Human Carotid Research Articles

MicroRNA-216a Promotes Endothelial Inflammation by Smad7/IκBα Pathway in Atherosclerosis.

Background The endothelium is the first line of defence against harmful microenvironment risks, and microRNAs (miRNAs) involved in vascular inflammation may be promising therapeutic targets to modulate atherosclerosis progression. In this study, we aimed to investigate the mechanism by which microRNA-216a (miR-216a) modulated inflammation activation of endothelial cells. Methods. A replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established, and population-doubling levels (PDLs) were defined during passages. PDL8 HUVECs were transfected with miR-216a mimics/inhibitor or small interfering RNA (siRNA) of SMAD family member 7 (Smad7). Real-time PCR and Western blot assays were performed to detect the regulatory role of miR-216a on Smad7 and NF-κB inhibitor alpha (IκBα) expression. The effect of miR-216a on adhesive capability of HUVECs to THP-1 cells was examined. MiR-216a and Smad7 expression in vivo were measured using human carotid atherosclerotic plaques of the patients who underwent carotid endarterectomy (n = 41). Results Luciferase assays showed that Smad7 was a direct target of miR-216a. Smad7 mRNA expression, negatively correlated with miR-216a during endothelial aging, was downregulated in senescent PDL44 cells, compared with young PDL8 HUVECs. MiR-216a markedly increased endothelial inflammation and adhesive capability to monocytes in PDL8 cells by promoting the phosphorylation and degradation of IκBα and then activating NF-κB signalling pathway. The effect of miR-216a on endothelial cells was consistent with that blocked Smad7 by siRNAs. When inhibiting endogenous miR-216a, the Smad7/IκBα expression was rescued, which led to decreased endothelial inflammation and monocytes recruitment. In human carotid atherosclerotic plaques, Smad7 level was remarkably decreased in high miR-216a group compared with low miR-216a group. Moreover, miR-216a was negatively correlated with Smad7 and IκBα levels and positively correlated with interleukin 1 beta (IL1β) expression in vivo. Conclusion In summary, our findings suggest a new mechanism of vascular endothelial inflammation involving Smad7/IκBα signalling pathway in atherosclerosis.

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability.

Aims6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158.Methods and ResultsPFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6–8 weeks old male Ldlr–/– mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization.ConclusionHigh PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis.

Atlas of the immune cell repertoire in human atherosclerotic plaques characterized by single cell RNA-sequencing and multi-color flow cytometry

Abstract Rationale Atherosclerosis is a chronic inflammatory disease that is driven by the accumulation of pro- and anti-inflammatory leukocytes in the intima of affected arteries. Yet, the cellular composition of human atherosclerotic plaques is only poorly understood. Here, we characterized immune cells to human carotid atherosclerotic plaques by multi-color flow cytometry and scRNAseq. Methods and results First, we compared a set of previously reported digestion protocols to liberate leukocytes from human carotid plaques after surgical thrombendarteriectomy. One digestion cocktail, containing Collagenase IV and DNase I, was superior regarding cell survival and cell surface marker preservation. Second, leukocytes from 56 surgical specimen were characterized by flow cytometry with a set of 16 parameters and cell surface markers capable of identifying principal hematopoietic leukocyte lineages. This protocol allowed to extract and analyze on average 4x103 viable CD45+ leukocytes from a mean of 988 mg plaque tissue. Surprisingly, we found that atherosclerotic plaques were dominated by T cells with 33.7±2.2% CD4+ T-helper cells and 25.6±2.5% CD8+ cytotoxic T cells. CD11b+ myeloid cells, including monocytes and macrophages, represented only 20.2±4.0% of all CD45+ leukocytes. CD19+B cells and CD56+ NK-cells accounted for 3.9±1.2 and 3.3±0.5%, respectively. TCR-g/d+ T cells and neutrophils were undetectable in atherosclerotic plaques. This cellular composition differed significantly from peripheral blood, but was not relevantly changed between different plaque locations, indicating that macrophage-rich necrotic cores mostly contain dead cells. We confirmed the principal composition of human plaques by single-cell RNA-sequencing from six patients. To allow an estimation of cellular heterogeneity independent of classical cell surface marker assignment, we performed an unsupervised cluster detection algorithm by t-distributed stochastic neighbor embedding (tSNE) and found more than 16 leukocyte clusters with unique cell surface marker expression, suggesting an unexpected high diversity of plaque leukocytes. Conclusion We developed an immune cell phenotyping protocol optimized for human carotid plaques. The definition of phenotypes and frequencies in atherosclerotic plaques will allow to build clinical associations between the immune cell composition and clinical outcomes in future. Funding Acknowledgement Type of funding source: None

Deficiency of the purinergic receptor P2X4 limits atherosclerosis in mice

Abstract Introduction Extracellular nucleotides like ATP promote inflammation as danger signals in various chronic diseases via purinergic receptors. In our previous work we identified P2X4 expression in murine atherosclerotic lesions. Therefore, we hypothesized a contribution of the ATP-P2X4 axis to vascular inflammation in atherosclerosis. Methods To investigate the functional role of P2X4 in atherogenesis, wild-type LDL-receptor deficient mice (LDLR−/−) and P2X4-deficient LDLR−/− mice (P2X4−/−LDLR−/−) were fed a high cholesterol diet for 16 weeks. Plaque progression in aortic arches was monitored by echography at intervals of 4 weeks, and leukocyte subsets in blood samples were analysed by flow cytometry. Atherosclerotic lesions were then assessed histologically in aortic root, arch, and abdominal aorta. In order to assess leukocyte recruitment, intravital microscopy was performed after injection of ATP in P2X4−/− or wildtype mice (WT). Regarding transferability to human disease, atherosclerotic plaque from carotid endarterectomy has been stained immunohistochemically for P2X4-receptor expression. Results After 16 weeks, P2X4-deficient mice showed significantly reduced atherosclerotic lesions in the aortic root (n=40, LDLR−/−: 0.47 mm2, P2X4−/−LDLR−/−: 0.39 mm2, p=0.04). Ly6C- monocyte count in peripheral blood was higher in P2X4−/−LDLR−/− (n=32, LDLR−/−: 241/μl, P2X4−/−LDLR−/−: 542/μl, p=0.0088), shifting the balance to a more anti-inflammatory subset. Memory-cell generation of CD4-T-cells is significantly higher in knockout-mice, suggesting an involvement of T-helper cells (n=25, LDLR−/−: 27%, P2X4−/−LDLR−/−: 46%, p=0.0003). Peritoneally injected ATP induced leukocyte rolling in WT, but not in P2X4-deficient mice. In human carotid arteries, atherosclerotic plaque shows higher staining for P2X4−/− receptor than not diseased areas. Conclusion P2X4-deficiency enhances anti-inflammatory leukocytes in peripheral blood and reduces atherosclerosis. Therefore, blocking the ATP-P2X4 axis may prevent leukocyte recruitment to atherosclerotic lesions and could present a potential new target for anti-atherogenic therapy. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This work was supported by a research grant of the German Research Foundation (DFG) to Peter Stachon. Sebastian König was supported by a research grant of the German Cardiac Society (DGK)

P.059 Identifying the role of trace amine-associated receptor 9 in behaviour, brain neurochemistry and blood biochemistry

Accelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined.We examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH.FOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and β-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls.FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Associations Between Plasma Kinin B1 Receptor Levels and the Presence and Severity of Coronary Artery Disease.

Aim: Kinin B1 receptor (KB1R) was shown to be up-regulated in human carotid atherosclerotic lesions. Serum KB1R levels were also reported to be high in patients with stroke. However, KB1R deficiency increased atherosclerotic lesions. Therefore, the role of KB1R in atherosclerosis remains unclear. Moreover, no study has reported blood KB1R levels in patients with coronary artery disease (CAD). Methods: We measured plasma KB1R levels in 375 patients undergoing coronary angiography. The severity of CAD was represented as the numbers of ＞50% stenotic vessels and segments and the severity score. Results: CAD was found in 197 patients, of whom 89 had 1-vessel disease (1-VD), 62 had 2-VD, and 46 had 3-VD. Plasma KB1R levels were higher in 197 patients with CAD than in 178 without CAD (median 83.3 vs. 73.7 pg/mL, p ＜0.01). A stepwise increase in KB1R levels was found depending on the number of stenotic vessels: 77.1 in 1-VD, 87.8 in 2-VD, and 88.5 pg/mL in 3-VD ( p ＜0.025). A high KB1R level (＞90.0 pg/mL) was present in 30% of patients with CAD(-), 39% of 1-VD, 50% of 2-VD, and 48% of 3-VD ( p ＜0.025). KB1R levels correlated with the number of stenotic segments and the severity score (r=0.14 and r=0.17, p ＜0.01). In multivariate analysis, KB1R levels were an independent factor associated with CAD. Odds ratio for CAD was 1.62 (95%CI=1.02-2.58) for high KB1R level ＞90.0 pg/mL. Conclusion: Plasma KB1R levels in patients with CAD were high and were associated with the presence and severity of CAD independent of atherosclerotic risk factors.

Reliability assessment on intravascular photoacoustic imaging of lipid: ex vivo animal and human sample validation.

Although the lipid-detecting IVPA imaging system has been developed in resolution, speed, and catheter size, there is no parameterization study of the reliability on the IVPA imaging for lipid diagnosis. Here, the sensitivity, specificity, and accuracy were calculated to assess the reliability of the IVPA imaging of lipid. Abdominal aortas from six rabbits with atherosclerosis, were subjected to the IVPA imaging and Oil Red O staining, and 75 groups of IVPA as well as corresponding histological images were obtained. Similarly, 125 groups of IVPA and histological results were obtained from five human carotid plaque samples. The sensitivity, specificity, and accuracy, calculated from the statistical data, were 96.8%, 83.3%, 94.6% and 97.3%, 72.7%, 95.2%, respectively. The numerical values of sensitivity, specificity, and accuracy demonstrated the reliability of IVPA imaging on distinguishing the lesions vessel with lipid-rich plaque, which provided the foundation for IVPA translation to clinical application.

Anti-Apolipoprotein A-1 IgG Influences Neutrophil Extracellular Trap Content at Distinct Regions of Human Carotid Plaques.

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.

On the flow characteristics in different carotid arteries

Hemodynamics plays an important role in the cause of atherosclerotic disease. In this work, we simulated the flow fields in six human carotids using the sharp-interface immersed boundary method. We compare the axial velocity, the secondary flow, the vortex structures, the area with reversed flows, and the standard deviation of velocity fluctuations for different carotids. The computed results show similarities among different carotids for the overall pattern of different flow characteristics. More importantly, differences are observed in terms of the details of the area of the reversed flow region, the size and shape of the region dominated by reversed flows, and the length of time with reversed flow or dominated by secondary flows, which are strongly correlated with the formation of plaques. Further analysis shows that patient-specific features of the carotid geometry, e.g., the flare and proximal curvature of the carotid, have significant effects on these flow characteristics. This suggests that a patient-specific program is needed for the prevention and treatment of atherosclerosis.

Spatiotemporal transfer of nitric oxide in patient-specific atherosclerotic carotid artery bifurcations with MRI and computational fluid dynamics modeling

Despite great progress in nitric oxide (NO) controlled releasing strategies, the in vivo spatiotemporal NO distribution of arteries is still unclear, which makes it impossible to assess the status of arteries and render NO-based therapies in vivo largely fruitless. Here, we presented personalized computational modelling to calculate the NO distribution on the endothelial surface and in the arterial wall of human atherosclerotic carotid artery bifurcations using models constructed from MRI. The computational results indicated the distribution of NO in the atherosclerotic artery is highly uneven. The volume-weighted average NO concentration (CV) in regions with lipid plaques (9.76 ± 2.82 nM) was about 22 times higher than that in the plaque-free regions. Regions where also the calcified plaque components and the intraplaque hemorrhages are present would increase and abate the CV, respectively. The dynamic blood flow during the cycle would directly affect the distribution of NO on the endothelial surface. The luminal NO distribution is closely related to hemodynamic indicators, including wall shear stress (WSS), time averaged wall shear stress (TAWSS), oscillating shear index (OSI) and relative residence time (RRT). In conclusion, atherosclerotic components determine the space-averaged NO concentration in arterial wall and blood flow controls the luminal NO concentration.

PROFOUND ENDOTHELIAL DYSFUNCTION AS A CAUSE OF STROKE IN COVID-19: A CASE REPORT

SESSION TITLE: Medical Student/Resident Signs and Symptoms of Chest Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: There are recent reports of neurological manifestations (NMs) from COVID-191. We present a healthy male who developed multiple strokes likely from direct endothelial dysfunction (ED). CASE PRESENTATION: 66M with GERD presents with 5 days of cough, fever, and chills. He was febrile and hypoxic. Work-up revealed CRP 19 mg/dL, ferritin 594 ng/mL, D-dimer 2.01 mg/L, positive COVID-19 PCR, and normal lipid panel. He had wheezing and no NMs. He was treated with antibiotics, enoxaparin, and oxygen. On day 14 he acutely developed altered mental status, aphasia, and unilateral weakness. CT head revealed acute left ischemic infarct. CTA head/neck revealed proximal left ICA dissection. Aspirin and Atorvastatin were given. Further studies revealed D-dimer 0.4 mg/L, no inheritable hypercoagulopathy, and negative TTE and lower extremity venous duplex. MRI brain confirmed multiple acute infarcts. Left ICA thrombectomy and stent placement were performed. It was found that there was a pseudo-dissection with a ruptured plaque and an overlying thrombus resulting in a 75% stenosis. DISCUSSION: Arterial complications from COVID-19 have recently been reported.3 Studies from Wuhan, China showed that 36.7% of COVID-19 patients had NMs, most commonly dizziness (16.8%) and headache (13.1%); 5.7% had acute ischemic stroke. These patients had severe respiratory symptoms and developed neurologic signs in 1-2 days.1 Another study revealed that stroke occurred in elevated CRP (51.1 vs 12.1 ng/L) and D-dimer (6.9 vs 0.5 mg/L)2. It is believed that the virus directly invades and injures endothelial cells (ECs), changes prothrombotic factors, and promotes a hypercoagulable state secondary to a profound inflammatory response4. ACE-2-receptors on ECs are used for viral adhesion. Viral replication causes EC apoptosis, which promotes vasoconstriction, organ ischemia, tissue edema, and microvascular prothrombotic sequelae.5 In our case, COVID-19 had profound ED distal to the carotid artery bifurcation. Arterial bifurcations are sites of turbulent blood flow that are athero-prone.6 The stroke occurred in the setting of moderate elevations of D-dimer, suggesting an alternate mechanism.2 Plaque rupture occurred with prophylactic anticoagulation and normal lipid panel. At the time of the stroke, D-dimer and inflammatory markers largely normalized. His late onset NMs indicate that prothrombotic sequelae and ED persist after the cytokine storm has improved.1 Further studies are needed to investigate the duration of cytokine storm and prothrombotic state, and appropriate screening measures. CONCLUSIONS: Clinicians must be cognizant of NMs from COVID-19 such as thromboembolic strokes from direct ED. Reference #1: 1. Mao L et al. Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China. JAMA Neurol. Epub 2020 Apr 10. Reference #2: 2. Li Y, Wang M, Zhou Y, Chang J. Acute Cerebrovascular Disease Following COVID-19: A Single Center, Retrospective, Observational Study. Available at SSRN: https://ssrn.com/abstract=3550025. 2020 Mar 3. Reference #3: 3. Oxley, Thomas et al. Large-Vessel Stroke as a Presenting Feature of Covid-19 in the Young. N Engl J Med. Epub 2020 Apr 28. 4. Connors JM, Levy JH. COVID-19 and its implications for thrombosis and anticoagulation. Blood. Epub 2020 Apr 27. 5. Ferrario CM et al. Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2. Circulation. 2005;111:2605-10. 6. Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low oscillating shear stress. Atherosclerosis. 1985 May-Jun; 5:293-302 DISCLOSURES: No relevant relationships by Krishna Akella, source=Web Response No relevant relationships by Luke Alessi, source=Web Response No relevant relationships by David Capone, source=Web Response No relevant relationships by Olga McAbee, source=Web Response No relevant relationships by Heidi Roppelt, source=Web Response No relevant relationships by Fredric Weinbaum, source=Web Response No relevant relationships by Daisy Young, source=Web Response

Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics.

Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4+ and CD8+ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death

Background and aimsA vulnerable plaque is an atherosclerotic plaque that is rupture-prone with a higher risk to cause cardiovascular symptoms such as myocardial infarction or stroke. Mimecan or osteoglycin is a small leucine-rich proteoglycan, important for collagen fibrillogenesis, that has been implicated in atherosclerotic disease, yet the role of mimecan in human atherosclerotic disease remains unknown. Methods196 human atherosclerotic carotid plaques were immunostained for mimecan. Smooth muscle cells, macrophages and intraplaque haemorrhage were also measured with immunohistochemistry. Neutral lipids were stained with Oil Red O and calcium deposits were quantified. Plaque homogenate levels of MCP-1, IL-6 and MIP-1β were measured using a Proximity Extension Assay and MMP-9 levels were measured using Mesoscale. Glycosaminoglycans, collagen and elastin were assessed by colorimetric assays and TGF-β1, β2 and β3 were measured using a multiplex assay. Mimecan gene expression in THP-1 derived macrophages was quantified by qPCR and protein expression in vitro was visualized with immunofluorescence. Cardiovascular events were registered using medical charts and national registers during follow-up. ResultsMimecan correlated positively with plaque area of lipids, macrophages, intraplaque haemorrhage and inversely with smooth muscle cell staining. Mimecan also correlated positively with plaque levels of MMP-9 and MCP-1. Mimecan was upregulated in THP-1 derived macrophages upon stimulation with MCP-1. Patients with high levels of mimecan (above median) had higher risk for cardiovascular death. ConclusionsThis study indicates that mimecan is associated with a vulnerable plaque phenotype, possibly regulated by plaque inflammation. In line, plaque levels of mimecan independently predict future cardiovascular death.

Иммуногистохимические особенности каротидного клубочка человека

Introduction. The carotid body is a chemoreceptor organ and the initial link of the reflex regulation of car-diovascular and respiratory systems. However, molecular genetic and immunohistochemical characteristics of the human carotid body remains underinvestigated. Although there are numerous studies of the second half of the 20th century devoted to the classical light-optical histology of the human organ, the immunohis-tochemical investigations are very few. The aim of our study was to clarify immunohistochemical features of the human carotid body in comparison with those of the most commonly used laboratory animals. Materials and methods. The study was performed on 10 carotid bodies of the adult human of different ages of both sexes using immunoperoxidase labeling with antibodies to bIII-tubulin, tyrosine hydroxylase, syn-aptophysin, PGP9.5, neurofilaments 200kDa, S100, and GFAP. Results. Nerve fibers passing between the lobules, as well as entering them, were positive for bIII-tubulin, tyrosine hydroxylase, PGP9.5 and neurofilaments. Type I cells had cytoplasmic reaction for bIII-tubulin and synaptophysin as well as cytoplasmic and nuclear staining for PGP9.5. At the same time, they had weaker reaction for tyrosine hydroxylase. Type II cells were positive for GFAP and S100. Conclusion. Immunohistochemical characteristics of the human carotid body were similar to those of rats and mice. The human carotid body cells and nerve fibers showed the same distribution of PGP9.5, bIII-tubulin, synaptophysin, neurofilaments, GFAP and S100 as rat and mouse carotid body cells. However, human carotid body reaction for tyrosine hydroxylase was much lower, which may indicate a smaller amount of synthesized catecholamines compared to the carotid body in rats and mice. Keywords: human carotid body, immunohistochemistry, sympathoadrenal system, tyrosine hydroxylase

Lack of RAC1 in macrophages protects against atherosclerosis.

The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis.

Neopterin derivatives - a novel therapeutic target rather than biomarker for atherosclerosis and related diseases.

This review provides an updated overview of the emerging roles of neopterin derivatives in atherosclerosis. Neopterin, a metabolite of guanosine triphosphate, is produced by interferon-γ-activated macrophages and is expressed at high levels in atheromatous plaques within the human carotid and coronary arteries as well as in the aorta. Plasma concentrations of neopterin are higher in patients with carotid, cerebral, and coronary artery diseases as well as aortic aneurysm. The concentration of neopterin is positively correlated with the severity of coronary artery disease. However, a prospective cohort study showed that neopterin contributes to protection against plaque formation in carotid arteries in patients with atherosclerosis. Moreover, using both in vitro and in vivo experiments, a recent study has shown the atheroprotective effects of neopterin. Neopterin suppresses the expression of monocyte chemotactic protein-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in endothelial cells, and thereby suppresses the adhesion of monocytes to endothelial cells. It also suppresses the inflammatory phenotype of monocyte-derived macrophages. In addition, neopterin suppresses oxidized low-density lipoprotein-induced foam cell formation in macrophages and the migration and proliferation of vascular smooth muscle cells. Neopterin injection into apolipoprotein E-deficient (Apoe-/-) mice suppresses the development of atherosclerotic lesions. A neopterin derivative tetrahydroneopterin (BH4), also known as a cofactor for nitric oxide (NO) synthases, suppresses atherosclerosis and vascular injury-induced neointimal hyperplasia in Apoe-/- mice. BH4 administration improves endothelial dysfunction in patients with coronary artery disease. These findings suggest that neopterin production may increase to counteract the progression of atherosclerosis, as neopterin contributes to atheroprotection. Otherwise, the increased neopterin levels in atherosclerosis may reflect a compensatory mechanism associated with inducible NO synthase upregulation in macrophages to supply BH4 for high output NO production caused by decreased endothelial NO synthase in atherosclerosis. Therefore, neopterin derivatives are a novel therapeutic target for atherosclerosis and related diseases.

Calcium-Binding Protein S100A4 Is Upregulated in Carotid Atherosclerotic Plaques and Contributes to Expansive Remodeling.

BackgroundCarotid plaques with expansive arterial remodeling are closely related to cerebral ischemic events. Although S100A4 (S100 calcium‐binding protein A4) is expressed in atherosclerotic lesions, its role in atherosclerotic plaque progression remains unknown. In this study, we examined the association between carotid arterial expansive remodeling and S100A4 expression.Methods and ResultsPreoperative high‐resolution magnetic resonance imaging was used to assess luminal stenosis and vascular remodeling in patients undergoing carotid endarterectomy. To examine murine carotid atherosclerosis, we induced experimental lesions by flow cessation in apolipoprotein E‐deficient mice fed a high‐fat diet. The role of S100A4 in plaque formation and smooth muscle cell proliferation was investigated in vivo and in vitro, respectively. Human carotid arterial expansive remodeling showed positive correlations with the expression of S100A4, MMP2, and MMP9. S100A4 mRNA levels were positively correlated with those of MMP2, MMP9, and MMP13. S100A4 was expressed in vascular smooth muscle cells (VSMCs) and VSMC‐derived foam cells in the plaque shoulder and marginal areas. S100A4 expression increased concomitantly with plaque formation in our animal model. Exogenous recombinant S100A4 protein enhanced the levels of Mmp2, Mmp9, and Mmp13 and the cell proliferation ability in VSMCs. A chemotaxis assay indicated that extracellular S100A4 functions as a chemoattractant for VSMCs.ConclusionsS100A4 expression was elevated in human carotid plaques and showed a positive correlation with the degree of expansive remodeling. S100A4‐positive VSMC‐derived cells are considered to play an important role in carotid expansive remodeling.

Cholesterol crystals use complement to increase NLRP3 signaling pathways in coronary and carotid atherosclerosis.

BackgroundDuring atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease.MethodsWe analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling.FindingsTranscripts of interleukin (IL)-1beta(β) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1β protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1β, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components.InterpretationWe demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.

CD163+ macrophages are associated with a vulnerable plaque phenotype in human carotid plaques

Macrophages are a functionally heterogeneous group of immune cells abundant in atherosclerotic plaques. Macrophages expressing CD163 are associated with intraplaque hemorrhage and have previously been considered atheroprotective. However, in a recent study CD163-deficient atherosclerotic ApoE−/− mice exhibited smaller and less complex plaques, suggesting a proatherogenic role of CD163. Previous smaller studies on CD163+ macrophages and plaque stability in humans have yielded diverging results. Here we assessed the association of CD163+ cells to plaque vulnerability in a large cohort of human carotid plaques. CD163 protein expression was analyzed by immunohistochemistry in 200 human carotid plaques removed by endarterectomy from 103 patients with and 93 patients without cerebrovascular symptoms. Furthermore, CD163 mRNA expression was analyzed in 66 of the plaques. Both protein and mRNA expression of CD163 was higher in plaques from symptomatic patients and in plaques with high vulnerability index. CD163+ macrophages were primarily found in shoulder regions and in the center of the plaques. The present data show that CD163 is associated with increased plaque vulnerability in human carotid plaques, supporting the notion that CD163+ macrophages could contribute to clinical events.

Effects of Hematocrit on Blood Flow Through A Stenosed Human Carotid Artery

In this paper, the effects of hematocrit of red blood cells on blood flow through a stenosed human carotid artery was considered by taking blood as a Newtonian fluid. The governing equations on blood flow were derived. The mathematical content involved in the equations are the variables of interest such as number of stenosis , percentage of hematocrit of red blood cells in the blood, flow rate, wall shear stress, and viscosity of the blood. Guided by medical data collected on the constraint of blood flow in stenosed human carotid arteries, the governing equations were used to check the effects of pressure gradient, wall shear stress, velocity, and volumetric flow rate of blood in the human carotid arteries. Also, the one-dimensional equation for the steady and axially symmetric flow of blood through an artery was transformed using Einstein’s coefficient of viscosity and hematocrit of red blood cells with the help of the boundary conditions. The effects of hematocrit on the blood flow characteristics are shown graphically and discussed briefly. It was discovered that the resistance increases as the level of hematocrit increases. Also, the wall shear stress decreases with the increase in the hematocrit level of the red blood cells.