Advanced Atherosclerotic Lesions Research Articles

Effect of Oral Administration of Collagen Peptide OG-5 on Advanced Atherosclerosis Development in ApoE-/- Mice.

Atherosclerosis is a chronic inflammatory disease of the arterial wall, which involves multiple cell types. Peptide OG-5 is identified from collagen hydrolysates derived from Salmo salar and exhibits an inhibitory effect on early atherosclerosis. The primary objective of this study was to investigate the impact of OG-5 on advanced atherosclerotic lesions as well as its stability during absorption. In this study, the ApoE-/- mice were employed to establish advanced atherosclerosis model to investigate the treatment effect of peptide OG-5. The results showed that oral administration of OG-5 at a dosage of 150 mg/kg bw resulted in a 30% reduction in the aortic plaque formation area in ApoE-/- mice with few bleeding risks. Specifically, intervention with a low dose of OG-5 (50 mg/kg bw), initiated in the early stage of atherosclerosis, continues to provide benefits into the middle and late stages without bleeding risks. Furthermore, treatment of OG-5 increased expression levels of contractile phenotype markers and reduced the accumulation of lipoprotein in VSMCs induced by ox-LDL. Peptide OG-5 could ensure transport across Caco-2 cell monolayers, exhibiting a Papp value of 1.80 × 10-5 cm/s, and exhibited a robust stability in plasma with remaining content >70% after 8 h incubation. In vivo studies revealed that OG-5 reached maximum concentration in blood after 120 min. The present results demonstrate the potential efficacy of peptide OG-5 as a promising agent for intervention in anti-atherogenesis strategies.

A penetrating aortic ulcer rapidly evolving into aortic dissection in a patient presenting with respiratory tract infection to the emergency department: an acute aortic syndrome case report

BackgroundPenetrating aortic ulcers (PAU) are life-threatening conditions which derive from severely advanced atherosclerotic lesions of the aorta. The clinical course is unpredictable; thus clinical vigilance should be maintained. It is very challenging to separate PAU from co-existing AAS as predisposing factors and findings overlap.Case presentationCase of 58-year-old gentleman, who presented for atypical chest pain in the setting of respiratory tract infection. Computed Tomographic angiography (CTA) of the chest showed a large PAU and intramural hematoma which rapidly progressed into an acute aortic dissection in the emergency department. Close follow up with cardiac point of care ultrasound one hour later detected an intimal flap which was not initially present on CTA. Patient underwent surgical aortic graft replacement and had an uneventful in-hospital stay.DiscussionThis case underlines the importance of broadening differential diagnoses in atypical presentations in patients with risk factors. Prompt intervention and careful management are imperative to optimize patient outcomes and prevent complications of aortic lesions. Cardiac point of care ultrasound can help in detecting progression of dynamic atherosclerotic diseases such as acute aortic syndrome.

Association between Vascular Calcification and Intraplaque Hemorrhage in Coronary Atherosclerosis from Autopsy: The Hisayama Study.

Vascular calcification is observed in advanced atherosclerotic lesions. Vascular calcification is considered to increase the risk of intraplaque hemorrhage and subsequent plaque destabilization; however, there is limited pathohistoological evidence of the association between vascular calcification and intraplaque hemorrhage. The aim of this study was to investigate the association between vascular calcification and intraplaque hemorrhage in the coronary arteries. We examined 374 coronary arteries obtained from the autopsy samples of 126 deceased individuals. The vascular calcification levels of each artery were categorized into no calcification and quintiles of calcification area size among the arteries with calcification. Macrophage infiltration and neovascularization were also evaluated. The association of the calcification area, macrophage area, or number of vessels with the presence of intraplaque hemorrhage in the coronary arteries was estimated using a logistic regression analysis. Calcification lesions were observed in 149 coronary arteries. Arteries in the fourth quintile of calcification area size had a significantly greater likelihood of intraplaque hemorrhage than the arteries without calcification, after adjusting for confounders: odds ratio 13.13 (95% confidence interval: 2.97-58.16). After evaluating the influence of macrophage infiltration, the highest odds ratio of intraplaque hemorrhage was associated with the combination of large macrophage area and moderately sized calicification areas. The odds ratio of intraplaque hemorrhage additively increased with the combination of calcification and the number of vessels. The present findings suggest that vascular calcification is significantly associated with intraplaque hemorrhage. The association between vascular calcification and intraplaque hemorrhage may decrease above a certain size of the calcification area.

Cyclophilin D induces necrotic core formation by mediating mitochondria-associated macrophage death in advanced atherosclerotic lesions

Background and aimsIn advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation. MethodsTo clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe−/−Ppif−/−) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development. ResultsDeletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe−/− mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. ConclusionsThese results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.

Postmortem diagnosis of arterial atherosclerosis in autopsy, postmortem computed tomography and histopathological examinations in medico-legal aspect.

The aim of this study is to provide a retrospective assessment of the possibility of determining the severity of atherosclerosis based on postmortem computed tomography (PMCT), autopsy protocols and histopathological examination results. In the first stage of the study, 200 cases were evaluated of persons over 40 years of age in whom postmortem computed tomography and autopsy were performed. In the second stage, the cases were divided into a study group (cardiovascular deaths) and a control group, as well as divided by age and, in addition to autopsy protocols and PMCT results, histopathological findings were evaluated. The results of stage I demonstrated that the best detection of atherosclerosis was in the advanced stage with a predominance of detection in PMCT. Atherosclerosis detection in autopsy was highest in the coronary arteries, aorta and cerebral arteries; while in PMCT it remained equal in all evaluated locations. Autopsy showed higher detection of advanced atherosclerotic lesions in the coronary arteries and aorta compared to PMCT. The results of stage II of the study revealed that attaching the results of the general histopathological examination to the retrospective evaluation does not provide an opportunity to increase the accuracy of the evaluation of atherosclerotic lesions. The results obtained indicate the need for prospective studies. Autopsy allows macroscopic evaluation of a very broad spectrum of atherosclerotic lesions, but often without precise determination of their nature, and with limited localization; PMCT allows accurate and reproducible evaluation of calcified atherosclerotic lesions in large and medium-sized vessels, but is unsuitable for the evaluation of non-calcified lesions and small vessels; targeted histopathological examinations allow very accurate, but local assessment of atherosclerotic lesions.

Drug-interacting gene expression levels differ between atherosclerotic plaque subtypes

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): The Dutch Heart foundation Recent transcriptomic-based clustering of human atherosclerotic plaques revealed that plaques have a diversity and complexity that cannot be accurately described by classical concepts. Previous plaque RNA sequencing that we performed in 654 lesions identified five major plaque types that reflect different mechanisms of progression of atherosclerotic disease. These include the following plaque subtypes: 0. Fibro-collagenous, 1. Intermediate, 2. Lipo-necrotic, 3. Fibro-inflammatory, and 4. Fibro-cellular. We hypothesize that these different plaque types may respond differentially upon drug treatment. Objective To assess differential expression levels of drug-responsive genes between the earlier described five plaque subtypes. For this in silico experiment, the differential expression of drug-responsive genes was examined after colchicine treatment in smooth muscle cells and endothelial cells. Methods To begin, a web-based search identified 46 published colchicine interacting genes. Then, colchicine was applied to cultured smooth muscle and endothelial cells to verify the publicly reported drug responsive genes. Next, we modelled the effect of colchicine on the five types of human atherosclerotic plaques for those genes that were considered drug responsive. This was achieved by projecting the in vitro derived expression changes onto the gene expression data from the carotid plaques of 654 patients who participated in the ATHERO-EXPRESS study. Besides colchicine, the same experimental approach was applied for atorvastatin and rofecoxib drug treatment. Finally, changes in drug responsive gene expression were validated with mice gene expression data. Results 34 out of the 46 publicly known colchicine interacting genes were differently expressed between plaque types (Figure 1). These genes included established determinants of cell motility and atherosclerosis development (SCD, ITGB1, Tubulins). Furthermore, in-silico colchicine treatment revealed colchicine's strong effect on the atherosclerotic plaques' transcriptome. We are currently assessing if these perturbations may lead to shifts in plaque subtypes and if these changes are reflected in mice transcriptomic data. Conclusion The pilot data demonstrated major differences in drug-interacting gene expression levels in advanced atherosclerotic lesions. Therefore, novel and standard drugs used to treat atherosclerosis may have varying effects depending on the atherosclerotic plaque subtype.

IL-1β Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1β or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1β antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1β antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. Taken together, IL-1β appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Alternate day fasting aggravates atherosclerosis through the suppression of hepatic ATF3 in Apoe−/− mice

Abstract Atherosclerosis is the major contributor to cardiovascular mortality worldwide. Alternate day fasting (ADF) has gained growing attention due to its metabolic benefits. However, the effects of ADF on atherosclerotic plaque formation remain inconsistent and controversial in atherosclerotic animal models. The present study was designed to investigate the effects of ADF on atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice. Eleven-week-old male Apoe−/− mice fed with Western diet (WD) were randomly grouped into ad libitum (AL) group and ADF group, and ADF aggravated both the early and advanced atherosclerotic lesion formation, which might be due to the disturbed cholesterol profiles caused by ADF intervention. ADF significantly altered cholesterol metabolism pathways and down-regulated integrated stress response (ISR) in the liver. The hepatic expression of activating transcription factor 3 (ATF3) was suppressed in mice treated with ADF and hepatocyte-specific overexpression of Aft3 attenuated the effects of ADF on atherosclerotic plaque formation in Apoe−/− mice. Moreover, the expression of ATF3 could be regulated by Krüppel-like factor 6 (KLF6) and both the expressions of ATF3 and KLF6 were regulated by hepatic cellular ISR pathway. In conclusion, ADF aggravates atherosclerosis progression in Apoe−/− mice fed on WD. ADF inhibits the hepatic ISR signaling pathway and decreases the expression of KLF6, subsequently inhibiting ATF3 expression. The suppressed ATF3 expression in the liver mediates the deteriorated effects of ADF on atherosclerosis in Apoe−/− mice. The findings suggest the potentially harmful effects when ADF intervention is applied to the population at high risk of atherosclerosis.

Secreted Protein Profiling of Human Aortic Smooth Muscle Cells Identifies Vascular Disease Associations.

Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

Intralesional pentraxin 3 increases with atherosclerotic disease progression, but may protect from thrombosis: Friend or foe?

AimTo investigate the role of pentraxin 3 (PTX3) in atherosclerotic disease progression and plaque destabilization, as well as in coronary restenosis after directional coronary atherectomy (DCA). Materials and methodsPTX3 contents of early and advanced atherosclerotic lesions of the aorta obtained at autopsy were determined by ELISA and Western blot. Also, coronary plaques of patients with acute coronary syndrome (ACS) or stable angina pectoris (SAP) obtained by DCA were analyzed by immunohistochemistry for PTX3. The effects of PTX3 on smooth muscle cells (SMCs) and thrombogenesis were investigated with cultured human coronary artery SMCs and a flow chamber system, respectively. ResultsAdvanced atherosclerotic lesions contained a significantly larger amount of PTX3 than early lesions (ELISA: 9.96 ± 2.77 ng/100 mg tissue, n = 8 vs 0.24 ± 0.18 ng/100 mg tissue, n = 6, P = 0.0097). Also, ACS plaques contained a significantly larger amount of PTX3 than SAP plaques (PTX3 immunohistochemistry–positive area percentage: 2.88 ± 0.53 %, n = 22 vs 0.67 ± 0.27 %, n = 23, P = 0.0009). Curiously, the patients who would remain free of post-DCA restenosis (n = 19) had plaques with a significantly higher PTX3 immunohistochemistry–positive area percentage than those who would develop restenosis (n = 12) (2.32 ± 0.49 % vs 0.49 ± 0.17 %, P = 0.002). In the mechanistic part of the study, PTX3 inhibited SMC proliferation and migration. PTX3 also inhibited platelet thrombus formation in the condition simulating arterial blood flow. ConclusionsPTX3 is increased in advanced (vs early) atherosclerotic lesions and unstable (vs stable) coronary plaques. The inhibitory effects of PTX3 on SMCs and thrombogenesis suggest that intraplaque PTX3 might have atheroprotective effects.

Autopsy Pathology of Heart and Coronary Vessels in Sudden Death: A Cross-sectional Study from Southern India

Introduction: Cardiac diseases emerge as the primary culprits behind sudden and unexplained deaths. Morphological evaluation through autopsy is invaluable in establishing the prevalence of cardiac lesions and coronary atherosclerosis within the general population. It provides healthcare researchers with valuable insights to develop preventive measures and early detection strategies crucial for improving survival rates. Aim: To estimate the prevalence of cardiac lesions in postmortem specimens of the heart from autopsies of sudden and unexplained deaths. Materials and Methods: The present cross-sectional study was conducted in the Department of Pathology, Government Medical College (a public sector hospital), Cochin, Kerala, India, from January to December 2021 on 140 specimens of the heart. Medicolegal autopsies were performed in the Department of Forensic Medicine, with specimens forwarded to the Pathology Department for histopathological examination. Hearts dissected by the modified Virchow’s method were examined for gross pathology in the cardiac wall, chambers, valves, coronaries and aorta. Microscopy findings were documented and the grading of coronary atherosclerosis was done using the Modified American Heart Association (AHA) criteria. Proportions were analysed from the compiled data using a Chi-square test. Statistical analysis was done using Statistical Package for the Social Sciences (SPSS) software version 21.0 software. Results: In the study encompassing 140 adult autopsies, the mean age of the study population was 45.98 with a Standard Deviation (SD) of 15.82 years and the male to female ratio was 3.5:1. The prevalence of cardiac lesions was as follows: coronary artery atherosclerosis (80.70%), Ischaemic Heart Disease (IHD) (44.28%), cardiac hypertrophy and hypertension (10%), pericarditis (6.42%) and myocarditis (3.50%). The male- to-female ratio for coronary atherosclerosis was 4.4:1, with triple vessel disease observed in 42.90% of cases. Atherosclerosis severity grades included early non atheromatous lesions (19.3%), Pathological Intimal Thickening (PIT) (23.6%), and advanced atherosclerotic lesions (37.80%). Among cases associated with Coronavirus Disease 2019 (COVID-19)-related deaths (10.7%), changes noted included myocarditis, microhaemorrhages, oedema, capillary dilatation, fibrin deposition and inflammatory infiltrates. Conclusion: The present study revealed a high prevalence of coronary atherosclerosis affecting all three vessels, particularly among the younger population. This underscores the significance of screening for cardiovascular risk factors and implementing preventive measures from an early age.

Unraveling the Role of Sex in Endothelial Cell Dysfunction: Evidence From Lineage Tracing Mice and Cultured Cells.

Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. We used human and mouse aortic EC as well as EC-lineage tracing (Cdh5-CreERT2 Rosa-YFP [yellow fluorescence protein]) atherosclerotic Apoe-/- mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. Bioinformatics analyses were performed on male and female mouse aortic EC and human lung and aortic EC. In vitro, female human and mouse aortic ECs showed more apoptosis and higher cellular reactive oxygen species levels than male EC. In addition, female mouse aortic EC had lower mitochondrial membrane potential (ΔΨm), lower TFAM (mitochondrial transcription factor A) levels, and decreased angiogenic potential (tube formation, cell viability, and proliferation) compared with male mouse aortic EC. In vivo, female mice had significantly higher lipid accumulation within the aortas, impaired glucose tolerance, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and endothelial-to-mesenchymal transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNA-seq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. Our study delineates critical sex-specific differences in EC relevant to proinflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biological basis for sex-specific proatherosclerotic mechanisms.

Drug-interacting gene expression levels differ between atherosclerotic plaque subtypes

Abstract Background Recent transcriptomic-based clustering of human atherosclerotic plaques revealed that plaques have a diversity and complexity that cannot be accurately described by classical concepts. Previous plaque RNA sequencing that we performed identified five major plaque types that reflect the severity of the atherosclerotic disease. These include the following plaque subtypes: 0. Fibro-collagenous, 1. Intermediate, 2. Lipo-necrotic, 3. Fibro-inflammatory, and 4. Fibro-cellular. We hypothesised that identifying these different plaque types could explain differential responses following drug treatment. Objective To assess differential expression levels of drug-responsive genes between the earlier described five plaque subtypes. For this pilot experiment, we selected genes that are differentially expressed after colchicine treatment in smooth muscle cells and endothelial cells. Methods A web-based search identified 46 published colchicine interacting genes. Colchicine was also applied to smooth muscle and endothelial cells to identify direct-effect genes via differential gene expression analysis. Differences in gene expression between plaque types were assessed for those genes that were considered drug-responsive. Finally, to model the effect of colchicine on an atherosclerotic plaque, we projected the in vitro derived expression changes onto the gene expression data from carotid plaques of 654 patients that participated in the ATHERO-EXPRESS study. Results 34 out of the 46 publicly known colchicine interacting genes were differently expressed between plaque types (Figure 1). These genes included established determinants of cell motility and atherosclerosis development (SCD, ITGB1, Tubulins). Furthermore, in-silico colchicine treatment revealed colchicine's strong effect on the atherosclerotic plaques' transcriptome. We are currently assessing if these perturbations may lead to changes in plaque subtypes. Conclusion The pilot data demonstrated major differences in drug-interacting gene expression levels in advanced atherosclerotic lesions. Therefore, novel and standard drugs used to treat atherosclerosis may have varying effects depending on the atherosclerotic plaque subtype.Figure 1

Caspase-11 deficiency attenuates neutrophil recruitment into the atherosclerotic lesion in apolipoprotein E-deficient mice

Caspase-11 is an inflammatory caspase that triggers an inflammatory response by regulating non-canonical NLRP3 inflammasome activation. Although the deficiency of both caspase-11 and caspase-1, another inflammatory caspase that functions as an executor of the inflammasome, prevents the development of atherosclerosis, the effect of caspase-11 deficiency alone on the development of atherosclerosis has not been fully evaluated. In the present study, we found that caspase-11 deficiency prevented the formation of the necrotic core, whereas it did not affect the development of atherosclerosis in Apoe-deficient mice. Notably, the infiltration of neutrophils into atherosclerotic lesions was attenuated by caspase-11 deficiency. RNA-seq analysis of stage-dependent expression of atherosclerotic lesions revealed that both upregulations of caspase-11 and neutrophil migration are common features of advanced atherosclerotic lesions. Furthermore, similar expression profiles were observed in unstable human plaque. These data suggest that caspase-11 regulates neutrophil recruitment and plaque destabilization in advanced atherosclerotic lesions.

Modulating Plaque Inflammation via Targeted mRNA Nanoparticles for the Treatment of Atherosclerosis.

Atherosclerosis is a common pathology present in many cardiovascular diseases. Although the current therapies (including statins and inhibitors of the serine protease PCSK9) can effectively reduce low-density lipoprotein (LDL) cholesterol levels to guideline-recommended levels, major adverse cardiovascular events still occur frequently. Indeed, the subendothelial retention of lipoproteins in the artery wall triggers multiple events of inflammation in macrophages and is a major contributor to the pathological progression of atherosclerosis. It has been gradually recognized that modulating inflammation is, therefore, an attractive avenue to forestall and treat atherosclerosis and its complications. Unfortunately, challenges with specificity and efficacy in managing plaque inflammation have hindered progress in atherosclerosis treatment. Herein, we report an NP-mediated mRNA therapeutic approach to target atherosclerotic lesional macrophages, modulating inflammation in advanced atherosclerotic lesions for the treatment of atherosclerosis. We demonstrated that the targeted NPs containing IL-10 mRNA colocalized with M2-like macrophages and induced IL-10 production in atherosclerotic plaques following intravenous administration to Western diet (WD)-fed Ldlr-/- mice. Additionally, the lesions showed a significantly alleviated inflammatory response, as evidenced by reduced oxidative stress and macrophage apoptosis, resulting in decreased lipid deposition, diminished necrotic areas, and increased fiber cap thickness. These results demonstrate the successful delivery of mRNA therapeutics to macrophage-enriched plaques in a preclinical model of advanced atherosclerosis, showing that this targeted NP inflammation management approach has great potential for translation into a wide range of clinical applications.

X chromosome inactivation skewing is common in advanced carotid atherosclerotic lesions in females and predicts secondary peripheral artery events

Background and aimSex differences in atherosclerosis have been described with female plaques being mostly perceived as stable and fibrous. Sex-specific mechanisms such as mosaic loss of the Y chromosome in men have been linked to cardiovascular health. In women, X-linked mechanisms such as X chromosome inactivation (XCI) skewing is common in several tissues. Yet, information on the role of XCI in female atherosclerotic plaques is lacking. Here, we investigated the presence of XCI skewing in advanced atherosclerotic lesions and its association with cardiovascular risk factors, histological plaque data, and clinical data.MethodsXCI skewing was quantified in 154 atherosclerotic plaque and 55 blood DNA samples of women included in the Athero-Express study. The skewing status was determined performing the HUMARA assay. Then, we studied the relationship of XCI skewing in female plaque and cardiovascular risk factors using regression models. In addition, we studied if plaque XCI predicted plaque composition, and adverse events during 3-years follow-up using Cox proportional hazard models.ResultsXCI skewing was detected in 76 of 154 (49.4%) plaques and in 27 of 55 (67%) blood samples. None of the clinical risk factors were associated with plaque skewing. Plaque skewing was more often detected in plaques with a plaque hemorrhage (OR [95% CI]: 1.44 [1.06–1.98], P = 0.02). Moreover, skewed plaques were not associated with a higher incidence of composite and major events but were specifically associated with peripheral artery events during a 3-year follow-up period in a multivariate model (HR [95%CI]: 1.46 [1.09–1.97]; P = 0.007).ConclusionsXCI skewing is common in carotid plaques of females and is predictive for the occurrence of peripheral artery events within 3 years after carotid endarterectomy.

Trib1 Deficiency Promotes Hyperlipidemia, Inflammation, and Atherosclerosis in LDL Receptor Knockout Mice.

Genetic variants at the TRIB1 gene locus are strongly associated with plasma lipid traits and the risk of coronary artery disease in humans. Here, we analyzed the consequences of Trib1 deficiency on lipid metabolism and atherosclerotic lesion formation in atherosclerosis-susceptible Ldlr-/- mice. Trib1-/- mice were crossed onto the Ldlr-/- background to generate double-knockout mice (Trib1-/-Ldlr-/-) and fed a semisynthetic, modified AIN76 diet (0.02% cholesterol and 4.3% fat) until 20 weeks of age. Trib1-/-Ldlr-/- mice had profoundly larger (5.8-fold) and more advanced atherosclerotic lesions at the aortic root as compared with Trib1+/+Ldlr-/- controls. Further, we observed significantly elevated plasma total cholesterol and triglyceride levels in Trib1-/-Ldlr-/- mice, resulting from higher VLDL (very-low-density lipoprotein) secretion. Lipidomics analysis revealed that loss of Trib1 altered hepatic lipid composition, including the accumulation of cholesterol and proinflammatory ceramide species, which was accompanied by signs of hepatic inflammation and injury. Concomitantly, we detected higher plasma levels of IL (interleukin)-6 and LCN2 (lipocalin 2), suggesting increased systemic inflammation in Trib1-/-Ldlr-/- mice. Hepatic transcriptome analysis demonstrated significant upregulation of key genes controlling lipid metabolism and inflammation in Trib1-/-Ldlr-/- mice. Further experiments suggested that these effects may be mediated through pathways involving a C/EPB (CCAAT/enhancer binding protein)-PPARγ (peroxisome proliferator-activated receptor γ) axis and JNK (c-Jun N-terminal kinase) signaling. We provide experimental evidence that Trib1 deficiency promotes atherosclerotic lesion formation in a complex manner that includes the modulation of lipid metabolism and inflammation.

Editor's Choice – Two Year Results of the Randomised DISCOVER Trial Comparing Covered Versus Bare Metal Stents in the Common Iliac Artery

It has been suggested that covered stents (CS) may lower restenosis rates compared with bare metal stents (BMS) after endovascular treatment of the common iliac artery. This trial aimed to provide additional evidence on the efficacy of CS vs. BMS in the common iliac artery. This multicentre, randomised, single blind controlled superiority trial compared balloon expandable CS and balloon expandable BMS for advanced atherosclerotic lesions in the common iliac artery; this was defined as a stenosis > 3 cm in length or occlusion. The primary end point was freedom from binary restenosis after two years of follow up. The study was conducted according to the principles of the Declaration of Helsinki (version: October 2008) and registered with the Dutch Trial register (NTR3381). One hundred and seventy-four limbs were included between 2012 and 2019 with 87 limbs in each group. Six patients crossed over from the BMS group to the CS group but were analysed according to an intention to treat principle. Freedom from binary restenosis after two years of follow up was 84.7% (95% CI 76.7 - 92.7%) in the BMS group and 89.1% (95% CI 82.4 - 95.8%) in the CS group (p= .40). Freedom from occlusion was 95.0% (95% CI 90.3 - 95.7%) in the BMS group and 96.4% (95% CI 92.5 - 100%) in the CS group (p= .66). Freedom from target lesion revascularisation was 91.1% (95% CI 84.8 - 97.3%) and 95.2% (95% CI 90.7 -99.7%), respectively (p= .31). Technical success, complications, haemodynamic success, and clinical success were also comparable between both groups. Per-protocol analysis did not affect the outcomes of the study. No difference was found between balloon expandable CS and BMS for treating advanced atherosclerotic lesions of the common iliac artery.

Blockade of the BLT1-LTB4 axis does not affect mast cell migration towards advanced atherosclerotic lesions in LDLr−/− mice

Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/− mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression.

Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin/heme-mediated vascular damage in atherosclerosis

Vulnerable atherosclerotic plaques with hemorrhage considerably contribute to cardiovascular morbidity and mortality. Calcification is the main characteristic of advanced atherosclerotic lesions and calcified aortic valve disease (CAVD). Lyses of red blood cells and hemoglobin (Hb) release occur in human hemorrhagic complicated lesions. During the interaction of cell-free Hb with plaque constituents, Hb is oxidized to ferric and ferryl states accompanied by oxidative changes of the globin moieties and heme release. Accumulation of both ferryl-Hb and metHb has been observed in atherosclerotic plaques. The oxidation hotspots in the globin chain are the cysteine and tyrosine amino acids associated with the generation of Hb dimers, tetramers and polymers. Moreover, fragmentation of Hb occurs leading to the formation of globin-derived peptides. A series of these pro-atherogenic cellular responses can be suppressed by hydrogen sulfide (H2S). Since H2S has been explored to exhibit a wide range of physiologic functions to maintain vascular homeostasis, it is not surprising that H2S may play beneficial effects in the progression of atherosclerosis. In the present review, we summarize the findings about the effects of H2S on atherosclerosis and CAVD with a special emphasis on the oxidation of Hb/heme in atherosclerotic plaque development and vascular calcification.