Atherosclerotic Plaque Research Articles

PCSK9 inhibitors and coronary atherosclerotic plaque modification: a meta-analysis

Abstract Introduction Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have emerged as a promising class of medications, primarily recognized for their potent cholesterol-lowering effects. Beyond their established role in reducing low-density lipoprotein cholesterol levels, recent studies suggest a potential impact of PCSK9 inhibitors on the modification of coronary atherosclerotic plaques. Purpose The purpose of this meta-analysis is to comprehensively synthesize data from relevant clinical studies and trials that have investigated the effects of PCSK9 inhibitors on coronary atherosclerotic plaque characteristics. Methods We performed a literature search for studies assessing the evolution of coronary atherosclerotic plaques after treatment with a PCSK9 inhibitor compared to a control group. We excluded reviews, editorials, case reports/case series, and studies that did not use PCSK9 inhibitors or did not have a control group for comparison. The main outcomes of interest were the changes in percent atheroma volume (PAV), total atheroma volume (TAV), minimal fibrous cap thickness (FCT), lipid arch, as well as the number of patients with improved PAV at follow-up. Effect sizes are presented as standardized mean difference (SMD) or risk ratio (RR) with their corresponding 95% confidence intervals (CIs) and were pooled based on a random-effects model. Publication bias was assessed by funnel plot inspection and egger’s regression test. Results The literature search yielded 189 results. After application of the exclusion criteria, a total of 8 studies were selected for data extraction and inclusion in the meta-analysis. Concerning the intravascular ultrasound findings PCSK9 inhibitors induced greater reductions in PAV (SMD -2.70, 95% CI -5.24 to -0.15, p=0.04) (Figure 1) and TAV (SMD -2.46, 95% CI -4.68 to -0.23, p=0.03) (Figure 2), with a greater proportion of patients exhibiting an improvement in PAV (RR 1.30, 95% CI 1.20 to 1.41, p<0.01). Moving to optical coherence tomography parameters, patients treated with PCSK9 inhibitors had an increase in minimal FCT (SMD 2.09, 95% CI 0.12 to 4.07, p=0.04) and a lower lipid arch (SMD -0.64, 95% CI -0.97 to -0.32, p<0.01). Funnel plot inspection and egger’s regression test did not indicate the presence of publication bias. Conclusion This meta-analysis suggests that PCSK9 inhibitors use is associated with a favorable coronary atherosclerotic plaque remodeling.Figure 1Figure 2

Coronary Computed Tomographic Angiography (CCTA) in non-ST elevation acute coronary syndromes

Abstract Background In patients presenting with an acute coronary syndrome (ACS), a diverse range of coronary artery disease is observed from structurally normal vessels to non-obstructive atherosclerosis and severe/obstructive coronary artery disease (CAD). In current clinical practice guidelines, the main diagnostic route for optimal management of patients with non-ST segment elevation acute coronary syndrome is invasive coronary angiography. However, in 30-40% of cases, no significant coronary lesions are evident. The purpose of this study is to describe the role of coronary computed tomographic angiography (CCTA) in non-ST elevation acute coronary syndromes in low and intermediate risk patients. Methods We describe the results of 504 patients evaluated with a protocol that includes the use of CCTA in patients with ACS. Patients over 18 years of age with a diagnosis of ACS without ST elevation who did not meet high-risk criteria (hemodynamic instability, dynamic changes in the ST segment or T wave, ventricular arrhythmia, GRACE score >140, refractory angina, heart failure or cardiorespiratory arrest) were included. They underwent CCTA as the initial coronary evaluation. Data collection was carried out between May 11, 2022 and February 20, 2024. Results 504 patients were included, with a mean age of 58 years, of which 44% were women and 56% men. Among the admission diagnoses, 26% were NSTEMI and 74% were unstable angina according to the 4th universal definition of acute myocardial infarction (AMI). The median high-sensitivity cardiac troponin T value was 7 nanograms per liter (interquartile range, 7 to 15) for the first troponin measurement. CCTA was performed in 93% of the patients. It was observed that 28% did not have any coronary lesions or plaques. 36% had non-obstructive coronary artery disease (plaques <50%). The rest had significant lesions in at least one vessel (27%), predominantly in the left anterior descending artery, or were nondiagnostic (9%). 31% had myocardial bridges, while in 13% of the patients a high-risk plaque was found. 38% of the patients included underwent invasive coronary angiography and 23% required revascularisation. Conclusion This study shows that the prevalence of ACS with coronary arteries without significant lesions by CCTA was 28%, which suggests CCTA could have a predominant role in the diagnostic algorithm of acute coronary syndromes without high-risk criteria, implying a decrease in the need of invasive studies in these patients. Only 38% required invasive coronary angiography, while revascularisation was performed in 23% of the patients. Furthermore, CCTA allowed a better detection of non-obstructive coronary artery disease (NOCAD) in 36% of the patients, and a more complete characterization of the atherosclerotic plaque, which could prompt to an initiation of preventive therapies or the optimization of medical treatment in these patients.Table 1CCTA findings

Macrophages overexpressing interleukin-10 target and prevent atherosclerosis: regression of plaque formation and reduction in necrotic core

Abstract Background Atherosclerosis, a slowly progressing inflammatory disease, is characterized by the presence of monocyte-derived macrophages. Interventions targeting the inflammatory characteristics of atherosclerosis hold promising potential. Although interleukin (IL)-10 is widely acknowledged for its anti-inflammatory effects, systemic administration of IL-10 has limitations due to its short half-life and significant systemic side effects. Purpose We aimed to investigate the effectiveness of an approach designed to overexpress IL-10 in macrophages and subsequently introduce these genetically modified cells into ApoE-/- mice to promote atherosclerosis regression. Methods We engineered RAW264.7 cells to overexpress IL-10 (referred to as IL-10M) using lentivirusvectors. The ApoE-/- mice were divided into two groups based on the timing of the intervention. The early intervention group (n = 45) received intravenous tail injections from the beginning of Western diet (WD) at six weeks old. The late intervention group (n = 45) started receiving injections at 16 weeks old, 2 months after being fed the WD. Both the early and late intervention group stopped receiving injections after 5 months of being fed the WD. Results The IL-10M exhibited robust IL-10 secretion, maintained phagocytic function, improved mitochondrial membrane potentials, reduced superoxide production and showed a tendency toward the M2 phenotype when exposed to inflammatory stimuli. IL-10M can selectively target plaques in ApoE-/- mice, and oil red staining of the entire aorta unequivocally demonstrated that intervention with IL-10M elicited a substantial reduction in plaque area. H&E staining revealed that mice treated with IL-10M in the early intervention group showed a significant reduction in the ratio of plaque area to lumen area. However, the late intervention group did not exhibit statistically significant differences. Notably, mice treated with IL-10M exhibited significantly smaller necrotic cores and reduction in matrix metalloproteinase 9 within the aortic plaques in both the early and late intervention groups. Additionally, the administration of IL-10M showed no obvious side effects. Conclusion We developed an anti-inflammatory protein delivery system based on engineered macrophages that can target atherosclerotic plaques and reduce the plaque area and necrotic core. This approach has a promising safety profile and has the potential to be a therapeutic strategy for the intervention of atherosclerosis.

Systemic inflammation and risk of death in patients with atherosclerotic cardiovascular disease and chronic kidney disease

Abstract Background Atherosclerotic cardiovascular disease (ASCVD) and chronic kidney disease stage 3 - 4 (CKD) affects 7% and 10% of the Danish population, respectively. Systemic inflammation (SI) contributes to destabilisation of atherosclerotic plaques and is characterized by elevated levels of C-reactive protein (CRP) in the blood. CKD is associated with SI and is independently associated with an increased risk of ASCVD. CRP levels ≥2 mg/L have been identified as a risk factor for mortality and major adverse cardiovascular events (MACE). Purpose To provide real-world evidence on the clinical burden of SI in patients diagnosed with ASCVD and CKD, using national health registers. Method Data from the Danish National Patient Registry from 1994-2022 was used to identify ASCVD patients by ICD-10 codes. CKD was identified by eGFR test results in the range [15 -59mL/min/1.73m2], in the Register of Laboratory Results for Research (RLRR). The latter diagnosis of ASCVD and CKD was set as index date. CRP-test results were extracted from RLRR and only individuals with at least two CRP tests were included. If two CRP-test results ≥2mg/L to < 20mg/L were observed less than six month apart, and within two years of index date, the individual was classified to have SI. The eGFR data from RLRR was available from 2011 onwards, with some regional differences in reporting. Individuals observed with CKD or ASCVD in the first year of eGFR availability were excluded to ensure a study of incident individuals. Individuals using select medications or diagnosed with selected chronic conditions thought to influence CRP levels were excluded. Additional population characteristics were extracted from corresponding national health and administrative registers. Hazard of all-cause mortality and MACE was analysed in a Cox proportional risk regression. Results From 2012 to 2022, a total of 19,162 individuals were eligible for the study population (SI: 13,039, No SI: 6,123). Median follow-up time was 4.1 and 8.2 years for individuals with or without SI, respectively. Median CRP two years before and after index date in the two groups was 7mg/L and 1mg/L. No clinically significant differences were found for demographic or socioeconomic characteristics. Patients with SI had more frequent history of hypertension, atrial fibrillation, COPD, heart failure and Type 2 diabetes before index, and higher risk of hypertension, atrial fibrillation, stroke, heart failure and type 2 diabetes after index. SI population had significantly increased hazard of mortality in a crude model (HR: 2.35, 95% CI: [2.22 – 2.50]), and in adjusted analysis HR: 2.06 ([1.92 - 2.21]). The corresponding hazard ratios for MACE were 1.9 ([1.81 – 2.01]) and 1.66 ([1.57 – 1.77]). The results were consistent across different model specifications and subgroups. Conclusion SI is associated with a significantly higher clinical burden of disease and a higher risk of all-cause mortality.Flowchart of inclusion/exclusionHazard ratio, overall and by subgroup

Blockade of foam cell formation by an AP-1 inhibitor attenuates atherosclerosis in diabetes

Abstract Background Atherosclerosis is a major contributor to cardiovascular disease (CVD) related premature deaths in people with diabetes. Residual increased risk of CVD remains even after treatment of standard risk factors including hyperglycaemia. Therefore, there is an unmet clinical need of novel therapies that can directly target the intrinsic pathobiology of atherosclerotic plaque development. Macrophage associated pro-atherosclerotic processes including impaired cholesterol efflux resulting in foam cell formation are important therapeutic targets in atherosclerosis. Expression of genes linked to impaired cholesterol efflux can be regulated by multiple factors including transcription factors (TFs) such as activator protein 1 (AP-1) complex. Although, previous studies have implicated the role of foam cells in atherosclerosis, the role of AP-1 TF complex in foam cell formation in diabetes induced atherosclerosis is not known. Methods Diabetes was induced with streptozotocin (STZ) in atheroprone Apoe-/- mice and 10 weeks later aortae were subjected to single cell RNA sequencing (scRNA-seq) analysis using 10X Genomics platform. In vitro model mimicking diabetes-induced foam cell formation was established in human monocytes THP-1 derived macrophages using high glucose (25mM) and ox-LDL ± a small molecular inhibitor of AP-1, T-5224. AP-1 activity, gene expression and foam cell formation were assessed with activity assay, RT PCR and Oil Red O staining respectively in this setting. Complementary in vitro AP-1 knockdown experiments using shRNA were also performed. Furthermore, preclinical intervention studies were also performed where Apoe-/- mice made diabetic with STZ were treated 5-weeks post diabetes with T-5224 (30 mg/kg body weight daily for 5 weeks). Results scRNA-seq analysis identified a well-defined cluster of foam cells within macrophages with a diabetes specific proatherogenic transcriptomic profile. Differential gene expression analysis showed increased expression of AP-1 members cFos, Atf3, Atf4 and Junb exclusively in foam cell cluster in diabetes (cut off = FDR<0.05, Log2FC -2/2). Comparison with the ENCODE dataset for AP-1 target genes using Harmonizome version 3 showed that indeed multiple differentially expressed genes in diabetes were targets of AP-1. AP-1 members were also upregulated in the THP-1-based in vitro model of high glucose induced foam cell formation. THP-1 derived foam cells showed increased AP-1 activity and lipid uptake. Interestingly, AP-1 inhibition by T-5224 attenuated foam cell formation. AP-1 knockdown experiments validated experimental findings of AP-1 inhibition studies in THP-1 derived macrophages. Importantly, T-5224 treatment blocked foam cell formation resulting in reduced atherosclerosis in diabetic Apoe-/- mice in our intervention study design. Conclusion This study identified the AP-1 inhibition with T-5224 as a potential treatment for the diabetes induced foam cell formation and associated atherosclerosis.

Coronary atherosclerotic plaque burden and characterization by spectral computed tomography scan in patients with high lipoprotein a and non-hemodynamically significant stenosis

Abstract Introduction Atherosclerotic coronary disease increases morbidity and mortality worldwide. Lipoprotein A (LpA) has emerged as a cardiovascular risk marker, but little is known of its direct influence in plaque volume and composition. Spectral computed tomography (CT) technology allows to characterize coronary plaques with new promising tools. Purpose To evaluate the association of LpA with the extent and composition of non-significant coronary plaques producing stenosis <50%. Methods We prospectively recruited 109 patients referred for noninvasive CT angiography (November 2023-January 2024) with suspected coronary artery disease. 20 had totally/partially non-calcified, non-significant coronary plaques. Plaques were segmented and volume was measured. Their composition was stablished as calcified (>200 UH), lipidic (<60 UH) and fibrous (60-200 UH). Spectral data-based plaque parameters were analyzed, including radiological attenuation on polyenergetic images (CTPI), on virtual low/high monoenergetic images (CT40KeV and CT140KeV, respectively), variation in attenuation across monoenergetic levels (slope40-140 KeV) and Z-effective value. CT scan images were acquired with a 128 detectors dual-layer spectral scan with prospective-retrospective/synchronization. Intravascular iodine-based contrast was administered. LpA was stablished as high (≥25 mg/dL) or low (<25 mg/dL). Results Mean age was 60 ± 11. Cardiovascular risk factor were equally distributed between groups. We analyzed 33 plaques. Total plaque volume was significantly higher in high LpA patients (125.9 mm3 vs 62.1 mm3). There were no differences in plaque composition by LpA (Table). Additionally, spectral data showed significantly higher plaque attenuation on CT140KeV images in high LpA patients (36.07 vs 21.32 HU; p = 0.05). There were no differences for the rest of spectral parameters. Conclusions Plaque volume was higher in patients with non-significant atherosclerotic disease and high LpA, but these plaques showed similar composition in high and low LpA patients. These patients might, therefore, respond to the same conventional treatment event though there is not specific treatment for high LpA yet. Additionally, spectral CT140KeV images could add more precision differentiating these plaques since radiological attenuation in these reconstructions is higher in patients with high LpA.TableFigure

Role of Galectin-3 in resident macrophages and effects on atherosclerosis

Abstract Background Arterial macrophages play an important role in atherosclerosis, exhibiting heterogeneity in their origins as either resident or inflammatory monocyte-derived macrophages. Resident macrophages originate predominantly from the yolk sac, and they are endowed with homeostatic functions and can have anti-inflammatory properties under inflammation [1]. Resident macrophages pre-existing within the tissue differentiate into foamy macrophages before monocytes appear in early atherosclerotic lesions [2]. Galectin-3 (Gal-3) encoded by the gene Lgals3, is a crucial effector molecule that is prominently expressed by macrophages [3]. Studies have provided conflicting evidence regarding the role of Gal-3 in atherosclerosis by affecting macrophage functions and monocyte recruitment [4,5]. Purpose This study aims to investigate the role of Gal-3 in arterial resident macrophages for atherosclerosis and reveal the underlying mechanisms. Methods General Lgals3 knockout (Lgals3-/-) and resident macrophage specific knockout (Lgals3-/-Rank-Cretg) mice on the Apoe-deficient background were generated and atherosclerotic lesion development (8 weeks of western diet) was compared. Results Our study revealed that Lgals3-/- mice exhibited increased numbers of monocytes and neutrophils in the blood, which was associated with the development of larger atherosclerotic plaques. Plasma Gal-3 concentration from Lgals3-/-Rank-Cretg mice was decreased by 60%, indicating that the majority of circulating Gal-3 is derived from resident macrophages. Surprisingly, Lgals3-/-Rank-Cretg mice did not show the observed increase in blood monocytes and neutrophils, suggesting that this effect was independent of circulating Gal-3. Consistent with a causal role of monocytosis and leukocytosis in atherogenesis, plaque formation in Lgals3-/-Rank-Cretg was not significantly affected. Additionally, Gal-3 deficiency promoted M1 polarization and reduced efferocytosis, phagocytosis and chemokine receptor expression in BMDMs, and the addition of exogenous Gal-3 restored the effect on phagocytosis and blocked CXCL12 and CCL5-induced leukocyte migration. Conclusion This study reveals a prominent protective role of Gal-3, primarily through inflammatory macrophages rather than resident macrophages for the development of atherosclerotic lesions. This protective effect is attributed to its homeostatic effects on monocyte and neutrophil numbers, as well as its influence on macrophage functions.

A direct comparison of non-invasive blood markers of liver fibrosis as associates of cardiovascular risk. The Athens cardiometabolic registry

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is associated with increased risk of cardiovascular disease (CVD). This risk increases further with advanced liver fibrosis. To date, non-invasive tests (NIT) for liver fibrosis risk have not been established as diagnostic or prognostic biomarkers of CVD. Purpose The purpose of the study was to compare NIT of liver fibrosis with respect to their associations with vascular injury and cardiovascular (CV) events. Methods Individuals without clinically overt CVD and patients with CVD (total n=1,692), consecutively recruited in the Athens Cardiometabolic Registry, were analysed in this study. We retrospectively evaluated the association of NIT (i.e. Fibrosis-4 index (FIB-4), NAFLD Fibrosis score (NFS), BARD score) with indices of subclinical arterial injury (i.e. carotid wall thickness and atherosclerotic plaque for subclinical atherosclerosis, pulse wave velocity (PWV), for arterial stiffness) and the incidence of CV events. Patients were followed-up for CV events for a median time of 39 months. Results Elevated FIB-4 (>2.67) was associated with vascular damage [adjusted odds ratio (aOR)=2.97, 95% confidence intervals (CI)= 1.71-5.78 for maximal wall thickness (maxWT) and aOR=4.58, 95% CI=2.44-8.60 for PWV] across the entire study population, including those with or without clinically overt CVD. NFS> 0.675 showed similar associations but lacked significant correlation with arterial stiffness in coronary artery disease patients. FIB-4> 2.67 and BARD> 2 were associated with increased risk of the composite endpoint of CV death, acute myocardial infarction, or coronary revascularization [adjusted hazard ratio, (aHR)=2.00, 95% CI= 1.12-3.55 and aHR=3.36, 95% CI=1.03-11.0 for FIB-4 (n=842) and BARD (n=887) respectively]. FIB-4 and BARD as continuous variables confered incremental prognostic value compared to European Systematic Coronary Risk Evaluation 2 (SCORE2). Conclusion In a population with a wide range of cardiometabolic risk, FIB-4 was associated with both markers of atherosclerotic disease and arterial stiffness as well as with adverse CV events. These data support further investigation of FIB-4 as a biomarker of CV risk.

AI-enhanced detection of coronary inflammation predicts 10-year risk for cardiac mortality and MACE in individuals with no or minimal coronary atherosclerosis on routine CCTA

Abstract Background Coronary CT angiogram (CCTA) is one of the first line investigations for patients with chest pain suspected of coronary artery disease (CAD). Current CCTA interpretations mainly focus on luminal obstruction, which is identified in the minority of patients, whereas the majority who have no or minimal atherosclerotic plaques visible on CCTA are often provided with reassurance. However, residual risk from inflammation that drives atherosclerosis and plaque vulnerability are not captured with luminal stenoses alone. Fat attenuation index (FAI) Score quantifies coronary inflammation on routine CCTA, and integrate with clinical risk factors/plaque burden in an artificial intelligence enabled risk algorithm (AI-Risk) that could help to risk stratify this patient group. Purpose To evaluate the prognostic value or FAI Score and AI-Risk in the risk stratification of patients with no or minimal atherosclerosis. Methods In a nested cohort within the Oxford Risk Factors and Non-invasive imaging (ORFAN) study, consecutive patients (n=3,393) who underwent routine clinical CCTA were followed up over a median(IQR) 7.7(6.4-9.1) years for cardiac mortality and major adverse cardiac events (MACE, including myocardial infarction, new onset heart failure, cardiac mortality). CADRADS 2.0 classification 0 or 1 were used to define no or minimal luminal stenoses. FAI Score was calculated for each of the 3 epicardial coronary arteries. Results A total of 1,678 patients had no or minimal atherosclerosis on CCTA [median (IQR) age 53(45-63), 49.8% male]. Cardiac mortality occurred in 3.1% and MACE occurred in 8.8% of the patients during the follow-up period. Patients at the lowest quartile of FAI Score or low/medium AI-Risk classification showed very low event rate during follow up. Patients with the highest quartile of LAD FAI Score showed 9 times higher risk of cardiac mortality (Panel A) and 5 times higher risk of MACE (Panel B) compared to those at lowest quartile. Similar findings were observed for LCX and RCA. Finally, integrating coronary inflammation with clinical risk factors and plaque burden, patients at very high AI-Risk classification showed 5 times higher risk of cardiac mortality (Panel C), and 4 times higher risk of MACE (Panel D) compared to those with low/medium risk classification. Conclusion Coronary artery inflammation measured by FAI Score could stratify the risk of patients with no or minimal coronary atherosclerosis on CCTA, and could help to guide early preventative treatments.

Aspirin inhibits atherosclerosis through macrophage pyroptosis by interacting with Lactobacillus johnsonii and its metabolite butyrate

Abstract Introduction Atherosclerotic cardiovascular disease (ASCVD) is a chronic progressive disease closely related to metabolism and inflammation. Aspirin inhibits cyclooxygenase (COX) and has antiplatelet aggregation effects, considered as the cornerstone of ASCVD treatment. Numerous studies have shown that gut microbiota (GM) and its metabolites affects host metabolism. Studies have also shown that people taking aspirin exhibit specific changes in the microbiota profile. However, whether the interaction of aspirin with GM and its metabolites affects the therapeutic efficacy of aspirin in treating atherosclerosis (AS) have not been reported. Purpose This study aimed to explore the interaction of aspirin with the GM and its metabolites in the treatment of AS, to reveal the specific mechanism of the GM-metabolite axis and provide potential new targets for the combined treatment of AS. Methods To establish the mouse model of atherosclerosis by high-fat diet, and the atherosclerotic plaque burden and composition were analyzed. The role of GM in aspirin anti-atherosclerosis was explored by antibiotics and fecal microbiota transplantation (FMT). The third generation 16S RNA sequencing was used to explore the effects of aspirin on species diversity and abundance of GM, and to screen differential microorganisms for intervention of AS mice. UHPLC-MS and GC-MS were used to explore the changes of intestinal metabolites induced by aspirin. The correlation between gut microbiota and metabolites was also investigated. Finally, in vitro and in vivo experiments were performed to confirm the effects and mechanisms of metabolites on macrophage pyroptosis and atherosclerosis. Results Aspirin has anti-atherosclerosis effect. After removing gut microbiota, the therapeutic effect of aspirin on atherosclerosis was decreased. Three generations of 16S rRNA gene sequencing results showed that aspirin supplementation alters the diversity of gut microbiota, and increased the relative abundance of Lactobacillus johnsonii(L.johnsonii) and Ligilactobacillus murinus(L.murinus). After gavage of L.johnsonii and L.murinus, the plaque burden and macrophage pyroptosis were significantly reduced in AS mice. Colonization with L.johnsonii and L.murinus significantly increased the abundance of probiotics and butyrate level in the feces of AS mice and L.johnsonii and L.murinus were positively correlated with butyrate. Supplementation of butyrate inhibited macrophage pyroptosis and atherosclerosis in vivo. In vitro, butyrate could inhibit the expression of NLRP3/Caspase-1/IL-1β and pyroptosome production, and this process mainly by activating butyrate receptors GPR41, GPR43 and GPR109A. Conclusions The gut microbiota may mediate the anti-AS effect of aspirin by promoting an increase in the abundance of L.johnsonii and L.murinus. Butyrate produced by Lactobacillus may mediate the anti-AS effect of aspirin, and inhibiting macrophage pyroptosis and the progression of AS.

Macrophage heterogeneity in cardiometabolic disease

Abstract Metabolic diseases such as obesity and atherosclerosis are characterised by chronic inflammation that leads to the accumulation of immune cells in affected organs. Macrophages in the obese adipose tissue and the atherosclerotic plaque show transcriptional, phenotypic and functional heterogeneity. The accumulation of CD11c+ macrophages in atherosclerosis and obesity suggests the presence of conserved macrophage phenotypes in metabolic tissues. We aimed to characterise shared and distinct features of monocytes and macrophages in the aorta and adipose tissue in the context of metabolic disease using single-cell techniques. Single-cell RNA sequencing of the blood, aorta and visceral adipose tissue during a time course of metabolic disease progression (steady state, 12w western diet, 18w western diet) and data integration with murine aortic and adipose tissue community datasets revealed conserved and tissue specific features of metabolic disease. Resident macrophages in the aorta and adipose tissue displayed tissue specific transcriptional programs, highlighting that cell maintenance and function of tissue resident macrophages differ between metabolic tissues. Further, macrophages in the aorta and adipose tissue undergo disease associated compositional changes. For example, we uncovered distinct CD11c+ macrophage subsets that are conserved in the aorta and adipose tissue across multiple metabolic disease models and share core transcriptional signatures between both metabolic tissues. However, CD11c+ macrophage subsets showed tissue specific dynamics during metabolic disease progression. Overall, our analysis underscores that tissue niche affects macrophage composition as well as activation and transcriptional regulation patterns. Understanding similarities and differences between the two tissues will be important to inform identification of new therapeutic targets for cardiometabolic diseases.

The evolution of carotid atherosclerotic plaque composition: a prospective study

Abstract Background The rupture of carotid atherosclerotic plaque is one of the major causes of ischemic stroke, and largely determined by the composition of the plaque. To date, most studies on carotid plaque composition focused on individual plaque components, whilst the interplay between different components within the same plaque may be a critical driving force for plaque evolution and rupture. Objectives To determine the interplay between carotid plaque components over time and describe the dynamics of plaque composition with a specific focus on age- and sex-specific changes in a population-based setting. Methods From a population-based cohort in Netherlands, 802 participants (mean age ± SD 68 ± 8 years, 43% women) with subclinical carotid atherosclerosis underwent repeat magnetic resonance imaging (MRI) of carotid plaque (first scan between 2007 and 2012, second scan between 2014 to 2017). We evaluated carotid plaque composition, including calcification, lipid-rich necrotic core (LRNC) and intraplaque hemorrhage (IPH). To assess the relationship between the incidence of one specific component and baseline plaque composition, plaques with this specific component present at the baseline were excluded. Generalized estimation equation models were used with adjustments for baseline plaque size and cardiovascular risk factors. We demonstrated the evolution of plaque composition along age and by sex. Results Baseline presence of calcification in carotid plaque is independently associated with a higher incidence of IPH (adjusted odds ratio: 2.25; 95% confidence interval: 1.40 to 3.63). No associations were found between baseline plaque composition and the new onset of calcification or LRNC. The proportion of complex plaques with multiple components significantly increased at follow-up (Figure 1a). The plaque composition dramatically evolved over time and the proportion of plaques with two or more components started with 10% around 55 years and increased to over 50% after 70 years of age (Figure 1b). Although the age distribution was comparable between men and women, men were more likely to have and develop plaques with multiple vulnerable plaque components such as IPH (Figure 2). Conclusions A higher incidence of IPH was found in plaques with baseline calcification. The pre-existing plaque composition strongly determines how the plaque will evolve over time. Given the interplay between individual components, the overall plaque composition exhibits significant age- and sex- specific characteristics.

The foam cell-derived exosomal miRNA Novel-3 drives neuroinflammation and ferroptosis during ischemic stroke.

Large artery atherosclerosis (LAA) is a prevalent cause of acute ischemic stroke (AIS). Understanding the mechanisms linking atherosclerosis to stroke is essential for developing appropriate intervention strategies. Here, we found that the exosomal miRNA Novel-3 is selectively upregulated in the plasma of patients with LAA-AIS. Notably, Novel-3 was predominantly expressed in macrophage-derived foam cells, and its expression correlated with atherosclerotic plaque vulnerability in patients undergoing carotid endarterectomy. Exploring the function of Novel-3 in a mouse model of cerebral ischemia, we found that Novel-3 exacerbated ischemic injury and targeted microglia and macrophages expressing ionized calcium-binding adapter molecule 1 in peri-infarct regions. Mechanistically, Novel-3 increased ferroptosis and neuroinflammation by interacting with striatin (STRN) and downregulating the phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin signaling pathway. Blocking Novel-3 activity or overexpressing STRN provided neuroprotection under ischemic conditions. Our findings suggest that exosomal Novel-3, which is primarily derived from macrophage-derived foam cells, targets microglia and macrophages in the brain to induce neuroinflammation and could serve as a potential therapeutic target for patients with stroke who have atherosclerosis.

Coronary CT angiography using photon counting CT for the detection of coronary artery stenoses and coronary atherosclerotic plaque: comparison of reconstructions with 0.2 mm and 0.4 mm slice thickness

Abstract Photon Counting CT allows the acquisition of high-resolution data sets with 0.2 mm reconstructed slice thickness. It is unknown whether the detection of coronary artery stenoses, of coronary plaque or of vulnerable plaque characteristics is improved in data sets that employ maximum resolution. We therefore compared the identification of above characteristics in data sets reconstructed with 0.2 mm and 0.4 mm slice thickness In 30 patients with suspected coronary artery disease, sinus rhythm, a heart rate ≤65 beats/min, and body weight ≤ 100 kg, high-resolution data sets were acquired by photon counting CT (60 ml contrast i.v. at 6 ml/s) after s.c. application of nitrates in inspiratory breathhold. Data were reconstructed with 0.2 mm slice thickness at 0.1 mm increment and 0.4 mm slice thickness at 0.2 mm increment. Next to quantitative analysis of image quality parameters, all data sets were evaluated by two independent expert reviewers concerning subjective image quality as well as the ability to determine the presence of coronary artery stenoses, calcified and non- calcified plaque, as well as parameters of plaque vulnerability (positive remodeling, necrotic core, density < 30 HU and napkin-ring sign) using a 5-point Likert scale (-2 to +2) and a dedicated 11-segment coronary model. Of the 30 patients, 47% were male (mean age 58±12 years, mean heart rate 53±6 bpm, mean body weight 80±15 kg). The median dose length product was 508 mGy*cm. Mean image noise was 42±7 HU vs. 34±7 HU for 0.2 mm vs. 0.4 mm reconstructions (p < 0.001). The median image quality score was significantly higher for 0.2 mm reconstructions (3.8±0.8 vs 3.6±0.7). Of 335 coronary segments, none were classified as "unevaluable" while 87% vs. 76% were classified as "perfect" in 0.2 mm vs. 0.4 mm reconstructions (p < 0.001). Full confidence to rule-in or rule out significant coronary stenoses was achieved in 93% of segments in both reconstructions. The number of non-calcified plaques identified in 0.2 mm reconstructions was numerically higher than in 0.4 mm reconstructions (77 vs. 71, p = n.s.). On the Likert scale, the confidence to identify parameters of plaque vulnerability was significantly higher for 0.2 mm reconstructions (1.93 vs. 1.85, p < 0.001). In a selected patient group, photon counting CT coronary angiography demonstrated excellent image quality and an extremely low rate of unevaluable coronary segments. High-resolution reconstructions improve subjective image quality as was as the assessment of vulnerable plaque characteristics.

NIR-II scattering gold superclusters for intravascular optical coherence tomography molecular imaging.

Currently, intravascular optical coherence tomography (IV-OCT) is limited to anatomical imaging, providing structural information about atherosclerotic plaque morphology, thrombus and dissection. Earlier detection and risk stratification would be possible through molecular characterization of endothelium but necessitates a purpose-engineered IV-OCT contrast agent. Here we developed gold superclusters (AuSCs) tailored to clinical instrumentation and integrated into clinically relevant workflows. AuSCs are aqueously dispersible clusters of closely packed small gold nanoparticles, affording plasmon hybridization to maximize light scattering at the IV-OCT laser line (~1,350 nm). A polymer coating fosters AuSC uniformity and provides a functionalizable handle, which we targeted to intravascular P-selectin, an early vascular endothelial marker of inflammation. In a rat model of intravascular inflammation, P-selectin-targeted AuSC facilitated IV-OCT molecular imaging, where the strength of the signal correlates with the severity of vascular inflammation.

Antiplatelet can improve the short-term prognosis of type 2 myocardial infarction caused by acute respiratory failure

Abstract Background Type 2 myocardial infarction (T2MI) is characterized by myocardial infarction resulting from an imbalance between myocardial oxygen supply and demand, without the rupture of acute atherosclerotic plaque. Antiplatelet therapy is the cornerstone of treatment for Type 1 myocardial infarction, however, its efficacy in T2MI remains unclear. Purpose This study aimed to explore the impacts of antiplatelets on prognosis in patients with T2MI caused by acute respiratory failure. Methods The data for this study were derived from the Health and Medical Big Data Superplatform, comprising a retrospective cohort of patients admitted to and discharged from 72 secondary and tertiary hospitals from 2010 to 2023. A total of 4708 patients with T2MI due to acute respiratory failure were included. Participants were divided into two groups: those receiving antiplatelet therapy and those not. T2MI caused by acute respiratory failure was defined as NSTEMI patients caused by respiratory failure without percutaneous coronary intervention (PCI). Propensity score matching (PSM) was used to balance covariates between the groups, followed by building a multivariable Cox regression model to further adjust for potential confounders. Covariates adjusted in multivariable Cox regression included antiplatelet, anticoagulant, and lipid-lowering drug use during hospitalization, age, sex, KILLIP classification, history of diabetes, hypertension, percutaneous coronary intervention, stroke, renal insufficiency, cerebral hemorrhage, and atrial fibrillation. One-to-one matching was performed based on propensity scores using the nearest available pair matching method with a caliper width of 0.01. The primary endpoint was Net Adverse Clinical Events (NACE) within 1 month, including myocardial infarction, cardiac death, stroke and Bleeding Academic Research Consortium (BARC) criteria type 3 or 5. Results Following 1:1 propensity score matching, a total of 1418 participants were enrolled in the final study sample, with 709 patients retained in each group. Patients using antiplatelet were more likely to use ACEI/ARB, CCB, and β-blockers during hospitalization, all p<0.05.Compared to non-antiplatelet therapy group, the use of antiplatelet was associated with a 0.69 times risk of 1-month NACE (aHR, 0.69; p<0.001), a 0.69 times risk of 1-month Major Adverse Cardiovascular and Cerebrovascular Events (MACCE) (aHR, 0.69; p<0.001), a 0.70 times risk of 1-month cardiac death (aHR, 0.70; p<0.001), and a 0.70 times risk of 1-month all-cause mortality (aHR, 0.70; p<0.001); with no statistical significance for other clinical outcomes (p > 0.05) (Figure 1). Conclusions The present study suggested that patients receiving antiplatelet therapy had a lower incidence of one-month NACE, primarily due to a reduction in major cardiac death and MACCE at 1 month.

Deficiency of transcription factor E4BP4 suppresses neointimal formation after vascular injury

Abstract Aim In addition to traditional risk factors such as hypertension, diabetes, and dyslipidemia, chronic inflammation is deeply involved in the onset and pathogenesis of atherosclerotic diseases. Furthermore, diurnal fluctuations are observed in the rupture of atherosclerotic plaques and the onset of myocardial infarction. There are still many patients for whom medical treatments such as statins and antihypertensive drugs do not respond adequately, and new treatment methods are expected. E4BP4 (also known as NFIL3) is a basic leucine zipper (bZIP)-type transcription factor associated with circadian rhythms and cell viability, and it has been reported that E4BP4 regulates inflammation. Therefore, to investigate how the circadian transcription factor E4BP4 which is involved in inflammation, we decided to examine the role of E4BP4 in neointimal formation after vascular injury. Methods and Results Using E4BP4-deficient (E4BP4−/−) and wild-type (WT) mice, we investigated neointimal formation 2 weeks after femoral artery injury induced by an external vascular cuff model. Intimal and medial area were measured, and the intima/media ratio was calculated. Systolic blood pressure was similar between groups (WT mice: 92 ± 5 mmHg [n = 5] vs. E4BP4−/− mice: 90 ± 4 mmHg [n = 5]). And there was no significant difference in baseline body weight between groups (WT mice: 22.4 ± 0.8 g [n = 5] vs. E4BP4−/− mice: 22.0 ± 1.1 g [n = 5]). The mean intimal area and the intima/media ratio of E4BP4−/− mice decreased by 86 % (WT mice: 3,683 ± 512 μm2 [n = 10] vs. E4BP4−/− mice: 521 ± 363 μm2 [n = 10]; P < 0.0001) and 97% (WT mice: 0.33 ± 0.05 vs. E4BP4−/− mice: 0.01 ± 0.03; P < 0.0001), compared with WT mice. However, we observed no significant difference in the medial area (WT mice: 11,019 ± 358 μm2 vs. E4BP4−/− mice: 11,281 ± 401 μm2; P = NS) between groups (Figure). Immunohistochemistry for NK cell (NKp46) revealed the percentage of NKp46 positive area in the adventitia of E4BP4−/− mice was significantly lower compared with WT mice at 14 days post-injury (P <0.05). Furthermore, the positive area of IL-6 (P<0.05), TNF-α (P<0.05) and IFN-γ (P<0.05) in the intima of E4BP4−/− mice tended to be much smaller than those of WT mice. Our findings indicate that E4BP4 might induce vascular inflammation following vascular injury. Conclusions Deficiency of E4BP4 reduced the inflammatory cytokines and suppressed neointimal formation after injury. We also observed a decrease in NKp46, suggesting that NK cell development is impaired by E4BP4 deficiency. The present study is the first to demonstrate that E4BP4 plays an important role in the increase of neointimal formation after injury in vivo, thus suggesting that E4BP4 inhibition may represent a useful strategy to inhibit vascular inflammation.Neointimal formation after cuff injury

Interscan reproducibility of computed tomography derived coronary plaque volume measurements using a semi-automated analysis software

Abstract Background Coronary computed tomography angiography (CCTA) enables detailed quantification and characterization of coronary atherosclerotic plaque, offering diagnostic and prognostic value. Despite the growing demand for accurate plaque analysis, studies on interscan reproducibility of automated plaque quantification are limited in number and size. Moreover, impact of clinical and technical factors on the reproducibility of plaque assessment tools are scarce. Purpose The objective of this study was to assess the interscan reproducibility of CCTA-derived plaque volume measurements and the implications of clinical and technical characteristics on interscan reproducibility. Methods A total of 101 patients with known coronary artery disease underwent two CCTA scans within hour interval using identical CT acquisition protocol. Coronary plaque volume measurements were quantified on a per-patient level using a contemporary semi-automated plaque analysis software. Results There was no statistically significant difference in median plaque volumes between the first and second scan. The direct correlation was excellent for volumes of non-calcified plaque (NCP), calcified plaque (CP), and total plaque (TP) across all analyses (Pearson’s coefficient 0.96 to 0.99), while moderate for low-density non-calcified plaque (LD-NCP) volumes (Pearson’s coefficient 0.77). Bland Altman analyses demonstrated high interscan agreement with narrow absolute and relative limits of agreement (LoA) across plaque subtypes, except for LD-NCP volumes (Table 1). Interscan reproducibility on CP volumes was affected by CT image quality with narrower LoA in scans with the highest image quality score (p = 0.003), or lowest image reconstructive iteration level (p <0.001). Limits of agreement were significantly narrower for NCP, CP, and TP volumes in lesions located in LAD compared to non-LAD lesions (p <0.001). However, no significant difference in LoA was present when comparing patients according to BMI (<27 and ≥27 kg/m2), heart rate (<65 and ≥65 beats per minute), or Agatston score (<193 and ≥193). Conclusions This comprehensive interscan reproducibility study revealed good agreement in the measurement of plaque subtypes. Adding new insight to existing literature, we have demonstrated that factors such as image quality, image reconstructive algorithms, and lesion location affect the reproducibility of plaque volume measurements. CT-derived plaque quantification using a semi-automated plaque analysis software holds potential for monitoring disease progression or regression in patients with CAD, provided high CCTA image quality.

Diesel exhaust particles activate macrophages and IRF5 expression in atherosclerosis

Abstract Introduction Over 20% of global deaths related to cardiovascular disease have been attributed in part to air pollution, particularly fine particulate matter (PM2.5) (1). In highly trafficked urban areas, smaller ultrafine diesel exhaust particles (DEP) represent a significant proportion of PM2.5. DEP is associated with the progression of atherosclerosis and increased plaque susceptibility to rupture (2). We have previously shown that interferon regulatory factor 5 (IRF5) has a key role in necrotic core formation, a distinguishing feature of vulnerable atherosclerotic plaques, by rendering macrophages defective at efferocytosis (3). Pronounced elevation of IRF5 is also seen in symptomatic carotid plaques, particularly in the rupture-prone shoulder regions (4). Purpose We examined the effect of DEP on macrophage phenotype and IRF5 expression in atherosclerosis. Methods Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice, differentiated with GM-CSF (to mimic inflammatory macrophages) or M-CSF (mimicking homeostatic resident macrophages) were incubated with PBS or DEP (10 µg/mL) in vitro for 18 hours. Expression of inflammatory cytokines (IL-6, IL-12α), macrophage polarisation markers CD11c (itgax, inflammatory) and CD206 (mrc1, resident anti-inflammatory) and IRF5 was quantified by real-time PCR. In addition, high fat-fed ApoE-/- mice were exposed to saline or DEP (35 µL at 1 mg/mL) twice weekly for 5 weeks by pulmonary administration. The pan-macrophage marker CD64, CD206 and IRF5 were quantified in paraffin-embedded sections of brachiocephalic artery lesions by immunohistochemistry. Results In M-CSF-differentiated murine macrophages incubated with DEP, significant fold increases in gene expression (relative to PBS control) were observed: IRF5 (1.4-fold), IL-6 (31.5-fold) and IL-12α (5.8-fold) while CD206 (0.8-fold) decreased (Figure 1a, n = 5, p<0.05, two-tailed paired t-tests). CD11c expression was unchanged. No statistically significant differences were detected in GM-CSF-differentiated macrophages. In the brachiocephalic arteries of DEP-exposed ApoE-/- mice we observed increases in CD64+ macrophages (28.2±7.4 vs 15.5±5.6) and IRF5 (16.6±3.6 vs 10.1±3.4) expression, while CD206 expression decreased (4.1±0.9 vs 11.0±1.2) in comparison to saline-exposed mice (Figure 1b mean±SD, n = 5, p<0.05, two-tailed unpaired t-tests, data expressed as % of intima area). Conclusion DEP promotes CD64+ macrophages and IRF5 expression in murine atherosclerosis, while inflammation and IRF5 is induced in vitro, but only in M-CSF-differentiated murine BMDMs. This suggests that DEP has no impact on inflammatory macrophages but could switch homeostatic resident macrophages to a more inflammatory phenotype via the activation of IRF5. Therefore, IRF5 is a promising candidate for further investigation into the initial macrophage response to inhaled particles that leads to the exacerbation of vascular disease.

Sex differences in coronary atherosclerotic plaque activity using 18F-sodium fluoride positron emission tomography

Abstract Background There are sex differences in the extent, severity, and outcomes of coronary artery disease. Purpose To assess the influence of sex on coronary atherosclerotic plaque activity measured using coronary 18F-sodium fluoride (18F-NaF) positron emission tomography (PET), and to determine whether 18F-NaF PET has prognostic value in both women and men. Methods In a post-hoc analysis of observational cohort studies of patients with coronary atherosclerosis who had undergone 18F-NaF PET CT angiography, we compared the coronary microcalcification activity (CMA) in women and men. Results Baseline 18F-NaF PET CT angiography was available in 999 participants (151 (15%) women) with 4282 patient-years of follow-up. Compared to men, women had lower coronary calcium scores (116 [interquartile range, 27-434] versus 205 [51-571] Agatston units; p=0.002) and CMA values (0.0 [0.0-1.12] versus 0.53 [0.0-2.54], p=0.01). Following matching for plaque burden by coronary calcium scores and clinical comorbidities, there was no sex-related difference in CMA values (0.0 [0.0-1.12] versus 0.0 [0.0-1.23], p=0.21) and similar proportions of women and men had no 18F-NaF uptake (53.0% (n=80) and 48.3% (n=73); p=0.42), or CMA values >1.56 (21.8% (n=33) and 21.8% (n=33); p=1.00). Over a median follow-up of 4.5 [4.0-6.0] years, myocardial infarction occurred in 6.6% of women (n=10) and 7.8% of men (n=66). Coronary microcalcification activity greater than 0 was associated with a similarly increased risk of myocardial infarction in both women (HR: 3.83; 95% CI:1.10-18.49; p=0.04) and men (HR: 5.29; 95% CI:2.28-12.28; p<0.001). Conclusions Although men present with more coronary atherosclerotic plaque than women, increased plaque activity is a strong predictor of future myocardial infarction regardless of sex.Sex differences in CAD activityCase examples