Human Atherosclerotic Plaques Research Articles

Abstract P157: Loss Of Nicotinamide Nucleotide Transhydrogenase Promotes Endothelial Activation In Atherosclerosis

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly, in vitro , the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co-treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease.

Human Atherosclerotic Plaque Progression Is Dependent on Apoptosis According to Bomb-Pulse 14C Dating

Individuals with rapidly progressing atherosclerotic plaques are at higher risk of experiencing acute complications. Currently, we lack knowledge regarding factors in human plaque that cause rapid progression. Using the 14C bomb-pulse dating method, we assessed the physical age of atherosclerotic plaques and which biological processes were associated with rapidly progressing plaques. Interestingly, increased apoptosis was the main component associated with a young physical plaque age, reflecting rapid plaque progression. Our findings in combination with recent advances in imaging techniques could guide future diagnostic imaging strategies to identify rapidly progressing plaques or therapeutic targets, halting plaque progression.

Long non-coding RNA RP11-490M8.1 inhibits lipopolysaccharide-induced pyroptosis of human umbilical vein endothelial cells via the TLR4/NF-κB pathway

Background and aimsPyroptosis is a relatively newly discovered form of programmed cell death that plays an important role in the development of atherosclerosis. Many studies have reported that lncRNAs participated in the regulation of atherosclerosis development. However, the regulatory mechanism of lncRNAs in pyroptosis must be studied further. MethodsIn a previous study, microarray analysis was used to detect the lncRNA expression profile in three human advanced atherosclerotic plaques and three normal arterial intimae. In the present research, in vitro assays were performed to investigate the role of lncRNA RP11-490M8.1 on pyroptosis. The relative gene mRNA and lncRNA expression levels were tested by quantitative real-time PCR, and protein levels were evaluated by western blot analysis. The RNA hybrid structure was analyzed using the DINAMelt server. ResultsThe lncRNA RP11-490M8.1 was significantly downregulated in atherosclerotic plaques and serum. Lipopolysaccharide (LPS) markedly reduced the expression of lncRNA RP11-490M8.1 and induced pyroptosis by increasingthe mRNA and protein levels of NLRP3, caspase-1, ASC, IL-1β, and IL-18 in HUVECs. The promotion effects ofLPS on pyroptosis were markedly suppressed by overexpression of lncRNA RP11-490M8.1. In addition, LPS increased the mRNA and protein levels ofTLR4 and NF-κB, which was also markedly offsetby overexpression of lncRNA RP11-490M8.1. ConclusionsThese findings indicated that lncRNA RP11-490M8.1 inhibited LPS-induced pyroptosis via the TLR4/NF-κB pathway. Thus, lncRNA RP11-490M8.1 may provide a therapeutic target to ameliorate atherosclerosis.

DNA methylome profiling reveals epigenetic regulation of lipoprotein-associated phospholipase A2 in human vulnerable atherosclerotic plaque

BackgroundAtherosclerotic plaque vulnerability is a key feature of atheroprogression and precipitating acute cardiovascular events. Although the pivotal role of epigenetic regulation in atherosclerotic plaque destabilization is being recognized, the DNA methylation profile and its potential role in driving the progression and destabilization of atherosclerotic cardiovascular disease remains largely unknown. We conducted a genome-wide analysis to identify differentially methylated genes in vulnerable and non-vulnerable atherosclerotic lesions to understand more about pathogenesis.ResultsWe compared genome-wide DNA methylation profiling between carotid artery plaques of patients with clinically symptomatic (recent stroke or transient ischemic attack) and asymptomatic disease (no recent stroke) using Infinium Methylation BeadChip arrays, which revealed 90,368 differentially methylated sites (FDR < 0.05, |delta beta|> 0.03) corresponding to 14,657 annotated genes. Among these genomic sites, 30% were located at the promoter regions and 14% in the CpG islands, according to genomic loci and genomic proximity to the CpG islands, respectively. Moreover, 67% displayed hypomethylation in symptomatic plaques, and the differentially hypomethylated genes were found to be involved in various aspects of inflammation. Subsequently, we focus on CpG islands and revealed 14,596 differentially methylated sites (|delta beta|> 0.1) located at the promoter regions of 7048 genes. Integrated analysis of methylation and gene expression profiles identified that 107 genes were hypomethylated in symptomatic plaques and showed elevated expression levels in both advanced plaques and ruptured plaques. The imprinted gene PLA2G7, which encodes lipoprotein-associated phospholipase A2 (Lp-PLA2), was one of the top hypomethylated genes with an increased expression upon inflammation. Further, the hypomethylated CpG site at the promoter region of PLA2G7 was identified as cg11874627, demethylation of which led to increased binding of Sp3 and expression of Lp-PLA2 through bisulfate sequencing, chromatin immunoprecipitation assay and enzyme-linked immunosorbent assay. These effects were further enhanced by deacetylase.ConclusionExtensive DNA methylation modifications serve as a new and critical layer of biological regulation that contributes to atheroprogression and destabilization via inflammatory processes. Revelation of this hitherto unknown epigenetic regulatory mechanism could rejuvenate the prospects of Lp-PLA2 as a therapeutic target to stabilize the atherosclerotic plaque and reduce clinical sequelae.

CCL3/CCR5 and CX3CL1/CX3CR1 chemokine interactions are involved in T-cell accumulation in human atherosclerotic plaques

Background and Aims: Immune cells, particularly T-lymphocytes, play a critical role in atherosclerotic plaques’ progression and destabilization leading to myocardial infarctions and strokes. Chemokine binding was proposed to drive T-cell accumulation in plaques. We analysed the chemokine receptor signatures of T-cells isolated from 17 atherosclerotic plaques from 13 patients and compared them with autologous peripheral blood T-cells. Also, the chemokine expression levels in plaque tissue were compared with those in peripheral blood cells.

Line-1 methylation in different vessels, vascular and blood cells in human atherosclerosis.

Background and Aims: Decreased LINE-1 methylation was associated with cardiovascular diseases. However, LINE-1 methylation in vascular cells is poorly investigated in atherosclerosis. Here we studied DNA methylation of LINE-1 promoter in human carotid atherosclerotic plaque, intact artery, and vein, as well as in vascular smooth muscle cells (VSMC) and macrophages (MPH) isolated from plaques, and in blood cells.

EV-associated cytokines in human atherosclerotic plaques cultured ex vivo

Background and Aims: Extracellular vesicles (EVs) have been shown to play a role in atherosclerotic plaque progression. However, the mechanisms of this process are poorly understood, partially, due to the absence of ex vivo models that faithfully reproduce the plaque’s structure and microenvironment. Using this model, we studied the distribution of cytokines between EV-associated and soluble fractions in the culture medium of our model of human atherosclerotic plaque culture that allows the investigation of intracellular interactions.

Sex-dependent gene regulation of human atherosclerotic plaques by DNA methylation and transcriptome integration points to smooth muscle cell involvement in women.

Background and Aims: Sex differences are evident in the clinical presentation and underlying histology of atherosclerotic disease with women developing more stable atherosclerotic lesions than men. It is unknown whether this is explained by sex differences in gene regulation in cellular compartments of atherosclerotic plaques.

ILF3 is responsible for hyperlipidemia-induced arteriosclerotic calcification by mediating BMP2 and STAT1 transcription

Calcification is common in atherosclerotic plaque and can induce vulnerability, which further leads to myocardial infarction, plaque rupture and stroke. The mechanisms of atherosclerotic calcification are poorly characterized. Interleukin enhancer binding factor 3 (ILF3) has been identified as a novel factor affecting dyslipidemia and stroke subtypes. However, the precise role of ILF3 in atherosclerotic calcification remains unclear. In this study, we used smooth muscle-conditional ILF3 knockout (ILF3SM-KO) and transgenic mice (ILF3SM-Tg) and macrophage-conditional ILF3 knockout (ILF3M-KO) and transgenic (ILF3M-Tg) mice respectively. Here we showed that ILF3 expression is increased in calcified human aortic vascular smooth muscle cells (HAVSMCs) and calcified atherosclerotic plaque in humans and mice. We then found that hyperlipidemia increases ILF3 expression and exacerbates calcification of VSMCs and macrophages by regulating bone morphogenetic protein 2 (BMP2) and signal transducer and activator of transcription 1 (STAT1) transcription. We further explored the molecular mechanisms of ILF3 in atherosclerotic calcification and revealed that ILF3 acts on the promoter regions of BMP2 and STAT1 and mediates BMP2 upregulation and STAT1 downregulation, which promotes atherosclerotic calcification. Our results demonstrate the effect of ILF3 in atherosclerotic calcification. Inhibition of ILF3 may be a useful therapy for preventing and even reversing atherosclerotic calcification.

Research on the correlation between activating transcription factor 3 expression in the human coronary artery and atherosclerotic plaque stability

BackgroundActivating transcription factor 3 (ATF3) is an early response gene that is activated in response to atherosclerotic stimulation and may be an important factor in inhibiting the progression of atherosclerosis. In this study, we directly measured the expression of ATF3 and inflammatory factors in human coronary atherosclerotic plaques to examine the relationship between ATF3 expression, inflammation and structural stability in human coronary atherosclerotic plaques.MethodsA total of 68 coronary artery specimens were collected from the autopsy group, including 36 cases of sudden death from coronary heart disease (SCD group) and 32 cases of acute death caused by mechanical injury with coronary atherosclerosis (CHD group). Twenty-two patients who had no coronary heart disease were collected as the control group (Con group). The histological structure of the coronary artery was observed under a light microscope after routine HE staining, and the intimal and lesion thicknesses, thickness of the fibrous cap, thickness of necrosis core, degree of lumen stenosis were assessed by image analysis software. Western blotting and immunohistochemistry were used to measure the expression and distribution of ATF3, inflammatory factors (CD45, IL-1β, TNF-α) and matrix metalloproteinase-9 (MMP-9) and vascular cell adhesion molecule 1 (VCAM1) in the coronary artery. The Pearson correlation coefficient was used to analyse the correlation between ATF3 protein expression and inflammatory factors and between ATF3 protein expression and structure-related indexes in the lesion group.ResultsCompared with those in the control group, the intima and necrotic core in the coronary artery were thickened, the fibrous cap became thin and the degree of vascular stenosis was increased in the lesion group, while the intima and necrotic core became thicker and the fibrous cap became thinner in the SCD group than in the CHD group (P < 0.05). There was no or low expression of ATF3, inflammatory factors, VCAM1 and MMP-9 in the control group, and the expression of inflammatory factors, VCAM1 and MMP-9 in the SCD group was higher than that in CHD group, while the expression of ATF3 in the SCD group was significantly lower than that in CHD group (P < 0.05). In the lesion group, the expression of ATF3 was negatively correlated with intimal and necrotic focus thickness, positively correlated with fibrous cap thickness (P < 0.01), and negatively correlated with inflammatory factors, VCAM1 and MMP-9 (P < 0.01).ConclusionsThe expression of ATF3 may be related to the progression and stability of atherosclerotic plaques, and may affect the structural stability of atherosclerotic plaques by regulating the inflammatory response, thus participating in the regulation of atherosclerotic progression.

Comprehensive analysis of dysregulated genes associated with atherosclerotic plaque destabilization.

Atherosclerotic plaque destabilization is a dominating cause of acute cardiovascular events such as myocardial infarction and stroke. This study aims to identify genetic biomarkers related to atherosclerotic plaque destabilization using bioinformatics. Three transcriptome datasets of human carotid atherosclerotic plaque samples were downloaded from ArrayExpress and Gene Expression Omnibus databases, including E-MATB-2055, E-TABM-190, and GSE120521. With Robust Rank Aggregation analysis, we documented 46 differentially expressed genes between stable and unstable/ruptured plaques. Functional enrichment analysis using DAVID tool demonstrated that these genes were mainly related to biological functions such as extracellular matrix disassembly, collagen catabolic process, response to mechanical stimulus, and PPAR signaling pathway. A protein-protein interaction network for the differentially expressed genes was constructed, and eight pivotal genes (ITGAM, MMP9, PLAUR, CCR1, CD163, CD36, ADAM8, and IL1RN) were obtained from the network with a connective degree > 5. The expression patterns of these hub differentially expressed genes could be verified in atherosclerotic plaque samples with intraplaque hemorrhage. Using gene set variation analysis, the eight genes were integrated to generate an atherosclerotic plaque destabilization score, which showed a high performance in not only discriminating individuals with myocardial infarction from those with stable coronary illness, but also in predicting future acute cardiovascular events in atherosclerotic patients. In conclusion, the findings of this study will enhance our knowledge on the pathological mechanisms involved in atherosclerotic plaque destabilization, and provide potential gene biomarkers for risk stratification of patients with atherosclerotic cardiovascular disease.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-β1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600–1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Immune cell profiling in atherosclerosis: role in research and precision medicine.

Inflammation is intimately involved at all stages of atherosclerosis and remains a substantial residual cardiovascular risk factor in optimally treated patients. The proof of concept that targeting inflammation reduces cardiovascular events in patients with a history of myocardial infarction has highlighted the urgent need to identify new immunotherapies to treat patients with atherosclerotic cardiovascular disease. Importantly, emerging data from new clinical trials show that successful immunotherapies for atherosclerosis need to be tailored to the specific immune alterations in distinct groups of patients. In this Review, we discuss how single-cell technologies — such as single-cell mass cytometry, single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing — are ideal for mapping the cellular and molecular composition of human atherosclerotic plaques and how these data can aid in the discovery of new precise immunotherapies. We also argue that single-cell data from studies in humans need to be rigorously validated in relevant experimental models, including rapidly emerging single-cell CRISPR screening technologies and mouse models of atherosclerosis. Finally, we discuss the importance of implementing single-cell immune monitoring tools in early phases of drug development to aid in the precise selection of the target patient population for data-driven translation into randomized clinical trials and the successful translation of new immunotherapies into the clinic.

Endonuclease V Regulates Atherosclerosis Through C-C Motif Chemokine Ligand 2-Mediated Monocyte Infiltration.

BackgroundIn cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine‐to‐inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine‐to‐inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking.Methods and ResultsQuantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine‐ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E‐deficient (ApoE−/− ) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE−/− mice.ConclusionsENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Chemically Modified Biomimetic Carbon-Coated Iron Nanoparticles for Stent Coatings: In Vitro Cytocompatibility and In Vivo Structural Changes in Human Atherosclerotic Plaques.

Atherosclerosis, a systematic degenerative disease related to the buildup of plaques in human vessels, remains the major cause of morbidity in the field of cardiovascular health problems, which are the number one cause of death globally. Novel atheroprotective HDL-mimicking chemically modified carbon-coated iron nanoparticles (Fe@C NPs) were produced by gas-phase synthesis and modified with organic functional groups of a lipophilic nature. Modified and non-modified Fe@C NPs, immobilized with polycaprolactone on stainless steel, showed high cytocompatibility in human endothelial cell culture. Furthermore, after ex vivo treatment of native atherosclerotic plaques obtained during open carotid endarterectomy surgery, Fe@C NPs penetrated the inner structures and caused structural changes of atherosclerotic plaques, depending on the period of implantation in Wistar rats, serving as a natural bioreactor. The high biocompatibility of the Fe@C NPs shows great potential in the treatment of atherosclerosis disease as an active substance of stent coatings to prevent restenosis and the formation of atherosclerotic plaques.

VEGF-Targeted Multispectral Optoacoustic Tomography and Fluorescence Molecular Imaging in Human Carotid Atherosclerotic Plaques.

Vulnerable atherosclerotic carotid plaques are prone to rupture, resulting in ischemic strokes. In contrast to radiological imaging techniques, molecular imaging techniques have the potential to assess plaque vulnerability by visualizing diseases-specific biomarkers. A risk factor for rupture is intra-plaque neovascularization, which is characterized by overexpression of vascular endothelial growth factor-A (VEGF-A). Here, we study if administration of bevacizumab-800CW, a near-infrared tracer targeting VEGF-A, is safe and if molecular assessment of atherosclerotic carotid plaques in vivo is possible using multispectral optoacoustic tomography (MSOT). Healthy volunteers and patients with symptomatic carotid artery stenosis scheduled for carotid artery endarterectomy were imaged with MSOT. Secondly, patients were imaged two days after intravenous administration of 4.5 bevacizumab-800CW. Ex vivo fluorescence molecular imaging of the surgically removed plaque specimen was performed and correlated with histopathology. In this first-in-human MSOT and fluorescence molecular imaging study, we show that administration of 4.5 mg bevacizumab-800CW appeared to be safe in five patients and accumulated in the carotid atherosclerotic plaque. Although we could visualize the carotid bifurcation area in all subjects using MSOT, bevacizumab-800CW-resolved signal could not be detected with MSOT in the patients. Future studies should evaluate tracer safety, higher doses of bevacizumab-800CW or develop dedicated contrast agents for carotid atherosclerotic plaque assessment using MSOT.

Calcification patterns in femoral and carotid atheromatous plaques: A comparative morphometric study.

This comparative study was designed to focus on the mineral patterns in human atherosclerotic plaques based on quantitative measurements of calcium deposits through the morphometric method. A total of 101 atherosclerotic plaques were harvested by conventional transluminal angioplasty from the carotid artery (CA) and different segments of the femoral-popliteal axis (FPA), fixed in formalin and sent for histological processing. The histological grade of the atherosclerotic plaque and the calcification pattern were evaluated, followed by a morphometric analysis of the mineral deposits. Regarding the localization, the advanced plaques (VII and VIII types) developed predominantly at the level of the superficial femoral artery (SFA) compared to the CA (P<0.001). This significant difference was maintained even if they were divided into low grade (IV and V) and high grade categories (VI, VII and VIII) (P<0.05). Compared with that in the carotid plaques, in the FPA plaques the mineralized surface increased in parallel with the narrowing of the vascular lumen diameter. The image analysis of the total pathological calcification score (pCS) showed a significant difference between the CA plaques and distal SFA (dSFA) plaques (P=0.038) and between the proximal SFA (pSFA) and dSFA plaques (P=0.013). In the case of the simple nodular pattern, calcification occupied significantly larger areas in the plaques developed in the dSFA and popliteal artery (PA) in comparison with the CA plaques (P=0.0007 and P=0.0009). pCSs calculated in plaques with extensive calcification pattern showed a lower value in the CA vs. the pSFA plaques (P=0.004). A less pronounced, but significant difference was observed between the pCS of pSFA and dSFA plaques (P=0.017). Femoral and carotid plaques exhibited different morphology and tendency for calcification. In parallel with the narrowing of the vascular lumen diameter, the mineralized surface increased at the level of different FPA segments. These results suggest that the mechanism is site-specific, and wall structure-dependent.

Integrative multiomics analysis of human atherosclerosis reveals a serum response factor-driven network associated with intraplaque hemorrhage.

BackgroundWhile single‐omics analyses on human atherosclerotic plaque have been very useful to map stage‐ or disease‐related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale‐intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model.MethodsIn this study, we compared protein and gene makeup of low‐ versus high‐risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method.ResultsWe identified a protein‐gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA−PDGFRα+ fibroblast‐like cell content (p = 2.4E‐05) and Arg1+ macrophage content (p = 2.2E‐04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia‐1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts.ConclusionsIn conclusion, our integrative omics study has identified an intraplaque hemorrhage‐associated cardiovascular signature that provides excellent stratification of low‐ from high‐risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF‐regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.

Material Decomposition in Low-Energy Micro-CT Using a Dual-Threshold Photon Counting X-Ray Detector

Material decomposition in computed tomography is a method for differentiation and quantification of materials in a sample and it utilizes the energy dependence of the linear attenuation coefficient. In this study, a post-image reconstruction material decomposition method is constructed for a low-energy micro-CT setup using a photon counting x-ray detector. The low photon energy range (4–11 keV) allows for K-edge contrast separation of naturally occurring materials in organic tissue without the need of additional contrast agents. The decomposition method was verified using a phantom and its capability to decompose biomedical samples was evaluated with paraffin embedded human atherosclerotic plaques. Commonly, the necessary dual energy data for material decomposition is obtained by manipulating the emitted x-ray spectrum from the source. With the photon counting detector, this data was obtained by acquiring two energy window images on each side of the K-edge of one material in the sample. The samples were decomposed into three materials based on attenuation values in manually selected regions. The method shows a successful decomposition of the verification phantom and a distinct distribution of iron, calcium and paraffin in the atherosclerotic plaque samples. Though the decompositions are affected by beam hardening and ring artifacts, the method shows potential for spectral evaluation of biomedical samples.