Integrated analysis of differentially m6A modified and expressed lncRNAs for biomarker identification in coronary artery disease.

N6-methyladenosine (m6A) is the most prevalent internal RNA modification in mammals. However, limited research has been conducted on the role of m6A in coronary artery disease (CAD). We conducted methylated RNA immunoprecipitation sequencing and RNA sequencing to obtain a genome-wide profile of m6A-modified long noncoding RNAs (lncRNAs) in human coronary artery smooth muscle cells either exposed to oxidized low-density lipoprotein treatment or not, and the characteristics of the expression profiles were explored using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The predictive effects of seven selected lncRNAs on CAD were evaluated in peripheral blood mononuclear cells (PBMCs). The differentially m6A-modified and expressed lncRNAs related genes were predominantly enriched in small GTPase-mediated signal transduction, ErbB signaling, and Rap1 signaling. Additionally, the expression levels of uc003pes.1, ENST00000422847, and NR_110155 were significantly associated with CAD, with uc003pes.1 identified as an independent risk factor and NR_110155 as an independent protective factor for CAD. NR_110155 and uc003pes.1 in PBMCs have the potential to serve as biomarkers for predicting CAD.

Exploration and bioinformatic prediction for profile of mRNA bound to circular RNA BTBD7_hsa_circ_0000563 in coronary artery disease.

BACKGROUND: As a novel circRNA, BTBD7_hsa_circ_0000563 has not been fully investigated in coronary artery disease (CAD). Our aim is to reveal the possible functional role and regulatory pathway of BTBD7_hsa_circ_0000563 in CAD via exploring genes combined with BTBD7_hsa_circ_0000563. METHODS: A total of 45 peripheral blood mononuclear cell (PBMC) samples of CAD patients were enrolled. The ChIRP-RNAseq assay was performed to directly explore genes bound to BTBD7_hsa_circ_0000563. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted to reveal possible functions of these genes. The interaction network was constructed by the STRING database and the Cytoscape software. The Cytoscape software were used again to identify clusters and hub genes of genes bound to BTBD7_hsa_circ_0000563. The target miRNAs of hub genes were predicted via online databases. RESULTS: In this study, a total of 221 mRNAs directly bound to BTBD7_hsa_circ_0000563 were identified in PBMCs of CAD patients via ChIRP-RNAseq. The functional enrichment analysis revealed that these mRNAs may participate in translation and necroptosis. Moreover, the interaction network showed that there may be a close relationship between these mRNAs. Eight clusters can be further subdivided from the interaction network. RPS3 and RPSA were identified as hub genes and hsa-miR-493-5p was predicted to be the target miRNA of RPS3. CONCLUSIONS: BTBD7_hsa_circ_0000563 and mRNAs directly bound to it may influence the initiation and progression of CAD, among which RPS3 and RPSA may be hub genes. These findings may provide innovative ideas for further research on CAD.

Circular RNA ZBTB46 depletion alleviates the progression of Atherosclerosis by regulating the ubiquitination and degradation of hnRNPA2B1 via the AKT/mTOR pathway.

BACKGROUND: CircZBTB46 has been identified as being associated with the risk of coronary artery disease (CAD) and has the potential to be a diagnostic biomarker for CAD. However, the specific function and detailed mechanism of circZBTB46 in CAD are still unknown. METHODS: The expression levels and properties of circRNAs were examined using qRT‒PCR, RNA FISH, and subcellular localization analysis. ApoE-/- mice fed a high-fat diet were used to establish an atherosclerosis model. HE, Masson, and Oil Red O staining were used to analyze the morphological features of the plaque. CCK-8, Transwell, and wound healing assays, and flow cytometric analysis were used to evaluate cell proliferation, migration, and apoptosis. RNA pull-down, silver staining, mass spectrometry analysis, and RNA-binding protein immunoprecipitation (RIP) were performed to identify the interacting proteins of circZBTB46. RESULTS: CircZBTB46 is highly conserved and is significantly upregulated in atherosclerotic lesions. Functional studies revealed that knockdown of circZBTB46 significantly decreased the atherosclerotic plaque area, attenuating the progression of atherosclerosis. In addition, silencing circZBTB46 inhibited cell proliferation and migration and induced apoptosis. Mechanistically, circZBTB46 physically interacted with hnRNPA2B1 and suppressed its degradation, thereby regulating cell functions and the formation of aortic atherosclerotic plaques. Additionally, circZBTB46 was identified as a functional mediator of PTEN-dependent regulation of the AKT/mTOR signaling pathway and thus affected cell proliferation and migration and induced apoptosis. CONCLUSION: Our study provides the first direct evidence that circZBTB46 functions as an important regulatory molecule for CAD progression by interacting with hnRNPA2B1 and regulating the PTEN/AKT/mTOR pathway.

Long non-coding RNA in coronary artery disease: the role of PDXDC1-AS1 and SFI1-AS1.

This study investigates the interaction between long non-coding RNAs (lncRNAs) and metabolic risk factors that contribute to coronary artery disease (CAD). A total transcriptome high throughput sequencing study was conducted on peripheral blood mononuclear cells from five patients with CAD and five healthy controls. Validation assay by qRT-PCR was conducted among 270 patients and 47 controls. Finally, to evaluate the lncRNAs' diagnostic value for CAD, the Spearman correlation test and receiver operating characteristic curve (ROC) analysis were utilized. Additionally, univariate and multivariate logistic regression along with crossover analyses were conducted to identify the interaction between lncRNA and environmental risk factors. A total of 2149 of 26,027 lncRNAs identified by RNA sequencing were differentially expressed in CAD patients compared to controls. Validation by qRT-PCR showed significantly different relative expression levels for lncRNAs PDXDC1-AS1, SFI1-AS1, RP13-143G15.3, DAPK1-IT1, PPIE-AS1, and RP11-362A1.1 between the two groups (all P<0.05). The area under the ROC values of PDXDC1-AS1 and SFI1-AS1 is 0.645 (sensitivity=0.443 and specificity=0.920) and 0.629 (sensitivity=0.571 and specificity=0.909), especially. Multivariate logistic regression analyses showed that lncRNAs PDXDC1-AS1 (OR=2.285, 95%CI=1.390-3.754, p=0.001) and SFI1-AS1 (OR=1.163, 95%CI=1.163-2.264, p=0.004) were protective factors against CAD. Under the additive model, cross-over analyses demonstrated significant interactions between lncRNAs PDXDC1-AS1 and smoking in relation to CAD risk (S=3.871, 95%CI=1.140-6.599). PDXDC1-AS1 and SFI1-AS1 were sensitive and specific biomarkers for CAD and exhibited synergistic effects with certain environmental factors. These results highlighted their potential use as CAD diagnostic biomarkers for future research.

Interaction between the expression of hsa_circRPRD1A and hsa_circHERPUD2 and classical coronary risk factors promotes the development of coronary artery disease.

BACKGROUND: Recent studies suggest that classical coronary risk factors play a significant role in the pathogenesis of coronary artery disease. Our study aims to explore the interaction of circRNA with classical coronary risk factors in coronary atherosclerotic disease. METHOD: Combined analysis of RNA sequencing results from coronary segments and peripheral blood mononuclear cells of patients with coronary atherosclerotic disease was employed to identify critical circRNAs. Competing endogenous RNA networks were constructed by miRanda-3.3a and TargetScan7.0. The relative expression quantity of circRNA in peripheral blood mononuclear cells was determined by qRT-PCR in a large cohort including 256 patients and 49 controls. Spearman's correlation test, receiver operating characteristic curve analysis, multivariable logistic regression analysis, one-way analysis of variance, and crossover analysis were performed. RESULTS: A total of 34 circRNAs were entered into our study, hsa_circRPRD1A, hsa_circHERPUD2, hsa_circLMBR1, and hsa_circDHTKD1 were selected for further investigation. A circRNA-miRNA-mRNA network is composed of 20 microRNAs and 66 mRNAs. The expression of hsa_circRPRD1A (P = 0.004) and hsa_circHERPUD2 (P = 0.003) were significantly down-regulated in patients with coronary artery disease compared to controls. The area under the curve of hsa_circRPRD1A and hsa_circHERPUD2 is 0.689 and 0.662, respectively. Univariate and multivariable logistic regression analyses identified hsa_circRPRD1A (OR = 0.613, 95%CI:0.380-0.987, P = 0.044) as a protective factor for coronary artery disease. Based on the additive model, crossover analysis demonstrated that there was an antagonistic interaction between the expression of hsa_circHERPUD2 and alcohol consumption in subjects with coronary artery disease. CONCLUSION: Our findings imply that hsa_circRPRD1A and hsa_circHERPUD2 could be used as biomarkers for the diagnosis of coronary artery disease and provide epidemiological support for the interactions between circRNAs and classical coronary risk factors.

Detailed profiling of m6A modified circRNAs and synergistic effects of circRNA and environmental risk factors for coronary artery disease.

The modification of N6-methyladenosine (m6A) modification is implicated in human diseases. However, considerable uncertainty is associated with the regulatory mechanisms of m6A circRNAs in coronary artery disease (CAD), which require further clarification. In this study, m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) was conducted to investigate m6A-modified circRNAs in human coronary artery smooth muscle cells (HCASMCs) and to identify potential biomarkers for CAD. A total of 830 and 331 up- and down-regulated m6A peaks, (corresponding to 463 and 243 up- and down-regulated circRNAs, respectively), were identified in HCASMCs in a pathological condition. Functional analysis suggested that these circRNAs appeared to participate in intracellular protein, histone deacetylase complex, ATP-dependent activity, autophagy, and AMPK signaling pathway. Four candidate circRNAs were selected for further evaluation in HCASMCs and human samples. The results suggested that hsa_circHECTD1 and hsa_circZBTB46 were significantly increased in patients with CAD (p-value = 0.039 and p-value = 0.014) and may act as potential diagnostic biomarkers of CAD. Furthermore, statistical results showed that hsa_circHECTD1 and hsa_circSEC62 were positively correlated with triglyceride (TG) (r = 0.213, p-value = 0.014) and Gensini Score (used to quantify the severity of CAD) (r = 0.349, p-value <0.001), respectively. Logistic regression revealed that hsa_circZBTB46 was strongly correlated with the incidence of CAD, and the synergistic effects of circRNAs and hypertension enhanced the risk of CAD. These results show that hsa_circHECTD1 and hsa_circZBTB46 may be new targets for further studies, and this study enhances our understanding of the effects of m6A-circRNAs on the pathogenesis of CAD.

Combination of D-dimer level and neutrophil to lymphocyte ratio predicts long-term clinical outcomes in acute coronary syndrome after percutaneous coronary intervention.

BACKGROUND: High D-dimer (DD) is associated with short-term adverse outcomes in patients with acute coronary syndrome (ACS). In ACS patients who underwent percutaneous coronary intervention (PCI), however, the value of DD (or combined with neutrophil to lymphocyte ratio [NLR]) to predict long-term major adverse cardiovascular events (MACEs) has not been fully evaluated. METHODS: Patients diagnosed with ACS and receiving PCI were included. The primary outcome was MACEs. Cox proportional hazards regression and logistic regression were used to illustrate the relationship between clinical risk factors, biomarkers and MACEs. Survival models were developed based on significant factors and evaluated by the Concordance-index (C-index). RESULTS: The final study cohort was comprised of 650 patients (median age, 64 years; 474 males), including 98 (15%) with MACEs during a median follow-up period of 40 months. According to the cut-off value of DD and NLR, the patients were separated into four groups: high DD or nonhigh DD with high or nonhigh NLR. After adjusting for confounding variables, DD (adjusted hazard ratio [aHR]: 2.39, 95% confidence interval [CI]: 1.52-3.76) and NLR (aHR: 2.71, 95% CI: 1.78-4.11) were independently associated with long-term MACEs. Moreover, patients with both high DD and NLR had a significantly higher risk in MACEs when considering patients with nonhigh DD and NLR as reference (aHR: 6.19, 95% CI: 3.30-11.61). The area under curve increased and reached 0.70 in differentiating long-term MACEs when DD and NLR were combined, and survival models incorporating the two exhibited a stronger predictive power (C-index: 0.75). CONCLUSIONS: D-dimer (or combined with NLR) can be used to predict long-term MACEs in ACS patients undergoing PCI.

Identification of circular RNA BTBD7_hsa_circ_0000563 as a novel biomarker for coronary artery disease and the functional discovery of BTBD7_hsa_circ_0000563 based on peripheral blood mononuclear cells: a case control study.

BACKGROUND: BTBD7_hsa_circ_0000563 is a novel circRNA and contains conserved binding sites with RNA-binding proteins. However, BTBD7_hsa_circ_0000563 has not been fully studied in coronary artery disease (CAD). We aimed to clarify the diagnostic value and the possible functional role of BTBD7_hsa_circ_0000563 in CAD. METHODS: A total of 276 human peripheral blood mononuclear cell (PBMC) samples were employed. The circularization of BTBD7_hsa_circ_0000563 was verified via Sanger sequencing. The expression level of BTBD7_hsa_circ_0000563 in CAD samples and control individuals was analysed via qRT-PCR. The diagnostic potential of BTBD7_hsa_circ_0000563 was evaluated using Spearman's analysis, univariate and multivariable logistic regression analysis, and receiver-operator characteristic (ROC) curve analysis. ChIRP-MS was performed to directly explore the proteins bound to BTBD7_hsa_circ_0000563. Bioinformatic analysis was conducted to investigate the possible functions and interactions of proteins bound to BTBD7_hsa_circ_0000563. RESULTS: In the present study, BTBD7_hsa_circ_0000563 was verified as a circular RNA in the PBMCs of CAD patients. The expression level of BTBD7_hsa_circ_0000563 in the CAD group was significantly lower than that in the control group. The area under the ROC curve was 0.690. ChIRP-MS found seven proteins that were directly bound to BTBD7_hsa_circ_0000563. Bioinformatic analysis of these seven proteins showed that the mitophagy and DNA repair pathways were enriched. These proteins interacted with each other to a certain extent. CONCLUSION: BTBD7_hsa_circ_0000563 may be a novel biomarker for the diagnosis of CAD and may influence the initiation and progression of CAD. These studies may reveal new possibilities for the diagnosis and treatment of CAD.

Exploration of N6-Methyladenosine Profiles of mRNAs and the Function of METTL3 in Atherosclerosis.

OBJECTIVES: N6-methylladenosine (m6A) modification has not been fully studied in atherosclerosis. The objectives of this study were to investigate differentially expressed m6A methylated peaks and mRNAs, along with the regulatory role of methyltransferase 3 (METTL3) in pathological processes of atherosclerosis. METHODS: The pathological models of human coronary artery smooth muscle cells (HCASMCs) were induced in vitro. The differentially expressed mRNAs and m6A peaks were identified by RNA-Seq and meRIP-Seq. The potential mechanisms were analyzed via bioinformatic assays. Methylases expression was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) in HCASMCs, and by immunohistochemical assays in 40 human coronary arteries. The knockdown of METTL3 expression in cells was performed by siRNA transfection, and cell proliferation and migration were detected after transfection. RESULTS: We identified 5121 m6A peaks and 883 mRNAs that were expressed differentially in the pathological processes of HCASMCs. Bioinformatic analyses showed that the different m6A peaks were associated with cell growth and cell adhesion, and the 883 genes showed that the extracellular matrix and PI3K/AKT pathway regulate the processes of HCASMCs. Additionally, 10 hub genes and 351 mRNAs with differential methylation and expression levels were found. METTL3 was upregulated in the arteries with atherosclerotic lesions and in the proliferation and migration model of HCASMCs, and pathological processes of HCASMCs could be inhibited by the knockdown of METTL3. The mechanisms behind regulation of migration and proliferation reduced by siMETTL3 are concerned with protein synthesis and energy metabolism. CONCLUSIONS: These results revealed a new m6A epigenetic method to regulate the progress of atherosclerosis, which suggest approaches for potential therapeutic interventions that target METTL3 for the prevention and treatment of coronary artery diseases.

Circular RNA expression profile of H2O2 induced ferroptosis model of human coronary artery endothelial cells.

Background and aims: Coronary artery disease (CAD) is among the most common type of cardiovascular diseases. The role of circular RNAs (circRNAs) and ferroptosis in CAD remains largely unexplored. The aim of this study was to evaluate the circRNAs expression profile in ferroptosis of human coronary artery endothelial cells (HCAECs). Methods: The ferroptosis induced by H2O2-stimulated oxidative stress in HCAECs and the role of Ferrostatin-1 (Fer-1) was assessed by the levels of CCK8, oxidized and reduced glutathione (GSSH and GSH), ferrous irons, lactate dehydrogenase (LDH), malondialdehyde (MDA), lipid reactive oxygen species (Lipid ROS), PTGS2 and GPX4. The expression profile of circRNAs was characterized by RNA sequencing. Results: LDH, MDA, Lipid ROS, ferrous ions, GSSH and PTGS2 were significantly increased, CCK8, GSH and GPX4 were significantly decreased in H2O2 induced cell damage. Moreover, Fer-1 increased CCK8, GSH and GPX4 levels and decreased LDH, MDA, Lipid ROS, GSSH and PTGS2 levels, which alleviated H2O2 induced cell damage. The circRNA-miRNA-mRNA network and circRNA-protein interactions network were constructed based on differentially expressed circRNAs. In total, 17 downregulated and 18 upregulated circRNAs were identified in H2O2 treated HCAECs by RNA sequencing. Parental genes of circRNAs were analyzed by KEGG and GO, detecting pathways related to ferroptosis. 10 differentially expressed circRNAs were verified by qRT-PCR. Conclusions: These results provide new sight to the character of circRNAs in the progress of HCAECs ferroptosis and contribute a significant data for further investigating the potential mechanisms of CAD.

Comprehensive Analysis of mRNA Expression Profiling and Identification of Potential Diagnostic Biomarkers in Coronary Artery Disease.

The aim of this study is to investigate mRNA expression profiling by RNA sequencing (RNA-seq) in patients with coronary artery disease (CAD) and validate differentially expressed genes (DEGs) as novel biomarkers for CAD. Transcriptome-wide mRNA expression analysis of peripheral blood mononuclear cells was performed in five CAD patients and five controls. Functional enrichment analyses, protein-protein interaction network construction, and hub gene selection were further conducted. Relative expression levels of hub genes were validated by quantitative reverse transcription PCR in larger cohorts. Spearman correlation test and multiple linear regression analysis were applied to examine the relationship between confounding factors with severity of coronary artery atherosclerosis. Receiver operating characteristic (ROC) curve analysis was adopted to identify potentially diagnostic biomarkers for CAD. A total of 527 upregulated and 653 downregulated mRNAs were identified as DEGs in CAD patients. The relative expression levels of beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), F-box and leucine-rich repeat protein 4 (FBXL4), ubiquitin conjugating enzyme E2 D2 (UBE2D2), and ankyrin repeat and SOCS box containing 1 (ASB1) were significantly different between two groups (all p ≤ 0.05). The severity of coronary artery atherosclerosis was negatively associated with the BTRC gene relative expression level (r = -0.323, p < 0.001) and positively with UBE2D2 (r = 0.285, p < 0.001). ROC analysis of BTRC and UBE2D2 genes showed that the areas under the curve were 0.782 (95% CI: 0.720-0.845, p < 0.001) and 0.753 (95% CI: 0.681-0.824, p < 0.001), respectively. We described the characteristics of mRNA expression in the peripheral blood of CAD patients and controls by RNA-seq. Combined with Spearman correlation analysis and ROC analyses, BTRC and UBE2D2 genes had significantly diagnostic values, which may have potential to act as novel diagnostic biomarkers and therapeutic targets for CAD.

Shexiang Baoxin Pills Inhibited Proliferation and Migration of Human Coronary Artery Smooth Muscle Cells via PI3K/AKT/mTOR Pathway.

Background: Proliferation and migration of smooth muscle cells in the coronary artery contribute to the deterioration of coronary artery disease (CAD). Aim: This research was designed to study the function of Shexiang Baoxin pills (SBPs) on the proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and their mechanism. Methods: Oxidized low-density lipoprotein (ox-LDL) was applied to stimulate the proliferation and migration of HCASMCs. The function of ox-LDL and SBP on HCASMCs was evidenced by the cell counting kit-8 assay, cell cycle, and Transwell assay. Network pharmacology was employed to predict the potential targets and pathways of SBP on CAD. Western blot assay and molecular docking were conducted to validate the potential targets and pathways. Results: The current research revealed that 2.5 mg/L SBP significantly inhibited the proliferation and migration of HCASMCs. Besides, network pharmacology revealed 11 candidate targets. Molecular docking and Western blot assay validated that the activation of the top 2 targets STAT3 and MAPK14 was associated with the inhibition of HCASMCs. Moreover, the Western blot assay also detected that HCASMCs treated with ox-LDL promoted the phosphorylation of the PI3K/AKT/mTOR pathway, and SBP inhibited the activation of the PI3K/AKT/mTOR pathway in HCASMCs stimulated by ox-LDL. Conclusion: This study demonstrated that the treatment of CAD using SBP may result from the suppression of the proliferation and migration of HCASMCs. The mechanism of this function partly resulted from relieving the phosphorylation of targets STAT3 and MAPK14 and the PI3K/AKT/mTOR pathway. This study enhanced our comprehension of SBP and provides new targets for the treatment of CAD.

Effects of Bivalirudin and Unfractionated Heparin on Liver and Renal Function in Chinese Patients with Coronary Artery Disease Undergoing Coronary Angiography with/without Percutaneous Coronary Intervention.

BACKGROUND AND AIMS: Unfractionated heparin (UFH) and bivalirudin are widely used as anticoagulants in cardiovascular medicine, including for thrombosis prevention during coronary angiography (CAG) and percutaneous coronary intervention (PCI). Little is known of the effects of UFH and bivalirudin on liver and kidney function in patients subjected to these procedures. This study compared the effects of bivalirudin and UFH on liver/renal function in patients with coronary artery disease who underwent CAG, with or without PCI. METHODS: The study comprised 134 consecutive patients (40-89 years-old), who underwent CAG (or CAG and PCI); among them, 66 and 68 patients were subject to, respectively, bivalirudin or UFH. The following indicators of liver/renal function were measured before and after the procedures: plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen, estimated glomerular filtration rate (eGFR), creatinine clearance, and serum creatinine. Patients were further stratified by severity of chronic kidney disease (CKD), based on original eGFR. RESULTS: Relative to baseline, in the bivalirudin group, ALT and AST were higher after CAG (p=0.005, 0.025), while blood urea nitrogen and serum creatinine were lower (p=0.049, <0.001). In the UFH group, ALT, AST, eGFR, and creatinine clearance were lower after CAG (p≤0.001, all). Patients given bivalirudin with moderate or severe CKD, but not those with mild CKD, gained significant improvement in kidney function. CONCLUSIONS: Relative to UFH, bivalirudin may better safeguard the renal function of patients with coronary artery disease who undergo CAG, especially patients with moderate-to-severe renal insufficiency. UFH may cause less liver damage than bivalirudin.

Characteristics of circular RNAs expression of peripheral blood mononuclear cells in humans with coronary artery disease.

Circular RNAs (circRNAs) function as promising biomarkers and therapeutic targets for coronary artery disease due to their high stability, covalently closed structure, and potential gene regulation. We aimed to identify the expression profile and role of circular RNAs (circRNAs) in coronary artery disease (CAD). We performed RNA sequence analysis of circRNAs in peripheral blood mononuclear cells of five patients with CAD and five controls. Bioinformatics analyses were adopted to explore biological functions of differentially expressed circRNAs. The miRanda and TargetScan tools were used to predict the microRNA (miRNA)-targeting interactions and to construct a triple network of differentially expressed gene-circRNA-miRNA-mRNA. In total, 13,160 downregulated and 12,905 upregulated circRNAs were identified in CAD. A gene ontology annotation analysis showed that genes in the network were involved in organelle organization, cell cycle, mitotic cycle, and cellular metabolic process. Parental genes of the 10 dysregulated circRNAs were involved in metabolism and protein modification, and these circRNAs might regulate gene expression associated with CAD via miRNA sponges. As potential competing endogenous RNAs (ceRNAs), dysregulated circRNAs may be involved in the pathogenesis of CAD, which provides new insights into the diagnosis and prognosis of coronary artery disease.

LncRNA Landscape of Coronary Atherosclerosis Reveals Differentially Expressed LncRNAs in Proliferation and Migration of Coronary Artery Smooth Muscle Cells.

We aimed to investigate differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in atherosclerosis and validate the expression of lncRNAs and co-expressed target genes in proliferation and migration models of human coronary artery smooth muscle cells (HCASMCs). Ten coronary artery specimens from a subject who died from a heart attack were employed. The pathological analysis was analyzed by hematoxylin and eosin (H&E) staining, and the lncRNAs and mRNAs were identified by RNA sequencing. Bioinformatic analyses were performed to predict possible mechanisms. The proliferation and migration of HCASMCs were induced with oxidized low-density lipoprotein (ox-LDL). Differentially expressed lncRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, 68 lncRNAs and 222 mRNAs were identified differentially expressed in atherosclerosis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the Fanconi anemia pathway may be involved in atherosclerosis. GON4L was found to be the co-localized target gene of LNC_000439, and 14 genes had high correlations with the expression of seven lncRNAs. In addition, nine lncRNA-miRNA-mRNA networks were constructed, and 53 co-expressed gene modules were detected with weighted gene co-expression network analysis (WGCNA). LNC_000684, LNC_001046, LNC_001333, LNC_001538, and LNC_002115 were downregulated, while LNC_002936 was upregulated in proliferation and migration models of HCASMCs. In total, six co-expressed mRNAs were upregulated in HCASMCs. This study suggests that the differentially expressed lncRNAs identified by RNA sequencing and validated in smooth muscle cells may be a target for regulating HCASMC proliferation and migration in atherosclerosis, which will provide a new diagnostic basis and therapeutic target for the treatment of cardiovascular diseases.

Verification of ferroptosis and pyroptosis and identification of PTGS2 as the hub gene in human coronary artery atherosclerosis.

Ferroptosis and pyroptosis have not been fully studied in atherosclerosis. We aimed to investigate the expression of ferroptosis-related and pyroptosis-related proteins in human coronary arteries and analyse correlation with severity of atherosclerosis and clarify the interactions between proteins and possible mechanisms of atherosclerosis. 40 human coronary artery specimens were employed. The atherosclerotic lesions were characterized by Haematoxylin and Eosin (H&E) staining. The expression of prostaglandin-endoperoxide synthase 2 (PTGS2), anti-acyl-CoA synthetase long-chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), caspase-1, and NOD-like receptor protein 3 (NLRP3) were analysed by immunohistochemical assay. Correlations between expression of proteins and severity of atherosclerosis were assessed using Spearman correlation analysis. Bioinformatic and coexpression analyses were performed to study the possible pathways and interactions. In the present study, PTGS2, ACSL4, caspase-1, and NLRP3, were upregulated, while GPX4 was downregulated in the advanced stages of atherosclerosis. The severity of atherosclerosis was positively associated with the expression of PTGS2, ACSL4, caspase-1, and NLRP3 and negatively associated with the expression of GPX4. Biological processes of lipid metabolism and inflammation and C-type lectin receptor signaling pathway were enriched. The five proteins interacted with each other directly or indirectly and PTGS2 might be the hub gene of atherosclerosis. Ferroptosis and pyroptosis may regulate the occurrence and development of atherosclerosis. These findings may shed light on new ideas and potential targets for the prevention and treatment of coronary artery atherosclerosis and the proteins may be used as biomarkers for the severity of atherosclerosis.

The epidemiological characteristics of cluster transmission of coronavirus disease 2019 (COVID-19): a multi-center study in Jiangsu Province.

Coronavirus Disease 2019 (COVID-19) pandemic has rapidly spread across the globe while little multi-center research about the epidemiological characteristics of cluster transmission is conducted. To provide a more comprehensive description of the epidemiological characteristics of cluster transmission and the virulence of SARS-CoV-2 carried by asymptomatic carriers, we studied the epidemiological characteristics of 70 clusters. 70 clusters including 311 consecutive subjects from January 20, 2020, to March 10, 2020, were enrolled. Of 70 clusters, 5 were infected by asymptomatic or presymptomatic carriers. We gathered and analyzed information about their demographic, epidemiological, clinical, diagnostic classification, and cluster characteristics. Among the 66 asymptomatic carriers in Jiangsu Province, 49 asymptomatic were observed in 311 subjects distributed in 70 clusters. We demonstrated that there is a significance between the severity of cases infected by asymptomatic carriers and cases infected by symptomatic patients (P=0.033) and the former usually presented with milder symptoms. A significant difference was shown regarding the level distribution of age (P=0.006) and the frequency distribution of gender (P=0.014) and disease severity of COVID-19 (P=0.008) among the seven groups classified by the relationship with the index cases. The average age of infected medical staff was the youngest and the majority of infected medical are females while the infected patients were generally oldest and usually accompanied by severest symptoms. We concluded that asymptomatic carriers are mainly screened out of clusters and the patients infected by asymptomatic carriers present with milder symptoms than those infected by symptomatic patients, which indicated that the SARS-CoV-2 shares decreased virulence among asymptomatic carriers. Effective measures should be taken to prevent transmission in hospitals to protect doctors, nurses, and patients.

Clinical characteristics of asymptomatic carriers of novel coronavirus disease 2019: A multi-center study in Jiangsu Province.

Asymptomatic SARS-CoV-2-infected individuals are thought to play major roles in virus transmission. This study aimed to analyze the characteristics of asymptomatic carriers with COVID-19 to control the spread of the virus. We retrospectively investigated the clinical characteristics of 648 consecutive subjects who were enrolled in the study and were divided into asymptomatic carriers, mild cases, ordinary cases, severe or critical cases, and evaluated their impact on disease severity by means of Spearman correlation and multiple regression analyses. Receiver operating characteristic curve analysis was conducted to determine the optimum cutoff levels of laboratory findings for diagnostic predictors of asymptomatic carriers of COVID-19. In our study, a total of 648 subjects on admission with a mean age of 45.61 y including 345 males and 303 females were enrolled in our study. The leukocyte, lymphocyte, eosinophil, platelet, C-reactive protein, interleukin-6, CD3+, CD4+, and CD8 + T lymphocyte levels, and the erythrocyte sedimentation rate differed significantly among the groups (all p ≤ 0.05). Disease severity was negatively associated with the CD3+ (r = -0.340; p < 0.001), CD4+ (r = -0.290; p = 0.001) and CD8+ (r = -0.322; p < 0.001) T lymphocyte levels. The significant diagnostic predictors of asymptomatic carriers of COVID-19 included the blood cell, cytokine, and T lymphocyte subset levels. Inflammation and immune response may play important roles in disease progression. Hence, the laboratory parameters identified should be considered in clinical practice, which provide new insights into the identification of asymptomatic individuals and the prevention of virus transmission.

Association between circular RNA expression content and severity of coronary atherosclerosis in human coronary artery.

BACKGROUND: Circular RNAs (circRNAs) may act as biomarkers of coronary artery disease (CAD). However, the relationship between expression characteristics of circRNAs and coronary atherosclerosis has not been fully explored. The aim of this study was to determine and characterize the circRNAs from human coronary artery. METHODS: The coronary artery segments were obtained from an 81-year-old male patient with sudden death of myocardial infarction at autopsy. The coronary stenosis and atherosclerosis were evaluated by hematoxylin and eosin (H&E) staining, and the circRNAs expression profile was characterized by RNA sequencing (RNA-seq). The differentially expressed circRNAs were validated by qRT-PCR. RESULTS: The analysis of H&E staining indicated that coronary atherosclerosis grade and extent in the LM was more serious than that in other coronary arteries. Twenty-seven circRNAs were selected for expression validation in coronary artery. CircRNAs corresponding cyclization sites of 3 circRNAs (hsa_circ_0016868, hsa_circ_0001364, hsa_circ_0006731) have been verified by Sanger sequencing. CONCLUSION: The 3 circRNAs are suggested to play a pathological role underlying the coronary arteries atherosclerosis and may serve as a valuable resource as diagnostic or therapeutic targets against CAD.