cGAS-STING signaling in cardiovascular diseases

Sterile inflammation, characterized by a persistent chronic inflammatory state, significantly contributes to the progression of various diseases such as autoimmune, metabolic, neurodegenerative, and cardiovascular disorders. Recent evidence has increasingly highlighted the intricate connection between inflammatory responses and cardiovascular diseases, underscoring the pivotal role of the Stimulator of Interferon Genes (STING). STING is crucial for the secretion of type I interferon (IFN) and proinflammatory cytokines in response to cytosolic nucleic acids, playing a vital role in the innate immune system. Specifically, research has underscored the STING pathway involvement in unregulated inflammations, where its aberrant activation leads to a surge in inflammatory events, enhanced IFN I responses, and cell death. The primary pathway triggering STING activation is the cyclic GMP-AMP synthase (cGAS) pathway. This review delves into recent findings on STING and the cGAS-STING pathways, focusing on their regulatory mechanisms and impact on cardiovascular diseases. It also discusses the latest advancements in identifying antagonists targeting cGAS and STING, and concludes by assessing the potential of cGAS or STING inhibitors as treatments for cardiovascular diseases.


Introduction
Cardiovascular diseases (CVDs) represent a wide array of complex conditions, including coronary artery diseases, thoracic aortic aneurysms, heart failure (HF), cardiomyopathies, and congenital cardiac anomalies (1).Their global prevalence highlights a significant impact on health worldwide, contributing to a substantial number of fatalities (2).In 2015, CVDs affected 11 million people in European Society of Cardiology member countries, with a total prevalence of approximately 83.5 million (3).These conditions lead to over 17 million deaths globally each year, including 4 million in Europe alone (4,5).The interaction between immune responses and inflammation in CVDs' pathophysiology has become a focal point of recent research.Despite advances in surgery and pharmacology improving clinical outcomes, the overall prognosis for CVD patients remains concerning (6).Immune responses and inflammation are pivotal in the pathogenesis of CVDs, as demonstrated by both innate and adaptive immune responses playing critical roles in atherosclerosis progression.A landmark clinical trial in 2017 showed that targeting inflammation could significantly reduce cardiovascular events in high-risk patients (7,8).Under pathological conditions, cytoplasmic DNA accumulates through various mechanisms, including efflux from mitochondrial and nuclear DNA into the cytosol and release from dying cells (9,10).This accumulation triggers the cGAS pathway upon binding with double-stranded DNA, leading to STING activation in the endoplasmic reticulum (11,12).Activated STING initiates a cascade of immune responses, including the activation of interferon regulatory factor 3 (IRF3), TANK binding kinase 1 (TBK1), and nuclear factor-kB (NF-kB) (13)(14)(15).While this process upregulates interferons and inflammatory factors, excessive inflammation can lead to tissue damage and organ dysfunction, exacerbating various diseases (16)(17)(18).Inflammation is a key determinant in the onset, progression, and outcomes of CVDs, particularly in atherosclerosis and heart failure, where it significantly contributes to disease progression and adverse outcomes (5,19).The cGAS-STING signaling pathway has been identified as critical in CVD pathogenesis with recent evidence suggesting that targeting cGAS-STING-mediated inflammation holds promise as a treatment for CVDs (20)(21)(22).This comprehensive review explores the impact of cGAS-STING-mediated sterile inflammation on CVDs, including the pathway's intricate functions and its role in coordinating the IFN immune response.We also examine the landscape of suppressors targeting both cGAS and STING, highlighting potential therapeutic strategies to modulate this crucial pathway.

Overview of cGAS-STING pathway
DNA, recognized as a crucial damage-associated molecular pattern (DAMP), triggers the innate immune system by engaging corresponding receptors and initiating intracellular pathways (23).The sources of dsDNA include dying cells, DNA viruses, genomic instability, bacteria, damaged mitochondria, retroviruses, and DNA damage (24,25).Various DNA sensors, such as absent in melanoma 2 (AIM2), Toll-like receptor (TLR) 9, and cyclic GMP-AMP synthase (cGAS), play pivotal roles in mediating DNA-stimulated innate immune responses (26)(27)(28).Among pattern recognition receptors (PRRs), the TLR family, particularly TLR9, is notable (29).Located in the endosomal membrane, TLR9 is highly sensitive to hypomethylated cytidine-phosphate-guanosine regions in DNA from bacteria or viruses, activating the innate immune response (30).Discovered in 2009, AIM2 acts as a cytoplasmic DNA receptor, forming an interaction with dsDNA and associating with the adaptor molecule apoptosis-associated speck-like protein, which activates and recruit caspases (31).This interaction leads to inflammasome/pyroptosome formation, activating both NF-kB and caspase-1 (32).In 2013, Chen et al. identified cGAS, a nucleotidestransferase, that catalyzes the production of the second messenger cGAMP, which binds to STING on the endoplasmic reticulum (ER) membrane, triggering the IFN-I pathway (28).cGAS recognizes both exogenous DNA from pathogens and endogenous DNA from various subcellular locations, such as cytoplasmic chromatin, micronuclei, and mitochondria (33)(34)(35).Upon dsDNA recognition, cGAS forms a complex, inducing conformational changes to synthesize cGAMP from ATP and GTP (36,37).cGAMP binds to STING, causing a conformational change that activates the protein.STING then relocates from the ER to the ER-Golgi intermediate compartment and the Golgi apparatus, activating TBK1.TBK1 phosphorylates itself and STING, activating the IRF3 transcription factor.Dimerized IRF3 translocate into the nucleus, initiating IFN-I synthesis (38-40).Additionally, STING can activate IKK kinase, leading to the phosphorylation of NF-kB transcription factor inhibitors.This cascade ultimately stimulates the production of inflammatory cytokines, including TNF and IL-6 (41) (Figure 1).

The function of cGAS-STING pathway on cardiovascular diseases
Inflammation plays a pivotal role in the progression of cardiovascular diseases.Recent advancements in understanding the STING-mediated inflammatory response have made significant contributions to cardiovascular disease research.This focus has sharpened on the accumulation of cytoplasmic DNA and its role in activating the cGAS-STING-mediated inflammatory response within pathological cardiovascular environments.This section offers a comprehensive review of the pathological processes in cardiovascular diseases, highlighting the regulatory role of inflammation driven by the cGAS-STING pathway (Table 1).

Heart failure
Heart failure (HF) manifests as a syndrome characterized by symptoms such as dyspnea, edema, and lower limb weakness, often accompanied by higher pulmonary edema, peripheral edema, and jugular vein strain (48).It results from impaired heart structure or function, significantly contributing to rising global mortality rates (49).There is mounting evidence linking inflammatory pathways' activations to the progression of left ventricular remodeling and dysfunction in HF patients, suggesting a potential chronic inflammatory aspect to HF.However, the specific function of inflammation in HF remains incompletely understood.In a study investigating non-ischemic pressure-overload induced HF (transverse aortic constriction, TAC), characterized by cardiac dysfunction, hypertrophy, and fibrosis, elevated IFNa, IFNb, and STING expressions were observed (42).Remarkably, STING knockout (STING-KO) mice exhibited a return to baseline levels of these indicators, indicating a direct association (42).Similarly, angiotensin II-treated neonatal rat cardiomyocytes displayed heightened STING, IFN-a/-b expressions.Inhibiting STING via siRNA in these cells resulted in a significant reduction in TNFa, IFNa/b, and IL-6/-1b levels.Elevated IFNa/b and STING expressions were also noted in dilative and hypertrophic cardiomyopathies specimens from humans (42).Complementary findings emerged from another study reporting substantial increases in IFN, STING, cGAS, as well as IFN-induced chemokines ISG15, IFIT3, CXCL10 expressions, three days post TAC (43).cGAS silence using adeno-associated virus 9 (AAV9) led to a notable drop in left ventricular remodeling and fibrosis, underscoring the potential therapeutic relevance of the cGAS-STING pathway in HF (43).While scientific investigations have initiated exploration into the relationship between HF and the cGAS-STING signaling pathway, further research is imperative to gain a comprehensive understanding of its precise role in disease progression.

Myocardial infarction
Myocardial infarction (MI), a severe manifestation of coronary heart disease, significantly impacts patient survival, with the potential for ventricular remodeling, progressive deterioration in cardiac function, and eventual development of chronic heart failure (HF), resulting in a 10-year survival rate typically below 50% (50).Following cardiac cell injury and death during MI, the release of damage-associated molecular patterns (DAMPs) initiates the innate immune response.In a mouse model of MI, activation of interferon regulatory factor 3 (IRF3), a molecule downstream of STING, was found to amplify the release of interferon type I (IFN-I) and inflammation significantly.Disrupting IRF3 signaling markedly reduced the production of inflammatory markers (CXCL10 and IFNB1) and cell infiltration, improving survival and cardiac function post-MI (51).This suggests that inhibiting IRF3 signaling could provide cardiac protection after MI.Rech et al. found that pharmacologic inhibition of STING reduces infarction size and scarring, restoring ventricular systolic function to near-normal levels three weeks post-reperfusion in MI mice, with a noted decrease in myocardial hypertrophy (44).They assessed the inflammatory response by examining expressions of proinflammatory genes, uncovering significant differences in IFI44 and CXCL10 expressions between the inhibition and control groups.Hu et al. further confirmed the protective effect of silencing STING through the development of H-151, a selective STING pathway suppressor, both in vivo and in vitro.STING suppression mitigated the impact of MI by reducing cardiomyocyte apoptosis and fibrosis in fibroblasts induced by the STING pathway in dsDNA-triggered infiltrated macrophages (21).Additionally, inhibiting the macrophage-derived IFN-I signaling pathway, using antibodies against IFN, STING, or a cGAS inhibitor, led to a notable reduction in infarct size in mice subjected to myocardial ischemia- Overview of the cGAS-STING pathway in cardiovascular diseases.Initially, the cGAS-STING signaling pathway serves to protect host cells from pathogens.Further studies have highlighted its significance in the context of cardiovascular diseases, where cGAS detects DNA from both intracellular and extracellular origins.Upon DNA detection, cGAS converts its substrates, GTP and ATP, into cGAMP.STING, situated on the endoplasmic reticulum (ER), detects cGAMP and, when bound to it, becomes activated.This activation leads STING to recruit and phosphorylate TBK1 and IKKs.These kinases then phosphorylate IRF3 and IkBa, respectively.Phosphorylated IRF3 moves to the nucleus, initiating the transcription of type I interferon (IFN-I) genes.Concurrently, phosphorylated IkBa facilitates the recruitment of NF-kB to the nucleus, triggering the transcription of genes encoding proinflammatory cytokines.STING, stimulator of interferon genes; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; TBK1, TANK-binding kinase 1; IRF-3, interferon regulatory factor-3; IkBa: inhibitors of transcription factor NF-kB; type-I IFN, type-I interferon; IL-6, interleukin 6; TNF-a, tumor necrosis factor-a; GTP, guanosine triphosphate; ATP, adenosine triphosphate.reperfusion (52).These findings collectively suggest that DAMPs released during MI prompt a STING-mediated inflammatory response, contributing to the pathological process of MI.

Myocarditis
Myocarditis can arise from a variety of sources, including fungi, parasites, viruses, bacteria, autoimmune reactions, or as side effects of drugs (53).Viral infections play a crucial role in the initiation and progression of myocarditis, leading to inflammation that can cause cardiac cells to undergo necrosis or apoptosis, which are key aspects of the disease's pathophysiological process (54, 55).Recent research has shown that the cGAS-STING pathway has a dual role in viral myocarditis.Following a viral infection, the presence of viral DNA within the cytoplasm can lead to two contrasting outcomes: on one hand, it activates the cGAS-STING pathway, promoting the production of IFN-I, which helps the body combat viruses; on the other hand, this same pathway can exacerbate myocardial damage by inducing inflammation (56, 57).In addition to its role in viral myocarditis, the cGAS-STING pathway is also significant in noninfectious myocarditis and microbial infections.An experimental study using a lipopolysaccharide-induced myocardial infarction mouse model with sepsis observed a notable increase in STING and p-IRF3 expression.Remarkably, STING knockout (STING-KO) effectively reduced inflammation, apoptosis, and cardiomyocyte scarring induced by lipopolysaccharide, while simultaneously improving cardiac function and extending mice survival (45).Myocarditis following Trypanosoma cruzi infection, which activates the poly-ADP ribose polymerase 1-cGAS-NF-kB pathway, facilitating the conversion of inflammatory macrophages, has also been observed.In addition, blocking cGAS activation has proven effective in mitigating this inflammatory response (58).

Atherosclerosis
Atherosclerosis (AS) is recognized as a chronic inflammatory condition and a primary cause of clinical cardiovascular events.The presence of DNA damage and extracellular vesicles containing DNA at vascular sites contributes to vascular disorders, especially atherosclerosis, by promoting the progression from calcification to plaque formation, and subsequently thrombosis (59).DNA damage is a notable feature in various vascular diseases, including genetic vascular degenerative syndromes (59).While the presence of microbial DNA in human atherosclerotic plaques has been noted, the exact pathophysiological connection to vascular disease progression is yet to be fully understood (60,61).However, a correlation between the amount of bacterial DNA at plaque sites and the recruitment and accumulation of monocytes suggests a critical role for DNA-modulated monocyte recruitment in both vascular and systemic inflammation (62).Oxidized mitochondrial DNA from macrophages within atherosclerotic plaques is known to induce STING-dependent inflammation, exacerbating

Aortic aneurysms and dissections
Aortic aneurysm and dissection (AAD) are closely linked cardiovascular conditions that significantly increase morbidity and mortality risks (64).Currently, effective clinically proven medications to halt the progression of aortic degeneration are absent.AAD may manifest sporadically or in conjunction with genetic disorders.Sporadic AAD is characterized by a progressive loss of smooth muscle cells (SMCs), alongside fragmentation and depletion of the extracellular matrix (ECM), culminating in aneurysm development, dissection, and possible rupture (65).Luo et al. found that inducing sporadic AAD in Sting-deficient mice led to reduced aortic enlargement, decreased elastic fiber fragmentation, and a lower incidence of ADD compared to the wild-type group (47).This finding implies that the absence of STING contributes to the preservation of aortic structure and the smooth muscle layer, underscoring STING's critical role in the progression of sporadic AAD and aortic degeneration.Further analysis through single-cell transcriptomics of the aorta, GO analyses, and in vitro experiments highlighted STING's involvement in several biological processes, such as response to reactive oxygen species (ROS), DNA damage response, inflammatory response, and cell death in aortic smooth muscle (47).ROS-mediated DNA damage initiates the release of DNA fragments, activating the cGAS-STING-TBK1-IRF3 pathway, which in turn promotes the death of aortic SMCs.It was also discovered that tissue monocytes primarily detect and engulf dsDNA from damaged aortic SMCs, activating the STING-IRF3 pathway and increasing the expression of matrix metallopeptidase 9 (MMP-9), an enzyme that plays a role in ECM degradation.Importantly, the use of a pharmacological TBK1 suppressor to block phosphorylation at STING's Ser366 was effective in mitigating sporadic AAD.This approach helped maintain the aortic structure, improved survival rates, and reduced the incidence and severity of ADD (47).

Diabetic cardiomyopathy
Diabetes, a chronic condition, can lead to heart failure due to prolonged cardiac pressure overload (66).Recent research has revealed STING's role in diabetes-related processes, including cholesterol metabolism, liver inflammation, and islet cell damage (67,68).Inflammation is a key factor in the development of diabetic cardiomyopathy.Yan et al. demonstrated that activation of the cGAS-STING pathway leads to NLRP3 inflammasome-induced pyroptosis, exacerbating diabetic cardiomyopathy (20).In diabetic mice, hyperlipidemia induces the release of DNA from myocardial cells, which activates the cGAS-STING pathway, resulting in pyroptosis and inflammation.This contributes to myocardial hypertrophy and remodeling (20).However, studies have shown that blocking the Sting gene with AAV9 or pharmacologically suppressing STING can significantly reduce diabetic cardiomyopathy and myocardial inflammation (20,69).These findings highlight the critical role of cytosolic mtDNA-induced cGAS-STING activation in the pathogenesis of obesity-related diabetic cardiomyopathy (DCM) and provide preclinical evidence supporting the targeting of this pathway as a potential therapeutic strategy for DCM.

Cardiac aging
Inflammation is a fundamental hallmark of the cardiac aging process.Chronic low-grade inflammation, mediated through specific pathways, is a well-recognized precursor to the development of age-related CVDs (70).Central to this process is mitochondrial dysfunction, which plays a key role in the progression of senescence in CVD (71).One critical outcome of mitochondrial dysfunction is the release of mitochondrial DNA (mtDNA) into the cytoplasm (72).This event can activate various inflammatory pathways, including the cGAS pathway.cGAS, stimulated by the presence of cytosolic DNA, serves as a potent activator of the STING pathway.Upon activation, STING induces the transcription factor interferon-regulatory factor 3 (IRF3) to enter the nucleus, promoting the secretion of interferons and further enhancing the inflammatory response (73,74).Recent research by Wang et al. reported that nanoplastics cause cardiomyocytes senescence by triggering a series of senescencerelated marker molecules (75).Further experiments showed that nano-scaled microplastics may lead to ROS production by inducing calcium overload.The excessive accumulation of ROS leads to the release of mtDNA from mitochondria into the cytoplasm, which in turn activates the cGAS-STING signaling pathway, thus leading to cardiomyocyte senescence (75).In light of these findings, targeting the STING pathway may represent a novel therapeutic strategy in managing age-associated cardiovascular conditions by moderating the senescent inflammatory processes that significantly contribute to disease progression.

Discovery of cGAS-STINGtargeting drugs
As previously emphasized, the cGAS-STING signaling pathway plays a pivotal role in the progression of various sterile cardiovascular diseases (CVDs).This pathway has emerged as a critical therapeutic target not only for the induction of cytokines, including IFN-I, but also in addressing broader health challenges.Several small-molecule agonists targeting this pathway have been developed and are actively being tested in clinical trials (NCT03172936, NCT04144140) (76).These efforts highlight the potential of modulating this pathway.Currently, treatments such as the JAK inhibitor baricitinib and steroids are used clinically to manage patients with STING activation (77,78).However, it is necessary to develop novel drug interventions that more directly target cGAS and STING.This section will explore the use of cGAS and STING suppressors specifically in the context of cardiovascular diseases, analyzing their potential therapeutic benefits and implications.By targeting these molecular mechanisms, novel treatment strategies aim to offer more precise control over the inflammatory processes that contribute to the progression of CVDs, providing a promising avenue for therapeutic intervention (Figure 2) (Table 2).cGAS inhibitors cGAS acts as both a molecular alarm and an enzyme, catalyzing the production of cGAMP for STING signaling.The development of small molecule cGAS suppressors is based on three distinct mechanisms: The first is that mediating cGAS post-translational modification (92); The second is blocking the binding of DNA to cGAS (46); The third is targeting the catalytic pocket of cGAS (59).In the realm of cGAS inhibitors, the first mechanism focuses on the post-translational modifications of cGAS, with Aspirin being an example that targets cGAS acetylation at Lys 384/394/414 to effectively suppress autoimmunity caused by TREX1 genetic deletion (79).The second mechanism is aimed at interrupting the interaction between cGAS and dsDNA.Compounds such as A151, hydroxychloroquine (HCQ), Suramin, quinacrine (QC), and X6 prevent the formation of the cGAS-dsDNA complex by binding to the DNA minor groove at the cGAS/DNA interface (79)(80)(81)83).Suramin, a nucleic acid mimic, competes with dsDNA for the cGAS binding site, reducing the migration and proliferation of vascular smooth muscle cells (VSMCs) and decreasing neointima hyperplasia (82, 93,94).The third mechanism involves inhibitors that compete with ATP or GTP, the natural substrates of cGAS, or with cGAMP, the product.This category includes non-substratecompetitive suppressors like RU.521/365, which target essential residues in the catalytic site to reduce binding to ATP/GTP and, consequently, downregulate IFN expression.RU.521 specifically binds to the cGAS catalytic sites, inhibiting cGAMP synthesis and subsequent STING activation, and has been shown to protect against septic cardiomyopathy induced by LPS (84,95).Meanwhile, substrate-competitive inhibitors, such as PF-06928215, directly inhibit the catalytic activity of cGAS and have been proven to alleviate palmitic acid-induced contractile dysfunction in cardiomyocytes (85,96).Overall, cGAS inhibitors demonstrate protective effects in cardiovascular diseases.assembly of the STING polymer complex, reducing infarct dilation and scar formation, thus restoring left ventricular systolic function and reducing myocardial hypertrophy in a myocardial infarction mouse model (44,90).Additionally, targeting posttranslational modifications of STING and its associated kinases presents viable methods for inhibiting STING activity.For example, the FDAapproved drug amlexanox, known for its anti-inflammatory effects, is utilized in clinical settings to treat asthma and recurrent aphthous ulcers (103).Amlexanox effectively prevents complete activation of STING by blocking TBK1-induced phosphorylation at Ser366, due to its strong affinity and specificity for TBK1 (91).In summary, STING inhibitors have shown protective effects against cardiovascular diseases, presenting a novel strategy for treatment.

Conclusion and perspective
In the realm of cardiovascular diseases, the persistence of an inflammatory response significantly impacts initiation, development, and outcomes, often leading to adverse clinical consequences (19, 104).The cGAS-STING pathway, recognized for its role in mediating sterile inflammation, has increasingly become a focus of interest over the years (17).Pathological scenarios such as mitochondrial damage or cell death in immune cells, vascular endothelial cells, vascular smooth muscle cells, or cardiomyocytes can cause mitochondrial or nuclear DNA to be released into the cytoplasm (18).This cytoplasmic DNA then activates the cGAS-STING pathway, upregulating inflammatory factors, chemokines, and interferons, thereby exacerbating the inflammatory response (105).This review highlights the critical association between cardiac dysfunction and the cGAS-STING pathway, which is central to cell death and influences various physiological functions.The pathway is instrumental in the onset and progression of diverse cardiovascular disorders, such as MI, HF, and myocarditis, by mediating inflammation, regulating immunity, promoting autophagy, and contributing to aging.Animal studies have consistently shown that inhibiting this pathway can improve cardiac function in these diseases, also leading to increased survival rates.Given these promising findings, the cGAS-STING pathway is considered a potential therapeutic target for various cardiovascular disorders.Nonetheless, due to the complex and varied nature of cardiovascular diseases, further exploration and a deeper understanding of the cGAS-STING pathway's role in different cardiovascular conditions are essential.

FIGURE 1
FIGURE 1 STING is a critical signaling molecule in the cGAS-STING DNA-sensing pathway, with its activation essential for various intracellular signaling cascades.Development of STING inhibitors

FIGURE 2
FIGURE 2Therapeutical potential of cGAS-STING pathway targeting against inflammation in cardiovascular diseases.This figure provides an overview of different pharmacological agents to modulate the cGAS-STING signaling pathway by intervening at various targets.STING, stimulator of interferon genes; ER, endoplasmic reticulum; TBK1, TANK-binding kinase 1; IRF-3, interferon regulatory factor-3; IkBa, inhibitors of transcription factor NF-kB; type-I IFN, type-I interferon; IL-6, interleukin 6; TNF-a, tumor necrosis factor-a.

TABLE 1
The connection between cardiovascular diseases and the cGAS-STING pathway.

TABLE 2
Overview of cGAS-STING signaling pathway inhibitors.