LncRNA RP4-639F20.1 interacts with THRAP3 to attenuate atherosclerosis by regulating c-FOS in vascular smooth muscle cells proliferation and migration.

BACKGROUND AND AIMS: The aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) play an essential role in the pathogenesis of atherosclerosis (AS). Long noncoding RNAs (lncRNAs) have been reported as important regulators in a number of diseases. However, very little is known regarding the functional role of lncRNAs in governing proliferation and migration of VSMCs and AS development. METHODS: Both in vitro and in vivo assays were performed to investigate the role of lncRNA in the pathophysiology of AS. Our previous lncRNA arrays revealed that lncRNA RP4-639F20.1 was significantly decreased in atherosclerotic plaques. Lentivirus overexpressing RP4-639F20.1 and lncRNA RP4-639F20.1 silencing vectors (Si-lnc-RP4-639F20.1) were constructed and transfected in VSMCs. The in vitro functions of lncRNA were analyzed by CCK-8 assays, EdU assays, scratch wound assays, transwell assays, qRT-PCR and Western blot analyses. RNA fluorescence in situ hybridization, immunoprecipitation and mRNA microarrays were used to explore the underlying mechanism. Adeno-associated-virus-9 (AAV9) overexpressing RP4-639F20.1 was constructed and injected intravenously into ApoE-/- mice to explore the role of lncRNA in vivo. RESULTS: In vitro experiments showed that lncRNA RP4-639F20.1 interacted with THRAP3 and downregulated c-FOS expression. Both increase of lncRNA RP4-639F20.1 expression and knockdown of c-FOS inhibited the expression of MMP10 and VEGF-α in VSMCs and suppressed VSMCs proliferation and migration. In vivo experiments using ApoE-/- mice fed a high-fat diet demonstrated that lncRNA RP4-639F20.1 overexpression deterred atherosclerosis and decreased lipid levels in atherosclerotic lesions. Patients with coronary artery disease were found to have higher c-FOS levels than healthy individuals and c-FOS expression was positively correlated with the SYNTAX score of patients. CONCLUSIONS: Overall, these data indicated that lncRNA RP4-639F20.1/THRAP3/c-FOS pathway protects against the development of atherosclerosis by suppressing VSMCs proliferation and migration. LncRNA RP4-639F20.1 and c-FOS could represent potential therapeutic targets to ameliorate atherosclerosis-related diseases.

Making Aggregation-Induced Emission Luminogen More Valuable by Gold: Enhancing Anticancer Efficacy by Suppressing Thioredoxin Reductase Activity.

Gold complexes have been recognized as potential anticancer agents against various kinds of diseases due to their inherent suppressions of antioxidant thioredoxin reductase (TrxR) activity. Herein, a powerful aggregation-induced emission luminogen (AIEgen), TBP-Au, was designed and synthesized by integrating an anticancer Au(I) moiety with an AIE-active photosensitizer (TBP), in which both the production and consumption routes of reactive oxygen species (ROS) were elaborately considered simultaneously to boost the anticancer efficacy. It has been demonstrated that TBP-Au could realize superior two-photon fluorescence imaging in tumor tissues with high resolution and deep penetration as well as long-term imaging in live animals due to its AIE property. In addition, the introduction of a special Au(I) moiety could tune the organelle specificity and efficiently facilitate the ROS-determined photodynamic therapy (PDT). More impressively, TBP-Au could efficiently eliminate cancer cells under light irradiation through the preconceived synergetic approaches from the PDT and the effective suppression of TrxR, demonstrating that TBP-Au holds great potential for precise cancer theranostics.

A novel electrochemical biosensor for exosomal microRNA-181 detection based on a catalytic hairpin assembly circuit.

Exosomal microRNAs (miRNAs) derived from different cells are proposed to be important noninvasive biomarkers for the diagnosis of cardiovascular disease. Recently, sensitive and reliable sensing of exosomal miRNAs has been garnered significant attention. Herein, a novel electrochemical biosensor based on a step polymerization catalytic hairpin assembly (SP-CHA) circuit is designed for exosomal miR-181 detection. Exosomal miR-181 as a trigger, induced SP-CHA process and generated a large number of T shaped concatemers with different length on the electrode surface. These ultra-concatemers could provide a much enhanced signal-to-noise ratio with the linear range from 10 fM to 100 nM and the detection limit of 7.94 fM. Furthermore, this assay was successfully applied to the detection of exosomal miR-181 in serum samples of normal healthy controls and patients with coronary heart disease (CHD) and the results were consistent with those analysis collected from qRT-PCR. The assembly demonstrated great performance in differentiating CHD patients from healthy controls (AUC:0.9867). Collectively, this sensing system possessed high stability and sensitivity with ease of operation and cost efficiency, leading to great potential for exosomal miRNAs detection in cardiovascular disease.

LncRNA AC105942.1 Downregulates hnRNPA2/B1 to Attenuate Vascular Smooth Muscle Cells Proliferation.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) is crucial in the atherosclerosis. Although long noncoding RNAs (lncRNAs) are implicated in a variety of diseases, their roles in activation of VSMCs proliferation and vascular disorder diseases are not well understood. In addition, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) was reported to participate in lncRNAs-mediated function. Herein, we propose to investigate the role of lncRNA AC105942.1 and hnRNPA2/B1 in pathological VSMCs proliferation and the possible mechanisms in vitro. We have identified that lncRNA AC105942.1 was downregulated and hnRNPA2/B1 was upregulated in atherosclerotic plaques compared with normal artery tissues. Enhanced lncRNA AC105942.1 could noticeably inhibit Ang II-induced VSMCs proliferation. Further investigation suggested that lncRNA AC105942.1 could downregulate the expression of hnRNPA2/B1 and then regulate the level of CDK4 and p27. Taken together, our study indicated that lncRNA AC105942.1 downregulated hnRNPA2B1 to protect against the atherosclerosis by suppressing VSMCs proliferation. LncRNA AC105942.1 and hnRNPA2/B1 could represent potential therapeutic and diagnostic targets to atherosclerosis-related diseases.

EDTA-K2 Improves the Detection Sensitivity of SARS-CoV-2 IgM and IgG Antibodies by Chelating Colloidal Gold in the Immunochromatographic Assay.

OBJECTIVE: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). PATIENTS AND METHODS: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. RESULTS: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. CONCLUSION: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.

TM4SF19 aggravates LPS-induced attenuation of vascular endothelial cell adherens junctions by suppressing VE-cadherin expression.

Atherosclerosis is a chronic vascular inflammatory disease that initially starts from an arterial intima lesion and endothelial barrier dysfunction. The purpose of this study was to investigate the role of TM4SF19, a recently identified member of the transmembrane 4L six superfamily, in vascular endothelial cell adherens junctions. We found TM4SF19 expression was significantly increased in atherosclerotic plaques and sera of patients with coronary heart disease (CHD) compared with healthy people by immunohistochemistry and ELISA. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated by lipopolysaccharides (LPS). TM4SF19 and VE-cadherin expression as well as cell adherens junctions were assessed. Additionally, LPS could upregulate TM4SF19 expression and downregulate VE-cadherin expression in HUVECs in a concentration dependent manner. Overexpression of TM4SF19 substantially aggravated LPS-induced reduction of VE-cadherin expression and attenuation of vascular endothelial cell adherens junctions. However, both the decreased VE-cadherin expression and weakened cell adherens junctions induced by LPS could be dramatically reversed when the expression of TM4SF19 was depressed. This study is the first to reveal the effect of TM4SF19 on endothelial cell adherens junctions. Meanwhile, our results also provide novel therapeutic strategies for atherosclerotic diseases.

The emerging role of cell senescence in atherosclerosis.

Cell senescence is a fundamental mechanism of aging and appears to play vital roles in the onset and prognosis of cardiovascular disease, fibrotic pulmonary disease, liver disease and tumor. Moreover, an increasing body of evidence shows that cell senescence plays an indispensable role in the formation and development of atherosclerosis. Multiple senescent cell types are associated with atherosclerosis, senescent human vascular endothelial cells participated in atherosclerosis via regulating the level of endothelin-1 (ET-1), nitric oxide (NO), angiotensin II and monocyte chemoattractant protein-1 (MCP-1), senescent human vascular smooth muscle cells-mediated plaque instability and vascular calcification via regulating the expression level of BMP-2, OPN, Runx-2 and inflammatory molecules, and senescent macrophages impaired cholesterol efflux and promoted the development of senescent-related cardiovascular diseases. This review summarizes the characteristics of cell senescence and updates the molecular mechanisms underlying cell senescence. Moreover, we also discuss the recent advances on the molecular mechanisms that can potentially regulate the development and progression of atherosclerosis.

MLKL Aggravates Ox-LDL-Induced Cell Pyroptosis via Activation of NLRP3 Inflammasome in Human Umbilical Vein Endothelial Cells.

Atherosclerosis is a progressive chronic inflammation in the arterial walls. It is believed that the deposition of low-density lipoprotein (LDL) and its damage to endothelial cells play a vital role in atherosclerosis. Oxidized LDL (Ox-LDL) was confirmed to induce endothelial cell pyroptosis which plays an important role in intima inflammation and the development of atherosclerosis, but the underlying molecular mechanism needs to be explored. Here, we showed that ox-LDL upregulated the expression of mixed lineage kinase domain-like (MLKL) protein at both the mRNA and protein levels in endothelial cells, associated with the augment of pro-caspase-1 cleavage, interleukin-1ß (IL-1ß) maturation, pro-IL-1ß production, and lactate dehydrogenase (LDH) release. Overexpression of MLKL substantially aggravated ox-LDL-induced increasing levels of caspase-1, IL-1ß, pro-IL-1ß, and LDH. MLKL-induced caspase-1 activation and IL-1ß maturation were abolished by NLR family, pyrin domain-containing 3 (NLRP3) specific inhibitor MCC950, or extracellular high potassium concentration. Our findings indicated that MLKL is essential for regulation of ox-LDL-induced pyroptosis and inflammation through the activation of NLRP3 inflammasome, and suggested that MLKL could act as potential therapeutic targets to ameliorate atherosclerosis-related diseases.

Lipopolysaccharide Promotes Inflammatory Response via Enhancing IFIT1 Expression in Human Umbilical Vein Endothelial Cells.

Atherosclerosis is an immune inflammatory disease and a major cause of mortality and morbidity worldwide. It is generally considered that a number of potent proinflammatory cytokines have a great influence on its pathogenesis, including IL-1ß, IL-6, TNF-α, and NF-κB. A growing amount of empirical evidence indicates that the mechanism of cardiac dysfunction caused by lipopolysaccharide (LPS) is the activation of inflammation, but the exact mechanism in atherosclerosis is still unclear. Previous studies have shown that interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) participates in inflammation, but the effects and possible mechanism of action of IFIT1 on proinflammatory response remain largely unexplained. We found that LPS induced upregulation of IFIT1 expression in a time- and concentration-dependent manner in human umbilical vein endothelial cells (HUVECs). Overexpression of IFIT1 significantly upregulated LPS-induced expression of IL-1ß, IL-6, TNF-α, and NF-κB in HUVECs. IFIT1-siRNA treatment dramatically decreased LPS-induced expression of IL-1ß, IL-6, TNF-α, and NF-κB in HUVECs. The above results show that LPS induces expression of IL-1ß, IL-6, TNF-α, and NF-κB through upregulating IFIT1 expression in HUVECs, and suggested that IFIT1 could act as potential therapeutic target to ameliorate atherosclerosis-related diseases.

Impact of heat-inactivation on the detection of SARS-CoV-2 IgM and IgG antibody by ELISA.

BACKGROUND: To establish a safe and accurate method for detecting SARS-CoV-2 IgM and IgG, we assessed the impact of sera after heat-inactivation on the SARS-CoV-2 IgM and IgG levels measured by ELISA-immunoassay. METHODS: The serum samples of 62 patients with COVID-19 and 18 healthy controls were collected in Hankou's Hospital of Wuhan from February 27 to March 6, 2020. Before and after the samples were inactivated, the levels of IgM and IgG antibodies were measured. RESULTS: The indexes of antibodies after inactivated were significantly higher than those in fresh sera, while the positive rates in all participants or in patients with COVID-19 did not change. The positive coincidence rate, negative coincidence rate and total coincidence rate of IgM antibodies before and after inactivation were 100.00% (55/55), 96.00% (24/25) and 98.75% (79/80), respectively (κ = 0.971, P < 0.001), while those for IgG antibodies were 98.21% (55/56), 91.67% (22/24) and 98.75% (79/80) respectively (κ = 0.910, P < 0.001). These results showed a good consistency. CONCLUSIONS: Heating-activation does not decrease the diagnostic efficacy of SARS-CoV-2 IgM or IgG antibodies. Sera inactivated by heating at 56 °C for 30 min should be recommended to minimize the risk of virus contamination of laboratory staff.

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.