ABSTRACT
Atherosclerotic plaque instability contributes to ischaemic stroke and myocardial infarction. This study is to compare the abundance and difference of immune cell subtypes within unstable atherosclerotic tissues. CIBERSORT was used to speculate the proportions of 22 immune cell types based on a microarray of atherosclerotic carotid artery samples. R software was utilized to illustrate the bar plot, heat map and vioplot. The immune cell landscape in atherosclerosis was diverse, dominated by M2 macrophages, M0 macrophages, resting CD4 memory T cells and CD8 T cells. There was a significant difference in resting CD4 memory T cells (p = 0.032), T cells follicular helper (p = 0.033), M0 (p = 0.047) and M2 macrophages (p = 0.012) between stable and unstable atherosclerotic plaques. Compared with stable atherosclerotic plaques, unstable atherosclerotic plaques had a higher percentage of M2 macrophages. Moreover, correlation analysis indicated that the percentage of naïve CD4 T cells was strongly correlated with that of gamma delta T cells (r = 0.93, p < 0.001), while memory B cells were correlated with plasma cells (r = 0.85, p < 0.001). In summary, our study explored the abundance and difference of specific immune cell subgroups at unstable plaques, which would aid new immunotherapies for atherosclerosis.
Subject(s)
Atherosclerosis/immunology , Carotid Arteries/immunology , Carotid Artery Diseases/immunology , Myocardial Infarction/immunology , Plasma Cells/immunology , Brain Ischemia/immunology , CD8-Positive T-Lymphocytes/immunology , Humans , Macrophages/immunology , Memory B Cells/immunology , Memory T Cells/immunology , Plaque, Atherosclerotic/immunology , Stroke/immunologyABSTRACT
Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (Tmem). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of Tmem and/or other T-cell subsets. Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of Tmem, proportion of Tmem/cfPWV, CD28null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required. Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 Tmem in men (P ≤ 0.05) but not in women; proportions of CD4 Tmem/cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people. Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 Tmem, higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Carotid Arteries/immunology , Cytomegalovirus Infections/immunology , Immunologic Memory/immunology , Vascular Stiffness/immunology , Aged , Aorta/immunology , Aorta/virology , Atherosclerosis/immunology , Atherosclerosis/virology , Blood Pressure/immunology , CD28 Antigens/immunology , Cardiovascular Diseases/immunology , Cardiovascular Diseases/virology , Carotid Arteries/virology , Female , Humans , Male , Pulse Wave Analysis/methods , Risk FactorsABSTRACT
Since immune infiltration is closely associated with the progression and prognosis of atherosclerosis, we aimed to describe the abundance of 24 immune cell types within atherosclerotic tissues. In the current study, we used the Immune Cell Abundance Identifier (ImmuCellAI), a web-based tool, to estimate the abundance of 24 immune cells based on the microarray profiles of atherosclerotic carotid artery samples to analyze the proportions and the dysregulation of immune cell types within carotid atherosclerosis. We found that atherosclerotic immune cells had a diverse landscape dominated by T cells and myeloid cells and that macrophages and dendritic cells (DCs) showed different abundance in normal and atherosclerotic tissues. Moreover, the expression of macrophages was closely related to the level of the expression of DCs and of exhausted T cells, while the expression of T-helper type 1 (Th1) cells was strongly correlated with the expression of T-helper type 2 (Th2) cells and effector memory cells. Our data confirm a distinct profile of atherosclerosis-infiltrating immune cell subpopulations, which may inspire an immunological direction for research on atherosclerosis.
Subject(s)
Carotid Arteries , Carotid Artery Diseases , Dendritic Cells , Gene Expression Regulation/immunology , Macrophages , Th1 Cells , Th2 Cells , Aged , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Artery Diseases/immunology , Carotid Artery Diseases/pathology , Databases, Nucleic Acid , Dendritic Cells/immunology , Dendritic Cells/pathology , Female , Humans , Immunologic Memory , Macrophages/immunology , Macrophages/pathology , Male , Middle Aged , Th1 Cells/immunology , Th1 Cells/pathology , Th2 Cells/immunology , Th2 Cells/pathologyABSTRACT
ABSTRACT: It remains uncertain whether statin/ezetimibe combination therapy serves as a useful and equivalent alternative to statin monotherapy for reducing atherosclerotic plaque inflammation. The aim of the present study was to compare the effects of statin/ezetimibe combination therapy and statin monotherapy on carotid atherosclerotic plaque inflammation using 18F-fluorodeoxyglucose (18FDG) positron emission tomography (PET)/computed tomography (CT) imaging. Data were pooled from 2 clinical trials that used serial 18FDG PET/CT examination to investigate the effects of cholesterol-lowering therapy on carotid atherosclerotic plaque inflammation. The primary outcome was the percent change in the target-to-background ratio (TBR) of the index vessel in the most diseased segment (MDS) at 6-month follow-up. Baseline characteristics were largely similar between the 2 groups. At the 6-month follow-up, the MDS TBR of the index vessel significantly decreased in both groups. The percent change in the MDS TBR of the index vessel (primary outcome) did not differ significantly between the 2 groups (-8.41â±â15.9% vs -8.08â±â17.0%, respectively, Pâ=â.936). Likewise, the percent change in the whole vessel TBR of the index vessel did not differ significantly between the 2 groups. There were significant decreases in total and LDL cholesterol levels in both groups at follow-up (Pâ<â.001). There were no significant correlations between the percent changes in MDS TBR of the index vessel, changes in the lipid, and high-sensitive C-reactive protein levels. The reduction in carotid atherosclerotic plaque inflammation by statin/ezetimibe combination therapy was equivalent to that by the statin monotherapy.
Subject(s)
Acute Coronary Syndrome/drug therapy , Carotid Artery Diseases/drug therapy , Ezetimibe, Simvastatin Drug Combination/administration & dosage , Hydroxymethylglutaryl-CoA Reductase Inhibitors/administration & dosage , Plaque, Atherosclerotic/drug therapy , Acute Coronary Syndrome/blood , Acute Coronary Syndrome/immunology , Aged , C-Reactive Protein/analysis , Carotid Arteries/diagnostic imaging , Carotid Arteries/drug effects , Carotid Arteries/immunology , Carotid Artery Diseases/blood , Carotid Artery Diseases/complications , Carotid Artery Diseases/immunology , Cholesterol, LDL/blood , Clinical Trials as Topic , Datasets as Topic , Female , Follow-Up Studies , Humans , Inflammation/complications , Inflammation/drug therapy , Inflammation/immunology , Male , Middle Aged , Plaque, Atherosclerotic/blood , Plaque, Atherosclerotic/complications , Plaque, Atherosclerotic/immunology , Rosuvastatin Calcium/administration & dosage , Simvastatin/administration & dosageABSTRACT
Atherosclerosis is a systemic disease associated with inflammatory cell infiltration and activation of immune-related pathways. In our study, we aimed to uncover immune-related changes and explore novel immunological features in the development of carotid atherosclerotic plaques. First, we applied integrated bioinformatics methods, including CIBERSORT and gene set enrichment analysis (GSEA). The gene expression matrices GSE28829, GSE41571, and GSE43292 were obtained from the Gene Expression Omnibus (GEO) dataset. After a series of data pre-processing steps, the resulting combined expression matrices were analysed using the CIBERSORT, GSEA, and Cluster Profiler packages. After the comparison and analysis between the carotid atherosclerotic plaques in the early and advanced stages, we discovered that there is a higher percentage of activated memory CD4 T cells and a lower percentage of resting memory CD4 cells in advanced-stage plaques. Moreover, activation of memory CD4 T cells can promote the development of carotid atherosclerotic plaques. Additionally, FOXP3+ Treg cell maturation can also participate in the progression of carotid plaques.
Subject(s)
Carotid Arteries , Carotid Artery Diseases , Computational Biology , Databases, Nucleic Acid , Plaque, Atherosclerotic , T-Lymphocytes, Regulatory , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Artery Diseases/genetics , Carotid Artery Diseases/immunology , Carotid Artery Diseases/pathology , Female , Humans , Male , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/immunology , Plaque, Atherosclerotic/pathology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
BACKGROUND: Neutrophils accumulate in atherosclerotic plaques. Neutrophil extracellular traps (NET) were recently identified in experimental atherosclerosis and in complex human lesions. However, not much is known about the NET marker citrullinated histone-3 (H3Cit) expression and functionality in human carotid plaques. Moreover, the association between the proatherosclerotic autoantibody anti-apolipoprotein A-1 (anti-ApoA-1 IgG) and NET has never been investigated. METHODS: Atherosclerotic plaques have been obtained from 36 patients with severe carotid stenosis that underwent carotid endarterectomy for severe carotid stenosis. Samples were sectioned into upstream and downstream regions from the same artery segment. Plaque composition and expression of NET markers neutrophil elastase (NE) and H3Cit were quantified by immunohistochemistry. H3Cit expression and function was evaluated by immunofluorescence and confocal analysis in a subset of patients. RESULTS: Pathological features of vulnerable phenotypes were exacerbated in plaques developed at downstream regions, including higher accumulation of neutrophils and enhanced expression of NE and H3Cit, as compared to plaques from upstream regions. The H3Cit signal was also more intense in downstream regions, with significant extracellular distribution in spaces outside of neutrophils. The percentage of H3Cit colocalization with CD66b (neutrophils) was markedly lower in downstream portions of carotid plaques, confirming the extrusion of NET in this region. In agreement, the maximum distance of the H3Cit signal from neutrophils, extrapolated from vortex distance calculation in all possible directions, was also higher in downstream plaques. The serum anti-ApoA-1index positively correlated with the expression of H3Cit in downstream segments of plaques. Expression of the H3Cit signal outside of neutrophils and H3Cit maximal distance from CD66b-positive cells increased in plaques from serum positive anti-ApoA-1 patients compared with serum negative patients. CONCLUSION: NET elements are differentially expressed in upstream versus downstream regions of human carotid plaques and may be influenced by circulating levels of anti-ApoA-1 IgG. These findings could warrant the investigation of NET elements as potential markers of vulnerability.
Subject(s)
Apolipoprotein A-I/immunology , Carotid Arteries/immunology , Carotid Arteries/pathology , Extracellular Traps/metabolism , Immunoglobulin G/immunology , Plaque, Atherosclerotic/immunology , Aged , Apolipoprotein A-I/blood , Cohort Studies , Female , Histones/metabolism , Humans , Leukocyte Elastase/metabolism , Male , Plaque, Atherosclerotic/bloodSubject(s)
Anticoagulants/administration & dosage , Carotid Arteries/immunology , Carotid Artery Diseases/diagnosis , Inflammation/diagnosis , Neck Pain/diagnosis , Administration, Oral , Adult , Carotid Arteries/diagnostic imaging , Carotid Artery Diseases/complications , Carotid Artery Diseases/drug therapy , Carotid Artery Diseases/immunology , Computed Tomography Angiography , Humans , Inflammation/complications , Inflammation/drug therapy , Inflammation/immunology , Magnetic Resonance Imaging , Male , Neck Pain/drug therapy , Neck Pain/immunology , Treatment OutcomeABSTRACT
OBJECTIVE: Atherosclerosis is a chronic inflammatory process characterized by the accumulation and formation of lipid-rich plaques within the layers of the arterial wall. Although numerous studies have reported the underlying pathogenesis, no data-based studies have been conducted to analyze the potential genes and immune cells infiltration in the different stages of atherosclerosis via bioinformatics analysis. METHODS: In this study, we downloaded GSE100927 and GSE28829 from NCBI-GEO database. Gene ontology and pathway enrichment were performed via the DAVID database. The protein interaction network was constructed via STRING. Enriched hub genes were analyzed by the Cytoscape software. The evaluation of the infiltrating immune cells in the dataset samples was performed by the CIBERSORT algorithm. RESULTS: We identified 114 common upregulated differentially expressed genes and 22 common downregulated differentially expressed genes. (adjust p value < 0.01 and log FC ≥ 1). A cluster of 10 genes including CYBA, SLC11A1, FCER1G, ITGAM, ITGB2, CD53, ITGAX, VAMP8, CLEC5A, and CD300A were found to be significant. Through the deconvolution algorithm CIBERSORT, we analyzed the significant alteration of immune cells infiltration in the progression of atherosclerosis with the threshold of the Wilcoxon test at p value <0.05. CONCLUSIONS: These results may reveal the underlying correlations between genes and immune cells in atherosclerosis, which enable us to investigate the novel insights for the development of treatments and drugs.
Subject(s)
Carotid Arteries/immunology , Carotid Artery Diseases/genetics , Carotid Artery Diseases/immunology , Gene Regulatory Networks , Oligonucleotide Array Sequence Analysis , Transcriptome , Carotid Arteries/pathology , Carotid Artery Diseases/pathology , Databases, Genetic , Disease Progression , Gene Expression Regulation , Humans , Plaque, AtheroscleroticABSTRACT
Effector CD8 T cells infiltrate atherosclerotic lesions and are correlated with cardiovascular events, but the mechanisms regulating their recruitment and retention are not well understood. CD137 (4-1BB) is a costimulatory receptor induced on immune cells and expressed at sites of human atherosclerotic plaque. Genetic variants associated with decreased CD137 expression correlate with carotid-intimal thickness and its deficiency in animal models attenuates atherosclerosis. These effects have been attributed in part to endothelial responses to low and disturbed flow (LDF), but CD137 also generates robust effector CD8 T cells as a costimulatory signal. Thus, we asked whether CD8 T cell-specific CD137 stimulation contributes to their infiltration, retention, and IFNγ production in early atherogenesis. We tested this through adoptive transfer of CD8 T cells into recipient C57BL/6J mice that were then antigen primed and CD137 costimulated. We analyzed atherogenic LDF vessels in normolipidemic and PCSK9-mediated hyperlipidemic models and utilized a digestion protocol that allowed for lesional T-cell characterization via flow cytometry and in vitro stimulation. We found that CD137 activation, specifically of effector CD8 T cells, triggers their intimal infiltration into LDF vessels and promotes a persistent innate-like proinflammatory program. Residence of CD137+ effector CD8 T cells further promoted infiltration of endogenous CD8 T cells with IFNγ-producing potential, whereas CD137-deficient CD8 T cells exhibited impaired vessel infiltration, minimal IFNγ production, and reduced infiltration of endogenous CD8 T cells. Our studies thus provide novel insight into how CD137 costimulation of effector T cells, independent of plaque-antigen recognition, instigates their retention and promotes innate-like responses from immune infiltrates within atherogenic foci. NEW & NOTEWORTHY Our studies identify CD137 costimulation as a stimulus for effector CD8 T-cell infiltration and persistence within atherogenic foci, regardless of atherosclerotic-antigen recognition. These costimulated effector cells, which are generated in pathological states such as viral infection and autoimmunity, have innate-like proinflammatory programs in circulation and within the atherosclerotic microenvironment, providing mechanistic context for clinical correlations of cardiovascular morbidity with increased CD8 T-cell infiltration and markers of activation in the absence of established antigen specificity.
Subject(s)
Aorta, Abdominal/metabolism , Atherosclerosis/metabolism , CD8-Positive T-Lymphocytes/metabolism , Carotid Arteries/metabolism , Immunity, Innate , Lymphocyte Activation , Plaque, Atherosclerotic , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Adoptive Transfer , Animals , Aorta, Abdominal/immunology , Aorta, Abdominal/pathology , Atherosclerosis/immunology , Atherosclerosis/pathology , CD8-Positive T-Lymphocytes/immunology , Carotid Arteries/immunology , Carotid Arteries/pathology , Cells, Cultured , Diet, High-Fat , Disease Models, Animal , Gene Transfer Techniques , Hyperlipidemias/complications , Interferon-gamma/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Proprotein Convertase 9/genetics , Proprotein Convertase 9/metabolism , Signal Transduction , Tumor Necrosis Factor Receptor Superfamily, Member 9/deficiency , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunologyABSTRACT
Apolipoprotein-E (ApoE) has been implicated in Alzheimer's disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (KD~140-580 pM) in vitro, and C1q-ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aß plaques, and atherosclerosis in vivo. C1q-ApoE complexes in human ChPs, Aß plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aß-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.
Subject(s)
Antigen-Antibody Complex/immunology , Apolipoproteins E/immunology , Carotid Artery Diseases/immunology , Choroid Plexus/immunology , Cognitive Dysfunction/immunology , Complement C1q/immunology , Complement Pathway, Classical/immunology , Aged , Aged, 80 and over , Amyloid beta-Peptides/immunology , Animals , Aorta/immunology , Aorta/pathology , Atherosclerosis/immunology , Atherosclerosis/pathology , Brain/immunology , Brain/pathology , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Artery Diseases/pathology , Choroid Plexus/pathology , Cognitive Dysfunction/pathology , Complement C5 , Female , Humans , Leukocytes , Male , Mice, Knockout, ApoE , Microscopy, Fluorescence , Middle Aged , Plaque, Amyloid/immunology , Plaque, Amyloid/pathology , Protein Isoforms/immunology , RNA, Small InterferingABSTRACT
BACKGROUND: Specialized proresolving mediators from ω-3 polyunsaturated fatty acid may control resolution of inflammation. We evaluated the influence of two specialized proresolving mediators, resolvin D1 (RvD1) and protectin D1 isomer (PD1 iso) on neointimal hyperplasia after balloon injury. MATERIALS AND METHODS: Sprague Dawley male rats at 12-14 wk of age were injured as a model of balloon angioplasty. Then, 1 µg/rat of RvD1 or PD1 iso was administered intravenously via the tail vein immediately and 2 d after angioplasty. The proliferation of injured artery and the infiltration of leukocytes, monocytes, and macrophages at 3 d after injury were evaluated by immunostaining. The activity of the inflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the injured artery at 3 d after injury was evaluated using an enzyme-linked immuno sorbent assay kit. The proliferation of the neointima was evaluated by calculating the ratio of the neointimal and medial areas using specimens at 14 d after injury. RESULTS: RvD1 and PD1 iso attenuated proliferation of medial cells (P < 0.05) and infiltration of leukocytes (P < 0.05) and monocytes/macrophages (P < 0.01). Although both RvD1 and PD1 iso mitigated NFκB activity (P < 0.01), RvD1 attenuated this activity more strongly (P < 0.01). RvD1 decreased neointimal hyperplasia by 37.3% (P < 0.01), whereas PD1 iso decreased neointimal hyperplasia by 31.8% (P < 0.05) (RvD1 versus PD1 iso: P = 0.51). CONCLUSIONS: RvD1 and PD1 iso reduced the activity of inflammatory transcription factor NFκB within the injured artery and attenuated inflammatory cell infiltration, leading to a reduction in early inflammation and subsequent neointimal hyperplasia.
Subject(s)
Angioplasty, Balloon/adverse effects , Carotid Artery Injuries/drug therapy , Docosahexaenoic Acids/administration & dosage , Neointima/drug therapy , Animals , Carotid Arteries/drug effects , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Artery Injuries/etiology , Carotid Artery Injuries/pathology , Disease Models, Animal , Humans , Hyperplasia/drug therapy , Hyperplasia/etiology , Hyperplasia/pathology , Injections, Intravenous , Male , NF-kappa B/immunology , NF-kappa B/metabolism , Neointima/etiology , Neointima/pathology , Rats , Rats, Sprague-Dawley , Treatment Outcome , Tunica Intima/drug effects , Tunica Intima/immunology , Tunica Intima/pathologyABSTRACT
This study was aimed to observe the level of T helper 17 (Th17) cells and CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) and their related factors in carotid atherosclerosis (AS) patients and AS model rats to explore the influence of Th17 on the pathological process of AS and its specific mechanism. 60 cases with AS in our hospital from July 2012 to July 2014 were recruited for this study as the observation group, and 40 healthy people who came to the hospital for a physical examination were the control group. Flow cytometry (FCM) was used to detect the Th17 and Treg cells in the peripheral blood of the two groups, ELISA was used to detect IL-17 and transforming growth factor-ß (TGF-ß), and RT-PCR was used to test the RORγT mRNA and Foxp3 mRNA expression levels. An AS model was created in rats using high-fat+ VD3 to explore the mechanism of Th17 on AS. The Th17 count, serum level of IL-17, and RORγT mRNA level of the observation group were significantly higher than those of the control group (P<0.001). The Tregs count, serum TGF-ß level, and Foxp3 mRNA level were significantly lower in the observation group than in the control group (P<0.001). In addition, the findings of the AS model in rats showed that the Th17 cell count and serum level of IL-17 in high-fat rats were significantly higher than in the normal rats (P<0.05). The Treg count and TGF-ß levels of the observation rats were significantly lower than in the normal rats (P<0.05). The IL-17 level, serum total cholesterol, and triglyceride in the high-fat-feed rats decreased after being injected with the IL-17 neutralizing antibody, but TGF-ß levels increased, and the difference was significant (P<0.05). Th17 cells and their related factors can be involved in promoting the pathological process of AS, while Tregs and its related factors can be involved in the inhibition of AS. Blocking IL-17 can be one potential method of treating AS.
Subject(s)
Carotid Arteries/metabolism , Carotid Artery Diseases/blood , Cytokines/blood , Plaque, Atherosclerotic , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/metabolism , Adult , Aged , Animals , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Artery Diseases/genetics , Carotid Artery Diseases/immunology , Carotid Artery Diseases/pathology , Case-Control Studies , Cholesterol/blood , Cytokines/genetics , Cytokines/immunology , Diet, High-Fat , Disease Models, Animal , Female , Forkhead Transcription Factors/blood , Humans , Interleukin-17/blood , Male , Middle Aged , Nuclear Receptor Subfamily 1, Group F, Member 3/blood , Rats, Wistar , T-Lymphocytes, Regulatory/immunology , Th17 Cells/immunology , Transforming Growth Factor beta/blood , Triglycerides/bloodABSTRACT
Inflammatory cells play key roles in restenosis upon vascular surgical procedures such as bypass grafts, angioplasty and stent deployment but the molecular mechanisms by which these cells affect restenosis remain unclear. The p110δ isoform of phosphoinositide 3-kinase (PI3K) is mainly expressed in white blood cells. Here, we have investigated whether p110δ PI3K is involved in the pathogenesis of restenosis in a mouse model of carotid injury, which mimics the damage following arterial grafts. We used mice in which p110δ kinase activity has been disabled by a knockin (KI) point mutation in its ATP-binding site (p110δD910A/D910A PI3K mice). Wild-type (WT) and p110δD910A/D910A mice were subjected to longitudinal carotid injury. At 14 and 30 days after carotid injury, mice with inactive p110δ showed strongly decreased infiltration of inflammatory cells (including T lymphocytes and macrophages) and vascular smooth muscle cells (VSMCs), compared with WT mice. Likewise, PI-3065, a p110δ-selective PI3K inhibitor, almost completely prevented restenosis after artery injury. Our data showed that p110δ PI3K plays a main role in promoting neointimal thickening and inflammatory processes during vascular stenosis, with its inhibition providing significant reduction in restenosis following carotid injury. p110δ-selective inhibitors, recently approved for the treatment of human B-cell malignancies, therefore, present a new therapeutic opportunity to prevent the restenosis upon artery injury.
Subject(s)
Carotid Artery Injuries/enzymology , Carotid Stenosis/enzymology , Class I Phosphatidylinositol 3-Kinases/immunology , Inflammation/enzymology , Animals , Carotid Arteries/enzymology , Carotid Arteries/immunology , Carotid Arteries/metabolism , Carotid Arteries/pathology , Carotid Artery Injuries/genetics , Carotid Artery Injuries/immunology , Carotid Artery Injuries/pathology , Carotid Stenosis/genetics , Carotid Stenosis/immunology , Carotid Stenosis/pathology , Class I Phosphatidylinositol 3-Kinases/genetics , Disease Models, Animal , Gene Knock-In Techniques , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Male , Mice, Inbred C57BL , Neointima/enzymology , Neointima/genetics , Neointima/immunology , Neointima/pathology , Point MutationABSTRACT
Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.
Subject(s)
Atherosclerosis/genetics , Cholesterol/pharmacology , Endoplasmic Reticulum Stress/genetics , Lipoproteins/genetics , Plaque, Atherosclerotic/genetics , RNA, Messenger/genetics , Atherosclerosis/immunology , Atherosclerosis/pathology , Atherosclerosis/surgery , Carotid Arteries/drug effects , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Arteries/surgery , Crystallization , Endarterectomy, Carotid , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Gene Expression Regulation , Humans , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-8/genetics , Interleukin-8/immunology , Lipoproteins/antagonists & inhibitors , Lipoproteins/immunology , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Phenylbutyrates/pharmacology , Plaque, Atherosclerotic/immunology , Plaque, Atherosclerotic/pathology , Plaque, Atherosclerotic/surgery , Primary Cell Culture , RNA, Messenger/immunology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription Factor CHOP/genetics , Transcription Factor CHOP/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
OBJECTIVE: Both environmental and genetic factors are important in the development of antiphospholipid antibodies (aPL) in patients with antiphospholipid syndrome (APS). Currently, the only available data on predisposing genetic factors have been obtained from epidemiologic studies, without mechanistic evidence. Therefore, we studied the influence of major histocompatibility complex (MHC) class II alleles on the production of aPL in a mouse model of APS. METHODS: Three groups of mice, MHC class II-deficient (MHCII-/- ) mice, MHCII-/- mice transgenic for human HLA-DQ6 (DQ6), DQ8, or DR4 alleles, and the corresponding wild-type (WT) mouse strains were immunized; half were immunized with human ß2 -glycoprotein I (ß2 GPI), and the other half were immunized with control ovalbumin (OVA) protein. Thrombus formation in vivo, tissue factor activity in carotid and peritoneal macrophages, and serum levels of tumor necrosis factor (TNF), IgG anticardiolipin (aCL), antibodies, and anti-OVA antibodies were determined. RESULTS: Immunization with ß2 GPI induced significant production of aCL and anti-ß2 GPI in WT mice compared with control mice immunized with OVA (P < 0.001) but diminished aCL (P < 0.001) and anti-ß2 GPI (P = 0.016) production in MHCII-/- mice. Anti-ß2 GPI production was fully restored in DQ6 and DQ8 mice, while levels of anti-ß2 GPI in DR4 mice and aCL in all transgenic lines were only partially restored (P < 0.001 to P < 0.046). Thrombus size in WT mice was twice that in MHCII-/- mice (P < 0.001) but similar to that in all transgenic lines. Carotid and peritoneal macrophage tissue factor levels decreased by >50% in MHCII-/- mice compared with wild-type B6 mice and were restored in DQ8 mice but not DR4 mice or DQ6 mice. TNF levels decreased 4-fold in MHCII-/- mice (P < 0.001) and were not restored in transgenic mice. CONCLUSION: Our mechanistic study is the first to show that MHC class II alleles influence not only quantitative aPL production but also the pathogenic capacity of induced aPL.
Subject(s)
Antibodies, Antiphospholipid/immunology , Genes, MHC Class II/genetics , HLA-DQ Antigens/genetics , HLA-DR4 Antigen/genetics , Alleles , Animals , Antibodies, Anticardiolipin/immunology , Carotid Arteries/immunology , Disease Models, Animal , Humans , Immunization , Immunoglobulin G/immunology , Macrophages/immunology , Macrophages, Peritoneal/immunology , Mice , Mice, Knockout , Mice, Transgenic , Ovalbumin/immunology , Severity of Illness Index , Thrombosis , Tumor Necrosis Factor-alpha/immunology , beta 2-Glycoprotein I/immunologyABSTRACT
Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [11C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [11C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction of the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [11C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [11C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [11C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.
Subject(s)
Carotid Arteries/metabolism , Positron-Emission Tomography/methods , Receptors, Lysosphingolipid/metabolism , Vascular Diseases/metabolism , Animals , Carotid Arteries/immunology , Inflammation/immunology , Inflammation/metabolism , Rats , Rats, Sprague-Dawley , Sphingosine-1-Phosphate Receptors , Tomography, X-Ray Computed , Vascular Diseases/immunologyABSTRACT
Matrix metalloproteinase (MMP)-12 plays a key role in the development of aneurysm. Like other members of MMP family, MMP-12 is produced as a proenzyme, mainly by macrophages, and undergoes proteolytic activation to generate an active form. Accordingly, molecular imaging of the MMP-12 active form can inform of the pathogenic process in aneurysm. Here, we developed a novel family of fluorescent probes based on a selective MMP-12 inhibitor, RXP470.1 to target the active form of MMP-12. These probes were stable in complex media and retained the high affinity and selectivity of RXP470.1 for MMP-12. Amongst these, probe 3 containing a zwitterionic fluorophore, ZW800-1, combined a favorable affinity profile toward MMP-12 and faster blood clearance. In vivo binding of probe 3 was observed in murine models of sterile inflammation and carotid aneurysm. Binding specificity was demonstrated using a non-binding homolog. Co-immunostaining localized MMP-12 probe binding to MMP-12 positive areas and F4/80 positive macrophages in aneurysm. In conclusion, the active form of MMP-12 can be detected by optical imaging using RXP470.1-based probes. This is a valuable adjunct for pathophysiology research, drug development, and potentially clinical applications.
Subject(s)
Aneurysm/diagnostic imaging , Carotid Arteries/diagnostic imaging , Macrophages/metabolism , Matrix Metalloproteinase 12/metabolism , Matrix Metalloproteinase Inhibitors/metabolism , Optical Imaging/methods , Aneurysm/immunology , Aneurysm/metabolism , Aneurysm/pathology , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Carotid Arteries/immunology , Carotid Arteries/metabolism , Carotid Arteries/pathology , Disease Models, Animal , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Gene Expression , Humans , Inflammation , Macrophages/immunology , Macrophages/pathology , Matrix Metalloproteinase 12/genetics , Matrix Metalloproteinase Inhibitors/chemical synthesis , Mice , Mice, Inbred C57BL , Peptides/chemistry , Peptides/metabolism , Protein Binding , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/metabolism , Sulfonic Acids/chemistry , Sulfonic Acids/metabolismABSTRACT
Automated blood sampling through a vascular catheter is a frequently utilized technique in laboratory mice. The potential immunological and physiological implications associated with this technique have, however, not been investigated in detail. The present study compared plasma levels of the cytokines IL-1ß, IL-2, IL-6, IL-10, IL-17A, GM-CSF, IFN-γ and TNF-α in male NMRI mice that had been subjected to carotid artery catheterization and subsequent automated blood sampling with age-matched control mice. Body weight and histopathological changes in the surgical area, including the salivary glands, the heart, brain, spleen, liver, kidneys and lungs were compared. Catheterized mice had higher levels of IL-6 than did control mice, but other cytokine levels did not differ between the groups. No significant difference in body weight was found. The histology revealed inflammatory and regenerative (healing) changes at surgical sites of all catheterized mice, with mild inflammatory changes extending into the salivary glands. Several catheterized mice had multifocal degenerative to necrotic changes with inflammation in the heart, kidneys and livers, suggesting that thrombi had detached from the catheter tip and embolized to distant sites. Thus, catheterization and subsequent automated blood sampling may have physiological impact. Possible confounding effects of visceral damage should be assessed and considered, when using catheterized mouse models.
Subject(s)
Blood Specimen Collection/adverse effects , Catheterization/adverse effects , Inflammation/etiology , Interleukin-6/blood , Mice/immunology , Animals , Blood Specimen Collection/methods , Carotid Arteries/immunology , Carotid Arteries/pathology , Catheterization/methods , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Inflammation/blood , Inflammation/immunology , Inflammation/pathology , Interferon-gamma/blood , Interferon-gamma/immunology , Interleukin-6/immunology , Kidney/immunology , Kidney/pathology , Liver/immunology , Liver/pathology , Male , Mice, Inbred Strains , Myocardium/immunology , Myocardium/pathology , Salivary Glands/immunology , Salivary Glands/pathology , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/immunologyABSTRACT
BACKGROUND: Cardiovascular disease, a progressive manifestation of α-L-iduronidase deficiency or mucopolysaccharidosis type I, continues in patients both untreated and treated with hematopoietic stem cell transplantation or intravenous enzyme replacement. Few studies have examined the effects of α-L-iduronidase deficiency and subsequent glycosaminoglycan storage upon arterial gene expression to understand the pathogenesis of cardiovascular disease. METHODS: Gene expression in carotid artery, ascending, and descending aortas from four non-tolerized, non-enzyme treated 19 month-old mucopolysaccharidosis type I dogs was compared with expression in corresponding vascular segments from three normal, age-matched dogs. Data were analyzed using R and whole genome network correlation analysis, a bias-free method of categorizing expression level and significance into discrete modules. Genes were further categorized based on module-trait relationships. Expression of clusterin, a protein implicated in other etiologies of cardiovascular disease, was assessed in canine and murine mucopolysaccharidosis type I aortas via Western blot and in situ immunohistochemistry. RESULTS: Gene families with more than two-fold, significant increased expression involved lysosomal function, proteasome function, and immune regulation. Significantly downregulated genes were related to cellular adhesion, cytoskeletal elements, and calcium regulation. Clusterin gene overexpression (9-fold) and protein overexpression (1.3 to 1.62-fold) was confirmed and located specifically in arterial plaques of mucopolysaccharidosis-affected dogs and mice. CONCLUSIONS: Overexpression of lysosomal and proteasomal-related genes are expected responses to cellular stress induced by lysosomal storage in mucopolysaccharidosis type I. Upregulation of immunity-related genes implicates the potential involvement of glycosaminoglycan-induced inflammation in the pathogenesis of mucopolysaccharidosis-related arterial disease, for which clusterin represents a potential biomarker.