Function In Coronary Artery Disease Research Articles

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Topography of immune cell infiltration in different stages of coronary atherosclerosis revealed by multiplex immunohistochemistry.

Aim of this study was to investigate immune cells and subsets in different stages of human coronary artery disease with a novel multiplex immunohistochemistry (mIHC) technique. Human left anterior descending coronary artery specimens were analyzed: eccentric intimal thickening (N=11), pathological intimal thickening (N=10), fibroatheroma (N=9), and fibrous plaque (N=9). Eccentric intimal thickening was considered normal, and pathological intimal thickening, fibroatheroma, and fibrous plaque were considered diseased coronary arteries. Two mIHC panels, consisting of six and five primary antibodies, autofluoresence, and DAPI, were used to detect adaptive and innate immune cells. Via semi-automated analysis, (sub)types of immune cells in whole plaques and specific plaque regions were quantified. Increased numbers of CD3+ T cells (P<0.001), CD20+ B cells (P=0.013), CD68+ macrophages (P=0.003), CD15+ neutrophils (P=0.017), and CD31+ endothelial cells (P=0.024) were identified in intimas of diseased coronary arteries compared to normal. Subset analyses of T cells and macrophages showed that diseased coronary arteries contained an abundance of CD3+CD8- non-cytotoxic T cells and CD68+CD206- non-M2-like macrophages. Proportions of CD3+CD45RO+ memory T cells were similar to normal coronary arteries. Among pathological intimal thickening, fibroatheroma, and fibrous plaque, all immune cell numbers and subsets were similar. The type of immune response does not differ substantially between different stages of plaque development and may provide context for mechanistic research into immune cell function in atherosclerosis. We provide the first comprehensive map of immune cell subtypes across plaque types in coronary arteries demonstrating the potential of mIHC for vascular research.

Transcriptome-wide analysis reveals the coregulation of RNA-binding proteins and alternative splicing genes in the development of atherosclerosis

RNA-binding proteins (RBPs) are involved in the regulation of RNA splicing, stability, and localization. How RBPs control the development of atherosclerosis, is not fully understood. To explore the relevant RNA-binding proteins (RBPs) and alternative splicing events (ASEs) in atherosclerosis. We made a comprehensive work to integrate analyses of differentially expressed genes, including differential RBPs, and variable splicing characteristics related to different stages of atherosclerosis in dataset GSE104140. A total of 3712 differentially expressed genes (DEGs) were identified, including 2921 upregulated genes and 791 downregulated genes. Further analysis screened out 54 RBP genes, and 434 AS genes overlapped DEGs. We selected high expression ten RBP genes (SAMHD1, DDX60 L, TLR7, RBM47, MYEF2, RNASE6, PARP12, APOBEC3G, SMAD9, and RNASE1) for co-expression analysis. Meanwhile, we found seven regulated alternative splicing genes (RASGs) (ABI1, FXR1, CHID1, PLEC, PRKACB, BNIP2, PPP3CB) that could be regulated by RBPs. The co-expression network was used to further elucidate the regulatory and interaction relationship between RBPs and AS genes. Apoptotic process and innate immune response, revealed by the functional enrichment analysis of RASGs regulated by RBPs were closely related to atherosclerosis. In addition, 26 of the 344 alternative splicing genes regulated by the above 10 RBPs were transcription factors (TFs), We selected high expression nine TFs (TFDP1, RBBP7, STAT2, CREB5, ERG, ELF1, HMGN3, BCLAF1, and ZEB2) for co-expression analysis. The target genes of these TFs were mainly enriched in inflammatory and immune response pathways that were associated with atherosclerosis. indicating that AS abnormalities of these TFs may have a function in atherosclerosis. Furthermore, the expression of differentially expressed RBPs and the alternative splicing events of AS genes was validated by qRT-PCR in umbilical vein endothelial cells (HUVEC). The results showed that RBM47 were remarkedly difference in HUVEC treated with ox-LDL and the splicing ratio of AS in BCLAF1which is regulated by RBM47 significantly changed. In conclusion, the differentially expressed RBPs identified in our analysis may play important roles in the development of atherosclerosis by regulating the AS of these TF genes.

Extracellular traps from activated vascular smooth muscle cells drive the progression of atherosclerosis

Extracellular DNA traps (ETs) represent an immune response by which cells release essential materials like chromatin and granular proteins. Previous studies have demonstrated that the transdifferentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. This study seeks to investigate the interaction between CD68+ VSMCs and the formation of ETs and highlight its function in atherosclerosis. Here we show that ETs are inhibited, and atherosclerotic plaque formation is alleviated in male Myh11CrePad4flox/flox mice undergoing an adeno-associated-virus-8 (AAV8) mediating overexpression of proprotein convertase subtilisin/kexin type 9 mutation (PCSK9) injection and being challenged with a high-fat diet. Obvious ETs generated from CD68+ VSMCs are inhibited by Cl-amidine and DNase I in vitro. By utilizing VSMCs-lineage tracing technology and single-cell RNA sequencing (scRNA-seq), we demonstrate that the ETs from CD68+ VSMCs influence the progress of atherosclerosis by regulating the direction of VSMCs’ transdifferentiation through STING-SOCS1 or TLR4 signaling pathway.

TREATMENT OF ELDERLY PEOPLE WITH SENSORINEURAL HEARING LOSS

Age-related changes in auditory function in atherosclerosis are based on morphological changes in the inner ear. The problem of pathology of the inner ear, which occurs against the background of vascular atherosclerosis, has not been sufficiently studied, and therefore it is advisable to conduct further study of the state of cerebral circulation for the diagnosis and treatment of elderly patients. To achieve this goal, we examined 60 people aged 55 to 70 years. Taking into account the data on the state of auditory function, REG, ECG and other studies, patients suffering from sensorineural hearing loss on the background of atherosclerosis and osteochondrosis of the cervical spine were treated. In the presence of osteochondrosis of the cervical spine, patients were recommended physical therapy, massage of the cervical-thoracic spine up to 10-20 sessions (taking into account blood pressure), complamin, calcium pangamate, cerebro-lysine, relanium. After the therapy, all patients showed an improvement in the perception of whispered and spoken speech.

The intracellular signaling pathways governing macrophage activation and function in human atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and plaque formation in arterial vessel walls. Atherosclerotic plaques narrow the arterial lumen to increase the risk of heart attacks, ischemic stroke and peripheral vascular disease, which are major and worldwide health and economic burdens. Macrophage accumulation within plaques is characteristic of all stages of atherosclerosis and their presence is a potential marker of disease activity and plaque stability. Macrophages engulf lipids and modified lipoproteins to form foam cells that express pro-inflammatory and chemotactic effector molecules, stress inducing factors and reactive oxygen species. They control plaque stability and rupture through secretion of metalloproteinases and extracellular matrix degradation. Although macrophages can worsen disease by propagating inflammation, they can stabilize atherosclerotic plaques through tissue remodeling, promoting the formation of a fibrous cap, clearing apoptotic cells to prevent necrotic core formation and through vascular repair. In atherosclerosis, macrophages respond to dyslipidaemia, cytokines, dying cells, metabolic factors, lipids, physical stimuli and epigenetic factors and exhibit heterogeneity in their activation depending on the stimuli they receive. Understanding these signals and the pathways driving macrophage function within developing and established plaques and how they can be pharmacologically modulated, represents a strategy for the prevention and treatment of atherosclerosis. This review focusses on the current understanding of factors controlling macrophage heterogeneity and function in atherosclerosis. Particular attention is given to the macrophage intracellular signaling pathways and transcription factors activated by biochemical and biophysical stimuli within plaques, and how they are integrated to regulate plaque formation and stability.

Abstract 15273: Impact of Lipid Lowering Therapy on Progenitor Endothelial Cell Phenotype and Function in Coronary Artery Disease

Introduction: Early revascularization of infarcted zone is crucial to improve patient′s outcomes. Despite their primary actions, major lipid-lowering drugs may also reduce cardiovascular events by pleiotropic mechanisms. Hypothesis: Conventional (statin) and novel lipid-lowering therapies [(PSCK9-inhibitor (PCSK9i) and eicosatetraenoic acid (EPA)] can improve endothelial progenitor cells (EPC) phenotype and favor neovascularization. Methods: EPC were isolated from peripheral blood of patients with acute coronary syndrome (n=62) who were statin-naïve (n=34) or chronic statin treatment (n=28) at time of inclusion. A set of statin-naïve EPC and mature endothelial cells (HUVEC) were treated in vitro with statin (20nM), PCSKi (1μM) and EPA (3μM). EPC phenotype was determined as EPC colony number and time of appearance, proliferative rate, and ability to form capillary-like structures. Serum PCSK9 and markers of angiogenesis and endothelial function were determined by multiplexed protein assay. Results: Frequency of EPC appearance was higher in samples of statin group (78%) compared to naïve group (66%) (Fig A). They also appeared much earlier (Fig B), in greater quantity (4.7 vs 2.1 colony, p = 0.0009) and had higher proliferative rate than EPCs of naïve group. Besides they had increased potential of tube formation and shaped more branched vessel-like structures (Fig C). No differences in serum levels of biomarkers were found. In vitro treatment of EPC with statin, PCSK9i and EPA did not modify colonies behavior (Fig D). PCSK9 levels showed negative correlation with EPC proliferation (r -0.4150, p=0.004), and PCSK9i treatment increased angiogenic potential of HUVEC (Fig E). Conclusions: Statin and PCSK9i have pleiotropic effects on EPC, boosting their potential to form endothelial cells colonies and to induce angiogenesis. These effects could contribute to the progression and outcomes of coronary artery disease. Funding: ISCIII - FEDER-ERDF (PI19/00264)

Abstract 12293: Retinoic Acid Signaling Regulates Smooth Muscle Cell Phenotypic Switching in Atherosclerosis Through Epigenetic Modulation of Gene Expression

Background: Smooth muscle cells (SMCs) substantially contribute to atherosclerosis through “phenotypic switching.” Our previous work identified an intermediate SMC-derived cell type, termed “SEM” cells, which was multipotent and activated in inflammatory response. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuated atherosclerosis in mice coincident with dramatic suppression of SEM cell formation from SMCs. However, the regulatory mechanisms by which RA signaling modulates SMC transition to SEM cells are largely unknown. Methods: We employed molecular and cell biology techniques, SMC-linage tracing and atheroprone mouse models, and next-generation sequencing (e.g., RNA-seq, ChIP-seq) to reveal how RA signaling modulates SMC transition to SEM cells. Results: Activation of RA signaling with ATRA significantly reduced SEM cells in established atherosclerosis, as well as downregulated the expression of SEM cell marker genes (e.g., Ly6a , Ly6c1 ) and repressed inflammatory response in ex vivo SEM cells, whereas inhibition of the signaling with antagonist, BMS49334, showed opposite results. RARα occupied the promoter regions of SEM cell marker genes, and ATRA treatment significantly increased the enrichment of NCOR1 at promoters of these genes. These findings suggest that RA signaling suppresses SMC transition to SEM cells via directly repressing the expression of SEM cell marker genes. Furthermore, we found EZH2, one of the subunits of PRC2, physically interacted with RARα in SMCs and occupied the promoters of SEM cell marker genes, and its methyltransferase activity at the promoter regions was responsible for the repression of SEM cell maker genes. Moreover, activation of RA signaling inhibited SEM cell inflammatory response through LXR-mediated suppression of a series of inflammatory genes. Finally, multiple epigenetic signatures (e.g., H3K27me3, H3K27ac, H3K4me3, etc.) at TSS of SEM cell marker genes and inflammatory genes were extensively altered in response to the activation of RA signaling. Conclusions: Our findings indicate that RA signaling modulates maintenance of SEM cell identity and inflammatory function in atherosclerosis by epigenetic regulation of gene expression.

Integration of CAD-associated GWAS loci and deconvolution from human carotid plaques to study smooth muscle cell function in atherosclerosis

Background and Aims : A central role of smooth muscle cells (SMCs) in atherosclerosis has recently evolved implying genetic and causal links in the disease. Here, we hypothesized that coronary artery disease (CAD)-related gene polymorphisms influence SMC function in atherosclerosis and investigated the role of genes harboring such variants in SMCs in vitro.

Epigenetic Control of Vascular Smooth Muscle Cell Function in Atherosclerosis: A Role for DNA Methylation.

Atherosclerosis is a complex vascular inflammatory disease in which multiple cell types are involved, including vascular smooth muscle cells (VSMCs). In response to vascular injury and inflammatory stimuli, VSMCs undergo a "phenotypic switching" characterized by extracellular matrix secretion, loss of contractility, and abnormal proliferation and migration, which play a key role in the progression of atherosclerosis. DNA methylation modification is an important epigenetic mechanism that plays an important role in atherosclerosis. Studies investigating abnormal DNA methylation in patients with atherosclerosis have determined a specific DNA methylation profile, and proposed multiple pathways and genes involved in the etiopathogenesis of atherosclerosis. Recent studies have also revealed that DNA methylation modification controls VSMC function by regulating gene expression involved in atherosclerosis. In this review, we summarize the recent advances regarding the epigenetic control of VSMC function by DNA methylation in atherosclerosis and provide insights into the development of VSMC-centered therapeutic strategies.

Inflammation-induced arterial dysfunction in atherosclerosis; the modulating action of mesenchymal stem cell-derived extracellular vesicles

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): CNCS-UEFISCDI Background Atherosclerosis is a progressive, chronic inflammatory disease of the large arteries caused by the constant accumulation of cholesterol, followed by endothelial dysfunction and vascular inflammation. We hypothesised that delivery of extracellular vesicles (EVs), recognized for their potential as therapeutic targets and tools, could restore vascular function in atherosclerosis. Purpose The goal was to evaluate vascular consequences of inflammation along with its key molecular and cellular players as intermediate endpoints for cardiovascular disease (CVD), and to explore ‘in vivo’ the potential beneficial effect of EVs from subcutaneous adipose tissue (EVs-ADSCs) or bone marrow mesenchymal stem cells (EVs-MSCs). Methods Our experimental animal model of atherosclerosis (hypertensive-hyperlipidemic hamster-HH) was treated with EVs-ADSCs or EVs-MSCs transfected or not with Smad2/3siRNA. For comparison, healthy animals (control group) or HH group transfected with Smad2/3siRNA alone were used. Results The results showed that compared with the control group, HH group displayed: (1) a marked increase of plasmatic parameter levels (cholesterol, LDL-cholesterol, triglycerides); (2) an amplification of expression of inflammatory markers with role in vascular dysfunction (COL1A, α-SMA, Cx43, VCAM-1, MMP-2); (3) a massive infiltration of total/M1 macrophages and T-cells into thoracic aorta and carotid artery; (4) an increased level of cytosolic ROS production; (5) a significant increase in plasma concentration of TGF-β1 and Ang II proteins; (6) significant structural (thickening of the arterial wall and reduction of the inner diameter) and functional changes (reduced distensibility, increased pulse wave velocity and velocity time integral, and diminished contractile responses to noradrenaline (NA) and reduced relaxation to acetylcholine (ACh)) in both thoracic aorta and carotid artery; (7) an altered protein expression profile (Smad2/3, ATF-2, NF-kBp50/p65) and a significant increase in the expression level of the miRNA panel (miR21, miR-29a, miR-192, miR-200b, miR-210, miR-146a) associated with inflammation-induced endothelial dysfunction. In addition, by comparison with the HH group, all treatment groups showed a significant decrease in the levels of plasma parameters, but also a noticeable improvement in the structure and function of the investigated blood vessels along with a decrease in the key molecules that modulate the inflammatory response through soluble mediators and cellular components. Conclusion We can conclude that the administration for 4 months of atherogenic diet activated key regulatory molecules in the atherosclerotic process, respectively TGF-β1, Ang II and ROS, modulating main orchestrators of inflammation-mediated atherosclerotic CVD progression. Also, EV-based treatment of inflammation, especially the EV-ADSC-based one, led to regression of arterial dysfunction and its key molecular players.

Abstract 151: Macrophage Specific Ablation Of Bmal1 Regulate Cholesterol Metabolism And Atherosclerosis

Macrophages play a critical role in atherosclerosis. We have previously shown that Clock mutant and global or liver-specific Bmal1 deficient mice show enhanced atherosclerosis. Yet, the role of macrophage Bmal1 in cholesterol transport and atherosclerosis remains unknown. Here, we used global and macrophage-specific ablation of Bmal1 to clarify the role of Bmal1 in macrophage function in atherosclerosis. We found that macrophage-specific Bmal1 deficient mice show increased atherosclerosis. Biochemical studies showed that Bmal1 deficient macrophages exhibit increased expression of Lox-1 and Cd36 and take up more oxLDL. Furthermore, they show decreased expression of Abca1 and Abcg1 and reduced cholesterol efflux as well as in vivo reverse cholesterol transport. Thus, Bmal1 regulates macrophage cholesterol metabolism by regulating uptake of oxidized lipoproteins and cholesterol efflux pathways. Molecular studies showed that Bmal1 indirectly regulates Cd36 and Abca1/Abcg1 by modulating Nr1d1 and Znf202, respectively. We provide evidence that Bmal1 collaborates with Clock and interacts with Nr1d1 promoter. Furthermore, our studies show that Bmal1 interacts with both Clock and Npas2 to regulate Znf202. Thus, Bmal1 regulates two different transcription factors to regulate uptake of oxidized lipoproteins and cholesterol efflux. In summary, our data show that Bmal1 is a key regulator of cholesterol metabolism in macrophages and atherosclerosis. It regulates macrophage function by modulating the expression of different transcription factors. It is likely that augmentation of Bmal1 may prevent atherosclerosis.

Abstract 218: Integration Of Coronary Artery Disease GWAS With Bulk And Single-cell Transcriptomics From Atherosclerotic Plaques By Deconvolution, Reveals Novel Smooth Muscle Cell Genes

Background and Aim: A central role of vascular smooth muscle cells (SMCs) in atherosclerosis has recently evolved that suggests causal genetic links to disease processes. Single cell sequencing studies of atherosclerotic plaques have identified multiple mesenchymal transition cell populations within the plaques. Here, we correlated cell fractions from plaques to coronary artery disease (CAD) related gene polymorphisms to identify novel SMC targets and study their influence on SMC function in atherosclerosis. Methods: Deconvolution analysis was performed on bulk microarray data from carotid plaques in the Biobank of Karolinska Endarterectomies (BiKE, n=127) using single cell sequencing data from coronary plaques (n=5). CAD-associated GWAS loci associated with mesenchymal cell fractions were identified, followed by functional analyses of these genes in SMC in vitro using migration, proliferation and apoptosis assays. Results: We identified 5 mesenchymal cell-specific genetic variants associated with CAD, BiKE patient symptomatology and gene expression eQTLs in BiKE plaque tissue and GTex normal arteries. These variants were harbored in genetic loci of ARNTL, LDLR, MIA3, PAK1 and ARHGAP15 . Microarray analysis revealed increased expression of ARHGAP15 and PAK1 and decreased levels of LDLR in carotid plaques compared with normal arteries (n=127 vs. 10 respectively, student’s t-test). Immunohistochemistry demonstrated increased expression of corresponding proteins in the fibrous cap of plaques compared to normal arteries (n=5). To investigate their function in SMCs, the genes were silenced using siRNAs followed by migration, proliferation and apoptosis assays. Preliminary results indicated that silencing of MIA3, LDLR and ARNTL inhibited SMC proliferation. Conclusions: The results of this project may reveal novel SMC-specific genetic links to the disease, which may serve as therapeutic targets to be explored for improved treatment of atherosclerosis.

Sirtuin 3 Dependent and Independent Effects of NAD+ to Suppress Vascular Inflammation and Improve Endothelial Function in Mice

Atherosclerosis is initiated by endothelial cell dysfunction and vascular inflammation under the condition of hyperlipidemia. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD+)-dependent mitochondrial deacetylase, which plays a key role in maintaining normal mitochondrial function. The present study tested whether endothelial-selective SIRT3 deletion accelerates vascular inflammation and oxidative stress, and assessed the protective effect of NAD+ to alleviate these changes in endothelial cells and in mouse models of atherosclerosis. We found that the selective deletion of SIRT3 in endothelial cells further impaired endothelium-dependent vasodilatation in the aorta treated with IL-1β, which was accompanied by upregulation of vascular inflammation markers and mitochondrial superoxide overproduction. Excepting the dysfunction of endothelium-dependent vasodilatation, such effects could be attenuated by treatment with NAD+. In human umbilical vein endothelial cells, SIRT3 silencing potentiated the induction of inflammatory factors by IL-1β, including VCAM-1, ICAM-1, and MCP1, and the impairment of mitochondrial respiration, both of which were alleviated by NAD+ treatment. In ApoE-deficient mice fed with a high-cholesterol diet, supplementation with nicotinamide riboside, the NAD+ precursor, reduced plaque formation, improved vascular function, and diminished vascular inflammation. Our results support the SIRT3-dependent and -independent of NAD+ to improve endothelial function in atherosclerosis.

Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players.

Atherosclerosis is a progressive, chronic inflammatory disease of the large arteries caused by the constant accumulation of cholesterol, followed by endothelial dysfunction and vascular inflammation. We hypothesized that delivery of extracellular vesicles (EVs), recognized for their potential as therapeutic targets and tools, could restore vascular function in atherosclerosis. We explored by comparison the potential beneficial effects of EVs from subcutaneous adipose tissue stem cells (EVs (ADSCs)) or bone marrow mesenchymal stem cells (EVs (MSCs)) on the consequences of atherogenic diet on vascular health. Also, the influences of siRNA-targeting Smad2/3 (Smad2/3siRNA) on endothelial dysfunction and its key molecular players were analyzed. For this study, an animal model of atherosclerosis (HH) was transplanted with EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3siRNA. For controls, healthy or HH animals were used. The results indicated that by comparison with the HH group, the treatment with EVs(ADSCs) or EVs(MSCs) alone or in combination with Smad2/3siRNA of HH animals induced a significant decrease in the main plasma parameters and a noticeable improvement in the structure and function of the thoracic aorta and carotid artery along with a decrease in the selected molecular and cellular targets mediating their changes in atherosclerosis: 1) a decrease in expression of structural and inflammatory markers COL1A1, α-SMA, Cx43, VCAM-1, and MMP-2; 2) a slight infiltration of total/M1 macrophages and T-cells; 3) a reduced level of cytosolic ROS production; 4) a significant diminution in plasma concentrations of TGF-β1 and Ang II proteins; 5) significant structural and functional improvements (thinning of the arterial wall, increase of the inner diameter, enhanced distensibility, diminished VTI and Vel, and augmented contractile and relaxation responses); 6) a reduced protein expression profile of Smad2/3, ATF-2, and NF-kBp50/p65 and a significant decrease in the expression levels of miR-21, miR-29a, miR-192, miR-200b, miR-210, and miR-146a. We can conclude that 1) stem cell-derived EV therapies, especially the EVs (ADSCs) led to regression of structural and functional changes in the vascular wall and of key orchestrator expression in the atherosclerosis-induced endothelial dysfunction; 2) transfection of EVs with Smad2/3siRNA amplified the ability of EVs(ADSCs) or EVs(MSCs) to regress the inflammation-mediated atherosclerotic process.

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.

Targeting PIEZO1 Refines Macrophage Function in Atherosclerosis

Targeting PIEZO1 Refines Macrophage Function in Atherosclerosis

Use of Mouse Carotid Artery Ligation Model of Intimal Thickening to Probe Vascular Smooth Muscle Cell Remodeling and Function in Atherosclerosis.

The thickening of the intima is a critical underlying component of atherosclerosis. Consequently, robust and reproducible animal models of intimal thickening are essential for a greater understanding of the mechanisms underlying the process of intimal thickening and to evaluate new approaches for the reduction of intimal thickening and thereby atherosclerosis. The ligation of the carotid artery in the mouse causes the thickening of the intimal layer of the artery. This model is relatively simple and is reproducible and therefore is a preferred and well-established model of intimal thickening. Here, we detail a protocol for carotid artery ligation in the mouse and methods for histological examination and quantification of intimal thickening.

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of vascular damage and thought to drive atherosclerosis. A method to identify these changes in vivo would be desirable in the clinic. Magnetic resonance imaging (MRI)-based tools and other technologies have enabled a profound understanding of the role of the endothelium in cardiovascular diseases and risk in vivo. There is, however, a need for reproducible and simple approaches for extracting quantifiable data reflective of endothelial damage from a single imaging study. A non-invasive, easy-to-implement, and quantitative MRI workflow was developed to acquire and analyze images that allow the quantification of two imaging biomarkers of arterial endothelial damage (leakiness/permeability and dysfunction). Here, the protocol describes the application of this method in the brachiocephalic artery of atherosclerotic ApoE-/- mice using a clinical MRI scanner. First, late gadolinium enhancement (LGE) and Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping protocols to quantify endothelial leakage using an albumin-binding probe are described. Second, anatomic, and quantitative blood flow sequences to measure endothelial dysfunction, in response to acetylcholine are described. Importantly, the method outlined here allows the acquisition of high-spatial-resolution 3D images with large volumetric coverage enabling accurate segmentation of vessel wall structures to improve inter- and intra-observer variability and to increase reliability and reproducibility. Additionally, it provides quantitative data without the need for high-temporal resolution for complex kinetic modeling, making it model-independent and even allowing for imaging of highly mobile vessels (coronary arteries). Therefore, the approach simplifies and expedites data analysis. Finally, this method can be implemented on different scanners, can be extended to image different arterial beds, and is clinically applicable for use in humans. This method could be used to diagnose and treat patients with atherosclerosis by adopting a precision-medicine approach.

LKB1 Regulates Vascular Macrophage Functions in Atherosclerosis.

Liver kinase B1 (LKB1) is known to shape the regulation of macrophage function by participating in multiple processes including cell metabolism, growth, and polarization. However, whether LKB1 also affects the functional plasticity of macrophages in atherosclerosis has not attracted much attention. Abnormal macrophage function is a pathophysiological hallmark of atherosclerosis, characterized by the formation of foam cells and the maintenance of vascular inflammation. Mounting evidence supports that LKB1 plays a vital role in the regulation of macrophage function in atherosclerosis, including affecting lipid metabolism reprogramming, inflammation, endoplasmic reticulum stress, and autophagy in macrophages. Thus, decreased expression of LKB1 in atherosclerosis aggravates vascular injury by inducing excessive lipid deposition in macrophages and the formation of foam cells. To systematically understand the role and potential mechanism of LKB1 in regulating macrophage functions in atherosclerosis, this review summarizes the relevant data in this regard, hoping to provide new ideas for the prevention and treatment of atherosclerosis.