Potential Therapeutic Target For Atherosclerosis Research Articles

Effects of Proteasome Inhibitor on Catalase Expression and Intima-media Thickness in the Aorta of Atherosclerotic Rats

Various studies have been carried out to obtain proper management for atherosclerosis. Proteasome, a subcellular enzyme complex, is a potential therapeutic target for atherosclerosis. However, the effect of proteasome inhibitors on atherosclerosis still needs to be explored. It was an experimental study with a post-test-only control group design conducted at the Faculty of Medicine, Universitas Riau in Juni–November 2021. This study aimed to analyze the effects of proteasome inhibitors on catalase expression and intima-media thickness (IMT) in the thoracic aorta of atherosclerotic rats. Fifteen male Wistar rats were randomly divided into three groups (five rats per group), namely rats given standard feed (control, group I), rats induced atherosclerosis (group II), and rats induced atherosclerosis and given proteasome inhibitor (group III). The proteasome inhibitor, bortezomib, 50 µg/kgBW/day was given intraperitoneally on days one and three. After 4 days, rats were terminated, and the thoracic aorta was taken for the IMT analysis and catalase expression assessment using immunohistochemistry. Catalase expression was carried out quantitatively using Adobe Photoshop software. Analysis of variance test was used to compare the expression of catalase and IMT. A p value<0.05 was considered statistically significant. The results showed a significant decrease in IMT in group III compared to group II and an increase in catalase expression in group III compared to group II but not statistically significant. This study concludes that administration of bortezomib 50 µg/kg in atherosclerotic rats could inhibit thickening tunica intima-media in the thoracic aorta, although not significantly increasing the catalase expression.

The Role of Transcription Factors in Coronary Artery Disease and Myocardial Infarction.

Coronary artery disease (CAD) and its main complication, myocardial infarction (MI), is a complex disease caused by environmental and genetic factors and their interaction. Family-based linkage analysis and genome-wide association studies have indicated many of genetic variations related to CAD and MI in recent years. Some are in the coding sequence, which mediates the coding protein, while others are in the non-coding region, which affects the expression of adjacent genes and forms differential gene expression. These variants and differential expressions will have varying degrees of impact on the development of the cardiovascular system and normal heart electrical activity function, subsequently leading to CAD and MI. Among these affected genes, some Transcription Factors (TFs), as important means of transcriptional regulation, have a key role in the pathogenesis of coronary artery disease and myocardial infarction. The GATAs binding protein 2 (GATA2) enhances monocyte adhesion and promoted vessel wall permeabilization through vascular EC adhesion molecule 1 (VCAM-1) upregulation, further revealing its atherosclerosis-promoting role. Myocyte enhancer factor 2 (MEF2) has a role in fostering many functions of the atherosclerotic endothelium and is a potential therapeutic target for atherosclerosis, thrombosis, and inflammation. Nuclear factor-kappa B (NF-κB) is an important promoter of vascular endothelial growth factor (VEGF)-driven angiogenesis, and its pathway has a key role in atherosclerosis-related complications such as angiogenesis, inflammation, apoptosis, and immune effects. Activating transcription factor 3 (ATF3) may be a novel prognostic biomarker and therapeutic target for atherosclerosis. The important role of signal transducer and activator of transcription 3 (STAT3) (especially in mitochondria) in endothelial cells (EC) dysfunction, inflammation, macrophage polarization and immunity in atherosclerosis.

Abstract 13606: Deletion of CD47 Specifically in Lymphatic Endothelium Augments Arterial Lymphangiogenesis and Attenuates Atherosclerosis

Thrombospondin-1 (TSP1), an extracellular glycoprotein mediates differential physio- or pathological effects via interacting with cell surface receptors including CD36 and CD47. TSP1 is a well-known angiogenesis inhibitor, however, its role in regulating lymphangiogenesis is not clear. We recently reported that global CD47-deficient mice are protected from atherosclerosis, however, myeloid cell-specific CD47 loss augments lesion formation suggesting the importance of cell-specific CD47 blockade in atherosclerosis. Our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we herein investigated the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerotic lesion formation. Our immunoblotting data demonstrated elevated TSP1 expression in human and mouse atherosclerotic aortic tissue compared to non-atherosclerotic control tissue. TSP1 at pathological concentrations inhibited VEGF-C-stimulated in vitro lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF-C-induced Akt activation in LECs, leading to decreased eNOS phosphorylation and attenuated nitric oxide production. Further, TSP1-treated CD47-silenced LECs proliferate faster and have higher Akt and eNOS activation than TSP1-treated control cells. AAV8-PCSK9-injected LEC-specific CD47 knockout ( Cd47 f/f Lyve-1 Cre +/- ) mice had reduced atherosclerosis in both aorta and aortic root compared with control ( Cd47 f/f ) mice. Additional immunostaining experiments performed on aortic root cross-sections indicated improved lymphatic vessel density in Cd47 f/f Lyve-1 Cre +/- mice. These results demonstrate that TSP1-mediated LEC CD47 activation inhibits lymphangiogenesis and blockade of LEC CD47 signaling suppresses atherosclerosis. Altogether, these findings identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Gene-dosage effect of Pfkfb3 on monocyte/macrophage biology in atherosclerosis.

Macrophage-rich atherosclerotic arteries are highly active in glycolysis. PFKFB3, a key glycolytic enzyme, has emerged as a potential therapeutic target in atherosclerosis. Small-molecule inhibitors of PFKFB3, such as 3PO and PFK158, have demonstrated efficacy in hampering atherogenesis in preclinical models. However, genetic studies elucidating the role of Pfkfb3 in atherogenesis need to be conducted to validate pharmacological findings and to unveil potential pharmacological side effects. Apoe-/- mice with global heterozygous or myeloid cell-specific Pfkfb3 deficiency were fed a Western diet (WD), after which atherosclerosis development was determined. Monocyte subsets in atherosclerotic mice and patients were examined by flow cytometry. Monocyte infiltration was assayed by a Ly6Chi monocyte-specific latex labelling procedure. In situ efferocytosis was assessed on mouse aortic root sections. Additionally, metabolic status, macrophage motility, efferocytosis, and involved mechanisms were analysed in peritoneal macrophages. Global heterozygous or myeloid cell-specific Pfkfb3 deficiency reduced atherogenesis in Apoe-/- mice. Mechanistic studies showed that PFKFB3 controlled the proliferation and infiltration of proinflammatory monocytes. Moreover, PFKFB3 expression was associated with inflammatory monocyte expansion in patients with atherosclerotic coronary artery disease. Surprisingly, homozygous loss of Pfkfb3 impaired macrophage efferocytosis and exacerbated atherosclerosis in Apoe-/- mice. Mechanistically, PFKFB3-driven glycolysis was shown to be essential for actin polymerization, thus aiding the efferocytotic function of macrophages. Collectively, these findings suggest the existence of a double-edged sword effect of myeloid PFKFB3 on the pathogenesis of atherosclerosis and highlight the need for caution in developing anti-atherosclerotic strategies that target PFKFB3.

Macrophage polarization as a potential therapeutic target for atherosclerosis: a dynamic stochastic modelling study.

We proposed a dynamic stochastic mathematical model to evaluate the role of macrophage polarization in plaque development. The dynamic process of macrophages from proliferation to death was simulated under different lipid microenvironments. The probability of macrophage phenotypic switching was described using a Bernoulli distribution where the stochastic variable was determined by the local lipid level. Moreover, the interactions between macrophages and microenvironmental factors vary with macrophage phenotype. We investigated the distribution of key microenvironmental factors, the dynamics of macrophage polarization and its influence on foam cell formation. M1 macrophages were found to predominate in advanced plaque corresponding to the exacerbated inflammation observed in mice experiments. The imbalance between the deposition of oxidized low-density lipoprotein and phagocytic effects of macrophages governed the formation of foam cells. Furthermore, we simulated targeted therapies by either directly inhibiting the polarization probability to M1 macrophages or indirectly regulating macrophage polarization due to high-density lipoprotein levels. Comparison of simulation results with experimental findings in both therapies indicated that the intervention and regulation of macrophage polarization could influence plaque microenvironment and subsequently induce plaque regression, especially in the early stage. The proposed modelling system can facilitate the evaluation of novel therapies targeting macrophage polarization.

CircRNA_0050486 promotes cell apoptosis and inflammation by targeting miR-1270 in atherosclerosis.

BackgroundAtherosclerosis (AS) is a chronic inflammatory disease that plays a major role in cardiovascular disease. Circular RNAs (circRNAs) are related to the pathogenesis of AS, including the inflammatory response. This study aimed to explore the underlying mechanisms of circRNAs and how they regulate the inflammatory response in AS.MethodsAnalyzed the expression profile of circRNAs in three oxidized low-density lipoprotein (oxLDL) treated macrophage samples and three macrophage control samples using bioinformatics methods. Expression and biological function of circRNA were verified in oxLDL-induced THP-1 macrophages. MiRNAs and target genes of circRNA were predicted by functional enrichment analysis. Expression and function of circRNA target miRNAs were explored in oxLDL-induced THP-1 macrophages. Finally, we predicted and analyzed the function of circRNAs-miRNAs target genes in AS.ResultsWe identified nine upregulated circRNAs and found that circ_0050486 was significantly upregulated in a THP-1 + PMA + oxLDL group compared with a THP-1 + PMA group. Additionally, circ_0050486 knockdown markedly inhibited IL-6 and TNF-α concentrations and the cell death rates in oxLDL-induced THP-1 macrophages. Furthermore, circ_0050486 targeted and inhibited miR-145 and miR-1270. Upregulated miR-1270 markedly inhibited IL-6 and TNF-α levels and the cell death rates in oxLDL-induced THP-1 macrophages. Finally, the target genes of miR-1270 and miR-145 were predicted by the miRDB, miRWalk, and Targetscan databases, and a functional analysis network of the target genes was constructed by Cytoscape GlueGO, including the regulation of the immune response and monocyte chemotaxis. The common target genes of miR-145 and miR-1270 were established by Cytoscape and included NF1A, among others.ConclusionsOur study suggested that circ_0050486 knockdown inhibited inflammation and apoptosis by targeting miR-1270 in oxLDL-induced THP-1 macrophages. This finding may provide a potential therapeutic target for atherosclerosis.

BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation.

Atherosclerosis is a chronic systemic inflammatory disease that causes severe cardiovascular events. B cell lymphoma 2-associated athanogene (BAG3) was proven to participate in the regulation of tumor angiogenesis, neurodegenerative diseases, and cardiac diseases, but its role in atherosclerosis remains unclear. Here, we aim to investigate the role of BAG3 in atherosclerosis and elucidate the potential molecular mechanism. In this study, ApoE−/− mice were given a tail-vein injection of BAG3-overexpressing lentivirus and fed a 12-week high-fat diet (HFD) to investigate the role of BAG3 in atherosclerosis. The overexpression of BAG3 reduced plaque areas and improved atherosclerosis in ApoE−/− mice. Our research proves that BAG3 promotes autophagy in vitro, contributing to the suppression of EndMT in human umbilical vein endothelial cells (HUVECs). Mechanically, autophagy activation is mediated by BAG3 via the interaction between BAG3 and its chaperones HSP70 and HSPB8. In conclusion, BAG3 facilitates autophagy activation via the formation of the chaperone-assisted selective autophagy (CASA) complex interacting with HSP70 and HSPB8, leading to the inhibition of EndMT during the progression of atherosclerosis and indicating that BAG3 is a potential therapeutic target for atherosclerosis.

MiR-550a-3p restores damaged vascular smooth muscle cells by inhibiting thrombomodulin in an in vitro atherosclerosis model.

Thrombomodulin (TM) is involved in the pathological process of atherosclerosis; however, the underlying mechanism remains unclear. Oxidised low-density lipoprotein (Ox-LDL; 100 μg/mL) was used to induce human vascular smooth muscle cells (HVSMCs) into a stable atherosclerotic cell model. The expression levels of miR-550a-3p and TM were detected by real-time reverse transcription-polymerase chain reaction. Cell proliferation was estimated using CCK8 and EDU assays. Wound scratch and transwell assays were used to measure the ability of cells to invade and migrate. Propidium iodide fluorescence-activated cell sorting was used to detect apoptosis and cell cycle changes. A dual-luciferase reporter assay was performed to determine the binding of miR-550a-3p to TM. Our results suggested the successful development of a cellular atherosclerosis model. Our data revealed that TM overexpression significantly promoted the proliferation, invasion, migration, and apoptosis of HVSMCs as well as cell cycle changes. Upregulation of miR-550a-3p inhibited the growth and metastasis of HVSMCs. Furthermore, miR-550a-3p was confirmed to be a direct target of TM. Restoration of miR-550a-3p expression rescued the effects of TM overexpression. Thus, miR-550a-3p might play a role in atherosclerosis and, for the first time, normalised the function of injured vascular endothelial cells by simultaneous transfection of TM and miR-550a-3p. These results suggest that the miR-550a-3p/TM axis is a potential therapeutic target for atherosclerosis.

CircRNA-PTPRA Knockdown Inhibits Atherosclerosis Progression by Repressing ox-LDL-Induced Endothelial Cell Injury via Sponging of miR-671-5p.

Atherosclerosis (AS) is a chronic inflammatory disease with high morbidity and mortality rates worldwide. This study aimed to investigate the role of circular RNA protein tyrosine phosphatase receptor type A (circRNA_PTPRA) in oxidizedlow-densitylipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVECs) injury and its underlying molecular mechanism. The expression of circRNA-PTPRA and microRNA (miR)-671-5p was assessed by quantitative reverse transcription PCR (qRT-PCR). The interaction between circRNA-PTPRA and miR-671-5p was predicted using bioinformatic analysis. Cell viability and apoptosis were determined using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Inflammation in HUVECs was analyzed by measuring the secretion of tumornecrosisfactoralpha (TNF-α), interleukin-1beta(IL-1β), and IL-6 using enzyme-linkedimmunosorbentassay(ELISA). Cleaved-caspase-3 expression was assessed using western blotting. The results indicated that circRNA-PTPRA expression was significantly increased and miR-671-5p expression was decreased in the serum of patients with AS and in ox-LDL-treated HUVECs. The interaction between circRNA-PTPRA and miR-671-5p was verified by dual luciferase reporter and RNA pull-down assays. In HUVECs, downregulation of circRNA-PTPRA reversed ox-LDL-induced reduction in cell viability, increase in apoptosis, and enhanced inflammation, whereas all these effects mediated by circRNA-PTPRA downregulation in ox-LDL-treated HUVECs were abolished by miR-671-5p downregulation. In conclusion, circRNA-PTPRA downregulation protects against ox-LDL-induced HUVECs injury by upregulating miR-671-5p, thereby providing potential therapeutic targets for AS.

Focused ultrasound-induced targeted delivery of miR-155-5p inhibitor employing biomimetic microbubbles reduces NADPH oxidase expression and inflammation in atherosclerotic ApoE-/- mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): UEFISCDI Background MicroRNAs are important biomarkers and potential therapeutic targets in atherosclerosis. MiR-155-5p amplifies inflammation in atherosclerosis by targeting negative regulators of NF-kB signaling pathway. Inhibition of pro-atherogenic miRNAs or mimicking the function of selected anti-atherogenic miRNAs have emerged as prospective oligonucleotide-base therapeutic interventions in order to correct the expression of key genes that are mechanistically linked to atherosclerotic lesion formation. Purpose The aim of this study was to develop a strategy to reduce oxidative stress and inflammation in atherosclerotic mice employing sterically stabilized biomimetic microbubbles (MB) as focused ultrasound-responsive targeted delivery systems of miR-155-5p inhibitor. Methods ApoE-/- mice were fed a normal (control) or a high-fat, cholesterol-rich diet for 14 weeks to accelerate de development of aortic atherosclerotic lesions. FAM-labelled miRNA negative control inhibitor, miRNA negative control inhibitor or miR-155-5p inhibitor were charge-coupled to VCAM1-targeted MB. Targeted MB were delivered to mice in a single dose via retro-orbital injection. Ultrasound imaging interrogation of atherosclerotic lesions in ApoE-/- mice was performed using a conventional 30 MHz transducer (Vevo 2100 System). The Sonidel SP100 sonoporator was used for ultrasound-induced targeted release of miRNA inhibitors in mice. The biodistribution of MB was monitored by high-resolution fluorescence imaging. Atherosclerotic lesions were assessed by Oil Red O staining. Real time PCR and Western blot were employed to determine the expression of NADPH oxidase (Nox) subtypes, a major source of oxidative stress, and markers of oxidative stress inflammation (4-HNE-protein adducts, inducible nitric oxide synthase/NOS2, CD68). Results Epi-fluorescence analysis demonstrated the enhanced uptake of FAM-labelled miRNA control inhibitor by lungs and the atherosclerotic aorta of ApoE-/- mice following ultrasound wave exposure (1 MHz, 2.0 w/cm2, 50% duty cycles, 15 minutes). The mRNA/protein levels of Nox1-4 subtypes, 4-HNE-protein adducts, NOS2, and macrophage infiltration marker CD68 were found significantly elevated in the atherosclerotic aorta of ApoE-/- mice. The expression levels of Nox isoforms, 4-HNE-protein adducts, NOS2, and CD68 were significantly decreased in the atherosclerotic aorta of ApoE-/- mice 1-week after administration of targeted MB carrying miR-155-5p inhibitor as compared with atherosclerotic ApoE-/- mice treated with targeted MB carrying miRNA negative control inhibitor. Conclusion Systemic administration of miR-155-5p inhibitor conjugated with VCAM1-targeted MB reduces the expression of oxidative stress and inflammation-related markers in experimental atherosclerosis. Ultrasound-induced targeted delivery of miRNA-oriented oligonucleotides could become an important theranostic strategy combining imaging and treatment procedures in clinical atherosclerosis.

TRPM2 deficiency in mice protects against atherosclerosis by inhibiting TRPM2-CD36 inflammatory axis in macrophages.

Atherosclerosis is the major cause of ischemic heart disease and stroke, the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable transient receptor potential melastatin 2 (TRPM2) plays a critical role in atherogenesis. Both global and macrophage-specific Trpm2 deletion protect Apoe−/− mice against atherosclerosis. Trpm2 deficiency reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, thereby minimizing macrophage infiltration, foam cell formation and inflammatory responses. Activation of the oxLDL receptor CD36 induces TRPM2 activity, and vice versa. In cultured macrophages, TRPM2 is activated by CD36 ligands oxLDL and thrombospondin-1 (TSP1), and deleting Trpm2 or inhibiting TRPM2 activity suppresses the activation of CD36 signaling cascade induced by oxLDL and TSP1. Our findings establish the TRPM2-CD36 axis as a molecular mechanism underlying atherogenesis, and suggest TRPM2 as a potential therapeutic target for atherosclerosis.

Specialized Proresolving Lipid Mediators: A Potential Therapeutic Target for Atherosclerosis.

Cardiovascular disease (CVD) is a global public health issue due to its high morbidity, mortality, and economic impact. The implementation of innovative therapeutic alternatives for CVD is urgently required. Specialized proresolving lipid mediators (SPMs) are bioactive compounds derived from ω-3 and ω-6 fatty acids, integrated into four families: Lipoxins, Resolvins, Protectins, and Maresins. SPMs have generated interest in recent years due to their ability to promote the resolution of inflammation associated with the pathogeneses of numerous illnesses, particularly CVD. Several preclinical studies in animal models have evidenced their ability to decrease the progression of atherosclerosis, intimal hyperplasia, and reperfusion injury via diverse mechanisms. Large-scale clinical trials are required to determine the effects of SPMs in humans. This review integrates the currently available knowledge of the therapeutic impact of SPMs in CVD from preclinical and clinical studies, along with the implicated molecular pathways. In vitro results have been promising, and as such, SPMs could soon represent a new therapeutic alternative for CVD.

DNA Methylation Aberrant in Atherosclerosis.

Atherosclerosis (AS) is a pathological process involving lipid oxidation, immune system activation, and endothelial dysfunction. The activated immune system could lead to inflammation and oxidative stress. Risk factors like aging and hyperhomocysteinemia also promote the progression of AS. Epigenetic modifications, including DNA methylation, histone modification, and non-coding RNA, are involved in the modulation of genes between the environment and AS formation. DNA methylation is one of the most important epigenetic mechanisms in the pathogenesis of AS. However, the relationship between the progression of AS and DNA methylation is not completely understood. This review will discuss the abnormal changes of DNA methylation in AS, including genome-wide hypermethylation dominating in AS with an increase of age, hypermethylation links with methyl supply and generating hyperhomocysteinemia, and the influence of oxidative stress with the demethylation process by interfering with the hydroxyl-methylation of TET proteins. The review will also summarize the current status of epigenetic treatment, which may provide new direction and potential therapeutic targets for AS.

Silencing of IGF2BP1 restrains ox-LDL-induced lipid accumulation and inflammation by reducing RUNX1 expression and promoting autophagy in macrophages.

Atherosclerosis (AS) is a chronic inflammatory disease with the formation and accumulation of macrophage-derived foam cells in the subendothelial space of blood vessels as one major characteristic. Insulin-like growth factor 2 messenger RNA (mRNA) binding protein 1 (IGF2BP1) is an RNA-binding factor and its elevation has been reported to be associated with macrophage infiltration into the atherosclerotic vascular wall. This study aims to investigate the roles of IGF2BP1 in AS-associated foam cell formation. Herein, ApoE-/- mice fed with high-fat diet developed atherosclerotic lesions in the aorta, where IGF2BP1 expression was upregulated and autophagy was impaired. IGF2BP1 expressed in F4/80+ macrophages and coexisted with p62. In vitro, IGF2BP1 expression was upregulated in RAW264.7 macrophages exposed to oxidized low-density lipoprotein (ox-LDL) (100 μg/ml). Interestingly, silencing of IGF2BP1 ameliorated ox-LDL-induced lipid accumulation and inflammation, and enhanced autophagic flux in macrophages. Furthermore, the expression of RUNX family transcription factor 1 (RUNX1), a gene that is able to inhibit autophagy in multiple cell types, was elevated in atherosclerotic aortas and in ox-LDL-treated macrophages. In addition, RNA immunoprecipitation results revealed that IGF2BP1 is bound to RUNX1 mRNA. Alterations induced by IGF2BP1 knockdown in ox-LDL-treated macrophages were abolished by RUNX1 overexpression. Furthermore, afterautophagy inhibitor 3-methyladenine administration, silencing of IGF2BP1-reduced lipid accumulation and inflammation were recovered in RAW264.7 cells. In summary, our study demonstrated that silencing of IGF2BP1 restrained ox-LDL-induced lipid accumulation and inflammation by reducing RUNX1 expression and facilitating autophagy in macrophages. IGF2BP1/RUNX1 axis may be considered as a potential therapeutic target in AS.

The lncRNA DANCR promotes development of atherosclerosis by regulating the miR-214-5p/COX20 signaling pathway

BackgroundAlthough long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) has been reported to be involved in atherosclerosis (AS) development, its specific mechanism remains unclear.MethodsDANCR expression levels in blood samples of AS patients and oxidized low-density lipoprotein (ox-LDL) treated vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The small interfering RNA targeting DANCR (si-DANCR) was used to silence DANCR expression. Cell viability was assessed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry. Levels of inflammatory cytokines, anti-oxidative enzyme superoxide dismutase (SOD) activity, and malonaldehyde (MDA) were detected by specific commercial kits. An animal AS model was established to confirm the role of DANCR/microR-214-5p/COX20 (the chaperone of cytochrome c oxidase subunit II COX2) in AS development.ResultsDANCR was significantly increased in the blood samples of AS patients and ox-LDL treated VSMCs and HUVECs. DANCR downregulation obviously increased viability and reduced apoptosis of ox-LDL-treated VSMCs and HUVECs. Meanwhile, DANCR downregulation reduced the levels of inflammatory cytokines, including interleukin (IL)-6 (IL-6), IL-1beta (IL-1β), IL-6 and tumor necrosis factor (TNF)-alpha (TNF-α) and MDA while increasing the SOD level in ox-LDL-treated VSMCs and HUVECs. DANCR regulated COX20 expression by acting as a competing endogenous RNA (ceRNA) of miR-214-5p. Rescue experiments demonstrated that miR-214-5p downregulation obviously attenuated si-DANCR-induced protective effects on ox-LDL-caused endothelial injury.ConclusionsOur results revealed that DANCR promoted AS progression by targeting the miR-214-5p/COX20 axis, suggesting that DANCR might be a potential therapeutic target for AS.

Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

Statins play a major role in reducing circulating cholesterol levels and are widely used to prevent coronary artery disease. Although they are recently confirmed to up-regulate mitophagy, little is known about the molecular mechanisms and its effect on endothelial progenitor cell (EPC). Here, we explore the role and mechanism underlying statin (pitavastatin, PTV)-activated mitophagy in EPC proliferation. ApoE−/− mice are fed a high-fat diet for 8 weeks to induce atherosclerosis. In these mice, EPC proliferation decreases and is accompanied by mitochondrial dysfunction and mitophagy impairment via the PINK1-PARK2 pathway. PTV reverses mitophagy and reduction in proliferation. Pink1 knockout or silencing Atg7 blocks PTV-induced proliferation improvement, suggesting that mitophagy contributes to the EPC proliferation increase. PTV elicits mitochondrial calcium release into the cytoplasm and further phosphorylates CAMK1. Phosphorylated CAMK1 contributes to PINK1 phosphorylation as well as mitophagy and mitochondrial function recover in EPCs. Together, our findings describe a molecular mechanism of mitophagy activation, where mitochondrial calcium release promotes CAMK1 phosphorylation of threonine177 before phosphorylation of PINK1 at serine228, which recruits PARK2 and phosphorylates its serine65 to activate mitophagy. Our results further account for the pleiotropic effects of statins on the cardiovascular system and provide a promising and potential therapeutic target for atherosclerosis.

Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence

Accumulation of senescent cells with age is an important driver of aging and age-related diseases. However, the mechanisms and signaling pathways that regulate senescence remain elusive. In this report, we performed post-genome-wide association studies (GWAS) functional studies on the CDKN2A/B locus, a locus known to be associated with multiple age-related diseases and overall human lifespan. We demonstrate that transcription factor CUX1 (Cut-Like Homeobox 1) specifically binds to an atherosclerosis-associated functional single-nucleotide polymorphism (fSNP) (rs1537371) within the locus and regulates the CDKN2A/B-encoded proteins p14ARF, p15INK4b and p16INK4a and the antisense noncoding RNA in the CDK4 (INK4) locus (ANRIL) in endothelial cells (ECs). Endothelial CUX1 expression correlates with telomeric length and is induced by both DNA-damaging agents and oxidative stress. Moreover, induction of CUX1 expression triggers both replicative and stress-induced senescence via activation of p16INK4a expression. Thus, our studies identify CUX1 as a regulator of p16INK4a-dependent endothelial senescence and a potential therapeutic target for atherosclerosis and other age-related diseases.

Knockdown of NCK1-AS1 inhibits the development of atherosclerosis by targeting miR-1197/COX10 axis

BackgroundAlthough long non-coding RNA (lncRNA) NCK1-AS1 plays important roles in human cancer, its function in atherosclerosis (AS) remains unclear.MethodThe expression of NCK1-AS1 in AS blood samples was detected by qRT-PCR. Oxidized low-density lipoprotein (ox-LDL) was used to construct the AS cell model, and quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to evaluate NCK1-AS1 level. Cell phenotypes including proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. The malondialdehyde level was measured to evaluate oxidative stress. The expression of apoptosis-related proteins was evaluated by western blot. The expression of inflammatory cytokines (IL-1β, IL-6 and TNK-α) was measured by qRT-PCR and ELISA assays. The relationship among NCK1-AS1, miR-1197 and COX10 was determined by bioinformatic analysis and luciferase reporter assay.ResultsNCK1-AS1 was significantly upregulated in AS blood samples and ox-LDL stimulated vascular smooth muscle cells (VSMCs). Knockdown of NCK1-AS1 increased cell viability, reduced cell apoptosis and MDA level, and also inhibited the expression of inflammatory cytokines (IL-1β, IL-6 and TNK-α) in ox-LDL stimulated VSMCs. NCK1-AS1 could positively regulate COX10 expression by directly sponging miR-1197. Moreover, co-transfection of sh-NCK1-AS1 and miR-1197 inhibitor, or co-transfection of sh-NCK1-AS1 and pc-COX10 (COX10 overexpressing plasmid) obviously reduced cell viability, promoted cell apoptosis, and increased MDA level in VSMCs followed by ox-LDL treatment for 24 h compared to that in sh-NCK1-AS1 transfected VSMCs.ConclusionOur study revealed that knockdown of NCK1-AS1 attenuated the development of AS by regulating miR-1197/COX10 axis, suggesting that this lncRNA might be a potential therapeutic target for AS.

GALNT4 primes monocytes adhesion and transmigration by regulating O-Glycosylation of PSGL-1 in atherosclerosis

Atherosclerosis is a major underlying cause of cardiovascular disease. Genome wide association studies have predicted that GalNAc-T4 (GALNT4), which responsible for initiating step of mucin-type O-glycosylation, plays a causal role in the susceptibility to cardiovascular diseases, whereas the precise mechanism remains obscure. Thus, we sought to determine the role and mechanism of GALNT4 in atherosclerosis. Firstly, we found the expression of GALNT4 and protein O-glycosylation were both increased in plaque as atherosclerosis progressed in ApoE−/− mice by immunohistochemistry. And the expression of GALNT4 was also increased in human monocytes treated with ACS (acute coronary syndrome) sera and subjected to LPS and ox-LDL in vitro. Moreover, silencing expression of GALNT4 by shRNA lentivirus alleviated atherosclerotic plaque formation and monocyte/macrophage infiltration in ApoE−/− mice. Functional investigations demonstrate that GALNT4 knockdown inhibited P-selectin-induced activation of β2 integrin on the surface of monocytes, decreased monocytes adhesion under flow condition with P-selectin stimulation, as well as suppressed monocytes transmigration triggered by monocyte chemotactic protein- 1(MCP-1). In contrast, GALNT4 overexpression enhanced monocytes adhesion and transmigration. Furthermore, Vicia Villosa Lectin (VVL) pull down and PSGL-1 immunoprecipitation assays showed that GALNT4 overexpression increased O-Glycosylation of PSGL-1 and P-selectin induce phosphorylation of Akt/mTOR and IκBα/NFκB on monocytes. Conversely, knockdown of GALNT4 decreased VVL binding and attenuated the activation of Akt/mTOR and IκBα/NFκB. Additionally, mTOR inhibitor rapamycin blocked these effects of GALNT4 overexpression on monocytes. Collectively, GALNT4 catalyzed PSGL-1 O-glycosylation that involved in P-selectin induced monocytes adhesion and transmigration via Akt/mTOR and NFκB pathway. Thus, GALNT4 may be a potential therapeutic target for atherosclerosis.

Abstract 11936: Macrophage-Specific DNase II Deficiency Stimulates Atherosclerosis in Apolipoprotein E-deficient Mice

Background: Atherosclerosis is a chronic inflammatory disease of the walls of blood vessels. Macrophage activated by endogenous ligands plays an important role in chronic inflammation of blood vessels. Endogenous DNA fragments derived from vascular cells in the presence of various risk factors attract much attention because endogenous DNA fragments, as well as exogenous DNA fragments induce inflammatory responses through the activation of DNA sensor, including Toll-like receptor 9 (TLR9). We hypothesized that DNase II (DN2) in macrophages degradates incorporated DNA fragments, suppressing pro-inflammatory activation of this cell-type and vascular inflammation. Therefore, we investigated the role of DN2 in macrophages in atherogenesis in apolipoprotein e-deficient (ApoE KO). Methods and Results: Macrophage-specific DN2 deleted ApoE KO mice were generated by cross breeding of DN2-flox mice, LysMCre mice, and ApoE KO mice. Mice obtained by cross breeding of DN2-flox mice and ApoE KO mice were used as the control. All mice were fed a western-type diet from 7 weeks of age and received subcutaneous angiotensin II infusion (1000 ng/kg per minute) from 8 weeks of age for 4 weeks. Macrophage-specific DNase II deletion significantly accelerated atherosclerotic lesion development in the aortic arch in ApoE KO mice (P < 0.05), accompanied with the increase in the expression of MMP-9 and TNF-α (P < 0.05, respectively) in the aorta. DN2-deletion significantly promoted the expression of inflammatory molecules such as MCP-1 (P < 0.01) in macrophages in the response to CpG-ODN 1826, a TLR9 agonist, or mitochondrial DNA. mtDNA promoted IkBα degradation in DN2-deficient macrophages, suggesting that NF-kB signal was related to DNA-induced macrophage activation. Conclusion: Our results suggested that macrophage-specific DN2 deletion accelerated pro-inflammatory activation of macrophages in the response to DNA fragments, leading to the development of atherogenesis in ApoE KO mice. DN2 may be a potential therapeutic target for atherosclerosis.