Mast cells participate in smooth muscle cell reprogramming and atherosclerotic plaque calcification.

BACKGROUND: Calcification, a key feature of advanced human atherosclerosis, is positively associated with vascular disease burden and adverse events. We showed that macrocalcification can be a stabilizing factor for carotid plaque molecular biology, due to inverse association with immune processes. Mast cells (MCs) are important contributors to plaque instability, but their relationship with macrocalcification is unexplored. With a hypothesis that MC activation negatively associates with carotid plaque macrocalcification, we aimed to investigate the link between MCs and carotid plaque vulnerability, and study MC role in plaque calcification via smooth muscle cells (SMCs). METHODS: Pre-operative computed tomography angiographies of patients (n = 40) undergoing surgery for carotid stenosis were used to characterize plaque morphology. Plaque microarrays (n = 40 and n = 126) were used for bioinformatic deconvolution of immune cell populations. Tissue microarrays (n = 103) were used to histologically validate the contribution of activated and resting MCs in plaques. RESULTS: Activated MCs and their typical markers were negatively correlated with macrocalcification. The ratio of activated vs. resting MCs was increased in low-calcified plaques from symptomatic patients. There was no modulating effect of medication on MC ratios. In vitro experiments showed that SMC calcification attenuated MC activation, while both active and resting MCs stimulated SMC calcification and induced dedifferentiation towards a pro-inflammatory-, osteochondrocyte-like phenotype, without modulating their migro-proliferative function. CONCLUSIONS: Integrative analyses from human plaques showed that MC activation is inversely associated with macrocalcification and positively with parameters of plaque vulnerability. Mechanistically, MCs induce SMC osteogenic reprograming, while matrix calcification in turn attenuates MC activation, offering new therapeutic avenues for exploration.

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.

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.

The resolvin D1 receptor GPR32 transduces inflammation resolution and atheroprotection.

Chronic inflammation is a hallmark of atherosclerosis and results from an imbalance between proinflammatory and proresolving signaling. The human GPR32 receptor, together with the ALX/FPR2 receptor, transduces biological actions of several proresolving mediators that stimulate resolution of inflammation. However, since no murine homologs of the human GPR32 receptor exist, comprehensive in vivo studies are lacking. Using human atherosclerotic lesions from carotid endarterectomies and creating a transgenic mouse model expressing human GPR32 on a Fpr2×ApoE double-KO background (hGPR32myc×Fpr2-/-×Apoe-/-), we investigated the role of GPR32 in atherosclerosis and self-limiting acute inflammation. GPR32 mRNA was reduced in human atherosclerotic lesions and correlated with the immune cell markers ARG1, NOS2, and FOXP3. Atherosclerotic lesions, necrotic core, and aortic inflammation were reduced in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice as compared with Fpr2-/-×Apoe-/- nontransgenic littermates. In a zymosan-induced peritonitis model, the hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice had reduced inflammation at 4 hours and enhanced proresolving macrophage responses at 24 hours compared with nontransgenic littermates. The GPR32 agonist aspirin-triggered resolvin D1 (AT-RvD1) regulated leukocyte responses, including enhancing macrophage phagocytosis and intracellular signaling in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice, but not in Fpr2-/-×Apoe-/- nontransgenic littermates. Together, these results provide evidence that GPR32 regulates resolution of inflammation and is atheroprotective in vivo.

TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is a common age-related disease characterized by active calcification of the leaflets of the aortic valve. How innate immune cells are involved in disease pathogenesis is not clear. In this study we investigate the role of the pattern recognition receptor Toll-like receptor 7 (TLR7) in CAVS, especially in relation to macrophage subtype. Human aortic valves were used for mRNA expression analysis, immunofluorescence staining, or ex vivo tissue assays. Response to TLR7 agonist in primary macrophages and valvular interstitial cells (VICs) were investigated in vitro. In the aortic valve, TLR7 correlated with M2 macrophage markers on mRNA levels. Expression was higher in the calcified part compared with the intermediate and healthy parts. TLR7+ cells were co-stained with M2-type macrophage receptors CD163 and CD206. Ex vivo stimulation of valve tissue with the TLR7 ligand imiquimod significantly increased secretion of IL-10, TNF-α, and GM-CSF. Primary macrophages responded to imiquimod with increased secretion of IL-10 while isolated VICs did not respond. In summary, in human aortic valves TLR7 expression is associated with M2 macrophages markers. Ex vivo tissue challenge with TLR7 ligand led to secretion of immunomodulatory cytokine IL-10. These results connect TLR7 activation in CAVS to reduced inflammation and improved clearance.

Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells.

The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity and overexpression of CARD8 mRNA was previously identified in atherosclerosis. However, very little is known about the regulation of CARD8 in endothelial cells and atherosclerosis. The aim of this study was to investigate CARD8 in the regulation of cytokine and chemokine expression in endothelial cells. Sections of human atherosclerotic lesions and non-atherosclerotic arteries were immunostained for CARD8 protein. Expression of CARD8 was correlated to mediators of inflammation in atherosclerotic lesions using Biobank of Karolinska Endarterectomies microarray data. The CARD8 mRNA was knocked-down in human umbilical vein endothelial cells (HUVECs) in vitro, followed by quantitative RT-PCR analysis and OLINK Proteomics. Endothelial and smooth muscle cells in arterial tissue expressed CARD8 and CARD8 correlated with vWF, CD163 and the expression of inflammatory genes, such as CXCL1, CXCL6 and PDGF-A in plaque. Knock-down of CARD8 in HUVECs significantly altered proteins involved in inflammatory response, such as CXCL1, CXCL6, PDGF-A, MCP-1 and IL-6. The present study suggest that CARD8 regulate the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions, suggesting that CARD8 plays a significant role in endothelial activation.

Low TLR7 gene expression in atherosclerotic plaques is associated with major adverse cardio- and cerebrovascular events.

AIMS: Processes in the development of atherosclerotic lesions can lead to plaque rupture or erosion, which can in turn elicit myocardial infarction or ischaemic stroke. The aims of this study were to determine whether Toll-like receptor 7 (TLR7) gene expression levels influence patient outcome and to explore the mechanisms linked to TLR7 expression in atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques were removed by carotid endarterectomy (CEA) and subjected to gene array expression analysis (n = 123). Increased levels of TLR7 transcript in the plaques were associated with better outcome in a follow-up study over a maximum of 8 years. Patients with higher TLR7 transcript levels had a lower risk of experiencing major cardiovascular and cerebrovascular events (MACCE) during the follow-up period after CEA (hazard ratio: 2.38, P = 0.012, 95% CI 1.21-4.67). TLR7 was expressed in all plaques by T cells, macrophages and endothelial cells in capillaries, as shown by immunohistochemistry. In short-term tissue cultures, ex vivo treatment of plaques with the TLR7 ligand imiquimod elicited dose-dependent secretion of IL-10, TNF-α, GM-CSF, and IL-12/IL-23p40. This secretion was blocked with a TLR7 inhibitor. Immunofluorescent tissue analysis after TLR7 stimulation showed IL-10 expression in T cells, macrophages and vascular smooth muscle cells. TLR7 mRNA levels in the plaques were correlated with IL-10 receptor (r = 0.4031, P < 0.0001) and GM-CSF receptor A (r = 0.4354, P < 0.0001) transcripts. CONCLUSION: These findings demonstrate that TLR7 is abundantly expressed in human atherosclerotic plaques. TLR7 ligation elicits the secretion of pro-inflammatory and anti-inflammatory cytokines, and high TLR7 expression in plaques is associated with better patient outcome, suggesting that TLR7 is a potential therapeutic target for prevention of complications of atherosclerosis.