Atherosclerotic Plaque Destabilization Research Articles

The novel bispecific anti-NLRP3 inflammasome antibody InflamAb inhibits atherosclerosis in apolipoprotein E-deficient mice

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Background/Introduction Atherosclerosis is the underlying pathology of many cardiovascular diseases and is characterized by chronic inflammation in the larger arteries. Activation of the NLRP3 inflammasome is one of the drivers of inflammation during atherosclerosis. Purpose Therefore, we aimed to determine the ability of the novel bispecific anti-NLRP3 inflammasome antibody, called InflamAb, to inhibit atherosclerosis. Methods and Results In vitro treatment of bone marrow derived macrophages with 25 ng/mL InflamAb effectively inhibited the IL-1β release induced by NLRP3 inflammasome activation with LPS and Aluminium hydroxide (P<0.05). In addition, InflamAb administration in western-type diet fed Apoe-/- mice significantly reduced circulating IL-1β at 4 hours post inflammasome activation (P<0.05), thereby showing in vivo efficacy of InflamAb. Subsequently, we assessed the effect of InflamAb on both the development of atherosclerosis and on pre-existing plaques. Treatment of western-type diet fed female Apoe-/- mice with 100 µg InflamAb or isotype control antibody (3x per week i.p) significantly inhibited collar-induced atherosclerotic plaque development in the carotid artery from 59±8*10^3 µm^2 in control mice to 36±5*10^3 µm^2 (P<0.05), which coincided with a reduction in relative macrophage (control: 36±2% versus InflamAb: 28±3%, P<0.05) and necrotic core content (control: 16±2% versus InflamAb: 8±1%, P<0.05). InflamAb treatment of male Apoe-/- with pre-existing atherosclerosis did not affect lesion size, but improved plaque stability parameters as illustrated by a reduced relative macrophage (control: 48±2% versus InflamAb: 42±2%, P<0.05) and necrotic core (control: 21±1% versus InflamAb: 18±1%, P<0.05) content. In addition, we observed a trend towards increased collagen levels upon InflamAb treatment (control: 39±2% versus InflamAb: 44±2%, P=0.08). Conclusion To conclude, NLRP3 inflammasome inhibition by the bispecific antibody InflamAb shows promising efficacy in inhibiting atherosclerotic plaque development and destabilization.

Secondary Prevention of Multifocal Atherosclerosis Combined with Chronic Obstructive Pulmonary Disease

The problem of secondary prevention of multifocal atherosclerosis (MAS) combined with chronic obstructive pulmonary disease (COPD), is a leading cause of death in the world. Therefore, the search for drugs capable of affecting the destructive mechanisms underlying both MAS and COPD is an extremely urgent problem today. Objective — to reduce the risks of destabilization of atherosclerotic plaques by reducing the levels of metallomatrix metalloproteases (MMP)-2 and MMP-9 in patients on MAS combined with COPD. Materials and methods. The study included 62 men (68.1 ± 4.2) years old with MAS. All patients with MAS had clinical and functional signs of injury in coronary, cerebral and femoral vascular territories, 30 of these patients (group MAS-2) additionally had clinical and functional signs of COPD (GOLD-2). The control group (CG) consisted of 18 practically healthy men, aged (65.4 ± 3.7) years. Examination of the patients included echocardiography, dopplerography of the vessels of the neck and arteries of the lower extremities, determination of walking distance and ankle-brachial index, Holter ECG monitoring, spirography and determination of MMP-2 and MMP-9 levels in blood. Patients of both groups were prescribed cilostazol (50 mg twice a day) and GABA aminalon (250 mg twice a day) on the background of basic treatment. The course of treatment lasted 16 weeks. Results and discussion. During the initial examination of patients, MAS-2 group showed significantly (p < 0.001) lower volumetric blood flow in the studied arteries, and significantly (p < 0.01) higher levels of MMP-2 and MMP-9, as compared to CG, as well as with patients of the MAS-1 group. After 16 weeks of treatment, with the addition of cilostazol and aminalon, the level of MMP in the blood significantly (p < 0.05) decreased in both groups, in particular, in the MAS-2 group, in patients with the combined pathology of MAS and COPD, a significant decrease was found MMP-2 by 23.6 % (p < 0.01), and MMP-9 by 12.1 % (p < 0.05). Volumetric blood flow indicators increased significantly (p < 0.05) in all studied vascular territories, which led to improvement in clinical manifestations of the disease — a decrease in the number of painful and painless episodes of myocardial ischemia, and an increase in walking distance. Conclusions. The use of a complex therapy of statins, cilostazol and GABA in patients with MAS combined with COPD allows to reduce significantly the levels of MMP-2 and MMP-9, which ensures the stability of atheromatous plaques and significantly improves blood supply in vascular territories with atherosclerotic lesions.

Integrating machine learning algorithms and single-cell analysis to identify gut microbiota-related macrophage biomarkers in atherosclerotic plaques.

The relationship between macrophages and the gut microbiota in patients with atherosclerosis remains poorly defined, and effective biological markers are lacking. This study aims to elucidate the interplay between gut microbial communities and macrophages, and to identify biomarkers associated with the destabilization of atherosclerotic plaques. The goal is to enhance our understanding of the underlying molecular pathways and to pave new avenues for diagnostic approaches and therapeutic strategies in the disease. This study employed Weighted Gene Co-expression Network Analysis (WGCNA) and differential expression analysis on atherosclerosis datasets to identify macrophage-associated genes and quantify the correlation between these genes and gut microbiota gene sets. The Random Forest algorithm was utilized to pinpoint PLEK, IRF8, BTK, CCR1, and CD68 as gut microbiota-related macrophage genes, and a nomogram was constructed. Based on the top five genes, a Non-negative Matrix Factorization (NMF) algorithm was applied to construct gut microbiota-related macrophage clusters and analyze their potential biological alterations. Subsequent single-cell analyses were conducted to observe the expression patterns of the top five genes and the interactions between immune cells. Finally, the expression profiles of key molecules were validated using clinical samples from atherosclerosis patients. Utilizing the Random Forest algorithm, we ultimately identified PLEK, IRF8, CD68, CCR1, and BTK as gut microbiota-associated macrophage genes that are upregulated in atherosclerotic plaques. A nomogram based on the expression of these five genes was constructed for use as an auxiliary tool in clinical diagnosis. Single-cell analysis confirmed the specific expression of gut microbiota-associated macrophage genes in macrophages. Clinical samples substantiated the high expression of PLEK in unstable atherosclerotic plaques. Gut microbiota-associated macrophage genes (PLEK, IRF8, CD68, CCR1, and BTK) may be implicated in the pathogenesis of atherosclerotic plaques and could serve as diagnostic markers to aid patients with atherosclerosis.

Levels of metalloproteinases and adipose tissue hormones in men with coronary atherosclerosis

Aim. To study the effect of adipose tissue hormones on the level of metalloproteinases in men with verified coronary atherosclerosis and to assess associations between the studied biomarkers and abdominal obesity.Materials and methods. The study included 96 men aged 58.9 ± 5.1 years: 80 men with angiographically verified atherosclerosis and class II–III angina pectoris and 16 men without atherosclerosis. Anthropometric parameters were measured in all patients, and their blood was taken on an empty stomach. The blood levels of adiponectin, leptin, resistin, adipsin, amylin, and metalloproteinases (MMPs) -1, -2, -3, -7, -9, -10, -12, -13 were determined by the multiplex analysis. Statistical processing of the results was carried out using the SPSS 13.0 software.Results. In patients with severe atherosclerosis, lipocalin, MMP-1, MMP-7, and MMP-12 levels were higher than in the control group. The blood concentration of adiponectin in patients with atherosclerosis was reduced. Inverse correlations were revealed between waist circumference and concentrations of MMP-1 and MMP-12, as well as between body mass index and MMP-1. A moderate direct relationship was revealed between resistin and MMP-2 and MMP-3; between amylin and MMP-9; between adiponectin and MMP-12; between leptin and MMP-7.Conclusion. The results obtained suggest a relationship between the level of damage markers and adipose tissue hormones, which lead to complications of cardiovascular diseases and explain the effect of obesity on atherosclerotic plaque destabilization.

Proteomic analysis of the extracellular matrix of human atherosclerotic plaques shows marked changes between plaque types

Cardiovascular disease is the leading cause of death, with atherosclerosis the major underlying cause. While often asymptomatic for decades, atherosclerotic plaque destabilization and rupture can arise suddenly and cause acute arterial occlusion or peripheral embolization resulting in myocardial infarction, stroke and lower limb ischaemia. As extracellular matrix (ECM) remodelling is associated with plaque instability, we hypothesized that the ECM composition would differ between plaques. We analyzed atherosclerotic plaques obtained from 21 patients who underwent carotid surgery following recent symptomatic carotid artery stenosis. Plaques were solubilized using a new efficient, single-step approach. Solubilized proteins were digested to peptides, and analyzed by liquid chromatography-mass spectrometry using data-independent acquisition. Identification and quantification of 4498 plaque proteins was achieved, including 354 ECM proteins, with unprecedented coverage and high reproducibility. Multidimensional scaling analysis and hierarchical clustering indicate two distinct clusters, which correlate with macroscopic plaque morphology (soft/unstable versus hard/stable), ultrasound classification (echolucent versus echogenic) and the presence of hemorrhage/ulceration. We identified 714 proteins with differential abundances between these groups. Soft/unstable plaques were enriched in proteins involved in inflammation, ECM remodelling, and protein degradation (e.g. matrix metalloproteinases, cathepsins). In contrast, hard/stable plaques contained higher levels of ECM structural proteins (e.g. collagens, versican, nidogens, biglycan, lumican, proteoglycan 4, mineralization proteins). These data indicate that a single-step proteomics method can provide unique mechanistic insights into ECM remodelling and inflammatory mechanisms within plaques that correlate with clinical parameters, and help rationalize plaque destabilization. These data also provide an approach towards identifying biomarkers for individualized risk profiling of atherosclerosis.

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.

Extracellular Vesicles in Atherosclerosis: State of the Art.

Atherosclerosis is a chronic inflammatory disease driven by lipid accumulation in the arteries, leading to narrowing and thrombosis that causes mortality. Emerging evidence has confirmed that atherosclerosis affects younger people and is involved in the majority of deaths worldwide. EVs are associated with critical steps in atherosclerosis, cholesterol metabolism, immune response, endothelial dysfunction, vascular inflammation, and remodeling. Endothelial cell-derived EVs can interact with platelets and monocytes, thereby influencing endothelial dysfunction, atherosclerotic plaque destabilization, and the formation of thrombus. EVs are potential diagnostic and prognostic biomarkers in atherosclerosis (AS) and cardiovascular disease (CVD). Importantly, EVs derived from stem/progenitor cells are essential mediators of cardiogenesis and cardioprotection and may be used in regenerative medicine and tissue engineering.

Abstract 14313: A Proteomic Atlas of Human Coronary Artery Reveals the Pathological Progression of Atherosclerosis

Introduction: The key molecular mechanisms associated with human atherosclerotic plaque progression, atherosclerotic lesion initiation and plaque destabilization are not fully understood. Hypothesis: Different stages of atherosclerosis (AS) progression have distinct pathological processes. Some key proteins induce AS progression. Methods: We employed 20 coronary artery samples among 4 explanted hearts from coronary heart disease patients. Each heart contains stages of unaffected control (NC), pathological intimal thickening (PIT), fibroatheroma (FA), transition to thin-cap fibroatheroma (TCFA) and ruptured plaque (RP), which were validated by hematoxylin and eosin (H&E) staining. We performed quantitative proteomics in each of the samples based on tandem-mass-tags and liquid chromatography-tandem mass spectrometry. Furthermore, we measured proteins in human coronary artery tissue via immunofluorescence. Results: Proteomics revealed that extracellular matrix (ECM) organization was the most significantly enriched pathway in PIT. FA exhibited an enrichment of immunologic processes, such as neutrophil degranulation, myeloid leukocyte activation and innate immune response. Molecular profiles of TCFA and RP are similar, with complement and coagulation cascades pathway as a hallmark of these stages. Clustering analysis of DEPs displayed 4 clusters, notably cluster 2 had a precipitous elevation from FA to TCFA. Within this cluster, we identified a high-rank molecule: macrophage-capping protein (CAPG). Immunohistochemistry and immunofluorescence revealed that CAPG is highly elevated in plaque compared to NC and is specifically expressed in the plaque. Conclusions: ECM organization was continuously dysregulated in AS progression. Immune response might initiate the atherosclerotic plaque. Complement and coagulation cascades pathway was a hallmark of TCFA and RP. CAPG might be a therapeutic target for AS progression.

Protocatechuic aldehyde increases pericyte coverage and mitigates pericyte damage to enhance the atherosclerotic plaque stability

Pericyte dysfunction and loss contribute substantially to the destabilization and rupture of atherosclerotic plaques. Protocatechuic aldehyde (PCAD), a natural polyphenol, exerts anti-atherosclerotic effects. However, the effects and mechanisms of this polyphenol on pericyte recruitment, coverage, and pericyte function remain unknown. We here treated apolipoprotein E-deficient mice having high-fat diet-induced atherosclerosis with PCAD. PCAD achieved therapeutic effects similar to rosuvastatin in lowering lipid levels and thus preventing atherosclerosis progression. With PCAD administration, plaque phenotype exhibited higher stability with markedly reduced lesion vulnerability, which is characterized by reduced lipid content and macrophage accumulation, and a consequent increase in collagen deposition. PCAD therapy increased pericyte coverage in the plaques, reduced VEGF-A production, and inhibited intraplaque neovascularization. PCAD promoted pericyte proliferation, adhesion, and migration to mitigate ox-LDL-induced pericyte dysfunction, which thus maintained the capillary network structure and stability. Furthermore, TGFBR1 silencing partially reversed the protective effect exerted by PCAD on human microvascular pericytes. PCAD increased pericyte coverage and impeded ox-LDL-induced damages through TGF-β1/TGFBR1/Smad2/3 signaling. All these novel findings indicated that PCAD increases pericyte coverage and alleviates pericyte damage to improve the stability of atherosclerotic plaques, which is accomplished by regulating TGF-β1/TGFBR1/Smad2/3 signaling in pericytes.

Features of the myocardial infarction course in convalescents of the new coronavirus infection COVID-19

Acute myocardial injury is one of the possible complications of the new coronavirus infection COVID-19 and can be diagnosed both in the acute phase of the infection, and also after stabilization or clinical improvement of the patient's condition. This review is devoted to the actual problem of the acute myocardial infarction development during the period of COVID-19 convalescence. The pathophysiological mechanisms of acute myocardial infarction during recovery from COVID-19 are diverse. The key role belongs to the state of hypercoagulation and the systemic inflammatory response, which can provoke destabilization and rupture of unstable atherosclerotic cardiac plaques. Type 2 acute myocardial infarction is most often diagnosed in COVID-19 convalescents against the background of intact coronary arteries. In this case, acute myocardial infarction develops due to an imbalance between increased myocardial oxygen demand (increased levels of cytokines in the blood, hypercatecholaminemia, hyperthermia and tachycardia) and a decrease in oxygen supply to cardiomyocytes due to hypoxemia and hypotension. Subclinical myocardial injury may be present despite normal coronary arteries on angiography and intact left ventricular ejection fraction. There is no clear relationship between the development of acute myocardial infarction during the period of COVID-19 convalescence and the severity of the infection, the time from the initial diagnosis, and the presence of traditional risk factors for cardiovascular diseases in the patient. Cardiac complaints in COVID-19 convalescents are often interpreted as a post-COVID syndrome, especially given the absence of a history of coronary heart disease in many patients, which can lead to late diagnosis. Further study of the features of acute myocardial infarction during the period of COVID-19 convalescence is needed.

Predictive role of MMP-9 and MPO in patients with reduced glomerular filtration rate after acute coronary syndrome

Background. Coronary artery disease (CAD) persistently remains the leading cause of mortality globally. Given the severity and impact of this condition, researchers have been meticulously studying the pathogenesis of athe-rosclerosis, a principal cause behind CAD. The pathogenesis stages are complex and multifaceted, including factors such as lipid accumulation, inflammation, and plaque formation. A particular area of active exploration pertains to the influence and role of different biomarkers, including matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO), on these processes. These biomarkers have been associated with the progression and destabilization of athe-rosclerotic plaques, which are central to CAD. However, the use of these biomarkers in the context of comorbidities, such as chronic kidney disease, remains an open area of research, especially in patients after myocardial infarction. Materials and methods. In our study, 96 patients who had acute coronary syndrome and subsequently undergone percutaneous coronary intervention were enrolled. They were stratified into groups (A and B) based on respective glomerular filtration rates. The primary endpoint of the study was all-cause mortality and major adverse cardiovascular and cerebrovascular events. Results. Our analysis revealed that serum levels of MPO in group B were insignificantly higher than those in group A. Conversely, the area under the receiver operating characteristic (ROC) curve for MMP-9 in group A exhibited a significant difference, standing at 0.8 (95% confidence interval 0.609–0.991; p = 0.039). However, the ROC curve for MPO did not yield a significant result in any group. A combined ROC curve was also generated, with the area under this curve showing a significantly higher value of 0.890 (95% confidence interval 0.805–0.975; p < 0.001). Conclusions. We found that plasma levels of the above-mentioned biomarkers do not seem to influence a decrease in glomerular filtration rate. Nonetheless, MMP-9 levels offered significant prognostic information regarding predicted outcomes.

The Roles of Periodontal Bacteria in Atherosclerosis.

Atherosclerosis (AS) is an inflammatory vascular disease that constitutes a major underlying cause of cardiovascular diseases (CVD) and stroke. Infection is a contributing risk factor for AS. Epidemiological evidence has implicated individuals afflicted by periodontitis displaying an increased susceptibility to AS and CVD. This review concisely outlines several prevalent periodontal pathogens identified within atherosclerotic plaques, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. We review the existing epidemiological evidence elucidating the association between these pathogens and AS-related diseases, and the diverse mechanisms for which these pathogens may engage in AS, such as endothelial barrier disruption, immune system activation, facilitation of monocyte adhesion and aggregation, and promotion of foam cell formation, all of which contribute to the progression and destabilization of atherosclerotic plaques. Notably, the intricate interplay among bacteria underscores the complex impact of periodontitis on AS. In conclusion, advancing our understanding of the relationship between periodontal pathogens and AS will undoubtedly offer invaluable insights and potential therapeutic avenues for the prevention and management of AS.

Imaging Carotid Plaque Inflammation Using Positron Emission Tomography: Emerging Role in Clinical Stroke Care, Research Applications, and Future Directions.

Atherosclerosis is a chronic systemic inflammatory condition of the vasculature and a leading cause of stroke. Luminal stenosis severity is an important factor in determining vascular risk. Conventional imaging modalities, such as angiography or duplex ultrasonography, are used to quantify stenosis severity and inform clinical care but provide limited information on plaque biology. Inflammatory processes are central to atherosclerotic plaque progression and destabilization. 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is a validated technique for quantifying plaque inflammation. In this review, we discuss the evolution of FDG-PET as an imaging modality to quantify plaque vulnerability, challenges in standardization of image acquisition and analysis, its potential application to routine clinical care after stroke, and the possible role it will play in future drug discovery.

The role of acetylsalicylic acid in the treatment of diseases caused by atherosclerosis

Arterial involvement in atherosclerosis is systemic in nature and is not limited to a single vessel causing ischemic symptoms. Atherosclerotic plaques at various stages of development are simultaneously present in arteries of a patient with atherosclerosis, and many of them undergo cycles of rupture and repair without any clinical manifestations. The chronic use of antithrombotic drugs is one of the ways to prevent the development of clinically significant thrombosis after atherosclerotic plaque destabilization. For this purpose antiplatelet agents, are used in patients with diseases caused by atherosclerosis, who do not have indications for long-term use of high (therapeutic) doses of anticoagulants. At present, acetylsalicylic acid maintains a leading position of the main antithrombotic drug in monotherapy and a must component of composite antithrombotic therapy. Recent studies have limited its role only in certain clinical settings: (1) a prolonged (more than 1 month) use after coronary stenting in patients requiring a long-term use of high (therapeutic) doses of oral anticoagulants; (2) a long-term use after coronary stenting in some patients receiving acetylsalicylic acid combined with ticagrelor; (3) use as first-line drug monotherapy in patients with symptomatic atherosclerosis of arteries of the lower extremities; (4) a long-term use as first-line drug monotherapy after ischemic non-cardioembolic stroke. The use of acetylsalicylic acid requires adequate patient adherence to treatment and sufficient bioavailability of the active substance. When there are doubts on proper bioavailability, it is reasonable to use nonenteric-coated dosage forms of acetylsalicylic acid.

Influence of PCSK9 inhibition on biomarkers of atherosclerotic plaque destabilization release in patient with dyslipidemia

Influence of PCSK9 inhibition on biomarkers of atherosclerotic plaque destabilization release in patient with dyslipidemia

Hypoglycemia imitating cardiac ischemic symptoms A case report and review of literature

Ischemic heart disease is the most common cause of mortality in all low and high-income countries. Diabetes mellitus is also a major public health challenge increasing rapidly worldwide and is reported as an equivalent to coronary heart disease (CHD) in some studies. Hypoglycemia is a common complication in diabetic patients that can stimulate the sympathetic nervous system in its severe form, eventually leading to the destabilization of atherosclerotic plaques and cardiovascular events. Therefore, preventing hypoglycemia is crucial in diabetic patients, especially those with a previous history of CAD, in order to prevent cardiovascular events. In this study, we report a diabetic patient presented with multiple hypoglycemic episodes and chest pain.

Bruton's Tyrosine Kinase inhibition by Acalabrutinib does not affect early or advanced atherosclerotic plaque size and morphology in Ldlr-/- mice

Atherosclerosis is characterized by the accumulation of lipids and immune cells, including mast cells and B cells, in the arterial wall. Mast cells contribute to atherosclerotic plaque growth and destabilization upon active degranulation. The FcεRI-IgE pathway is the most prominent mast cell activation route. Bruton's Tyrosine Kinase (BTK) is involved in FcεRI-signaling and may be a potential therapeutic target to limit mast cell activation in atherosclerosis. Additionally, BTK is crucial in B cell development and B-cell receptor signaling. In this project, we aimed to assess the effects of BTK inhibition on mast cell activation and B cell development in atherosclerosis.In human carotid artery plaques, we showed that BTK is primarily expressed on mast cells, B cells and myeloid cells. In vitro, BTK inhibitor Acalabrutinib dose-dependently inhibited IgE mediated activation of mouse bone marrow derived mast cells. In vivo, male Ldlr−/− mice were fed a high-fat diet for eight weeks, during which mice were treated with Acalabrutinib or control solvent. In Acalabrutinib treated mice, B cell maturation was reduced compared to control mice, showing a shift from follicular II towards follicular I B cells. Mast cell numbers and activation status were not affected. Acalabrutinib treatment did not affect atherosclerotic plaque size or morphology. In advanced atherosclerosis, where mice were first fed a high-fat diet for eight weeks before receiving treatment, similar effects were observed.Conclusively, BTK inhibition by Acalabrutinib alone did neither affect either mast cell activation nor early- and advanced atherosclerosis, despite the effects on follicular B cell maturation.

Destabilization of Atherosclerotic Plaque by Bilirubin Deficiency.

The rupture of atherosclerotic plaque contributes significantly to cardiovascular disease. Plasma concentrations of bilirubin-a byproduct of heme catabolism-inversely associate with risk of cardiovascular disease, although the link between bilirubin and atherosclerosis remains unclear. To assess the role of bilirubin in atherosclerotic plaque stability, we crossed Bvra-/- with Apoe-/- mice and used the tandem stenosis model of plaque instability. Human coronary arteries were obtained from heart transplant recipients. Analysis of bile pigments, heme metabolism, and proteomics were performed by liquid chromatography tandem mass spectrometry. MPO (myeloperoxidase) activity was determined by in vivo molecular magnetic resonance imaging, liquid chromatography tandem mass spectrometry analysis, and immunohistochemical determination of chlorotyrosine. Systemic oxidative stress was evaluated by plasma concentrations of lipid hydroperoxides and the redox status of circulating Prx2 (peroxiredoxin 2), whereas arterial function was assessed by wire myography. Atherosclerosis and arterial remodeling were quantified by morphometry and plaque stability by fibrous cap thickness, lipid accumulation, infiltration of inflammatory cells, and the presence of intraplaque hemorrhage. Compared with Bvra+/+Apoe-/- tandem stenosis littermates, Bvra-/-Apoe-/- tandem stenosis mice were deficient in bilirubin, showed signs of increased systemic oxidative stress, endothelial dysfunction, as well as hyperlipidemia, and had a higher atherosclerotic plaque burden. Heme metabolism was increased in unstable compared with stable plaque of both Bvra+/+Apoe-/- and Bvra-/-Apoe-/- tandem stenosis mice and in human coronary plaques. In mice, Bvra deletion selectively destabilized unstable plaque, characterized by positive arterial remodeling and increased cap thinning, intraplaque hemorrhage, infiltration of neutrophils, and MPO activity. Proteomic analysis confirmed Bvra deletion enhanced extracellular matrix degradation, recruitment and activation of neutrophils, and associated oxidative stress in unstable plaque. Bilirubin deficiency, resulting from global Bvra deletion, generates a proatherogenic phenotype and selectively enhances neutrophil-mediated inflammation and destabilization of unstable plaque, thereby providing a link between bilirubin and cardiovascular disease risk.

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Meet the First Authors.

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.