Advanced Atherosclerotic Lesions Research Articles

NOX4 mRNA correlates with plaque stability in patients with carotid artery stenosis

Carotid artery stenosis (CAS) develops from atherosclerotic lesions and plaques. Plaque rupture or stenosis may result in occlusion of the carotid artery. Accordingly, the asymptomatic disease becomes symptomatic, characterized by ischemic stroke or transient ischemic attacks, indicating an urgent need for better understanding of the underlying molecular mechanisms and eventually prevent symptomatic CAS. NOX4, a member of the NADPH oxidase family, has anti-atherosclerotic and anti-inflammatory properties in animal models of early atherosclerosis. We hypothesized that NOX4 mRNA expression is linked to protective mechanisms in CAS patients with advanced atherosclerotic lesions as well. Indeed, NOX4 mRNA expression is lower in patients with symptomatic CAS. A low NOX4 mRNA expression is associated with an increased risk of the development of clinical symptoms. In fact, NOX4 appears to be linked to plaque stability, apoptosis and plaque hemorrhage. This is supported by cleaved caspase-3 and glycophorin C and correlates inversely with plaque NOX4 mRNA expression. Even healing of a ruptured plaque appears to be connected to NOX4, as NOX4 mRNA expression correlates to fibrous cap collagen and is reciprocally related to MMP9 activity. In conclusion, low intra-plaque NOX4 mRNA expression is associated with an increased risk for symptomatic outcome and with reduced plaque stabilizing mechanisms suggesting protective effects of NOX4 in human advanced atherosclerosis.

Abstract P3124: Treatment Of Apoe -/- Mice With Advanced Atherosclerosis With The Senolytic Agent Abt-263 Caused Increased Mortality And Was Associated With Increased Endo-mt But Reduced Acta2 Cap Thickness And Collagen Content

Background: Plaque rupture and erosion of advanced atherosclerotic lesions resulting in ischemic heart disease or stroke are the leading worldwide cause of death. Multiple studies have shown that senescent cells are present in human atherosclerotic lesions, and recent animal studies have claimed that removing senescent cells from the lesions using senolytic agents has some beneficial effects but also limitations. Although these mouse studies have important implications for using senolytic agents to treat atherosclerosis, due to the lack of lineage tracing models and relying on markers not specific for various lesion cell types, it is difficult to ascertain the mechanisms that underline the beneficial effects shown by previous studies. Hypothesis: Treatment of western diet (WD) fed Apoe -/- mice with the senolytic agent ABT-263 (Navitoclax) will reduce lesion size and induce changes consistent with increased plaque stability including a thicker collagen-rich ACTA2 fibrous cap. Methods: SMC (Myh11-ERT2-Cre-eYFP+) and EC (Cdh5-ERT2-Cre-eYFP+) lineage tracing Apoe -/- were fed a WD for 18 weeks, followed by ABT-263 (100MG or 50MG /KG/BW) treatment for six to nine weeks on WD and analyzed for the indices of stability. Results: ABT-263 treatment did not result in changes in lesion size, lumen area, or outward remodeling of the brachiocephalic artery (BCA). However, surprisingly, ABT-263 treatment was associated with an 80% reduction in SMC (Myh11-eYFP+) but a 50-60% increase in endothelial cell (EC) contributions to lesions via EC to mesenchymal transition (EndoMT) (Chd5-eYFP+). ABT-263 treatment also reduced ACTA2 cap thickness by 50-75% and collagen content by 50%, but increased mortality by 50-60%. Conclusions: Taken together, contrary to our hypothesis, the senolytic agent ABT-263 treatment on advanced atherosclerosis showed multiple detrimental effects, including reduced indices of stability, increased Endo-MT, and increased mortality.

Telomere length in atherosclerotic lesions of human aortic intima

Background and Aims : The early studies of atherosclerotic plaques have demonstrated that the cells in advanced atherosclerotic lesions display changes of senescence, and telomere shortening is generally believed to be the molecular mechanism that triggers the onset of cellular senescence. This study was undertaken to test the hypothesis on the association of telomere length and the type of atherosclerotic lesion of human aorta.

ILRUN Promotes Atherosclerosis Through Lipid-Dependent and Lipid-Independent Factors.

Common genetic variation in close proximity to the ILRUN gene are significantly associated with coronary artery disease as well as with plasma lipid traits. We recently demonstrated that hepatic inflammation and lipid regulator with ubiquitin-associated domain-like and NBR1-like domains (ILRUN) regulates lipoprotein metabolism in vivo in mice. However, whether ILRUN, which is expressed in vascular cells, directly impacts atherogenesis remains unclear. We sought to determine the role of ILRUN in atherosclerosis development in mice. For our study, we generated global Ilrun-deficient (IlrunKO) male and female mice on 2 hyperlipidemic backgrounds: low density lipoprotein receptor knockout (LdlrKO) and apolipoprotein E knockout (ApoeKO; double knockout [DKO]). Compared with littermate control mice (single LdlrKO or ApoeKO), deletion of Ilrun in DKO mice resulted in significantly attenuated both early and advanced atherosclerotic lesion development, as well as reduced necrotic area. DKO mice also had significantly decreased plasma cholesterol levels, primarily attributable to non-HDL (high-density lipoprotein) cholesterol. Hepatic-specific reconstitution of ILRUN in DKO mice on the ApoeKO background normalized plasma lipids, but atherosclerotic lesion area and necrotic area remained reduced in DKO mice. Further analysis showed that loss of Ilrun increased efferocytosis receptor MerTK expression in macrophages, enhanced in vitro efferocytosis, and significantly improved in situ efferocytosis in advanced lesions. Our results support ILRUN as an important novel regulator of atherogenesis that promotes lesion progression and necrosis. It influences atherosclerosis through both plasma lipid-dependent and lipid-independent mechanisms. These findings support ILRUN as the likely causal gene responsible for genetic association of variants with coronary artery disease at this locus and suggest that suppression of ILRUN activity might be expected to reduce atherosclerosis.

Increased Remnant Lipoproteins in Apo E Deficient Mice Induce Coronary Atherosclerosis following Transverse Aortic Constriction and Aggravate the Development of Pressure Overload-Induced Cardiac Hypertrophy and Heart Failure.

Murine coronary arteries are very resistant to the development of atherosclerosis, which may be related to their intramyocardial course. Blood pressure promotes atherosclerotic plaque formation by acting as a physical force that potentiates the migration of pro-atherogenic lipoproteins across the endothelium. C57BL/6N apolipoprotein (apo) E deficient mice have increased remnant lipoproteins that are a risk factor for coronary atherosclerosis. In this study, our aim was to quantify coronary atherosclerosis and artery remodeling following transverse aortic constriction (TAC) in C57BL/6N apo E−/− mice and to evaluate the impact of increased remnant lipoproteins on the development of pressure overload-induced cardiac hypertrophy and heart failure. Advanced atherosclerotic lesions were observed in the left coronary artery of C57BL/6N apo E−/− TAC mice but not in C57BL/6N TAC mice. Pressure overload resulted in markedly increased cardiac hypertrophy and more pronounced heart failure in C57BL/6N apo E−/− TAC mice in comparison to C57BL/6N TAC mice. Pathological hypertrophy, as evidenced by increased myocardial fibrosis and capillary rarefaction, was more prominent in C57BL/6N TAC apo E−/− than in C57BL/6N TAC mice and led to more marked cardiac dysfunction. In conclusion, TAC in apo E deficient mice induces coronary atherosclerosis and aggravates the development of pathological cardiac hypertrophy and heart failure.

Effect of type 2 diabetes on A disintegrin and metalloprotease 10.

BackgroundAs a type 1 transmembrane protein, a disintegrin and metalloprotease 10 (ADAM10) is responsible for the cleavage of a variety of cell surface molecules and has been implicated in the pathogenesis of Alzheimer disease, atherosclerosis, and inflammatory and neoplastic disorders. It has been suggested that systemic ADAM10 concentration may potentially be used as a prognostic biomarker. Since high glucose can upregulate ADAM10 expression in vitro, we investigated whether serum levels of ADAM10 and its substrate, the lectin‐like oxidized low‐density lipoprotein receptor 1 (LOX‐1), can be influenced by type 2 diabetes.MethodsA total of 1091 individuals with type 2 diabetes and 358 age‐matched healthy control subjects were recruited. Serum concentrations of ADAM10 and the soluble form of LOX‐1 (sLOX‐1) released by cleavage of LOX‐1 by ADAM were measured by enzyme‐linked immunosorbent assay kits (ELISA).ResultsSerum ADAM10 was increased in subjects with diabetes compared with control (40.5 ng/mL [22.3‐65.7] vs 10.3 ng/mL [7.0‐17.9], respectively; P < .01); the highest levels were seen in insulin‐treated subjects. On multiple linear regression analysis, glycosylated hemoglobin, age, body mass index, and insulin use were independent determinants of ADAM10 level. The increase in serum ADAM10 levels in diabetes was accompanied by changes in serum sLOX‐1. Subjects with diabetes had higher serum sLOX‐1 than the control (110 pg/mL [89‐153] vs 104 pg/mL [85‐138], respectively; P < .01), and there was a significant correlation between serum ADAM10 and sLOX‐1 (r = 0.26, P < .01).ConclusionsSerum concentration of ADAM10 is increased in type 2 diabetes and is associated with glycemia and insulin therapy, which may potentially affect the specificity of systemic ADAM10 level as a biomarker.

Increased frequency of proangiogenic tunica intima endothelial kinase 2 (Tie2) expressing monocytes in individuals with type 2 diabetes mellitus

BackgroundIndividuals with type 2 diabetes mellitus (T2DM) have an increased risk for developing macrovascular disease (MVD) manifested by atherosclerosis. Phenotypically and functionally different monocyte subsets (classical; CD14++CD16−, non-classical; CD14+CD16++, and intermediate; CD14++CD16+) including pro-angiogenic monocytes expressing Tie2 (TEMs) can be identified. Here we investigated monocyte heterogeneity and its association with T2DM and MVD.MethodsIndividuals with (N = 51) and without (N = 56) T2DM were recruited and allocated to "non-MVD" or "with MVD" (i.e., peripheral or coronary artery disease) subgroups. Blood monocyte subsets were quantified based on CD14, CD16 and Tie2 expression levels. Plasma levels of Tie2-ligands angiopoietin-1 and angiopoietin-2 were determined using ELISA. Carotid endarterectomy samples from individuals with (N = 24) and without (N = 22) T2DM were stained for intraplaque CD68+ macrophages (inflammation) and CD34+ (angiogenesis), as plaque vulnerability markers.ResultsMonocyte counts were similar between individuals with T2DM and healthy controls (non-diabetic, non-MVD). Non-classical monocytes were reduced (p < 0.05) in T2DM, whereas the percentage of TEMs within the intermediate subset was increased (p < 0.05). T2DM was associated with increased angiopoietin-1 (p < 0.05) and angiopoietin-2 (p = 0.0001) levels. Angiopoietin-2 levels were higher in T2DM individuals with MVD compared with non-MVD (p < 0.01). Endarterectomized plaques showed no differences in macrophage influx and microvessel number between individuals with and without T2DM.ConclusionsMonocyte subset distribution is altered in T2DM with reduced non-classical monocytes and increased TEM percentage in the intermediate monocyte subset. Increased angiopoietin-2 levels together with increased frequency of TEMs might promote plaque vulnerability in T2DM which could however not be confirmed at tissue level in advanced atherosclerotic lesions.

Proximal vertebral artery surgery. Results of treatment of ischemia of the rhombencephalon (hindbrain). Simultaneous carotid and vertebral artery decompression procedures.

Surgical treatment of hindbrain ischemia is becoming the accepted surgical procedure in cases of stenosis of extracranial segments of vertebral arteries in the course of advanced atherosclerotic lesions in their initial segments (V1) or caused by external compression of these arteries in the canal formed by the transverse processes of cervical vertebrae. The latter is referred to as vertebrobasilar insufficiency (VBI). The transposition of the left vertebral artery to the common carotid artery in cases where stent graft implantation is necessary with coverage of the subclavian artery and possible blood supply disorders of both the hindbrain and the spinal cord has become important. In the Department of Vascular Surgery at the Medical University in Wrocław , in 58 out of 76 patients treated for ischemia of the hindbrain, the procedures were performed in the distal vertebral artery. The authors present the diagnosis of 18 patients treated surgically for stenosis or kinking of vertebral arteries in their first segment. In 12 cases, the procedure was performed with simultaneous unblocking of significantly narrowed left internal carotid artery. The material includes patients operated on between 1994 and 2018, most of whom had a good or very good outcome with zero mortality.

The Impact of Gclm on Efferocytosis and Atherosclerotic Progression

Atherosclerosis is the leading cause of coronary artery disease (CAD) and stroke which are the primary contributors to cardiovascular disease related deaths in the world. This is an inflammatory disease characterized by the accumulation of lipid and fibrous elements in large arteries resulting in the development of a plaque. Progression of the disease leads to the accumulation of apoptotic cells and formation of a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion resulting in stroke or myocardial infarction (MI). One of the main contributors to a large necrotic core is macrophages that are unable to clear the accumulated apoptotic cells and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap (characteristic of an unstable plaque) and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability due to a deletion on the modifier subunit of Glutamate Cysteine Ligase (GCLM). The objective of this study is to identify the role and mechanism of GSH during the formation of the necrotic core in atherosclerosis. We hypothesize that availability of GSH is important in macrophages for adequate efferocytosis of apoptotic cells during atherosclerosis progression.MethodsTriglycerides, total cholesterol, and HDL levels were measured on PCSK9 virus driven atherosclerosis in wild type and heterozygous GCLM mice. Sections of the aortic root from these mice were stained with Movat Pentachrome to measure plaque area and percent necrosis. An efferocytic index was measured using PHK26 labelled apoptotic cells (AC). AC were added to cultured bone marrow derived macrophages (BMDM) allowing for their uptake. Percent positive macrophages containing a fluorescently labelled AC were quantified. BMDM from WT, Het, and KO GCLM mice were collected for total protein, surface receptor expression, and mRNA analysis.ResultsEfferocytosis was affected in KO mice as compared to WT and Het BMDM. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression.ConclusionThese results suggest that a loss of GCLM and GSH during efferocytosis impairs adequate efferocytosis.

Abstract 453: Investigating The Role Of Glutathione During Macrophage Apoptotic Cell Clearance

Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis. Results: Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression. Conclusion: These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.

A Transcriptional Regulation Bioinformatics Pipeline to Predict Co‐Regulated Genes in Vascular Smooth Muscle Cell Phenotypic Transitions During Atherosclerosis

The rupture of vulnerable or advanced atherosclerotic lesions contributes to myocardial infarctions and strokes, the leading causes of death globally. During all stages of atherosclerosis, vascular smooth muscle cells (VSMCs) are recruited as a major source of plaque cells within lesions, where they undergo phenotypic modulation and give rise to both plaque‐stabilizing and destabilizing phenotypes. Because most atherosclerosis studies test the functional role of single transcription factors, genes, and proteins in VSMCs, many aspects of how VSMC plasticity can be clinically exploited to prevent the rupture of vulnerable atherosclerotic lesions remain unclear. Systems biology approaches have emerged as a promising avenue for characterizing cellular dynamics at the organismal level, but they remain under‐utilized in the field of cardiovascular medicine. This study proposes a novel systems biology pipeline that integrates experimental and computational tools with large datasets measuring the transcriptomes and epigenomes of phenotypically transitioning VSMCs. Our pipeline allows users to identify candidate transcription factors that co‐regulate mouse or human gene sets controlling specific VSMC phenotypic transitions during atherosclerosis. The computational algorithms developed in this workflow integrate an array of publicly available databases, such as the Ensembl genome database and the JASPAR transcription factor database. To demonstrate the translational potential of our pipeline, we used it to test the hypothesis that the murine mesenchymal marker stem cell antigen‐1 Sca1(Ly6a) has a human ortholog, which has not been previously validated as a target in clinical therapies. To test the hypothesis that there are sets of genes co‐regulated with Ly6ain human VSMCs which, when expressed in concert, promote a distinct phenotypic state marked by Ly6ain mice, we integrated genomic and ATAC‐seq analyses of VSMCs to identify murine genes with human orthologs that bind Ly6a‐specific transcription factors. Specifically, we predict that the transcription factors ETS1, KLF5, GATA6, and SOX10 co‐regulate sets of human genes with Ly6a. While prior studies have associated these transcription factors with VSMC proliferation and phenotypic modulation, we specifically predict these Ly6a‐specific transcription factor co‐regulate the following human gene sets: PHACTR1, EFHC1, LAMC2(ETS1); TTC33, RUNX1, KIAA1217(KLF5); TMEM50A(GATA6); and ZBTB20, MYLK2(SOX10). To validate these predictions, we will determine if these transcription factors and their co‐regulated genes are expressed in single cell RNA‐sequencing analyses of human carotid arteries and can be detected with immunofluorescence staining of human coronary artery sections. We will also use siRNA to target these candidate transcription factors and genes in cultured VSMCs. The results of our pipeline will help identify novel transcriptional regulatory networks in VSMCs that can be therapeutically targeted for the treatment of advanced atherosclerosis.

Tenascin-X Mediates Flow-Induced Suppression of EndMT and Atherosclerosis.

Endothelial-to-mesenchymal transition (EndMT) has been identified as a critical driver of vascular inflammation and atherosclerosis, and TGF-β (transforming growth factor β) is a key mediator of EndMT. Both EndMT and atherosclerosis are promoted by disturbed flow, whereas unidirectional laminar flow limits EndMT and is atheroprotective. How EndMT and endothelial TGF-β signaling are regulated by different flow patterns is, however, still poorly understood. Flow chamber experiments in vitro and endothelium-specific knockout mice were used to study the role of tenascin-X in the regulation of EndMT and atherosclerosis as well as the underlying mechanisms. In human endothelial cells as well as in human and mouse aortae, unidirectional laminar flow but not disturbed flow strongly increased endothelial expression of the extracellular matrix protein TN-X (tenascin-X) in a KLF4 (Krüppel-like factor 4) dependent manner. Mice with endothelium-specific loss of TN-X (EC-Tnxb-KO) showed increased endothelial TGF-β signaling as well as increased endothelial expression of EndMT and inflammatory marker genes. When EC-Tnxb-KO mice were subjected to partial carotid artery ligation, we observed increased vascular remodeling. EC-Tnxb-KO mice crossed to low-density lipoprotein receptor-deficient mice showed advanced atherosclerotic lesions after being fed a high-fat diet. Treatment of EC-Tnxb-KO mice with an anti-TGF-beta antibody or additional endothelial loss of TGF-beta receptors 1 and 2 normalized endothelial TGF-beta signaling and prevented EndMT. In in vitro studies, we found that TN-X through its fibrinogen-like domain directly interacts with TGF-β and thereby interferes with its binding to the TGF-β receptor. In summary, we show that TN-X is a central mediator of flow-induced inhibition of EndMT, endothelial inflammation and atherogenesis, which functions by binding to and by blocking the activity of TGF-β. Our data identify a novel mechanism of flow-dependent regulation of vascular TGF-β, which holds promise for generating new strategies to prevent vascular inflammation and atherosclerosis.

Role of Extracellular Vesicles in the Pathogenesis of Vascular Damage.

Extracellular vesicles (EVs) are nanosized membrane-bound structures released by cells that are able to transfer nucleic acids, protein cargos, and metabolites to specific recipient cells, allowing cell-to-cell communications in an endocrine and paracrine manner. Endothelial, leukocyte, and platelet-derived EVs have emerged both as biomarkers and key effectors in the development and progression of different stages of vascular damage, from earliest alteration of endothelial function, to advanced atherosclerotic lesions and cardiovascular calcification. Under pathological conditions, circulating EVs promote endothelial dysfunction by impairing vasorelaxation and instigate vascular inflammation by increasing levels of adhesion molecules, reactive oxygen species, and proinflammatory cytokines. Platelets, endothelial cells, macrophages, and foam cells secrete EVs that regulate macrophage polarization and contribute to atherosclerotic plaque progression. Finally, under pathological stimuli, smooth muscle cells and macrophages secrete EVs that aggregate between collagen fibers and serve as nucleation sites for ectopic mineralization in the vessel wall, leading to formation of micro- and macrocalcification. In this review, we summarize the emerging evidence of the pathological role of EVs in vascular damage, highlighting the major findings from the most recent studies and discussing future perspectives in this research field.

Diverse ultrastructural landscape of atherosclerotic endothelium

Background and aimsThe endothelium plays a major role in atherosclerosis, yet the endothelial plaque surface is a largely uncharted territory. Here we hypothesize that atherosclerosis-driven remodeling of the endothelium is a dynamic process, involving both damaging and regenerative mechanisms. MethodsUsing scanning electron microscopy (SEM) and immuno-SEM, we studied endothelial junction ultrastructure, endothelial openings and immune cell-endothelium interactions in eight apoe−/− mice and two human carotid plaques. ResultsThe surface of early mouse plaques (n = 11) displayed a broad range of morphological alterations, including junctional disruptions and large transcellular endothelial pores with the average diameter between 0.6 and 3 μm. The shoulder region of advanced atherosclerotic lesions (n = 7) had a more aggravated morphology with 8 μm-size paracellular openings at two-fold higher density. In contrast, the central apical surface of advanced plaques, i.e., the plaque body (n = 7), displayed endothelial normalization, as shown by a significantly higher frequency of intact endothelial junctions and a lower incidence of paracellular pores. This normalized endothelial phenotype correlated with low immune cell density (only 5 cells/mm2).The human carotid plaque surface (n = 2) displayed both well-organized and disrupted endothelium with similar features as described above. In addition, they were accompanied by extensive thrombotic areas. ConclusionsOur study unveils the spectrum of endothelial abnormalities associated with the development of atherosclerosis. These were highly abundant in early lesions and in the shoulder region of advanced plaques, while normalized at the advanced plaque's body. Similar endothelial features were observed in human atherosclerotic plaques, underlining the versatility of endothelial transformations in atherosclerosis.

The Initial Human Atherosclerotic Lesion and Lipoprotein Modification-A Deep Connection.

Atherosclerosis research typically focuses on the evolution of intermediate or advanced atherosclerotic lesions rather than on prelesional stages of atherogenesis. Yet these early events may provide decisive leads on the triggers of the pathologic process, before lesions become clinically overt. Thereby, it is mandatory to consider extracellular lipoprotein deposition at this stage as the prerequisite of foam cell formation leading to a remarkable accumulation of LDL (Low Density Lipoproteins). As progression of atherosclerosis displays the characteristic features of a chronic inflammatory process on the one hand and native LDL lacks inflammatory properties on the other hand, the lipoprotein must undergo biochemical modification to become atherogenic. During the last 25 years, evidence was accumulated in support of a different concept on atherogenesis proposing that modification of native LDL occurs through the action of ubiquitous hydrolytic enzymes (enzymatically modified LDL or eLDL) rather than oxidation and contending that the physiological events leading to macrophage uptake and reverse transport of eLDL first occur without inflammation (initiation with reversion). Preventing or reversing initial atherosclerotic lesions would avoid the later stages and therefore prevent clinical manifestations. This concept is in accordance with the response to retention hypothesis directly supporting the strategy of lowering plasma levels of atherogenic lipoproteins as the most successful therapy for atherosclerosis and its sequelae. Apart from but unquestionable closely related to this concept, there are several other hypotheses on atherosclerotic lesion initiation favoring an initiating role of the immune system (‘vascular-associated lymphoid tissue’ (VALT)), defining foam cell formation as a variant of lysosomal storage disease, relating to the concept of the inflammasome with crystalline cholesterol and/or mitochondrial DAMPs (damage-associated molecular patterns) being mandatory in driving arterial inflammation and, last but not least, pointing to miRNAs (micro RNAs) as pivotal players. However, direct anti-inflammatory therapies may prove successful as adjuvant components but will likely never be used in the absence of strategies to lower plasma levels of atherogenic lipoproteins, the key point of the perception that atherosclerosis is not simply an inevitable result of senescence. In particular, given the importance of chemical modifications for lipoprotein atherogenicity, regulation of the enzymes involved might be a tempting target for pharmacological research.

Long Noncoding RNA MIAT Controls Advanced Atherosclerotic Lesion Formation and Plaque Destabilization.

Long noncoding RNAs (lncRNAs) are important regulators of biological processes involved in vascular tissue homeostasis and disease development. The present study assessed the functional contribution of the lncRNA myocardial infarction-associated transcript (MIAT) to atherosclerosis and carotid artery disease. We profiled differences in RNA transcript expression in patients with advanced carotid artery atherosclerotic lesions from the Biobank of Karolinska Endarterectomies. The lncRNA MIAT was identified as the most upregulated noncoding RNA transcript in carotid plaques compared with nonatherosclerotic control arteries, which was confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. Experimental knockdown of MIAT, using site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (SMCs) but also increased their apoptosis. MIAT mechanistically regulated SMC proliferation through the EGR1 (Early Growth Response 1)-ELK1 (ETS Transcription Factor ELK1)-ERK (Extracellular Signal-Regulated Kinase) pathway. MIAT is further involved in SMC phenotypic transition to proinflammatory macrophage-like cells through binding to the promoter region of KLF4 and enhancing its transcription. Studies using Miat-/- and Miat-/-ApoE-/- mice, and Yucatan LDLR-/- mini-pigs, as well, confirmed the regulatory role of this lncRNA in SMC de- and transdifferentiation and advanced atherosclerotic lesion formation. The lncRNA MIAT is a novel regulator of cellular processes in advanced atherosclerosis that controls proliferation, apoptosis, and phenotypic transition of SMCs, and the proinflammatory properties of macrophages, as well.

Deletion of Fam172a accelerates advanced atherosclerosis and induces plaque instability

Background and aimsVascular smooth muscle cells (VSMCs) play a critical role in atherosclerosis. The family with sequence similarity 172, member A (FAM172A) is a novel protein and its role in atherosclerosis has not been explored so far. Therefore, our aim is to investigate whether FAM172A affects atheroprogression through VSMCs and its possible mechanism. MethodsFam172a−/− mice were generated using CRISPR/Cas9 technology. Fam172a−/− and Apoe−/− double knockout (Fam172a−/−/Apoe−/−) mice and their littermates (Fam172a+/+/Apoe−/−) were fed with a Western diet for 18 weeks to induce advanced atherosclerotic lesions. The role and mechanism of Fam172a in phenotypic switching, proliferation and migration of VSMCs were investigated through in vivo and in vitro experiments. ResultsCompared with Fam172a+/+/Apoe−/− mice, Fam172a−/−/Apoe−/− mice showed increased atherosclerotic lesion size and plaque instability such as increased necrotic core area and decreased fiber deposition. Additionally, knockout of Fam172a promoted expression of CD68 and KLF4 and decreased expression of α-SMA and SM22α in atherosclerotic lesions. Furthermore, overexpression of Fam172a promoted Movas cells proliferation and migration, increased expression of α-SMA and SM22α and decreased expression of KLF4. Meanwhile, knockdown of Fam172a in Movas cells and deletion of Fam172a in VSMCs from Fam172a−/−/Apoe−/− mice showed opposite phenotypes. Similar phenotypes were also observed in human aortic smooth muscle cells. ConclusionsOur results provide the first direct evidence that Fam172a has a protective role in advanced atherosclerosis by increasing atherosclerotic plaque stability and inhibiting transition of VSMCs from contractile to synthetic phenotype, which may be through KLF4-dependent pathway.

The regulation of Ero1-alpha in homocysteine-induced macrophage apoptosis and vulnerable plaque formation in atherosclerosis

Background and aimsHyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis and plaque vulnerability. Macrophage apoptosis mediated by endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of HHcy-aggravated atherosclerosis. Endoplasmic reticulum oxidoreductase 1α (Ero1α) is critical for ER stress-induced apoptosis. We hypothesized that Ero1α may contribute to ER-stress induced macrophage apoptosis and plaque stability in advanced atherosclerotic lesions by HHcy. MethodsApoe−/− mice were maintained on drinking water containing homocysteine (Hcy, 1.8 g/L) to establish HHcy atherosclerotic models. The role of Ero1α in atherosclerotic plaque stability, macrophage apoptosis and ER stress were monitored in the plaque of aortic roots in HHcy Apoe−/− mice with or without silence or overexpression of Ero1α through lentivirus. Mouse peritoneal macrophages were used to confirm the regulation of Ero1α on ER stress dependent apoptosis in the presence of HHcy. ResultsAtherosclerotic plaque vulnerability and macrophage apoptosis were promoted in Apoe−/− mice by high Hcy diet, accompanied by the upregulation of Ero1α expression and ER stress. Inhibition of Ero1α prevented macrophage apoptosis and atherosclerotic plaque vulnerability, and vice versa. Consistently, in mouse peritoneal macrophages, ER stress and apoptosis were attenuated by Ero1α deficiency, but enhanced by Ero1α overexpression. ConclusionsHcy, via upregulation of Ero1α expression, activates ER stress-dependent macrophage apoptosis to promote vulnerable plaque formation in atherosclerosis. Ero1α may be a potential therapeutic target for atherosclerosis induced by Hcy.

Telomere damage promotes vascular smooth muscle cell senescence and immune cell recruitment after vessel injury

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2T188A) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease.

Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMβ2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.