Atherosclerosis is a complex and progressive vascular disease characterized by the accumulation of lipids, inflammatory cells, and extracellular matrix components within the arterial wall [55, 56, 57, 58, 59, 60]. The development of atherosclerosis is initiated by endothelial injury, which promotes the retention and oxidation of LDL in the subendothelial space [61, 62, 63, 64]. This injury disrupts the homeostasis of the arterial wall and triggers a series of pathological events, including lipid deposition, smooth muscle cell proliferation, and immune cell infiltration [65, 66, 67, 68, 69, 70]. The retention of oxidized LDL (ox-LDL) in the intima activates endothelial cells, which secrete pro-inflammatory cytokines and adhesion molecules that recruit circulating monocytes to the site of injury [63, 71, 72]. Once monocytes are recruited, they differentiate into macrophages, which engulf ox-LDL and transform into foam cells [73, 74]. These foam cells contribute to the formation of a lipid-rich core within the plaque. As the plaque matures, smooth muscle cells migrate into the intima, forming a fibrous cap that stabilizes the plaque. However, in the presence of sustained inflammation, the plaque can become unstable, leading to rupture and thrombosis, which is a major cause of acute cardiovascular events such as myocardial infarction and stroke [75, 76].

Recent research has elucidated the crucial role of immune responses in the pathogenesis of atherosclerosis [68, 77]. The inflammatory process is driven by the activation of innate and adaptive immune cells, which orchestrate the recruitment and activation of additional inflammatory mediators. Central to the regulation of inflammation in atherosclerosis are the inflammasomes, multiprotein complexes that mediate the activation of pro-inflammatory cytokines such as IL-1$\beta$ and IL-18 [78, 79]. Among the different inflammasomes, the NLRP3 inflammasome is considered one of the most important in atherosclerosis, as its activation is implicated in the chronic inflammation that accelerates plaque progression [80, 81].

Immune cells play a central role in the development and progression of atherosclerosis [82]. The disease is marked by the infiltration of various immune cell types, including monocytes, macrophages, dendritic cells, T lymphocytes, and B lymphocytes, all of which contribute to the inflammatory microenvironment of the atherosclerotic plaque [83, 84, 85, 86, 87, 88].

Monocytes and macrophages are the most prominent immune cells involved in atherosclerosis [84, 89]. In response to endothelial damage and lipid accumulation, monocytes migrate to the plaque and differentiate into macrophages [90, 91]. These macrophages, in turn, become foam cells by ingesting ox-LDL, contributing to the formation of the lipid-rich core of the plaque [73, 92]. Foam cells also release a range of pro-inflammatory cytokines, such as IL-1$\beta$, IL-6, and Tumor Necrosis Factor (TNF)-$\alpha$, which further propagate the inflammatory response and promote plaque growth [93, 94]. Moreover, the presence of macrophages and foam cells in the plaque is associated with increased oxidative stress, which exacerbates endothelial dysfunction and amplifies the inflammatory cycle [95, 96]. T lymphocytes, particularly CD4⁺ T helper cells, are also critical in the immune response to atherosclerosis [97]. Th1 cells, which produce Interferon (IFN)-$\gamma$, promote the activation of macrophages and the secretion of pro-inflammatory cytokines, thereby enhancing plaque inflammation [98, 99]. Conversely, regulatory T cells (Tregs) exert an anti-inflammatory effect by secreting cytokines such as IL-10, which dampens the immune response and promotes plaque stability [100, 101]. An imbalance between pro-inflammatory Th1 cells and anti-inflammatory Tregs can lead to the destabilization of atherosclerotic plaques, increasing the risk of rupture [87, 102]. Additionally, the contribution of dendritic cells to atherosclerosis has become an area of increasing interest. Dendritic cells are important antigen-presenting cells that activate T lymphocytes, influencing the adaptive immune response within the plaque [103, 104]. They have been shown to promote both pro-inflammatory and anti-inflammatory responses depending on their activation state, and their role in plaque development and progression remains an active area of investigation.

The inflammatory process in atherosclerosis is tightly regulated by several signaling pathways, with inflammasomes playing a pivotal role in modulating the immune response [105]. Inflammasomes are large, multi-protein complexes that respond to pathogen- or damage-associated molecular patterns and activate the pro-inflammatory cytokines IL-1$\beta$ and IL-18 [21]. Among the various inflammasomes, the NLRP3 inflammasome has been extensively studied in the context of atherosclerosis due to its critical involvement in both innate immunity and the chronic inflammation seen in atherosclerotic lesions [80, 106]. Upon activation, the NLRP3 inflammasome triggers the maturation of IL-1$\beta$ and IL-18, which further amplify the inflammatory cascade by promoting immune cell recruitment, activation, and cytokine production [107]. Elevated levels of IL-1$\beta$ in the plaque microenvironment have been associated with increased plaque instability, making the plaque more prone to rupture [9, 108]. The release of these cytokines also leads to the recruitment of additional immune cells to the plaque, thereby perpetuating the inflammatory cycle and accelerating plaque progression.

In addition to the NLRP3 inflammasome, other inflammasomes, such as the Absent in Melanoma 2 (AIM2) and NLR Family CARD Domain-Containing Protein 4 (NLRC4) inflammasomes, are also implicated in atherosclerosis, though their roles are less well understood [109]. Importantly, these inflammasomes can be modulated by various endogenous factors, such as lipids and oxidative stress, both of which are elevated in atherosclerosis. The activation of these inflammasomes within the plaque provides a critical link between lipid metabolism, immune activation, and inflammation in the pathogenesis of atherosclerosis.

The NLRP3 inflammasome is one of the most well-characterized inflammasomes involved in atherosclerosis, and it is tightly regulated by various cellular components, including CARD8 [45, 46]. CARD8, a negative regulator of NLRP3, plays a crucial role in limiting the overactivation of the inflammasome [110]. By binding to NLRP3, CARD8 inhibits its activation and prevents excessive production of IL-1$\beta$ and other inflammatory mediators [111, 112]. This regulatory function of CARD8 is important for maintaining a balanced immune response in atherosclerotic plaques, as excessive inflammasome activation can lead to uncontrolled inflammation, plaque destabilization, and the progression of atherosclerosis [51, 53].

The balance between CARD8 and NLRP3 activity is critical in controlling the inflammatory environment in atherosclerotic plaques. Increased expression of CARD8 in macrophages and other immune cells within the plaque may serve as a compensatory mechanism to counteract the harmful effects of excessive inflammasome activation [50]. This highlights the potential of CARD8 as a therapeutic target for modulating inflammasome activity and reducing the chronic inflammation that accelerates atherosclerotic progression (Table 1).

CARD8 is a member of the CARD family of proteins, which includes other well-known inflammasome regulators such as NLRP3, ASC, and CASP1 [113]. The CARD8 gene is located on chromosome 1, and the protein contains a CARD at its N-terminus, which facilitates interactions with other CARD-containing proteins [51]. CARD8 does not possess caspase-like protease activity but plays a crucial role in regulating the activation of inflammasomes, particularly the NLRP3 inflammasome [46].

The protein structure of CARD8 consists of a CARD domain at the N-terminal, followed by a central coiled-coil domain and a C-terminal domain [114]. The CARD domain allows CARD8 to interact with other CARD-containing proteins, such as NLRP3 and ASC, thereby modulating inflammasome activation [115]. The coiled-coil domain is thought to mediate protein-protein interactions, and the C-terminal domain is involved in the regulation of CARD8 stability and function [115]. Importantly, the structure of CARD8 allows it to function as a negative regulator of the NLRP3 inflammasome, which is crucial in controlling the inflammatory response in atherosclerosis and other inflammatory diseases.

CARD8 is primarily involved in modulating the innate immune response, particularly through its interaction with the NLRP3 inflammasome [116]. The NLRP3 inflammasome is a multi-protein complex that is activated in response to a variety of danger signals, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [117, 118]. When activated, NLRP3 triggers the activation of caspase-1, which subsequently processes pro-IL-1$\beta$ and pro-IL-18 into their active forms [119, 120]. These cytokines are released into the extracellular space, where they drive inflammation and immune cell recruitment to sites of injury or infection. CARD8 serves as a negative regulator of NLRP3 by interacting with the CARD domain of NLRP3 and preventing its activation [46]. In this way, CARD8 helps to suppress the excessive release of IL-1$\beta$ and IL-18, which are potent pro-inflammatory cytokines involved in chronic inflammation, tissue damage, and atherosclerosis progression. By inhibiting NLRP3 activation, CARD8 helps to maintain a balanced immune response, ensuring that the inflammatory process does not become dysregulated and lead to excessive tissue damage or plaque instability. In addition to its role in regulating inflammasomes, CARD8 has been shown to interact with other signaling pathways, including the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-$\kappa$B) pathway, which is involved in the production of pro-inflammatory cytokines. Through these interactions, CARD8 further contributes to the resolution of inflammation and the prevention of chronic inflammatory diseases such as atherosclerosis.

CARD8 is expressed in a variety of immune cells, including macrophages, dendritic cells, and T lymphocytes [44, 51, 121]. The expression of CARD8 is tightly regulated in response to inflammatory stimuli, and its levels can be upregulated in response to danger signals such as ox-LDL and other DAMPs [122]. Studies have shown that the expression of CARD8 is particularly high in macrophages, where it plays a critical role in controlling the inflammatory response within atherosclerotic plaques [123]. In these cells, CARD8 interacts with the NLRP3 inflammasome to limit the activation of IL-1$\beta$ and IL-18, thereby reducing the inflammatory burden in the plaque [124]. In addition to macrophages, CARD8 expression has been detected in other immune cells involved in atherosclerosis, such as dendritic cells and T lymphocytes [43]. Dendritic cells are important antigen-presenting cells that activate T lymphocytes and regulate adaptive immune responses in atherosclerotic lesions [123]. The expression of CARD8 in dendritic cells suggests that it may play a role in modulating the adaptive immune response in atherosclerosis, potentially influencing the balance between pro-inflammatory and regulatory T cells [43].

The regulation of CARD8 expression is also influenced by various transcription factors, including NF-$\kappa$B and Activator Protein (AP)-1, which are activated in response to inflammatory signals [125]. These transcription factors promote the expression of CARD8 in immune cells, particularly during times of inflammation [125]. Moreover, CARD8 itself can be regulated by post-translational modifications, such as phosphorylation, which can alter its stability and activity [126]. The precise mechanisms that regulate CARD8 expression and activity in atherosclerosis remain an active area of research [43].

CARD8 plays a critical role in modulating the immune response in atherosclerosis by regulating the activation of the NLRP3 inflammasome and other inflammatory pathways. In atherosclerotic plaques, the interaction between CARD8 and NLRP3 helps to suppress excessive inflammasome activation, thereby reducing the release of IL-1$\beta$ and IL-18 [6]. This, in turn, helps to limit the recruitment and activation of immune cells, such as macrophages and T lymphocytes, which are responsible for driving the chronic inflammation in the plaque [127].

In macrophages, CARD8 limits the production of pro-inflammatory cytokines, which helps to stabilize the plaque and prevent plaque rupture [43]. In addition, CARD8 modulates the activation of T lymphocytes, particularly Tregs, which play a key role in suppressing inflammation and promoting plaque stability [128]. By influencing both the innate and adaptive immune responses, CARD8 contributes to the resolution of inflammation in atherosclerosis and plays a protective role in maintaining plaque stability [113]. Furthermore, CARD8’s regulatory function extends beyond inflammasome inhibition [113]. While CARD8 predominantly acts as a negative regulator of NLRP3 inflammasome activation in atherosclerosis by binding to NLRP3 and inhibiting its oligomerization with ASC, thereby limiting IL-1$\beta$ and IL-18 release to prevent excessive inflammation in plaques [113], emerging evidence reveals a context-dependent duality where CARD8 can promote pro-inflammatory responses under specific conditions, such as in macrophage activation and foam cell formation. This functional switch is triggered by molecular cues like pathogen-derived proteases or dysregulated proteostasis, where CARD8 undergoes autoproteolytic processing at its Function to Find Domain (FIIND) domain, releasing a bioactive C-terminal fragment (UPA-CARD) that directly recruits and activates caspase-1, independent of ASC [50, 115]. For instance, in lipid-laden environments mimicking foam cell formation, the T60 isoform of CARD8—prevalent in immune cells—senses Human Immunodeficiency Virus (HIV)-1 protease activity, leading to N-terminal cleavage, proteasome-mediated degradation of the inhibitory fragment, and subsequent CARD8 inflammasome assembly, which induces pyroptosis and IL-1$\beta$ secretion in macrophages [129]. This pro-inflammatory activation may exacerbate plaque instability by amplifying cytokine-driven immune cell recruitment and necrotic core expansion, particularly in the presence of viral co-infections or oxidative stress that unfolds CARD8’s disordered N-terminal region, enhancing its susceptibility to degradation and sensor function [114]. Genetic variants, such as rs2043211 (C10X), further modulate this duality; the minor allele disrupts the T48 isoform’s inhibitory binding to NLRP3, potentially shifting toward pro-inflammatory CARD8 activation in heterozygous individuals, as observed in inflammatory diseases with atherosclerotic overlap [130]. These mechanisms underscore CARD8’s isoform- and stimulus-specific roles, highlighting the need for targeted therapies that preserve its anti-inflammatory function while mitigating pro-inflammatory activation in advanced plaques. CARD8 has been implicated in regulating the migration and activation of immune cells, particularly macrophages and T lymphocytes, within the atherosclerotic plaque [43]. By controlling immune cell function and limiting excessive inflammation, CARD8 helps to reduce plaque progression and the risk of thrombosis [111].

A study by Paramel Varghese et al. [106] examined CARD8 mRNA expression in atherosclerotic vascular tissue and compared it to transplant donor arterial tissue. They found that CARD8 mRNA was highly expressed in atherosclerotic plaques compared to the expression in transplant donor vessels [51]. Another research used immunohistochemistry to examine CARD8 expression in non-atherosclerotic arteries and carotid lesions. In non-atherosclerotic vessels, CARD8 expression was primarily detected in endothelial cells and smooth muscle cells in the tunica media. In atherosclerotic carotid lesions, CARD8 was detected in the endothelial layer, smooth muscle cells, and CD68⁺ macrophages, suggesting that immune cells, along with vascular cells, contribute to the increased expression of CARD8 in human atherosclerotic lesions [43]. These studies indicate that CARD8 expression indeed varies between normal and atherosclerotic tissues. However, more research is needed to comprehensively understand its expression differences across diverse atherosclerosis models, such as those induced by different risk factors (e.g., high- fat diet - induced, oxidized LDL - induced).

Atherosclerosis is a chronic inflammatory disease in which immune cell activation, lipid deposition, and extracellular matrix remodeling contribute to the thickening of the arterial walls and the formation of plaques [131]. The inflammatory response plays a pivotal role in all stages of atherosclerosis, from the initiation of endothelial injury to the destabilization and rupture of advanced plaques [131]. As a negative regulator of the NLRP3 inflammasome, CARD8 has emerged as a key player in controlling the balance between inflammation and tissue repair in atherosclerosis [113].

The expression of CARD8 in atherosclerotic lesions has been shown to influence plaque stability and progression [43]. In animal models, the overexpression of CARD8 in macrophages leads to the suppression of IL-1$\beta$ and IL-18 production, reducing the inflammatory burden within the plaque [113]. This results in the stabilization of the plaque, as lower levels of inflammatory cytokines reduce immune cell recruitment and smooth muscle cell proliferation [132]. Conversely, a lack of CARD8 or its inhibition exacerbates the inflammatory response, promoting plaque progression and destabilization, which increases the risk of rupture and subsequent cardiovascular events [43]. Several studies have demonstrated that CARD8 interacts with other inflammatory pathways in atherosclerosis [43]. For example, CARD8 can modulate the NF-$\kappa$B signaling pathway, which is activated in response to inflammatory stimuli [113]. By inhibiting excessive NF-$\kappa$B activation, CARD8 helps to limit the production of pro-inflammatory cytokines such as TNF-$\alpha$ and IL-6, further reducing inflammation within the plaque [43]. In addition to its direct modulation of NF-$\kappa$B, CARD8 engages in potential crosstalk with other key inflammatory pathways, such as mitogen-activated protein kinase (MAPK) and Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT), to regulate cytokine production and immune cell function in atherosclerosis. CARD8 inhibits NF-$\kappa$B activation via interaction with the I$\kappa$B kinase complex (I$\kappa$B kinase $\gamma$ subunit (IKK$\gamma$)/NF-$\kappa$B essential modulator (NEMO)), suppressing downstream cytokine expression (e.g., TNF-$\alpha$, IL-6) in endothelial cells and macrophages, which may indirectly influence JAK-STAT signaling given IL-6’s role in activating JAK/STAT3 to induce monocyte chemoattractant protein-1 (MCP-1) in vascular endothelial cells [43]. Although direct interactions with MAPK are not well-established, CARD8’s regulation of inflammasome activity could intersect with MAPK pathways, as NLRP3 inhibition by CARD8 limits p38 MAPK-mediated inflammatory responses in immune cells, potentially reducing cytokine-driven plaque progression [113]. In atherosclerotic lesions, this network integration helps maintain immune homeostasis by dampening macrophage activation and T cell polarization, with CARD8 overexpression in ApoE^-⁣/- models attenuating IL-1$\beta$ and IL-18 release, which otherwise amplifies NF-$\kappa$B-JAK-STAT crosstalk to exacerbate foam cell formation and plaque instability [43]. These mechanisms underscore CARD8’s multifaceted role in orchestrating inflammatory signaling, though further studies are needed to elucidate direct crosstalk with MAPK and JAK-STAT in human plaques. This highlights CARD8’s multifaceted role in controlling inflammation and plaque stability, making it a potential therapeutic target in atherosclerosis.

Experimental studies have provided valuable insights into the role of CARD8 in atherosclerosis. In ApoE^-⁣/- mice, a commonly used animal model of atherosclerosis, CARD8 expression is upregulated in the aortic tissues, particularly in macrophages and other immune cells within atherosclerotic plaques [43]. Studies have shown that the deletion of CARD8 in these mice leads to increased levels of pro-inflammatory cytokines such as IL-1$\beta$ and IL-18, along with greater immune cell infiltration into the plaque [112]. These findings support the notion that CARD8 acts as a negative regulator of inflammation and plaque development.

In contrast, the overexpression of CARD8 in macrophages has been shown to attenuate the inflammatory response in these animal models, leading to a reduction in plaque size and improved plaque stability [43]. This protective effect is attributed to CARD8’s ability to inhibit NLRP3 inflammasome activation, preventing the excessive release of IL-1$\beta$ and IL-18, which are known to drive inflammation in atherosclerotic lesions [133]. The overexpression of CARD8 also leads to a decrease in the number of foam cells in the plaque, suggesting that CARD8 may influence lipid metabolism and foam cell formation [43]. Immunohistochemical analysis of human atherosclerotic plaques reveals that CARD8 is expressed in macrophages and smooth muscle cells within the plaque [43]. Notably, the expression of CARD8 is inversely correlated with the levels of IL-1$\beta$ in the plaque, suggesting that CARD8 acts to dampen the inflammatory response in human atherosclerosis as well [43]. Furthermore, elevated CARD8 expression has been associated with a more stable plaque phenotype, characterized by a thicker fibrous cap and fewer signs of inflammation (Table 2, Ref. [43, 80, 113, 133, 134]) [43, 134]. Differential expression of CARD8 across atherosclerosis models highlights its context-specific roles in regulating inflammation and plaque stability. In ApoE^-⁣/- mice, CARD8 is significantly upregulated in aortic macrophages and smooth muscle cells within atherosclerotic lesions, correlating with reduced IL-1$\beta$ and IL-18 levels, which mitigates plaque progression and enhances stability [43]. Conversely, in Low-Density Lipoprotein Receptor (LDLR)^-⁣/- mice, CARD8 expression is generally lower under hyperlipidemic conditions, potentially due to heightened oxidative stress and lipid accumulation, leading to increased NLRP3 inflammasome activity and more severe plaque inflammation [79]. Human atherosclerotic plaques exhibit heterogeneous CARD8 expression, predominantly in macrophages and foam cells, with higher levels in stable plaques compared to unstable ones, inversely correlating with IL-1$\beta$ expression [43]. A 2024 study further revealed that the CARD8 rs2043211 polymorphism modulates expression in human monocytes, with the minor allele reducing CARD8 mRNA levels in males, potentially exacerbating inflammatory responses in early atherosclerosis [135]. In vitro, human endothelial cells exposed to ox-LDL show induced CARD8 expression, which suppresses adhesion molecule expression (e.g., Intercellular Adhesion Molecule (ICAM)-1) to limit monocyte recruitment, contrasting with the macrophage-centric anti-inflammatory role in animal models [43]. These variations suggest that CARD8 expression is influenced by model-specific factors, such as lipid metabolism, genetic background, and inflammatory stimuli, underscoring the need for tailored therapeutic strategies targeting CARD8 in atherosclerosis [113].

The role of CARD8 in immune cells, particularly macrophages, has been extensively studied in the context of atherosclerosis. Macrophages are the primary immune cells involved in the formation and progression of atherosclerotic plaques. Upon recruitment to the site of injury, monocytes differentiate into macrophages, where they play a key role in phagocytosing ox-LDL and forming foam cells [136]. Foam cells contribute to plaque formation and progression by secreting pro-inflammatory cytokines that perpetuate the inflammatory cycle [137].

CARD8 has been shown to regulate the inflammatory response in macrophages by inhibiting NLRP3 inflammasome activation [113]. In macrophages from CARD8-deficient mice, there is a marked increase in IL-1$\beta$ production and foam cell formation, leading to more severe plaque progression [138]. This suggests that CARD8 plays a crucial role in maintaining macrophage function and preventing excessive inflammation in the plaque [139]. Conversely, macrophages overexpressing CARD8 exhibit reduced levels of IL-1$\beta$ and IL-18, along with a decrease in foam cell formation, leading to a more stable plaque [113].

CARD8 is also expressed in other immune cells, including monocytes and T lymphocytes, which are involved in the adaptive immune response in atherosclerosis. In monocytes, CARD8 regulates the activation of the NLRP3 inflammasome and helps to control the production of IL-1$\beta$, which is essential for monocyte migration and differentiation into macrophages [113]. In T cells, particularly T helper cells, CARD8 may influence the balance between pro-inflammatory Th1 cells and anti-inflammatory Tregs, which has implications for plaque stability and immune regulation [42] (Table 3). Beyond its role in monocytes and macrophages, CARD8 significantly influences dendritic cell (DC) and T lymphocyte functions, modulating adaptive immune responses critical to atherosclerotic plaque dynamics. In DCs, CARD8 is expressed at moderate levels and regulates antigen presentation by limiting NLRP3 inflammasome activation, which reduces IL-1$\beta$-driven DC maturation and subsequent T cell priming [43]. This dampening effect promotes tolerogenic DC phenotypes, enhancing the induction of Tregs over pro-inflammatory Th1 cells in atherosclerotic lesions, as evidenced by reduced IFN-$\gamma$ and increased IL-10 expression in CARD8-overexpressing DC-T cell co-cultures [113]. In T lymphocytes, particularly CD4⁺ T cells, CARD8 modulates polarization by inhibiting caspase-1-mediated pyroptosis, preserving Treg survival and function, which is critical for maintaining immune homeostasis and plaque stability [42]. A 2023 study demonstrated that CARD8 expression in T cells from human atherosclerotic plaques correlates with higher Treg/Th1 ratios, suggesting a protective role against excessive Th1-driven inflammation [87]. Furthermore, CARD8’s interaction with NF-$\kappa$B pathways in DCs suppresses pro-inflammatory cytokine production (e.g., IL-12), which otherwise skews T cell differentiation toward Th1 cells, exacerbating plaque progression [43]. These findings highlight CARD8’s role in DC-T cell crosstalk, where it fosters an anti-inflammatory microenvironment by balancing antigen presentation and T cell polarization, offering potential therapeutic avenues to enhance plaque stability through targeted modulation of adaptive immunity.

Beyond its role in immune regulation, CARD8 may also influence lipid deposition within the atherosclerotic plaque. Foam cell formation is a key event in atherosclerosis, and macrophages play a central role in the uptake of ox-LDL, which contributes to lipid accumulation in the plaque [140]. Recent studies suggest that CARD8 may modulate lipid metabolism in macrophages by regulating the expression of genes involved in lipid uptake and efflux [43]. In particular, CARD8 has been shown to inhibit the expression of pro-inflammatory lipid receptors, such as scavenger receptors, which are responsible for the uptake of ox-LDL into macrophages [140]. In addition, CARD8 may influence plaque inflammation through its effects on smooth muscle cells. Smooth muscle cells contribute to the formation of the fibrous cap in advanced plaques, and their proliferation is driven by inflammatory signals [141]. By suppressing the activation of pro-inflammatory pathways in smooth muscle cells, CARD8 may help maintain plaque stability and prevent plaque rupture [141].

Clinical studies investigating the role of CARD8 in human atherosclerosis have provided further evidence of its involvement in plaque stability. Immunohistochemical analysis of human atherosclerotic plaques reveals that CARD8 expression is significantly higher in stable plaques compared to unstable plaques [43]. Moreover, higher levels of CARD8 in plaque macrophages are associated with lower levels of IL-1$\beta$ and reduced immune cell infiltration [43]. These clinical observations are supported by immunohistochemical (IHC) and gene expression analyses from well-characterized patient cohorts. In the Biobank of Karolinska Endarterectomies (BiKE) study, carotid atherosclerotic plaques were obtained from 126 patients undergoing endarterectomy for ischemic cerebrovascular disease (advanced symptomatic atherosclerosis), with non-atherosclerotic control vessels from transplant donors [43]. Comorbidities in this cohort included hypertension, diabetes, and hyperlipidemia, common in advanced atherosclerosis, though specific prevalence rates were not stratified for CARD8 analysis. CARD8 protein expression was assessed via IHC on formalin-fixed, paraffin-embedded sections (4 µm) using anti-CARD8 antibodies, visualized with 3,3^′-diaminobenzidine (DAB), and semi-quantitatively scored based on staining intensity and cellular localization in macrophages and smooth muscle cells, revealing higher expression in stable plaques with thicker fibrous caps. Complementary microarray analysis (Affymetrix HG-U133 plus 2.0) on RNA from 106 BiKE plaques (normalized via robust multi-array average on log2 scale) showed CARD8 mRNA upregulation in plaques versus controls, with inverse correlations to IL-1$\beta$ levels after Benjamini-Hochberg false discovery rate adjustment [51]. These methods underscore CARD8’s association with reduced inflammation in stable plaques, though larger cohorts with quantitative digital pathology scoring could further validate translational applicability. These findings suggest that CARD8 may serve as a marker of plaque stability and may help predict the risk of plaque rupture in patients with atherosclerosis.

Despite promising associations with plaque stability, translating CARD8 as a biomarker into clinical practice faces several hurdles, including assay development for detection in blood versus plaque tissue and establishing correlations with clinical endpoints like major adverse cardiovascular events (MACE). CARD8 is primarily an intracellular protein, complicating its detection in circulation; current methods rely on IHC analysis of plaque tissue or mRNA quantification via microarray in cohorts like the BiKE study, where CARD8 expression inversely correlates with IL-1$\beta$ but lacks direct blood-based assays such as ELISA due to low solubility and absence of secreted forms [43]. Genetic polymorphisms, such as rs2043211 (C10X variant), serve as potential proxies for CARD8 function, with the minor allele associated with reduced CARD8 activity and increased inflammatory markers in healthy individuals, but their predictive value for atherosclerosis progression remains limited without large-scale validation [135]. Correlation with MACE is underexplored; in abdominal aortic aneurysms, CARD8 variants interact with NLRP3 to influence disease risk, but no direct links to cardiovascular events like rupture or infarction have been established, highlighting the need for prospective studies integrating CARD8 genetics with imaging or circulating inflammation markers [47]. Additional challenges include assay sensitivity for low-abundance proteins in blood, variability due to comorbidities (e.g., hypertension, diabetes), and the requirement for standardized quantitation methods to enable routine clinical use [46, 113]. Overcoming these barriers could position CARD8 as a viable biomarker for risk stratification in atherosclerosis.

The therapeutic potential of CARD8 as a target for atherosclerosis treatment is supported by its ability to modulate inflammation and immune cell function within plaques [43]. By enhancing CARD8 expression or activity, it may be possible to reduce the inflammatory burden in atherosclerosis, prevent plaque destabilization, and improve patient outcomes [113]. However, further studies are needed to determine the exact mechanisms by which CARD8 influences plaque biology and to evaluate its potential as a therapeutic target in clinical settings [39].

As a negative regulator of the NLRP3 inflammasome, CARD8 has emerged as a promising therapeutic target for atherosclerosis, a disease primarily driven by inflammation. The idea of modulating CARD8 expression or function to control the inflammatory response in atherosclerotic lesions offers a novel approach to treatment [113]. Given that atherosclerosis is characterized by chronic inflammation, which leads to plaque instability, targeted therapies aimed at restoring the regulatory function of CARD8 could potentially halt or even reverse disease progression [142].

Preclinical studies have highlighted the potential of CARD8 as a therapeutic target. By inhibiting excessive activation of the NLRP3 inflammasome, CARD8 can reduce the production of IL-1$\beta$ and IL-18, which are critical cytokines driving inflammation and atherosclerotic plaque formation. The ability to restore CARD8 function in macrophages and other immune cells could lead to the stabilization of plaques, reducing the likelihood of plaque rupture and the risk of cardiovascular events, such as myocardial infarction and stroke [111].

There are several strategies for targeting CARD8 in the context of atherosclerosis treatment. One promising approach is the use of small molecules that can enhance CARD8 expression or activate its function [137]. Alternatively, the development of monoclonal antibodies that mimic CARD8’s regulatory effects on the NLRP3 inflammasome could provide a more targeted and specific therapeutic intervention [111].

Although direct therapeutic strategies targeting CARD8 are still in the early stages of development, several approaches have been explored in related inflammatory diseases, which could provide insight into potential therapies for atherosclerosis [143].

One potential strategy involves the use of small molecules that modulate the activity of the inflammasome. For example, inhibitors of the NLRP3 inflammasome, such as MCC950, have shown promise in reducing systemic inflammation and preventing disease progression in models of atherosclerosis [144]. These inhibitors could work synergistically with CARD8, either by increasing its activity or by restoring its function in cases of CARD8 deficiency [145]. As the role of CARD8 in inflammasome regulation becomes clearer, drugs that specifically target CARD8’s interaction with NLRP3 could be developed to provide more precise modulation of the immune response [146].

Another potential approach involves gene therapy to enhance the expression of CARD8 in atherosclerotic plaques. Using viral vectors or mRNA-based delivery systems, the therapeutic delivery of CARD8 could help restore its normal function in immune cells, particularly macrophages, where its effect on inflammasome regulation is most significant [147]. Preclinical studies in animal models of atherosclerosis have demonstrated the potential for gene therapy to reduce plaque size and improve plaque stability, with CARD8 playing a central role in this effect [111].

Additionally, monoclonal antibodies or recombinant proteins that mimic CARD8’s anti-inflammatory effects could offer a more targeted approach to modulating the inflammasome in atherosclerosis [111]. These therapies would be designed to activate CARD8’s role in inhibiting NLRP3 activation, thereby reducing IL-1$\beta$ and IL-18 levels and limiting the inflammatory processes that drive atherosclerosis [148] (Table 4). Preliminary preclinical data on candidate molecules and delivery platforms provide insights into targeting CARD8’s regulatory function in atherosclerosis. For small molecules, MCC950, a selective NLRP3 inhibitor, synergizes with CARD8’s inhibitory role by blocking NLRP3 activation at nanomolar concentrations, reducing IL-1$\beta$ release in human monocyte-derived macrophages [149]. In ApoE^-⁣/- mouse models of atherosclerosis, MCC950 administered intraperitoneally hindered plaque development, attenuating macrophage pyroptosis and inflammation, with a 30–40% reduction in aortic lesion area and lowered serum IL-1$\beta$ levels, demonstrating dose-dependent efficacy without significant toxicity [150]. Monoclonal antibodies targeting downstream pathways, such as canakinumab (anti-IL-1$\beta$), mimic CARD8’s anti-inflammatory effects; in preclinical rabbit models of atherosclerosis, subcutaneous dosing stabilized plaques by reducing IL-1$\beta$-driven inflammation, with a 25% decrease in macrophage infiltration and improved fibrous cap thickness [151]. These examples highlight progress in CARD8-related therapeutics, though direct CARD8 agonists remain under development, emphasizing the need for CARD8-specific lead optimization.

A variety of preclinical studies have investigated the role of CARD8 in atherosclerosis and its potential as a therapeutic target [152]. In animal models, the inhibition or deletion of CARD8 exacerbates plaque formation and inflammation, leading to unstable plaques that are prone to rupture [153]. These findings suggest that restoring CARD8 function could stabilize plaques and reduce the incidence of cardiovascular events. Gene knockout studies in ApoE^-⁣/- mice have demonstrated that CARD8 deficiency accelerates the development of atherosclerotic lesions and increases the inflammatory cytokine production in the plaques [148]. Restoring CARD8 expression in these animals significantly reduces plaque size and stabilizes the lesions. Additionally, the modulation of CARD8 activity through gene therapy or small molecules has been shown to reduce the inflammatory response, improving plaque stability and reducing the risk of plaque rupture [111].

In vitro studies using macrophage and endothelial cell cultures have further confirmed the role of CARD8 in regulating the inflammatory response in atherosclerosis. For instance, activating CARD8 in macrophages has been shown to inhibit NLRP3 inflammasome activation, reduce IL-1$\beta$ secretion, and prevent foam cell formation [111]. These cellular models have provided valuable insights into the mechanisms by which CARD8 modulates inflammation and plaque progression, and they will be essential for developing targeted therapies [152].

While the therapeutic potential of CARD8 in atherosclerosis is promising, several challenges must be addressed before it can be translated into clinical practice [154]. One major challenge is the need for specific and safe targeting of CARD8. Given the complexity of the immune system and the fact that CARD8 is involved in regulating multiple immune responses, any therapeutic intervention must be carefully designed to avoid unintended effects on other aspects of immune function [143].

Another challenge lies in the delivery of CARD8-targeting therapies to the atherosclerotic lesions. Efficient and targeted delivery of therapeutic agents to specific cells, such as macrophages in plaques, remains a significant hurdle in the field of cardiovascular disease treatment [155]. Advances in nanotechnology and targeted drug delivery systems may help overcome these obstacles, but further research is needed to optimize these approaches [156].

Finally, while preclinical studies have demonstrated the potential benefits of CARD8 modulation, clinical trials in humans are required to assess the safety and efficacy of such therapies [153]. The success of clinical trials will depend on the ability to identify suitable biomarkers for CARD8 activity and monitor the effects of treatment on plaque progression and stability [157]. Additionally, the potential for off-target effects and long-term safety must be thoroughly evaluated before CARD8-targeting therapies can be introduced into clinical practice [158].

Long-term safety challenges for CARD8-targeted therapies, which primarily inhibit NLRP3 inflammasome activation, include unintended immunosuppression, off-target effects on other inflammasomes such as AIM2 and NLRC4, and compromised host defense mechanisms, potentially increasing infection risk in chronic inflammatory conditions like atherosclerosis [159]. Unintended immunosuppression arises from NLRP3’s role in innate immunity; chronic inhibition may impair pathogen clearance, as evidenced by increased susceptibility to bacterial and viral infections in preclinical models treated with NLRP3 inhibitors like MCC950, where long-term dosing reduced IL-1$\beta$ but elevated infection rates in mice challenged with pathogens [160]. Off-target effects on AIM2 and NLRC4 are a concern, as CARD8 mutations marginally impact AIM2 activation without affecting NLRC4 or pyrin, potentially leading to dysregulated DNA-sensing (AIM2) or bacterial defense (NLRC4) in immune cells, exacerbating opportunistic infections or autoimmune flares. Impacts on host defense are highlighted in studies showing NLRP3’s beneficial early-stage role in infection recognition; selective inhibitors preserve some immune functions but risk broader immunosuppression in vulnerable populations, such as those with comorbidities, necessitating monitoring for MACE and infections in trials [161]. These risks underscore the importance of developing CARD8-specific agents with minimal off-target activity to ensure clinical feasibility.

Given the complex pathophysiology of atherosclerosis, combination therapies targeting CARD8 along with other inflammatory pathways could offer a more effective treatment strategy [162]. For example, combining CARD8 modulation with existing anti-inflammatory therapies, such as IL-1$\beta$ inhibitors, may provide synergistic effects in reducing systemic inflammation and stabilizing plaques [163]. Moreover, combining CARD8-targeted therapies with lipid-lowering agents, such as statins, could further enhance the therapeutic benefits by addressing both the inflammatory and lipid components of atherosclerosis [164]. The combination of CARD8-targeting strategies with lifestyle interventions, such as diet and exercise, could also play a role in managing atherosclerosis and reducing the need for invasive procedures [165]. Further research into the mechanisms of action of CARD8 in atherosclerosis will help define the best strategies for combination therapies and improve patient outcomes [166].