| Background: Atherosclerosis is the important pathological basis for cardiovascular disease,and it has been reported that atherosclerotic cardiovascular disease(ASCVD)has become the leading cause of death worldwide.It is widely accepted that lipid infiltration theory is the pathogenesis of atherosclerosis.However,numerous experimental studies and many clinical observations have shown that hyperlipidemia alone is not sufficient to cause atherosclerosis unless inflammation is present.Therefore,atherosclerosis is considered as a chronic progressive process driven by a combination of lipid and inflammation.Research on atherosclerosis-related inflammatory mechanisms may contribute to looking for novel antiinflammatory agents for anti-atherosclerotic therapy,thus strengthening the defense against ASCVD.Although deoxyribonucleic acid(DNA)is essential as important genetic information in life,mis-localized self-DNA and damaged DNA,as well as DNA released from the dead cell can act as the trigger for endogenous inflammation,leading to the onset and development of atherosclerosis.For example,TLR9 and AIM2,which can be activated by DNA,have been confirmed to contribute to atherosclerosis by promoting inflammatory response.Cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS),as an important DNA sensor,was initially identified as a critical part of anti-infective immune defense.Nowadays,it has been found that by sensing mis-localized self-DNA,cGAS is also involved in a range of non-infectious settings including tumors,cellular senescence,and inflammatory diseases.Although the relationship between cGAS and atherosclerosis is not clear,studies have confirmed that interferon(IFN)signaling,downstream of cGAS,can bring a deleterious effect on atherosclerosis.Treatment with IFNβ can accelerate the plaque lesions in atherosclerotic mouse models and induce macrophages recruitment to lesions.Blocking IFN signaling can prevent the macrophages’ recruitment to lesions and the formation of necrotic cores.Also,cGAS-STING signaling has been demonstated to contribute to high-fat diet(HFD)-induced inflammation in both adipocytes and macrophages.It is knowledge that macrophages are of great importance in the initiation,progression,and regression of atherosclerosis and that different subtypes of macrophages affect the outcome of atherosclerosis.Classically,the pro-inflammatory M1 macrophage phenotype is responsible for atherosclerotic plaque vulnerability,whereas the antiinflammatory M2 macrophage phenotype can increase atherosclerotic plaque stability.It has been found that the cytoplasmic DNA of macrophages induces the expression of M1 markers as well as CXCL10 by activating cGAS,and knockdown of cGAS in THP-1 cells using CRISPR-Cas9 technology resulted in elevated expression of M2 markers including CD163,IL-10,and CCL17.Taken together,a speculation that cGAS contribute to atherosclerosis maybe reasonable.This study focused on exploring the relationship between cGAS and atherosclerosis as well as the possible underlying mechanisms.Objective:(1)To investigate whether cGAS is involved in atherosclerosis.(2)To investigate whether cGAS promotes macrophage-derived foam cell formation.(3)To investigate whether cGAS modulates the progression of atherosclerosis by mediating the macrophage inflammatory signaling pathway.Methods and Results: Part I:(1)Plasma was collected from people with or without atherosclerosis,and the levels of plasma ds DNA were measured by Quant-i T? Pico Green ? ds DNA quantification kit.In addition,Eight-week-old male Apo E-/-mice on a C57BL/6 background were fed a western diet(containing high fat)or a chow diet for 8 and 16 weeks,respectively.The levels of ds DNA in plasma were measured by Quant-i T? Pico Green ? ds DNA quantification kit.Results showed that human atherosclerotic plasma contained higher concentrations of ds DNA than those in people with non-atherosclerosis.Furthermore,ds DNA was detected in the plasma of Apo E-/-mice fed for both 8 and 16 weeks,and plasma ds DNA levels in mice fed for 16 weeks were significantly higher than that in mice fed for 8 weeks.(2)Apo E-/-male mice were kept on a western-type or chow diet for 16 weeks,respectively.Hearts and aortas were harvested for assessment of atherosclerotic burden.Immunofluorescence staining was used to analyze the DNA damage in plaque lesions.In accordance with previous findings,immunofluorescence revealed that 8-Oxo-2′-deoxyguanosine(8-OH-d G),a marker for oxidative damage to DNA,can be detected in the plaque of Apo E-/-mice,and strong 8-OH-d G staining was discovered mainly in mitochondria,irrespective of diet.(3)In the present study,the dataset GSE40156 from the Gene Expression Omnibus(GEO)was re-analyzed by the GEO2 R online tool and it was found that cGAS expressions in Apo E-/-mice were significantly increased with age.(4)The peripheral blood was collected from people with or without atherosclerosis,and the expression of cGAS was detected by q RT-PCR.Results displayed that the m RNA levels of cGAS in the peripheral blood of people with atherosclerosis were significantly higher than that of people with non-atherosclerosis.Part II:(1)Apo E-/-male mice were kept on a western-type or chow diet for 16 weeks,respectively.Hearts and aortas were harvested for assessment of atherosclerotic burden.Immunofluorescence staining,Oil Red O staining,BODIPY staining as well as HE staining were used to analyze the relationship between cGAS and plaque lesion.Results demonstrated that cGAS was expressed in the plaque of Apo E-/-mice regardless of diet,and was positively correlated with plaque area and lipid deposition,and mainly distributed in macrophages.whereas,the protein expression of cGAS in the aorta of Apo E-/-mice fed a western-type diet was higher than that of Apo E-/-mice fed a chow diet.(2)Raw264.7 macrophages were stimulated with cGAS inhibitor RU.521(2 ug/m L)for 12 h,and then incubated with ox-LDL(100 ug/m L)for 24 h,followed by Oil Red O and BODIPY staining.The results showed that inhibition of cGAS reduced macrophage foam cell formation and lipid deposition when compared with the control group(non-RU.521 intervention).(3)Raw264.7 macrophages were stimulated with cGAS inhibitor RU.521(2ug/m L)for 24 h,and then incubated with Dil-LDL for 2 h.The cholesterol uptake of Raw264.7 macrophages was observed by fluorescence staining.It was found that inhibition of cGAS could reduce the cholesterol uptake of macrophages.(4)After labeled with NBD-labeled cholesterol(5 umol/L)for 12 h,Raw264.7 macrophages were incubated with RU.521(2 μg/m L)for 24 h,followed by HDL(50 μg/m L)incubation for 6 h.The efflux was calculated according to the fluorescence intensity of NBD under 485 nm excitation and 535 nm emission.Results showed that inhibition of cGAS did not affect cholesterol efflux.Part III:(1)In order to have a better view of the mechanisms of atherosclerosis mediated by cGAS,we performed RNA-seq transcriptomic analysis of the underlying differentiallyexpressed genes(DEGs)and the mechanisms in Raw264.7 macrophages treated for 12 h with or without RU.521.The genes expressed were sequenced using the Illumina platform.DEGs between the cGAS inhibition group and the control group were detected.The differential expression was considered significant when a Padj < 0.05 and | log2 FC | ≥ 2.GO enrichment and KEGG pathway were obtained using DAVID online tool.At the same time,STRING was applied to obtain protein–protein interactions(PPI).Cytoscape and its plug-in MCODE were used to visualize the PPI networks and find top hub genes.A total of 275 differentially encoded protein genes related to atherosclerosis were identified,of which functional enrichments mainly occurred in the inflammatory response,immune response,and cytokinecytokine receptor interaction signaling pathways.Stat1,Irf7 were considered as the top hub genes with MCC method.Raw264.7 macrophages were stimulated with cGAS inhibitor RU.521(2 ug/m L)for 12 h,and then RNA was extracted.The m RNA levels of inflammationrelated factors were detected using q RT-PCR.It was found that the m RNA levels of proinflammatory factors such as Il1 b and Il18 were significantly down-regulated when cGAS inhibition,while m RNA levels of anti-inflammatory factors such as Il10,Arg1 as well as Il1 rn were significantly up-regulated.In addition,m RNA levels of Msr1 contribute to cholesterol uptake were also down-regulated when cGAS inhibition.Similarly,the m RNA levels of Stat1 and Irf7 were also significantly down-regulated when cGAS inhibition.(2)The dataset GSE57614 from the GEO was re-analyzed by the GEO2 R online tool and it was found that there was a significant increase in cGAS expression in the M1 phenotype of macrophages.After stimulation with cGAS inhibitor RU.521(2 ug/m L)for 12 h,Raw264.7 macrophages were continued to incubate for 6 h using LPS + IFNγ(10 ng/m L,20 ng/m L,macrophage M1 phenotype induction method).RNA was extracted,and q RTPCR was used to detect m RNA levels.Results showed elevated m RNA levels of macrophage M1 phenotype markers(Il1b,Il6,Tnfa,and Cd86)when inhibition of cGAS,compared with the control group.(3)Raw264.7 macrophages were stimulated with cGAS inhibitor RU.521(2 ug/m L)+ STAT1 enhancer 2-NP(10 u M/L)for 12 h,and q RT-PCR was used to detect m RNA levels of macrophage M1 phenotype markers.Compared with the control group(without STAT1 enhancer),stimulating STAT1 activity prevented the m RNA levels of M1 phenotype markers(Il1b,Il6,Tnfa and Cd86)induced by cGAS inhibition.Conclusion:(1)cGAS may promote the development of atherosclerosis by mediating macrophage cholesterol uptake.(2)Mechanistically,cGAS may mediate macrophage inflammatory response through STAT1,and promote M1 phenotype transformation,thus promoting the development of atherosclerosis.This study reveals the relationship between cGAS and atherosclerosis,and elucidates its mechanism,thus facilitating looking for novel anti-inflammatory therapy for atherosclerosis. |
