Enrichment of type 1 innate lymphoid cells in the course of human atherosclerotic plaque development suggests contribution to atherogenesis.

Introduction: Innate lymphoid cells (ILCs) have been implicated in multiple pathologic conditions, including atherogenesis, as documented in experimental mice studies, however, their role in atherosclerosis in humans remains unexplored. Methods: Here, we identify ILCs and their dynamics in early, advanced, and complicated human carotid- and aortic atherosclerotic plaques, using a multiplex immunohistochemical quadruple-staining technique with prototypic transcription factors T-bet, GATA3, or RORgt for identification of the ILC1, ILC2 and ILC3 subsets, respectively, in combination with lineage markers CD3, CD20/ CD79a and CD56 to exclude other lymphoid cell types. ILC subsets were quantified, and to put this in perspective, their numbers were expressed as percentage of the total number of infiltrated lymphoid cells and related to the frequency of conventional T cells, B cells, NK cells, and NKT cells. Results: All ILC subsets were present in every different stage of atherogenesis. ILC1s were the most abundant ILC subset, and their numbers significantly increased in the course of plaque development, but paradoxically, their relative frequency was reduced because of a higher increment of T cells and B cells. The numbers of ILC2s and ILC3s also gradually increased, but this trend did not achieve significance. T cell subsets always significantly outnumbered their ILC counterparts, except for the early lesions where the proportion of ILC1s was markedly higher, albeit not significant. Discussion: The high abundance of ILC1s in the early stages and further significant enrichment in later stages, suggest they may participate in the initiation and development of atherogenesis, and thus, may represent a novel target to prevent or treat atherosclerosis.

Prognostic Value of Histopathological Thrombus Age in Large Vessel Occlusion-Related Stroke.

INTRODUCTION: Acute mechanical thrombectomy (MT) is the preferred treatment for large vessel occlusion-related stroke. Histopathological research on the obtained occlusive embolic thrombus may provide information regarding the aetiology and pathology of the lesion to predict prognosis and propose possible future acute ischaemic stroke therapy. METHODS: A total of 75 consecutive patients who presented to the Amphia Hospital with acute large vessel occlusion-related stroke and underwent MT were included in the study. The obtained thrombus materials were subjected to standard histopathological examination. Based on histological criteria, they were considered fresh (<1 day old) or old (>1 day old). Patients were followed for 2 years for documentation of all-cause mortality. RESULTS: Thrombi were classified as fresh in 40 patients (53%) and as older in 35 patients (47%). Univariate Cox regression analysis showed that thrombus age, National Institutes of Health Stroke Scale at hospital admission, and patient age were associated with long-term mortality (p < 0.1). Multivariable Cox hazards and Kaplan-Meier analysis demonstrated that after extensive adjustment for clinical and procedural variables, thrombus age persisted in being independently associated with higher long-term mortality (hazard ratio: 3.34; p = 0.038, log-rank p = 0.013). CONCLUSION: In this study, older thromboemboli are responsible for almost half of acute large ischaemic strokes. Moreover, the presence of an old thrombus is an independent predictor of mortality in acute large vessel occlusion-related stroke. More research is warranted regarding future therapies based on thrombus composition.

Etosis, rather than apoptosis or cell proliferation, typifies thrombus progression - An immunohistochemical study of coronary aspirates.

BACKGROUND: Coronary thrombosis is a process with unpredictable clinical outcome. Changes of thrombus composition overtime influence tissue repair and stabilization. We investigated rates of cell deaths and cell proliferation at different time points after initiation of thrombosis. METHODS: Thrombectomy aspirates of 55 myocardial infarction patients were selected and histomorphologically classified as fresh (25), lytic (25), partially fibrocellular (10), completely fibrocellular (10). Paraffin sections were immunostained with anti-(cleaved) caspase-3/Casp3 (apoptosis), Citrullinated histone/CitH 3 (etosis), C-reactive protein/CRP and Ki67 (proliferation) in combination with either Feulgen counterstaining (DNA) or cell markers for granulocytes, macrophages, SMCs, platelets and endothelium. Rates of apoptosis, etosis and proliferation were measured as a percentage of total number of immunopositive pixels versus total number of DNA positive pixels, while co-localization with cell markers was assessed by digital image analysis. RESULTS: Positive staining of CitH3 was observed more frequently (93%) than Casp3 (70%), Ki67 (79%) or CRP (59%) (p < 0.05). Moreover, rate of etosis, found in granulocytes and macrophages, differed significantly among thrombi of different age, being higher in lytic (12.82) than in fresh (8.52) and late-organized (2.75) (p < 0.05). Such differences were not observed for the rates of apoptosis or cell proliferation related to thrombus age. CRP staining was present in fresh, lytic and organized thrombi, but did not reliably identify necrotic areas. CONCLUSIONS: Different patterns of cell death and cell proliferation are noticed during progression of coronary thrombus overtime, but with significant differences for only etosis. Etosis could potentially serve as a biomarker for thrombus instability with clinical significance.

Ischemia Reperfusion Injury Produces, and Ischemic Preconditioning Prevents, Rat Cardiac Fibroblast Differentiation: Role of KATP Channels.

Ischemic preconditioning (IPC) and activation of ATP-sensitive potassium channels (KATP) protect cardiac myocytes from ischemia reperfusion (IR) injury. We investigated the influence of IR injury, IPC and KATP in isolated rat cardiac fibroblasts. Hearts were removed under isoflurane anesthesia. IR was simulated in vitro by application and removal of paraffin oil over pelleted cells. Ischemia (30, 60 and 120 min) followed by 60 min reperfusion resulted in significant differentiation of fibroblasts into myofibroblasts in culture (mean % fibroblasts ± SEM in IR vs. time control: 12 ± 1% vs. 63 ± 2%, 30 min ischemia; 15 ± 3% vs. 71 ± 4%, 60 min ischemia; 8 ± 1% vs. 55 ± 2%, 120 min ischemia). IPC (15 min ischemia, 30 min reperfusion) significantly attenuated IR-induced fibroblast differentiation (52 ± 3%) compared to 60 min IR. IPC was mimicked by opening KATP with pinacidil (50 µM; 43 ± 6%) and by selectively opening mitochondrial KATP (mKATP) with diazoxide (100 µM; 53 ± 3%). Furthermore, IPC was attenuated by inhibiting KATP with glibenclamide (10 µM; 23 ± 5%) and by selectively blocking mKATP with 5-hydroxydecanoate (100 µM; 22 ± 9%). These results suggest that (a) IR injury evoked cardiac fibroblast to myofibroblast differentiation, (b) IPC attenuated IR-induced fibroblast differentiation, (c) KATP were involved in IPC and (d) this protection involved selective activation of mKATP.

Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis.

Extracellular traps generated by neutrophils contribute to thrombus progression in coronary atherosclerotic plaques. It is not known whether other inflammatory cell types in coronary atherosclerotic plaque or thrombus also release extracellular traps. We investigated their formation by macrophages, mast cells, and eosinophils in human coronary atherosclerosis, and in relation to the age of thrombus of myocardial infarction patients. Coronary arteries with thrombosed or intact plaques were retrieved from patients who died from myocardial infarction. In addition, thrombectomy specimens from patients with myocardial infarction were classified histologically as fresh, lytic or organised. Neutrophil and macrophage extracellular traps were identified using sequential triple immunostaining of CD68, myeloperoxidase, and citrullinated histone H3. Eosinophil and mast cell extracellular traps were visualised using double immunostaining for eosinophil major basic protein or tryptase, respectively, and citrullinated histone H3. Single- and double-stained immunopositive cells in the plaque, adjacent adventitia, and thrombus were counted. All types of leucocyte-derived extracellular traps were present in all thrombosed plaques, and in all types of the in vivo-derived thrombi, but only to a much lower extent in intact plaques. Neutrophil traps, followed by macrophage traps, were the most prominent types in the autopsy series of atherothrombotic plaques, including the adventitia adjacent to thrombosed plaques. In contrast, macrophage traps were more numerous than neutrophil traps in intact plaques (lipid cores) and organised thrombi. Mast cell and eosinophil extracellular traps were also present, but sparse in all instances. In conclusion, not only neutrophils but also macrophages, eosinophils, and mast cells are sources of etosis involved in evolving coronary thrombosis. Neutrophil traps dominate numerically in early thrombosis and macrophage traps in late (organising) thrombosis, implying that together they span all the stages of thrombus progression and maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Neutrophil Extracellular Traps Participate in All Different Types of Thrombotic and Haemorrhagic Complications of Coronary Atherosclerosis.

Acute coronary syndromes can be initiated by either atherosclerotic fibrous cap ruptures, superficial plaque erosions or intraplaque haemorrhages (IPHs). Since neutrophil extracellular traps (NETs) display pro-inflammatory and pro-thrombotic properties, we investigated the presence, extent and distribution of neutrophils and NETs in different types of plaque complications in relation to the age of overlying thrombus mass or haemorrhage. Sixty-four paraffin-embedded coronary plaque segments of 30 acute myocardial infarction patients were retrieved from the autopsy archives, which contained 44 complicated plaques (17 IPHs, 9 erosions and 18 ruptures) and 20 intact plaques. Complicated plaques were further categorized according to the histological age of thrombus or haemorrhage. Immunohistochemistry was performed to visualize neutrophils (anti-myeloperoxidase, anti-elastase and anti-CD177) and NETs (anti-citrullinated histone-3 and anti-peptidyl-arginine-deiminase-4). The results were scored semi-quantitatively. Neutrophils and NETs were abundantly present in all types of complicated, but not in intact, plaques (p < 0.05). They were found in thrombus, haemorrhages and at the thrombus-plaque interface, with no significant differences in extent between ruptures, erosions and IPHs. Interestingly, adjacent perivascular tissue of complicated, but not of intact plaques, also contained high numbers of neutrophils and NETs (p < 0.05). In thrombus and haemorrhage of different age, neutrophils and NETs were more frequently present in non-organized (fresh) thrombi and in on-going IPHs. In conclusion, netosis is a prominent pro-thrombotic participant in all distinct types of atherothrombosis, which may facilitate the progression of thrombotic or haemorrhagic complications and thus the onset of ensuing clinical coronary ischemic syndromes.

Immunophenotypic analysis of the chronological events of tissue repair in aortic medial dissections.

Acute medial dissection of aorta can occur in the context of a sudden and unexpected death. For medico-legal reasons it is important to estimate as accurately the histological age of dissections. We evaluated the additional value of a systematic application of immunohistochemistry, compared with conventional histology only, in determining chronological steps of injury and repair. Thirty two paraffin embedded specimens of aortic dissection were retrospectively allocated to one of four defined stages: acute (I), subacute (II), early organizing (III) and scarring (IV) using Hematoxylin and Eosin and Elastica van Gieson stained sections. Subsequent immunohistochemically staining was performed with the following markers: (myeloperoxidase (neutrophils), citrullinated-Histone 3 (neutrophil extracellular traps), CD68 (macrophages), CD3 (T-cells), CD31 and CD34 (endothelial cells), and smooth muscle actin. Immune stained sections were scored semi-quantitatively. Histologically, five cases were identified as stage I, 16 as II, 7 as III and 4 as IV. Additional immunostaining for smooth muscle cells and endothelial cells altered the classification in 25% of cases (all in groups II and III). Immunostaining and semi-quantitative grading of involvement of neutrophils, macrophages and NETs also provided specific distribution patterns over the 4 age categories, including unexpected involvement of the peri adventitial fat tissue. In conclusion, it appears that semi-quantitative immunohistochemistry of resident vascular wall cells, inflammatory cells and NETS represents a useful adjunct in detailed histopathological grading of the chronological age of aortic dissections.