ABSTRACT
OBJECTIVE: COMP (cartilage oligomeric matrix protein) is abundantly expressed in the cardiovascular system, cartilage, and atherosclerotic plaques. We investigated if the total COMP (COMPtotal) and COMP neoepitope (COMPneo) with other cardiovascular markers and clinical parameters could identify symptomatic carotid stenosis. Approach and Results: Blood samples were collected from patients with symptomatic carotid stenosis (stenosis, n=50), patients with stroke without carotid stenosis but small plaques (plaque, n=50), and control subjects (n=50). COMPtotal and COMPneo were measured using an ELISA. Ninety-two cardiovascular disease markers were measured by the Olink CVD kit. The presence of native COMP and COMPneo was determined by immunohistochemistry. The concentration of COMPneo was higher and COMPtotal was lower in the stenosis group. When the concentration was compared between the stenosis and control groups, IL-1ra (interleukin-1 receptor antagonist protein), IL6 (interleukin-6), REN (Renin), MMP1 (matrix metalloproteinase-1), TRAIL-R2 (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), ITGB1BP2 (integrin beta 1 binding protein 2), and COMPneo were predictive of stenosis. Conversely, KLK6 (kallikrein-6), COMPtotal, NEMO (nuclear factor-kappa-B essential modulator), SRC (Proto-oncogene tyrosine-protein kinase Src), SIRT2 (SIR2-like protein), CD40 (cluster of differentiation 40), TF (tissue factor), MP (myoglobin), and RAGE (receptor for advanced glycation end-products) were predictive of the control group. Model reproducibility was good with the receiver operating characteristic plot area under the curve being 0.86. When comparing the plaque group and stenosis group, COMPneo, GAL (galanin), and PTX3 (pentraxin-related protein PTX3) were predictive of stenosis. Model reproducibility was excellent (receiver operating characteristic plot area under the curve 0.92). COMPneo was detected in smooth muscle-, endothelial-, and foam-cells in carotid stenosis. CONCLUSIONS: Degradation of COMP may be associated with atherosclerosis progression and generation of a specific COMP fragment-COMPneo. This may represent a novel biomarker that together with COMPtotal and other risk-markers could be used to identify symptomatic carotid stenosis. Graphic Abstract: A graphic abstract is available for this article.
Subject(s)
Carotid Stenosis/blood , Cartilage Oligomeric Matrix Protein/blood , Cartilage Oligomeric Matrix Protein/immunology , Epitopes/blood , Aged , Biomarkers/blood , Biomarkers/metabolism , Carotid Stenosis/immunology , Cartilage Oligomeric Matrix Protein/metabolism , Case-Control Studies , Disease Progression , Epitopes/metabolism , Female , Humans , Male , Middle Aged , Models, Cardiovascular , Plaque, Atherosclerotic/blood , Plaque, Atherosclerotic/immunology , Plaque, Atherosclerotic/metabolism , Proto-Oncogene Mas , Stroke/blood , Stroke/immunologyABSTRACT
BACKGROUND AND PURPOSE: erosion of vulnerable atherosclerotic plaques may cause life-threatening thromboembolic complications. There is indeed an urgent need to recognize a clear-cut biomarker able to identify vulnerable plaques. Here, we focused on circulating proteins belonging to the lectin pathway (LP) of complement activation. METHODS: we analyzed mannose-binding lectin (MBL), ficolin-1, -2 and -3 (LP initiators) levels by ELISA in sera from n = 240 of an already published cohort of patients undergoing endarterectomy for severe carotid stenosis and followed-up until 18 months after surgery. Immunofluorescence followed by confocal and polarized light microscopy was used to detect LP initiator intraplaque localization. Spearman's rank test was drawn to investigate correlation between serum LP levels and circulating inflammatory proteins or intraplaque components. Survival analyses were then performed to test the predictive role of LP on long-term adverse outcome. RESULTS: ficolins, but not MBL, correlated positively with 1) high circulating levels of inflammatory markers, including MPO, MMP-8, MMP-9, ICAM-1, osteopontin, neutrophil elastase, and; 2) immune cell intraplaque recruitment. Immunofluorescence showed ficolins in calcified plaques and ficolin-2 in cholesterol-enriched plaque regions in association with macrophages. In the multivariate survival analysis, ficolin-2 serum levels predicted a major adverse cardiovascular event during the follow-up, independently of symptomatic status and inflammatory markers (hazard ratio 38.6 [95 % CI 3.9-385.2]). CONCLUSIONS: ficolins support intraplaque immune cell recruitment and inflammatory processes ultimately leading to plaque vulnerability. Especially for ficolin-2 a strong predictive value toward adverse cardiovascular events was demonstrated. This evidence offers potentially new pharmacological target to dampen the inflammatory mechanisms leading to plaque vulnerability.
Subject(s)
Acute Coronary Syndrome/blood , Carotid Stenosis/blood , Lectins/blood , Acute Coronary Syndrome/complications , Acute Coronary Syndrome/immunology , Aged , Carotid Stenosis/complications , Carotid Stenosis/immunology , Complement Activation , Female , Humans , Inflammation/blood , Inflammation/complications , Inflammation/immunology , Lectins/immunology , Male , Prognosis , FicolinsABSTRACT
BACKGROUND: Inflammatory mechanisms play an important role in both atherosclerosis and stroke. There are several inflammatory peripheral blood count markers associated with carotid artery stenosis degree, symptomatic carotid artery lesions and carotid artery stent restenosis that reported in previous studies. However, the prognostic role of the blood cell counts and their ratios in predicting in-hospital and long-term outcomes in patients undergoing carotid artery stenting (CAS) has not been comprehensively investigated. Systemic immune-inflammation index (SII) proved its' efficiency in patients with solid tumors and its' role was rarely examined in cardiovascular disorders and stroke. The current study evaluated the effect of this novel risk index on in-hospital and long-term outcomes in a large patient population who underwent CAS. METHOD: A total of 732 patients with carotid artery stenosis who underwent CAS were enrolled to the study. SII was calculated using the following formula: neutrophil-to-lymphocyte ratio × total platelet count in the peripheral blood (per mm3) and the patients were stratified accordingly: T1, T2 and T3. In-hospital and 5-year outcomes were compared between the tertiles of SII. RESULTS: During the hospitalization, major stroke, ipsilateral stoke, myocardial infarction, death and major adverse cardiovascular events (MACE) rates were significantly higher in high SII level (T3) compared to SII levels (T1 and 2). In long-term outcomes, ipsilateral stroke, major stroke, transient ischemic attack, death, and MACE were significantly higher in the patients with higher SII level (T3). The 5-year Kaplan-Meier overall survival for T1, T2, and T3 were 97.5%, 96.7% and 86.0% respectively. In-hospital and 5-year regression analyses demonstrated that high SII was independently associated with MACE and mortality. CONCLUSION: SII was independently associated with in-hospital and long-term clinical outcomes in patients undergoing CAS. Immune and inflammation status, as assessed easily and quickly using SII, has a good discriminative value in these patients.
Subject(s)
Blood Platelets , Carotid Stenosis/therapy , Endovascular Procedures/instrumentation , Lymphocytes , Neutrophils , Stents , Aged , Carotid Stenosis/diagnosis , Carotid Stenosis/immunology , Carotid Stenosis/mortality , Endovascular Procedures/adverse effects , Endovascular Procedures/mortality , Female , Hospital Mortality , Humans , Ischemic Attack, Transient/etiology , Lymphocyte Count , Male , Middle Aged , Myocardial Infarction/etiology , Platelet Count , Predictive Value of Tests , Retrospective Studies , Risk Assessment , Risk Factors , Stroke/etiology , Time Factors , Treatment OutcomeABSTRACT
Background and Purpose- Focal cerebral arteriopathy is monophasic inflammatory stenosis of the distal internal carotid artery or the proximal segment of the middle cerebral artery. It is one of the most common causes of acute arterial ischemic stroke in young children but is a less familiar entity for adult neurologists. Methods- We retrospectively reviewed stroke service radiology records at a tertiary referral center from January 2013 to December 2014. Focal cerebral arteriopathy was defined as nonprogressive unifocal and unilateral stenosis/irregularity of the distal internal carotid artery or its proximal branches. Only patients aged 16 to 55 years with stroke were included. Results- There were 5 cases of focal cerebral arteriopathy: 2 males and 3 females. Three cases were from the cohort of 123 acute presentations of young stroke, and 2 cases were outpatient referrals. The mean age (range) was 43 (32-55) years. The majority presented with recurrent transient ischemic attacks/minor strokes within a single vascular territory over days to weeks. All cases had characteristic radiological features. Interval imaging demonstrated resolution in 1 case and improvement in 3 cases. Functional outcome was excellent with discharge modified Rankin Scale score ranging from 0 to 1. Recurrence occurred in 1 case. Conclusions- Focal cerebral arteriopathy is a rare cause of arterial ischemic stroke in young adults. Follow-up intracranial imaging is essential to differentiate from progressive arteriopathies. Evidence-based treatment warrants further investigation. Prognosis is favorable.
Subject(s)
Carotid Artery, Internal/diagnostic imaging , Carotid Stenosis/diagnostic imaging , Cerebral Arterial Diseases/diagnostic imaging , Middle Cerebral Artery/diagnostic imaging , Stroke/diagnostic imaging , Acyclovir/therapeutic use , Adult , Antibodies, Viral/immunology , Antiviral Agents/therapeutic use , Aspirin/therapeutic use , Carotid Stenosis/complications , Carotid Stenosis/drug therapy , Carotid Stenosis/immunology , Cerebral Angiography , Cerebral Arterial Diseases/complications , Cerebral Arterial Diseases/drug therapy , Cerebral Arterial Diseases/immunology , Computed Tomography Angiography , Dual Anti-Platelet Therapy , Female , Glucocorticoids/therapeutic use , Herpesvirus 3, Human/immunology , Humans , Immunoglobulin G , Immunoglobulin M , Ischemic Attack, Transient/diagnosis , Ischemic Attack, Transient/etiology , Male , Middle Aged , Platelet Aggregation Inhibitors/therapeutic use , Recurrence , Retrospective Studies , Stroke/drug therapy , Stroke/etiologyABSTRACT
Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.
Subject(s)
Atherosclerosis/immunology , Atherosclerosis/physiopathology , Complement Activation , Disease Progression , Lectins/metabolism , Mannose-Binding Lectin/metabolism , Calcium/metabolism , Carotid Stenosis/immunology , Cholesterol/chemistry , Cholesterol/immunology , Cholesterol/metabolism , Cholesterol/pharmacology , Complement C4/metabolism , Crystallization , Fluorescent Antibody Technique , Glycoproteins/immunology , Glycoproteins/metabolism , Humans , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunoglobulin M/immunology , Immunoglobulin M/metabolism , Inflammation , Lectins/immunology , Mannose-Binding Lectin/immunology , Microscopy, Fluorescence , Receptors, Pattern Recognition/immunology , Receptors, Pattern Recognition/metabolism , Recombinant Proteins/metabolism , FicolinsABSTRACT
BACKGROUND AND PURPOSE: This study was aimed to assess the hypothesis that unstable plaque formation in the carotid artery is one of phenotypes of chronic and systemic inflammation. METHODS: This study included 8 symptomatic patients with internal carotid stenosis (ICS) and 7 healthy controls. All subjects underwent 18F-fluorodeoxyglucos positron emission tomography (18F-FDG PET) of whole body. Plaque vulnerability was evaluated on magnetic resonance imaging (MRI). On 18F-FDG PET, the maximum standardized uptake (SUVmax) value was measured in the carotid plaque, aorta, spleen, liver, and bone marrow. The SUVmax ratio of the spleen or bone marrow to the liver was also calculated. These values were compared between 2 groups. All 8 patients in ICS group underwent carotid endarterectomy, and surgical specimens were subjected to immunohistochemistry. RESULTS: All 8 patients in ICS group had unstable plaque on MRI. The mean SUVmax of carotid plaque was 2.5 ± .2 in ICS group. The SUVmax of spleen was significantly higher in ICS group than in the controls (3.20 ± â.25 and 2.51 ± â.40, respectively; Pâ¯=â¯.003). The SUVmax ratio (spleen/liver) was also significantly higher in ICS group than in the controls (1.12 ± â.06 and .85 ± â.12, respectively; Pâ¯=â¯.001). The SUVmax of aorta was also significantly higher in ICS group than in the controls (2.16 ± â.27 and 1.48 ± â.15, respectively; Pâ¯=â¯.001). However, there were no significant differences in the SUVmax in the bone marrow and SUVmax ratio (bone marrow/liver) between the 2 groups (Pâ¯=â¯.811 and Pâ¯=â¯.731, respectively). Histological examination showed that the plaque strongly expressed endothelial progenitor cells, microvessels, and M1 macrophages. CONCLUSIONS: These data strongly suggest the inflammation coupling between the spleen and unstable carotid plaque, and may be useful to develop novel therapeutic strategies against systemic inflammation in patients with ICS.
Subject(s)
Carotid Artery, Internal/diagnostic imaging , Carotid Stenosis/diagnostic imaging , Fluorodeoxyglucose F18/administration & dosage , Inflammation/diagnostic imaging , Plaque, Atherosclerotic , Positron-Emission Tomography , Radiopharmaceuticals/administration & dosage , Spleen/diagnostic imaging , Aged , Carotid Artery, Internal/immunology , Carotid Artery, Internal/pathology , Carotid Artery, Internal/surgery , Carotid Stenosis/immunology , Carotid Stenosis/pathology , Carotid Stenosis/surgery , Case-Control Studies , Endarterectomy, Carotid , Humans , Immunohistochemistry , Inflammation/immunology , Magnetic Resonance Imaging , Male , Predictive Value of Tests , Rupture, Spontaneous , Spleen/immunologyABSTRACT
Changes in immune and inflammatory responses may play a crucial role in the development and progression of atherosclerosis, as an autoimmune, chronic and progressive inflammatory disease. Immunological activity and vascular inflammation during atherosclerosis can be modulated by autoimmune responses against self-antigens, according to changeable risk factors (cholesterol, oxidized low-density lipoprotein (ox-LDL) in the vascular wall, fatty acids, etc.), and accompanied by accumulation of leucocytes and proinflammatory cytokines, which stimulate the transcription of matrix metalloproteinases (MMPs), whose concentration are increased in foam cell-rich regions. Regulatory T cells (Tregs) represent a unique subpopulation of T cells specialized in the regulation of immune response and in the suppression of proatherogenic T cells. The aim of our study was to examine the interactions between the concentration of enzyme matrix metalloproteinases 2 and 9 (MMP-2 and 9) in urine and the percentage of Tregs in peripheral blood of two groups of patients: with carotid artery stenosis (CAS), undergoing surgery and with mild atherosclerosis (A) from general practice. The method of enzyme immunoassay (ELISA) was used to determine enzyme MMP expression, and Tregs was examined by flow cytometric analysis. Our data have showed a large increase in the enzyme MMP-2 and 9 in the urine of CAS and A patients in comparison with healthy controls and indicated this method as an easy marker for the monitoring of the development of atherosclerosis. Simultaneously, the diminished number of Tregs in the same patients pointed the importance of these regulatory mechanisms in the etiopathogenesis of atherosclerosis and possible Tregs-mediated therapy.
Subject(s)
Atherosclerosis/immunology , Matrix Metalloproteinase 2/immunology , Matrix Metalloproteinase 9/immunology , T-Lymphocytes, Regulatory/immunology , Aged , Aged, 80 and over , Atherosclerosis/blood , Atherosclerosis/urine , Carotid Stenosis/blood , Carotid Stenosis/immunology , Carotid Stenosis/urine , Cholesterol/immunology , Cholesterol/metabolism , Cytokines/immunology , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Global Burden of Disease/statistics & numerical data , Humans , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Lipoproteins, LDL/immunology , Lipoproteins, LDL/metabolism , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 2/urine , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase 9/urine , Middle Aged , Protein Binding , Risk FactorsABSTRACT
RATIONALE: Proinflammatory adaptive immune responses are recognized as major drivers of atherosclerotic lesion formation. Although CD8(+) T cells have recently been proposed as a proatherogenic cell subset, their full scope of actions remains to be elucidated. OBJECTIVE: We here addressed the contribution of CD8(+) T cells to monocyte trafficking in atherosclerosis. METHOD AND RESULTS: We observed that CD8(+) T cells express proinflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-12) within atherosclerotic lesions and spleens of high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Antibody-mediated CD8(+) T-cell depletion in high-fat diet-fed Ldlr(-/-) mice decreased atherosclerotic plaque formation, associated with decreased macrophage accumulation within lesions. Despite a reduction in vascular chemokine (CC-motif) ligand 2 and chemokine (CXC-motif) ligand 1 expression, CD8(+) T-cell depletion did not directly affect monocyte recruitment to inflamed vessels. However, CD8(+) T-cell depletion decreased chemokine (CC-motif) ligand serum concentrations and circulating Ly6C(high) monocyte counts. We further evidenced that CD8(+) T-cell depletion decreased levels of mature monocytes and myeloid granulocyte-monocyte progenitors in the bone marrow and spleen of hypercholesterolemic mice, effects that were partially reproduced by interferon-γ neutralization, showing a role for interferon-γ. CONCLUSIONS: These data suggest that CD8(+) T cells promote atherosclerosis by controlling monopoiesis and circulating monocyte levels, which ultimately contributes to plaque macrophage burden without affecting direct monocyte recruitment, identifying this cell subset as a critical regulator of proatherogenic innate immune cell responses in atherosclerosis.
Subject(s)
Antigens, Ly/analysis , Atherosclerosis/immunology , CD8-Positive T-Lymphocytes/immunology , Monocytes/immunology , Myelopoiesis/immunology , Animals , Antilymphocyte Serum/therapeutic use , Atherosclerosis/etiology , Bone Marrow/metabolism , Bone Marrow/pathology , CD8-Positive T-Lymphocytes/metabolism , Carotid Stenosis/immunology , Carotid Stenosis/pathology , Cells, Cultured , Chemotaxis, Leukocyte/immunology , Cytokines/biosynthesis , Cytokines/genetics , Cytokines/metabolism , Diet, Atherogenic/adverse effects , Dietary Fats/toxicity , Endarterectomy, Carotid , Gene Expression Regulation/immunology , Humans , Hypercholesterolemia/complications , Hypercholesterolemia/etiology , Interferon-gamma/physiology , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, LDL/deficiency , Receptors, LDL/geneticsABSTRACT
BACKGROUND AND PURPOSE: Transient cerebral hypoxia may induce neuronal injury through an ischemia-reperfusion (I/R) response, with a subsequent activation of inflammation and coagulation-fibrinolysis. During carotid endarterectomy (CEA), the artery is clamped, which might impair the regional cerebral perfusion and initiate a local I/R response. Data suggest that the CD40-CD40 ligand dyad acts as a modulator in the induced activation. The aim of this study was to locally measure soluble CD40 ligand (sCD40L), in conjunction with inflammation and coagulation activation markers, during CEA. SUBJECTS AND METHODS: This is a prospective study of 18 patients undergoing CEA. Blood samples from the venous jugular bulb (JB) and the radial artery (RA) were drawn at baseline and during the procedure. Measurements of sCD40L, interleukin-6 (IL-6), fragment 1 + 2 (F1 + 2), plasminogen activator inhibitor-1 (PAI-1), and d-dimer were analyzed. Comparisons during CEA were made between levels: baselines versus JB, JB versus RA, and sequential JB measurements. Fifty cardiovascular healthy patients were the reference group for the sCD40L baseline comparison. RESULTS: Increased cerebral IL-6 levels were demonstrated throughout the procedure, as well as the temporal influence in F1 + 2, PAI-1, and d-dimer values. sCD40L remained unchanged throughout the procedure . This indicates a local cerebral inflammatory reaction together with an activation of coagulation-fibrinolysis, but it does not appear to primarily involve the CD40-CD40 ligand dyad. CONCLUSIONS: Signs of a local inflammatory reaction and activation of coagulation were observed during CEA, but levels of sCD40L remained stable, unaffected by carotid artery clamping and reperfusion.
Subject(s)
Carotid Artery, Internal , Carotid Stenosis/blood , Carotid Stenosis/surgery , Endarterectomy, Carotid , Aged , Biomarkers/blood , CD40 Ligand/blood , Carotid Stenosis/immunology , Female , Fibrin Fibrinogen Degradation Products/metabolism , Humans , Interleukin-6/blood , Jugular Veins , Male , Middle Aged , Plasminogen Activator Inhibitor 1/blood , Prospective StudiesABSTRACT
BACKGROUND: Atherosclerosis is an inflammatory disease of the vessel wall promoted by different immune cells and inflammatory mediators. METHODS: In this study, 26 human plaques and 12 control vessels without atherosclerosis were immunohistochemically stained to analyze the emergence of mast cells dependent on plaque morphology and to correlate mast cell occurrence with the emergence of myeloid as well as plasmacytoid dendritic cells. Also, mast cell emergence was correlated with the number of pro-inflammatory T cells. For this, plaques were classified as stable or unstable according to established histological criteria. RESULTS: As expected, atherosclerotic lesions showed significantly higher numbers of tryptase+, chymase+, and cathepsin G+ mast cells compared to control vessels, particularly in lesions with unstable morphology. As a novel finding, we detected significant correlations between mast cells and myeloid dendritic cells (fascin, CD83, r > 0.3, p < 0.01), but not plasmacytoid dendritic cells (CD123, CD304). Also, we observed significant correlations of mast cells and different subgroups of pro-inflammatory T cells (CD3, CD8, CD161, CD25; r > 0.35, p < 0.05). CONCLUSIONS: Overall, the higher number of mast cells in plaques, particularly with unstable morphology, suggests that mast cells might be involved in the progression of atherosclerosis. The correlation of mast cells with other immune cells that are pivotal in atherogenesis, e.g., myeloid dendritic cells and pro-inflammatory T cells, also suggests an interplay leading to plaque destabilization. Therefore, modulating local mast cell function and invasion into the plaque might be a therapeutic tool for plaque stabilization.
Subject(s)
Carotid Arteries/immunology , Carotid Stenosis/immunology , Dendritic Cells/immunology , Femoral Artery/immunology , Inflammation/immunology , Mast Cells/immunology , Myeloid Cells/immunology , Peripheral Arterial Disease/immunology , Plaque, Atherosclerotic , Aged , Biomarkers/analysis , Carotid Arteries/enzymology , Carotid Arteries/pathology , Carotid Stenosis/enzymology , Carotid Stenosis/pathology , Case-Control Studies , Dendritic Cells/enzymology , Dendritic Cells/pathology , Disease Progression , Female , Femoral Artery/enzymology , Femoral Artery/pathology , Humans , Inflammation/enzymology , Inflammation/pathology , Male , Mast Cells/enzymology , Mast Cells/pathology , Middle Aged , Myeloid Cells/enzymology , Myeloid Cells/pathology , Peripheral Arterial Disease/enzymology , Peripheral Arterial Disease/pathology , Prognosis , Rupture, SpontaneousABSTRACT
BACKGROUND AND PURPOSE: Interleukin-16 (IL-16) functions as a regulator of T-cell growth and acts as an inducer of cell migration. The aim of this study was to determine whether IL-16 measured in human carotid plaques was associated with symptoms (eg, stroke, transient ischemic attack, or amaurosis fugax), markers of plaque stability, and postoperative cardiovascular events. METHODS: Plaques obtained from patients who had ≥1 cerebrovascular ischemic events within 1 month before endarterectomy (n=111) were compared with plaques from patients without symptoms (n=95). Neutral lipids, smooth muscle cell, and macrophage contents were evaluated histologically, and collagen, elastin, and caspase-3 activity were measured biochemically. IL-16, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were measured in plaque homogenates using a multiplex immunoassay. IL-16, CD3, CD4, and FoxP3 mRNA expressions in carotid plaques were analyzed with quantitative real-time polymerase chain reaction. RESULTS: Carotid plaques from asymptomatic patients had higher levels of IL-16 mRNA. High plaque IL-16 protein levels (above median) were associated with reduced incidence of postoperative cardiovascular events during a mean follow-up of 21 months (hazard ratio, 0.47; 95% confidence interval, 0.22-0.99; P=0.047). IL-16 levels correlated with the plaque-stabilizing components: elastin, collagen, matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2 and FoxP3 mRNA. CONCLUSIONS: This study shows that high levels of IL-16 are associated with asymptomatic carotid plaques, expression of factors contributing to plaque stability, and decreased risk of new cardiovascular events during a 2-year period after surgery, suggesting that IL-16 might have a protective role in human atherosclerotic disease.
Subject(s)
Carotid Stenosis/complications , Carotid Stenosis/immunology , Interleukin-16/biosynthesis , Intracranial Arteriosclerosis/epidemiology , Ischemic Attack, Transient/epidemiology , Stroke/epidemiology , Aged , Biomarkers/analysis , Female , Humans , Interleukin-16/analysis , Intracranial Arteriosclerosis/etiology , Ischemic Attack, Transient/etiology , Male , Middle Aged , Polymerase Chain Reaction , Risk Factors , Stroke/etiologyABSTRACT
OBJECTIVE: Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Unstable atherosclerotic plaques are characterized by a large necrotic core. In this study we investigated the distribution and interaction between gene polymorphisms encoding proinflammatory molecules in an Italian population with internal carotid artery stenosis (ICAS). We also evaluated whether reciprocal interaction between these gene polymorphisms increased the risk of plaque vulnerability. METHODS: In this genetic association study, 11 proinflammatory gene polymorphisms were analyzed in 933 individuals comprising 344 patients with ICAS who underwent carotid endarterectomy and 589 controls without ultrasound evidence of atherosclerosis or intimal thickening. RESULTS: We found that interleukin (IL) 6 (IL-6), IL-1ß, monocyte chemoattractant protein-1 (CCL2) macrophage inflammatory protein-1α (CCL3), E-selectin (SELE), intercellular adhesion molecule 1 (ICAM1), and matrix metalloproteinase-3 (MMP-3), and 9 (MMP-9) gene variants were independently and significantly associated with ICAS. The association remained significant even after the Bonferroni correction. We also found a genetic profile associated with different risks for ICAS, depending on the number of high-risk genotypes simultaneously present in an individual. Furthermore, proinflammatory genetic profiles are significantly more common in individuals with unstable carotid plaque. CONCLUSIONS: Our study shows, for the first time, a reciprocal interaction between proinflammatory genotypes for the development and progression of ICAS.
Subject(s)
Carotid Artery, Internal/pathology , Carotid Stenosis/genetics , Inflammation Mediators , Inflammation/genetics , Plaque, Atherosclerotic , Polymorphism, Single Nucleotide , Aged , Carotid Artery, Internal/diagnostic imaging , Carotid Artery, Internal/surgery , Carotid Intima-Media Thickness , Carotid Stenosis/diagnosis , Carotid Stenosis/immunology , Carotid Stenosis/surgery , Case-Control Studies , Disease Progression , Endarterectomy, Carotid , Female , Gene Frequency , Genetic Association Studies , Genetic Markers , Genetic Predisposition to Disease , Humans , Inflammation/diagnostic imaging , Inflammation/immunology , Italy , Male , Phenotype , Predictive Value of Tests , Risk Factors , Rupture, SpontaneousABSTRACT
OBJECTIVE: Accumulating evidence shows that immune cells play an important role in atherosclerosis. Most attention has focused on the role of different T cell subsets, whereas the possible involvement of B cells has been less studied. In this study, we assessed the association of 2 different B cell subsets, CD19(+)CD40(+) and CD19(+)CD86(+) B cells, with risk for development of acute cardiovascular events. APPROACH AND RESULTS: The prospective study included 700 subjects randomly selected from the cardiovascular cohort of the Malmö Diet and Cancer study. Mononuclear leukocytes, stored at -140(â)C at the baseline investigation in 1991-1994, were thawed and B cell subsets analyzed by flow cytometry. Cytokine release from CD3/CD28-stimulated mononuclear leukocytes was measured with multiplex ELISA. Baseline carotid intima-media thickness and stenosis were assessed by ultrasonography, and clinical events were monitored through validated national registers during a median/mean follow-up time of 15 years. The subjects in the highest tertile of CD19(+)CD40(+) B cells had a significantly lower risk of incident stroke after adjustment for other risk factors. In contrast, CD19(+)CD86(+) B cells were associated with higher risk for development of a stroke event and increased release of proinflammatory cytokines from mononuclear leukocytes. CONCLUSIONS: These observations provide evidence for an involvement of B cells in the incidence of stroke and suggest that both pathogenic and protective B cell subsets exist.
Subject(s)
B-Lymphocyte Subsets/immunology , B7-2 Antigen/blood , CD40 Antigens/blood , Carotid Stenosis/immunology , Stroke/immunology , Aged , Antigens, CD19/blood , Biomarkers/blood , Carotid Intima-Media Thickness , Carotid Stenosis/blood , Carotid Stenosis/diagnosis , Carotid Stenosis/epidemiology , Cells, Cultured , Chi-Square Distribution , Cytokines/metabolism , Disease-Free Survival , Female , Humans , Incidence , Inflammation Mediators/metabolism , Kaplan-Meier Estimate , Linear Models , Male , Middle Aged , Proportional Hazards Models , Prospective Studies , Risk Factors , Stroke/blood , Stroke/diagnosis , Stroke/epidemiology , Sweden/epidemiology , Time FactorsABSTRACT
RATIONALE: Nuclear factor of activated T-cells (NFAT) is importantly implicated in pathological cardiac remodeling and vascular lesion formation. NFAT functionality is mainly regulated by calcineurin, a Ca(2+)-dependent multi-effector phosphatase. Calcineurin inhibitors such as cyclosporine A (CsA) were shown to be effective in the treatment of restenosis and vascular inflammation but with adverse side effects. OBJECTIVE: This prompted the design of more selective inhibitors such as VIVIT and inhibitors of NFAT-calcineurin association, which unfortunately have a poor potency precluding clinical use. METHODS AND RESULTS: Here, we describe the rational design of a potent bipartite inhibitor of NFAT-calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs. Modeling, site-directed mutagenesis, and functional studies demonstrated that MCV1 acts by allosteric modulation of calcineurin. Comparable to CsA, MCV1 prevents NFAT activation at nanomolar potency without impairing calcineurin phosphatase activity, nuclear factor-κB nuclear import, and general cell signaling. In contrast, CsA but not MCV1-activated basal level extracellular signal-regulated kinases activity and prevented nuclear import of calcineurin, independent of NFAT activation. In vivo MCV1 abrogated NFAT-mediated T-cell activation in a model of PMA-elicited peritonitis, whereas topical application of MCV1 markedly reduced neointima formation in a mouse model of restenosis. CONCLUSIONS: We designed a bipartite NFAT inhibitor that is more potent than VIVIT and more selective than CsA. MCV1 constitutes not only a powerful tool to unravel NFAT function but also a potential drug candidate for the treatment of diseases implicating NFAT activation.
Subject(s)
Carotid Artery Injuries/drug therapy , Carotid Artery, Common/drug effects , Carotid Stenosis/drug therapy , Cell Proliferation/drug effects , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , NFATC Transcription Factors/antagonists & inhibitors , Peptides/pharmacology , Peritonitis/drug therapy , T-Lymphocytes/drug effects , Amino Acid Motifs , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , COS Cells , Calcineurin/metabolism , Carotid Artery Injuries/immunology , Carotid Artery Injuries/metabolism , Carotid Artery Injuries/pathology , Carotid Artery, Common/immunology , Carotid Artery, Common/metabolism , Carotid Artery, Common/pathology , Carotid Stenosis/immunology , Carotid Stenosis/metabolism , Carotid Stenosis/pathology , Chlorocebus aethiops , Cyclosporine/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Design , HEK293 Cells , Humans , Hyperplasia , Immunosuppressive Agents/chemistry , Jurkat Cells , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Peptides/chemistry , Peritonitis/immunology , Peritonitis/metabolism , Recurrence , Signal Transduction/drug effects , Structure-Activity Relationship , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TransfectionABSTRACT
BACKGROUND AND PURPOSE: Several studies have reported moyamoya syndrome associated with thyroid disease, and the mechanism involved in this relationship is unknown. This study aimed to clarify the involvement of thyroid antibodies and thyroid function in intracranial arterial stenosis. METHODS: The study included 30 patients <65 years of age with intracranial arterial steno-occlusion. Patients with definitive moyamoya disease were excluded. Thyroid function and thyroid antibody levels were evaluated. The steno-occlusive site and the presence of moyamoya vessels were evaluated using digital subtraction angiography. The characteristics of intracranial arterial lesions were compared between patients with and without elevated thyroid antibody levels, and between patients with increased thyroid function and those with normal thyroid function. RESULTS: Five patients had increased thyroid function and seven had elevated thyroid antibody levels. Four were diagnosed with Graves' disease, 13 with atherosclerotic intracranial stenosis, two with intracranial arterial dissection, one with vasculitis syndrome and 10 with intracranial stenosis of unknown cause. All patients with Graves' disease and patients with elevated antithyroid peroxidase antibody levels had steno-occlusion in the terminal portion of the internal carotid arteries, whereas most of the patients with normal thyroid function or without elevated thyroid antibody levels had stenosis in the middle cerebral arteries. CONCLUSIONS: In young and middle-aged patients, a lesion in the terminal portion of the internal carotid artery was associated with elevated thyroid antibody levels and increased thyroid function. Stenoses found in the terminal portion of the internal carotid artery and immune-mediated thyroid diseases may share a common background.
Subject(s)
Autoantibodies/blood , Carotid Artery, Internal/pathology , Carotid Stenosis/immunology , Moyamoya Disease/immunology , Thyroid Diseases/immunology , Adult , Carotid Stenosis/blood , Carotid Stenosis/pathology , Female , Humans , Male , Middle Aged , Moyamoya Disease/blood , Moyamoya Disease/pathology , Thyroid Diseases/blood , Thyroid Diseases/pathologyABSTRACT
OBJECTIVE: Carotid plaque instability is a major cause of ischemic stroke, but detailed knowledge about underlying molecular pathways is still lacking. Here, we evaluated large-scale transcriptomic and protein expression profiling in a biobank of carotid endarterectomies followed by characterization of identified candidates, as a platform for discovery of novel proteins differentially regulated in unstable carotid lesions. APPROACH AND RESULTS: Genes highly upregulated in symptomatic versus asymptomatic plaques were selected from Affymetrix microarray analyses (n=127 plaques), and tissue microarrays constructed from 34 lesions were assayed for 21 corresponding proteins by immunohistochemistry. Quantification of stainings demonstrated differential expression of CD36, CD137, and DOCK7 (P<0.05) in unstable versus stable lesions and the most significant upregulation of a proprotein convertase, PCSK6 (P<0.0001). Increased expression of PCSK6 in symptomatic lesions was verified by quantitative real-time polymerase chain reaction (n=233), and the protein was localized to smooth muscle α-actin positive cells and extracellular matrix of the fibrous cap by immunohistochemistry. PCSK6 expression positively correlated to genes associated with inflammation, matrix degradation, and mitogens in microarrays. Stimulation of human carotid smooth muscle cells in vitro with cytokines caused rapid induction of PCSK6 mRNA. CONCLUSIONS: Using a combination of transcriptomic and tissue microarray profiling, we demonstrate a novel approach to identify proteins differentially expressed in unstable carotid atherosclerosis. The proprotein convertase PCSK6 was detected at increased levels in the fibrous cap of symptomatic carotid plaques, possibly associated with key processes in plaque rupture such as inflammation and extracellular matrix remodeling. Further studies are needed to clarify the role of PCSK6 in atherosclerosis.
Subject(s)
Carotid Stenosis/enzymology , Carotid Stenosis/genetics , Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis , Proprotein Convertases/genetics , Proprotein Convertases/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Tissue Array Analysis , Asymptomatic Diseases , Carotid Stenosis/immunology , Carotid Stenosis/pathology , Cells, Cultured , Cytokines/metabolism , Extracellular Matrix/metabolism , Fibrosis , Humans , Immunohistochemistry , Inflammation Mediators/metabolism , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/immunology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/immunology , Plaque, Atherosclerotic , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Rupture, Spontaneous , Up-RegulationABSTRACT
BACKGROUND: Inflammation has been closely linked to auto-immunogenic processes in atherosclerosis. Plasmacytoid dendritic cells (pDCs) are specialized to produce type-I interferons in response to pathogenic single-stranded nucleic acids, but can also sense self-DNA released from dying cells or in neutrophil extracellular traps complexed to the antimicrobial peptide Cramp/LL37 in autoimmune disease. However, the exact role of pDCs in atherosclerosis remains elusive. METHODS AND RESULTS: Here we demonstrate that pDCs can be detected in murine and human atherosclerotic lesions. Exposure to oxidatively modified low-density lipoprotein enhanced the capacity of pDCs to phagocytose and prime antigen-specific T cell responses. Plasmacytoid DCs can be stimulated to produce interferon-α by Cramp/DNA complexes, and we further identified increased expression of Cramp and formation of neutrophil extracellular traps in atherosclerotic arteries. Whereas Cramp/DNA complexes aggravated atherosclerotic lesion formation in apolipoprotein E-deficient mice, pDC depletion and Cramp-deficiency in bone marrow reduced atherosclerosis and anti-double-stranded DNA antibody titers. Moreover, the specific activation of pDCs and interferon-α treatment promoted plaque growth, associated with enhanced anti-double-stranded-DNA antibody titers. Accordingly, anti-double-stranded DNA antibodies were elevated in patients with symptomatic versus asymptomatic carotid artery stenosis. CONCLUSIONS: Self-DNA (eg, released from dying cells or in neutrophil extracellular traps) and an increased expression of the antimicrobial peptide Cramp/LL37 in atherosclerotic lesions may thus stimulate a pDC-driven pathway of autoimmune activation and the generation of anti-double-stranded-DNA antibodies, critically aggravating atherosclerosis lesion formation. These key factors may thus represent novel therapeutic targets.
Subject(s)
Atherosclerosis/immunology , Autoantigens/immunology , DNA/immunology , Dendritic Cells/immunology , Proteins/genetics , Proteins/immunology , Animals , Atherosclerosis/genetics , Atherosclerosis/metabolism , Autoantigens/genetics , Carotid Stenosis/genetics , Carotid Stenosis/immunology , Carotid Stenosis/metabolism , DNA/genetics , Dendritic Cells/metabolism , Female , Humans , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
BACKGROUND: The findings of experimental studies suggest that the immune system plays a key role in atherosclerosis, but the clinical importance of different immune cells in cardiovascular disease remains poorly characterized. In this study we investigated the association between CD8(+) T cells and carotid disease as well as development of cardiovascular disease events. METHODS: The study cohort comprised 700 subjects from the cardiovascular arm of the Malmö Diet and Cancer Study. Peripheral blood mononuclear cells, obtained at the 1991-1994 baseline investigation and stored at -140 °C, were thawed and the different CD8(+) T-cell populations analysed by flow cytometry. Baseline carotid intima-media thickness and stenosis were assessed by ultrasonography and clinical events were monitored through validated national registers. RESULTS: Subjects with a high fraction of CD8(+) T cells were characterized by decreased cytokine release from activated leucocytes, metabolic signs of insulin resistance and increased incidence of coronary events; hazard ratios (95% confidence intervals) for the second and third tertiles of CD8(+) T cells were 2.57 (1.16, 5.67) and 2.61 (1.19, 5,71), respectively, in a Cox proportional hazards regression model. Correlations were found between the fraction of CD8(+) CD25(+) T cells and the degree of carotid stenosis (r = 0.11, P < 0.01), and between the CD8(+) CD56(-) IFN-γ(+) T-cell fraction and the degree of stenosis (r = -0.18, P < 0.005). The association between CD8(+) CD56(-) IFN-γ(+) T cells and carotid stenosis remained significant after controlling for major cardiovascular disease risk factors. CONCLUSION: This study provides prospective clinical evidence for a role of CD8(+) T cells in cardiovascular disease and suggests the existence of CD8(+) T-cell subsets with different pathological functions.
Subject(s)
CD8-Positive T-Lymphocytes , Carotid Intima-Media Thickness , Carotid Stenosis/immunology , Coronary Artery Disease/immunology , Stroke/immunology , Aged , Biomarkers/blood , CD56 Antigen/analysis , CD8-Positive T-Lymphocytes/immunology , Cardiovascular Diseases/immunology , Carotid Stenosis/diagnostic imaging , Carotid Stenosis/epidemiology , Carotid Stenosis/physiopathology , Cohort Studies , Coronary Artery Disease/diagnostic imaging , Coronary Artery Disease/epidemiology , Coronary Artery Disease/physiopathology , Cytokines/metabolism , Female , Flow Cytometry , Humans , Incidence , Interleukin-2 Receptor alpha Subunit/analysis , Kaplan-Meier Estimate , Leukocytes/immunology , Male , Middle Aged , Odds Ratio , Predictive Value of Tests , Proportional Hazards Models , Prospective Studies , Registries , Risk Assessment , Risk Factors , Severity of Illness Index , Stroke/diagnostic imaging , Stroke/epidemiology , Stroke/physiopathology , Sweden/epidemiologyABSTRACT
Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 ± 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 ± 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 ± 16 vs. 146 ± 19 pg/ml; TNFα 3.8 ± 1.0 vs. 6.6 ± 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFα and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 ± 4 vs. 7 ± 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.
Subject(s)
Apoptosis , Carotid Arteries/enzymology , Carotid Stenosis/enzymology , Cell Differentiation , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Inflammation/enzymology , Macrophage Activation , Macrophages/enzymology , Carotid Arteries/immunology , Carotid Arteries/pathology , Carotid Arteries/surgery , Carotid Stenosis/immunology , Carotid Stenosis/pathology , Carotid Stenosis/surgery , Cell Differentiation/drug effects , Dipeptidases/metabolism , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Endarterectomy, Carotid , Humans , Inflammation/immunology , Inflammation/pathology , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Protease Inhibitors/pharmacology , RNA Interference , Time Factors , Transfection , Tumor Necrosis Factor-alpha/metabolism , U937 CellsABSTRACT
OBJECTIVE: Severe carotid stenosis is a frequent cause of stroke in both men and women. Whereas several sex-related comparisons are available on coronary atherosclerosis, there are few data appraising gender-specific features of carotid plaques. We aimed to systematically compare the pathology and inflammatory features of carotid plaques in men vs women. METHODS: Carotid plaque specimens were collected from patients undergoing surgical endarterectomy for asymptomatic or symptomatic carotid stenosis. Histologic analysis was performed, as well as measurements of plaque composition and inflammation. RESULTS: A total of 457 patients were included (132 women, 325 men). Baseline analyses showed a greater prevalence of hypercholesterolemia, hypertension, and former smoking status in women, despite a higher Framingham Heart Score in men (all P < .05). Women had a lower prevalence of thrombotic plaques, smaller percentage area of necrotic core, and hemorrhage extension (all P < .05). Plaque inflammation analysis showed a lower concentration of inflammatory and, in particular, of macrophage foam cells in the plaque cap of women (both P < .05). These differences were, however, no longer significant at multivariable analysis, including several baseline features, such as symptom status and stenosis severity. CONCLUSIONS: Carotid plaques seem significantly different in women and men, but the main drivers of such pathologic differences are baseline features, including stenosis severity and symptom status.