Dysregulation of micro-RNA 143-3p as a Biomarker of Carotid Atherosclerosis and the Associated Immune Reactions During Disease Progression.

Atherosclerosis commonly remains undiagnosed until disease manifestations occur. The disease is associated with dysregulated micro(mi)RNAs, but how this is linked to atherosclerosis-related immune reactions is largely unknown. A mouse model of carotid atherosclerosis, human APOB100-transgenic Ldlr-/- (HuBL), was used to study the spatiotemporal dysregulation of a set of miRNAs. Middle-aged HuBL mice with established atherosclerosis had decreased levels of miR-143-3p in their carotid arteries. In young HuBL mice, early atherosclerosis was observed in the carotid bifurcation, which had lower levels of miR-15a-5p, miR-143-3p, and miR-199a-3p, and higher levels of miR-155-5p. The dysregulation of these miRNAs was reflected by specific immune responses during atheroprogression. Finally, levels of miR-143-3p were 70.6% lower in extracellular vesicles isolated from the plasma of patients with carotid stenosis compared to healthy controls. Since miR-143-3p levels progressively decrease when transitioning between early and late experimental carotid atherosclerosis, we propose it as a biomarker for atherosclerosis.

The relationship with the stability between GRP78, CHOP and human carotid atherosclerotic plaque.

BACKGROUND: Current researches on human carotid atherosclerosis (AS) plaques are focused on vulnerable plaques, and various methods have been clinically used to detect vulnerable plaques to prevent adverse events. GRP78 and CHOP, as markers in the endoplasmic reticulum stress (ERS), have a certain relationship with the stability of plaque tissue. METHODS: In this study, 150 plaque specimens were obtained from carotid endarterectomy (CEA). According to pathology, they were divided into two groups: stable plaque and vulnerable plaque. Immunohistochemistry was used to semi-quantitate and localize the target molecule. Western blot and RT-qPCR were used to detect the expression of GRP78 and CHOP in the samples. The receiver operating characteristic curve (ROC curve) judges the significance of the target molecule as a biomarker for the diagnosis of vulnerable plaques. RESULTS: The results of immunohistochemistry showed that the target molecules of GRP78 and CHOP were mainly expressed in inflammatory cells and vascular endothelial cells; Western blot and RT-qPCR techniques were used to detect the expression of GRP78 and CHOP in different pathlogical types of plaques, which respectively indicated that there were differential expressions. The expression in vulnerable plaques was significantly higher than that in stable plaques (P < 0.05). analysis with ROC, the areas under curves (AUC) of the GRP78 and CHOP data were calculated as 0.792 and 0.850, respectively and the combination showed the largest AUC of 0.870. CONCLUSION: In endoplasmic reticulum stress, GRP78 and CHOP are significantly higher expressions in vulnerable plaques than stable's, which indicated that GRP78 and CHOP played a certain role in the occurrence and development of human carotid atherosclerosis and vulnerable plaques; GRP78 and CHOP are promising molecular biomarkers for identifying the endoplasmic reticulum stress situation, atherosclerosis and plaque stability. They also could provide a potential drug targets for the prevention and treatment of atherosclerosis.

Correlation between neutrophil-to-lymphocyte ratio and stability of carotid plaques.

OBJECTIVE: The neutrophil-to-lymphocyte ratio (NLR) has been proved to be a strong predictor of carotid atherosclerotic plaque, but the correlation between NLR and the stability of carotid plaque is not clear. Thus we conducted a study to evaluate the correlation between NLR and the stability of carotid atherosclerotic plaque, and to develop a new evaluation scale for rapid clinical evaluation of carotid plaque stability. METHODS: We recruited 528 patients with acute anterior circulation ischemic stroke who were in accordance with extracranial and intracranial large artery atherosclerosis of Chinese ischemic stroke subtype. Blood routine examination and carotid ultrasound examination were performed on admission. According to the ultrasonic characteristics, the patients were divided into plaque stabilization group and plaque instability group. RESULTS: There was significant difference in NLR between plaque stability and instability groups (P < 0.001). The risk of plaque instability increased with the increase of NLR (odds ratio (OR), 4.737; 95% confidence interval (CI), 3.404-6.592; P < 0.001). Receiver operating characteristic (ROC) curve showed that the critical point of NLR is 2.55 and the area under the curve (AUC) was 0.782 (95%CI, 0.740-0.823; P < 0.001). The best cut-off value of the evaluation scale was ≥ 4 points (sensitivity, 0.77; specificity, 0.75; accuracy, 0.76). CONCLUSION: There is a correlation between NLR and carotid plaque instability. NLR may be useful as a potential inflammation biomarker indicating the risk of unstable carotid plaques. The new scoring scale is a reliable index to predict the stability of carotid plaque.

The Predictive Role of a Novel Risk Index in Patients Undergoing Carotid Artery Stenting: Systemic Immune-Inflammation Index.

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.

Plasma cells in the carotid plaque: occurrence and significance.

OBJECTIVE: Atherosclerosis is one of the leading causes of disability and mortality worldwide. Inflammation, including monocytes, T and B cells, plays a key role in its pathogenesis. Our purpose was to evaluate plasma cells' presence in a large series of carotid artery plaques and the clinical association. PATIENTS AND METHODS: Forty-eight consecutive patients treated with carotid endarterectomy were retrospectively analyzed to assess plasma cells' presence inside the plaque. A semiquantitative grading score was applied, ranging from absence, scattered, clusters of 5-10, and sheets of >10 plasma cells. Plasma cell's location, as intraplaque, subendothelial or peri-adventitial, was also defined. RESULTS: In 75% of plaques analyzed, plasma cells were detected: scattered in 63.9%, in clusters in 22.2%, and in sheets in 13.9% of cases. In all cases, plasma cells were observed only inside the plaque. In 13.9% and in 11.1% of cases, plasma cells showed, respectively, a concomitant subendothelial or peri-adventitial distribution. In 5.6% of plaques, there was a simultaneous distribution in subendothelial, peri-adventitial layer, and intraplaque. Association between the presence of symptoms and plasma cells infiltrate was found. CONCLUSIONS: Our results suggest that plasma cells could be a key parameter linked to plaque instability. Some types of configurations are significantly associated with the occurrence of cerebrovascular symptoms.

COMP (Cartilage Oligomeric Matrix Protein) Neoepitope: A Novel Biomarker to Identify Symptomatic Carotid Stenosis.

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.

Ficolin-2 serum levels predict the occurrence of acute coronary syndrome in patients with severe carotid artery stenosis.

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.

Focal Cerebral Arteriopathy in Young Adult Patients With Stroke.

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.

Immune cells in carotid artery plaques: what can we learn from endarterectomy specimens?

INTRODUCTION: Endarterectomy specimens represent a unique opportunity to study atherosclerosis. This review aims to summarize the recent knowledge of atherogenesis from studies characterizing a cellular composition of carotid endarterectomy specimens. EVIDENCE ACQUISITION: A non-systematic literature review was carried out to summarize recent knowledge regarding ex vivo analysis of carotid artery plaque composition. Upon evaluation of their relevance, and elaborate forward and backward search, 95 articles were included in the review. EVIDENCE SYNTHESIS: Despite the significant advancement of in vivo imaging techniques, the stroke prediction based on carotid artery plaque morphology is not reliable. Besides analyses of plaque morphology, present studies focus on precise characterization of the different immune cell types and elucidation of their role in plaque development. Plaque content analyses revealed the presence of various immune cells in carotid artery plaques. Presence of different immune cells subpopulations can be connected to some undesirable changes in plaque stability. CONCLUSIONS: Since the destabilization of the atherosclerotic plaque is a multifactorial process, a combination of various methods should be used to characterize the unstable plaques more accurately. In this context, studies characterizing plaque content from a cellular point of view could elucidate some processes underlying the plaque progression. Together with morphological evaluation, these analyses could enable more precise assessment of plaque stability.

Low CD4 Cell Counts Are Associated with Carotid Plaque and Intima-Media Thickness in Virologically Suppressed HIV-Infected Asians Older Than 50 Years.

Information about the prevalence, and risk factors for subclinical atherosclerosis in an Asian HIV-infected population is limited. Carotid intima-media thickness (cIMT) is one predictor for the risk of cardiovascular disease (CVDs) and mortality. We evaluated the prevalence and risk factors related to carotid atherosclerosis among well-suppressed HIV-infected adults receiving long-term ART from Thailand. This was a cross-sectional study of HIV-infected adults >50 years of age and free from CVDs from Thailand during 1 March 2016 and 30 May 2017. Ultrasonography of the carotid was performed and read by cIMT experienced neurologists who were blinded from the patient care. Subclinical atherosclerosis was defined by carotid plaque or cIMT of the common carotid artery (CCA) >0.9 mm. Totally 316 HIV-infected adults (61% males) were included. Median age was 54.4 years and 15.8% were diabetic, 40.2% had hypertension, and 12.7% were current smokers. The median duration of ART was 16.3 years and 32% were currently on boosted protease inhibitor. The mean overall cIMT of the common carotid arteries were 0.63 (IQR 0.55-0.72) mm. Men had higher cIMT than women, 0.64 (IQR 0.56-0.76) vs. 0.60 (IQR 0.53-0.70), p = .03. Overall, 3.8% had cIMT >0.9 mm and 24.4% had carotid plaque. From the multivariate logistic regression analysis, age per 1 year increase [odds ratio (OR) 1.06; 95% confidence interval (CI) 1.003-1.12; p = .04] and nadir CD4 < 200 cells/mm3 (OR 1.8; 95%CI 1.02-3.18, p = .04) were significantly associated with subclinical atherosclerosis. High-sensitivity C-reactive protein was not associated with subclinical atherosclerosis. In this well-suppressed HIV-infected Aging Asian cohort with relatively low prevalence of current smokers, 26.9% of them had subclinical atherosclerosis. Advanced age and low nadir CD4 cell count were significantly associated with subclinical atherosclerosis. Given that approximately a quarter of the patients had carotid plaques, longitudinal studies to evaluate the development of future overt coronary artery disease and stroke are warranted.

Monocyte Subsets, Stanford-A Acute Aortic Dissection, and Carotid Artery Stenosis: New Evidences.

Monocytes are a heterogeneous cell population distinguished into three subsets with distinctive phenotypic and functional properties: "classical" (CD14++CD16-), "intermediate" (CD14++CD16+), and "nonclassical" (CD14+CD16++). Monocyte subsets play a pivotal role in many inflammatory systemic diseases including atherosclerosis (ATS). Only a low number of studies evaluated monocyte behavior in patients affected by cardiovascular diseases, and data about their role in acute aortic dissection (AAD) are lacking. Thus, the aim of this study was to investigate CD14++CD16-, CD14++CD16+, and CD14+CD16++ cells in patients with Stanford-A AAD and in patients with carotid artery stenosis (CAS). Methods. 20 patients with carotid artery stenosis (CAS group), 17 patients with Stanford-A AAD (AAD group), and 17 subjects with traditional cardiovascular risk factors (RF group) were enrolled. Monocyte subset frequency was determined by flow cytometry. Results. Classical monocytes were significantly increased in the AAD group versus CAS and RF groups, whereas intermediate monocytes were significantly decreased in the AAD group versus CAS and RF groups. Conclusions. Results of this study identify in AAD patients a peculiar monocyte array that can partly explain depletion of T CD4+ lymphocyte subpopulations observed in patients affected by AAD.

Lag3+ regulatory T lymphocytes in critical carotid artery stenosis.

The aim of this study was to evaluate CD25+ and Lag3+ T regulatory subpopulations in patients with critical carotid artery stenosis (CAS) and Stanford-A acute aortic dissection (AAD). CD25+ and Lag3+ were measured in 36 patients affected by CAS and 24 patients with Stanford type A AAD. Based on neurological symptoms, patients affected by CAS were further divided in 25 asymptomatic (CAS-A) and 11 symptomatic (CAS-S) subjects. Twenty-five patients with traditional cardiovascular risk factors (RF), matched for age and sex, were used as control group. Interleukin (IL)-10, IL-6 and transforming growth factor-ß-levels were also measured. CD25+ T cells were significantly increased in CAS-S versus CAS-A (p > 0.05), AAD (p > 0.05) and RF (p > 0.05). Moreover, a significant increase in Lag3+ Tregs was observed in CAS e CAS-S versus AAD (p < 0.05) and RF (p < 0.05), whereas no significant difference was observed between CAS-S and CAS-A. IL-6 was higher in AAD compared to the other groups. Patients with neurological symptoms display a peculiar expansion of CD25+ T cells, strongly confirming a relationship between ischemic brain damage and this regulatory subpopulation, whereas Lag3+ Tregs early distinguish CAS from AAD and probably exert protective actions against aortic wall rupture throughout their anti-inflammatory functions.

Inflammation Coupling Between Unstable Carotid Plaque and Spleen-A 18F-Fluorodeoxyglucos Positron Emission Tomography Study.

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 ±  .25 and 2.51 ±  .40, respectively; P = .003). The SUVmax ratio (spleen/liver) was also significantly higher in ICS group than in the controls (1.12 ±  .06 and .85 ±  .12, respectively; P = .001). The SUVmax of aorta was also significantly higher in ICS group than in the controls (2.16 ±  .27 and 1.48 ±  .15, respectively; P = .001). However, there were no significant differences in the SUVmax in the bone marrow and SUVmax ratio (bone marrow/liver) between the 2 groups (P = .811 and P = .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.

Augmented Th17 differentiation in Trim21 deficiency promotes a stable phenotype of atherosclerotic plaques with high collagen content.

Aims: Patients with hyperlipidemia are at risk of atherosclerosis, but not all develop cardiovascular disease, highlighting the importance of other risk factors such as inflammation. Both the innate and adaptive arms of the immune system have been suggested in the initiation and propagation of plaque formation. Tri-partite motif (TRIM) 21 is a regulator of tissue inflammation and pro-inflammatory cytokine production, and has been implicated in chronic inflammatory disease. Here, we investigate a potential role for TRIM21 in coronary artery disease. Methods and results: Trim21-deficient or wild-type bone marrow was transplanted into Ldlr-/- mice fed a hypercholesterolemic diet. The Trim21-/-->Ldlr-/- mice developed larger atherosclerotic plaques, with significantly higher collagen content compared to mice transplanted with wild-type cells. High collagen content of the atheroma is stabilizing, and has recently been linked to IL-17. Interestingly, Trim21-/-->Ldlr-/- mice had elevated CD4 and IL-17 mRNA expression in plaques, and increased numbers of activated CD4+ T cells in the periphery. An increased differentiation of naïve T cells lacking Trim21 into Th17 cells was confirmed in vitro, with transcriptomic analysis revealing upregulation of genes of a non-pathogenic Th17 phenotype. Also, decreased expression of matrix metalloproteinases (MMPs) was noted in aortic plaques. Analysis of human carotid plaques confirmed that TRIM21 expression negatively correlates with the expression of key Th17 genes and collagen, but positively to MMPs also in patients, linking our findings to a clinical setting. Conclusion: In this study, we demonstrate that TRIM21 influences atherosclerosis via regulation of Th17 responses, with TRIM21 deficiency promoting IL-17 expression and a more fibrous, stable, phenotype of the plaques.

Inhibition of p110δ PI3K prevents inflammatory response and restenosis after artery injury.

Inflammatory cells play key roles in restenosis upon vascular surgical procedures such as bypass grafts, angioplasty and stent deployment but the molecular mechanisms by which these cells affect restenosis remain unclear. The p110δ isoform of phosphoinositide 3-kinase (PI3K) is mainly expressed in white blood cells. Here, we have investigated whether p110δ PI3K is involved in the pathogenesis of restenosis in a mouse model of carotid injury, which mimics the damage following arterial grafts. We used mice in which p110δ kinase activity has been disabled by a knockin (KI) point mutation in its ATP-binding site (p110δD910A/D910A PI3K mice). Wild-type (WT) and p110δD910A/D910A mice were subjected to longitudinal carotid injury. At 14 and 30 days after carotid injury, mice with inactive p110δ showed strongly decreased infiltration of inflammatory cells (including T lymphocytes and macrophages) and vascular smooth muscle cells (VSMCs), compared with WT mice. Likewise, PI-3065, a p110δ-selective PI3K inhibitor, almost completely prevented restenosis after artery injury. Our data showed that p110δ PI3K plays a main role in promoting neointimal thickening and inflammatory processes during vascular stenosis, with its inhibition providing significant reduction in restenosis following carotid injury. p110δ-selective inhibitors, recently approved for the treatment of human B-cell malignancies, therefore, present a new therapeutic opportunity to prevent the restenosis upon artery injury.

Carotid Endarterectomy Induces the Release of Inflammatory Markers and the Activation of Coagulation as Measured in the Jugular Bulb.

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.

Crosstalk Between Enzyme Matrix Metalloproteinases 2 and 9 and Regulatory T Cell Immunity in the Global Burden of Atherosclerosis.

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.

IL-33 stimulates the release of procoagulant microvesicles from human monocytes and differentially increases tissue factor in human monocyte subsets.

Monocytes and monocyte-derived microvesicles (MVs) are the main source of circulating tissue factor (TF). Increased monocyte TF expression and increased circulating levels of procoagulant MVs contribute to the formation of a prothrombotic state in patients with cardiovascular disease. Interleukin (IL)-33 is a pro-inflammatory cytokine involved in atherosclerosis and other inflammatory diseases, but its role in regulating thrombosis is still unclear. The aim of the present study was to investigate the effects of IL-33 on the procoagulant properties of human monocytes and monocyte-derived MVs. IL-33 induced a time- and concentration-dependent increase of monocyte TF mRNA and protein levels via binding to the ST2-receptor and activation of the NF-κB-pathway. The IL-33 treated monocytes also released CD14+TF+ MVs and IL-33 was found to increase the TF activity of both the isolated monocytes and monocyte-derived MVs. The monocytes were classified into subsets according to their CD14 and CD16 expression. Intermediate monocytes (IM) showed the highest ST2 receptor expression, followed by non-classical monocytes (NCM), and classical monocytes (CM). IL-33 induced a significant increase of TF only in the IM (p<0.01), with a tendency in NCM (p=0.06), but no increase was observed in CM. Finally, plasma levels of IL-33 were positively correlated with CD14+TF+ MVs in patients undergoing carotid endarterectomy (r=0.480; p=0.032; n=20). We hereby provide novel evidence that the proinflammatory cytokine IL-33 induces differential TF expression and activity in monocyte subsets, as well as the release of procoagulant MVs. In this manner, IL-33 may contribute to the formation of a prothrombotic state characteristic for cardiovascular disease.

Regulatory T CD4 + CD25+ lymphocytes increase in symptomatic carotid artery stenosis.

BACKGROUND: Atherosclerosis is a multifactorial disease characterized by an immune-inflammatory remodeling of the arterial wall. Treg and Th17 subpopulations are detectable inside atherosclerotic plaque; however, their behavior in symptomatic carotid artery stenosis (CAS) is not fully elucidated. The aim of this study was to evaluate Th17 and Treg subsets and their ratio in patients affected by symptomatic and asymptomatic CAS. METHODS: 14 patients with symptomatic CAS (CAS-S group), 41 patients with asymptomatic CAS (CAS-A group), 32 subjects with traditional cardiovascular risk factors (RF group), and 10 healthy subjects (HS group) were enrolled. Th17 and Treg frequency was determined by flow cytometry and by histology and immunohistochemistry. Interleukin (IL)-10, IL-17, and metalloproteinase (MMP)-12 levels were measured by ELISA. RESULTS: Th17 were significantly increased in CAS-A versus RF and versus HS. Tregs were significantly increased in CAS-S versus CAS-A. Tregs/Th17 ratio was significantly reduced in CAS-A versus RF and versus HS, whereas it was significantly increased in CAS-S versus CAS-A. CONCLUSIONS: The results of this study suggest that Th17 are related to the late stages of CAS but not to plaque instability. Moreover, Treg expansion seems to represent a specific cellular pattern displayed by patients with symptomatic CAS and associated with brain injury. KEY MESSAGES Tregs expansion seems to represent a specific cellular pattern displayed by patients with symptomatic CAS and associated with CD4+ effector depletion and brain ischemic injury. Th17 lymphocytes are related to the late stages of CAS but not to plaque instability.