Unstable Plaque Phenotype Research Articles

Abstract 1044: Single Cell RNA-sequencing Of Endothelial Cells In Atherosclerosis Reveals A Single Phenotypic Transition To A Mesenchymal Cell State

Introduction: Coronary artery disease (CAD) is the leading cause of mortality in the United States. Multiple cell types contribute to the atherosclerotic plaques causing CAD, influencing disease burden. Endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells (ECs) acquire characteristics of mesenchymal cells and loose canonical endothelial features, is correlated with an unstable plaque phenotype, although this transition remains poorly characterized. Hypothesis: A specific population of ECs may undergo EndMT, potentially contributing to the progression of atherosclerosis. Methods: Endothelial-specific lineage-tracing was induced in Tg Cdh5-CreERT2 , ApoE -/- mice, followed by 16 weeks of high-fat diet (HFD) to produce atherosclerosis. Aortic roots were collected from male and female disease-condition animals (n=13) and a baseline cohort that was not fed HFD (n=6). Single cell RNA sequencing was performed using 10X Genomics and analyzed with the Seurat package. RNAscope staining of sections (n=10 per genotype) was performed to characterize the lesion. Results: Multiple endothelial subtypes in the aortic root were identified. One population of arterial ECs, characterized by Klk10 expression, appears to give rise to a disease-specific population of ECs expressing Edn1 . These ECs appear to acquire a mesenchymal phenotype, consistent with the occurrence of EndMT. RNAscope staining revealed Edn1 + cells at the luminal surface and deep within the plaque. Upstream regulator analysis comparing Edn1+ and Klk10+ populations predict EndMT-associated transcription regulators including SNAI2, TWIST2, STAT3, SNAI1 and YAP1 as drivers of this transition. This analysis predicts upregulation of both fibrotic and cytokine signaling. In contrast to a previous study of carotid ECs under disturbed flow that identified multiple disparate EC phenotypes, we observed a single disease-associated EC transition. Conclusion: We identified ECs undergoing EndMT in a HFD-induced mouse model of atherosclerosis and determined that this transition is the primary EC cell state transition during disease. Thus, the process of EndMT may account for both mesenchymal and inflammatory changes observed in ECs during atherosclerosis.

Abstract 18799: Edn1 is a Disease-Specific Marker in Endothelial Cells During Atherosclerosis

Introduction: Coronary artery disease (CAD) is the leading cause of mortality in the United States. Multiple cell types contribute to the atherosclerotic plaques causing CAD, influencing its growth and propensity to rupture. Endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells (ECs) acquire characteristics of mesenchymal cells and loose canonical endothelial features, is correlated with an unstable plaque phenotype, although this transition remains poorly characterized. Hypothesis: A specific population of ECs may undergo EndMT, potentially contributing to the progression of atherosclerosis. Methods: We induced endothelial-specific lineage-tracing in Tg Cdh5-CreERT2 , ApoE -/- mice, followed by 16 weeks of high-fat diet (HFD) to produce atherosclerosis. Aortic roots were collected from male and female disease-condition animals (n=13) and a baseline cohort that was not fed HFD (n=6). Single cell RNA sequencing was performed and analyzed using the Seurat package. RNAscope staining of transverse sections (n=10 per genotype per sex) was performed to characterize the lesion. Results: We identified multiple endothelial subtypes within the aortic root. One population, characterized by the marker Klk10, appears to give rise to a disease-specific population characterized by expression of Edn1. Upstream regulator analysis of differentially expressed genes between Edn1+ and Klk10+ populations predict this EC transition to be driven by EndMT-associated transcription regulators including SNAI2, TWIST2, STAT3, SNAI1, YAP1, and TEAD2. This analysis also predicted upregulation of pulmonary fibrosis idiopathic signaling, wound healing signaling, pathogen induced cytokine storm signaling, and hepatic fibrosis signaling pathways. This EndMT process, characterized by a single, linear trajectory, was the only disease-associated EC transition state identified in the atherosclerotic aortic root. Conclusions: We identified ECs undergoing EndMT in a mouse model of atherosclerosis and determined that this transition is the primary EC cell state transition during disease. We localized this process in situ in the atherosclerotic lesion and identified potential key transcriptional drivers of this process.

Granzyme B+ CD4+ T cells associate with an unstable plaque phenotype in advanced human atherosclerosis

Background and Aims : Cytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease1, however their occurrence in atherosclerotic plaques and their association with the pathophysiology of atherosclerosis has not been established.

Cardiovascular changes after pneumonia in a dual disease mouse model

Residual inflammation in cardiovascular organs is thought to be one of the catalysts for the increased risk of cardiovascular complications seen following pneumonia. To test this hypothesis, we investigated changes in plaque characteristics and inflammatory features in ApoE−/− mouse aorta and heart following pneumonia. Male ApoE−/− mice were fed a high fat diet for 8 weeks before intranasal inoculation with either Streptococcus pneumoniae serotype 4 (test group) or phosphate buffered saline (control group). Mice were sacrificed at 2-, 7- and 28-days post-challenge. Changes in plaque burden and characteristics in aortic root and thoracic aorta were characterized by Oil red O and Trichrome stains. Inflammatory changes were investigated by FDG-PET imaging and immunofluorescence staining. We found TIGR4-infected mice present with increased plaque presence in the aortic root and thoracic aorta at 2- and 28-days post-inoculation, respectively. Aortic wall remodelling was also more pronounced in mice challenged with pneumococci at 28 days post-inoculation. Aortic root plaques of infected mice had reduced collagen and smooth muscle cells, consistent with an unstable plaque phenotype. Pneumonia alters plaque burden, plaque characteristics, and aortic wall remodelling in ApoE−/− mice. These effects caused by Streptococcus pneumoniae TIGR4, may contribute to the increased risk of cardiovascular complications seen in survivors of this infection.

Interfering in the ALK1 Pathway Results in Macrophage-Driven Outward Remodeling of Murine Vein Grafts.

AimsVein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-β via TGFβR2 or BMP9/10 via BMPR2, which is an important pathway in fibrotic, inflammatory, and angiogenic processes in vascular diseases. The role of the TGF-β pathway in VGD is previously reported, however, the contribution of ALK1 signaling is not known. Therefore, we investigated ALK1 signaling in VGD in a mouse model for vein graft disease using either genetic or pharmacological inhibition of the Alk1 signaling.Methods and ResultsMale ALK1 heterozygous (ALK1+/−), control C57BL/6, as well as hypercholesterolemic ApoE3*Leiden mice, underwent vein graft surgery. Histologic analyses of ALK1+/− vein grafts demonstrated increased outward remodeling and macrophage accumulation after 28 days. In hypercholesterolemic ApoE3*Leiden mice receiving weekly ALK1-Fc injections, ultrasound imaging showed 3-fold increased outward remodeling compared to controls treated with control-Fc, which was confirmed histologically. Moreover, ALK1-Fc treatment reduced collagen and smooth muscle cell accumulation, increased macrophages by 1.5-fold, and resulted in more plaque dissections. No difference was observed in intraplaque neovessel density. Flow cytometric analysis showed increased systemic levels of Ly6CHigh monocytes in ALK1-Fc treated mice, supported by in vitro increased MCP-1 and IL-6 production of LPS-stimulated and ALK1-Fc-treated murine monocytes and macrophages.ConclusionReduced ALK1 signaling in VGD promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype.Translational PerspectiveVein graft disease (VGD) severely hampers patency rates of vein grafts, necessitating research of key disease-driving pathways like TGF-β. The three-dimensional nature of VGD together with the multitude of disease driving factors ask for a comprehensive approach. Here, we combined in vivo ultrasound imaging, histological analyses, and conventional in vitro analyses, identifying the ambiguous role of reduced ALK1 signaling in vein graft disease. Reduced ALK1 signaling promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype in murine vein grafts. Characterization of in vivo vascular remodeling over time is imperative to monitor VGD development and identify new therapies.

Smooth muscle cells affect differential nanoparticle accumulation in disturbed blood flow-induced murine atherosclerosis.

Atherosclerosis is a lipid-driven chronic inflammatory disease that leads to the formation of plaques in the inner lining of arteries. Plaques form over a range of phenotypes, the most severe of which is vulnerable to rupture and causes most of the clinically significant events. In this study, we evaluated the efficacy of nanoparticles (NPs) to differentiate between two plaque phenotypes based on accumulation kinetics in a mouse model of atherosclerosis. This model uses a perivascular cuff to induce two regions of disturbed wall shear stress (WSS) on the inner lining of the instrumented artery, low (upstream) and multidirectional (downstream), which, in turn, cause the development of an unstable and stable plaque phenotype, respectively. To evaluate the influence of each WSS condition, in addition to the final plaque phenotype, in determining NP uptake, mice were injected with NPs at intermediate and fully developed stages of plaque growth. The kinetics of artery wall uptake were assessed in vivo using dynamic contrast-enhanced magnetic resonance imaging. At the intermediate stage, there was no difference in NP uptake between the two WSS conditions, although both were different from the control arteries. At the fully-developed stage, however, NP uptake was reduced in plaques induced by low WSS, but not multidirectional WSS. Histological evaluation of plaques induced by low WSS revealed a significant inverse correlation between the presence of smooth muscle cells and NP accumulation, particularly at the plaque-lumen interface, which did not exist with other constituents (lipid and collagen) and was not present in plaques induced by multidirectional WSS. These findings demonstrate that NP accumulation can be used to differentiate between unstable and stable murine atherosclerosis, but accumulation kinetics are not directly influenced by the WSS condition. This tool could be used as a diagnostic to evaluate the efficacy of experimental therapeutics for atherosclerosis.

Clinical Implications of IL-32, IL-34 and IL-37 in Atherosclerosis: Speculative Role in Cardiovascular Manifestations of COVID-19.

Atherosclerosis, which is a primary cause of cardiovascular disease (CVD) deaths around the world, is a chronic inflammatory disease that is characterised by the accumulation of lipid plaques in the arterial wall, triggering inflammation that is regulated by cytokines/chemokines that mediate innate and adaptive immunity. This review focuses on IL-32, -34 and -37 in the stable vs. unstable plaques from atherosclerotic patients. Dysregulation of the novel cytokines IL-32, -34 and -37 has been discovered in atherosclerotic plaques. IL-32 and -34 are pro-atherogenic and associated with an unstable plaque phenotype; whereas IL-37 is anti-atherogenic and maintains plaque stability. It is speculated that these cytokines may contribute to the explanation for the increased occurrence of atherosclerotic plaque rupture seen in patients with COVID-19 infection. Understanding the roles of these cytokines in atherogenesis may provide future therapeutic perspectives, both in the management of unstable plaque and acute coronary syndrome, and may contribute to our understanding of the COVID-19 cytokine storm.

Regularities of plaque stabilization in various scenarios of neointimal calcification and vascularization

Aim. To study the relationships between phenotypes of extracranial arteries' plaques (stable/unstable), their calcification and its causes, in particular, vascularization.Material and methods. The study included 88 patients: patients (n=44) with ischemic stroke and those (n=44) with chronic brain ischemia. In all subjects, the parameters of systemic mineral homeostasis were assessed (total and ionized calcium, phosphate, total protein, albumin, and calcification propensity). Atherosclerotic plaques have been obtained during carotid endarterectomy, fixed in formalin, postfixed in 1% osmium tetroxide, stained in 2% osmium tetroxide, dehydrated in ascending ethanol series and acetone, stained with 2% alcoholic uranyl acetate and embedded into epoxy resin with its further polymerization. Epoxy resin blocks were grinded, polished, counterstained with Reynolds' lead citrate and sputter coated with carbon. Sample visualization was performed employing backscattered scanning electron microscopy. Number and area of calcium deposits and neointimal vessels were quantified using ImageJ. Statistical analysis was carried out using Mann-Whitney U-test and Spearman's rank correlation coefficient.Results. It was found that area of neointimal calcification, but not number of calcium deposits, was associated with the stable plaque phenotype. The stabilizing effect of calcification was manifested in retarding stenosis associated with plaque rupture and stroke. Calcification extent directly correlated with total and local plaque vascularization, which have been associated with unstable and stable plaque phenotype, respectively. In addition, plaque calcification negatively correlated with total protein and albumin, thereby reflecting the impaired systemic mineral homeostasis.Conclusion. Atherosclerotic plaque calcification and active local vascularization reduce stenosis extent and stabilize plaque. In contrast, total plaque calcification contributes to the atherosclerosis progression and promotes major acute cardiovascular events.

Mitochondrial quality control is altered in ATG7-deficient vascular smooth muscle cells and linked to unstable atherosclerotic plaque phenotype

Mitochondrial quality control is altered in ATG7-deficient vascular smooth muscle cells and linked to unstable atherosclerotic plaque phenotype

CD163 deficiency increases foam cell formation and plaque progression in atherosclerotic mice.

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/- /CD163-/- compared with ApoE-/- /CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression.

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) drives atherosclerosis in mice and is associated with an unstable plaque phenotype and cerebrovascular events in humans.

GITR-a co-stimulatory immune checkpoint protein-is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr-/-Apoe-/- mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr-/-Apoe-/- and Apoe-/- monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr-/-Apoe-/- monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity. Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.

Nicotine in Senescence and Atherosclerosis.

Cigarette smoke is a known exacerbator of age-related pathologies, such as cardiovascular disease (CVD), atherosclerosis, and cellular aging (senescence). However, the role of nicotine and its major metabolite cotinine is yet to be elucidated. Considering the growing amount of nicotine-containing aerosol use in recent years, the role of nicotine is a relevant public health concern. A number of recent studies and health education sites have focused on nicotine aerosol-induced adverse lung function, and neglected cardiovascular (CV) impairments and diseases. A critical review of the present scientific literature leads to the hypothesis that nicotine mediates the effects of cigarette smoke in the CV system by increasing MAPK signaling, inflammation, and oxidative stress through NADPH oxidase 1 (Nox1), to induce vascular smooth muscle cell (VSMC) senescence. The accumulation of senescent VSMCs in the lesion cap is detrimental as it increases the pathogenesis of atherosclerosis by promoting an unstable plaque phenotype. Therefore, nicotine, and most likely its metabolite cotinine, adversely influence atherosclerosis.

Altered mitochondrial quality control in autophagy-deficient vascular smooth muscle cells is linked to high-risk atherosclerotic plaque phenotype

Atherosclerosis is one of the most common causes of stroke worldwide. Intracranial or extracranial atherosclerosis may occur concomitantly with systemic atherosclerosis involving other arterial beds, such as coronary, or peripheral arteries. A major mechanism of stroke, involves unstable plaque as described in the coronary model. Vascular smooth muscle cells (VSMCs) regulate various aspects of vessel homeostasis, including contraction, dilation, and vessel remodeling. Consequently, VSMCs are one of the main cellular determinants in arterial pathology and several evidence indicates that apoptosis of VSMCs is associated with features of high-risk/vulnerable atherosclerotic lesions. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using a mouse model of atherosclerosis carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we observed that impaired VSMC autophagy promotes an unstable plaque phenotype as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate the apoptosis of VSMCs and plaque vulnerability. Overall, our results indicate that mitochondrial quality control could be a promising therapeutic target to stabilize atherosclerotic plaques and to decrease the risk of stroke.

Plaque Calcification: Do Lipoproteins Have a Role?

Vascular calcification (VC) is strongly associated with all-cause mortality and is an independent predictor of cardiovascular events. Resulting from its complex, multifaceted nature, targeted treatments for VC have not yet been developed. Lipoproteins are well characterized in the pathogenesis of atherosclerotic plaques, leading to the development of plaque regressing therapeutics. Although their roles in plaque progression are well documented, their roles in VC, and calcification of a plaque, are not well understood. In this review, early in vitro data and clinical correlations suggest an inhibitory role for HDL (high-density lipoproteins) in VC, a stimulatory role for LDL (low-density lipoprotein) and VLDL (very low-density lipoprotein) and a potentially causal role for Lp(a) (lipoprotein [a]). Additionally, after treatment with a statin or PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitor, plaque calcification is observed to increase. With the notion that differing morphologies of plaque calcification associate with either a more stable or unstable plaque phenotype, uncovering the mechanisms of lipoprotein-artery wall interactions could produce targeted therapeutic options for VC.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE−/−) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

Abstract 599: Exposure to Biodiesel Exhaust Triggers Atherosclerotic Plaque Destabilisation through Increased Apoptosis of Vascular Smooth Muscle Cells in the Arterial Vessel Wall

Introduction: Long-term exposure to air pollutants increases cardiovascular morbidity and mortality. Particulate matter (PM) derived from diesel exhaust has been documented to be pro-atherogenic in animal studies. Biodiesels are widely introduced as new fuels with improved emission characteristics. However, biodiesel contains relatively more polycyclic aromatic carbohydrates (PAH) compared to diesel. Therefore, we hypothesise that exposure to biodiesel as compared to diesel exhaust results in increased atherosclerosis. Methods and Results: In a carotid cuff atherosclerosis model, the effects of exposure to exhaust PM of biodiesel vs. diesel PM vs. saline (control) on atherosclerosis were evaluated. Both common carotid arteries in LDLR -/- mice were cuffed at week 2 in the course of an 8-week high-fat diet regimen. All mice were intratracheally instilled with saline, PM biodiesel or biodiesel once-weekly for 5 times. Immunohistochemistry and primary human vascular smooth muscle cells (hVSMC) were used for evaluation. Results: Exposure to both biodiesel and diesel didn’t exacerbate atherosclerosis development, however, biodiesel affected plaque composition towards a more vulnerable phenotype as compared to diesel with decreased total collagen content and hVSMC (respectively -100%, p=0.09 and -339%, p<0.01 and tend to increase necrotic core volumes (37.5% vs 28.9 % p=0.2). Exposure to biodiesel PM triggered loss of hVSMC in tunica-media, strongly correlating with apoptosis in the vessel wall (Pearson r=0.7, p<0.01). These findings were supported by dose-dependent apoptosis of hVSMCs upon 2-hour PAH treatment. Conclusions: This study demonstrates that exposure to biodiesel exhaust PM dramatically modulates atherosclerotic plaque composition, resulting in an unstable plaque phenotype through enhanced pro-apoptotic mechanisms.

Noncanonical NF-κB signaling in microvessels of atherosclerotic lesions is associated with inflammation, atheromatous plaque morphology and myocardial infarction

Background and aimsNeovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. MethodsUsing cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. ResultsNIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (p < .05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (p < .05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (p < .05). NIK+EC were increased in lesions with lipid content >40% (p < .05) and more abundant in coronary artery lesions implicated in MI (p < .05). These vessels also associated with atheromatous rather than fibrous plaque morphology (p < .05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (p < .05). ConclusionsNIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.

170 Vasa Vasorum Activities in Human Carotid Atherosclerosis is Associated with Plaque Development and Vulnerability

Abstract INTRODUCTION Carotid endarterectomy (CEA) has been shown to be beneficial in patients with high-grade symptomatic carotid artery stenosis. Subintimal and intraplaque hemorrhages are frequently seen during CEA in the absence of any visible breach in the intima, as these changes are derived from the vasa vasorum system rather than from blood in the vessel lumen. Imaging modalities to characterize unstable, vulnerable plaques are strongly needed for better risk stratification in these patients. The aim of this study was to investigate the correlation between the activities of the carotid vasa vasorum and carotid plaque vulnerability using indocyanine green-video angiography (ICG-VA) during CEA, focusing on how the carotid artery vasa vasorum is depicted. METHODS Sixty-nine patients (mean age, 68.5 ± 2.5 years; mean degree of stenosis, 78.9 ± 3.8) who underwent CEA were enrolled prospectively from September 2013 to December 2014. ICG was injected intravenously as a bolus before and after resecting the atheroma during CEA. We also performed immunohistochemistry using CD68 (macrophage), CD117 (mast cell), CD4 (T-cell), and CD8 (T-cell) antibodies for resected plaque specimens. RESULTS &gt;Active vasa vasorum density was observed in all patients on ICG-VA (n = 69). The vasa vasorum externa (VVE) and interna (VVI) were seen in 11 (16%) and 57 patients (82.6%), respectively. The types of VVE were strongly associated with preoperative angiographic instability (90.0%, P = 0.005) and carotid plaque vulnerability (100%, P = 0.007) macroscopically. In contrast, the types of VVI were less associated with angiographic instability (36.1%) and plaque vulnerability (49.1%, P = 0.003). CD86- and CD117-stained macrophages and mast cells were observed more frequently in unstable plaque, compared to those in stable plaque (P &lt; 0.0001, P = 0.002, respectively). CONCLUSION The early appearance of VVE on ICG-VA was strongly associated with unstable carotid plaque and many microvessel channels that provided nutrients to the developing and expanding intima and potentially created an unstable hemorrhagic environment prone to rupture. Macrophages and mast cells were involved in the formation of microvessels in the atherogenic plaque and accelerated plaque progression into an unstable plaque phenotype.

Defective autophagy in vascular smooth muscle cells promotes an unstable atherosclerotic plaque phenotype and increased expression of mitophagy markers in Apo E-/- mice

Defective autophagy in vascular smooth muscle cells promotes an unstable atherosclerotic plaque phenotype and increased expression of mitophagy markers in Apo E-/- mice

P677Defective autophagy in vascular smooth muscle cells promotes an unstable atherosclerotic plaque phenotype and increased expression of mitophagy markers in Apo E−/− mice

P677Defective autophagy in vascular smooth muscle cells promotes an unstable atherosclerotic plaque phenotype and increased expression of mitophagy markers in Apo E−/− mice