL-1β in macrophages.32 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib32) In high-fat diet–fed ApoE2.Ki mice, ARG markedly decreased plasma IL-1β concentrations and lowered plasma total cholesterol and triglyceride levels. Ultimately, ARG significantly attenuated atherosclerotic lesion size to a degree comparable with that observed in Nlrp3−/− animals.32 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib32) Although ARG is clinically used in cancer therapy,64 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib64) its potential application in cardiac hypertrophy and remodeling requires further exploration. In the present study, we demonstrated that ARG treatment significantly reduced myocardial fibrosis and inflammatory cell infiltration while attenuating cardiac hypertrophy and dysfunction in TAC-induced PO mice. Bulk RNA sequencing analysis pinpointed downregulation of genes involved in ECM organization as core mechanisms mediating the cardioprotective effects of ARG, with SPP1 identified as a critical downstream effector. Mechanistically, ARG treatment markedly suppressed NLRP3 inflammasome activation and p65 nuclear translocation in activated macrophages. Furthermore, our functional validation via IL-1β rescue experiments demonstrated that ARG modulates NF-κB indirectly through NLRP3 inhibition. Collectively, we identified a novel mechanistic cascade underlying ARG's cardioprotective effects: ARG targets the HMGB1-driven NLRP3/IL-1β/NF-κB positive feedback loop, thereby suppressing macrophage-derived SPP1 and alleviating PO-induced cardiac remodeling and dysfunction. Furthermore, our findings support the potential clinical translation of ARG for the management of PO-related cardiac diseases. Study Limitations Although our study provides compelling evidence supporting the mechanistic role of the HMGB1-NLRP3-SPP1 axis in PO-induced cardiac remodeling, several knowledge gaps remain to be addressed. First, the cellular sources and stage-specific functional contributions of SPP1 require further dissection. Although we delineated the dynamic changes and pathological contribution of macrophage-derived SPP1 in the TAC model, the remaining elevated myocardial SPP1 protein expression observed at 5 weeks after TAC, even after cardiac immune cell populations returned to baseline, together with previous reports indicating that cardiomyocytes represent the major source of SPP1 in human idiopathic or ischemic cardiomyopathy,22 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib22) suggests that additional cellular sources of SPP1 (eg, cardiomyocytes) may modulate disease progression or tissue repair during late-stage disease progression. However, the spatiotemporal distribution of SPP1 across cell types during different stages of cardiac hypertrophy progression remains incompletely characterized in the present study. Second, the precise mechanism linking HMGB1 to NLRP3 inflammasome activation warrants deeper investigation. Although prior studies have established that HMGB1 can activate the NLRP3 inflammasome via NF-κB signaling, the concomitant suppression of the NF-κB pathway upon Nlrp3 knockdown suggests that in the present experimental system, NLRP3 may function upstream of NF-κB signaling—or indicate the existence of a regulatory loop. In this context, our previous work revealing that HMGB1 can trigger lysosomal membrane rupture and cathepsin B release, thereby directly activating the NLRP3 inflammasome, may offer a complementary explanatory mechanism.65 (https://www.jacc.org/doi/10.1016/j.jacbts.2026.101623#core-collateral-bib65) This specific mode of HMGB1-driven inflammasome activation, however, was not explored in the current model and merits further exploration. In addition, when analyzing the inhibitory effect of ARG on Spp1 gene expression in cardiac macrophages, the flow cytometry gating strategy (CD45+CD11b+) may also include neutrophils and other myeloid subsets. Therefore, we cannot fully exclude the potential contribution of these cell populations to the observed changes in SPP1 expression. Third, although clinical translation is a key motivation, direct validation in human myocardial tissues from PO-induced hypertrophy remains limited. In lieu of myocardial samples from patients with hypertensive or aortic valve–related HF, we leveraged publicly available scRNA-seq data from patients with DCM. The consistent identification of SPP1-expressing macrophages in this data set provides encouraging translational relevance and nominates SPP1 as a candidate therapeutic target. Nonetheless, future studies utilizing well-phenotyped human specimens of PO-related cardiomyopathy will be essential to confirm the clinical validity and disease-stage specificity of our preclinical observations. Conclusions Our study demonstrates that under PO, a specific macrophage subset expressing SPP1 mediates pathological cardiac remodeling. Mechanistically, stressed cardiomyocytes release HMGB1, which triggers NLRP3 inflammasome activation in macrophages and subsequent SPP1 production via NF-κB p65. Targeting this HMGB1-NLRP3-SPP1 axis with ARG, an emerging NLRP3 inhibitor, effectively suppressed myocardial SPP1 expression, restricted proinflammatory immune cell infiltration and cardiac fibrosis, and thereby alleviated adverse myocardial remodeling induced by PO. These results not only elucidate a crucial cellular cross talk underlying pathological hypertrophy but also position the HMGB1-NLRP3-SPPl axis as a promising therapeutic target for treating PO-induced cardiac remodeling. Declaration of Generative AI and AI-Assisted Technologies During the preparation of this work, the authors used the AI tool DeepSeek for grammar revision, spelling checks, and language refinement and enhancement to improve the readability of the article. After using this tool, the authors reviewed and edited the content as needed, and they take full responsibility for the content of the published article. Perspectives • *COMPETENCY IN MEDICAL KNOWLEDGE:** Pathological cardiac hypertrophy, a critical precursor to HF, is a multicellular process in which immune responses play a pivotal regulatory role. This study identifies a pivotal role for a specific subset of macrophages expressing SPP1 in mediating adverse cardiac remodeling under PO. We establish a novel mechanism wherein stress-induced cardiomyocyte release of HMGB1 activates the NLRP3 inflammasome in macrophages, leading to NF-κB-dependent SPP1 expression. These SPP1+ macrophages orchestrate proinflammatory monocyte recruitment and direct fibroblast activation, driving the progression of cardiac fibrosis, hypertrophy, and dysfunction. Critically, we demonstrate that myeloid-specific deletion of Spp1, pharmacological inhibition of SPP1 via the natural compound ARG, or upstream disruption of the HMGB1-NLRP3 axis all confer significant protection against PO-induced maladaptive remodeling. These findings indicate that targeting pathogenic axes specific to defined macrophage subsets represents a promising therapeutic strategy for pathological hypertrophy and HF. • *TRANSLATIONAL OUTLOOK:** This work identifies the HMGB1-NLRP3-SPP1 axis in macrophages as a pharmacologically targetable pathway and positions ARG as a promising candidate for clinical translation in PO-associated heart disease. Future studies should prioritize validation of this axis in human myocardial samples from patients with hypertensive heart disease or aortic stenosis to confirm its clinical relevance. Key translational steps include optimizing the pharmacokinetic properties, delivery strategies, and long-term safety profile of ARG in preclinical models. Moreover, given the stage- and cell-type-specific functions of SPP1, future therapeutic approaches should aim for precise spatiotemporal modulation, such as macrophage-targeted delivery systems, to preserve its reparative roles while suppressing its pathological effects. Evaluation of ARG, alone or in combination with current HF therapies, in advanced large-animal models may represent a critical step toward advancing mechanistic findings into potential clinical solutions for this condition with limited therapeutic options. Abbreviations and Acronyms AMCF adult mouse cardiac fibroblast ARG arglabin CCR2 C-C chemokine receptor 2 DCM dilated cardiomyopathy DEG differentially expressed gene ECM extracellular matrix EF ejection fraction FS fractional shortening HF heart failure HFpEF heart failure with preserved ejection fraction HMGB1 high-mobility group box 1 HW/BW heart weight–to–body weight ratio LV left ventricular LVIDs left ventricular internal dimension at end-systole LVPWd left ventricular posterior wall thickness at end-diastole NMCM neonatal mouse cardiomyocyte PM peritoneal macrophage PO pressure overload scRNA-seq single-cell RNA sequencing SPP1 secreted phosphoprotein 1 Spp1 f/f LoxP-flanked Spp1 Spp1 MyeKO myeloid-specific Spp1-knockout TAC transverse aortic constriction Acknowledgments The authors thank Jun Liu from Jiangxi Miaoyong Multi-Omics Technology Co Ltd for performing scRNA-seq bioinformatics analysis and providing technical support. Funding Support and Author Disclosures This work was supported by the National Natural Science Foundation of China (82270441 to Dr Niu, 82300406 to Dr Sun), the “Pioneer” and “Leading Goose” R&D Program of Zhejiang (2023C03030 to Dr Niu), the Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China (LHDMY23H070001 to Dr Niu), the Zhejiang Provincial Natural Science Foundation of China (LQ24H020008 to Dr Sun), and the Zhejiang Traditional Chinese Medicine Science and Technology Program Project (2025ZL620 to Ms Shen). The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Footnote The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the Author Center (https://www.jacc.org/author-center). Supplementary Material Supplemental Material 1(mmc1.docx) • Download (https://www.jacc.org/doi/suppl/10.1016/j.jacbts.2026.101623/suppl_file/mmc1.docx) • 2.09 MB Supplemental Material 2(mmc2.pdf) • Download (https://www.jacc.org/doi/suppl/10.1016/j.jacbts.2026.101623/suppl_file/mmc2.pdf) • 14.69 MB References 1. Martin S.S., Aday A.W., Allen N.B., et al. 2025 Heart Disease and Stroke Statistics: a report of US and global data from the American Heart Association. Circulation. 2025;151:e41-e660. 2. Bundy J.D., Li C., Stuchlik P., Bu X., He J. Systolic blood pressure reduction and risk of cardiovascular disease and mortality: a systematic review and network meta-analysis. JAMA Cardiol. 2017;2:775-781. 3. Nakamura M., Sadoshima J. Mechanisms of physiological and pathological cardiac hypertrophy. Nat Rev Cardiol. 2018;15:387-407. 4. Schiattarella G.G., Hill J.A. Inhibition of hypertrophy is a good therapeutic strategy in ventricular pressure overload. Circulation. 2015;131:1435-1447. 5. 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