LncRNA PSMB8-AS1 Instigates Vascular Inflammation to Aggravate Atherosclerosis.

BACKGROUND: Increasing evidence suggests that long noncoding RNAs play significant roles in vascular biology and disease development. One such long noncoding RNA, PSMB8-AS1, has been implicated in the development of tumors. Nevertheless, the precise role of PSMB8-AS1 in cardiovascular diseases, particularly atherosclerosis, has not been thoroughly elucidated. Thus, the primary aim of this investigation is to assess the influence of PSMB8-AS1 on vascular inflammation and the initiation of atherosclerosis. METHODS: We generated PSMB8-AS1 knockin and Apoe (Apolipoprotein E) knockout mice (Apoe-/-PSMB8-AS1KI) and global Apoe and proteasome subunit-ß type-9 (Psmb9) double knockout mice (Apoe-/-Psmb9-/-). To explore the roles of PSMB8-AS1 and Psmb9 in atherosclerosis, we fed the mice with a Western diet for 12 weeks. RESULTS: Long noncoding RNA PSMB8-AS1 is significantly elevated in human atherosclerotic plaques. Strikingly, Apoe-/-PSMB8-AS1KI mice exhibited increased atherosclerosis development, plaque vulnerability, and vascular inflammation compared with Apoe-/- mice. Moreover, the levels of VCAM1 (vascular adhesion molecule 1) and ICAM1 (intracellular adhesion molecule 1) were significantly upregulated in atherosclerotic lesions and serum of Apoe-/-PSMB8-AS1KI mice. Consistently, in vitro gain- and loss-of-function studies demonstrated that PSMB8-AS1 induced monocyte/macrophage adhesion to endothelial cells and increased VCAM1 and ICAM1 levels in a PSMB9-dependent manner. Mechanistic studies revealed that PSMB8-AS1 induced PSMB9 transcription by recruiting the transcription factor NONO (non-POU domain-containing octamer-binding protein) and binding to the PSMB9 promoter. PSMB9 (proteasome subunit-ß type-9) elevated VCAM1 and ICAM1 expression via the upregulation of ZEB1 (zinc finger E-box-binding homeobox 1). Psmb9 deficiency decreased atherosclerotic lesion size, plaque vulnerability, and vascular inflammation in Apoe-/- mice in vivo. Importantly, endothelial overexpression of PSMB8-AS1-increased atherosclerosis and vascular inflammation were attenuated by Psmb9 knockout. CONCLUSIONS: PSMB8-AS1 promotes vascular inflammation and atherosclerosis via the NONO/PSMB9/ZEB1 axis. Our findings support the development of new long noncoding RNA-based strategies to counteract atherosclerotic cardiovascular disease.

EPSTI1 promotes monocyte adhesion to endothelial cells in vitro via upregulating VCAM-1 and ICAM-1 expression.

Atherosclerosis is a chronic inflammatory disease of arterial wall, and circulating monocyte adhesion to endothelial cells is a crucial step in the pathogenesis of atherosclerosis. Epithelial-stromal interaction 1 (EPSTI1) is a novel gene, which is dramatically induced by epithelial-stromal interaction in human breast cancer. EPSTI1 expression is not only restricted to the breast but also in other normal tissues. In this study we investigated the role of EPSTI1 in monocyte-endothelial cell adhesion and its expression pattern in atherosclerotic plaques. We showed that EPSTI1 was dramatically upregulated in human and mouse atherosclerotic plaques when compared with normal arteries. In addition, the expression of EPSTI1 in endothelial cells of human and mouse atherosclerotic plaques is significantly higher than that of the normal arteries. Furthermore, we demonstrated that EPSTI1 promoted human monocytic THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs) via upregulating VCAM-1 and ICAM-1 expression in HUVECs. Treatment with LPS (100, 500, 1000 ng/mL) induced EPSTI1 expression in HUVECs at both mRNA and protein levels in a dose- and time-dependent manner. Knockdown of EPSTI1 significantly inhibited LPS-induced monocyte-endothelial cell adhesion via downregulation of VCAM-1 and ICAM-1. Moreover, we revealed that LPS induced EPSTI1 expression through p65 nuclear translocation. Thus, we conclude that EPSTI1 promotes THP-1 cell adhesion to endothelial cells by upregulating VCAM-1 and ICAM-1 expression, implying its potential role in the development of atherosclerosis.

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.

VNN1 promotes atherosclerosis progression in apoE-/- mice fed a high-fat/high-cholesterol diet.

Accumulated evidence shows that vanin-1 (VNN1) plays a key part in glucose metabolism. We explored the effect of VNN1 on cholesterol metabolism, inflammation, apoptosis in vitro, and progression of atherosclerotic plaques in apoE(-/-) mice. Oxidized LDL (Ox-LDL) significantly induced VNN1 expression through an ERK1/2/cyclooxygenase-2/PPARα signaling pathway. VNN1 significantly increased cellular cholesterol content and decreased apoAI and HDL-cholesterol (HDL-C)-mediated efflux by 25.16% and 23.13%, respectively, in THP-1 macrophage-derived foam cells (P < 0.05). In addition, VNN1 attenuated Ox-LDL-induced apoptosis through upregulation of expression of p53 by 59.15% and downregulation of expression of B-cell lymphoma-2 127.13% in THP-1 macrophage (P < 0.05). In vivo, apoE(-/-) mice were divided randomly into two groups and transduced with lentivirus (LV)-Mock or LV-VNN1 for 12 weeks. VNN1-treated mice showed increased liver lipid content and plasma levels of TG (124.48%), LDL-cholesterol (119.64%), TNF-α (148.74%), interleukin (IL)-1ß (131.81%), and IL-6 (156.51%), whereas plasma levels of HDL-C (25.75%) were decreased significantly (P < 0.05). Consistent with these data, development of atherosclerotic lesions was increased significantly upon infection of apoE(-/-) mice with LV-VNN1. These observations suggest that VNN1 may be a promising therapeutic candidate against atherosclerosis.

RP5-833A20.1/miR-382-5p/NFIA-dependent signal transduction pathway contributes to the regulation of cholesterol homeostasis and inflammatory reaction.

OBJECTIVE: Cardiovascular disease caused by atherosclerosis is the number one cause of death in Western countries and threatens to become the major cause of morbidity and mortality worldwide. Long noncoding RNAs are emerging as new players in gene regulation, but how long noncoding RNAs operate in the development of atherosclerosis remains unclear. APPROACH AND RESULTS: Using microarray analysis, we found that long noncoding RNA RP5-833A20.1 expression was upregulated, whereas nuclear factor IA (NFIA) expression was downregulated in human acute monocytic leukemia macrophage-derived foam cells. Moreover, we showed that long noncoding RNA RP5-833A20.1 may decreases NFIA expression by inducing hsa-miR-382-5p expression in vitro. We found that the RP5-833A20.1/hsa-miR-382-5p/NFIA pathway is essential to the regulation of cholesterol homeostasis and inflammatory responses in human acute monocytic leukemia macrophages. Lentivirus-mediated NFIA overexpression increased high-density lipoprotein cholesterol circulation, reduced low-density lipoprotein cholesterol, and very-low-density lipoprotein cholesterol circulation, decreased circulation of inflammatory cytokines, including interleukin-1ß, interleukin-6, tumor necrosis factor-α, and C-reactive protein, enhanced reverse cholesterol transport, and promoted regression of atherosclerosis in apolipoprotein E-deficient mice. CONCLUSIONS: Our findings indicated that the RP5-833A20.1/miR-382-5p/NFIA pathway was essential to the regulation of cholesterol homeostasis and inflammatory reactions and suggested that NFIA may represent a therapeutic target to ameliorate cardiovascular disease.

Dihydrocapsaicin down-regulates apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells.

BACKGROUND: Apolipoprotein M (apoM), as a novel apolipoprotein which is mainly expressed in liver and kidney tissues, is associated with development and progression of atherosclerosis and diabetes. Our group have recently shown that Dihydrocapsaicin(DHC)can significantly decrease atherosclerotic plaque formation in apoE-/- mice. However, the effect and possible mechanism of DHC on apoM expression remain unclear. METHODS: HepG2 cells were treated with 0 µM, 25 µM, 50 µM and 100 µM DHC for 24 h or were treated with 100 µM DHC for 0, 6, 12, and 24 h, respectively. The mRNA levels and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively. RESULTS: We found that DHC markedly decreased expression of apoM at both mRNA and protein level in HepG2 cells in a dose-dependent and time-dependent manner. Expression of Foxa2 was decreased while expression of LXRα was increased by DHC treatment in HepG2 cells. In addittion, overexpression of Foxa2 markedly compensated the inhibition effect induced by DHC on apoM expression. LXRα small interfering RNA significantly abolished the inhibition effect which induced by DHC on apoM expression. The liver of C57BL/6 mice treated with DHC had significantly lower expression of apoM. Furthermore, the liver had lower expression of Foxa2 while had higher expression of LXRα. CONCLUSIONS: DHC could down-regulate apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells.

LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1.

Long non-coding RNAs (lncRNAs) are emerging as important players in gene regulation and cardiovascular diseases. However, the roles of lncRNAs in atherosclerosis are poorly understood. In the present study, we found that the levels of NIPA1-SO were decreased while those of NIPA1 were increased in human atherosclerotic plaques. Furthermore, NIPA1-SO negatively regulated NIPA1 expression in human umbilical vein endothelial cells (HUVECs). Mechanistically, NIPA1-SO interacted with the transcription factor FUBP1 and the NIPA1 gene. The effect of NIPA1-SO on NIPA1 protein levels was reversed by the knockdown of FUBP1. NIPA1-SO overexpression increased, whilst NIPA1-SO knockdown decreased BMPR2 levels; these effects were enhanced by the knockdown of NIPA1. The overexpression of NIPA1-SO reduced while NIPA1-SO knockdown increased monocyte adhesion to HUVECs; these effects were diminished by the knockdown of BMPR2. The lentivirus-mediated-overexpression of NIPA1-SO or gene-targeted knockout of NIPA1 in low-density lipoprotein receptor-deficient mice reduced monocyte-endothelium adhesion and atherosclerotic lesion formation. Collectively, these findings revealed a novel anti-atherosclerotic role for the lncRNA NIPA1-SO and highlighted its inhibitory effects on vascular inflammation and intracellular cholesterol accumulation by binding to FUBP1 and consequently repressing NIPA1 expression.

Long non-coding RNA RP11-490M8.1 inhibits lipopolysaccharide-induced pyroptosis of human umbilical vein endothelial cells via the TLR4/NF-κB pathway.

BACKGROUND AND AIMS: Pyroptosis is a relatively newly discovered form of programmed cell death that plays an important role in the development of atherosclerosis. Many studies have reported that lncRNAs participated in the regulation of atherosclerosis development. However, the regulatory mechanism of lncRNAs in pyroptosis must be studied further. METHODS: In a previous study, microarray analysis was used to detect the lncRNA expression profile in three human advanced atherosclerotic plaques and three normal arterial intimae. In the present research, in vitro assays were performed to investigate the role of lncRNA RP11-490M8.1 on pyroptosis. The relative gene mRNA and lncRNA expression levels were tested by quantitative real-time PCR, and protein levels were evaluated by western blot analysis. The RNA hybrid structure was analyzed using the DINAMelt server. RESULTS: The lncRNA RP11-490M8.1 was significantly downregulated in atherosclerotic plaques and serum. Lipopolysaccharide (LPS) markedly reduced the expression of lncRNA RP11-490M8.1 and induced pyroptosis by increasingthe mRNA and protein levels of NLRP3, caspase-1, ASC, IL-1ß, and IL-18 in HUVECs. The promotion effects ofLPS on pyroptosis were markedly suppressed by overexpression of lncRNA RP11-490M8.1. In addition, LPS increased the mRNA and protein levels ofTLR4 and NF-κB, which was also markedly offsetby overexpression of lncRNA RP11-490M8.1. CONCLUSIONS: These findings indicated that lncRNA RP11-490M8.1 inhibited LPS-induced pyroptosis via the TLR4/NF-κB pathway. Thus, lncRNA RP11-490M8.1 may provide a therapeutic target to ameliorate atherosclerosis.

[Changes of iron metabolism indices in children with various genotypes of thalassema].

OBJECTIVE: To study the value of iron metabolism indices, serum iron (SI), total iron blinding capacity (TIBC) and transferring (Tf), in thalassema. METHODS: The serum samples from 9 children with silent alpha thalassema, 56 with standard alpha thalassema, 26 with HbH disease, 40 with beta+ thalassema, 56 with beta0 thalassema, 45 with iron deficiency anemia (IDA) and 70 healthy children were detected for SI, TIBC and Tf levels. RESULTS: The SI level increased (p<0.01), while the TIBC level decreased significantly in the beta0 thalassema group compared with those in the other groups (p<0.05 or 0.01), but the Tf level was not different. The Tf level of both the silent alpha thalassema and the standard alpha thalassema groups was statistically lower than that of the healthy group (p<0.01), but the levels of SI and TIBC were similar to the healthy group. Though the SI level of the HbH disease group was similar to the healthy group, the TIBC and Tf levels were statistically lower (p<0.01). CONCLUSIONS: Compared with Tf, SI and TIBC are better indices for monitoring iron loading in children with thalassema. The increased SI level and decreased TIBC level are two indices for the diagnosis of beta(0) thalassema in children with cellule anaemia.

TTDA inhibited apoptosis by regulating the p53-Bax/Bcl2 axis in glioma.

The trichothiodystrophy group A protein (TTDA) functions in nucleotide excision repair and basal transcription. TTDA plays a role in cancers and serves as a prognostic and predictive factor in high-grade serous ovarian cancer; however, its role in human glioma remains unknown. Here, we found that TTDA was overexpressed in glioma tissues. In vitro experiments revealed that TTDA overexpression inhibited apoptosis of glioma cells and promoted cell growth, whereas knockdown of TTDA had the opposite effect. Increased TTDA expression significantly decreased the Bax/Bcl2 ratio and the level of cleaved-caspase3. TTDA interacted with the p53 gene at the -1959 bp and -1530 bp region and regulated its transcription, leading to inhibition of the p53-Bax/Bcl2 mitochondrial apoptosis pathway in glioma cells. These results indicate that TTDA is an upstream regulator of p53-mediated apoptosis and acts as an oncogene, suggesting its value as a potential molecular target for the diagnosis and treatment of glioma.

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.

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.

Ox-LDL upregulates CRP expression through the IGF2 pathway in THP-1 macrophages.

C-reactive protein (CRP) is an acute-phase reactant protein that not only plays a predictive role in determining atherogenesis risk but also represents an active participant in atherogenesis onset and progression. Moreover, an increasing number of studies have reported that oxidized low-density lipoprotein (Ox-LDL) plays a significant role in the initiation and progression of atherosclerosis. However, the effect and underlying mechanism of Ox-LDL on CRP expression remains unclear. THP-1 macrophages were treated with 0, 25, 50, or 100 µg/mL of Ox-LDL for 48 h, or 50 µg/mL of Ox-LDL for 0, 12, 24, and 48 h, respectively. Messenger RNA (mRNA) and protein levels were measured by real-time quantitative PCR and Western blot analysis, respectively. We found that Ox-LDL markedly increased insulin-like growth factor 2 (IGF2) and CRP mRNA and protein levels in a dose- and time-dependent manner in THP-1 macrophages. Treatment with Ox-LDL increased CRP protein expression, and this effect was completely abolished by siRNA-mediated silencing of IGF2 in THP-1 macrophages. Moreover, treatment with pcDNA3.1-IGF2 significantly enhanced CRP protein expression in Ox-LDL-stimulated THP-1 macrophages. CRP expression is upregulated by Ox-LDL through the IGF2 pathway in THP-1 macrophages.

An agomir of miR-144-3p accelerates plaque formation through impairing reverse cholesterol transport and promoting pro-inflammatory cytokine production.

AIMS: ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to lipid-poor apolipoproteins, which then form nascent HDL, a key step in the mechanism of reverse cholesterol transport (RCT). While a series of microRNAs (miRNAs) have been identified as potent post-transcriptional regulators of lipid metabolism, their effects on ABCA1 function and associated mechanisms remain unclear. METHODS AND RESULTS: ABCA1 was identified as a potential target of miR-144-3p, based on the results of bioinformatic analysis and the luciferase reporter assay, and downregulated after transfection of cells with miR-144-3p mimics, as observed with real-time PCR and western blot. Moreover, miR-144-3p mimics (agomir) enhanced the expression of inflammatory factors, including IL-1ß, IL-6 and TNF-α, in vivo and in vitro, inhibited cholesterol efflux in THP-1 macrophage-derived foam cells, decreased HDL-C circulation and impaired RCT in vivo, resulting in accelerated pathological progression of atherosclerosis in apoE-/- mice. Clinical studies additionally revealed a positive correlation of circulating miR-144-3p with serum CK, CK-MB, LDH and AST in subjects with AMI. CONCLUSIONS: Our findings clearly indicate that miR-144-3p is essential for the regulation of cholesterol homeostasis and inflammatory reactions, supporting its utility as a potential therapeutic target of atherosclerosis and a promising diagnostic biomarker of AMI.