Plaque Tissue Research Articles

Comprehensive analysis of molecular mechanisms underlying kidney stones: gene expression profiles and potential diagnostic markers

BackgroundThe study aimed to investigate the molecular mechanisms underlying kidney stones by analyzing gene expression profiles. They focused on identifying differentially expressed genes (DEGs), performing gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, and screening optimal feature genes using various machine learning algorithms.MethodsData from the GSE73680 dataset, comprising normal renal papillary tissues and Randall’s Plaque (RP) tissues, were downloaded from the GEO database. DEGs were identified using the limma R package, followed by GSEA and WGCNA to explore functional modules. Functional enrichment analysis was conducted using KEGG and Disease Ontology. Various machine learning algorithms were used for screening the most suitable feature genes, which were then assessed for their expression and diagnostic significance through Wilcoxon rank-sum tests and ROC curves. GSEA and correlation analysis were performed on optimal feature genes, and immune cell infiltration was assessed using the CIBERSORT algorithm.Results412 DEGs were identified, with 194 downregulated and 218 upregulated genes in kidney stone samples. GSEA revealed enriched pathways related to metabolic processes, immune response, and disease states. WGCNA identified modules correlated with kidney stones, particularly the yellow module. Functional enrichment analysis highlighted pathways involved in metabolism, immune response, and disease pathology. Through machine learning algorithms, KLK1 and MMP10 were identified as optimal feature genes, significantly upregulated in kidney stone samples, with high diagnostic value. GSEA further elucidated their biological functions and pathway associations.ConclusionThe study comprehensively analyzed gene expression profiles to uncover molecular mechanisms underlying kidney stones. KLK1 and MMP10 were identified as potential diagnostic markers and key players in kidney stone progression. Functional enrichment analysis provided insights into their roles in metabolic processes, immune response, and disease pathology. These results contribute significantly to a better understanding of kidney stone pathogenesis and may inform future diagnostic and therapeutic strategies.

Atherectomy Techniques: Rotablation, Orbital and Laser

Coronary artery disease remains the leading cause of morbidity and mortality worldwide despite advancements in percutaneous coronary intervention (PCI). With an increasing ageing population, there is a significant challenge in addressing severe calcification in atherosclerotic plaque during angioplasty. This review article focuses on atherectomy strategies such as rotational atherectomy (RA), orbital atherectomy (OA) and excimer laser coronary angioplasty (ELCA) aimed at modifying calcified lesions and improving PCI outcomes. RA modifies plaque through rotational ablation, OA uses eccentrically mounted diamond-coated crown and has a reduced entrapment risk compared to RA. ELCA uses pulsatile laser energy to precisely ablate plaque tissue. This review provides insights into the mechanisms, procedural techniques and clinical outcomes associated with these calcium modification techniques. The selection of appropriate devices and adequate training are crucial for optimising lesion modification and enhancing procedural success. Further research and standardised protocols are required to overcome challenges associated with using these devices and expand their usage in clinical practice.

Monocyte Count as a Predictor of Major Adverse Limb Events in Aortoiliac Revascularization.

Background/Objectives: Atherosclerosis is a leading cause of death, especially in the developed world, and is marked by chronic arterial inflammation and lipid accumulation. As key players in its progression, monocytes contribute to plaque formation, inflammation, and tissue repair. Understanding monocyte involvement is crucial for developing better therapeutic approaches. The objective of this study is to assess the prognostic value of monocytes for limb-related outcomes following revascularization for complex aortoiliac lesions, thereby emphasizing the central role of monocytes in atherosclerosis. Methods: This prospective cohort study-enrolled patients who had undergone elective aortoiliac revascularization at two hospitals between January 2013 and December 2023. Patients with TASC II type D lesions were included, excluding those with aneurysmal disease. Demographic, clinical, and procedural data were gathered, and patients were monitored for limb-related outcomes. Preoperative complete blood counts were analyzed, and statistical analyses, including multivariable Cox regression, were conducted to identify predictors of major adverse limb events (MALE). Results: The study included 135 patients with a mean age of 62.4 ± 9.20 years and predominantly male (93%). Patients were followed for a median of 61 IQR [55.4-66.6] months. Smoking history (91%) was the most prevalent cardiovascular risk factor. Preoperative monocyte count >0.720 × 109/L was associated with worse 30-day limb-related outcomes (MALE: OR 7.138 95% CI: 1.509-33.764, p = 0.013) and long-term outcomes, including secondary patency (p = 0.03), major amputation (p = 0.04), and MALE (p = 0.039). Cox regression analysis confirmed an elevated monocyte count as an independent predictor of MALE (adjusted hazard ratio 2.149, 95% CI: 1.115-4.144, p = 0.022). Conclusions: This study demonstrated that patients with a higher absolute monocyte count may be more exposed to the risk of MALE in patients with aortoiliac TASC II type D lesions undergoing revascularization, with predictive accuracy in both the short and long term. Additionally, it was an independent predictor of major amputation. This new marker has the potential to serve as a cost-effective and easily available tool for risk stratification, helping identify patients at higher risk of MALE.

Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques.

Citrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. To investigate features and patterns of citrullination in atherosclerotic plaques. We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Biomolecules of Adipose Tissue in Atherosclerotic Plaques of Men With Coronary Atherosclerosis.

To study metabolic molecules (adiponectin, adipsin, resistin, glucagon-like peptide-1 (GLP-1), glucagon, secretin) of adipose tissue in atherosclerotic plaques (AP) and their associations with AP instability in men with coronary atherosclerosis. Metabolic molecules (adipocytokines and metabolic hormones) of adipose tissue can act as enzymes, hormones or growth factors in modulating insulin resistance and lipid and glucose metabolism and indirectly influence the course of the atherosclerotic process. This study included 48 men from whom 139 coronary artery (CA) samples were collected during coronary artery bypass grafting, after obtaining the informed consent. According to the histological conclusion, 84 (60.4%) CA plaques were stable, 44 (31.7%) were unstable, and 11 histological samples had a conditionally unchanged CA intima (7.9%). The concentrations of adiponectin, adipsin, resistin, GLP-1, glucagon, and secretin were measured in AP homogenates by multiplex analysis using the Human Metabolic Hormone V3 panel (MILLIPLEX, Germany). During the study, demographic and anthropometric characteristics, medical history, and presence of chronic diseases were recorded. The glucagon concentration in the conditionally unchanged intima was 16.7% lower and in the fragments of unstable atherosclerotic plaques 41.2% lower than in fragments of stable APs. However, the glucagon concentration in stable APs was 28% higher than in unstable APs. The secretin concentration in the conditionally unchanged intima was also lower than in stable APs by 41.2%, while in stable APs, the secretin concentration was 20% higher than in unstable APs. The adiponectin concentrations were directly correlated with serum high-density lipoprotein cholesterol (HDL-C) concentrations (r=0.286; p=0.002), while the secretin concentrations were inversely correlated with serum HDL-C concentrations (r= -0.199; p=0.038). The probability of having an unstable AP (in relation to conditionally unchanged intima) increases by 35.8% with an increase in the AP glucagon concentration by 1 pg/mg protein. The probability of having a stable AP (in relation to unchanged intima) increases by 29.4% with an increase in the AP glucagon concentration by 1 pg/mg protein and by 10.1% with an increase in the AP secretin concentration by 1 pg/mg protein. The AP adiponectin concentration directly correlates and the AP secretin concentration inversely correlates with the serum concentration of HDL-C. The presence of both stable and unstable APs is directly associated with the AP glucagon concentration in men with coronary atherosclerosis. The AP secretin concentration is directly associated with plaque stability in men with coronary atherosclerosis. Further thorough study of the identified markers in atherosclerotic lesions will allow using them as potential targets for therapy.

An Unsupervised Learning Tool for Plaque Tissue Characterization in Histopathological Images.

Stroke is the second leading cause of death and a major cause of disability around the world, and the development of atherosclerotic plaques in the carotid arteries is generally considered the leading cause of severe cerebrovascular events. In recent years, new reports have reinforced the role of an accurate histopathological analysis of carotid plaques to perform the stratification of affected patients and proceed to the correct prevention of complications. This work proposes applying an unsupervised learning approach to analyze complex whole-slide images (WSIs) of atherosclerotic carotid plaques to allow a simple and fast examination of their most relevant features. All the code developed for the present analysis is freely available. The proposed method offers qualitative and quantitative tools to assist pathologists in examining the complexity of whole-slide images of carotid atherosclerotic plaques more effectively. Nevertheless, future studies using supervised methods should provide evidence of the correspondence between the clusters estimated using the proposed textural-based approach and the regions manually annotated by expert pathologists.

Paired Transcriptomic Analyses of Atheromatous and Control Vessels Reveal Novel Autophagy and Immunoregulatory Genes in Peripheral Artery Disease.

Peripheral artery disease (PAD), a significant health burden worldwide, affects lower extremities due to atherosclerosis in peripheral vessels. Although the mechanisms of PAD have been well studied, the molecular milieu of the plaques localized within peripheral arteries are not well understood. Thus, to identify PAD-lesion-specific gene expression profiles precluding genetic, environmental, and dietary biases, we studied the transcriptomic profile of nine plaque tissues normalized to non-plaque tissues from the same donors. A total of 296 upregulated genes, 274 downregulated genes, and 186 non-coding RNAs were identified. STAG1, SPCC3, FOXQ1, and E2F3 were key downregulated genes, and CD93 was the top upregulated gene. Autophagosome assembly, cellular response to UV, cytoskeletal organization, TCR signaling, and phosphatase activity were the key dysregulated pathways identified. Telomerase regulation and autophagy were identified as novel interacting pathways using network analysis. The plaque tissue was predominantly composed of immune cells and dedifferentiated cell populations indicated by cell-specific marker-imputed gene expression analysis. This study identifies novel genes, non-coding RNAs, associated regulatory pathways, and the cell composition of the plaque tissue in PAD patients. The autophagy and immunoregulatory genes may drive novel mechanisms, resulting in atheroma. These novel interacting networks and genes have potential for PAD-specific therapeutic applications.

Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque

BackgroundThe heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition.MethodsWeighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria.ResultsThe CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice.ConclusionsThe results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

Lipoprotein(a) and Long-Term Plaque Progression, Low-Density Plaque, and Pericoronary Inflammation

Lipoprotein(a) (Lp[a]) is a causal risk factor for cardiovascular disease; however, long-term effects on coronary atherosclerotic plaque phenotype, high-risk plaque formation, and pericoronary adipose tissue inflammation remain unknown. To investigate the association of Lp(a) levels with long-term coronary artery plaque progression, high-risk plaque, and pericoronary adipose tissue inflammation. This single-center prospective cohort study included 299 patients with suspected coronary artery disease (CAD) who underwent per-protocol repeated coronary computed tomography angiography (CCTA) imaging with an interscan interval of 10 years. Thirty-two patients were excluded because of coronary artery bypass grafting, resulting in a study population of 267 patients. Data for this study were collected from October 2008 to October 2022 and analyzed from March 2023 to March 2024. The median scan interval was 10.2 years. Lp(a) was measured at follow-up using an isoform-insensitive assay. CCTA scans were analyzed with a previously validated artificial intelligence-based algorithm (atherosclerosis imaging-quantitative computed tomography). The association between Lp(a) and change in percent plaque volumes was investigated in linear mixed-effects models adjusted for clinical risk factors. Secondary outcomes were presence of low-density plaque and presence of increased pericoronary adipose tissue attenuation at baseline and follow-up CCTA imaging. The 267 included patients had a mean age of 57.1 (SD, 7.3) years and 153 were male (57%). Patients with Lp(a) levels of 125 nmol/L or higher had twice as high percent atheroma volume (6.9% vs 3.0%; P = .01) compared with patients with Lp(a) levels less than 125 nmol/L. Adjusted for other risk factors, every doubling of Lp(a) resulted in an additional 0.32% (95% CI, 0.04-0.60) increment in percent atheroma volume during the 10 years of follow-up. Every doubling of Lp(a) resulted in an odds ratio of 1.23 (95% CI, 1.00-1.51) and 1.21 (95% CI, 1.01-1.45) for the presence of low-density plaque at baseline and follow-up, respectively. Patients with higher Lp(a) levels had increased pericoronary adipose tissue attenuation around both the right coronary artery and left anterior descending at baseline and follow-up. In this long-term prospective serial CCTA imaging study, higher Lp(a) levels were associated with increased progression of coronary plaque burden and increased presence of low-density noncalcified plaque and pericoronary adipose tissue inflammation. These data suggest an impact of elevated Lp(a) levels on coronary atherogenesis of high-risk, inflammatory, rupture-prone plaques over the long term.

The association of carotid plaque burden and composition and the coronary artery calcium score in intermediate cardiovascular risk patients.

Both the carotid ultrasound and coronary artery calcium (CAC) score quantify subclinical atherosclerosis and are associated with cardiovascular disease and events. This study investigated the association between CAC score and carotid plaque quantity and composition. Adult participants (n = 43) without history of cardiovascular disease were recruited to undergo a carotid ultrasound. Maximum plaque height (MPH), total plaque area (TPA), carotid intima-media thickness (CIMT), and plaque score were measured. Grayscale pixel distribution analysis of ultrasound images determined plaque tissue composition. Participants then underwent CT to determine CAC score, which were also categorized as absent (0), mild (1-99), moderate (100-399), and severe (400+). Spearman correlation coefficients between carotid variables and CAC scores were computed. The mean age of participants was 63 ± 11 years. CIMT, TPA, MPH, and plaque score were significantly associated with CAC score (ρ = 0.60, p < 0.0001; ρ = 0.54, p = 0.0002; ρ = 0.38, p = 0.01; and ρ = 0.49, p = 0.001). Echogenic composition features %Calcium and %Fibrous tissue were not correlated to a clinically relevant extent. There was a significant difference in the TPA, MPH, and plaque scores of those with a severe CAC score category compared to lesser categories. While carotid plaque burden was associated with CAC score, plaque composition was not. Though CAC score reliably measures calcification, carotid ultrasound gives information on both plaque burden and composition. Carotid ultrasound with assessment of plaque features used in conjunction with traditional risk factors may be an alternative or additive to CAC scoring and could improve the prediction of cardiovascular events in the intermediate risk population.

Association of angiogenesis-associated genes with atherosclerotic plaque progression, intraplaque hemorrhage, and immune infiltration

Mounting evidence suggests that intraplaque angiogenesis is associated with the progression of atherosclerotic plaques and the development of intraplaque hemorrhage. The specificity of intraplaque immune cell infiltration may be associated with abnormalities in the structure and function of the nascent capillaries. Here, we analyzed expression levels of angiogenesis-associated genes in early and advanced carotid atheromatous plaque tissues as well as in stable and intraplaque hemorrhage plaques. Expression profiles of advanced arterial plaques based on angiogenesis-associated genes were classified into subtypes by performing a consensus clustering analysis. The correlation between the immune microenvironment of plaques and expression of angiogenesis-associated genes was also explored using the single sample gene set enrichment analysis method and the CIBERSORT algorithm. We identified hub angiogenesis-associated genes showing similar expression patterns throughout plaque adverse progression, and constructed a prediction model using the random forest algorithm. Receiver operating curves were constructed to evaluate efficacy in identification of intraplaque hemorrhage in a plaque. Our results suggest that heterogeneity of angiogenesis-related genes may promote the malignant development of plaques and cause plaque rupture. In conclusion, we propose a model based on expression of angiogenesis-related genes to predict the risk of plaque rupture.

LY86 facilitates ox-LDL-induced lipid accumulation in macrophages by upregulating SREBP2/HMGCR expression

LY86, also known as MD1, has been implicated in various pathophysiological processes including inflammation, obesity, insulin resistance, and immunoregulation. However, the role of LY86 in cholesterol metabolism remains incompletely understood. Several studies have reported significant up-regulation of LY86 mRNA in atherosclerosis; nevertheless, the regulatory mechanism by which LY86 is involved in this disease remains unclear. In this study, we aimed to investigate whether LY86 affects ox-LDL-induced lipid accumulation in macrophages. Firstly, we confirmed that LY86 is indeed involved in the process of atherosclerosis and found high expression levels of LY86 in human atherosclerotic plaque tissue. Furthermore, our findings suggest that LY86 may mediate intracellular lipid accumulation induced by ox-LDL through the SREBP2/HMGCR pathway. This mechanism could be associated with increased cholesterol synthesis resulting from enhanced endoplasmic reticulum stress response.

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.

Downregulation of MYBL1 in endothelial cells contributes to atherosclerosis by repressing PLEKHM1-inducing autophagy

MYBL1 is a strong transcriptional activator involved in the cell signaling. However, there is no systematic study on the role of MYBL1 in atherosclerosis. The aim of this study is to elucidate the role and mechanism of MYBL1 in atherosclerosis. GSE28829, GSE43292 and GSE41571 were downloaded from NCBI for differentially expressed analysis. The expression levels of MYBL1 in atherosclerotic plaque tissue and normal vessels were detected by qRT-PCR, Western blot and Immunohistochemistry. Transwell and CCK-8 were used to detect the migration and proliferation of HUVECs after silencing MYBL1. RNA-seq, Western blot, qRT-PCR, Luciferase reporter system, Immunofluorescence, Flow cytometry, ChIP and CO-IP were used to study the role and mechanism of MYBL1 in atherosclerosis. The microarray data of GSE28829, GSE43292, and GSE41571 were analyzed and intersected, and then MYBL1 were verified. MYBL1 was down-regulated in atherosclerotic plaque tissue. After silencing of MYBL1, HUVECs were damaged, and their migration and proliferation abilities were weakened. Overexpression of MYBL1 significantly enhanced the migration and proliferation of HUVECs. MYBL1 knockdown induced abnormal autophagy in HUVEC cells, suggesting that MYBL1 was involved in the regulation of HUVECs through autophagy. Mechanistic studies showed that MYBL1 knockdown inhibited autophagosome and lysosomal fusion in HUVECs by inhibiting PLEKHM1, thereby exacerbating atherosclerosis. Furthermore, MYBL1 was found to repress lipid accumulation in HUVECs after oxLDL treatment. MYBL1 knockdown in HUVECs was involved in atherosclerosis by inhibiting PLEKHM1-induced autophagy, which provided a novel target of therapy for atherosclerosis.Graphical abstract

A novel protein encoded by circLARP1B promotes the proliferation and migration of vascular smooth muscle cells by suppressing cAMP signaling

Background and aimsCircular RNA (circRNA) is closely related to atherosclerosis (AS) incidence and progression, but its regulatory mechanism in AS needs further elucidation. AS development is significantly influenced by abnormal vascular smooth muscle cells (VSMCs) growth and migration. This study explored the potential protein role of circLARP1B in VSMC proliferation and migration. MethodsWe performed whole-transcriptome sequencing in human normal arterial intima and advanced atherosclerotic plaques to screen for differentially expressed circRNAs. The sequencing results were combined with database analysis to screen for circRNAs with coding ability. Real-time quantitative polymerase chain reaction was utilized to assess circLARP1B expression levels in atherosclerotic plaque tissues and cells. circLARP1B-243aa function and pathway in VSMCs growth and migration were studied by scratch, transwell, 5-ethynyl-2′-deoxyuridine, cell counting kit-8, and Western blot experiments. ResultsWe found that circLARP1B was downregulated in atherosclerotic plaque tissue and promoted the proliferation and migration of VSMCs. circLARP1B encodes a novel protein with a length of 243 amino acids. Through functional experiments, we confirmed the role of circLARP1B-243aa in enhancing VSMCs migration and proliferation. Mechanistically, circLARP1B-243aa promotes VSMCs migration and growth by upregulating phosphodiesterase 4C to inhibit the cyclic adenosine monophosphate signaling pathway. ConclusionsOur results suggested that circLARP1B could promote VSMCs growth and migration through the encoded protein circLARP1B-243aa. Therefore, it could be a treatment target and biomarker for AS.

Abstract 1028: Cytotoxic CD4+ T Cells: Unraveling The Residual Risk In Lipid Lowering For Type 2 Diabetes Patients

Type 2 Diabetes (T2D) significantly increases the risk of cardiovascular diseases (CVDs), marked by severe plaque pathology resistant to standard cholesterol-lowering treatments. While the roles of the innate immune system and macrophages in CVDs are well-established, T cell alterations, particularly involving cytotoxic CD4 T cells (CD4 CTLs), remain unexplored in human atherosclerotic plaques. In our study, we aimed to identify the phenotype and function of CD4 CTLs in blood &amp; plaque tissue, in the context of T2D. Using unbiased single-cell RNA sequencing (scRNA-seq) analysis, we examined over 70,000 immune cells from 22 plaques of patients undergoing carotid endarterectomy (T2D: n=12, Non-Diabetic (ND): n=10), &amp; peripheral blood mononuclear cells (PBMCs) from the CHORD study (NCT04369664) (T2D: n=12, ND: n=4). scRNA-seq analysis revealed a diverse population of CD4 T cells, like naïve, central memory (CM), effector memory (EM), tissue resident memory (Trm), T regulatory (Tregs), &amp; two clusters of cytotoxic (CTL) T cells expressing canonical cytotoxicity-related genes. Cluster 5 expressed GZMB, GNLY, PRF1 , whereas Cluster 10 expressed GZMA, GZMK . Cell-cell communication analysis demonstrated that both CTL clusters received signaling from dendritic cells and macrophages, including TREM2+ macrophages. CTL T cells reciprocally communicated with clusters of EM and EMRA CD8 T cells, underscoring their central role in plaque cell-cell communication. Notably, Cluster 5, one of these two CTL clusters, was expanded in T2D plaque samples (p=0.032) and expressed genes involved in chemotaxis, IL-2 signaling, &amp; transcription factors (e.g., RUNX3, TBX21 ) involved in T cell activation. The equivalent of CD4 CTL phenotype was found to be expanded in T2D individuals compared to controls, &amp; these elevated levels persisted in T2D patients following intensive lipid-lowering therapy, indicating resistance to the treatment regimen. In summary, our study emphasizes the crucial role of CD4 CTLs in the immune dynamics of atherosclerotic plaques. The observed resistance of CTLs to lipid-lowering interventions underscores the necessity for targeted therapeutic strategies to address the distinctive immune changes in T2D-associated cardiovascular diseases.

The MMP-9 promoter genetic variant rs3918242, mRNA and protein expression in advanced carotid plaque tissue.

The MMP-9 is a known player in atherosclerosis, yet associations of the MMP-9 -1562C/T variant (rs3918242) with various atherosclerotic phenotypes and tissue mRNA expression are still contradictory. This study aimed to investigate the MMP-9 -1562C/T variant, its mRNA and protein expression in carotid plaque (CP) tissue, as a risk factor for CP presence and as a marker of different plaque phenotypes (hyperechoic and hypoechoic) in patients undergoing carotid endarterectomy. The MnSOD as an MMP-9 negative regulator was also studied in relation to CP phenotypes. Genotyping of 770 participants (285 controls/485 patients) was done by tetra-primer ARMS PCR. The MMP-9 mRNA expression in 88 human CP tissues was detected by TaqMan® technology. The protein levels of MMP-9 and MnSOD were assessed by Western blot analysis. The MMP-9 -1562C/T variant was not recognized as a risk factor for plaque presence or in predisposing MMP-9 mRNA and protein levels in plaque tissue. Patients with hypoechoic plaques had significantly lower MMP-9 mRNA and protein levels than those with hyperechoic plaque (p = 0.008, p = 0.003, respectively). MnSOD protein level was significantly higher in hypoechoic plaque compared to hyperechoic (p = 0.039). MMP-9 protein expression in CP tissue was significantly affected by sex and plaque type interaction (p = 0.009). Considering the differences of MMP-9 mRNA and protein expression in CP tissue regarding different plaque phenotypes and the observed sex-specific effect, the role of MMP-9 in human atherosclerotic plaques should be further elucidated.

Computational Investigation of Vessel Injury Due to Catheter Tracking During Transcatheter Aortic Valve Replacement.

Catheter reaction forces during transcatheter valve replacement (TAVR) may result in injury to the vessel or plaque rupture, triggering distal embolization or thrombosis. In vitro test methods represent the arterial wall using synthetic proxies to determine catheter reaction forces during tracking, but whether they can account for reaction forces within the compliant aortic wall tissue in vivo is unknown. Moreover, the role of plaque inclusions is not well understood. Computational approaches have predicted the impact of TAVR positioning, migration, and leaflet distortion, but have not yet been applied to investigate aortic wall reaction forces and stresses during catheter tracking. In this study, we investigate the role that catheter design and aorta and plaque mechanical properties have on the risk of plaque rupture during TAVR catheter delivery. We report that, for trackability testing, a rigid test model provides a reasonable estimation of the peak reaction forces experienced during catheter tracking within compliant vessels. We investigated the risk of rupture of both the aortic tissue and calcified plaques. We report that there was no risk of diseased aortic tissue rupture based on an accepted aortic tissue stress threshold (4.2MPa). However, we report that both the aortic and plaque tissue exceed a rupture stress threshold (300kPa) with and without the presence of stiff and soft plaque inclusions. We also highlight the potential risks associated with shorter catheter tips during catheter tracking and demonstrate that increasing the contact surface will reduce peak contact pressures experienced in the tissue.

CircHIPK3 targets DRP1 to mediate hydrogen peroxide-induced necroptosis of vascular smooth muscle cells and atherosclerotic vulnerable plaque formation

IntroductionNecroptosis triggered by H2O2 is hypothesized to be a critical factor in the rupture of atherosclerotic plaques, which may precipitate acute cardiovascular events. Nevertheless, the specific regulatory molecules of this development remain unclear. We aims to elucidate a mechanism from the perspective of circular RNA. ObjectivesThere are few studies on circRNA in VSMCs necroptosis. The objective of our research is to shed light on the intricate roles that circHIPK3 plays in the process of necroptosis in VSMCs and the development of atherosclerotic plaques that are prone to rupture. Our study elucidates the specific molecular mechanisms by which circHIPK3 regulates necroptosis and atherosclerotic vulnerable plaque formation through targeted proteins. Identifying this mechanism at the cellular level offers a molecular framework for understanding plaque progression and stability regulation, as well as a potential biomarker for the prognosis of susceptible atherosclerotic plaques. MethodsWe collected clinical vascular tissue for HE staining and Masson staining to determine the presence and stability of plaques. Then, NCBI database was used to screen out circRNA with elevated expression level in plaque tissue, and the up-regulated circRNA, circHIPK3, was verified by qRT-PCR and FISH. Further, we synthesized circHIPK3′s small interference sequence and overexpressed plasmid in vitro, and verified its regulation effect on necroptosis of VSMCs under physiological and pathological conditions by WB, qRT-PCR and PI staining. Through RNA pull down, mass spectrometry and RNA immunoprecipitation, DRP1 was identified as circHIPK3 binding protein and was positively regulated by circHIPK3. Meanwhile, on the basis of silencing of DRP1, the regulation of circHIPK3 on necroptosis is verified to be mediated by DRP1. Finally, we validated the regulation of circHIPK3 on vulnerable plaque formation in ApoE-/- mice. ResultsWe investigated that circHIPK3 was highly expressed in vulnerable plaques, and the increase in expression level promoted H2O2 induced necroptosis of VSMCs. CircHIPK3 targeted the protein DRP1, leading to an elevation in mitochondrial division rate, resulting in increased reactive oxygen species and impaired mitochondrial function, ultimately leading to necroptosis of VSMCs and vulnerable plaque formation. ConclusionCircHIPK3 interact with DRP1 involve in H2O2 induced Mitochondrial damage and necroptosis of VSMCs, and Silencing circHIPK3 in vivo can reduce atherosclerotic vulnerable plaque formation. Our research findings may have applications in providing diagnostic biomarkers for vulnerable plaques.

Orosomucoid 2 as a biomarker of carotid artery atherosclerosis plaque vulnerability through its generation of reactive oxygen species and lipid accumulation in vascular smooth muscle cells

BackgroundOrosomucoid (ORM) has been reported as a biomarker of carotid atherosclerosis, but the role of ORM 2, a subtype of ORM, in carotid atherosclerotic plaque formation and the underlying mechanism have not been established. MethodsPlasma was collected from patients with carotid artery stenosis (CAS) and healthy participants and assessed using mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) technology to identify differentially expressed proteins. The key proteins and related pathways were identified via western blotting, immunohistochemistry, and polymerase chain reaction of carotid artery plaque tissues and in vitro experiments involving vascular smooth muscle cells (VSMCs). ResultsWe screened 33 differentially expressed proteins out of 535 proteins in the plasma. Seventeen proteins showed increased expressions in the CAS groups relative to the healthy groups, while 16 proteins showed decreased expressions during iTRAQ and bioinformatic analysis. The reactive oxygen species metabolic process was the most common enrichment pathway identified by Gene Ontology analysis, while ORM2, PRDX2, GPX3, HP, HBB, ANXA5, PFN1, CFL1, and S100A11 were key proteins identified by STRING and MCODE analysis. ORM2 showed increased expression in patients with CAS plaques, and ORM2 was accumulated in smooth muscle cells. Oleic acid increased the lipid accumulation and ORM2 and PRDX6 expressions in the VSMCs. The recombinant-ORM2 also increased the lipid accumulation and reactive oxygen species (ROS) in the VSMCs. The expressions of ORM2 and PRDX-6 were correlated, and MJ33 (an inhibitor of PRDX6-PLA2) decreased ROS production and lipid accumulation in VSMCs. ConclusionORM2 may be a biomarker for CAS; it induced lipid accumulation and ROS production in VSMCs during atherosclerosis plaque formation. However, the relationships between ORM2 and PRDX-6 underlying lipid accumulation-induced plaque vulnerability require further research.