Microarray profiling analysis and validation of novel long noncoding RNAs and mRNAs as potential biomarkers and their functions in atherosclerosis.

Long noncoding (lnc)RNAs have been implicated in the development and progression of atherosclerosis. However, the expression and mechanism of action of lncRNAs in atherosclerosis are still unclear. We implemented microarray analysis in human advanced atherosclerotic plaques and normal arterial intimae to detect the lncRNA and mRNA expression profile. Gene Ontology functional enrichment and pathway analyses were applied to explore the potential functions and pathways involved in the pathogenesis of atherosclerosis. A total of 236 lncRNAs and 488 mRNAs were selected for further Ingenuity Pathway Analysis. Moreover, quantitative RT-PCR tests of most selected lncRNAs and mRNAs with high fold changes were consistent with the microarray data. We also performed ELISA to investigate the corresponding proteins levels of selected genes and showed that serum levels of SPP1, CD36, ATP6V0D2, CHI3L1, MYH11, and BDNF were differentially expressed in patients with coronary heart disease compared with healthy subjects. These proteins correlated with some biochemical parameters used in the diagnosis of cardiovascular diseases. Furthermore, receiver operating characteristic analysis showed a favorable diagnostic performance. The microarray profiling analysis and validation of differentially-expressed lncRNAs and mRNAs in atherosclerosis not only provide new insights into the pathogenesis of this disease but may also reveal new biomarkers for its diagnosis and treatment.

LncRNA AC096664.3/PPAR-γ/ABCG1-dependent signal transduction pathway contributes to the regulation of cholesterol homeostasis.

Atherosclerosis is a complex inflammatory disease that involves disrupted cellular cholesterol levels and formation of foam cells. Studies about long noncoding RNA (lncRNA) have revealed its function in the development of atherosclerosis, by mediating reverse cholesterol transport and formation of foam cells. In this study, we found that oxidized low-density lipoprotein (ox-LDL) markedly decreased lncRNA AC096664.3 in vascular smooth muscle cells (VSMCs) and THP-1 macrophages. We also found that ox-LDL reduced ATP-binding cassette (ABC) G1 through inhibiting lncRNA AC096664.3 in VSMCs. Further experiments showed that the downregulation of lncRNA AC096664.3 reduced ABCG1 expression through inhibiting the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and that ox-LDL reduced ABCG1 expression through inhibiting the expression of PPAR-γ. Furthermore, we discovered that ox-LDL inhibited ABCG1 via the lncRNA AC096664.3/PPAR-γ/ABCG1 pathway, which led to an increase in total and free cholesterol in VMSCs. Thus, we confirmed that ox-LDL induces cholesterol accumulation via the lncRNA AC096664.3/PPAR-γ/ABCG1 pathway in VSMCs, indicating a promising novel therapy in protecting against atherosclerosis.

LncRNA-RP11-714G18.1 suppresses vascular cell migration via directly targeting LRP2BP.

Atherosclerotic cardiovascular disease is considered as the leading cause of mortality and morbidity worldwide. Accumulating evidence supports an important role for long noncoding RNA (lncRNA) in the pathogenesis of atherosclerosis. Nevertheless, the role of lncRNA in atherosclerosis-associated vascular dysfunction and the underlying mechanism remain elusive. Here, using microarray analysis, we identified a novel lncRNA RP11-714G18.1 with significant reduced expression in human advanced atherosclerotic plaque tissues. We demonstrated in both human vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) that RP11-714G18.1 impaired cell migration, reduced the adhesion of ECs to monocytes, suppressed the neoangiogenesis, decreased apoptosis of VSMCs and promoted nitric oxide production. Mechanistically, RP11-714G18.1 could directly bind to its nearby gene LRP2BP and increased the expression of LRP2BP. Moreover, we showed that RP11-714G18.1 impaired cell migration through LRP2BP-mediated downregulation of matrix metalloproteinase (MMP)1 in both ECs and VSMCs. In atherosclerotic patients, the serum levels of LRP2BP were positively correlated with high-density lipoprotein cholesterol, but negatively correlated with cardiac troponin I. Our study suggests that RP11-714G18.1 may play an athero-protective role by inhibiting vascular cell migration via RP11-714G18.1/LRP2BP/MMP1 signaling pathway, and targeting the pathway may provide new therapeutic approaches for atherosclerosis.

Atorvastatin inhibits pyroptosis through the lncRNA NEXN-AS1/NEXN pathway in human vascular endothelial cells.

BACKGROUND AND AIMS: Many clinical trials have demonstrated that statins convey protective effects against atherosclerosis independent of cholesterol-lowering capacities. Other evidence indicates that pyroptosis, a type of programmed cell death, is likely involved in atherosclerosis, but the effects and mechanisms of statins on pyroptosis must be further revealed. METHODS: Here, we explored the effects and mechanisms of atorvastatin on pyroptosis in human vascular endothelial cells by quantitative real-time polymerase chain reaction and Western blot analyses. RESULTS: Atorvastatin upregulated long non-coding RNA (lncRNA) NEXN-AS1 and the expression of NEXN at both the mRNA and protein levels in a concentration- and time-dependent manner. Atorvastatin inhibited pyroptosis by decreasing the expression levels of the canonical inflammasome pathway biomarkers NLRP3, caspase-1, GSDMD, IL-1ß, and IL-18 at both the mRNA and protein levels. The promotion effects of atorvastatin on NEXN-AS1 and NEXN expression could be significantly abolished by knockdown of lncRNA NEXN-AS1 or NEXN, and its inhibitory effects on pyroptosis were also markedly offset by knock-down of lncRNA NEXN-AS1 or interference of NEXN. CONCLUSIONS: These results demonstrated that atorvastatin regulated pyroptosis via the lncRNA NEXN-AS1-NEXN pathway, which provides a new insight into the mechanism of how atorvastatin promotes non-lipid-lower effects against the development of atherosclerosis and gives new directions on how to reverse atherosclerosis.

Inhibitory Effect of Two Traditional Chinese Medicine Monomers, Berberine and Matrine, on the Quorum Sensing System of Antimicrobial-Resistant Escherichia coli.

The quorum sensing (QS) system controls bacterial biofilm formation, which is highly related to the virulence and resistance of pathogens. In the present study, the effect of two traditional Chinese medicine (TCM) monomers, berberine and matrine, on biofilm formation and QS-related gene expression of antimicrobial-resistant (AMR) Escherichia coli strains was investigated by laser scanning confocal microscopy (LSCM) observation and real-time PCR. The results indicated a roughly positive relationship between biofilm formation ability and antimicrobial resistance. LSCM observation showed that berberine and matrine inhibited biofilm formation of AMR E. coli strains at 1/2 minimal inhibitory concentration (MIC) (1/2 MIC berberine at OD630: 0.1020; 1/2 MIC matrine: OD630: 0.1045); furthermore, abnormal cell morphology such as rounded and elongated cells was also observed. This finding was consistent with the downregulation of QS-related genes: luxS, pfS, sdiA, hflX, motA, and fliA. At 1/2 MIC and 1/4 MIC concentrations of berberine, a significant downregulation of luxS, pfS, hflX, ftsQ, and ftsE was observed. The results indicate that berberine and matrine can inhibit biofilm formation by inhibiting the QS system and that berberine is more effective than matrine.

Forkhead Box C2 Attenuates Lipopolysaccharide-Induced Cell Adhesion via Suppression of Intercellular Adhesion Molecule-1 Expression in Human Umbilical Vein Endothelial Cells.

Atherosclerosis is a chronic vascular inflammatory disease that involves diverse cell types and circulating regulatory factors, including intercellular adhesion molecule (ICAM)-1, a proinflammatory cytokine. Lipopolysaccharides (LPS) increase ICAM-1 expression and promote cell adhesion, but the mechanism is not clear. We found that LPS induced time- and dose-regulated upregulation of ICAM-1 expression and downregulation of forkhead box protein C2 (Foxc2) expression in human umbilical vein endothelial cells (HUVECs). Overexpression of Foxc2 significantly inhibited both LPS-induced ICAM-1 expression in HUVECs and LPS-induced adhesion of THP-1 cells to HUVECs. Foxc2 siRNA dramatically increased both LPS-induced ICAM-1 expression and LPS-induced adhesion of THP-1 human monocytes cells to HUVECs. We conclude that Foxc2 inhibited LPS-induced adhesion of THP-1 cells to HUVECs by suppressing ICAM-1 expression in HUVECs.

The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy flux and inflammation through mTOR-dependent signaling.

Atherosclerosis is a progressive, chronic inflammation in arterial walls. Long noncoding RNAs (lncRNAs) participate in inflammation, but the exact mechanism in atherosclerosis is unclear. Our microarray analyses revealed that the levels of lncRNA-FA2H-2 were significantly decreased by oxidized low-density lipoprotein (OX-LDL). Bioinformatics analyses indicated that mixed lineage kinase domain-like protein (MLKL) might be regulated by lncRNA-FA2H-2. In vitro experiments showed that lncRNA-FA2H-2 interacted with the promoter of the MLKL gene, downregulated MLKL expression, and the binding sites between -750 and 471 were necessary for lncRNA-FA2H-2 responsiveness to MLKL. Silencing lncRNA-FA2H-2 and overexpression of MLKL could activate inflammation and inhibited autophagy flux. Both lncRNA-FA2H-2 knockdown and overexpression of MLKL could significantly aggravate inflammatory responses induced by OX-LDL. We found that the 3-methyladenine (3-MA) and Atg7-shRNA enhanced inflammatory responses induced by knockdown of lncRNA-FA2H-2 and overexpression of MLKL. We demonstrated that the effects of MLKL on autophagy might be associated with a mechanistic target of rapamycin (mTOR)-dependent signaling pathways. In vivo experiments with apoE knockout mice fed a western diet demonstrated that LncRNA-FA2H-2 knockdown decreased microtubule-associated expression of microtubule-associated protein 1 light chain 3 II and lysosome-associated membrane protein 1, but increased expression of sequestosome 1 (p62), MLKL, vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, and interleukin-6 in atherosclerotic lesions. Our findings indicated that the lncRNA-FA2H-2-MLKL pathway is essential for regulation of autophagy and inflammation, and suggested that lncRNA-FA2H-2 and MLKL could act as potential therapeutic targets to ameliorate atherosclerosis-related diseases.

Long noncoding RNA NEXN-AS1 mitigates atherosclerosis by regulating the actin-binding protein NEXN.

Noncoding RNAs are emerging as important players in gene regulation and disease pathogeneses. Here, we show that a previously uncharacterized long noncoding RNA, nexilin F-actin binding protein antisense RNA 1 (NEXN-AS1), modulates the expression of the actin-binding protein NEXN and that NEXN exerts a protective role against atherosclerosis. An expression microarray analysis showed that the expression of both NEXN-AS1 and NEXN was reduced in human atherosclerotic plaques. In vitro experiments revealed that NEXN-AS1 interacted with the chromatin remodeler BAZ1A and the 5' flanking region of the NEXN gene and that it also upregulated NEXN expression. Augmentation of NEXN-AS1 expression inhibited TLR4 oligomerization and NF-κB activity, downregulated the expression of adhesion molecules and inflammatory cytokines by endothelial cells, and suppressed monocyte adhesion to endothelial cells. These inhibitory effects of NEXN-AS1 were abolished by knockdown of NEXN. In vivo experiments using ApoE-knockout mice fed a Western high-fat diet demonstrated that NEXN deficiency promoted atherosclerosis and increased macrophage abundance in atherosclerotic lesions, with heightened expression of adhesion molecules and inflammatory cytokines, whereas augmented NEXN expression deterred atherosclerosis. Patients with coronary artery disease were found to have lower blood NEXN levels than healthy individuals. These results indicate that NEXN-AS1 and NEXN represent potential therapeutic targets in atherosclerosis-related diseases.

Pyroptosis and its relationship to atherosclerosis.

Pyroptosis is a pro-inflammatory form of regulated cell death and is dependent on the enzymatic activity of inflammatory proteases that belong to the family of cysteine-dependent aspartate-specific proteases (caspases). Pyroptosis is morphologically, mechanistically, and pathophysiologically distinct from other forms of cell death, including apoptosis and necrosis. Pyroptosis is characterized by rapid plasma membrane rupture, with the consequent release of intracellular contents and pro-inflammatory mediators, including interleukin (IL)-1ß, IL-18, and the alarmin HMGB-1.Recent studies have shown that pyroptosis may be involved in atherosclerosis and play an important role in atherosclerotic lesion instability. Here, we review the progress made in understanding the morphological, molecular, and pathophysiological mechanisms of pyroptosis and its potential role in atherosclerosis.