CD8+ T Cells Drive Plaque Smooth Muscle Cell Dedifferentiation in Experimental Atherosclerosis.

BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of an osteoblast-like, procalcifying cell phenotype. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.

Animal Models of Atherosclerosis-Supportive Notes and Tricks of the Trade.

Atherosclerotic cardiovascular disease is a major cause of death among humans. Animal models have shown that cholesterol and inflammation are causatively involved in the disease process. Apolipoprotein B-containing lipoproteins elicit immune reactions and instigate inflammation in the vessel wall. Still, a treatment that is specific to vascular inflammation is lacking, which motivates continued in vivo investigations of the immune-vascular interactions that drive the disease. In this review, we distill old notions with emerging concepts into a contemporary understanding of vascular disease models. Pros and cons of different models are listed and the complex integrative interplay between cholesterol homeostasis, immune activation, and adaptations of the vascular system is discussed. Key limitations with atherosclerosis models are highlighted, and we suggest improvements that could accelerate progress in the field. However, excessively rigid experimental guidelines or limiting usage to certain animal models can be counterproductive. Continued work in improved models, as well as the development of new models, should be of great value in research and could aid the development of cardiovascular disease diagnostics and therapeutics of the future.

Platelets enhance CD4+ central memory T cell responses via platelet factor 4-dependent mitochondrial biogenesis and cell proliferation.

Platelets regulate multiple aspects of CD4+ T cell immunity, and may exert distinct regulations among different T cell subsets. Our aim was to investigate how platelets regulate CD4+ central memory T cell (Tcm) responses. αCD3/αCD28-stimulated human CD4+ Tcm cells were cultured without or with platelets or platelet-derived mediators. Polyclonal stimulation induced cell proliferation and Th1 and Treg cell activation of Tcm cells. Platelet factor 4/PF4 neutralization abolished platelet-enhanced Tcm effector responses, whilst TGFß neutralization only partially inhibited platelet-enhanced Treg cell activation. PF4 supplementation mimicked the effects of platelet co-cultures, while PF4 receptor CXCR3 blockade and CXCR3 knockdown with siRNAs inhibited or abolished PF4-enhanced Th1 and Treg cell responses. Platelet co-cultures or PF4-treatment increased Tcm cell proliferation, whilst CXCR3 blockade counteracted. PF4-enhanced Tcm proliferation and effector cell responses were associated with mitochondrial biogenesis. Overexpression of mitochondrial transcription factor A (TFAM) mimicked PF4 effects, and PF4 treatment attenuated Akt phosphorylation of activated Tcm cells, leading to mitochondrial biogenesis. Impacts of platelets and PF4 on Tcm proliferation were further confirmed by that CXCR3 knockdown/blockade counteracted PF4-enhanced Tcm cell proliferation. In conclusion, platelets enhance Th1 and Treg cell responses of CD4+ Tcm cells, via PF4-dependent mitochondrial biogenesis and cell proliferation of Tcm cells.

Genetic Deficiency of Indoleamine 2,3-dioxygenase Aggravates Vascular but Not Liver Disease in a Nonalcoholic Steatohepatitis and Atherosclerosis Comorbidity Model.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe-/-and Apoe-/-Ido1-/- mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe-/-Ido1-/- mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe-/-Ido1-/- mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe-/- and Apoe-/-Ido1-/mice-. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues.

Prevention of radiotherapy-induced arterial inflammation by interleukin-1 blockade.

AIMS: Radiotherapy-induced cardiovascular disease is an emerging problem in a growing population of cancer survivors where traditional treatments, such as anti-platelet and lipid-lowering drugs, have limited benefits. The aim of the study was to investigate vascular inflammatory patterns in human cancer survivors, replicate the findings in an animal model, and evaluate whether interleukin-1 (IL-1) inhibition could be a potential treatment. METHODS AND RESULTS: Irradiated human arterial biopsies were collected during microvascular autologous free tissue transfer for cancer reconstruction and compared with non-irradiated arteries from the same patient. A mouse model was used to study the effects of the IL-1 receptor antagonist, anakinra, on localized radiation-induced vascular inflammation. We observed significant induction of genes associated with inflammasome biology in whole transcriptome analysis of irradiated arteries, a finding supported by elevated protein levels in irradiated arteries of both, pro-caspase and caspase-1. mRNA levels of inflammasome associated chemokines CCL2, CCL5 together with the adhesion molecule VCAM1, were elevated in human irradiated arteries as was the number of infiltrating macrophages. A similar pattern was reproduced in Apoe-/- mouse 10 weeks after localized chest irradiation with 14 Gy. Treatment with anakinra in irradiated mice significantly reduced Ccl2 and Ccl5 mRNA levels and expression of I-Ab. CONCLUSION: Anakinra, administered directly after radiation exposure for 2 weeks, ameliorated radiation induced sustained expression of inflammatory mediators in mice. Further studies are needed to evaluate IL-1 blockade as a treatment of radiotherapy-induced vascular disease in a clinical setting.

Low-Density Lipoprotein-Reactive T Cells Regulate Plasma Cholesterol Levels and Development of Atherosclerosis in Humanized Hypercholesterolemic Mice.

BACKGROUND: Atherosclerotic cardiovascular disease is a chronic inflammatory process initiated when cholesterol-carrying low-density lipoprotein (LDL) is retained in the arterial wall. CD4+ T cells, some of which recognize peptide components of LDL as antigen, are recruited to the forming lesion, resulting in T-cell activation. Although these T cells are thought to be proatherogenic, LDL immunization reduces disease in experimental animals. These seemingly contradictory findings have hampered the development of immune-based cardiovascular therapy. The present study was designed to clarify how activation of LDL-reactive T cells impacts on metabolism and vascular pathobiology. METHODS: We have developed a T-cell receptor-transgenic mouse model to characterize the effects of immune reactions against LDL. Through adoptive cell transfers and cross-breeding to hypercholesterolemic mice expressing the antigenic human LDL protein apolipoprotein B-100, we evaluate the effects on atherosclerosis. RESULTS: A subpopulation of LDL-reactive T cells survived clonal selection in the thymus, developed into T follicular helper cells in lymphoid tissues on antigen recognition, and promoted B-cell activation. This led to production of anti-LDL immunoglobulin G antibodies that enhanced LDL clearance through immune complex formation. Furthermore, the cellular immune response to LDL was associated with increased cholesterol excretion in feces and with reduced vascular inflammation. CONCLUSIONS: These data show that anti-LDL immunoreactivity evokes 3 atheroprotective mechanisms: antibody-dependent LDL clearance, increased cholesterol excretion, and reduced vascular inflammation.

ERV1/ChemR23 Signaling Protects Against Atherosclerosis by Modifying Oxidized Low-Density Lipoprotein Uptake and Phagocytosis in Macrophages.

BACKGROUND: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein-coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. METHODS: Lipidomic plasma analysis were performed after EPA supplementation in Apoe-/- mice. Erv1/Chemr23-/- xApoe-/- mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. RESULTS: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe-/- mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1-mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. CONCLUSIONS: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Hypercholesterolemia Induces Differentiation of Regulatory T Cells in the Liver.

RATIONALE: The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. OBJECTIVE: To investigate hepatic T-cell subsets upon hypercholesterolemia. METHODS AND RESULTS: We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-ß1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr-/-) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr-/- mice led to intrahepatic Th1 cell differentiation and CD11b+CD11c+ leukocyte accumulation. CONCLUSIONS: Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4+ T-cell pool in the atherosclerotic aorta.

Acute Loss of Apolipoprotein E Triggers an Autoimmune Response That Accelerates Atherosclerosis.

Objective- Dyslipidemia is a component of the metabolic syndrome, an established risk factor for atherosclerotic cardiovascular disease, and is also observed in various autoimmune and chronic inflammatory conditions. However, there are limited opportunities to study the impact of acquired dyslipidemia on cardiovascular and immune pathology. Approach and Results- We designed a model system that allows for the conversion to a state of acute hyperlipidemia in adult life, so that the consequences of such a transition could be observed, through conditionally deleting APOE (apolipoprotein E) in the adult mouse. The transition to hypercholesterolemia was accompanied by adaptive immune responses, including the expansion of T lymphocyte helper cell 1, T follicular helper cell, and T regulatory subsets and the formation of germinal centers. Unlike steady-state Apoe-/- mice, abrupt loss of APOE induced rapid production of antibodies recognizing rheumatoid disease autoantigens. Genetic ablation of the germinal center reduced both autoimmunity and atherosclerosis, indicating that the immune response that follows loss of APOE is independent of atherosclerosis but nevertheless promotes plaque development. Conclusions- Our findings suggest that immune activation in response to hyperlipidemia could contribute to a wide range of inflammatory autoimmune diseases, including atherosclerosis.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Lipid-driven immunometabolic responses in atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease in which subendothelial infiltration of lipoproteins leads to inflamed lesions in arteries. Despite improvements in secondary prevention, most cardiovascular events cannot be avoided with current therapies. This review focuses on novel mechanistic insights on lipid-driven immune activation, which could pave the way for new anti-inflammatory treatments for atherosclerosis. RECENT FINDINGS: Immunometabolic interactions can shape the immune response. Within atherosclerotic plaques, macrophages and T cells are the dominant immune cell populations. Using multiple mechanisms, lipoprotein-derived components activate both the innate and adaptive immune systems. Cholesterol crystals and apolipoprotein B-peptides have been shown to activate macrophages and T cells, respectively. Lipoproteins are also important modulators of regulatory T cells that can hamper vascular inflammation. In the liver, T cells can influence hepatic inflammation and lipoprotein metabolism. Hence, there is an intricate crosstalk between the immune system and lipoprotein metabolism. SUMMARY: Novel treatments are needed to prevent clinical manifestations of atherosclerosis. Improved understanding of lipid-driven immunometabolic responses is likely to reveal new therapeutic targets.

Increased Carotid Artery Lesion Inflammation Upon Treatment With the CD137 Agonistic Antibody 2A.

BACKGROUND: Increased inflammatory activity destabilizes the atherosclerotic lesion and may lead to atherothrombosis and symptomatic cardiovascular disease. Co-stimulatory molecules, such as CD137, are key regulators of inflammation, and CD137 activity regulates inflammation in experimental atherosclerosis. Here, we hypothesized that CD137 activation promotes carotid artery inflammation and atherothrombosis.Methods and Results:In a model of inducible atherothrombosis with surgical ligation of the right carotid artery and a subsequent placement of a polyethene cuff, elevated levels of CD137 and CD137 ligand mRNA in atherothrombotic vs. non-atherothrombotic murine carotid lesions was observed. Mice treated with the CD137 agonistic antibody 2A showed signs of increased inflammation in the aorta and a higher proportion of CD8+T cells in spleen and blood. In carotid lesions of 2A-treated mice, significantly higher counts of CD8+and major histocompatibility (MHC)-class II molecule I-Ab+cells were observed. Treatment with the CD137 agonistic antibody 2A did not significantly affect the atherothrombosis frequency in 16-week-old mice in this model. CONCLUSIONS: Levels of CD137 and CD137 ligand mRNA were higher in advanced atherosclerotic disease compared to control vessels, and treatment with the CD137 agonistic antibody 2A, in a murine model for inducible atherothrombosis promoted vascular inflammation, but had no significant effect on atherothrombosis frequency at this early disease stage.

Dysregulation of micro-RNA 143-3p as a Biomarker of Carotid Atherosclerosis and the Associated Immune Reactions During Disease Progression.

Atherosclerosis commonly remains undiagnosed until disease manifestations occur. The disease is associated with dysregulated micro(mi)RNAs, but how this is linked to atherosclerosis-related immune reactions is largely unknown. A mouse model of carotid atherosclerosis, human APOB100-transgenic Ldlr-/- (HuBL), was used to study the spatiotemporal dysregulation of a set of miRNAs. Middle-aged HuBL mice with established atherosclerosis had decreased levels of miR-143-3p in their carotid arteries. In young HuBL mice, early atherosclerosis was observed in the carotid bifurcation, which had lower levels of miR-15a-5p, miR-143-3p, and miR-199a-3p, and higher levels of miR-155-5p. The dysregulation of these miRNAs was reflected by specific immune responses during atheroprogression. Finally, levels of miR-143-3p were 70.6% lower in extracellular vesicles isolated from the plasma of patients with carotid stenosis compared to healthy controls. Since miR-143-3p levels progressively decrease when transitioning between early and late experimental carotid atherosclerosis, we propose it as a biomarker for atherosclerosis.

Transforming growth factor-ß2 is associated with atherosclerotic plaque stability and lower risk for cardiovascular events.

AIMS: Transforming growth factor-beta (TGF-ß) exists in three isoforms TGF-ß1, -ß2, and -ß3. TGF-ß1 has been suggested to be important for maintaining plaque stability, yet the role of TGF-ß2 and -ß3 in atherosclerosis remains to be investigated.This study explores the association of the three isoforms of TGF-ß with plaque stability in the human atherosclerotic disease. METHODS AND RESULTS: TGF-ß1, -ß2, and -ß3 proteins were quantified in 223 human carotid plaques by immunoassays. Indications for the endarterectomy were: symptomatic carotid plaque with stenosis >70% or without symptoms and >80% stenosis. Plaque mRNA levels were assessed by RNA sequencing. Plaque components and extracellular matrix were measured histologically and biochemically. Matrix metalloproteinases and monocyte chemoattractant protein-1 (MCP-1) was measured with immunoassays. The effect of TGF-ß2 on inflammation and protease activity was investigated in vitro using THP-1 and RAW264.7 macrophages. Patients were followed longitudinally for cardiovascular (CV) events.TGF-ß2 was the most abundant isoform and was increased at both protein and mRNA levels in asymptomatic plaques. TGF-ß2 was the main determinant separating asymptomatic plaques in an Orthogonal Projections to Latent Structures Discriminant Analysis. TGF-ß2 correlated positively to features of plaque stability and inversely to markers of plaque vulnerability. TGF-ß2 was the only isoform inversely correlated to the matrix-degrading matrix metalloproteinase-9 and inflammation in the plaque tissue. In vitro, TGF-ß2 pre-treatment reduced MCP-1 gene and protein levels as well as matrix metalloproteinase-9 gene levels and activity. Patients with plaques with high TGF-ß2 levels had a lower risk to suffer from future CV events. CONCLUSIONS: TGF-ß2 is the most abundant TGF-ß isoform in human plaques and may maintain plaque stability by decreasing inflammation and matrix degradation.

Pyruvate dehydrogenase kinase regulates vascular inflammation in atherosclerosis and increases cardiovascular risk.

AIMS: Recent studies have revealed a close connection between cellular metabolism and the chronic inflammatory process of atherosclerosis. While the link between systemic metabolism and atherosclerosis is well established, the implications of altered metabolism in the artery wall are less understood. Pyruvate dehydrogenase kinase (PDK)-dependent inhibition of pyruvate dehydrogenase (PDH) has been identified as a major metabolic step regulating inflammation. Whether the PDK/PDH axis plays a role in vascular inflammation and atherosclerotic cardiovascular disease remains unclear. METHODS AND RESULTS: Gene profiling of human atherosclerotic plaques revealed a strong correlation between PDK1 and PDK4 transcript levels and the expression of pro-inflammatory and destabilizing genes. Remarkably, the PDK1 and PDK4 expression correlated with a more vulnerable plaque phenotype, and PDK1 expression was found to predict future major adverse cardiovascular events. Using the small-molecule PDK inhibitor dichloroacetate (DCA) that restores arterial PDH activity, we demonstrated that the PDK/PDH axis is a major immunometabolic pathway, regulating immune cell polarization, plaque development, and fibrous cap formation in Apoe-/- mice. Surprisingly, we discovered that DCA regulates succinate release and mitigates its GPR91-dependent signals promoting NLRP3 inflammasome activation and IL-1ß secretion by macrophages in the plaque. CONCLUSIONS: We have demonstrated for the first time that the PDK/PDH axis is associated with vascular inflammation in humans and particularly that the PDK1 isozyme is associated with more severe disease and could predict secondary cardiovascular events. Moreover, we demonstrate that targeting the PDK/PDH axis with DCA skews the immune system, inhibits vascular inflammation and atherogenesis, and promotes plaque stability features in Apoe-/- mice. These results point toward a promising treatment to combat atherosclerosis.

The Spectrum of B Cell Functions in Atherosclerotic Cardiovascular Disease.

B cells are a core element of the pathophysiology of atherosclerotic cardiovascular disease (ASCVD). Multiple experimental and epidemiological studies have revealed both protective and deleterious functions of B cells in atherosclerotic plaque formation. The spearhead property of B cells that influences the development of atherosclerosis is their unique ability to produce and secrete high amounts of antigen-specific antibodies that can act at distant sites. Exposure to an atherogenic milieu impacts B cell homeostasis, cell differentiation and antibody production. However, it is not clear whether B cell responses in atherosclerosis are instructed by atherosclerosis-specific antigens (ASA). Dissecting the full spectrum of the B cell properties in atherosclerosis will pave the way for designing innovative therapies against the devastating consequences of ASCVD.

Inhibition of IL17A Using an Affibody Molecule Attenuates Inflammation in ApoE-Deficient Mice.

The balance between pro- and anti-inflammatory cytokines released by immune and non-immune cells plays a decisive role in the progression of atherosclerosis. Interleukin (IL)-17A has been shown to accelerate atherosclerosis. In this study, we investigated the effect on pro-inflammatory mediators and atherosclerosis development of an Affibody molecule that targets IL17A. Affibody molecule neutralizing IL17A, or sham were administered in vitro to human aortic smooth muscle cells (HAoSMCs) and murine NIH/3T3 fibroblasts and in vivo to atherosclerosis-prone, hyperlipidaemic ApoE-/- mice. Levels of mediators of inflammation and development of atherosclerosis were compared between treatments. Exposure of human smooth muscle cells and murine NIH/3T3 fibroblasts in vitro to αIL-17A Affibody molecule markedly reduced IL6 and CXCL1 release in supernatants compared with sham exposure. Treatment of ApoE-/- mice with αIL-17A Affibody molecule significantly reduced plasma protein levels of CXCL1, CCL2, CCL3, HGF, PDGFB, MAP2K6, QDPR, and splenocyte mRNA levels of Ccxl1, Il6, and Ccl20 compared with sham exposure. There was no significant difference in atherosclerosis burden between the groups. In conclusion, administration of αIL17A Affibody molecule reduced levels of pro-inflammatory mediators and attenuated inflammation in ApoE-/- mice.

AMPA-Type Glutamate Receptors Associated With Vascular Smooth Muscle Cell Subpopulations in Atherosclerosis and Vascular Injury.

Objectives and Aims: Vascular smooth muscle cells (VSMCs) are key constituents of both normal arteries and atherosclerotic plaques. They have an ability to adapt to changes in the local environment by undergoing phenotypic modulation. An improved understanding of the mechanisms that regulate VSMC phenotypic changes may provide insights that suggest new therapeutic targets in treatment of cardiovascular disease (CVD). The amino-acid glutamate has been associated with CVD risk and VSMCs metabolism in experimental models, and glutamate receptors regulate VSMC biology and promote pulmonary vascular remodeling. However, glutamate-signaling in human atherosclerosis has not been explored. Methods and Results: We identified glutamate receptors and glutamate metabolism-related enzymes in VSMCs from human atherosclerotic lesions, as determined by single cell RNA sequencing and microarray analysis. Expression of the receptor subunits glutamate receptor, ionotropic, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)-type subunit 1 (GRIA1) and 2 (GRIA2) was restricted to cells of mesenchymal origin, primarily VSMCs, as confirmed by immunostaining. In a rat model of arterial injury and repair, changes of GRIA1 and GRIA2 mRNA level were most pronounced at time points associated with VSMC proliferation, migration, and phenotypic modulation. In vitro, human carotid artery SMCs expressed GRIA1, and selective AMPA-type receptor blocking inhibited expression of typical contractile markers and promoted pathways associated with VSMC phenotypic modulation. In our biobank of human carotid endarterectomies, low expression of AMPA-type receptor subunits was associated with higher content of inflammatory cells and a higher frequency of adverse clinical events such as stroke. Conclusion: AMPA-type glutamate receptors are expressed in VSMCs and are associated with phenotypic modulation. Patients suffering from adverse clinical events showed significantly lower mRNA level of GRIA1 and GRIA2 in their atherosclerotic lesions compared to asymptomatic patients. These results warrant further mapping of neurotransmitter signaling in the pathogenesis of human atherosclerosis.

Molecular Imaging of Inflammation in a Mouse Model of Atherosclerosis Using a Zirconium-89-Labeled Probe.

BACKGROUND: Beyond clinical atherosclerosis imaging of vessel stenosis and plaque morphology, early detection of inflamed atherosclerotic lesions by molecular imaging could improve risk assessment and clinical management in high-risk patients. To identify inflamed atherosclerotic lesions by molecular imaging in vivo, we studied the specificity of our radiotracer based on maleylated (Mal) human serum albumin (HSA), which targets key features of unstable atherosclerotic lesions. MATERIALS AND METHODS: Mal-HSA was radiolabeled with a positron-emitting metal ion, zirconium-89 (89Zr4+). The targeting potential of this probe was compared with unspecific 89Zr-HSA and 18F-FDG in an experimental model of atherosclerosis (Apoe-/- mice, n=22), and compared with wild-type (WT) mice (C57BL/6J, n=21) as controls. RESULTS: PET/MRI, gamma counter measurements, and autoradiography showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe-/- mice. The maximum standardized uptake values (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe-/- mice than in control WT mice, whereas no difference in SUVmax was observed for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta, as evaluated by a gamma counter 48 h postinjection, was 32% higher (P<0.01) for Apoe-/- mice than in WT mice, and the aorta-to-blood ratio was 8-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA-based probes were mainly distributed to the liver, spleen, kidneys, bone, and lymph nodes. The phosphor imaging autoradiography (PI-ARG) results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe-/- mice than in WT mice (9.4±1.4 vs 0.8±0.3%; P<0.001). CONCLUSION: 89Zr radiolabeling of Mal-HSA probes resulted in detectable activity in atherosclerotic lesions in aortas of Apoe-/- mice, as demonstrated by quantitative in vivo PET/MRI. 89Zr-Mal-HSA appears to be a promising diagnostic tool for the early identification of macrophage-rich areas of inflammation in atherosclerosis.