Associations of serum levels of cGAMP in the context of COVID-19 infection, atherosclerosis, sterile inflammation, and functional endothelial biomarkers in patients with coronary heart disease and healthy volunteers

Introduction

A viral infection may have harmful effects on the cardiac function due to transcriptional and posttranslational modifications that disrupt the gap junctions, influence immune response, or mediate the development of cardiomyopathy [1]. The cyclic dinucleotide 2′3′-cyclic GMP-AMP (cGAMP) is considered a marker of pattern-recognition receptor (PRR) activation and sterile inflammation. cGAMP is produced by the cytosolic double-stranded DNA (dsDNA) sensor enzyme cGAMP synthase (cGAS) in response to the presence of aberrant dsDNA in the cytoplasm. In general, cytosolic dsDNA is associated with intracellular invasion of DNA viruses or certain bacterial or with cellular DNA damage. cGAMP acts as a second messenger to activate the stimulator of interferon genes (STING), which is the central hub of cytosolic dsDNA sensing, to eventually induce type-I interferon and proinflammatory cytokine responses against infections, cancer, or cellular stress [2].

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is RNA-containing coronavirus, which is the pathogen of COVID-19. SARS-CoV-2 is not expected to influence the activity of cGAS, accumulation of cGAMP, or activation of STING directly since this virus does not contain dsDNA. However, infection with SARS-CoV-2 is known to trigger autophagy that involves intracellular degradation process to maintain cellular homeostasis and combat the invading pathogen. Autophagy antagonizes interferon production possibly by promoting STING degradation [3]. On the other hand, genetic deletion of STING in apolipoprotein E-deficient (ApoE^−/−) mice was shown to reduce atherosclerotic lesions spontaneously developing in these animals [4]. Moreover, a recent study demonstrated direct STING-independent cGAMP-dependent effects of cGAS activation by dsDNA in the endothelium on vascular smooth muscle relaxation [5].

The mechanisms and targets of sterile inflammation in patients with coronary heart disease (CHD) and healthy subjects after COVID-19 are poorly understood. The aim of the present study was to elucidate the associations of serum cGAMP levels with endothelial dysfunction markers and the presence of antibodies against the spike protein receptor-binding domain (S1 RBD) of SARS-CoV2 (IgG-SARS-CoV-2) in apparently healthy subjects with elevated cardiovascular risk assessed by systematic coronary risk evaluation by European Society of Cardiology (SCORE) and in patients with coronary atherosclerosis, which represents the advanced stage of endothelial dysfunction.

Patients and methods

Results

The study included a total of 256 men and women randomly selected from two different cohorts to represent various stages of endothelial dysfunction evaluated as elevated SCORE (varied from 0 to 20) in healthy volunteers of cohort 1 [6] or as coronary atherosclerosis evaluated using Gensini score [19] (varied from 0 to 197) in CHD patients of cohort 2 [11], 12]. Patients with higher Gensini score corresponded to advanced clinical stages of endothelial dysfunction. Functional markers, including NOx and endothelin-1, were used as indicators of endothelial dysfunction.

Cohort 1 was enrolled in a microcirculation study in 2019–2021, and the participants were gradually infected with SARS-CoV-2 during the course of the COVID-19 pandemic [6]. All participants of cohort 1 had no complaints for pre-existing cardiovascular diseases, thrombotic events, or other serious comorbidities. General characteristics of the group selected from cohort 1 are listed in Table 1. Clinical and biochemical parameters of these participants varied within the corresponding normal ranges, indicating that the participants were apparently healthy. IgG-SARS antibodies were assayed in the blood samples of cohort 1, and serum cGAMP level was measured in 168 samples of cohort 1. This selected group of cohort 1 (n=168) was divided into two subgroups according to the IgG-SARS status: positive (+) (n=56) or negative (−) (n=112). All tested parameters were compared between these subgroups using Mann-Whitney U test (Table 1). The levels of tested parameters were similar between the subgroups of healthy volunteers with SARS(+) and SARS(−) status, except sex (Table 1).

The groups selected from cohort 1 and 2 were not balanced in sex or age. Thus, associations of sex and age with various tested parameters were estimated, including serum cGAMP, NOx, and endothelin-1 in combined cohorts 1 and 2. The results indicated the lack of associations with sex and age (data not shown).

Cohort 2 comprised patients with signs of CHD who underwent coronary angiography. The group randomly selected from cohort 2 (n=88) was divided into two equal subgroups according to the severity of coronary stenosis lesions: presence of coronary lesions corresponding to advanced endothelial dysfunction (n=44) and absence of coronary lesions corresponding to moderate endothelial dysfunction (n=44). Various parameters were compared between the two patient subgroups using a post-hoc test for Kruskal-Wallis, the Dunn’s test (Table 1). The levels of routine biomarkers, such as CRP and glucose, were increased in patients with coronary stenosis compared with those in patients without coronary lesions (Table 1). Total and HDL-cholesterol levels were decreased in patients with coronary stenosis compared with those in patients without coronary stenosis apparently because of statin treatment routinely prescribed to patients in the subgroup with coronary lesions.

A total of 145 healthy subjects with low SCORE index (0–1) and SARS(−) status were selected from cohort 1 to represent normal endothelial function, and associations between endothelial markers (endothelin-1 and NOx) with cGAMP were evaluated (Table 2). In these subjects, the levels of cGAMP were not correlated with endothelial markers (Table 2). This subgroup of healthy subjects was expanded by combining with the subgroup of healthy subjects with low SCORE (0–1) and IgG-SARS(+) status (Table 3). In this case, the IgG-SARS-CoV-2 status was correlated with endothelial markers (NOx and endothelin-1) but there were no correlations with cGAMP. Serum endothelin-1 and NOx levels were decreased in IgG-SARS(+) subjects compared with those in IgG-SARS(−) subjects: 1.77 ± 0.96 pg/mL vs. 1.38 ± 1.1 pg/mL (p=0.018) and 30.99 ± 22.98 μmol/L vs. 24.47 ± 12.67 μmol/L (p=0.05), respectively.

Thus, COVID-19 was linked to endothelial function, while cGAMP was not involved in these associations in apparently healthy individuals. Additionally, the cGAMP levels were not associated with IgG-SARS status in the group selected from cohort 1 with variable SCORE (Tables 1 and 4). However, serum endothelin-1 was directly correlated with SCORE in subjects with SARS(−) status with r=0.38 (p=0.0001) and in subjects with SARS(+) status with r=0.47 (p=0.0001) (Table 4). The NOx levels were directly correlated with SCORE but only in SARS(+) participants (r=0.43; p=0.001) (Table 4). Thus, higher SCORE was linked to higher levels of endothelin-1 regardless of IgG-SARS status, and only IgG-SASR(+) subjects displayed an association of SCORE with NOx.

In contrast to cohort 1, serum levels of cGAMP were associated with advanced level of endothelial dysfunction in cohort 2 in patients with atherosclerosis, and cGAMP was lower in patients with coronary stenosis compared with that in patients without coronary stenosis (Table 1). The data of receiver operating characteristic (ROC) analysis (AUC=0.69; 95 % CI 0.587–0.806; p=0.001) confirmed the associations between cGAMP, as a continuous variable, with coronary lesions (presence/absence), as a binary variable, in CHD patients of cohort 2 (Figure 1).

Figure 1:

ROC analysis of the associations (A) of cGAMP (continuous variable) with coronary lesions (binary variable; 0, without coronary lesions corresponding to Gensini score 0 vs. 1 with any coronary lesions; AUC=0.69; 95 % CI 0.59–0.81; p=0.001) and (B) NOx (continuous variable) with coronary lesions (binary variable; 0, without coronary lesions corresponding to Gensini score 0 vs. 1 with any coronary lesions; AUC=0.69; 95 % CI 0.62–0.77; p=0.0001).

Coronary stenosis expressed as Gensini score (continuous variable) in patients with advanced endothelial dysfunction was inversely correlated with serum NOx levels in CHD patients of cohort 2 (Table 5). This association was confirmed by the data of ROC analysis when coronary lesions were presented as a binary variable (no lesions/stenosis of any degree) and NOx (continuous variable) (AUC=0.69; 95 % CI 0.62–0.77; p=0.0001) (Figure 1). However, serum endothelin-1 was not associated with the presence of coronary lesions in CHD-patients (Table 5).

Advanced endothelial dysfunction in CHD-patients (presence of coronary stenosis) and apparently healthy subjects without endothelial dysfunction with SARS(+) status was associated with lower NOx levels. Higher extent of endothelial dysfunction corresponding to higher SCORE was associated with higher levels of serum NOx and endothelin-1. Healthy subjects with IgG-SARS(−) status demonstrated no correlations of SCORE with NOx or endothelin-1.

Thus, these results demonstrated that cGAMP, as a marker of sterile inflammation, was able to discriminate severe coronary stenosis in CHD-patients and was not associated with early stages of endothelial dysfunction in subjects with elevated SCORE. Moreover, cGAMP was not linked to the IgG SARS(+) and (−) immune status in healthy participants and was not associated with SCORE.

Discussion

Recognition of microbial and viral nucleic acids is a major mechanism of pathogen detection and neutralization by the immune system [20]. SARS-CoV-2 still poses a global threat due to several highly transmissible variants and long COVID-19 [20]. SARS-CoV-2 can trigger autophagy associated with lysosomal degradation of STING that mediates type I interferon production in response to infection. Autophagy is an intracellular degradation process that maintains cellular homeostasis required for efficient defense against pathogens [4].

STING is one of the key mediators of the cGAS-cGAMP-STING pathway. cGAS produces cGAMP in response to cytosolic dsDNA to activate innate immune responses mediated by STING [2], 21]. Additionally, the cGAS–cGAMP-STING pathway is able to detect almost any dsDNA that is present in the cytosol as a result of cellular DNA damage in the case of abnormal DNA damage response similar to the processes detected in many tumor cells.

Replication of pathogens inside the cell engages various cytosolic sensors, depending on the pathogen structure. These sensors are known as pattern-recognition receptors. For example, NOD-like receptor (NLR) family is involved in the detection of a variety of microbial toxins and general cellular damage to trigger certain downstream signaling pathways mediated by inflammasomes. Receptors of the RLR family detect viral RNA in the cytosol to trigger a signaling cascade that leads to the production of type I interferons and inflammatory cytokines [2], 22].

SARS-CoV-2 is the member of the Orthocoronavirinae subfamily of RNA-containing coronaviruses, with its genome encoding for 29 nonstructural, structural, and accessory proteins [23]. The data of the present study indicated that serum cGAMP did not discriminate between the SARS(+) and SARS(−) statuses in apparently healthy individuals because SARS-CoV-2 is a single-stranded RNA virus that does not influence the cGAS–cGAMP-STING pathway due to the lack of dsDNA. However, cGAMP may be linked to certain signaling pathways regulated by the NLR/RLR systems, which detect the presence of viral RNA in the cytosol [2], 22]. The NLR family pyrin domain-containing three inflammasome is known to be the key mediator in sterile inflammatory responses [24]. On the other hand, endogenous DNA fragments produced in the cytosol due to aberrant DNA damage provoke sterile inflammation through cGAS. Thus, STING has been actively studied in atherogenesis [4]. For example, hypercholesterolemic apolipoprotein E-deficient (ApoE^−/−) mice fed a western-type diet show an increase in STING expression and in markers of DNA damage, such as single-stranded DNA accumulation in macrophages in the aorta. Moreover, the levels of cGAMP, an STING agonist, in the aorta are higher in ApoE^−/− mice, and genetic deletion of STING in ApoE^−/− mice reduces atherosclerotic lesions and the expression of inflammatory mediators in the aorta. The authors concluded that STING stimulates the activation of proinflammatory macrophages, leading to the development of atherosclerosis, and STING is a potential therapeutic target for atherosclerosis [4]. The data of the present study are generally consistent with these observations because serum cGAMP was able to discriminate between CHD patients with and without coronary lesions.

Inflammation plays an important role in cardiometabolic diseases. Sterile inflammation is a non-infectious innate immune response largely induced by damage-associated molecular patterns (DAMP) via PRR and is closely associated with the development and progression of various cardiovascular diseases [24]. Thus, we aimed to study the associations between serum cGAMP and endothelial dysfunction that represents one of the initial stages of cardiovascular lesions in atherosclerosis. We used serum NOx and endothelin-1 as surrogate markers of endothelial function. Healthy subjects of cohort 1 with SCORE indexes 0 and 1 were used as the reference. In the present study, associations of serum endothelin-1 or NOx were detected only if SCORE index was elevated in healthy subjects or when the subjects were IgG-SARS positive (Table 4) and in patients with CHD. However, the data indicated that cGAMP was not associated with endothelial function in healthy subjects of cohort 1.

Severe Covid-19 infection may induce specific changes in typical responses of T and B cells manifested as a delay in over-response of T cells, which may contribute to functional exhaustion and premature senescence of T cells. These delays were hypothesized to contribute to pneumonitis and a delay in cytokine secretion. Similar patterns are observed in animal models and patients with STING hyperactivation that can be triggered only by indirect transfer of cGAMP from SARS-CoV-2-infected antigen-presenting cells because normal T cells do not express the main SARS-CoV receptor and are not infected by the virus [25]. However, these data are not particularly relevant to the present study because we did not include patients with current severe Covid-19.

A study in cell culture models demonstrated that infection with high doses of SARS-CoV-2 or fusion of the cells expressing SARS-CoV-2 spike protein with the cells expressing its main receptor ACE2 induces an increase in the levels of cytosolic chromatin, which is sensed by the cGAS-STING pathway, thus contributing to an antiviral response. Moreover, an STING activator was able to suppress viral replication in mice infected with the virus. However, these observations have not been extended to Covid-19 in patients [26]. We think that this scenario is not applicable for mild or asymptomatic Covid-19 since the scenario requires massive tissue damage and cell death associated with fusion or release of high amounts of dsDNA. These effects are secondary and are relevant to the cGAMP-STING pathway only in the most severe cases of Covid-19, and these cases have not been included in the present study.

These considerations are supported by a study in patients with long Covid-19 who had fibrotic changes in the lung tissue detected by chest CT scans. Comparison of peripheral blood mononuclear cells isolated from these patients with those from patients without fibrotic lesions in the lung indicated significant changes in the secretion of interferons. The authors concluded that these changes are not due to the differences in cGAMP signaling but are mediated by another pathway associated with activation of the Absent in melanoma-2 protein [27].

On the other hand, activation of the cGAMP-STING pathway by using cGAMP analogs, which directly stimulate STING, has been shown to provide nonspecific protection against infection by various RNA viruses in cell culture and animal models [28]. These results suggest that the cGAMP-STING pathway may be an important target for the development of antiviral agents with broad specificity to inhibit replication of RNA viruses. Currently, it is not known whether these mechanisms are relevant to actual inflammatory processes in human tissues during or after Covid-19. Nevertheless, these approaches to antiviral therapy are being explored to create nanoparticle-based formulations for control of severe respiratory infections in animal models [29] emphasizing critical roles of the pathway in nonspecific immune response.

Conclusions

Serum cGAMP level was similar in IgG-SARS(+) and IgG-SARS(−) healthy individuals of cohort 1. In contrast, serum levels of cGAMP were decreased in patients with coronary stenosis in cohort 2 compared with that in patients without coronary stenosis. Endothelial dysfunction was associated with cGAMP in CHD patients but endothelial function was not associated with cGAMP in healthy subjects. Thus, low serum cGAMP was associated with coronary stenosis in patients with advanced endothelial dysfunction.