The influence of apical periodontitis on circulatory inflammatory mediators in peripheral blood: A prospective case-control study.

AIM: To explore the influence of apical periodontitis (AP) on inflammatory markers in blood of otherwise healthy individuals and to depict the inflammatory profile of the healing after dental extraction. METHODOLOGY: This is a prospective case-control intervention study, during which, individuals with a diagnosis of AP of one affected tooth were included, along with a control group matched for age and gender. A broad panel of blood inflammatory mediators was examined longitudinally in all subjects during six visits. In the case of the AP subjects, the tooth with AP was extracted at the third visit. Results were analysed by linear regression analyses and linear mixed-model analyses. RESULTS: A total of 53 subjects were included in the study, 27 with AP and 26 without. Fifteen females and 12 males were included in the AP group, and 14 females and 12 males in the control group. At baseline, granulocyte colony-stimulating factor (p < .001), interleukin (IL)-1ß (p = .03) and IL-4 (p = .01) were significantly lower in AP subjects than in controls. Comparison of the differences between baseline and the last visit, i.e. 3 months after the tooth extraction, showed a significant reduction in IL-10 (p = .03) and IL-12p70 (p = .01). CONCLUSIONS: The immunologic profile of chronic AP in one tooth and its healing profile reveals a systemic low-grade inflammation through compensatory immunosuppression. A larger lesion or multiple lesions could disrupt the balance that the system is trying to maintain, resulting in loss of homeostasis.

Monocyte-Chemoattractant Protein-1 Levels in Human Atherosclerotic Lesions Associate With Plaque Vulnerability.

[Figure: see text].

Dysregulated Innate and Adaptive Immune Responses Discriminate Disease Severity in COVID-19.

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity.

Pre-Operative Plasma Extracellular Vesicle Proteins are Associated with a High Risk of Long Term Secondary Major Cardiovascular Events in Patients Undergoing Carotid Endarterectomy.

OBJECTIVE: Patients undergoing carotid endarterectomy (CEA) maintain a substantial residual risk of major cardiovascular events (MACE). Improved risk stratification is warranted to select high risk patients qualifying for secondary add on therapy. Plasma extracellular vesicles (EVs) are involved in atherothrombotic processes and their content has been related to the presence and recurrence of cardiovascular events. The association between pre-operative levels of five cardiovascular disease related proteins in plasma EVs and the post-operative risk of MACE was assessed. METHODS: In 864 patients undergoing CEA from 2002 to 2016 included in the Athero-Express biobank, three plasma EV subfractions (low density lipoprotein [LDL], high density lipoprotein [HDL], and tiny extracellular vesicles [TEX]) were isolated from pre-operative blood samples. Using an electrochemiluminescence immunoassay, five proteins were quantified in each EV subfraction: cystatin C, serpin C1, serpin G1, serpin F2, and CD14. The association between EV protein levels and the three year post-operative risk of MACE (any stroke, myocardial infarction, or cardiovascular death) was evaluated using multivariable Cox proportional hazard regression analyses. RESULTS: During a median follow up of three years (interquartile range 2.2 - 3.0), 137 (16%) patients developed MACE. In the HDL-EV subfraction, increased levels of CD14, cystatin C, serpin F2, and serpin C1 were associated with an increased risk of MACE (adjusted hazard ratios per one standard deviation increase of 1.30, 95% confidence interval [CI] 1.15-1.48; 1.22, 95% CI 1.06-1.42; 1.36, 95% CI 1.16-1.61; and 1.29, 95% CI 1.10-1.51; respectively), independently of cardiovascular risk factors. No significant associations were found for serpin G1. CD14 improved the predictive value of the clinical model encompassing cardiovascular risk factors (net re-classification index = 0.16, 95% CI 0.08-0.21). CONCLUSION: EV derived pre-operative plasma levels of cystatin C, serpin C1, CD14, and serpin F2 were independently associated with an increased long term risk of MACE after CEA and are thus markers for residual cardiovascular risk. EV derived CD14 levels could improve the identification of high risk patients who may benefit from secondary preventive add on therapy in order to reduce future risk of MACE.

Increased circulating IgG levels, myocardial immune cells and IgG deposits support a role for an immune response in pre- and end-stage heart failure.

The chronic inflammatory response plays an important role in adverse cardiac remodelling and the development of heart failure (HF). There is also evidence that in the pathogenesis of several cardiovascular diseases, chronic inflammation is accompanied by antibody and complement deposits in the heart, suggestive of a true autoimmune response. However, the role of antibody-mediated immune responses in HF progression is less clear. We assessed whether immune cell infiltration and immunoglobulin levels are associated with HF type and disease stage, taking sex differences into account. We found IgG deposits and increased infiltration of immune cells in the affected myocardium of patients with end-stage HF with reduced ejection fraction (HFrEF, n = 20). Circulating levels of IgG1 and IgG3 were elevated in these patients. Furthermore, the percentage of transitional/regulatory B cells was decreased (from 6.9% to 2.4%) compared with healthy controls (n = 5). Similarly, increased levels of circulating IgG1 and IgG3 were observed in men with left ventricular diastolic dysfunction (LVDD, n = 5), possibly an early stage of HF with preserved EF (HFpEF). In conclusion, IgG deposits and infiltrates of immune cells are present in end-stage HFrEF. In addition, both LVDD patients and end-stage HFrEF patients show elevated levels of circulating IgG1 and IgG3, suggesting an antibody-mediated immune response upon cardiac remodelling, which in the early phase of remodelling appear to differ between men and women. These immunoglobulin subclasses might be used as marker for pre-stage HF and its progression. Future identification of auto-antigens might open possibilities for new therapeutic interventions.

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles.

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood.

Intraluminal abdominal aortic aneurysm thrombus is associated with disruption of wall integrity.

OBJECTIVE: An association of intraluminal thrombus (ILT) with abdominal aortic aneurysm (AAA) growth has been suggested. Previous in vitro experiments have demonstrated that aneurysm-associated thrombus may secrete proteolytic enzymes and may develop local hypoxia that might lead to the formation of tissue-damaging reactive oxygen species. In this study, we assessed the hypothesis that ventral ILT thickness is associated with markers of proteolysis and with lipid oxidation in the underlying AAA vessel wall. METHODS: Ventral AAA tissue was collected from asymptomatic patients at the site of maximal diameter during open aneurysm repair. Segments were divided, one part for biochemical measurements and one for histologic analyses. We measured total cathepsin B, cathepsin S levels, and matrix metalloproteinase (MMP)-2 and MMP-9 activity. Myeloperoxidase and thiobarbituric acid reactive substances were determined as measures of lipid oxidation. Histologic segments were analyzed semiquantitatively for the presence of collagen, elastin, vascular smooth muscle cells (VSMCs), and inflammatory cells. Preoperative computed tomography angiography scans of 83 consecutive patients were analyzed. A three-dimensional reconstruction was obtained, and a center lumen line of the aorta was constructed. Ventral ILT thickness was measured in the anteroposterior direction at the level of maximal aneurysm diameter on the orthogonal slices. RESULTS: Ventral ILT thickness was positively correlated with aortic diameter (r=0.25; P=.02) and with MMP-2 levels (r=0.27; P=.02). No biochemical correlations were observed with MMP-9 activity or cathepsin B and S expression. No correlation between ventral ILT thickness and myeloperoxidase or thiobarbituric acid reactive substances was observed. Ventral ILT thickness was negatively correlated with VSMCs (no staining, 18.5 [interquartile range, 12.0-25.5] mm; minor, 17.6 [10.7-22.1] mm; moderate, 14.5 [4.6-21.7] mm; and heavy, 8.0 [0.0-12.3] mm, respectively; P=.01) and the amount of elastin (no staining, 18.6 [12.2-30.0] mm; minor, 16.5 [9.0-22.1] mm; moderate, 11.7 [2.5-15.3] mm; and heavy 7.7 [0.0-7.7] mm, respectively; P=.01) in the medial aortic layer. CONCLUSIONS: ILT thickness appeared to be associated with VSMCs apoptosis and elastin degradation and was positively associated with MMP-2 concentrations in the underlying wall. This suggests that ILT thickness affects AAA wall stability and might contribute to AAA growth and rupture. ILT thickness was not correlated with markers of lipid oxidation.

Osteoprotegerin is associated with aneurysm diameter and proteolysis in abdominal aortic aneurysm disease.

OBJECTIVE: Serum osteoprotegerin (OPG) concentrations have previously been associated with growth of abdominal aortic aneurysms (AAAs). In vitro experiments showed that OPG promotes matrix metalloprotease (MMP) release from monocytes and vascular smooth muscle cells. We hypothesized that OPG expression is increased in human AAAs and is associated with proteolysis. METHODS AND RESULTS: AAA biopsies were collected from 329 patients. We assessed the concentrations of OPG, cathepsins A, B, and S as well as the activity of MMP-2 and MMP-9. The AAA wall infiltration by macrophages, lymphocytes, and plasma cells was estimated by immunohistochemistry. The concentration of OPG correlated positively with aortic diameter (<55 mm: 16.1 [5.8-28.7], 55-70 mm: 21.9 [10.2-36.0], >70 mm: 24.0 [13.5-52.9] ng OPG/mg total amount of protein, P=0.020), cathepsin A (r=0.221, P=0.005), B (r=0.384, P<0.001), and S (r=0.467, P<0.001), MMP-2 (r=0.180, P<0.001), MMP-9 (r=0.178, P<0.001), and the number of lymphocytes (P<0.001) and plasma cells (P=0.001). OPG immunostaining was predominantly demonstrated in plasma cells. CONCLUSIONS: The concentration of aortic wall OPG is positively associated with established markers of AAA severity and pathogenesis. OPG appeared to be associated with lymphocytes and plasma cells. These human data support previous experimental data suggesting a role for OPG in AAA pathogenesis.

Analysis of C-reactive protein from finger stick dried blood spot to predict high risk of cardiovascular disease.

C-reactive protein (CRP) is an acute-phase protein involved in inflammation. Furthermore, CRP is an important biomarker used in diagnostics to predict risk of cardiovascular disease (CVD) in addition to monitoring bacterial and viral infections. To measure plasma CRP, venipuncture is still necessitated and has to be performed by trained phlebotomists. As a solution, dried blood spots (DBS) are used for minimally invasive at-home sampling of blood and can be send to diagnostic laboratories by regular mail. In this study, we included 53 patients that presented to the outpatient clinic of the University Medical Center Utrecht. Capillary finger stick was used to spot blood on a filter paper card and allowed to dry. After extraction of DBS, CRP was analyzed on an automated high-throughput chemistry analyzer. Additional validation steps regarding stability, effect of hematocrit, precision, and limits of blank and quantitation were conducted according to corresponding Clinical and Laboratory Standards Institute standards. An excellent regression analysis of R2 (95% confidence interval) = 0.986 (0.982-0.989) was found. This enabled correct classification for high CVD risk of all 25 cases with sensitivity (95% CI) of 1.00 (1.00-1.00) and specificity (95% CI) of 0.96 (0.89-1.03) and correct diagnosis of inflammation of 12/13 cases with sensitivity (95% CI) of 0.92 (0.77-1.07) and specificity (95% CI) of 1.00 (1.00-1.00). Furthermore, CRP was found to be stable for 31 days and observed hematocrit variation amongst patients was clinically acceptable. CRP from DBS can be accurately measured on an automated high-throughput chemistry analyzer and used to diagnose inflammation and classify high CVD risk. This method enables individuals to engage in at-home sampling of blood on DBS for (tele)diagnostics, screening programs, patient follow-up, and medication management.

A Preliminary Analysis of Thyrotropin Measurement from Finger Stick Dried Blood Spot with an Automated High-Throughput Immunoassay Analyzer.

Background: Hyper- and hypothyroidism are prevalent in Western countries and often go unnoticed for long periods. Thyrotropin (TSH) as a biomarker of thyroid dysfunction is regularly measured in venous plasma/serum. In newborn screening for congenital hypothyroidism, TSH is measured from dried blood spots (DBSs). DBS enables minimally invasive (at-home) sampling of a small blood volume that can be sent to diagnostic laboratories by regular mail. Methods: In this study, we included 109 patients who presented to the outpatient clinic of the University Medical Center Utrecht. Capillary finger stick was used to spot blood on a filter paper card and was dried. After extraction of TSH from DBS, method comparison with venous TSH was performed on an automated high-throughput immunoassay analyzer. Additional validation steps regarding stability, effect of hematocrit (Hct), precision, and limits of blank and quantitation were conducted according to corresponding Clinical and Laboratory Standards Institute evaluation protocol. Results: Method comparison of TSH from venous plasma versus finger stick DBSs showed an R2 [95% confidence interval] = 0.988 [0.986-0.990]. This enabled correct diagnosis of hypothyrotropinemia and hypothyroidism in 12 of 14 and 6 of 7 cases, respectively, with no false positives. Furthermore, TSH from DBS was stable for at least 4 days at temperatures between -20°C and +30°C, and the maximum decrease of eluate TSH was 1.13% for 1% increase in Hct. Conclusions: TSH from DBS may be accurately measured on an automated high-throughput immunoassay analyzer and could be used to diagnose hypothyroidism and, for the first time, hypothyrotropinemia. This method, when confirmed in larger field studies, may enable individuals to engage in (at-home) sampling of blood on DBSs for telediagnostics, screening programs, patient follow-up, and medication management.

Adipocyte fatty acid binding protein in atherosclerotic plaques is associated with local vulnerability and is predictive for the occurrence of adverse cardiovascular events.

AIMS: There is an increasing need for translational studies identifying molecular targets contributing to atherosclerotic plaque destabilization. Local molecular plaque markers that are related to plaque vulnerability may hold predictive value to identify patients who are at increased risk to suffer from cardiovascular events. Animal studies revealed that adipocyte fatty acid binding protein (FABP4) is associated with the progression of atherosclerosis; however, FABP4 expression studies in human atherosclerotic plaques are lacking. We investigated FABP4 expression in carotid atherosclerotic lesions in relation to plaque composition and future cardiovascular events. METHODS AND RESULTS: Atherosclerotic plaques were obtained from 561 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid core, smooth-muscle cells, collagen, calcification, and intraplaque haemorrhage. Patients were followed for 3 years after CEA. The primary outcome was defined as the composite of vascular death, vascular event, and surgical or percutaneous vascular intervention. Fatty acid binding protein levels correlated with unstable plaque characteristics and symptomatic lesions. Patients with increased FABP4 plaque levels showed a two-fold increased risk [HR = 1.99, 95% confidence interval (95% CI) (1.30-3.04)] (P = 0.005) to reach the primary outcome during follow-up. Increased FABP4 levels related to primary outcome, independent from general cardiovascular risk factors [HR = 1.33, 95% CI (1.08-1.65)] (P = 0.008). CONCLUSION: FABP4 levels in atherosclerotic lesions are associated with an unstable plaque phenotype and an increased risk for cardiovascular events during follow-up. Besides risk stratification for adverse future cardiovascular events, the outcome of the present study supports the relevance of exploring FABP4 antagonists as a potential pharmaceutical intervention to treat atherosclerotic disease progression.

Arteriogenesis requires toll-like receptor 2 and 4 expression in bone-marrow derived cells.

Adaptive collateral growth (arteriogenesis) is an important protective mechanism against ischemic injury in patients with cardiovascular disease. Arteriogenesis involves enlargement of pre-existent arterial anastomoses and shares many mechanistic similarities with inflammatory processes. Although infusion of the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) has shown to result in a significant stimulation of arteriogenesis and both Toll-like receptor 2 and 4 are involved in structural arterial adaptations, the requirement for TLRs in arteriogenesis has not yet been established. We therefore subjected TLR 2 null and TLR 4 defective mice to unilateral femoral artery occlusion. At 7 days, both TLR 2 null and TLR 4 defective mice showed a significant reduction (~35%) of collateral perfusion. Histological staining showed that TLR 2 and TLR 4 expression during arteriogenesis is mostly restricted to infiltrating leukocytes. To distinguish between the functional importance of vascular and leukocytic TLRs in arteriogenesis, cross-over bone marrow transplantation was performed 6 weeks before femoral artery occlusion. Perfusion measurements showed that transplantation of wild-type bone marrow into TLR 2 null and TLR 4 defective mice rescued the impaired arteriogenesis, while injection of TLR 2 null and TLR 4 defective bone marrow into wild-type mice significantly reduced collateral vessel growth to levels of TLR null/defective mice. RT-PCR analysis demonstrated a significant upregulation of two endogenous TLR ligands EDA and Hsp60 (91.7 fold and 1.9 fold respectively) in regions of collateral vessel formation. This study illustrates the involvement of TLR 2 and TLR 4 in adaptive collateral artery growth and shows the importance of TLR 2 and 4 expression by bone-marrow derived cells for this process.

Toll-like receptor 2 and 4 stimulation elicits an enhanced inflammatory response in human obese patients with atherosclerosis.

The innate immune response elicited by activation of TLRs (Toll-like receptors) plays an important role in the pathogenesis of atherosclerosis. We hypothesized that cardiovascular risk factors are associated with the activation status of the innate immune system. We therefore assessed the responsiveness of TLRs on circulating cells in two groups of patients with established atherosclerosis and related this to the presence of cardiovascular risk factors. TNF (tumour necrosis factor)-α release induced by TLR2 and TLR4 activation was measured in patients with established coronary [PCI (percutaneous coronary intervention) study, n=78] or carotid artery disease [CEA (carotid endarterectomy) study, n=104], by stimulating whole blood samples with lipopolysaccharide (TLR4 ligand) and Pam3CSK4 [tripalmitoylcysteinylseryl-(lysyl)4; TLR2 ligand]. As an early activation marker, CD11b expression was measured by flow cytometry on CD14+ cells. Obesity was the 'only' risk factor that correlated with the TLR response. In both studies, obese patients had significantly higher TNF-α levels after stimulation of TLR2 compared with non-obese patients [16.9 (7.7-49.4) compared with 7.5 (1.5-19.2) pg/ml (P=0.008) in coronary artery disease and 14.6 (8.1-28.4) compared with 9.5 (6.1-15.7) pg/ml (P=0.015) in carotid artery disease; values are medians (interquartile range)]. Similar results were obtained following TLR4 stimulation. The enhanced inflammatory state in obese patients was also confirmed by a significant increased expression of the activation marker CD11b on circulating monocytes. In conclusion, obesity is associated with an enhanced TLR response in patients suffering from established atherosclerotic disease.

Local atherosclerotic plaques are a source of prognostic biomarkers for adverse cardiovascular events.

OBJECTIVE: Atherosclerotic cardiovascular disease is a major burden to health care. Because atherosclerosis is considered a systemic disease, we hypothesized that one single atherosclerotic plaque contains ample molecular information that predicts future cardiovascular events in all vascular territories. METHODS AND RESULTS: AtheroExpress is a biobank collecting atherosclerotic lesions during surgery, with a 3-year follow-up. The composite primary outcome encompasses all cardiovascular events and interventions, eg, cardiovascular death, myocardial infarction, stroke, and endovascular interventions. A proteomics search identified osteopontin as a potential plaque biomarker. Patients undergoing carotid surgery (n=574) served as the cohort in which plaque osteopontin levels were examined in relation to their outcome during follow-up and was validated in a cohort of patients undergoing femoral endarterectomy (n=151). Comparing the highest quartile of carotid plaque osteopontin levels with quartile 1 showed a hazard ratio for the primary outcome of 3.8 (95% confidence interval, 2.6-5.9). The outcome did not change after adjustment for plaque characteristics and traditional risk factors (hazard ratio, 3.5; 95% confidence interval, 2.0-5.9). The femoral validation cohort showed a hazard ratio of 3.8 (95% confidence interval 2.0 to 7.4) comparing osteopontin levels in quartile 4 with quartile 1. CONCLUSIONS: Plaque osteopontin levels in single lesions are predictive for cardiovascular events in other vascular territories. Local atherosclerotic plaques are a source of prognostic biomarkers with a high predictive value for secondary manifestations of atherosclerotic disease.

High levels of osteoprotegerin are associated with coronary artery calcification in patients suspected of a chronic coronary syndrome.

Plasma osteoprotegerin (OPG) and vascular smooth muscle cell (VSMC) derived extracellular vesicles (EVs) are important regulators in the process of vascular calcification (VC). In population studies, high levels of OPG are associated with events. In animal studies, however, high OPG levels result in reduction of VC. VSMC-derived EVs are assumed to be responsible for OPG transport and VC but this role has not been studied. For this, we investigated the association between OPG in plasma and circulating EVs with coronary artery calcium (CAC) as surrogate for VC in symptomatic patients. We retrospectively assessed 742 patients undergoing myocardial perfusion imaging (MPI). CAC scores were determined on the MPI-CT images using a previously developed automated algorithm. Levels of OPG were quantified in plasma and two EV-subpopulations (LDL and TEX), using an electrochemiluminescence immunoassay. Circulating levels of OPG were independently associated with CAC scores in plasma; OR 1.39 (95% CI 1.17-1.65), and both EV populations; EV-LDL; OR 1.51 (95% CI 1.27-1.80) and EV-TEX; OR 1.21 (95% CI 1.02-1.42). High levels of OPG in plasma were independently associated with CAC scores in this symptomatic patient cohort. High levels of EV-derived OPG showed the same positive association with CAC scores, suggesting that EV-derived OPG mirrors the same pathophysiological process as plasma OPG.

Extracellular Vesicle cystatin c is associated with unstable angina in troponin negative patients with acute chest pain.

BACKGROUND: Despite the use of high-sensitive cardiac troponin there remains a group of high-sensitive cardiac troponin negative patients with unstable angina with a non-neglectable risk for future adverse cardiovascular events, emphasising the need for additional risk stratification. Plasma extracellular vesicles are small bilayer membrane vesicles known for their potential role as biomarker source. Their role in unstable angina remains unexplored. We investigate if extracellular vesicle proteins are associated with unstable angina in patients with chest pain and low high-sensitive cardiac troponin. METHODS: The MINERVA study included patients presenting with acute chest pain but no acute coronary syndrome. We performed an exploratory retrospective case-control analysis among 269 patients. Cases were defined as patients with low high-sensitive cardiac troponin and proven ischemia. Patients without ischemia were selected as controls. Blood samples were fractionated to analyse the EV proteins in three plasma-subfractions: TEX, HDL and LDL. Protein levels were quantified using electrochemiluminescence immunoassay. RESULTS: Lower levels of (adjusted) EV cystatin c in the TEX subfraction were associated with having unstable angina (OR 0.93 95% CI 0.88-0.99). CONCLUSION: In patients with acute chest pain but low high-sensitive cardiac troponin, lower levels of plasma extracellular vesicle cystatin c are associated with having unstable angina. This finding is hypothesis generating only considering the small sample size and needs to be confirmed in larger cohort studies, but still identifies extracellular vesicle proteins as source for additional risk stratification.

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure.

Upon myocardial damage, the release of cardiac proteins induces a strong antibody-mediated immune response, which can lead to adverse cardiac remodeling and eventually heart failure (HF). Stem cell therapy using mesenchymal stromal cells (MSCs) or cardiomyocyte progenitor cells (CPCs) previously showed beneficial effects on cardiac function despite low engraftment in the heart. Paracrine mediators are likely of great importance, where, for example, MSC-derived extracellular vesicles (EVs) also show immunosuppressive properties in vitro. However, the limited capacity of MSCs to differentiate into cardiac cells and the sufficient scaling of MSC-derived EVs remain a challenge to clinical translation. Therefore, we investigated the immunosuppressive actions of endogenous CPCs and CPC-derived EVs on antibody production in vitro, using both healthy controls and end-stage HF patients. Both MSCs and CPCs strongly inhibit lymphocyte proliferation and antibody production in vitro. Furthermore, CPC-derived EVs significantly lowered the levels of IgG1, IgG4, and IgM, especially when administered for longer duration. In line with previous findings, plasma cells of end-stage HF patients showed high production of IgG3, which can be inhibited by MSCs in vitro. MSCs and CPCs inhibit in vitro antibody production of both healthy and end-stage HF-derived immune cells. CPC-derived paracrine factors, such as EVs, show similar effects, but do not provide the complete immunosuppressive capacity of CPCs. The strongest immunosuppressive effects were observed using MSCs, suggesting that MSCs might be the best candidates for therapeutic targeting of B-cell responses in HF.

LDL extracellular vesicle coagulation protein levels change after initiation of statin therapy. Findings from the METEOR trial.

BACKGROUND: Statins are thought to have pleiotropic properties, including anticoagulant effects, in addition to reducing lipoprotein (LDL) levels. Plasma extracellular vesicles (EVs) are small bilayer membrane vesicles involved in various biological processes including coagulation. Since subsets of EVs in the LDL plasma fraction (LDL-EVs) correlate with thrombin activity, we hypothesized that changes in LDL-EVs after statin therapy may differ from that of serum levels of coagulation proteins, providing insight into the effects of statins on coagulation. METHODS: The study was conducted in 666 subjects with available serum from the METEOR trial, a trial of the effect of rosuvastatin versus placebo in patients with subclinical atherosclerosis. Changes in protein levels of von Willebrand Factor (VWF), SerpinC1 and plasminogen were measured in serum and in LDL-EVs, and were compared between the rosuvastatin and placebo groups. RESULTS: LDL-EV levels of plasminogen and VWF increased with rosuvastatin treatment compared to placebo (mean change of 126 ±â€¯8 versus 17 ±â€¯12 µg/mL for plasminogen (p < 0.001) and 310 ±â€¯60 versus 64 ±â€¯55 µg/mL for VWF (p = 0.015)). There was no difference between groups for change in LDL-EV-SerpinC1. In contrast, serum plasminogen levels increased to a lesser extent with rosuvastatin compared to placebo (23 ±â€¯29 versus 67 ±â€¯17 µg/mL, p = 0.024) and serum VWF levels showed no significant difference between both groups. CONCLUSIONS: Rosuvastatin increases LDL-EV coagulation proteins plasminogen and VWF in patients with subclinical atherosclerosis, an effect that is different from the effect of rosuvastatin on the same proteins in serum. This identifies LDL-EVs as a newly detected possible intermediate between statin therapy and coagulation.

Loss of Y Chromosome in Blood Is Associated With Major Cardiovascular Events During Follow-Up in Men After Carotid Endarterectomy.

BACKGROUND: Recent studies found an immune regulatory role for Y chromosome and a relationship between loss of Y chromosome (LOY) in blood cells and a higher risk of cancer and mortality. Given involvement of immune cells in atherosclerosis, we hypothesized that LOY is associated with the severity of atherosclerotic plaque characteristics and outcome in men undergoing carotid endarterectomy. METHODS AND RESULTS: LOY was quantified in blood and plaque from raw intensity genotyping data in men within the Athero-Express biobank study. Plaques were dissected, and the culprit lesions used for histology and the measurement of inflammatory proteins. We tested LOY for association with (inflammatory) atherosclerotic plaque phenotypes and cytokines and assessed the association of LOY with secondary events during 3-year follow-up. Of 366 patients with carotid endarterectomy, 61 exhibited some degree of LOY in blood. LOY was also present in atherosclerotic plaque lesions (n=8/242, 3%). LOY in blood was negatively associated with age (ß=-0.03/10 y; r2=0.07; P=1.6×10-7) but not with cardiovascular disease severity at baseline. LOY in blood was associated with a larger atheroma size (odds ratio, 2.15; 95% confidence interval, 1.06-4.76; P=0.04); however, this association was not significant after correction for multiple testing. LOY was independently associated with secondary major cardiovascular events (hazard ratio=2.28; 95% confidence interval, 1.11-4.67; P=0.02) in blood when corrected for confounders. CONCLUSIONS: In this hypothesis-generating study, LOY in blood is independently associated with secondary major cardiovascular events in a severely atherosclerotic population. Our data could indicate that LOY affects secondary outcome via other mechanisms than inflammation in the atherosclerotic plaque.

Leukocyte TLR5 deficiency inhibits atherosclerosis by reduced macrophage recruitment and defective T-cell responsiveness.

Toll-like receptors (TLR) provide a critical link between innate and adaptive immunity, both important players in atherosclerosis. Since evidence for the role of TLR5 is lacking, we aimed to establish this in the immune axis of atherosclerosis. We assessed the effect of the TLR5-specific ligand Flagellin on macrophage maturation and T-cell polarisation. Next, we generated TLR5-/-LDLr-/- chimeras to study the effect of hematopoietic TLR5 deficiency on atherosclerosis formation. Flagellin stimulation did not influence wildtype or TLR5-/- macrophage maturation. Only in wildtype macrophages, Flagellin exposure increased MCP-1 and IL6 expression. Flagellin alone reduced T-helper 1 proliferation, which was completely overruled in the presence of T-cell receptor activation. In vivo, hematopoietic TLR5 deficiency attenuated atherosclerotic lesion formation by ≈25% (1030*103 ± 63*103 vs. 792*103 ± 61*103 µm2; p = 0.013) and decreased macrophage area (81.3 ± 12.0 vs. 44.2 ± 6.6 µm2; p = 0.011). In TLR5-/- chimeric mice, we observed lower IL6 plasma levels (36.4 ± 5.6 vs. 15.1 ± 2.2 pg/mL; p = 0.003), lower (activated) splenic CD4+ T-cell content (32.3 ± 2.1 vs. 21.0 ± 1.2%; p = 0.0018), accompanied by impaired T-cell proliferative responses. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T-cell responsiveness.