Plaque Stability Research Articles

Early and short-term use of proprotein convertase anti-subtilisin-kexin type 9 inhibitors on coronary plaque stability in acute coronary syndrome.

Proprotein convertase anti-subtilisin-kexin type 9 inhibitors (PCSK9Is) improve plaque volume and composition and reduce major adverse coronary events in chronic coronary artery disease. We evaluated the effects of the short-term use of PCSK9Is on coronary plaque stability in patients with acute coronary syndrome (ACS) using optical coherence tomography (OCT). This is a multicentre, open-label randomized controlled trial. The enrolled 80 subjects met the inclusion criteria. Of these, 52 patients (age 60 ± 11 years, 38 men, 14 women) with ST-elevated ACS had undergone successful primary percutaneous coronary intervention with LDL-cholesterol (LDL-C) levels > 70 mg/dL while receiving high-intensity statins. Participants were randomly assigned to the PCSK9I group (evolocumab 420 mg for 3 months, n = 29) or the standard of care (SoC) group (n = 23). Optical coherence tomography was performed at baseline (BL) and 3 and 9 months after randomization to assess lipid-rich plaques in non-culprit lesions. The change in the minimum fibrous cap thickness (MFCT) from BL to 9 months was the primary endpoint. The percentage change in LDL-C levels from BL to 3 months was significantly greater in the PCSK9I group (-67.8 ± 21.5% in the PCSK9I group vs. -16.3 ± 21.8% in the SoC group; P < 0.0001), and the difference between the two groups disappeared from BL to 9 months (-20.0 ± 37.8% in the PCSK9I group vs. -6.7 ± 34.2% in the SoC group; P = 0.20). The changes in MFCT from BL to 9 months were significantly greater in the PCSK9I group, even after PCSK9I discontinuation {100 μm [interquartile range (IQR): 45-180 μm] vs. 50 μm [IQR: 0-110 μm]; P = 0.032}. Combination treatment with PCSK9Is and statins resulted in more marked plaque stabilization after ACS than SoC alone, and this effect persisted for 6 months after PCSK9I discontinuation. Adage-Joto study, UMIN ID No. 26516.

Effect of Serum IL-6 Levels on the Progression of Non-Target Lesions in Patients after Coronary Stenting.

percutaneous coronary intervention (PCI) has become the mainstay of treatment for atherosclerotic cardiovascular disease (ASCVD). Inflammatory factors have been shown to be involved in the initiation and progression of ASCVD. After PCI, the persistence of inflammation, especially the inflammation released at the target lesion, may affect the stability of non-target lesion plaques. Interleukin-6 (IL-6) is one of the most common inflammatory factors, however studies about the influence of IL-6 on the progression of non-target lesions (NTLs) of coronary artery are limited. This study investigated whether serum IL-6 levels can affect the progression of NTLs after coronary stent implantation. We performed a retrospective cohort study including 441 patients undergoing coronary angiography (CAG) and stent implantation, who had at least one NTL, between January 2019 and December 2021. They underwent followup CAG 9 to 12 months after PCI. Quartile grouping was based on serum IL-6 levels following readmission. The relationship between serum IL-6 levels and the progression of NTLs after coronary stent implantation was analyzed by using logistic regression analysis and restricted cubic spline regression. Predictive value of IL-6 on NTL progression was evaluated using the receiver operating characteristic (ROC) curve. When compared to the first quartile (Q1) group, the probability of NTL progression was increased in Q2 (adjusted odds ratio (aOR) 3.06, 95% CI 1.29-7.29), Q3 (aOR 3.55, 95% CI 1.52-8.26), and Q4 group (aOR 7.51, 95% CI 3.30-17.05), with a trend test p 0.001. With the increase of IL-6 levels, the risk of progression of NTLs gradually increased, and there was a non-linear relationship between IL-6 and progression of NTLs (p 0.001). The ROC curve showed that the critical value of the serum IL-6 level was 12.652 pg/mL (area under the curve is 0.673, sensitivity is 54.5%, specificity is 70.9%, p 0.05). A high serum IL-6 level is an independent risk factor for the progression of NTLs after coronary stent implantation, and has certain predictive value for the progression of NTLs.

20( S )-Protopanaxatriol Improves Atherosclerosis by Inhibiting Low-Density Lipoprotein Receptor Degradation in ApoE KO Mice.

Atherosclerosis (AS) is a chronic progressive disease caused by various factors and causes various cerebrovascular and cardiovascular diseases (CVDs). Reducing the plasma levels of low-density lipoprotein cholesterol is the primary goal in preventing and treating AS. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in regulating low-density lipoprotein cholesterol metabolism. Panax notoginseng has potent lipid-reducing effects and protects against CVDs, and its saponins induce vascular dilatation, inhibit thrombus formation, and are used in treating CVDs. However, the anti-AS effect of the secondary metabolite, 20( S )-protopanaxatriol (20( S )-PPT), remains unclear. In this study, the anti-AS effect and molecular mechanism of 20( S )-PPT were investigated in vivo and in vitro by Western blotting, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence staining, and other assays. The in vitro experiments revealed that 20( S )-PPT reduced the levels of PCSK9 in the supernatant of HepG2 cells, upregulated low-density lipoprotein receptor protein levels, promoted low-density lipoprotein uptake by HepG2 cells, and reduced PCSK9 mRNA transcription by upregulating the levels of forkhead box O3 protein and mRNA and decreasing the levels of HNF1α and SREBP2 protein and mRNA. The in vivo experiments revealed that 20( S )-PPT upregulated aortic α-smooth muscle actin expression, increased the stability of atherosclerotic plaques, and reduced aortic plaque formation induced by a high-cholesterol diet in ApoE -/- mice (high-cholesterol diet-fed group). Additionally, 20( S )-PPT reduced the aortic expression of CD68, reduced inflammation in the aortic root, and alleviated the hepatic lesions in the high-cholesterol diet-fed group. The study revealed that 20( S )-PPT inhibited low-density lipoprotein receptor degradation via PCSK9 to alleviate AS.

Ghrelin Expression in Atherosclerotic Plaques and Perivascular Adipose Tissue: Implications for Vascular Inflammation in Peripheral Artery Disease.

Atherosclerosis, a leading cause of peripheral artery disease (PAD), is driven by lipid accumulation and chronic inflammation within arterial walls. Objectives: This study investigates the expression of ghrelin, an anti-inflammatory peptide hormone, in plaque morphology and inflammation in patients with PAD, highlighting its potential role in age-related vascular diseases and metabolic syndrome. Methods: The analysis specifically focused on the immunohistochemical expression of ghrelin in atherosclerotic plaques and perivascular adipose tissue (PVAT) from 28 PAD patients. Detailed immunohistochemical staining was performed to identify ghrelin within these tissues, comparing its presence in various plaque types and assessing its association with markers of inflammation and macrophage polarization. Results: Significant results showed a higher prevalence of calcification in fibro-lipid plaques (63.1%) compared to fibrous plaques, with a notable difference in inflammatory infiltration between the two plaque types (p = 0.027). Complicated plaques exhibited increased ghrelin expression, suggesting a modulatory effect on inflammatory processes, although this did not reach statistical significance. The correlation between ghrelin levels and macrophage presence, especially the pro-inflammatory M1 phenotype, indicates ghrelin's involvement in the inflammatory dynamics of atherosclerosis. Conclusions: The findings propose that ghrelin may influence plaque stability and vascular inflammation, pointing to its therapeutic potential in managing atherosclerosis. The study underlines the necessity for further research to clarify ghrelin's impact on vascular health, particularly in the context of metabolic syndrome and age-related vascular alterations.

A multi-target and responsive nanoplatform (MM/ZnS/ATV) combining H2S and atorvastatin for enhanced atherosclerosis therapy

Atherosclerosis, a chronic inflammatory disease characterized by the formation of atherosclerotic plaques in the arterial walls. The pathological process of atherosclerosis mainly contains lipid metabolism disorder, endothelial dysfunction, and progressive inflammation. Given the complexity of pathological process, treatments based on a single target might have very limited therapeutic efficacy. Thus, developing a nanoplatform to obtain an enhanced multi-target therapy is promising. Hydrogen sulfide (H2S) has various functions including anti-inflammation, anti-oxidation and endothelial protection. In this study, a responsive multi-target nanoplatform (MM/ZnS/ATV) combining the functions of H2S and atorvastatin was developed. The acidic microenvironment of the plaque can accelerate ZnS hydrolysis to trigger H2S and atorvastatin release. The responsive H2S release from ZnS can overcome the shortcomings of rapid H2S release from H2S donors in vivo. Furthermore, the ZnS hydrolysis-mediated H+ consumption in the microenvironment can reduce acidity to some extent. Results showed macrophage membranes (MM) coating reduced the phagocytosis of nanoparticles and increased the bioavailability. In vitro studies demonstrated that H2S release from MM/ZnS/ATV inhibited ROS-induced endothelial cell apoptosis and repaired the damaged endothelium. Moreover, atorvastatin released from MM/ZnS/ATV regulated lipid metabolism and reduced the blood lipid content. Both in vitro and in vivo studies confirmed MM/ZnS/ATV exhibited excellent therapeutic efficacy. Specifically, in vivo results showed that H2S combined with atorvastatin effectively reduced inflammatory infiltration, and promoted plaque regression and stabilization. Our study provides a promising strategy for atherosclerosis therapy.

Resolvin D1 delivery to lesional macrophages using antioxidative black phosphorus nanosheets for atherosclerosis treatment.

The buildup of plaques in atherosclerosis leads to cardiovascular events, with chronic unresolved inflammation and overproduction of reactive oxygen species (ROS) being major drivers of plaque progression. Nanotherapeutics that can resolve inflammation and scavenge ROS have the potential to treat atherosclerosis. Here we demonstrate the potential of black phosphorus nanosheets (BPNSs) as a therapeutic agent for the treatment of atherosclerosis. BPNSs can effectively scavenge a broad spectrum of ROS and suppress atherosclerosis-associated pro-inflammatory cytokine production in lesional macrophages. We also demonstrate ROS-responsive, targeted-peptide-modified BPNS-based carriers for the delivery of resolvin D1 (an inflammation-resolving lipid mediator) to lesional macrophages, which further boosts the anti-atherosclerotic efficacy. The targeted nanotherapeutics not only reduce plaque areas but also substantially improve plaque stability in high-fat-diet-fed apolipoprotein E-deficient mice. This study presents a therapeutic strategy against atherosclerosis, and highlights the potential of BPNS-based therapeutics to treat other inflammatory diseases.

A deep learning algorithm to identify carotid plaques and assess their stability.

Carotid plaques are major risk factors for stroke. Carotid ultrasound can help to assess the risk and incidence rate of stroke. However, large-scale carotid artery screening is time-consuming and laborious, the diagnostic results inevitably involve the subjectivity of the diagnostician to a certain extent. Deep learning demonstrates the ability to solve the aforementioned challenges. Thus, we attempted to develop an automated algorithm to provide a more consistent and objective diagnostic method and to identify the presence and stability of carotid plaques using deep learning. A total of 3,860 ultrasound images from 1,339 participants who underwent carotid plaque assessment between January 2021 and March 2023 at the Shanghai Eighth People's Hospital were divided into a 4:1 ratio for training and internal testing. The external test included 1,564 ultrasound images from 674 participants who underwent carotid plaque assessment between January 2022 and May 2023 at Xinhua Hospital affiliated with Dalian University. Deep learning algorithms, based on the fusion of a bilinear convolutional neural network with a residual neural network (BCNN-ResNet), were used for modeling to detect carotid plaques and assess plaque stability. We chose AUC as the main evaluation index, along with accuracy, sensitivity, and specificity as auxiliary evaluation indices. Modeling for detecting carotid plaques involved training and internal testing on 1,291 ultrasound images, with 617 images showing plaques and 674 without plaques. The external test comprised 470 ultrasound images, including 321 images with plaques and 149 without. Modeling for assessing plaque stability involved training and internal testing on 764 ultrasound images, consisting of 494 images with unstable plaques and 270 with stable plaques. The external test was composed of 279 ultrasound images, including 197 images with unstable plaques and 82 with stable plaques. For the task of identifying the presence of carotid plaques, our model achieved an AUC of 0.989 (95% CI: 0.840, 0.998) with a sensitivity of 93.2% and a specificity of 99.21% on the internal test. On the external test, the AUC was 0.951 (95% CI: 0.962, 0.939) with a sensitivity of 95.3% and a specificity of 82.24%. For the task of identifying the stability of carotid plaques, our model achieved an AUC of 0.896 (95% CI: 0.865, 0.922) on the internal test with a sensitivity of 81.63% and a specificity of 87.27%. On the external test, the AUC was 0.854 (95% CI: 0.889, 0.830) with a sensitivity of 68.52% and a specificity of 89.49%. Deep learning using BCNN-ResNet algorithms based on routine ultrasound images could be useful for detecting carotid plaques and assessing plaque instability.

Urolithin A promotes atherosclerotic plaque stability by limiting inflammation and hypercholesteremia in Apolipoprotein E-deficient mice.

Urolithin A (UroA), a dietary phytochemical, is produced by gut bacteria from fruits rich in natural polyphenols ellagitannins (ETs). The efficiency of ETs metabolism to UroA in humans depends on gut microbiota. UroA has shown a variety of pharmacological activities. In this study we investigated the effects of UroA on atherosclerotic lesion development and stability. Apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat and high-cholesterol diet for 3 months to establish atherosclerosis model. Meanwhile the mice were administered UroA (50 mg·kg-1·d-1, i.g.). We showed that UroA administration significantly decreased diet-induced atherosclerotic lesions in brachiocephalic arteries, macrophage content in plaques, expression of endothelial adhesion molecules, intraplaque hemorrhage and size of necrotic core, while increased the expression of smooth muscle actin and the thickness of fibrous cap, implying features of plaque stabilization. The underlying mechanisms were elucidated using TNF-α-stimulated human endothelial cells. Pretreatment with UroA (10, 25, 50 μM) dose-dependently inhibited TNF-α-induced endothelial cell activation and monocyte adhesion. However, the anti-inflammatory effects of UroA in TNF-α-stimulated human umbilicalvein endothelial cells (HUVECs) were independent of NF-κB p65 pathway. We conducted RNA-sequencing profiling analysis to identify the differential expression of genes (DEGs) associated with vascular function, inflammatory responses, cell adhesion and thrombosis in UroA-pretreated HUVECs. Human disease enrichment analysis revealed that the DEGs were significantly correlated with cardiovascular diseases. We demonstrated that UroA pretreatment mitigated endothelial inflammation by promoting NO production and decreasing YAP/TAZ protein expression and TEAD transcriptional activity in TNF-α-stimulated HUVECs. On the other hand, we found that UroA administration modulated the transcription and cleavage of lipogenic transcription factors SREBP1/2 in the liver to ameliorate cholesterol metabolism in ApoE-/- mice. This study provides an experimental basis for new dietary therapeutic option to prevent atherosclerosis.

Notoginsenoside R1 decreases intraplaque neovascularization by governing pericyte-endothelial cell communication via Ang1/Tie2 axis in atherosclerosis.

Atherosclerosis represents the major cause of mortality worldwide and triggers higher risk of acute cardiovascular events. Pericytes-endothelial cells (ECs) communication is orchestrated by ligand-receptor interaction generating a microenvironment which results in intraplaque neovascularization, that is closely associated with atherosclerotic plaque instability. Notoginsenoside R1 (R1) exhibits anti-atherosclerotic bioactivity, but its effect on angiogenesis in atherosclerotic plaque remains elusive. The aim of our study is to explore the therapeutic effect of R1 on vulnerable plaque and investigate its potential mechanism against intraplaque neovascularization. The impacts of R1 on plaque stability and intraplaque neovascularization were assessed in ApoE-/- mice induced by high-fat diet. Pericytes-ECs direct or non-direct contact co-cultured with VEGF-A stimulation were used as the in vitro angiogenesis models. Overexpressing Ang1 in pericytes was performed to investigate the underlying mechanism. In vivo experiments, R1 treatment reversed atherosclerotic plaque vulnerability and decreased the presence of neovessels in ApoE-/- mice. Additionally, R1 reduced the expression of Ang1 in pericytes. In vitro experiments demonstrated that R1 suppressed pro-angiogenic behavior of ECs induced by pericytes cultured with VEGF-A. Mechanistic studies revealed that the anti-angiogenic effect of R1 was dependent on the inhibition of Ang1 and Tie2 expression, as the effects were partially reversed after Ang1 overexpressing in pericytes. Our study demonstrated that R1 treatment inhibited intraplaque neovascularization by governing pericyte-EC association via suppressing Ang1-Tie2/PI3K-AKT paracrine signaling pathway. R1 represents a novel therapeutic strategy for atherosclerotic vulnerable plaques in clinical application.

Novel Mouse Model of Myocardial Infarction, Plaque Rupture, and Stroke Shows Improved Survival With Myeloperoxidase Inhibition.

Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI∆CT/∆CT/Ldlr-/-). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI∆CT/∆CT/Ldlr-/- mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. SR-BI∆CT/∆CT/Ldlr-/- mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.

The Effect of Eicosapentaenoic and Docosahexaenoic Acid Supplementation on Coronary Artery Calcium Progression in Subjects With Diabetes and Coronary Artery Disease: A Secondary Analysis of a Randomized Trial

Higher coronary artery calcium (CAC) scores and progression of CAC are associated with higher mortality. We previously reported that subjects with coronary artery disease randomly allocated to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation or none had similar significant increases in CAC score over 30 months. Whether these findings are influenced by diabetes status is unknown. A total of 242 subjects with coronary artery disease who were on statin therapy were randomly allocated to to 1.86 g EPA and 1.5 g DHA daily or none (control). The CAC score was measured at baseline and 30-month follow-up using noncontrast, cardiac computed tomography. A significant interaction term between diabetes status and treatment arm was noted in the prediction of the CAC score (p <0.001). A total of 176 subjects (85.8% men) had no diabetes and 66 subjects (80.3% men) had diabetes. The mean age was 62.9 ± 7.9 versus 63.2 ± 7.1 years, respectively. The mean low-density lipoprotein cholesterol and median triglyceride levels were not significantly different between those without and with diabetes: 77.7 ± 25.9 versus 77.1 ± 30.2 mg/100 ml, respectively, and 117.0 (78.0 to 158.0) versus 119.0 (84.5 to 201.5) mg/100 ml, respectively. Subjects with diabetes on EPA+DHA had a greater increase in CAC score than subjects with diabetes in the control group (median 380.7 vs 183.5, respectively, p = 0.021). In contrast, no difference occurred between the EPA+DHA and control groups in subjects without diabetes (175.7 vs 201.1, respectively, p = 0.41). In conclusion, EPA+DHA supplementation was associated with greater CAC progression in subjects with diabetes than subjects with diabetes in the control group over a 30-month period; whether this indicates progression of the disease burden or plaque stabilization requires further study.

Detection and Stabilisation of Vulnerable Plaques in the Coronary Arteries

Vulnerable atherosclerotic plaques (AP) can be assessed by coronary artery imaging techniques. Intravascular ultrasound allows real-time assessment of the longitudinal and transverse dimensions of the vessel, the severity of its remodelling, and the size and structure of the AP. The resolution of optical coherent tomography makes it possible to accurately identify vulnerable APs according to the following characteristic parameters: a large necrotic nucleus, a thin fibrous capsule, neoangiogenesis and inflammatory changes in the AP cap. Percent atheroma volume, total atheroma volume, maximum lipid core burden index within 4 mm, minimal fibrous cap thickness, maximum lipid arc, lipid length and macrophage grade are the main characteristics of vulnerable AP. Lipid-lowering therapy (LLT) has a stabilising effect on AP. Databases searched were PubMed and Web of Science up to April 2023. In total, eight relevant articles (original clinical studies) were selected. In the ODISSEY J-IVUS study, there was a decrease of total AP volume by 3.1% in the monotherapy group and 4.8% in the combined LLT group (p=0.23). In the GLAGOV trial, atheroma volume decreased by 0.9 mm3 in the placebo group and 5.8 mm3 in the evolocumab group (p&lt;0.001). Plaque regression was observed in 64.3% of patients in the evolocumab group and 47.3% in the placebo group. The dynamics of the minimum thickness of the fibrous cap varied within 18.0–62.67 µm on combined LLT and 13.2–33.19 µm on monotherapy (PACMAN-AMI). Regression of the lipid arc was also observed in the HUYGENS study. The development of cardiovascular imaging has made it possible to expand understanding of the morphology of vulnerable AP.

LncRNA CDKN2B-AS1 regulates collagen expression

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10− 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10− 25). The inflammatory “TNFA signaling via NFKB” gene set was downregulated the most (Padj=1 × 10− 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10− 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10− 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.

A Multi-Dimensional Aggregation Network Guided by Key Features for Plaque Echo Classification Based on Carotid Ultrasound Video

ObjectiveUnstable plaques can cause acute cardiovascular and cerebrovascular diseases. The stability and instability of plaque are related to the plaque echo status in ultrasound. Carotid videos provide detailed plaque information compared to static images. Ultrasound-based plaque echo classification is challenging due to noise, interference frames, small targets (plaques), and complex shape changes. MethodsThis study proposes a Multi-dimensional Aggregation Network (MA-Net) guided by key features for plaque diagnosis based on carotid ultrasound video, which uses only video-level labels. MA-Net consists of Key-Feature (KF) and Temporal-Channel-Spatial (TCS) modules. The KF module learns the contribution of each frame to the classification at the feature level, adaptively infers the importance score of each frame, thereby reducing the influence of interference frames. The TCS module includes the Temporal-Channel (TC) and Temporal-Spatial (TS) sub-modules. In addition to studying the temporal dimension, it delves into the relationship between the channel and spatial dimensions. TC analyses the temporal dependencies among the channels and filters noise. Moreover, TS extracts features more accurately through the spatio-temporal information contained in the surrounding environment of the plaque. ResultsThe performance of MA-Net on the SHU-Ultrasound-Video-2020 dataset is better than that of the state-of-the-art models of video classification, showing at least a 5% increase in accuracy, with an accuracy rate of 87.36%. ConclusionThe outstanding diagnostic capability of the proposed model will help provide a more robust and reproducible diagnostic process with a lower labour cost for clinical carotid plaque diagnosis.

Crosstalk between efferocytic myeloid cells and T-cells and its relevance to atherosclerosis.

The interplay between myeloid cells and T-lymphocytes is critical to the regulation of host defense and inflammation resolution. Dysregulation of this interaction can contribute to the development of chronic inflammatory diseases. Important among these diseases is atherosclerosis, which refers to focal lesions in the arterial intima driven by elevated apolipoprotein B-containing lipoproteins, notably low-density lipoprotein (LDL), and characterized by the formation of a plaque composed of inflammatory immune cells, a collection of dead cells and lipids called the necrotic core, and a fibrous cap. As the disease progresses, the necrotic core expands, and the fibrous cap becomes thin, which increases the risk of plaque rupture or erosion. Plaque rupture leads to a rapid thrombotic response that can give rise to heart attack, stroke, or sudden death. With marked lowering of circulating LDL, however, plaques become more stable and cardiac risk is lowered-a process known as atherosclerosis regression. A critical aspect of both atherosclerosis progression and regression is the crosstalk between innate (myeloid cells) and adaptive (T-lymphocytes) immune cells. Myeloid cells are specialized at clearing apoptotic cells by a process called efferocytosis, which is necessary for inflammation resolution. In advanced disease, efferocytosis is impaired, leading to secondary necrosis of apoptotic cells, inflammation, and, most importantly, defective tissue resolution. In regression, efferocytosis is reawakened aiding in inflammation resolution and plaque stabilization. Here, we will explore how efferocytosing myeloid cells could affect T-cell function and vice versa through antigen presentation, secreted factors, and cell-cell contacts and how this cellular crosstalk may contribute to the progression or regression of atherosclerosis.

OPA1 affects lipid metabolism and atherosclerosis progression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): European Atherosclerosis Society Introduction Mitochondria are involved in cellular metabolism, energy generation, calcium homeostasis, sterol and bile acids (BAs) production. Mitochondria continuously undergo biogenesis, fusion, fission and mitophagy, maintaining a continuous balance between all forms. Purpose On these premises, we test the impact of OPA1, an inner mitochondria membrane fusion protein, on mitochondrial tethering on lipid metabolism in the liver and the atherosclerotic plaque. Methods OPA1HepKO and OPA1 TG on LDLR KO background were fed with a Western-type diet (WTD) respectively for 12 weeks. Inverse calorimetry, GTT, and LipidToleranceTest (LTT) were performed. Paraffin-embedded tissues were used for histological analysis, frozen tissues were used for OMICs analysis. VSMCs were isolated and cultured with VLDL (50 µg/ml). Results While OPA1HepKO mice display altered systemic metabolism with reduced body weight and reduced circulating lipid levels OPA1 overexpression on LDLR KO background showed significantly high cholesterol levels. OPA1 deficiency impair hepatic bile acid conjugation with significant accumulation of primary unconjugated bile acids in the liver. On the contrary OPA1 overexpression leads to a reduction of unconjugated bile acids and higher percentage of conjugated bile acids. OPA1 reduction leads to a reduced atherosclerotic plaque while, on the contrary, its overexpression doesn’t affect atherosclerosis despite an increase in circulating lipid and lipoprotein levels. OPA1 overexpression is therefore promoting plaque stabilization with VSMCs that are less prone to change their metabolism to synthetic. Conclusion Hepatic Opa1 deficiency alters bile acids production and therefore circulating lipid profile with a consequent reduction in atherosclerotic plaque formation. OPA1 overexpression in the aorta affects plaque stability despite the highest levels of circulating lipids.

Anti-interleukin-18 as therapy against atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Established Investigator of the Dutch Heart Foundation Background Interleukin-18 (IL-18) is a pro-inflammatory cytokine and is secreted by macrophages after NLRP3-inflammasome activation. IL-18 is important for the induction of the Th1 response and overexpression of IL-18 aggravates atherosclerosis, suggesting that inhibition of IL-18 may act atheroprotective. In this study, we investigated the therapeutic potential of an IL-18 neutralizing antibody (anti-IL18) on atherosclerosis development. Methods and results Female ApoE-/- mice were fed a Western-type diet for 8 weeks and simultaneously received an antibody against IL-18 (100 µg/mouse; n=15) or a rat IgG2a control (100 µg/mouse; n=15) 3x per week intraperitoneally, after which the mice were sacrificed. First, we assessed macrophage phenotype in the peritoneal cavity. Expression of the proinflammatory markers CD80 (control: 46.3±1.4 *103; p&amp;lt;0.05 vs. anti-IL18: 40.4 ±1.7 *103) and iNOS (control: 15.5±1.8 *102 vs. anti-IL18: 8.0±1.9 *102; p&amp;lt;0.01) were reduced. In the aorta, MFIs of anti-inflammatory macrophage markers Arginase-1 (control: 389±99 vs. anti-IL18: 654±85; p=0.06) and CD206 (control: 55.2±2.4 *103 vs. anti-IL18: 62.8±3.7 *103; p=0.1) tended to be increased expression in the ascending aorta of anti-IL18 mice compared to controls. In addition, anti-IL18 treatment promoted the number of CD4 T cells (control: 20±1% vs. anti-IL18: 28±1%; p&amp;lt;0.0001) in the mediastinal lymph node and increased it’s regulatory phenotype (Tregs; Foxp3+) of CD4 (control: 23±1% vs. anti-IL18: 26±1%; p=0.07) and CD8 T cells (control: 0.4±0.02% vs. anti-IL18: 0.9±0.09%; p&amp;lt;0.0001). These effects however, did not translate into an effect on plaque size in the aortic root, as measured using an Oil-Red-O staining, which did not differ between the control and anti-IL18 treatment group (control: 47.1±2.4 *104 µm2 vs. anti-IL18: 43.4±2.3 *104 µm2). Conclusions Anti-IL18 treatment reduced the proinflammatory phenotype of peritoneal macrophages and promoted Tregs in the mediastinal lymph node. Further analyses on plaque stability and intraplaque macrophage subsets are currently being performed.

Exploring PD-1+ and tissue resident memory T cells in atherosclerotic diseases

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Health Holland Visual Sonics Background Checkpoint inhibitor therapy (ICI), a potent oncologic treatment, has been associated with an enhanced risk for atherosclerotic cardiovascular events and increased plaque progression. Among the key players affected by ICI are tissue resident memory T cells (Trm), a subset residing in tissues and characterized by CD44, CD69, PD-1, and CD103. Although Trms have been associated with chronic inflammatory conditions, their role in atherosclerosis and accelerated atherosclerosis, as observed in postinterventional diseases such as vein graft disease, remains elusive Here, we present an investigation into the role of PD-1 positive cells and Trms in accelerated atherosclerosis as found in vein graft disease. Methods Human carotid atherosclerotic plaques were stained with a MOVAT stain and scored to determine plaque stability. 25 Stable and 25 unstable plaques were stained for CD3+ T cells and PD-1. Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery and were sacrificed at t7, t14, t28 (n=6 per time point). The vein grafts were processed for flow cytometry to quantify the Trms population, using markers including CD4+, CD8+, CD44+, CD25-, CD69+, CD103+ and PD-1+. Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery. Biweekly, mice received ICI (anti-PD-L1) or control antibody (n=7). After 28 days vein graft morphology was assed using MOVAT staining and vein graft inflammation by staining MAC3 and CXCL10. Results Within human atherosclerotic plaques, PD-1-expressing T cells were predominantly located in the shoulder regions of the necrotic core. Unstable lesions exhibited significantly higher levels of PD-1 expressing CD3+ T cells compared to stable lesions (p=0.001). Furthermore, the percentage of PD-1 expression positively correlated with immune cell infiltration scores (p=0.019). In murine vein grafts, Trms were identified through flow cytometry. A trend was observed indicating an increase in the number of CD4 and CD8 Trms at t14 compared to t7 Trms (CD8+, CD44+, CD69+/CD103+). At t28, the number of CD4 and CD8 Trms decreased compared to t14, with PD1+ expressing Trms showing a similar trend, peaking at t14. Vein grafts in mice receiving anti-PD-L1 mAb showed no morphological differences compared to the control group. However, a significant difference in inflammation was noted in the anti-PD-L1 treated group, characterized by a higher number of macrophages (p=0.02) and increased expression of CXCL10 (p=0.008). Conclusion Here we demonstrate the association of PD-1+ T cells with unstable lesions and reveals that Trms increase during the inflammatory phase and decrease during the stabilizing phase of accelerated atherosclerosis. Trms emerge as potential detrimental targets of ICI therapy, which is characterized by increased inflammation.

Asporin expression is associated with human atherosclerotic plaque integrity

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): This work was supported by the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds Background Calcification serves as a clinical marker of atherosclerotic plaque burden. Recent studies have also shed light on its intricate association with plaque vulnerability, likely depend on calcification structures and the surrounding environment. Asporin (ASPN) is a member of the small-leucine-rich proteoglycans that contains a unique and well-conserved stretch of aspartate (D) residues, which mimic the role of glycosaminoglycans. Via its D-repeat, ASPN can interact with calcium and several other extracellular matrix molecules, thus influencing collagen mineralization and TGF-β signaling. ASPN expression has been shown to be higher in asymptomatic compared to symptomatic atherosclerotic plaques, suggesting a potential protective role. However, the role of ASPN in maintaining plaque integrity remains unknown. Purpose Here we aimed to explore the role of ASPN in atherosclerosis, by investigating if ASPN was expressed in human carotid plaques and by determining if ASPN is associated with risk for postoperative cardiovascular events and death. Methods ASPN and TGF-β protein levels were measured by ELISA and Milliplex in 176 human plaque tissue homogenates obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque tissue sections were stained for ASPN, destabilizing (CD68, oil red O, glycophorin A) and stabilizing plaque components (Movat, α-actin). Plaque tissue RNA expression levels were assessed in 82 plaques using bulk RNA sequencing. Plaque levels of phosphate, elastin and collagen were measured using ELISA. Results ASPN protein levels were higher in plaques from asymptomatic patients compared to symptomatic patients (p=0.011). ASPN RNA levels correlated positively to RNA levels of the smooth muscle cell markers ACTA2, MYH11 and TAGLN. Moreover, protein levels correlated with plaque levels of phosphate (r=0.258, p=0.001), elastin (r=0.423, p&amp;lt;0.001), collagen (r=0.248, p=0.002) and stability factors like the TGF-β family members, TGF-β1 (r=0.176, p=0.035), TGF-β2 (r=0.581, p&amp;lt;0.001) and TGF-β3 (r=0.396, p&amp;lt;0.001). Furthermore, plaque ASPN protein concentration, correlated inversely with a histological plaque vulnerability index (r=-0.265, p=0.0029), which is ratio between the plaque area of destabilizing (macrophages, lipids, intra-plaque hemorrhage) and stabilizing plaque components (collagen and smooth muscle cells). Additionally, above median ASPN levels were associated with lower risk for postoperative cardiovascular events (p=0.008) and death (p=0.009). Conclusion Our findings suggest that elevated levels of ASPN might have a crucial role in maintaining plaque stability, affecting plaque calcification.

Photodynamic Therapy for Atherosclerosis: Past, Present, and Future.

This review paper examines the evolution of photodynamic therapy (PDT) as a novel, minimally invasive strategy for treating atherosclerosis, a leading global health concern. Atherosclerosis is characterized by the accumulation of lipids and inflammation within arterial walls, leading to significant morbidity and mortality through cardiovascular diseases such as myocardial infarction and stroke. Traditional therapeutic approaches have primarily focused on modulating risk factors such as hypertension and hyperlipidemia, with emerging evidence highlighting the pivotal role of inflammation. PDT, leveraging a photosensitizer, specific-wavelength light, and oxygen, offers targeted treatment by inducing cell death in diseased tissues while sparing healthy ones. This specificity, combined with advancements in nanoparticle technology for improved delivery, positions PDT as a promising alternative to traditional interventions. The review explores the mechanistic basis of PDT, its efficacy in preclinical studies, and the potential for enhancing plaque stability and reducing macrophage density within plaques. It also addresses the need for further research to optimize treatment parameters, mitigate adverse effects, and validate long-term outcomes. By detailing past developments, current progress, and future directions, this paper aims to highlight PDT's potential in revolutionizing atherosclerosis treatment, bridging the gap from experimental research to clinical application.