Caspase Recruitment Domain Family Member 8: A Favorable Target in the Pathogenesis of Atherosclerosis

Abstract

Atherosclerosis, a lipid-driven chronic inflammatory disease, is the primary pathological basis of cardiovascular diseases, characterized by endothelial injury, lipid deposition, immune cell infiltration, and chronic inflammation. The NOD-like Receptor Pyrin Domain-Containing 3 (NLRP3) inflammasome has emerged as a crucial mediator of inflammation in atherosclerosis, with caspase recruitment domain family member 8 (CARD8) acting as a key regulatory component. Indeed, CARD8, a member of the caspase recruitment domain family, regulates immune responses by modulating inflammasome activity, particularly NLRP3. Recent studies suggest that CARD8 influences various aspects of atherosclerotic development, including lipid accumulation, oxidative stress, vascular inflammation, smooth muscle cell proliferation, and plaque instability. Thus, this review summarizes the latest findings on the role of CARD8 in the pathogenesis of atherosclerosis, with a focus on the regulatory effects of this component on immune cells and inflammatory pathways. We also discuss the potential of targeting CARD8 as a therapeutic strategy for atherosclerosis, exploring the current preclinical and clinical evidence.

Graphical abstract

Keywords

CARD8 / atherosclerosis / cardiovascular disease / therapeutic potential / molecular mechanisms

1. Introduction

Atherosclerosis is a complex, multifactorial disease that is characterized by the chronic accumulation of lipids, inflammatory cells, and extracellular matrix within the arterial wall [1]. This disease is a leading cause of cardiovascular morbidity and mortality worldwide, contributing to major conditions such as coronary artery disease (CAD), stroke, and peripheral artery disease [2, 3]. The pathogenesis of atherosclerosis involves a series of processes, including endothelial injury, lipid accumulation, smooth muscle cell migration, and the formation of atherosclerotic plaques [4, 5]. Despite the widespread use of statins and other lipid-lowering therapies, the incidence of cardiovascular events remains high, and many patients still experience recurrent cardiovascular events even after treatment, underscoring the need for more effective and targeted therapies to address the underlying disease processes. Historically, atherosclerosis was primarily considered a disease of lipid accumulation; however, recent research has revealed that immune cells play a pivotal role in the development and progression of the disease [6]. The immune response in atherosclerosis is initiated by the retention of low-density lipoprotein (LDL) particles in the arterial intima, which undergo oxidative modifications [7]. These modified lipoproteins are recognized by immune cells, primarily macrophages, which engulf them to form foam cells [8]. Foam cell formation, in turn, triggers a cascade of inflammatory events that contribute to plaque instability [9, 10, 11, 12]. Furthermore, T lymphocytes and dendritic cells, which infiltrate the atherosclerotic lesions, release pro-inflammatory cytokines and chemokines, thus perpetuating the inflammatory response and contributing to lesion progression [13, 14, 15, 16, 17, 18]. It is now well-established that inflammation within the arterial wall is a central driver of atherosclerosis and that modulation of the immune response may offer potential therapeutic avenues for treatment [19]. Chronic low-grade inflammation in atherosclerosis is not only responsible for plaque formation but also for the destabilization of plaques, leading to acute cardiovascular events such as myocardial infarction and stroke [20]. Therefore, understanding the mechanisms that regulate immune responses within atherosclerotic plaques is critical for the development of novel therapies aimed at controlling inflammation and stabilizing plaques.

Inflammasomes are multi-protein complexes that are formed in response to cellular stress and injury [21, 22, 23, 24]. These complexes play a crucial role in innate immunity by sensing pathogens, danger signals, and cellular damage. The NOD-like Receptor Pyrin Domain-Containing 3 (NLRP3) inflammasome, one of the best-studied inflammasomes, is composed of the sensor protein NLRP3, the adapter protein ASC, and the effector protein caspase-1 [25, 26, 27, 28]. Upon activation, NLRP3 inflammasome triggers the processing and release of pro-inflammatory cytokines, particularly Interleukin (IL)-1\beta and IL-18, which are involved in the promotion of inflammation and the recruitment of immune cells to the site of injury [29, 30, 31, 32]. In the context of atherosclerosis, NLRP3 inflammasome activation plays a central role in the development of inflammatory responses within the atherosclerotic plaques [33, 34]. Studies have shown that NLRP3 inflammasome activation in macrophages, endothelial cells, and smooth muscle cells contributes to the local inflammatory environment that accelerates plaque progression and destabilization [35, 36, 37, 38]. For example, the release of IL-1\beta from activated macrophages leads to further recruitment of immune cells, which exacerbates the inflammatory response and promotes the formation of necrotic cores in the plaque. As a result, inhibiting NLRP3 inflammasome activity has emerged as a promising therapeutic strategy for reducing inflammation and preventing plaque rupture in atherosclerosis.

Caspase Recruitment Domain Family Member 8 (CARD8) is a recently identified member of the caspase recruitment domain (CARD) family of proteins that has been shown to play an important role in the regulation of inflammasome activation [39, 40, 41]. CARD8 is highly expressed in immune cells such as macrophages, dendritic cells, and neutrophils, where it interacts with the NLRP3 inflammasome to modulate its activation [42, 43, 44]. Unlike other inflammasome components that promote inflammasome activation, CARD8 acts as a negative regulator by inhibiting the excessive activation of NLRP3 [45, 46, 47]. CARD8 achieves this through interactions with NLRP3 and caspase-1, preventing the activation of the inflammasome under basal conditions. In addition to its role in inflammasome regulation, CARD8 is also involved in the modulation of other immune pathways, such as apoptosis and the regulation of cytokine production [42, 48]. The dual role of CARD8 in immune regulation has made it an attractive candidate for further investigation in the context of atherosclerosis. Some studies suggest that CARD8 expression is upregulated in macrophages within atherosclerotic lesions, where it helps control the inflammatory response by inhibiting excessive inflammasome activation [49, 50, 51, 52]. However, the precise role of CARD8 in atherosclerosis remains unclear, as some studies have also suggested that CARD8 may have pro-inflammatory effects under certain conditions, particularly in the context of macrophage activation and foam cell formation. Given these conflicting findings, further research is needed to elucidate the full spectrum of CARD8’s function in atherosclerotic disease and its potential as a therapeutic target.

In recent years, the exploration of CARD8 as a therapeutic target in atherosclerosis has gained attention. Targeting CARD8 could potentially modulate inflammasome activation, reduce inflammation, and stabilize atherosclerotic plaques [43, 51, 53, 54]. The development of small molecules or biologics that can selectively target CARD8 expression or its interactions with inflammasome components holds great promise for therapeutic intervention in atherosclerosis. However, the clinical translation of CARD8-based therapies faces several challenges. For instance, the specificity and selectivity of CARD8-targeting agents need to be carefully evaluated to avoid unintended effects on other immune pathways. Additionally, the potential for off-target effects and the long-term safety of such therapies must be addressed before clinical application. Despite these challenges, the promise of CARD8 as a therapeutic target in atherosclerosis underscores the need for further research to refine and optimize strategies that target this protein. This review aims to provide a comprehensive overview of the current understanding of CARD8’s role in atherosclerosis, with a focus on its function in regulating the NLRP3 inflammasome and its impact on the inflammatory processes driving atherosclerotic disease. We will examine the molecular mechanisms by which CARD8 influences immune cell activation and cytokine production, as well as its potential as a therapeutic target in atherosclerosis. Additionally, we will discuss current research on CARD8-targeted therapies and explore the challenges and opportunities for translating these findings into clinical practice.

2. Literature Search and Methodology

Web of Science is a globally authoritative retrieval system operated by Clarivate, widely cited and utilized in the academic community. It boasts an extensive subject coverage, encompassing 256 disciplines across diverse fields such as science and technology, social sciences, arts, and humanities, serving as a comprehensive resource library. Its literature resources are remarkably abundant: as of December 2024, the Web of Science Core Collection has included 79 million records, with the total number of records across the entire platform reaching 171 million. Additionally, the system contains citation information for each article.

Web of Science excels in retrieval functionality, featuring a variety of retrieval methods and rules. It also possesses unique citation indexes, in-depth analysis tools, academic influence evaluation functions, and personalized services. These capabilities provide support to researchers in multiple research aspects, such as understanding the development context of research topics, assessing influence, and tracking the progress of research projects.

Our team used the Web of Science Core Collection as the data source, and the configured retrieval formula was “(ALL = CARD8) OR (ALL = Caspase Recruitment Domain Family Member 8)”. Through this retrieval process, a total of 603 pieces of literature were obtained, including 491 research articles, 76 reviews, and 36 pieces of literature of other types. To enhance the accuracy of the content, all literature has been downloaded, and a comprehensive full-text review has been conducted. The flow chart of the screening process is shown in Fig. 1.

3. The Immunological Landscape of Atherosclerosis

3.1 Pathogenesis of Atherosclerosis: From Endothelial Dysfunction to Plaque Formation

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].

3.2 Immune Cells and Their Roles in Atherosclerosis

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.

3.3 Inflammation and Inflammasomes: Central to Atherosclerosis Pathogenesis

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).

4. CARD8: An Emerging Key Regulator in Inflammatory Responses

4.1 Basic Characteristics of CARD8

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.

4.2 Function of CARD8 in Immune Response

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-\kappaB) 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.

4.3 Expression and Regulation of CARD8: Key to Understanding its Function

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-\kappaB 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].

4.4 CARD8 in Atherosclerosis: Implications for Disease Progression

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).

5. CARD8 in Atherosclerosis: From Bench to Bedside

5.1 The Role of CARD8 in Atherosclerosis Progression

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-\kappaB signaling pathway, which is activated in response to inflammatory stimuli [113]. By inhibiting excessive NF-\kappaB 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-\kappaB, 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-\kappaB activation via interaction with the I\kappaB kinase complex (I\kappaB kinase \gamma subunit (IKK\gamma)/NF-\kappaB 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-\kappaB-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.

5.2 Expression and Functional Data of CARD8 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].

5.3 Immune Cells in Atherosclerosis: CARD8’s Impact on Macrophages, Monocytes, and T Cells

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-\kappaB 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.

5.4 CARD8 in Lipid Deposition and Plaque Inflammation

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].

5.5 Clinical Insights: Evidence for CARD8 as a Diagnostic and Therapeutic Target

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].

6. Therapeutic Potential of Targeting CARD8 in Atherosclerosis

6.1 Targeting CARD8: A Promising Strategy for Atherosclerosis Therapy

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].

6.2 Current Approaches and Therapeutic Strategies for Targeting CARD8

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.

6.3 Preclinical Insights: Experimental Models of CARD8 Inhibition

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].

6.4 Clinical Challenges and Future Considerations

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.

6.5 Synergistic Approaches: Combining CARD8 Inhibition With Other Therapeutic Modalities

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].

7. Conclusion and Future Directions

Atherosclerosis remains a leading cause of cardiovascular morbidity and mortality worldwide, and despite significant advancements in therapeutic strategies, the disease continues to pose a major public health challenge. Chronic inflammation is a hallmark of atherosclerosis, and targeting key regulatory pathways involved in immune responses holds considerable promise for novel therapeutic interventions. Among the various immune modulators, CARD8 has emerged as a critical player in regulating the NLRP3 inflammasome, a central driver of inflammation in atherosclerosis.

Through its inhibitory effect on NLRP3 activation, CARD8 regulates the production of key pro-inflammatory cytokines, such as IL-1\beta and IL-18, which contribute to plaque formation, instability, and rupture. Furthermore, emerging evidence suggests that CARD8 plays a key role in immune cell function, particularly in macrophages, where it helps modulate the inflammatory microenvironment within atherosclerotic plaques. These findings highlight CARD8’s potential as a therapeutic target for atherosclerosis, as its modulation could help stabilize plaques, reduce inflammation, and potentially prevent adverse cardiovascular events (Fig. 2).

Despite the promising preclinical data, several challenges remain in translating CARD8-targeted therapies into clinical practice. A comprehensive understanding of the molecular mechanisms by which CARD8 influences immune cell responses, along with the identification of reliable biomarkers, is essential for the development of targeted therapies. Additionally, overcoming obstacles related to the delivery of CARD8-modulating agents to atherosclerotic lesions and ensuring their safety and efficacy in long-term clinical trials are critical steps toward the successful clinical application of CARD8-targeted therapies.

Looking ahead, future research should focus on addressing these challenges by expanding our understanding of the role of CARD8 in immune regulation across different stages of atherosclerosis and in various immune cell populations. Moreover, the development of combination therapies that target CARD8 alongside other inflammatory pathways, such as IL-1\beta, may provide more effective treatment options for patients with atherosclerosis. Personalized medicine approaches, guided by genetic and immunological profiles, will be essential in optimizing therapeutic strategies for individual patients. In conclusion, CARD8 holds significant promise as a novel therapeutic target in atherosclerosis. Further research is needed to fully elucidate its molecular mechanisms and to validate its potential in clinical settings. The translation of CARD8-based therapies from bench to bedside could lead to more effective treatments for atherosclerosis, ultimately improving patient outcomes and reducing the burden of cardiovascular disease.

Despite the promising role of CARD8 in atherosclerosis and its potential as a therapeutic target, several important research gaps remain. First, the precise molecular mechanisms by which CARD8 regulates the NLRP3 inflammasome and how this influences atherosclerosis progression are not fully understood. While CARD8 has been established as an inhibitor of NLRP3 inflammasome activation, its detailed role in the modulation of other immune signaling pathways, such as those involved in macrophage polarization or endothelial dysfunction, requires further investigation. Understanding the broader context in which CARD8 functions could unveil additional therapeutic opportunities, especially in the context of other cardiovascular diseases that also involve chronic inflammation. Second, the role of CARD8 in different types of immune cells in the context of atherosclerosis needs to be more thoroughly explored. Although CARD8 has been predominantly studied in macrophages, its role in other immune cells, such as dendritic cells, T cells, and smooth muscle cells, is less well understood. These cells contribute to various stages of plaque development and stability, and their response to CARD8 modulation could have significant therapeutic implications. Another major challenge is the heterogeneity of atherosclerosis itself. Atherosclerotic plaques are highly variable, with different stages of progression and varying degrees of inflammation, lipid accumulation, and vascular remodeling. The development of biomarkers to identify patients at different stages of disease, and the role of CARD8 in these stages, would be crucial for determining the optimal timing and strategy for therapeutic intervention. Moreover, the effects of CARD8 modulation on plaque stability, rupture, and cardiovascular events need to be clarified through long-term clinical trials.

Future research should prioritize specific gaps to advance CARD8’s therapeutic potential in atherosclerosis. First, elucidating CARD8’s role in early versus advanced atherosclerotic lesions is critical, as its expression is elevated in stable plaques with thicker fibrous caps but less clear in early lipid-driven lesions, where NLRP3-driven inflammation may dominate [43]. Studies in ApoE^-⁣/- mice suggest CARD8 upregulation in advanced plaques reduces IL-1\beta, but its function in early endothelial dysfunction remains underexplored [135]. Second, sex-specific differences in CARD8 regulation, such as the rs2043211 polymorphism’s impact on reducing inflammatory markers (e.g., Chemokine (C-C motif) ligand 20 (CCL20), IL-6) in males but not females, potentially due to estrogen-mediated effects, warrant further investigation to tailor therapies for diverse populations [135]. Third, evaluating combination therapies integrating CARD8 modulation with statins could enhance efficacy; preclinical data on NLRP3 inhibitors like MCC950 show synergistic reductions in plaque size when combined with atorvastatin, suggesting CARD8 agonists could similarly augment lipid-lowering and anti-inflammatory effects [150]. These priorities—stage-specific functions, sex differences, and combination strategies—require targeted studies, including longitudinal human cohorts and genetic models, to validate CARD8’s clinical applicability and optimize personalized treatment approaches [46].

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Funding

Youth Research Foundation Project of General Hospital of Hunan University of Medicine(QNJJ202501)

Health Commission of Hunan Province 2023 National Clinical Key Specialty Major Scientific Research Project(Z2023005)

Hunan Provincial People's Hospital Medical Alliance Special Research Fund Project(LY-2024-27)