Advances in the study of S100A9 in cardiovascular diseases

Abstract Cardiovascular disease (CVD) is a group of diseases that primarily affect the heart or blood vessels, with high disability and mortality rates, posing a serious threat to human health. The causative factors, pathogenesis, and characteristics of common CVD differ, but they all involve common pathological processes such as inflammation, oxidative stress, and fibrosis. S100A9 belongs to the S100 family of calcium‐binding proteins, which are mainly secreted by myeloid cells and bind to the Toll‐like receptor 4 and receptor for advanced glycation end products and is involved in regulating pathological processes such as inflammatory response, fibrosis, vascular calcification, and endothelial barrier function in CVD. The latest research has found that S100A9 is a key biomarker for diagnosing and predicting various CVD. Therefore, this article reviews the latest research progress on the diagnostic and predictive, and therapeutic value of S100A9 in inflammatory‐related CVD such as atherosclerosis, myocardial infarction, and arterial aneurysm and summarizes its molecular mechanisms in the progression of CVD, aiming to explore new predictive methods and to identify potential intervention targets for CVD in clinical practice.


| INTRODUCTION
Cardiovascular disease (CVD) is a global health problem that seriously endangers human health.The incidence of CVD in the globe continues to rise and is the leading cause of mortality. 1[5][6] Currently, inflammatory response is a crucial triggering factor in the occurrence and development of CVD. 7 The elevated levels of inflammatory markers have been demonstrated to predict future cardiovascular events. 80][11] This process also increases reactive oxygen species (ROS), which contributes to the growth of cardiac fibroblasts and the activation of matrix metalloproteinases, causing interstitial fibrosis in the heart. 11Furthermore, inflammation prompts cardiac fibroblasts to transform into myofibroblasts.[11] Furthermore, the inflammatory response of the vascular endothelium is closely related to CVD. 12 Damaged and activated endothelial cells trigger leukocytes adhesion by releasing IL-8, chemokines, adhesion molecules, and other cytokines.9][20] Moreover, under physiological circumstances, S100A9 can non-covalently bind with S100A8 to form a 24.5 kDa heterodimer, termed as calprotectin (S100A8/A9 or MRP-8/14), which is present in vivo. 21,22S100A9 and its heterodimer are effective ligands for the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4). 23But the affinity of S100A9 binding with RAGE and TLR4 is significantly higher than that of S100A8/A9, a phenomenon that might be associated with its biological function being regulated in intricate regulatory mechanisms. 235][26][27][28] It has been reported that S100A9 can induce myocardial cell apoptosis through mediating ROS production and activating complement proteins C3 and C5 to produce anaphylatoxins C3a and C5a. 29Furthermore, S100A9 can activate the inflammatory response by modulating specific signalling pathways, which prompts disruption of endothelial barrier function.1][32][33] These suggest that S100A9 is a key stimulator of CVD.Thus, this review primarily focuses on elucidating the biological effects of S100A9 and its regulatory mechanisms in CVD.Specifically, considering the intimate association between CVD and inflammation, with S100A9 playing a prominent role as an inflammatory factor, we aim to summarize the intricate involvement of S100A9 in inflammation-associated CVD, including atherosclerosis, myocardial infarction (MI), pulmonary arterial hypertension (PAH), aortic aneurysm, peripheral arterial disease (PAD), and so on (Table 1).We mainly elucidate the role of S100A9 in modulating inflammatory responses, amplifying tissue fibrosis, promoting vascular calcification, inducing endothelial dysfunction, and serving as a pivotal biomarker in inflammation-associated CVD.This review is designed to furnish novel predictive indicator for CVD within clinical settings while exploring untapped therapeutic target.

| INTRODUCTION TO S100A9
S100A9 is a calcium-and zinc-binding protein consisting of 114 amino acids with a molecular weight of approximately 13 kDa. 34,35It possesses two EF-hand domains, which form a highly conserved helixloop-helix structure. 35And the gene encoding S100A9 is located on chromosome 1q21.3. 36S100A9 is mainly expressed in the immune system such as spleen, bone marrow, lymph nodes, lung and skin. 3700A9 is predominantly located in the cytoplasm; however, in response to elevated intracellular calcium levels, it can translocate to the cytoskeleton and cell membrane or be secreted extracellularly. 18,3800A9 levels exhibit a notable elevation in pathological states, with the precise mechanism underlying its release remaining incompletely elucidated.However, research has shown that S100A9 release is regulated by E-selectin. 39Specifically, E-selectin mediates the formation of N-terminal gasdermin D pores in neutrophils in an NLRP3-dependent manner, leading to the release of S100A9. 3900A9 is also regulated by p53. 29P53 is activated by the release of isoproterenol-inducesd ROS, further upregulating the transcription of the S100A9 gene. 29An increase in S100A9 levels triggers an inflammatory response, which in turn promotes the release of ROS, forming a ROS/p53/S100A9 positive feedback loop. 29der physiological conditions, S100A9 is generally underexpressed, primarily found in immune cells such as neutrophils. 40While under pathological conditions, S100A9 is released extracellularly by immune cells such as neutrophils, monocytes, macrophages, and dendritic cells, as well as necrotic myocardial cells, endothelial cells, and cancer cells, thereby performing corresponding functions such as modulating immune responses 27,[41][42][43][44][45] (Figure 1).Interestingly, the expression levels of S100A9 in aged male mice are higher than those in young mice in all organs, a phenomenon possibly related to ageassociated inflammation. 4600A9 exhibits a dual regulatory role in inflammatory responses.
Under various stress stimuli, the substantial increase in S100A9 levels subsequently promotes the activation and migration of leukocytes, leading to a substantial recruitment of leukocytes to the site of inflammatory injury. 47,48These recruited leukocytes, in turn, secrete various pro-inflammatory cytokines, ROS, and other substances, thereby initiating a detrimental cycle of inflammation. 47,48However, in abnormal systemic inflammation, S100A9 can exhibit an anti-inflammatory action to avoid tissue damage caused by overwhelming inflammation. 49Other studies suggest that S100A8/A9 heterodimer and S100A8/S100A9-tetramers also exert anti-inflammatory effects, in the context of MI, psoriasis and arthritis lesions. 50,51Furthermore, S100A9 has implicated in regulating cell proliferation, migration, and invasion in the context of chronic inflammation associated with cancer. 52,53sides regulating inflammation, S100A9 also plays a vital role in oxidative stress.In the context of oxidative stress, the expression of S100A9 significantly increases, leading to the activation of complement proteins C3 and C5 and subsequent myocardial injury. 29Li et al. 54 confirmed that S100A9 can significantly induce cell apoptosis.When transiently transfected pEGFPC1-S100A9 into p53 À/À and p53 +/+ cells respectively, p53 +/+ cells exhibited more severe apoptosis, suggesting that S100A9 induces apoptosis in a p53-dependent manner. 54Further, Boteanu et al. 47 found that inhibiting S100A9 can increase the expression of proteins related to the apoptosis process, such as NOL3, SOD2, T A B L E 1 Evidence supporting the important role of S100A9 in cardiovascular disease.2).This activation leads to the release of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IL-8, thus exerting potent pro-inflammatory effects. 25,55,56S100A9 induces NLRP3 inflammasome activation via TLR4-Myd88 and releases IL-1β, the latter interacts with the IL-1R on myeloid progenitors to stimulate monocytes and neutrophils production, leading to an exacerbation of inflammation. 57Interestingly, although TLR4 is generally considered pro-inflammatory, its interaction with S100A9 can normalize ketogenesis in diabetic mice by activating mTORC1, exhibiting anti-inflammatory effects. 58reover, TLR4 induces the disassembly of F-actin, ZO-1, and occludin by activating downstream p38 and ERK1/2, significantly increasing endothelial cell permeability and disrupting endothelial barrier function, leading to tissue edema. 30,59The activated S100A9/ TLR4/MAPK/NF-κB signalling pathway can also reduce caspase 9 and caspase 3 activity by inducing monocytes to secrete cytokines MCP-1, IL-6, and IL-8 which act on cytokine receptors on the surface of neutrophils, thereby inhibiting cell apoptosis. 60In the early stages of MI, some S100A9 enters myocardial cells or other heart cells via TLR4 and combines with GMP exchange factors, which transforms Rac1/2 into activated Rho GTPases, and then Rac1/2 activates the MAPK signal pathway and PI3K-AKT signal pathway, thereby directly regulating cell survival. 61Both signalling pathways can also indirectly govern autophagy and apoptosis via the mTOR signal pathway. 61other classic receptor of S100A9 is RAGE.S100A9 binds to RAGE on the cell membrane surface, which further binds to its adaptor protein Dia-1, activating NF-κB and leading to an increase in the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α, thereby activating a systemic inflammatory response. 62It is worth mentioning that similar to TLR4, RAGE can also induce endothelial dysfunction via F-actin, ZO-1, and occludin. 30,59The S100A9-RAGE axis, activated by high glucose levels, induces an upregulation of NF-κB activity and a downregulation of Nrf-2 activity. 63This heightened NF-κB activity leads to elevated expression of pro-inflammatory cytokines from macrophages, while the reduced Nrf-2 activity results in an increase in the expression of calcification-promoting factors, such as osteocalcin, osteopontin, BMP-2/4, ALP, and Runx-2, in macrophage-derived extracellular vesicles. 63This process promotes microcalcification of extracellular vesicles, thereby playing a role in regulating calcium homeostasis and contributing to vascular calcification, which links inflammation to microcalcification. 63The collective findings highlight the pivotal role of the S100A9-RAGE axis in CVD, as it exhibits pro-inflammatory properties, induces endothelial dysfunction, and contributes to vascular calcification (Figure 3). to tumour exacerbation. 64,65Further investigation is needed to determine whether S100A9 directly activates these pathways or indirectly through TLR4 and RAGE exerts its effects.

| ROLE OF S100A9 IN ATHEROSCLEROSIS
[68] Both innate immune responses and adaptive immune responses have been found to be activated in atherosclerosis. 69,70Specifically, innate immune cells intake lipids via scavenger receptors or TLRs, triggering intracellular signalling cascades, which results in a series of gene expressions encoding pro-inflammatory factors, thus inducing vascular inflammatory responses. 69Meanwhile, the induction of adaptive immune responses by T lymphocytes and B lymphocytes further exacerbates atherosclerosis. 70udies have indicated that S100A9, as an inflammatory mediator, is noticeably increased in atherosclerosis and contributes to its development. 31,71,72By quantifying the concentration of the S100A9 protein in carotid plaque samples from 186 patients and analysing the proportion of S100A9-expressing macrophages, Ionita et al. 72 found that S100A9 and S100A9-positive macrophages were highly expressed in unstable plaques compared with stable ones and correlated strongly with the pathological features and inflammatory status of unstable plaques.This suggests that S100A9 is a biomarker for rupture-prone plaques. 72Langley et al. 73  Bruneck study confirmed a positive correlation between high levels of S100A9 and the progression of atherosclerosis, the incidence of cardiovascular and cerebrovascular accidents during 10-year follow-up, suggesting S100A9 is a valuable biomarker for assessing the risk of unstable atherosclerosis, highlighting its importance in indicating atherosclerotic conditions.
Independent risk factor for atherosclerosis appears to include transient intermittent hyperglycaemia (TIH).This risk factor exerts its influence by stimulating extramedullary myelopoiesis, resulting in an elevated abundance of circulating inflammatory cells, such as Ly6C hi monocytes and neutrophils, which accelerates atherogenesis. 74Flynn et al. 74 reported that compared to mice transplanted with WT bone marrow, mice receiving S100A9 À/À bone marrow were shielded from myelopoiesis spurred on by TIH, leading to a reduction in circulating inflammatory cells, and ultimately attenuating atherosclerosis.This phenomenon may occur due to the blockade of the ROS-S100A9-RAGE axis induced by high blood glucose. 74Moreover, Hanssen et al. 75 constructed a mouse model to mimic TIH by intravenously injection with MGO (a reactive glucose metabolite), which increased atherosclerotic burden via induces circulating neutrophils and monocytes, with an increase of S100A9 and RAGE.This indicates the activation of the S100A9-RAGE axis by TIH leading to elevated levels of inflammatory factors, ultimately contributing to atherosclerosis. 75ese studies collectively provide evidence that S100A9 is a key regulatory factor in atherogenesis induced by hyperglycaemia.
Furthermore, diabetes can hasten the development of vascular calcification, serving as a significant risk factor for atherosclerosis. 63aakman et al. 76 have reported that the inhibition of S100A9 can reduce the formation of atherosclerosis in diabetic mice; in diabetic patients, inhibition of S100A9 bioactivity suppresses platelet production, which may help to reduce the incidence and severity of cardiovascular events.In plasma of fat-fed ApoE À/À mice, quantified levels of matrix vesicles were found to exhibit an upward trend compared to fat-fed WT mice, while the level of matrix vesicles in plasma of ApoE À/-S100A9 À/À mice fell to those of WT mice. 71This suggests that S100A9 may be a critical mediator of microcalcification in atherosclerosis. 71[80][81][82][83] Researchers have discovered numerous tissue peptides and proteins contributing to the diagnosis and prognosis of MI. 84 Healy et al. 85 employed a transcriptome analysis approach to compare the differential expression of platelet mRNA transcripts between patients with ST-segment elevation MI (STEMI) and those with stable coronary artery disease.They identified that S100A9 is a novel regulator of thrombus formation and found that S100A9, one of the strongest predictors in the microarray data analysis (p = 0.002), exhibited increased expression prior to STEMI occurrence. 85This suggests that S100A9 is a candidate biomarker for predicting the onset of MI.
The study has shown that compared to individuals with stable CVD or normal individuals, patients with ACS exhibit local and systemic S100A8/A9 levels that exceed the critical threshold of 8.0 mg/L within 3 h of symptom onset, making it a promising candidate for detecting ACS. 86Notably, Fraccarollo et al. 87 investigated the correlation between S100A9 and MI in 47 patients and found that MI patients exhibited high expression of S100A9 in circulating CD10 neg neutrophils compared to patients with stable angina.Furthermore, another study reported a significant elevation of S100A9 levels in platelets of MI patients, and the abundance of S100A9 in platelets showed a strong positive correlation with neutrophil count (R = 0.54, p = 0.0025), indicating that S100A9 in platelets originates from neutrophils. 88[88] The latest study reported that the application of LASSO regression and SVM-RFE algorithms could identify 11 overlapping genes, and ROC analysis of these 11 overlapping genes in the training sets GSE48060 and GSE66360 revealed that the genes with the highest area under the curve (AUC) reconciliation mean contained S100A9; further simplification of the diagnostic model demonstrated that the genes with over 90% accuracy in the training set contained S100A9, indicating that S100A9 is an effective biomarker for diagnosing MI. 89 S100A9 is associated with the prognosis of MI.Marinkovi c et al. 80 reported that patients with significantly elevated plasma S100A9 within 24 h after MI had an increased length of stay and an increased incidence of major adverse cardiovascular events due to heart failure during the follow-up.Sreejit et al. 90 and Li et al. 91 further showed that patients with higher neutrophils level following ACS, which secret S100A9, had a significantly higher incidence of major adverse cardiovascular events during the 1-year follow-up after revascularization.These findings suggest that the detection of elevated expression of S100A9 in early MI indicates a poor prognosis, and S100A9 levels or neutrophil counts could predict MI prognosis. 80,90,91ring the inflammatory phase of cardiac repair, S100A9 is mainly secreted by immune-activated cells, resulting in a rapidly increasing S100A9 levels in the blood and heart, and a peak 3-5 days post-MI. 19,80,91,92Elevated S100A9 can stimulate the production of myeloid cells and translocation to ischemic myocardium, thereby promoting cardiac inflammation. 80Furthermore, thinning of the infarcted wall is significantly associated with increased expression of S100A9, 92 which may contribute to post-MI complications such as ventricular aneurysm due to weakening of the cardiac structure.Interestingly, during the proliferative phase post-MI, S100A9 can mediate the transition from inflammatory Ly6C hi monocytes to reparative Ly6C lo macrophages by upregulating the levels and activity of Nur77 in macrophages and Ly6C hi/int monocytes, which facilitates myocardial repair after injury 78 (Figure 4).
Li et al. 91 further investigated the impact of S100A9 in myocardial ischaemia-reperfusion (MI/R) and found that compared to WT mice, S100A9 knockout mice exhibited markedly reduced infarct size and improved cardiac function, while transgenic mice overexpressing S100A9 showed opposite results.The underlying mechanism involves the rescue of mitochondrial function upon S100A9 knockout, which is associated with the restoration of the TLR4/ERK-mediated PGC-1α/ NRF1 signalling pathway. 91This restoration leads to the recovery of gene expression related to the mitochondrial complex I subunit NDUFs.Through these intricate processes, the study elucidates the specific mechanism behind early cardiomyocyte death in the context of MI, highlighting the pivotal pro-inflammatory role played by S100A9 (Figure 4).The study confirms that S100A9 critically regulates cardiomyocyte death and survival by impacting mitochondrial function, making it a key mediator in the progression towards cardiomyocyte death. 91wever, in models of MI induced by permanent coronary artery ligation, Marinkovi c et al. 78 observed that S100A9 À/À mice exhibited a decreased number of circulating monocytes and reduced infiltration of monocytes and macrophages into the heart on the 7th day after MI compared to WT mice, most importantly the numbers of repairing Ly6C lo MerTK hi macrophages were reduced by approximately 50% in the myocardium of S100A9 À/À mice; on the 21st day after MI, S100A9 À/À mice showed worse cardiac function.These findings suggest that the positive impact of S100A9 on cardiac repair after MI is ineffective during the acute phase of MI; meanwhile, indicating that early blockade of S100A9 is beneficial for cardiac repair, while prolonging the blockade may have adverse effects. 78,91

| ROLE OF S100A9 IN OTHER VASCULAR DISEASES
The pathological basis of vascular diseases is the narrowing or occlusion of the lumen of blood vessels due to multiple causes, leading to ischemic changes in organs or limbs, and some vascular diseases present local dilatation and tumour-like changes. 93,946][97][98] S100A9, a proinflammatory alarm factor, is also involved in the developmental process of these vascular diseases.
3][104] PAH has multifactorial aetiology and complex pathological mechanisms.[107] Research has shown that the pro-inflammatory mediator S100A9 is overexpressed in pulmonary arterial smooth muscle cells (PASMCs) of patients with PAH. 104Of note, Zeng et al. 108  aligning with its role as an inflammatory mediator. 108Additionally, a differential gene expression analysis showed that S100A9 is consistently present in both SARS-CoV-2 and PAH samples, with a heat map indicating a high-risk tendency propensity for S100A9 expression, implying an increased risk of PAH following SARS-CoV-2 infection. 109These findings highlight the crucial regulatory role of S100A9 in PAH-related inflammation.
As classical receptors for S100A9, both RAGE and TLR4 demonstrate consistent upregulation in PAH.Compared to healthy individuals, PAH patients exhibit excessive expression of RAGE in their plasma, pulmonary arteries, PASMCs, endothelial cells, and fibroblasts. 104,110This augmented expression of RAGE leads to the generation of pro-inflammatory cytokines and cellular proliferation, playing a pivotal role in the pathogenesis and progression of PAH.Moreover, in circulating monocytes and PASMCs of PAH patients, both TLR4 mRNA and protein levels are significantly elevated. 111,112Upregulation of TLR4 leads to increased PASMCs proliferation and decreased apoptosis, promoting vascular remodelling in PAH.This phenomenon is closely related to TLR4 being a target gene of miR-503 and regulated by the LncRNA MALAT1, the latter of which has been demonstrated to play a crucial role in vascular remodelling. 111Importantly, the increased expression of TLR4 activates the classical NF-κB inflammatory signalling pathway, thereby mediating endothelial cell inflammation within pulmonary arteries and emerging as one of the principal factors driving the pathogenesis of PAH. 112I G U R E 4 Role of the S100A9 in myocardial infarction.In MI, S100A9 promotes HSPCs proliferation and recruits inflammatory cells to the ischemic myocardium.Dying cardiomyocytes, neutrophils, monocytes, and macrophages further secrete S100A9.In the inflammatory phase, S100A9 binds to TLR4, activates ERK, downregulates PGC-1α, inhibits NRF1, and impairs mitochondrial complex I, causing cardiomyocyte death.
Considering the uniform upregulation of S100A9 and its classical receptors, RAGE and TLR4, in PAH, coupled with their ability to trigger inflammatory responses and perturb immune system homeostasis upon heightened expression, we speculate that the interplay between S100A9 and RAGE or TLR4 might constitute one of the underlying mechanisms driving the pathogenesis of PAH (Figure 5).
In other hypertensive disorders, S100A9 also plays a pivotal role.
Preeclampsia stands out as a prevalent hypertensive disorder during pregnancy, with its pathogenesis involving maternal infections and inflammation. 113Recent investigation has revealed heightened levels of S100A9 in the blood plasma and placenta of pregnant women affected by preeclampsia. 114S100A9 triggers the activation of the NLRP3 inflammasome in both the placenta and human umbilical vein endothelial cells, leading to the secretion of IL-1β and soluble endoglin. 114Moreover, the administration of exogenous S100A9 stimulates the secretion of soluble endoglin and the accumulation of neutrophils in pregnant mice, subsequently inducing maternal hypertension. 114These findings significantly imply that S100A9 potentially participates in the pathogenesis of pre-eclampsia. 114Furthermore, a study has found that upon the initial day of angiotensin II infusion in mice, there is a substantial upregulation of S100A9 expression, which consequently contributes to the development of hypertensioninduced cardiac injury caused by angiotensin II. 24These compelling findings underscore the potential of both S100A9 as critical indicators for monitoring the occurrence and progression of hypertension.
Aneurysm is a significant vascular disease whose development is also associated with S100A9.It has shown that compared to Porphyromonas gingivalis negative subjects, positive subjects exhibited a significant increase in the number of aneurysms, and a notable overexpression of S100A9 was detected on the smooth muscle cells surface of damaged vessels, indicating that P. gingivalis might promote the occurrence and development of aortic aneurysms through upregulating S100A9. 115ditionally, Nakaoka et al. 116 used gene expression profiling to reveal that the levels of S100A9 were significantly higher in ruptured aneurysms compared to unruptured intracranial aneurysms, and early stage ruptured aneurysms showed higher levels of S100A9 than late-stage ruptured aneurysms.Recently, a clinical study involving 63 patients reported that the median concentration of S100A8/A9 in the venous blood of aneurysm patients was 1257 ng/ml, significantly exceeding the median S100A8/A9 concentration of 390 ng/ml in healthy individuals (p ≤ 0.001); the median concentration of S100A8/A9 in ruptured aneurysms was 8530 ng/ml, which was significantly surpassed in unruptured aneurysms (p = 0.04). 117These findings suggest that S100A9 could serve as a molecular biomarker for diagnosing and distinguishing aneurysms prone to rupture and as a potential intervention target.
S100A9 is not only a marker for the diagnosis and differentiation of aneurysms, but also an important substance for predicting the prognosis of aneurysms.Lech et al. 118 found persistently elevated plasma S100A9 levels for months to decades in patients suffering from giant aneurysms after Kawasaki disease and monocytes that infiltrated the coronary arteries and cardiomyocytes continually expressed S100A9.Furthermore, a plasma S100A8/A9 concentration higher than 6020 pg/ml (sensitivity 53.57%, specificity 96.15%) within 48 h after the onset of aneurysmal subarachnoid haemorrhage is indicative of a poor prognosis. 119These demonstrate that S100A9 may be a marker for predicting the prognosis of aneurysms.Significantly, a recent study has reported an association between S100A9 and aortic dissection.Single-cell sequencing technology revealed an elevated proportion of pro-inflammatory macrophages within the arterial walls of patients suffering from aortic dissection, compared to the control group, with these macrophages demonstrating high expression of S100A9. 120This suggests that pro-inflammatory macrophages may through the secretion of S100A9 mediate vascular inflammation, contributing to the development of aortic dissection. 120wever, whether S100A9 serves as a target for the treatment of aneurysms and aortic dissection remains to be further investigated.In PAD-affected limbs, there has been confirmation of immune and inflammatory cell infiltration, with S100A9 showing elevated expression in these affected limbs. 121A recent report by Saenz-Pipaon et al. 122 revealed that S100A9 mRNA is abundant in extracellular vesicles and plasma of PAD patients and increased with the severity of PAD, suggesting S100A9 as a biomarker for predicting the severity of PAD.When S100A9 combines with the inflammatory marker hs-CRP or lipocalin-2, it best predicts the risk of amputation and major adverse cardiovascular events in PAD. 122,123This indicates that the approach of multiple marker combination is of superior value in predicting PAD risk.
M1 macrophages, known as classically activated macrophages, mainly participate in pro-inflammatory responses. 124A study has indicated that in preclinical PAD models, the increased expression of VEGF 165b in macrophages inhibits the phosphorylation of VEGFR1, leading to an increase in downstream S100A9, and an increase in calcium influx, thereby inducing MI-like polarization of macrophages, ultimately inhibiting angiogenesis and reperfusion recovery in ischemic muscles of PAD. 125This confirms that targeting S100A9 may be beneficial for restoring blood flow in the affected limb.

| ROLE OF S100A9 IN CARDIOMYOPATHY AND ATRIAL FIBRILLATION
[131] In HCM, Zhao et al. 132 identified immune infiltration-related genes through RNA sequencing, discovering that S100A9 is predominantly expressed by infiltrating M1 macrophages in the cardiac immune microenvironment, especially CCR2-M1 macrophages, and also confirmed that S100A9 is a potential biomarker for differentiating HCM from controls.But its specific molecular mechanism remains to be further explored.
In addition, Cai et al. 130 observed an upregulation in the transcriptional level of S100A9 in UCM rats.Furthermore, compared to UCM rats, the degree of myocardial cell hypertrophy and fibrosis was significantly improved in the UCM rats transduced with AAV-shS100A9, as evidenced by decreased mRNA and protein levels of fibrosis markers (TGFb1, α-SMA, collagen 4a1, and fibronectin) in myocardial tissue; and the expression of inflammatory cytokines (IL-6, TNF-α, and IL-1β) in both the local myocardium and systemic circulation was reduced. 130ese indicate that knockdown of S100A9 can alleviate the inflammatory response and tissue fibrosis in UCM, thereby improving cardiac function. 130 is a common arrhythmia caused by various factors such as inflammation and atrial fibrosis. 131Liu et al. 133 identified S100A9 as the most strongly associated factor with AF using the PPI network and LASSO model.Further analysis of the ROC curve determined the AUC of S100A9 in AF patients and controls. 133In the training cohort, the AUC was 0.9981 (95% CI, 0.993-1), while in the testing cohort, the AUC was 0.862 (95% CI, 0.7271-1), indicating the diagnostic value of S100A9 for AF and S100A9 is identifying as a potential AF biomarker. 133Chu et al. 134 also showed a significant correlation between S100A9 and AF using univariate logistic regression analysis, while the results of the binomial logistic regression analysis from the generalized linear model demonstrated a monotonic relationship between the two, proving that the risk of AF increases with the increase in S100A9 gene expression.In light of the fact that atrial inflammation can lead to atrial electrical remodelling and structural changes, resulting in the onset of AF, 135 and considering the observed positive correlation between S100A9 and AF, 133,134 we posit that the inflammatory response triggered by S100A9 may play a role in the pathogenesis of AF.
These studies have greatly enriched our understanding of the pro-inflammatory effects of S100A9, highlighting its essential role as a significant endogenous damage-associated molecular pattern.Moreover, these findings have provided compelling evidence supporting the potential significance of S100A9 as a key biomarker in cardiomyopathy and AF.Thus, the assessment and intervention of S100A9 expression hold promising value for the diagnosis and treatment of these specific disorders.

| ROLE OF S100A9 IN OTHER INFECTION-ASSOCIATED CVDs
Myocarditis is an important inflammatory disease of the myocardium,  136 In experimental research, increased expression of S100A9 mRNA and protein levels were likewise found in the heart and spleen of mice with myocarditis compared to control mice. 137Upregulation of S100A9 leads to increased secretion of its downstream pro-inflammatory cytokines, which in turn leads to myocardial inflammation, an effect that has been associated with increased cardiac viral load due to increased cardiac myeloid-derived suppressor cell counts. 137Furthermore, compared with myocarditis mice, the mRNA levels of RAGE and Dia-1 were significantly reduced in the hearts of CVB3-infected S100A9 À/À mice, while there was no statistical difference in MyD88, indicating that CVB3 may induce myocarditis by activating the S100A9-RAGE-Dia-1 pathway, and defective S100A9 gene leads to the suppression of this axis thereby attenuating myocardial inflammation. 62rrently, there is limited research exploring the connection between infective endocarditis (IE) and S100A9.Nonetheless, Xiao et al. 138 identified S100A9 as a key gene involved in IE through bioinformatics analysis and found that S100A9 plays a crucial role in the inflammation and immune response of IE, suggesting that S100A9 may be involved in the pathogenesis of IE.Furthermore, S100A9 neutralizing antibodies (nAb), botanical drugs, exercise training, and active vaccination against S100A9 have been confirmed to have cardiovascular protective effects (Figure 6).
Paquinimod and ABR-238901 primarily exert their effects by blocking the interaction between S100A9 and TLR4 and RAGE. 76,139quinimod has demonstrated affirmative efficacy and safety in phase II clinical trials for systemic sclerosis. 140Laquinimod, a quinoline-3-carboxamide, exhibits binding capability to S100A9, yet its precise mechanism of action remains to be elucidated. 23Some studies propose that laquinimod mainly functions through the reduction of NF-κB, direct inhibition of T cells, and modification of antigen-presenting cell subpopulations. 141,142Laquinimod has been subjected to phase III clinical trials for multiple sclerosis and phase II clinical trials for Crohn's disease, manifesting promising therapeutic outcomes. 143,144Tasquinimod, another S100A9 inhibitor, predominantly exerts its effects by blocking the interaction between S100A9 and TLR4. 145Although it has demonstrated progression-free survival during phase III clinical trials for prostate cancer, the anticipated effects were not observed in phase II studies for advanced hepatocellular carcinoma, gastric cancer, kidney cancer, and ovarian cancer. 146,147All of these drugs mentioned In addition, Li et al. 91 have developed an S100A9 nAb that has a neutralizing effect both in vitro and in vivo.In MI mice, this nAb can significantly reduce the infarct area, upregulate the activity of mitochondrial complex I, alleviate cardiac fibrosis and improve cardiac function. 91Nevertheless, the clinical application of this antibody remains pending, and its safety and efficacy as a viable therapeutic option necessitate further investigation and clinical trials.
Studies show that botanical drugs protect the heart by inhibiting macrophage activation, reducing the release of S100A9, and suppressing the expression of inflammatory factors such as IL-1β and TNFα.cardioprotection. 149,150It is worth noting that S100A9 nAb, botanical drugs, and exercise exert beneficial effects in CVD by reducing S100A9, further emphasizing S100A9 as a potential intervention target.
The tobacco mosaic virus (TMV) is an RNA virus that can bind to the S100A9 targeting peptide.If TMV can be conjugated with drugs, then utilizing S100A9-targeted TMV nanoparticles might assist in the development of new methods for clinical diagnosis and treatment of CVD. 1513][154] And with the continuous advancement of nanotechnology, the delivery efficiency of siRNA is expected to be significantly improved, 155,156 providing new methods and perspectives for targeting S100A9.
Therefore, S100A9 represents a crucial target for treating CVD.

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I G U R E 1 S100A9's influence on immune cell dynamics and cardiovascular function.S100A9 plays a pivotal role in modulating various aspects of immune cell behaviour and cardiovascular function.It regulates neutrophil infiltration, chemotaxis, migration, and recruitment, while also stimulating neutrophil production, activation, and degranulation.Additionally, S100A9 stimulates monocyte production, modulates monocyte infiltration, and induces monocyte migration, thereby indirectly influencing the transition from monocyte to macrophage.Moreover, S100A9 regulates macrophage phenotype transformation and infiltration, increases macrophage recruitment, and induces macrophage migration.Conversely, S100A9 inhibits dendritic cell differentiation and disrupts dendritic cell maturation.Furthermore, S100A9 promotes endothelial dysfunction and exerts detrimental effects on cardiomyocytes by inducing cardiomyocyte death, reducing contractility, suppressing autophagy, and inhibiting norepinephrine-induced myocyte hypertrophy.However, it remains unclear whether S100A9 has independent actions beyond the aforementioned receptors.A study has demonstrated that S100A9 expressed by testicular macrophages activates the PI3K-AKT signalling pathway, thus maintaining the M2 macrophages phenotype, which is associated with their immunosuppressive function.40S100A9 also exerts a role in promoting cancer cell proliferation, migration, invasion, and recurrence.These effects are closely associated with the activation of ALDH1A1/Retinoic Acid signal pathway and Wnt/β-catenin signal pathway.Activation of these pathways may result in immune evasion of cancer cells, ultimately contributing further performed a thorough proteomic analysis of the extracellular matrix in the plaques of atherosclerosis patients and identified elevated levels of S100A9 as potential biomarkers for atherosclerosis; further validation via the F I G U R E 2 S100A9 binds to Toll-like receptor 4 (TLR4) to activate downstream pathways for biological effects.S100A9 binds to TLR4, activating the MAPK pathway and subsequently NF-κB, resulting in pro-inflammatory cytokines release and pro-inflammatory effects.S100A9 induces NLRP3 inflammasome activation via TLR4-Myd88 and releases IL-1β, leading to an exacerbation of inflammation.MAPK activation disrupts endothelial barrier function by breakdown F-actin, ZO-1, and occludin.Simultaneously, NF-κB activation induces cytokine secretion and inhibits apoptosis through caspase 9/3 downregulation.S100A9-TLR4 promotes apoptosis and autophagy through Rac1/2 activation of MAPK and PI3K-AKT pathways.Additionally, their interaction normalizes ketogenesis by activating mTORC1 and inhibiting PPARα.p38, p38 mitogenactivated protein kinase; ERK1/2, extracellular regulated protein kinases1/2; JNK, jun N-terminal kinase; NF-κB, nuclear factor-κB; NLRP3, Nodlike-receptor family pyrin domain-containing 3; Myd88, Myeloid differentiation factor 88; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumour necrosis factor α; IL-8, interleukin-8; MAPK, mitogen-activated protein kinases; MCP-1, monocyte chemotactic protein-1; GEFs, GMP exchange factors; PI3K, phosphoinositide 3-Kinase; mTROC1, mammalian target of rapamycin C1; PPARα, peroxisome proliferator-activated receptoralpha; ZO-1, zonula occludens protein 1.
further validated the influence of macrophage-derived S100A9 on atherosclerosis and discovered that control mice had S100A9-positive macrophages in plaques, while almost none were found in plaques of siS100A9 mice, and the expression of inflammatory factors and vascular calcification markers were significantly reduced in splenic macrophages; in addition, colocalization of S100A9 with RAGE was observed in plaques.These findings confirm the involvement of the S100A9-RAGE axis in macrophagemediated matrix vesicle microcalcification in diabetes, suggesting inhibition of this axis may alleviate vascular calcification and inflammation, and thus ameliorate the progression of atherosclerosis.63 4 | ROLE OF S100A9 IN MIMI refers to a drastic reduction in myocardial blood and oxygen supply due to reduced or interrupted coronary blood flow, which subsequently induces ischemic necrosis of cardiac myocytes.77F I G U R E 3 Role of the S100A9-receptor for advanced glycation end products (RAGE) signalling pathway in cardiovascular disease.S100A9 binds to RAGE, activating NF-κB and increasing pro-inflammatory cytokines, leading to inflammation.It also activates p38 and ERK1/2, disrupting endothelial barrier function.Additionally, it decreases Nrf-2 activity, promoting microcalcification of extracellular vesicles and regulating calcium homeostasis.Dia-1, diaphanous-1; Nrf-2, nuclear factor 2 erythroid related factor 2; BMP-2/4, bone morphogenetic protein 2/4; ALP, alkaline phosphatase; Runx-2, runt-related transcription factor 2; OC, osteocalcin; OPN, osteopontin.Cardiomyocyte death is regulated by various complex processes in the early stages of MI.77During the initial hours post-MI, myocardial cells undergo swelling and necrosis, the interstitium of the myocardium becomes edematous and eosinophils infiltrate; in subsequent inflammatory phase, characterized by extensive infiltration of inflammatory cells (primarily neutrophils) into the necrotic area, activation of these inflammatory cells and death of cardiomyocytes leads to an elevation in the local and circulating concentrations of S100A9, which initiates an inflammatory cascade via triggering downstream signalling pathways and participating in the process of myocardial injury; concurrently, macrophages play a role from the inflammatory phase to the granulation tissue stage and are involved in both myocardial injury identified S100A9 as a promising biomarker for PAH using a combination of LASSO regression analysis and SVM-RFE algorithm.Subsequent evaluation of the diagnostic value of S100A9 in PAH revealed an impressive AUC of 0.931 (95% CI = 0.832-0.981) in GSE117621 and an AUC of 0.722 (95% CI = 0.457-0.907) in GSE4819, underscoring the significance of S100A9 in PAH diagnosis. 108S100A9 activation is positively correlated with immune cells, including neutrophils, monocytes, and T cells,

F I G U R E 5
Possible mechanism of vascular remodelling induction by S100A9.The expression of S100A9, TLR4, and RAGE is increased in PASMCs and ECs, and thus S100A9 is hypothesized to contribute to pulmonary vascular remodelling by binding to TLR4/RAGE.ECs, endothelial cells; PASMCs, pulmonary artery smooth muscle cells; LncRNA MALAT1, long-chain noncoding RNA metastasis-related lung adenocarcinoma transcript 1; MiR-503, microRNA-503.

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PERSPECTIVES ON THE CLINICAL APPLICATION OF S100A9 AS A THERAPEUTIC TARGET In CVD, S100A9 plays a regulatory role in inflammation, calcium balance, endothelial function, cell proliferation, autophagy, apoptosis, and cell death.Currently, S100A9 inhibitors, such as ABR-215757 (Paquinimod), ABR-215062 (Laquinimod), and ABR-215050 (Tasquinimod) have been used in various stages of clinical trials, and given the increased expression of S100A9 in the development of CVD; these inhibitors have potential for the treatment of CVD as well.
above are small molecule immunomodulators that work by affecting immune cells and inflammatory processes.While they have not yet been applied to clinical trials for CVD, their extensive application in animal models underscores their potential for future clinical trials and subsequent clinical therapeutic applications.Marinkovi c et al.80 reported that intraperitoneal injection of ABR-238901 into mice with MI for three consecutive days (at the onset of MI, 24 h post-MI, and 48 h post-MI) significantly improve ejection fraction and cardiac output.However, 21 days of treatment lead to progressive deterioration of cardiac function and ventricular remodelling.78It is indicated that short-term blockade of S100A9 can ameliorate post-MI inflammatory injury and promote cardiac repair, while extended treatment duration can lead to the opposite effect, correlating with a reduction in reparative macrophages due to long-term S100A9 blockade.Interestingly, compared to the control group, S100A9 dimer pretreatment enhances the anti-inflammatory effect of human amniotic mesenchymal stem cells at days 7, 14, and 28 post-MI, facilitating the recovery of cardiac function.50These findings reveal the dual pro-inflammatory and anti-inflammatory roles of S100A9 in the post-MI repair process, emphasizing the crucial importance of identifying the time window for blocking S100A9 for MI prognosis.

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CONCLUSIONInflammation serves as a fundamental initiator for various cardiovascular pathologies, and studies have revealed that monitoring immuneinflammatory responses holds paramount significance in diagnosing, predicting, and treating CVD.S100A9, a pivotal inflammatory modulator, has been reported to participate in the development and progression of diverse cardiovascular disorders.According to its physiological characteristics and mechanisms of action, S100A9 is prominently engaged in the regulation of inflammatory responses (including proinflammation and anti-inflammation), endothelial function, cellular proliferation, autophagy, apoptosis, and cell death.Through these intricate pathways, S100A9 assumes a pivotal role in driving the onset and advancement of CVD.Conversely, studies have revealed that the inhibition of S100A9 holds pivotal therapeutic significance in a plethora of CVD, encompassing atherosclerosis, MI, PAH, PAD, cardiomyopathy, and myocarditis.S100A9 inhibitors have already been employed as clinic trial medications for various inflammation-related disorders, implying their potential application value in CVD.However, considering the complexity of S100A9's actions and the unique characteristics of inflammatory responses in CVD, further research is warranted at different stages and with diverse formulations to better understand its role.