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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 R 2 (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., (© 2023. The Author(s).)

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (β=-0.03/10 y; r²=0.07; P=1.6x10-7) 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. [ABSTRACT FROM AUTHOR]

Background: Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome. Methods: Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls. Results: LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017). Conclusions: LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions. [ABSTRACT FROM AUTHOR]

Background Atherosclerosis is a process that begins in childhood, develops over decades and underlies the majority of cardiovascular events in adulthood. Previously, we demonstrated in adults with cardiovascular disease that levels of extracellular vesicle (EV) proteins CD14, Serpin F2 and cystatin C predict vascular outcome. Here, we study for the first time whether these EV proteins are related to vascular characteristics in healthy, young children. Methods and results In 141 eight-year old children of the Wheezing-Illnesses-Studie-LEidsche-Rijn birth cohort, anthropometrics and blood pressure were measured. In addition, common carotid intima-media thickness, carotid distensibility and carotid Young's elastic modulus were obtained non-invasively using ultrasound imaging. A fasting lipid spectrum was obtained and EVs were isolated from plasma. Levels of EV proteins CD14, Serpin F2 and cystatin C were measured using a multiplex assay. In a multivariable linear regression model we assessed the relation between these EV proteins and the selected vascular characteristics. Of the studied EV proteins, CD14 levels were positively related to common carotid intima-media thickness (log transformed, beta = 7.31 ln(mm)/(ng/mg) (1.24, 13.38), p = 0.02). EV proteins Serpin F2 and cystatin C were not related to common carotid intima-media thickness. In addition, we found no relation between all three EV proteins and carotid distensibility or carotid Young's elastic modulus. Conclusion In healthy eight-year old children, extracellular vesicle protein CD14 levels seem positively related to common carotid intima-media thickness. This would point towards inflammatory vascular alterations inflicted by extracellular vesicle protein CD14 already in early life and warrants further investigation. [ABSTRACT FROM AUTHOR]