Human Plaques Research Articles

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based μCT—A comparison with histology

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRμCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRμCT and histology. Phase-contrast SRμCT was performed with two different magnifications (voxel sizes 6.5 μm and 0.65 μm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 μm high-resolution SRμCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 μm low-resolution SRμCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRμCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method’s non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4<CD8), exhibiting clonal expansion of specific TCRs. Interestingly, we found that these plaque T cells had TCRs specific for influenza, coronavirus, and other viral epitopes, which share sequence homologies to proteins found on smooth muscle cells and endothelial cells, suggesting potential autoimmune-mediated T-cell activation in the absence of active infection. To better understand the potential function of these activated plaque T cells, we then interrogated their transcriptome at the single-cell level. Of the 3 T-cell phenotypic clusters with the highest expression of the activation marker HLA-DRA, 2 clusters expressed a proinflammatory and cytolytic signature characteristic of CD8 cells, while the other expressed AREG (amphiregulin), which promotes smooth muscle cell proliferation and fibrosis, and, thus, contributes to plaque progression. Taken together, these findings demonstrate that plaque T cells are clonally expanded potentially by antigen engagement, are potentially reactive to self-epitopes, and may interact with smooth muscle cells and macrophages in the plaque microenvironment.

Label-free analytic histology of carotid atherosclerosis by mid-infrared optoacoustic microscopy

Background and aimsAnalysis of atherosclerotic plaque composition is a vital tool for unraveling the pathological metabolic processes that contribute to plaque growth. MethodsWe visualize the constitution of human carotid plaques by mid-infrared optoacoustic microscopy (MiROM), a method for label-free analytic histology that requires minimal tissue preparation, rapidly yielding large field-of-view en-face images with a resolution of a few micrometers. We imaged endarterectomy specimens (n = 3, 12 sections total) at specific vibrational modes, targeting carbohydrates, lipids and proteins. Additionally, we recorded spectra at selected tissue locations. We identified correlations in the variability in this high-dimensional data set using non-negative matrix factorization (NMF). ResultsWe visualized high-risk plaque features with molecular assignment. Consistent NMF components relate to different dominant tissue constituents, dominated by lipids, proteins, and cholesterol and carbohydrates respectively. ConclusionsThese results introduce MiROM as an innovative, stain-free, analytic histology technology for the biochemical characterization of complex human vascular pathology.

Tetracycline-Loaded Electrospun Poly(l-lactide-co-ε-caprolactone) Membranes for One-Step Periodontal Treatment

In this research, a one-step periodontal membrane, with the required function and properties, has been designed as an alternative method of tissue regenerative treatments. Designed nanoporous prototypes from poly(l-lactide-co-ε-caprolactone) (PLCL, 70:30 mol %) were fabricated by electrospinning, denoted as S-PLCL. They were subsequently loaded with tetracycline (TC) in order to enhance periodontal regeneration and deliver an anti-inflammatory and antibiotic drug. It was found that TC loading did not have any significant effect on the fiber diameter but did increase hydrophilicity. With the increase in TC loading, the water vapor permeability (WVP) of the S-PLCL membrane decreased within the range of 31–56% when compared with neat S-PLCL membranes, while in the solvent-cast film (F-PLCL), no significant change in WVP was observed. Moreover, S-PLCL demonstrated a controllable slow release rate of TC. S-PLCL loaded with 1500 μg/mL of TC showed a release concentration of 30 ppm over a certain time period to promote greater levels of human oral fibroblast and human oral keratinocyte cell proliferation and plaque inhibition. In conclusion, a TC-loaded S-PLCL fibrous membrane has been designed and fabricated to provide the ideal conditions for cell proliferation and antibiotic activity during treatment, outperforming nonfibrous F-PLCL loaded with TC at the same concentration.

Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

Targeting platelet-derived CXCL12 impedes arterial thrombosis

AbstractThe prevention and treatment of arterial thrombosis continue to be clinically challenging, and understanding the relevant molecular mechanisms in detail may facilitate the quest to identify novel targets and therapeutic approaches that improve protection from ischemic and bleeding events. The chemokine CXCL12 augments collagen-induced platelet aggregation by activating its receptor CXCR4. Here we show that inhibition of CXCR4 attenuates platelet aggregation induced by collagen or human plaque homogenate under static and arterial flow conditions by antagonizing the action of platelet-secreted CXCL12. We further show that platelet-specific CXCL12 deficiency in mice limits arterial thrombosis by affecting thrombus growth and stability without increasing tail bleeding time. Accordingly, neointimal lesion formation after carotid artery injury was attenuated in these mice. Mechanistically, CXCL12 activated via CXCR4 a signaling cascade involving Bruton's tyrosine kinase (Btk) that led to integrin αIIbβ3 activation, platelet aggregation, and granule release. The heterodimeric interaction between CXCL12 and CCL5 can inhibit CXCL12-mediated effects as mimicked by CCL5-derived peptides such as [VREY]4. An improved variant of this peptide, i[VREY]4, binds to CXCL12 in a complex with CXCR4 on the surface of activated platelets, thereby inhibiting Btk activation and preventing platelet CXCL12-dependent arterial thrombosis. In contrast to standard antiplatelet therapies such as aspirin or P2Y12 inhibition, i[VREY]4 reduced CXCL12-induced platelet aggregation and yet did not prolong in vitro bleeding time. We provide evidence that platelet-derived CXCL12 is involved in arterial thrombosis and can be specifically targeted by peptides that harbor potential therapeutic value against atherothrombosis.

Lipocalin-2 and Calprotectin Potential Prognosis Biomarkers in Peripheral Arterial Disease

Peripheral arterial disease (PAD) is the most prevalent cardiovascular (CV) condition globally. Despite the high CV risk of PAD patients, no reliable predictors of adverse clinical evolution are yet available. In this regard, previous transcriptomic analyses revealed increased expression of calprotectin (S100A8/A9) and lipocalin-2 (LCN2) in circulating extracellular vesicles (EVs) of patients with PAD. The aim of this study was to determine the prognostic value of LCN2 and calprotectin for CV risk assessment in PAD. LCN2 and the S100A9 subunit of calprotectin were examined in human femoral plaques by immunohistochemistry and qPCR. LCN2 and calprotectin were determined by ELISA in PAD (CHN cohort, n= 331, Fontaine II-IV, serum), and PAD diagnosed by population based screening (VIVA trial, n= 413, the majority Fontaine 0-I, plasma). Patients were followed up for a mean of four years, recording the primary outcomes; CV death or amputation in the CHN cohort and CV death or major lower limb events (MALE) in the VIVA population. Secondary outcomes were all cause death or amputation, and all cause death or MALE, respectively. LCN2 and S100A9 were detected in human plaques in regions rich in inflammatory cells. LCN2 and calprotectin levels were 70% and 64% lower in plasma than in serum. In the CHN cohort, high serum levels of LCN2 and calprotectin increased the risk of primary and secondary outcomes 5.6 fold (p < .001) and 1.8 fold (p= .034), respectively, after covariable adjustment. Similarly, elevated plasma levels of LCN2 and calprotectin increased by three fold the risk of primary and secondary outcomes (p < .001) in the VIVA cohort. Moreover, addition of the combined variable to basal models, considering clinically relevant risk factors, improved reclassification for the primary outcome in both cohorts (p ≤ .024). Combined assessment of the inflammatory biomarkers LCN2 and calprotectin might be useful for risk stratification in advanced and early PAD.

Contribution of FBLN5 to Unstable Plaques in Carotid Atherosclerosis via mir128 and mir532–3p Based on Bioinformatics Prediction and Validation

FBLN5, a member of the short fibulins in the fibulin family of extracellular matrix/matricellular proteins, is involved in interactions with components of the basement membrane and extracellular matrix proteins. It plays key roles in endothelial tissues in many vascular diseases. In this study, the relationship between FBLN5 and carotid atherosclerotic plaque stability as well as the regulatory roles of miRNAs were evaluated. Differential gene expression analyses and weighted gene co-expression network analysis (WGCNA) based on the GSE163154 dataset (including 16 samples without intraplaque hemorrhage and 27 samples with intraplaque hemorrhage) in GEO revealed that FBLN5 is related to plaque stability and is the most significantly differentially expressed gene. LASSO regression was used to evaluate genes obtained from the intersection of differentially expressed genes and clinically significant modules identified by WGCNA. A prediction model based on eight genes, including FBLN5, was constructed and showed an accuracy of 0.951 based on an ROC analysis. Low FBLN5 expression in plaque tissues was confirmed by immunohistochemistry and western blotting. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that FBLN5 acted mainly by the maintenance of the cellular matrix and reactive oxygen species production. miRNAs upstream of these eight predictive genes, including FBLN5, were identified and used to construct a network diagram. These results revealed that hsa-mir-128 and hsa-mir-532–3p were upstream regulatory factors of FBLN5, as verified by PCR assays of human plaque tissues demonstrating that both miRNAs were significantly up-regulated. Therefore, FBLN5 may play an important role in carotid atherosclerosis via hsa-mir-128 and hsa-mir-532–3p as well as become an essential target for treatment.

Properties and fate of human mesenchymal stem cells upon miRNA let-7f-promoted recruitment to atherosclerotic plaques.

Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe-/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and two-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. Our findings indicate that let-7f promotes hMSC tropism towards atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis.

Markers of Carotid Plaque Destabilization in Patients With Sleep-Disordered Breathing.

Sleep-disordered breathing (SDB) is a nightly respiratory condition characterized by intermittent hypoxia, leading to oxidative stress, inflammation, and atherosclerosis. However, most cellular markers of human carotid plaques in SDB have not yet been assessed. We aimed at characterizing the cellular, inflammatory, and nitro-oxidative stress markers in carotid plaques obtained from 25 patients undergoing endarterectomy and screened for SDB. Sleep studies were performed during their preoperative hospitalization night using the Watch-PAT 100 device. Oxygen desaturation index (ODI) was used for dividing patients into two groups. Fourteen patients with ODI >5 were designated as SDB and 11 patients with ODI ≤ 5 as non-SDB. Demographics, comorbidities, cardiovascular risk factors, and medications were recorded. Cellular markers in plaques were analyzed by immunofluorescence using confocal microscopy. The expression of neutrophils was identified by CD66b+ and neutrophil elastase, macrophage-foam cells were identified by CD163+, and scavenger receptors by CD68+ and CD36+ expression. Additional markers included 3-nitrotyrosine, endothelial CD31, and smooth muscle cell-actin (SMC-actin). Plaques' lipids were determined by immunohistochemistry with Oil Red O staining. Notably, significantly higher values were found for SDB as compared to patients with non-SDB for 3-nitrotyrosine (p <0.004) and intracellular lipids' content (p <0.02), whereas SMC-actin was lower (p <0.006). There were no significant differences between patients with carotid-associated symptoms (symptomatic) and patients without carotid-associated symptoms (asymptomatic). However, a sub-group of symptomatic patients with co-existent SDB expressed the highest 3-nitrotyrosin, and intracellular lipids levels, and the lowest SMC-actin levels, whereas non-SDB/asymptomatic patients expressed the lowest 3-nitrotyrosin and lipids levels and the highest SMS-actin levels among all patients. Accordingly, ODI was lowest in non-SDB/asymptomatic patients and highest in SDB/symptomatic. In conclusion, plaques of patients with SDB were characterized by markedly increased levels of 3-nitrotyrosine and intracellular lipids content. Conversely, SMC-actin levels were significantly lower. These three markers, such as increased 3-nitrotyrosine and intracellular lipids and decreased SMC-actin are associated with plaque vulnerability and instability. These findings are in line with earlier reports demonstrating increased intima-media thickness in large cohorts of sleep apnea and patients with SDB, and thus, may indicate a higher susceptibility to plaque vulnerability and rapture in patients with SDB.

Interferon regulatory factor-5-dependent CD11c+ macrophages contribute to the formation of rupture-prone atherosclerotic plaques.

AimsInflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability.Methods and resultsBulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE−/− mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE−/−Irf5−/− mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts.ConclusionUsing complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture.

Osteomodulin Gene Expression Is Associated With Plaque Calcification, Stability, and Fewer Cardiovascular Events in the CPIP Cohort.

Stable atherosclerotic plaques are characterized by thick fibrous caps of smooth muscle cells, collagen, and macrocalcifications. Identifying factors of plaque stability is necessary to design drugs to prevent plaque rupture and symptoms. Osteomodulin, originally identified in bones, is expressed by bone synthesizing osteoblasts and involved in mineralization. In the present study, we analyzed osteomodulin expression in human carotid plaques, its link with plaque phenotype, calcification, and future cardiovascular events. Osteomodulin gene expression (OMD; n=82) was determined by RNA sequencing and osteomodulin protein levels by immunohistochemistry (n=45) in carotid plaques obtained by endarterectomy from patients with or without cerebrovascular symptoms from the CPIP (Carotid Plaque Imaging Project) cohort, Skåne University Hospital, Sweden. Plaque components were assessed by immunohistochemistry, RNA sequencing, and multiplex analysis. Patients were followed for cardiovascular events or cardiovascular death during a median of 57 or 70 months, respectively, using national registers. OMD levels were increased in plaques from asymptomatic patients compared to symptomatics. High OMD levels were associated with fewer cardiovascular events during follow-up. OMD correlated positively with smooth muscle α-actin (ACTA2; r=0.73, P=10-13) and collagen (COL1A2; r=0.4, P=0.0002), but inversely with CD68 gene expression (r=-0.67, P=10-11), lipids (r=-0.37, P=0.001), intraplaque hemorrhage (r=-0.32, P=0.010), inflammatory cytokine, and matrix metalloproteinase plaque contents. OMD was positively associated with MSX2 (Msh Homeobox 2) (r=0.32, P=0.003), a marker of preosteoblast differentiation, BMP4 (bone morphogenetic protein) (r=0.50, P=0.000002) and BMP6 (r=0.47, P=0.000007), plaque calcification (r=0.35, P=0.016), and was strongly upregulated in osteogenically stimulated smooth muscle cells, which was further increased upon BMP stimulation. Osteomodulin protein was present in calcified regions. Osteomodulin protein levels were associated with plaque calcification (r=0.41, P=0.006) and increased in macrocalcified plaques. These data show that osteomodulin mRNA and protein levels are associated with plaque calcification in human atherosclerosis. Furthermore, osteomodulin mRNA, but not protein levels, is associated with plaque stability.

The Correlation Between Wall Shear Stress and Plaque Composition in Advanced Human Carotid Atherosclerosis.

The role of wall shear stress (WSS) in atherosclerotic plaque development is evident, but the relation between WSS and plaque composition in advanced atherosclerosis, potentially resulting in plaque destabilization, is a topic of discussion. Using our previously developed image registration pipeline, we investigated the relation between two WSS metrics, time-averaged WSS (TAWSS) and the oscillatory shear index (OSI), and the local histologically determined plaque composition in a set of advanced human carotid plaques. Our dataset of 11 carotid endarterectomy samples yielded 87 histological cross-sections, which yielded 511 radial bins for analysis. Both TAWSS and OSI values were subdivided into patient-specific low, mid, and high tertiles. This cross-sectional study shows that necrotic core (NC) size and macrophage area are significantly larger in areas exposed to high TAWSS or low OSI. Local TAWSS and OSI tertile values were generally inversely related, as described in the literature, but other combinations were also found. Investigating the relation between plaque vulnerability features and different combinations of TAWSS and OSI tertile values revealed a significantly larger cap thickness in areas exposed to both low TAWSS and low OSI. In conclusion, our study confirmed previous findings, correlating high TAWSS to larger macrophage areas and necrotic core sizes. In addition, our study demonstrated new relations, correlating low OSI to larger macrophage areas, and a combination of low TAWSS and low OSI to larger cap thickness.

Vascular smooth muscle cell-derived hydrogen sulfide promotes atherosclerotic plaque stability via TFEB (transcription factor EB)-mediated autophagy

ABSTRACT Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H2S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H2S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2+ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68+ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = −0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H2S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H2S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H2S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H2S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H2S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H2S may act as a warning biomarker for vulnerable plaque.

Highly Bright AIE Nanoparticles by Regulating the Substituent of Rhodanine for Precise Early Detection of Atherosclerosis and Drug Screening.

Fluorescent probes capable of precise detection of atherosclerosis (AS) at an early stage and fast assessment of anti-AS drugs in animal level are particularly valuable. Herein, a highly bright aggregation-induced emission (AIE) nanoprobe is introduced by regulating the substituent of rhodanine for early detection of atherosclerotic plaque and screening of anti-AS drugs in a precise, sensitive, and rapid manner. With dicyanomethylene-substituted rhodanine as the electron-withdrawing unit, the AIE luminogen named TPE-T-RCN shows the highest molar extinction coefficient, the largest photoluminescence quantum yield, and the most redshifted absorption/emission spectra simultaneously as compared to the control compounds. The nanoprobes are obtained with an amphiphilic copolymer as the matrix encapsulating TPE-T-RCN molecules, which are further surface functionalized with anti-CD47 antibody for specifically binding to CD47 overexpressed in AS plaques. Such nanoprobes allow efficient recognition of AS plaques at different stages in apolipoprotein E-deficient (apoE-/- ) mice, especially for the recognition of early-stage AS plaques prior to micro-computed tomography (CT) and magnetic resonance imaging (MRI). These features impel to apply the nanoprobes in monitoring the therapeutic effects of anti-AS drugs, providing a powerful tool for anti-AS drug screening. Their potential use in targeted imaging of human carotid plaque is further demonstrated.

CD74 in Apoptotic Macrophages Is Associated with Inflammation, Plaque Progression and Clinical Manifestations in Human Atherosclerotic Lesions.

The aim of this study was to investigate whether CD74 levels in atherosclerotic lesions are associated with inflammation, apoptosis, plaque severity, and clinical symptoms among patients with carotid atherosclerosis. We further studied whether CD74 expression is associated with apoptosis in macrophages induced by 7ketocholesterol (7keto). Sixty-one carotid samples (39 males and 22 females) were immunostained with macrophages, smooth muscle cells, CD74, ferritin, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), and thrombin receptors. Double immunocytochemistry of CD74 and caspase 3 or CD74 and Annexin V was performed on THP-1 macrophages exposed to 7keto. In human carotid plaques, CD74 expression is lesion-dependently increased and is associated with necrotic core formation and plaque rupture, clinical symptoms, macrophage apoptosis, ferritin, and thrombin receptors. CD74 levels were inversely correlated to high-density lipoproteins and statin treatment, and positively correlated to triglycerides. In THP-1 macrophages, 7keto induced a significant increase in levels of CD74, ferritin, and apoptotic cell death. This study suggests that CD74 in apoptotic macrophages is linked to inflammation and thrombosis in progression of human atherosclerotic plaques, lipid metabolism, and clinical manifestation in atherosclerosis. Surface CD74 in apoptotic macrophages and ferritin production induced by oxidized lipids may contribute to inflammation and plaque vulnerability in atherosclerosis.

Single-Cell RNA-Seq Reveals a Population of Smooth Muscle Cells Responsible for Atherogenesis.

Understanding the regional propensity differences of atherosclerosis (AS) development is hindered by the lack of animal models suitable for the study of the disease process. In this paper, we used 3S-ASCVD dogs, an ideal large animal human-like models for AS, to interrogate the heterogeneity of AS-prone and AS-resistant arteries; and at the single-cell level, identify the dominant cells involved in AS development. Here we present data from 3S-ASCVD dogs which reliably mimic human AS pathophysiology, predilection for lesion sites, and endpoint events. Our analysis combined bulk RNA-seq with single-cell RNA-seq to depict the transcriptomic profiles and cellular atlas of AS-prone and AS-resistant arteries in 3S-ASCVD dogs. Our results revealed the integral role of smooth muscle cells (SMCs) in regional propensity for AS. Notably, TNC+ SMCs were major contributors to AS development in 3S-ASCVD dogs, indicating enhanced extracellular matrix remodeling and transition to myofibroblasts during the AS process. Moreover, TNC+ SMCs were also present in human AS-prone carotid plaques, suggesting a potential origin of myofibroblasts and supporting the relevance of our findings. Our study provides a promising large animal model for pre-clinical studies of ASCVD and add novel insights surrounding the regional propensity of AS development in humans, which may lead to interventions that delay or prevent lesion progression and adverse clinical events.

Imaging High-Risk Atherothrombosis Using a Novel Fibrin-Binding Positron Emission Tomography Probe.

High-risk atherosclerosis is an underlying cause of cardiovascular events, yet identifying the specific patient population at immediate risk is still challenging. Here, we used a rabbit model of atherosclerotic plaque rupture and human carotid endarterectomy specimens to describe the potential of molecular fibrin imaging as a tool to identify thrombotic plaques. Atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury (N=13). Pharmacological triggering was used in a group of rabbits (n=9) to induce plaque disruption. Animals were grouped into thrombotic and nonthrombotic plaque groups based on gross pathology (gold standard). All animals were injected with a novel fibrin-specific probe 68Ga-CM246 followed by positron emission tomography (PET)/magnetic resonance imaging 90 minutes later. 68Ga-CM246 was quantified on the PET images using tissue-to-background (back muscle) ratios and standardized uptake value. Both tissue-to-background (back muscle) ratios and standardized uptake value were significantly higher in the thrombotic versus nonthrombotic group (P<0.05). Ex vivo PET and autoradiography of the abdominal aorta correlated positively with in vivo PET measurements. Plaque disruption identified by 68Ga-CM246 PET agreed with gross pathology assessment (85%). In ex vivo surgical specimens obtained from patients undergoing elective carotid endarterectomy (N=12), 68Ga-CM246 showed significantly higher binding to carotid plaques compared to a D-cysteine nonbinding control probe. We demonstrated that molecular fibrin PET imaging using 68Ga-CM246 could be a useful tool to diagnose experimental and clinical atherothrombosis. Based on our initial results using human carotid plaque specimens, in vivo molecular imaging studies are warranted to test 68Ga-CM246 PET as a tool to stratify risk in atherosclerotic patients.

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype.

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation

Background: Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. Methods and Results: Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E–knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. Conclusions: PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.