Diacylglycerols and Lysophosphatidic Acid, Enriched on Lipoprotein(a), Contribute to Monocyte Inflammation.

BACKGROUND: Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). METHODS: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. RESULTS: Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1ß after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). CONCLUSIONS: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.

Atherosclerotic Plaque Epigenetic Age Acceleration Predicts a Poor Prognosis and Is Associated With Endothelial-to-Mesenchymal Transition in Humans.

BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.

Decreased serpin C1 in extracellular vesicles predicts response to methotrexate treatment in patients with pulmonary sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown etiology primarily affecting the lungs. Treatment is needed when disease symptoms worsen and organ function deteriorates. In pulmonary sarcoidosis, prednisone and methotrexate (MTX) are the most common anti-inflammatory therapies. However, there is large inter-patient variability in response to treatment, and predictive response markers are currently lacking. OBJECTIVE: In this study, we investigated the predictive potential of biomarkers in extracellular vesicles (EVs) isolated from biobanked serum of patients with pulmonary sarcoidosis stored prior to start of therapy. METHODS: Protein concentrations of a four-protein test panel of inflammatory proteins were measured in a discovery (n = 16) and replication (n = 129) cohort of patients with sarcoidosis and 47 healthy controls. Response to therapy was defined as an improvement of the absolute score of > 5% forced vital capacity (FVC) and/or > 10% diffusion lung of carbon monoxide (DLCO) after 24 weeks compared to baseline (before treatment). RESULTS: Serum protein levels differed between EV fractions and serum, and between sarcoidosis cases and controls. Serpin C1 concentrations in the low density lipid particle EV fraction were lower at baseline in the group of patients with a good response to MTX treatment in both the discovery cohort (p = 0.059) and in the replication cohort (p = 0.032). EV Serpin C1 showed to be a significant predictor for response to treatment with MTX (OR 0.4; p = 0.032). CONCLUSION: This study shows that proteins isolated from EVs harbor a distinct signal and have potential as new predictive therapy response biomarkers in sarcoidosis.

Endothelial Damage Arising From High Salt Hypertension Is Elucidated by Vascular Bed Systematic Profiling.

BACKGROUND: Considerable evidence links dietary salt intake with the development of hypertension, left ventricular hypertrophy, and increased risk of stroke and coronary heart disease. Despite extensive epidemiological and basic science interrogation of the relationship between high salt (HS) intake and blood pressure, it remains unclear how HS impacts endothelial cell (EC) and vascular structure in vivo. This study aims to elucidate HS-induced vascular pathology using a differential systemic decellularization in vivo approach. METHODS: We performed systematic molecular characterization of the endothelial glycocalyx and EC proteomes in mice with HS (8%) diet-induced hypertension versus healthy control animals. Isolation of eGC and EC compartments was achieved using differential systemic decellularization in vivo methodology. Altered protein expression in hypertensive compared to normal mice was characterized by liquid chromatography tandem mass spectrometry. Proteomic results were validated using functional assays, microscopic imaging, and histopathologic evaluation. RESULTS: Proteomic analysis revealed a significant downregulation of eGC and associated proteins in HS diet-induced hypertensive mice (among 1696 proteins identified in this group, 723 were markedly decreased in abundance, while only 168 were increased in abundance. Bioinformatic analysis indicated substantial derangement of the eGC layer, which was subsequently confirmed by fluorescent and electron microscopy assessment of vessel damage ex vivo. In the EC fraction, HS-induced hypertension significantly altered protein mediators of contractility, metabolism, mechanotransduction, renal function, and the coagulation cascade. In particular, we observed dysregulation of integrin subunits α2, α2b, and α5, which was associated with arterial wall inflammation and substantial infiltration of CD68+ monocyte-macrophages. Consequently, HS-induced hypertensive mice also displayed reduced vascular integrity of multiple organs including lungs, kidneys, and heart. CONCLUSIONS: These findings provide novel molecular insight into HS-induced structural changes in eGC and EC composition that may increase cardiovascular risk and potentially guide the development of new diagnostics and therapeutic interventions.

Female Gene Networks Are Expressed in Myofibroblast-Like Smooth Muscle Cells in Vulnerable Atherosclerotic Plaques.

BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.

Plasma Extracellular Vesicle Serpin G1 and CD14 Levels are Associated with Major Adverse Cardiovascular Events and Major Adverse Limb Events in Patients Undergoing Femoral Endarterectomy.

OBJECTIVE: Plasma extracellular vesicles (EV) are an emerging source of biomarkers for diagnosis and prognosis of cardiovascular disease (CVD). Risk stratification for common adverse events such as major adverse limb events (MALE) and major adverse cardiovascular events (MACE) by an EV blood sample could improve healthcare management by individualising drug therapy or improving informed decision making regarding revascularisations in patients with peripheral artery disease (PAD). As such, this study investigated the associations between plasma EV proteins and prospectively registered MALE and MACE in consecutive patients undergoing femoral endarterectomy. METHODS: Using the Athero-Express biobank study, four EV proteins (Cystatin C, CD14, Serpin C1, and Serpin G1) were measured in the high density lipoprotein subfraction isolated from plasma of 317 PAD patients undergoing arterial revascularisation. Multivariable Cox proportional hazard regression was used to investigate the association between plasma EV protein levels and MACE and MALE in the three year post-operative period. RESULTS: Most patients were treated for claudication (Fontaine II, 52.8%), although rest pain (Fontaine III, 30.1%) and ischaemic wounds (Fontaine IV, 17.1%) were common in this cohort. Within three years 51 patients died, amongst whom 25 deaths were due to CVD, 39 patients experienced a MACE, and 125 patients experienced a MALE. Multivariable regression models, based on statistically proven covariables and literature, showed a significant association of Serpin G1 (HR 1.49; 95% CI 1.08 - 2.06; p = .016) and CD14 (HR 1.40; 1.03 - 1.90; p = .029) with MACE, and of Serpin G1 (HR 1.29; 1.07 - 1.57; p = .009) with MALE. CONCLUSION: Serpin G1 and CD14 plasma EV protein levels are associated with future MACE and MALE in patients with severe PAD.

Plasma Lipoprotein Lipase Is Associated with Risk of Future Major Adverse Cardiovascular Events in Patients Following Carotid Endarterectomy.

INTRODUCTION: Carotid plaque intraplaque haemorrhage (IPH) is associated with future cardiovascular events. It was hypothesised that plasma proteins associated with carotid plaque IPH are also likely to be associated with major adverse cardiovascular events (MACE) after carotid endarterectomy (CEA). METHODS: In pre-operative blood samples from patients undergoing CEA within the Athero-Express biobank, proteins involved in cardiovascular disease were measured using three OLINK proteomics immunoassays. The association between proteins and IPH was analysed using logistic regression analyses. Subsequently, the association between the IPH associated plasma proteins and the three year post-operative risk of MACE (including stroke, myocardial infarction, or cardiovascular death) was analysed. RESULTS: Within the three year follow up, 130 patients (18.9%) of 688 symptomatic and asymptomatic patients undergoing CEA developed MACE. Six of 276 plasma proteins were found to be significantly associated with IPH, from which only lipoprotein lipase (LPL) was associated with the post-operative risk of MACE undergoing CEA. Within the 30 day peri-operative period, high plasma LPL was independently associated with an increased risk of MACE (adjusted hazard ratio [HR] per standard deviation [SD] 1.60, 1.10 - 2.30), p = .014). From 30 days to three years, however, high LPL was associated with a lower risk of MACE (adjusted HR per SD 0.80, 0.65 - 0.99, p= .036). CONCLUSION: High LPL concentrations were found to be associated with a higher risk of MACE in the first 30 post-operative days but with a lower risk MACE between 30 days and three years, meaning that LPL has different hazards at different time points.

Targeted proteomics improves cardiovascular risk prediction in secondary prevention.

AIMS: Current risk scores do not accurately identify patients at highest risk of recurrent atherosclerotic cardiovascular disease (ASCVD) in need of more intensive therapeutic interventions. Advances in high-throughput plasma proteomics, analysed with machine learning techniques, may offer new opportunities to further improve risk stratification in these patients. METHODS AND RESULTS: Targeted plasma proteomics was performed in two secondary prevention cohorts: the Second Manifestations of ARTerial disease (SMART) cohort (n = 870) and the Athero-Express cohort (n = 700). The primary outcome was recurrent ASCVD (acute myocardial infarction, ischaemic stroke, and cardiovascular death). Machine learning techniques with extreme gradient boosting were used to construct a protein model in the derivation cohort (SMART), which was validated in the Athero-Express cohort and compared with a clinical risk model. Pathway analysis was performed to identify specific pathways in high and low C-reactive protein (CRP) patient subsets. The protein model outperformed the clinical model in both the derivation cohort [area under the curve (AUC): 0.810 vs. 0.750; P < 0.001] and validation cohort (AUC: 0.801 vs. 0.765; P < 0.001), provided significant net reclassification improvement (0.173 in validation cohort) and was well calibrated. In contrast to a clear interleukin-6 signal in high CRP patients, neutrophil-signalling-related proteins were associated with recurrent ASCVD in low CRP patients. CONCLUSION: A proteome-based risk model is superior to a clinical risk model in predicting recurrent ASCVD events. Neutrophil-related pathways were found in low CRP patients, implying the presence of a residual inflammatory risk beyond traditional NLRP3 pathways. The observed net reclassification improvement illustrates the potential of proteomics when incorporated in a tailored therapeutic approach in secondary prevention patients.

Atherogenic Lipoprotein(a) Increases Vascular Glycolysis, Thereby Facilitating Inflammation and Leukocyte Extravasation.

RATIONALE: Patients with elevated levels of lipoprotein(a) [Lp(a)] are hallmarked by increased metabolic activity in the arterial wall on positron emission tomography/computed tomography, indicative of a proinflammatory state. OBJECTIVE: We hypothesized that Lp(a) induces endothelial cell inflammation by rewiring endothelial metabolism. METHODS AND RESULTS: We evaluated the impact of Lp(a) on the endothelium and describe that Lp(a), through its oxidized phospholipid content, activates arterial endothelial cells, facilitating increased transendothelial migration of monocytes. Transcriptome analysis of Lp(a)-stimulated human arterial endothelial cells revealed upregulation of inflammatory pathways comprising monocyte adhesion and migration, coinciding with increased 6-phophofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)-3-mediated glycolysis. ICAM (intercellular adhesion molecule)-1 and PFKFB3 were also found to be upregulated in carotid plaques of patients with elevated levels of Lp(a). Inhibition of PFKFB3 abolished the inflammatory signature with concomitant attenuation of transendothelial migration. CONCLUSIONS: Collectively, our findings show that Lp(a) activates the endothelium by enhancing PFKFB3-mediated glycolysis, leading to a proadhesive state, which can be reversed by inhibition of glycolysis. These findings pave the way for therapeutic agents targeting metabolism aimed at reducing inflammation in patients with cardiovascular disease.

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

[Figure: see text].

Delayed Development of Aneurysmal Dilatations in Patients with Extracranial Carotid Artery Dissections.

OBJECTIVE: Dissection of the carotid artery (CaAD) may result in aneurysm formation. The present study was undertaken to evaluate the time of onset of post-dissection extracranial carotid artery aneurysms (ECAA) following CaAD, and to analyse independent risk factors for the development of these aneurysms. METHODS: From four European stroke centres, 360 patients with extracranial CaAD were included. The time between the estimated dissection onset and aneurysm formation was analysed, and the clinical risk factors increasing the probability of aneurysm were assessed. RESULTS: The median duration of follow up was 5.2 months (range 0 - 24 months). A total of 75 post-dissection ECAAs were identified in 70 patients (19.4%, 95% confidence interval [CI] 15.7 - 23.8). In 52 of 70 (74%) patients, the ECAA was diagnosed at the initial clinical work up of CaAD diagnosis, with the median estimated time of dissection onset to ECAA diagnosis being six days (interquartile range [IQR] 0 - 25). In the remaining 18 (26%) patients who had normal carotid arteries at the initial imaging, the aneurysm diagnosis was made a median of 6.2 months (189 days) from the original imaging (IQR 128 - 198). A Cox proportional hazards model showed that both multiple artery dissections (hazard ratio [HR] 2.58, 95% CI 1.54 - 4.33) and arterial tortuosity (HR 1.79, 95% CI 1.08 - 2.95) were associated with presence of ipsilateral ECAA. CONCLUSION: This post hoc cohort analysis showed substantially delayed development of ipsilateral ECAA in patients with CaAD, months after baseline. Multiple dissections and arterial tortuosity are associated with the presence of ECAA and can be used in future prediction models of ECAA development in patients with CaAD.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.

Mast Cell Distribution in Human Carotid Atherosclerotic Plaque Differs Significantly by Histological Segment.

OBJECTIVE: Mast cells (MCs) are important contributors to atherosclerotic plaque progression. For prospective studies on mast cell contributions to plaque instability, the distribution of intraplaque MCs needs to be elucidated. Plaque stability is generally histologically assessed by dividing the plaque specimen into segments to be scored on an ordinal scale. However, owing to competitive use, studies may have to deviate to adjacent segments, yet intersegment differences of plaque characteristics, especially MCs, are largely unknown. Therefore, the hypothesis that there is no segment to segment difference in MC distribution between atherosclerotic plaque segments was tested, and intersegment associations between MCs and other plaque characteristics was investigated. METHODS: Twenty-six carotid atherosclerotic plaques from patients undergoing carotid endarterectomy included in the Athero-Express Biobank were analysed. The plaque was divided in 5 mm segments, differentiating between the culprit lesion (segment 0), adjacent segments (-1/+1) and more distant segments (-2/+2) for the presence of MCs. The associations between the intersegment distribution of MCs and smooth muscle cells, macrophage content, and microvessel density in the culprit lesion were studied. RESULTS: A statistically significant difference in MCs/mm2 between the different plaque segments (p < .001) was found, with a median of 2.79 (interquartile range [IQR] 1.63 - 7.10) for the culprit lesion, 1.34 (IQR 0.26 - 4.45) for the adjacent segment, and 0.62 (0.14 - 2.07) for the more distant segment. Post hoc analyses showed that intersegment differences were due to differences in MCs/mm2 between the culprit and adjacent segment (p = .037) and between the culprit lesion and the more distant segment (p < .001). MCs/mm2 in multiple different segments were positively correlated with microvessel density and macrophage content in the culprit lesion. CONCLUSION: MC numbers reveal significant intersegment differences in human carotid plaques. Future histological studies on MCs should use a standardised segment for plaque characterisation as plaque segments cannot be used interchangeably for histological MC analyses.

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.

Experimental parameters and infarct size in closed chest pig LAD ischemia reperfusion models; lessons learned.

BACKGROUND: Preclinical models that resemble the clinical setting as closely as possible are essential in translating promising therapies for the treatment of acute myocardial infarction. Closed chest pig left anterior descending coronary artery (LAD) ischemia reperfusion (I/R) models are valuable and clinically relevant. Knowledge on the influence of experimental design on infarct size (IS) in these models is a prerequisite for suitable models. To this end, we investigated the impact of several experimental features (occlusion and follow-up time and influence of area at risk (AAR)) on IS. METHODS: A total of fifty-one female Landrace pigs were subjected to closed chest LAD balloon occlusion and evaluated in three substudies with varying protocols. To assess the relationship between time of occlusion and the IS, 18 pigs were subjected to 60-, 75- and 90 min of occlusion and terminated after 24 h of follow-up. Influence of prolonged follow-up on IS was studied in 18 pigs after 75 min of occlusion that were terminated at 1, 3 and 7 days. The relation between AAR and IS was studied in 28 pigs after 60 min of occlusion and 24 h of follow-up. The relation between VF, number of shocks and IS was studied in the same 28 pigs after 60 min of occlusion. RESULTS: Increasing occlusion time resulted in an increased IS as a ratio of the AAR (IS/AAR). This ranged from 53 ± 23% after 60 min of occlusion to 88 ± 2.2% after 90 min (P = 0.01). Increasing follow-up, from 1 to 3 or 7 days after 75 min of occlusion did not effect IS/AAR. Increasing AAR led to a larger IS/AAR (r2 = 0.34, P = 0.002), earlier VF (r2 = 0.32, P = 0.027) and a higher number of shocks (r2 = 0.29, P = 0.004) in pigs subjected to 60 min of occlusion. CONCLUSIONS: These experiments describe the association of occlusion time, follow-up duration, AAR and VF with IS in closed chest pig LAD I/R models. These results have important implications for future I/R studies in pigs and can serve as a guideline for the selection of appropriate parameters and the optimal experimental design.

Ventricular TLR4 Levels Abrogate TLR2-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice.

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1ß, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

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.

The TAXINOMISIS Project: A multidisciplinary approach for the development of a new risk stratification model for patients with asymptomatic carotid artery stenosis.

INTRODUCTION: Asymptomatic carotid artery stenosis (ACAS) may cause future stroke and therefore patients with ACAS require best medical treatment. Patients at high risk for stroke may opt for additional revascularization (either surgery or stenting) but the future stroke risk should outweigh the risk for peri/post-operative stroke/death. Current risk stratification for patients with ACAS is largely based on outdated randomized-controlled trials that lack the integration of improved medical therapies and risk factor control. Furthermore, recent circulating and imaging biomarkers for stroke have never been included in a risk stratification model. The TAXINOMISIS Project aims to develop a new risk stratification model for cerebrovascular complications in patients with ACAS and this will be tested through a prospective observational multicentre clinical trial performed in six major European vascular surgery centres. METHODS AND ANALYSIS: The risk stratification model will compromise clinical, circulating, plaque and imaging biomarkers. The prospective multicentre observational study will include 300 patients with 50%-99% ACAS. The primary endpoint is the three-year incidence of cerebrovascular complications. Biomarkers will be retrieved from plasma samples, brain MRI, carotid MRA and duplex ultrasound. The TAXINOMISIS Project will serve as a platform for the development of new computer tools that assess plaque progression based on radiology images and a lab-on-chip with genetic variants that could predict medication response in individual patients. CONCLUSION: Results from the TAXINOMISIS study could potentially improve future risk stratification in patients with ACAS to assist personalized evidence-based treatment decision-making.

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Cerebral Small Vessel Disease in Standard Pre-operative Imaging Reports Is Independently Associated with Increased Risk of Cardiovascular Death Following Carotid Endarterectomy.

OBJECTIVE: Cerebral white matter lesions (WMLs) and lacunar infarcts are surrogates of cerebral small vessel disease (SVD). WML severity as determined by trained radiologists predicts post-operative stroke or death in patients undergoing carotid endarterectomy (CEA). It is unknown whether routine pre-operative brain imaging reports as part of standard clinical practice also predict short and long term risk of stroke and death after CEA. METHODS: Consecutive patients from the Athero-Express biobank study that underwent CEA for symptomatic high degree stenosis between March 2002 and November 2014 were included. Pre-operative brain imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) reports were reviewed for reporting of SVD, defined as WMLs or any lacunar infarcts. The primary outcome was defined as any stroke or any cardiovascular death over three year follow up. The secondary outcome was defined as the 30 day peri-operative risk of stroke or cardiovascular death. RESULTS: A total of 1038 patients were included (34% women), of whom 659 (63.5%) had CT images and 379 (36.5%) MRI images available. Of all patients, 697 (67%) had SVD reported by radiologists. Patients with SVD had a higher three year risk of cardiovascular death than those without (6.5% vs. 2.1%, adjusted HR 2.52 [95% CI 1.12-5.67]; p = .026) but no association was observed for the three year risk of stroke (9.0% vs. 6.7%, for patients with SVD vs. those without, adjusted HR 1.24 [95% CI 0.76-2.02]; p = .395). No differences in 30 day peri-operative risk were observed for stroke (4.4% vs. 2.9%, for patients with vs. those without SVD; adjusted HR 1.49 [95% CI 0.73-3.05]; p = .28), and for the combined stroke/cardiovascular death risk (4.4% vs. 3.5%, adjusted HR 1.20 [95% CI 0.61-2.35]; p = .59). CONCLUSION: Presence of SVD in pre-operative brain imaging reports can serve as a predictor for the three year risk of cardiovascular death in symptomatic patients undergoing CEA but does not predict peri-operative or long term risk of stroke.