Abdominal aortic aneurysm (AAA) is characterized by a permanent, localized dilation of the abdominal aorta that exceeds its normal diameter by 50%, or reaches a maximal diameter of 30 mm [1]. The majority of AAAs are located in the infrarenal aorta, proximal to the aortic bifurcation. The risk of AAA rupture escalates with increasing aortic diameter [2]. Non-syndromic AAA is a major cause of cardiovascular mortality due to the high risk of aortic rupture. The diagnosis of this condition is challenging because most aneurysms remain asymptomatic until rupture occurs [3, 4]. The incidence of AAA rupture in the American population between 2005 and 2012 was 7.29 per 100,000, accounting for 4%–5% of sudden death cases. Approximately 50% of patients who undergo AAA rupture reach hospital. The operative mortality rate is around 50%, although the exact figure is difficult to determine [4]. In 2017, AAA was responsible for more than 167,000 deaths globally, and 3 million disability-adjusted life years [5, 6]. In 2019, 35.12 million cases of AAA were reported world-wide among individuals aged 30–79 years (0.92%) [7]. Endovascular aneurysm repair (EVAR) and open surgery are the two surgical interventions for AAA repair [8]. While minimally invasive devices for EVAR have improved greatly, the durability of this treatment remains problematic, and the potential for rupture remains [9]. The development of novel therapies, including stem cell therapies, has faced significant challenges. Consequently, a strong imperative is to investigate the mechanisms underlying AAA formation and identify novel specific biomarkers or therapeutic approaches for diagnosing and treating AAA in its early stages.

Substantial evidence indicates the major contributors to AAA are chronic inflammation and dysregulation of the extracellular matrix (ECM), composed primarily of elastin and collagen proteins, and the loss of vascular smooth muscle cells (VSMCs) [10, 11]. The high-mobility group box (HMGB) family is comprised of four members (HMGB1-4) that play an important role in various inflammatory diseases and have the capacity to modulate innate immunity [12]. Research indicates that elevated HMGB1 levels are present in the aneurysmal tissue of human AAA and in murine experimental models of AAA [13]. The studies to date have predominantly focused on HMGB1, which drives pro-inflammatory signaling via receptors such as toll-like receptor (TLR) and advanced glycation end product (RAGE) [14, 15, 16]. Despite sharing $>$80% structural homology with HMGB1, HMGB2 exhibits distinct functional properties, including different expression patterns and interactions with immune pathways such as triggering receptor expressed on myeloid cells-1 (TREM-1) [17]. Previous clinical studies have demonstrated that elevated HMGB2 expression correlates with the severity of myocardial infarction (MI), major adverse cardiovascular events (MACEs) at one month, and in-stent restenosis [18, 19]. However, the role of HMGB2 in AAA requires further investigation. Wu et al. [20] reported increased HMGB2 levels in angiotensin-II-treated VSMCs, and in an angiotensin II-induced mouse model of AAA. Inhibition of HMGB2-regulated ferroptosis and inflammation in angiotensin-II-treated VSMCs may protect against AAA by inactivating nuclear factor-kappa B (NF-$\kappa$$\beta$) signaling. Despite these observations, there is a paucity of mechanistic evidence that directly connects HMGB2 to AAA development or rupture. TREM-1 is an immunoglobulin (Ig) superfamily member containing an Ig-like extracellular domain. It is closely associated with inflammatory reactions and various pathologies [21, 22]. Previous research has demonstrated that TREM-1 can exacerbate experimental AAA by modulating angiotensin II-induced monocyte trafficking and vascular wall inflammation [23]. Although the specific ligands for TREM-1 have yet to be definitively identified, several studies have suggested that HMGB1 could be a potential TREM-1 ligand involved in the inflammatory response [24, 25]. However, the interplay between HMGB2 and TREM-1, a key amplifier of inflammatory responses in AAA, has not yet been investigated.

The present study aims to address this gap in knowledge by investigating the specific contribution of HMGB2 to the pathogenesis of AAA, including the potential involvement of the HMGB2-TREM pathway. This should elucidate the potential value of HMGB2 for early detection and therapeutic targeting of AAA.

AAA is associated with a high mortality rate following aortic rupture and with severe effects on human health [32]. No specific biomarkers or effective drugs are currently available for the prevention, early identification and treatment of AAA [33]. Substantial evidence indicates that chronic inflammation and dysregulation of the ECM are key factors in the development of AAA, thus presenting a possible therapeutic strategy for controlling its progression [34]. Our study found that elevated serum HMGB2 and HMGB1 levels were both independently associated with the incidence and rupture of AAA in males. The Firth-corrected estimates aligned closely with the results from multiple logistic regression, demonstrating the robustness of the significant associations observed in the standard analysis. Compared to the first quartile of serum HMGB2 and HMGB1 levels, the odds ratio for AAA in the fourth quartile were increased by 6.92-fold and 8.62-fold, respectively. We also found that decreased levels of sTREM-1 were significantly associated with an increased risk of AAA. Moreover, sTREM-1 was positively correlated with serum HMGB2 levels, especially in the lower three HMGB2 quartiles, indicating that disruption of the HMGB2-TREM pathway may play a critical role in the pathogenesis of AAA.

HMGB1 is expressed in inflammatory cells, VSMCs, and endothelial cells, with previous studies also showing high abundance in human AAA lesions [13, 35, 36, 37]. Blocking HMGB1 with antibodies reduces pro-inflammatory cytokines and proteinases, and inhibits CaCl₂-induced AAA formation [13]. The current evidence suggests that HMGB1 activation of TLR4 amplifies TLR4 signaling, thereby promoting the release of interleukin (IL) 6 and monocyte chemoattractant protein-1 (MCP-1) from VSMCs and contributing to AAA formation and progression [14]. TLRs and HMGB1 can also induce signaling cascades via RAGE [15]. The HMGB1-RAGE signaling pathway is essential for maintaining chronic inflammation during the development of AAA [16]. Our study also found that serum HMGB1 levels are independently associated with a higher risk of AAA, confirming findings from previous research. However, information on the role of HMGB2 in AAA is still limited. Previous studies have shown that it is important for both the development of atherosclerosis and coronary artery in-stent restenosis by promoting neointimal hyperplasia in mice with femoral artery injury, and for the proliferation and migration of VSMCs [19, 38]. HMGB2 exacerbates myocardial ischemic injury via reactive oxygen species (ROS)-mediated apoptosis, aberrant autophagy, and the inflammatory response [18]. Wu et al. [20] observed increased HMGB2 levels in an angiotensin II-induced mouse model of AAA. Moreover, these authors reported that a potential therapeutic strategy for AAA may be the inhibition of HMGB2-regulated ferroptosis and inflammation in angiotensin-II-treated VSMCs through inactivation of NF-$\kappa$$\beta$ signaling. In line with previous research, we also found that elevated serum HMGB2 and HMGB1 levels were independently associated with the incidence of AAA. Our study is the first to report that serum HMGB2 levels were positively correlated with AAA diameter, with incremental increases in AAA diameter corresponding to elevated HMGB2 levels. Furthermore, we observed that higher HMGB2 levels were associated with an elevated risk of AAA rupture (OR: 1.117, 95% CI: 1.005–1.241, p 0.05). Therefore, our research provides novel insights into the association between HMGB2 and AAA. HMGB2 and HMGB1 share more than 80% identity at the amino acid sequence level and possess similar biochemical properties [17]. This could account for the positive correlation observed between HMGB1 and HMGB2 levels in our study. Although HMGB1 and HMGB2 share similar structural and biochemical characteristics, they are not completely identical [39]. He et al. [19] demonstrated that in mice with an arterial wire injury, HMGB2 promotes neointimal hyperplasia via RAGE-mediated activation of ROS, independently of TLRs. Previous research found that HMGB2 expression in the myocardium is low under normoxic conditions, but increases 60-fold at 12 to 24 h following MI. In contrast, HMGB1 expression is moderate under normoxic conditions, and increases 3-fold after MI [18]. Furthermore, the study found that recombinant HMGB1 could dose-dependently induce HMGB2 in H9C2 cardiomyocytes. This indicates a positive feedback loop between the two HMGBs, and implies that induction of HMGB2 following MI may be partially dependent on HMGB1. ROC curve analysis in the current study found that HMGB2 and HMGB1 had diagnostic value for the detection of AAA, with good specificity but limited sensitivity. The latter may be attributed to several factors: (1) the relatively small sample size of our study, requiring larger-scale validation; (2) the complex etiology and heterogeneity of AAA, which may affect biomarker performance; (3) potential confounding influences due to population characteristics, including the severity distribution of AAA, and control group composition; (4) biological variability in biomarker expression due to various physiological and pathological factors other than AAA. While these biomarkers are promising, particularly given their high specificity, their moderate sensitivity suggests they may be more effective when used in combination with other diagnostic methods. Further large-scale investigation is required to fully elucidate the roles of HMGB2 and HMGB1 in the pathogenesis of AAA, as well as their potential application in diagnosis.

Our study found a correlation between low serum sTREM-1 levels and the occurrence of AAA, suggesting that it may offer protection against AAA. Our results contrast to those of Vandestienne et al. [23], who reported elevated TREM1 mRNA expression in human aortic aneurysm tissues, and increased serum sTREM-1 levels in AAA patients. Their research indicated that TREM-1 could control angiotensin II-induced monocyte activity and promote experimental AAA. The reasons for the discrepancies between their results and the current findings are unclear, but may be attributable to several factors. First, Gibot et al. [40] reported that non-survivors exhibited reduced sTREM-1 levels on the first day after admission compared to survivors. sTREM-1 plasma concentrations in non-survivors remained stable or increased over time, but decreased in survivors, indicating that a high baseline sTREM-1 level is an independent protective factor in severe inflammatory conditions. Only the baseline level of sTREM-1 was assessed in our study, and hence any potential changes that occur subsequently require investigation at later follow-up times. Second, Giamarellos-Bourboulis et al. [41] reported that sTREM-1 functions as an anti-inflammatory mediator in sepsis, as evidenced by its positive correlations and similar kinetics with the anti-inflammatory cytokine IL-10. A decreased sTREM-1/tumor necrosis factor (TNF) ratio may promote the progression from sepsis to severe sepsis, and potentially to septic shock. Dai et al. [42] found that sTREM-1 plays a protective role in endothelial inflammation by inhibiting the expression of IL-1b, IL-6, TNF-$\alpha$, vascular cell adhesion protein-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs). Third, as a member of the Ig superfamily, TREM-1 is widely expressed by myeloid cells in both membrane-bound (mTREM-1) and soluble (sTREM-1) forms. sTREM-1 contains an Ig-like domain, which plays a crucial role in antigen recognition and also competes with mTREM-1 for the same ligands [43]. Consequently, sTREM-1 acts as a decoy receptor, obstructing ligands from binding to TREM-1 receptors during inflammatory responses, thereby downregulating the activation of inflammatory cytokines and exhibiting anti-inflammatory properties [44]. The protective effect of sTREM-1 in AAA patients is compromised due to its reduced levels.

Previous studies have confirmed that HMGB1 is one of the ligands for TREM-1 [25, 45, 46]. However, the relationship between HMGB2 and TREM-1 and the potential involvement of the HMGB2-TREM pathway in the pathogenesis of AAA has not been previously reported. The present findings indicate that sTREM-1 is positively correlated with HMGB2. Analysis of quartile groups based on HMGB2 levels revealed that serum sTREM-1 levels increased significantly in a concentration-dependent manner as the HMGB2 levels rose more moderately. Interestingly, a statistically significant decrease in the serum sTREM-1 level was observed in the upper quartile group of HMGB2. While sTREM-1 generally correlates positively with HMGB2, we hypothesize that there is a critical threshold beyond which this relationship inverts. Our analyses revealed a biphasic relationship between HMGB2 and sTREM-1 levels. Previous studies have also reported on the role of TREM-1 in amplifying the inflammatory response [47, 48]. We hypothesize that TREM-1 mediates the activation of its ligands, such as HMGB2, as well as inflammatory cell receptors (RAGE, TLR-4 and TLR-2), thereby initiating downstream signaling pathways that ultimately lead to AAA. The release of sTREM-1 depends on the activation and cleavage of mTREM-1 [43]. A mild to moderate increase in HMGB2 can stimulate sTREM-1 production, which acts as a decoy receptor and antagonist to mTREM-1, thus conferring anti-inflammatory properties. However, a negative feedback regulation between HMGB2 and sTREM-1 occurs when HMGB2 is highly expressed, thereby reducing its anti-inflammatory effect and contributing to the development of AAA. TREM-2 serves as a negative regulator of the inflammatory response [49]. Nonetheless, we did not observe any significant associations between sTREM-2 and AAA or HMGB2 in the current study.

Our findings indicate that current smoking and hypertension are independent risk factors for AAA, whereas the use of statins and the presence of diabetes confer protection against AAA, consistent with previous research. There is substantial evidence that smoking is a significant risk factor for AAA, with current smokers exhibiting a 5-fold increased risk and former smokers a 2-fold increase risk compared to individuals who have never smoked. Furthermore, a positive dose-response relationship was observed between the daily quantity of cigarettes smoked and the risk of developing AAA, as well as cumulative pack-years smoked and risk of AAA [50]. Although our study did not identify a significant association between hyperlipidemia and AAA, a negative correlation was observed between statin use and the incidence of AAA. Multiple Mendelian randomization analyses have implicated elevated LDL-C and reduced HDL-C levels as contributing factors to the pathogenesis of AAA [51]. Specifically, small dense LDL shows a strong association with AAA [3]. Despite these associations, there is no evidence that dyslipidemia is associated with the risk of AAA growth or rupture [52]. Several studies suggest that most statins can decrease or prevent AAA progression through various mechanisms, including regulation of endoplasmic reticulum stress, antioxidant activity, ECM synthesis, and inhibition of matrix-metalloproteinase (MMP) [53]. The increased use of statins among hyperlipidemic patients in the control group of our study may account for the observed lack of association between hyperlipidemia and AAA. Our study found a negative correlation between diabetes and AAA, consistent with results from previous research. The relationship between diabetes and a slower rate of AAA growth is well-documented [54]. However, it remains uncertain whether this effect is attributable to the diabetes itself, or to pharmacological treatments associated with its condition. In this regard, metformin, a widely used diabetes medication, has been found to mitigate matrix remodeling and inflammation in AAA [55]. The patient cohort in our study included individuals with hyperlipidemia, diabetes, or hypertension. Relevant pharmacological agents were administered during the study to treat these conditions, which may have influenced the observed outcomes.

This research has several limitations. First, it was a preliminary, retrospective, and cross-sectional study aimed at investigating the relationship between the HMGB2-TREM pathway and AAA from an inflammatory perspective. The association between the HMGB2-TREM pathway and AAA is still not fully elucidated and warrants further research. Second, we conducted a single-center, small-scale cohort study comprised of male participants only to eliminate possible sex-related confounders. The small sample size reduced the statistical power, increased the risk of Type II errors, and limited the subgroup analyses. The single-center design also introduces potential selection bias, as the study cohort may not accurately represent all populations. The exclusion of female participants, while intended to control for sex-specific confounders, prevents extrapolation of the findings to women, who exhibit distinct AAA risk profiles and pathophysiological mechanisms. In the quartile-based regression analysis, the wide confidence intervals observed for the 4th quartile of HMGB1 and HMGB2 are likely because of the small sample size of these subgroups, thus reducing the estimate precision. While point estimates suggest a strong association, the broad intervals indicate uncertainty and warrant a cautious interpretation. However, the consistent and statistically significant trend seen across quartiles supports the robustness of our findings. Due to corona virus disease 2019 (COVID-19), this study experienced difficulties with the recruitment of patients, further restricting sample diversity and potentially skewing the results toward individuals with more severe or accessible AAA cases. Additionally, only the baseline level of HMGB2 was assessed in our study. It remains to be determined whether subsequent changes in this level during follow-up could mask its role as a prognostic biomarker for the growth, rupture, or repair of AAA. Finally, our study did not investigate the mechanistic and pathological roles of the HMGB2-TREM pathway in AAA.

Given the limitations of our study, there are many issues concerning AAA that still need to be addressed in future research work. One major issue is the uncertain pathological mechanism of AAA. Further in vitro and in vivo experiments are needed to elucidate the mechanistic and pathological roles of the HMGB2-TREM pathway in AAA. These should focus on the impact of this pathway on inflammatory responses, VSMC apoptosis, and ECM degradation. Various AAA animal models and gene knockout or overexpression models could also be used to study the role of the HMGB2-TREM pathway on AAA development. The second major issue to be addressed is the difficulty of early diagnosis. To explore the potential of HMGB2 as a biomarker for the early diagnosis and prognostic assessment of AAA, dynamic changes in the serum level of HMGB2 in relation to AAA progression should be evaluated. The third issue to be addressed is the lack of effective treatment. This requires screening and development of small molecule inhibitors or agonists targeting the HMGB2-TREM pathway, followed by an evaluation of their efficacy for the prevention and treatment of AAA. Early-stage clinical trials that assess the safety and efficacy of HMGB2-TREM pathway-targeted therapies in human patients will be required. These efforts should help to identify specific biomarkers and therapeutic strategies for the early diagnosis, prognosis, and treatment of AAA patients.

In conclusion, elevated serum HMGB2 levels are independently associated with the incidence of AAA. Disruption of the HMGB2-TREM pathway may have a significant impact on the pathogenesis of AAA. The HMGB2-TREM pathway therefore represents a potentially novel therapeutic target for the treatment and prevention of AAA.

The data that support the findings of this study are not publicly available due to their containing information that could compromise subsequent unfinished research. The data of this study can not be disclosed until the results of the follow-up study are published.

LP and JC contributed equally to this work. LP contributed to data analyses and drafting of the manuscript. JC contributed to data collection and collation. YS was responsible for the supply of materials and samples. FW was responsible for the conception, design and supervision of the manuscript. All authors contributed to the conception and editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All procedures were performed according to the principles of the Declaration of Helsinki and approved by the local Research and Ethics Committee of the Ruijin Hospital (ethical approval number: 2023 (No.70)). Informed written consent was obtained from all participants.

This study was supported by Chinese National Nature Science Foundation (Grant no. 81900413), Hainan Provincial Health Commission (Grant no. 22A200174) and National Key Research and Development Program of China (Grant no. 2021YFC2500600, Grant no. 2021YFC2500602). Three-Year Action Plan for Promoting Clinical Skills and Clinical Innovation in Municipal Hospitals (SHDC2022CRS037). The funders had no role in the design, data collection, data analysis, and reporting of this study.

The authors declare no conflict of interest.