Ceramides and phospholipids in plasma extracellular vesicles are associated with high risk of major cardiovascular events after carotid endarterectomy.

Ceramides and phosphatidylcholines (PCs) are bioactive lipids and lipid bilayer membrane components. Distinct ceramides/PCs (ratios) predict cardiovascular outcome in patients with coronary artery disease. Extracellular vesicles (EVs) are proposed biomarkers for cardiovascular disease and contain ceramides/PCs. Ceramides/PCs have not been studied in patients undergoing carotid endarterectomy (CEA) nor in EVs. We therefore investigated whether levels of ceramides/PCs in plasma and EVs are associated with postoperative risk of major adverse cardiovascular events (MACE) following CEA. In 873 patients undergoing CEA of the Athero-Express biobank, we quantitatively measured seven ceramides/PCs in preoperative blood samples: Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) in plasma and two plasma EV-subfractions (LDL and TEX). We analyzed the association of ceramides, PCs and their predefined ratios with the three-year postoperative risk of MACE (including stroke, myocardial infarction and cardiovascular death). A total of 138 patients (16%) developed MACE during the three-year follow-up. In the LDL-EV subfraction, higher levels of Cer(d18:1/24:1) and Cer(d18:1/16:0)/PC(16:0/22:5) ratio were significantly associated with an increased risk of MACE (adjusted HR per SD [95% CI] 1.24 [1.01-1.53] and 1.26 [1.04-1.52], respectively). In the TEX-EV subfraction, three ratios Cer(d18:1/16:0)/Cer(d18:1/24:0), Cer(d18:1/18:0)/Cer(d18:1/24:0) and Cer(d18:1/24:1)/Cer(d18:1/24:0) were positively associated with MACE (adjusted HR per SD 1.34 [1.06-1.70], 1.24 [1.01-1.51] and 1.31 [1.08-1.58], respectively). In conclusion, distinct ceramides and PCs in plasma EVs determined in preoperative blood were independently associated with an increased 3-year risk of MACE after CEA. These lipids are therefore potential markers to identify high-risk CEA patients qualifying for secondary preventive add-on therapy.

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.

Elevated Lp(a) (Lipoprotein[a]) Levels Increase Risk of 30-Day Major Adverse Cardiovascular Events in Patients Following Carotid Endarterectomy.

BACKGROUND AND PURPOSE: General population studies have shown that elevated Lp(a) (lipoprotein[a]) levels are an emerging risk factor for cardiovascular disease and subsequent cardiovascular events. The role of Lp(a) for the risk of secondary MACE in patients undergoing carotid endarterectomy (CEA) is unknown. Our objective is to assess the association of elevated Lp(a) levels with the risk of secondary MACE in patients undergoing CEA. METHODS: Lp(a) concentrations were determined in preoperative blood samples of 944 consecutive patients with CEA included in the Athero-Express Biobank Study. During 3-year follow-up, major adverse cardiovascular events (MACE), consisting of myocardial infarction, stroke, and cardiovascular death, were documented. RESULTS: After 3 years follow-up, Kaplan-Meier cumulative event rates for MACE were 15.4% in patients with high Lp(a) levels (>137 nmol/L; >80th cohort percentile) and 10.2% in patients with low Lp(a) levels (≤137 nmol/L; ≤80th cohort percentile; log-rank test: P=0.047). Cox regression analyses adjusted for conventional cardiovascular risk factors revealed a significant association between high Lp(a) levels and 3-year MACE with an adjusted hazard ratio of 1.69 (95% CI, 1.07-2.66). One-third of MACE occurred within 30 days after CEA, with an adjusted hazard ratio for the 30-day risk of MACE of 2.05 (95% CI, 1.01-4.17). Kaplan-Meier curves from time point 30 days to 3 years onward revealed no significant association between high Lp(a) levels and MACE. Lp(a) levels were not associated with histological carotid plaque characteristics. CONCLUSIONS: High Lp(a) levels (>137 nmol/L; >80th cohort percentile) are associated with an increased risk of 30-day MACE after CEA. This identifies elevated Lp(a) levels as a new potential risk factor for secondary cardiovascular events in patients after carotid surgery. Future studies are required to investigate whether Lp(a) levels might be useful in guiding treatment algorithms for carotid intervention.

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.

Plasma extracellular vesicle proteins are associated with stress-induced myocardial ischemia in women presenting with chest pain.

Diagnosing stable ischemic heart disease (IHD) is challenging, especially in females. Currently, no blood test is available. Plasma extracellular vesicles (EV) are emerging as potential biomarker source. We therefore aimed to identify stress induced ischemia due to stable IHD with plasma extracellular vesicle protein levels in chest pain patients. We analyzed 450 patients suspected for stable IHD who were referred for 82Rb PET/CT in the outpatient clinic. Blood samples were collected before PET/CT and plasma EVs were isolated in 3 plasma subfractions named: TEX, HDL, LDL. In total 6 proteins were quantified in each of these subfractions using immuno-bead assays. CD14 and CystatinC protein levels were independent significant predictors of stress-induced ischemia in the LDL and the HDL subfraction and SerpinC1 and SerpinG1 protein levels in the HDL fraction. Subgroup-analysis on sex revealed that these associations were completely attributed to the associations in women. None of the significant EV proteins remained significant in men. Plasma EV proteins levels are associated with the presence of stable IHD in females presenting with chest pain. This finding, if confirmed in larger cohort studies could be a crucial step in improving diagnostic assessment of women with suspected IHD.

Flotillins in the intercalated disc are potential modulators of cardiac excitability.

BACKGROUND: The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. METHODS: Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. RESULTS: First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. CONCLUSIONS: In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.