Morten Hjuler Nielsen

Diagnostic and prognostic potential of extracellular vesicles in peripheral blood

PURPOSEnExtracellular vesicles (EVs) are small, membrane-enclosed entities released from cells in many different biological systems. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. The complement of biomolecules reflects the parent cell, and their characterization may provide information about the presence of an aberrant process. Peripheral blood is a rich source of circulating EVs, which are easily accessible through a blood sample. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic and prognostic value. The objectives of this review are to briefly present the current knowledge about EVs and to introduce a toolbox of selected techniques, which can be used to rapidly characterize clinically relevant properties of EVs from peripheral blood.nnnMETHODSnSeveral techniques exist to characterize the different features of EVs, including size, enumeration, RNA cargo, and protein phenotype. Each technique has a number of advantages and pitfalls. However, with the techniques presented in this review, a possible platform for EV characterization in a clinical setting is outlined.nnnFINDINGSnAlthough EVs have great diagnostic and prognostic potential, a lack of standardization regarding EV analysis hampers the full use of this potential. Nevertheless, the analysis of EVs in peripheral blood has several advantages compared with traditional analyses of many soluble molecules in blood.nnnIMPLICATIONSnOverall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential.

Elevated atherosclerosis-related gene expression, monocyte activation and microparticle-release are related to increased lipoprotein-associated oxidative stress in familial hypercholesterolemia.

Objective Animal and in vitro studies have suggested that hypercholesterolemia and increased oxidative stress predisposes to monocyte activation and enhanced accumulation of oxidized LDL cholesterol (oxLDL-C) through a CD36-dependent mechanism. The aim of this study was to investigate the hypothesis that elevated oxLDL-C induce proinflammatory monocytes and increased release of monocyte-derived microparticles (MMPs), as well as up-regulation of CD36, chemokine receptors and proinflammatory factors through CD36-dependent pathways and that this is associated with accelerated atherosclerosis in subjects with heterozygous familial hypercholesterolemia (FH), in particular in the presence of Achilles tendon xanthomas (ATX). Approach and Results We studied thirty FH subjects with and without ATX and twenty-three healthy control subjects. Intima-media thickness (IMT) and Achilles tendon (AT) thickness were measured by ultrasonography. Monocyte classification and MMP analysis were performed by flow cytometry. Monocyte expression of genes involved in atherosclerosis was determined by quantitative PCR. IMT and oxLDL-C were increased in FH subjects, especially in the presence of ATX. In addition, FH subjects had elevated proportions of intermediate CD14++CD16+ monocytes and higher circulating MMP levels. Stepwise linear regression identified oxLDL-C, gender and intermediate monocytes as predictors of MMPs. Monocyte expression of pro-atherogenic and pro-inflammatory genes regulated by oxLDL-C-CD36 interaction was increased in FH, especially in ATX+ subjects. Monocyte chemokine receptor CX3CR1 was identified as an independent contributor to IMT. Conclusions Our data support that lipoprotein-associated oxidative stress is involved in accelerated atherosclerosis in FH, particularly in the presence of ATX, by inducing pro-inflammatory monocytes and increased release of MMPs along with elevated monocyte expression of oxLDL-C-induced atherosclerosis-related genes.

The Impact of Lipoprotein-Associated Oxidative Stress on Cell-Specific Microvesicle Release in Patients with Familial Hypercholesterolemia.

Objective. Microvesicles (MVs) are small cell-derived particles shed upon activation. Familial hypercholesterolemia (FH) particularly when associated with Achilles tendon xanthomas (ATX) predisposes to atherosclerosis, possibly through oxLDL-C interaction with the CD36 receptor. To investigate the hypothesis that MVs derived from cells involved in atherosclerosis are increased in FH and that CD36 expressing MVs (CD36+ MVs) may be markers of oxLDL-C-induced cell activation, cell-specific MVs were measured in FH patients with and without ATX and their association with atherogenic lipid profile was studied. Approach and Results. Thirty FH patients with and without ATX and twenty-three controls were included. Plasma concentrations of MVs and CD36+ MVs derived from platelets (PMVs), erythrocytes (ErytMVs), monocytes (MMVs), and endothelial cells (EMVs), as well as tissue factor-positive cells (TF+ MVs), were measured by flow cytometry. Total MVs, MMVs, EMVs, ErytMVs, and TF+ MVs were significantly increased in FH patients, compared to controls. CD36+ MVs derived from endothelial cells and monocytes were significantly higher in FH patients and oxLDL-C predicted all the investigated cell-specific CD36+ MVs in FH patients with ATX. Conclusions. MVs derived from cells involved in atherosclerosis were increased in FH and may contribute to elevated atherothrombosis risk. The increased cell-specific CD36+ MVs observed in FH may represent markers of oxLDL-C-induced cell activation.

In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry

Microvesicles (MVs) are submicron vesicles with sizes of 0.1–1.0u2009μm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxidized low-density lipoprotein (oxLDL), a platelet ligand, induces platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study, we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5u2009μm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5u2009μm limit. MV size distribution was analyzed in plasma from 11 healthy volunteers (four females and seven males). MVs were identified as <1u2009μm and positive for lactadherin binding and cell-specific markers. Platelet-rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41+ and CD41+CD36+ MVs increased by six to eightfold in PRP, when left at room temperature, and the presence of cell-specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large interindividual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that procoagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great interindividual variability in size distribution of platelet-derived MVs and thereby stress the importance for generation of standardized protocols for MV quantification by flow cytometry.

Microvesicles Correlated with Components of Metabolic Syndrome in Men with Type 2 Diabetes Mellitus and Lowered Testosterone Levels But Were Unaltered by Testosterone Therapy

Aims. To investigate how circulating microvesicle phenotypes correlate with insulin sensitivity, body composition, plasma lipids, and hepatic fat accumulation. We hypothesized that changes elicited by testosterone replacement therapy are reflected in levels of microvesicles. Methods. Thirty-nine type 2 diabetic males with lowered testosterone levels were assigned to either testosterone replacement therapy or placebo and evaluated at baseline and after 24 weeks. Microvesicles were analysed by flow cytometry and defined as lactadherin-binding particles within the 0.1–1.0u2009μm gate. Microvesicles of platelet, monocyte, and endothelial cell origin were identified by cell-specific markers and their expression of CD36 was investigated. Results. Triglycerides correlated positively with all investigated microvesicle phenotypes in this study (p < 0.05), and indicators of hepatic fat accumulation, alanine aminotransferase, and gamma glutamyltransferase correlated with platelet and endothelial microvesicles and CD36-expressing microvesicles from platelets and monocytes (p < 0.05). BMI, waist circumference, and fat percentage correlated with CD36-expressing monocyte microvesicles (p < 0.05), while insulin sensitivity did not correlate with any microvesicle phenotypes. Microvesicle levels were unaffected by testosterone therapy. Conclusions. Metabolic syndrome components and hepatic fat accumulation correlated with microvesicle phenotypes, supporting the involvement of especially CD36 on monocytes in metabolic syndrome pathogenesis. Although testosterone therapy improved body composition measures, microvesicle phenotype levels were unaffected. This trial was registered at ClinicalTrials.gov (NCT01560546).

Postprandial Increase in Blood Plasma Levels of Tissue Factor–Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

Bariatric surgery reduces CD36-bearing microvesicles of endothelial and monocyte origin

BackgroundBariatric surgery is a widely adopted treatment for obesity and its secondary complications. In the past decade, microvesicles (MVs) and CD36 have increasingly been considered as possible biomarkers for obesity, the metabolic syndrome (MetSy), type 2 diabetes mellitus (T2DM). Thus, the purpose of this study was to investigate how weight loss resulting from bariatric surgery affects levels of specific MV phenotypes and their expression of CD36 scavenger receptor. Additionally, we hypothesised that subjects with MetSy had higher baseline concentrations of investigated MV phenotypes.MethodsTwenty individuals undergoing Roux-en-Y gastric bypass surgery were evaluated before and 3xa0months after surgery. MVs were characterised by flow cytometry at both time points and defined as lactadherin-binding particles within a 100-1000xa0nm size gate. MVs of monocyte (CD14) and endothelial (CD62E) origin were defined by cell-specific markers, and their expression of CD36 was investigated.ResultsFollowing bariatric surgery, subjects incurred an average BMI reduction (delta) of −u20098.4u2009±u20091.4 (pu2009<u20090.0001). Significant reductions were observed for the total MVs (−u200966.55%, pu2009=u20090.0017) and MVs of monocyte (−u200936.11%, pu2009=u20090.0056) and endothelial (−u200940.10%, pu2009=u20090.0007) origins. Although the bulk of CD36-bearing MVs were unaltered, significant reductions were observed for CD36-bearing MVs of monocyte (−u200960.04%, pu2009=u20090.0192) and endothelial (−u200954.93%, pu2009=u20090.04) origin. No differences in levels of MVs were identified between subjects who presented with MetSy at baseline (nu2009=u200913) and those that did not (nu2009=u20097).ConclusionBariatric surgery resulted in significantly altered levels of CD36-bearing MVs of monocyte and endothelial origin. This likely reflects improvements in ectopic fat distribution, plasma lipid profile, low-grade inflammation, and oxidative stress following weight loss. Conversely, however, the presence of MetSy at baseline had no impact on MV phenotypes.

Novel triggering threshold strategy for discovery of rare microvesicle phenotypes on flow cytometers dedicated to small particle analysis

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

The phenotypical changes of plasma EVs over time in healthy donors

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

Flow cytometers dedicated to the analysis of small particles: a powerful tool for EV characterisation

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414