Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular... Show morePreviously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m(2)), and patients with DN (eGFR <30 mL/min/1.73 m(2)). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132). Show less
Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular... Show morePreviously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m2), and patients with DN (eGFR <30 mL/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132). Show less
Microparticles (MPs) have important physiological and pathological roles in blood coagulation, inflammation and tumor progression. In recent years MPs also have been recognized to participate in... Show moreMicroparticles (MPs) have important physiological and pathological roles in blood coagulation, inflammation and tumor progression. In recent years MPs also have been recognized to participate in important biological processes, such as in signaling and in the horizontal transfer of their specific proteins and mRNAs. However, studies of MPs have been hampered by the lack of methods for the sensitive detection and accurate quantification of MPs. Thus, we have developed a new methodology by using atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) to detect, quantify and characterize MPs in plasma. We have shown that AFM detects 1000-fold more platelet derived-MPs than a conventional flow cytometry does. These MPs have diameters ranging from 10-475 nm with a peak at 67.5 nm, which is clearly far below the detection limit of flow cytometry. By using cryo-EM we found that the number of lipoprotein particles exceeds that of MPs or exosomes in plasma. We also demonstrated that by using immuno-magnetic beads selected subset of MPs could directly be captured/depleted from plasma and assessed for MP-associated tissue factor activity. In the future the measurement of MPs will perhaps serve as a diagnostic tool to identify and predict diseases, like cancer. Show less
Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic (VTE) complications. Aim of this study was to measure microparticle-associated tissue factor (MP-TF) activity in... Show moreMultiple myeloma (MM) is associated with an increased risk of venous thromboembolic (VTE) complications. Aim of this study was to measure microparticle-associated tissue factor (MP-TF) activity in patients with newly diagnosed MM before and after chemotherapy and to investigate whether MP-TF activity is associated with VTE. MP-TF activity was assessed in 122 newly diagnosed MM patients who were eligible for combination chemotherapy. MP-TF activity levels (17.6 fM Xa/min [8.6-33.2] (median [IQR]) were higher in untreated MM patients compared to normal healthy volunteers (4.1 fM Xa/min [2.3-6.6], p <0.001). MP-TF activity prior to the start of treatment was not different between patients who developed a VTE during follow-up (n=15) and those who did not (n=107). In 75 patients in whom plasma was obtained before and after chemotherapy, MP-TF activity decreased significantly (from 17.4 [10.2-32.8] to 12.0 [7.0-18.5] fM Xa/min, P=0.006). MP-TF activity remained, however, elevated in patients who developed VTE (15.1 [10.3-25.2]), in contrast to patients not developing VTE (11.4 [7.0-25.2], P<0.001). In conclusion, MP-TF activity is increased in patients with MM. Whether MP-TF activity has a pathogenetic role in VTE in MM patients remains to be established in future studies. Show less
Results of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in... Show moreResults of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in pre-analytical conditions. In this review we will discuss the pre-analytical variables related to plasma and MP preparation which may affect MP analysis. Additionally we will address several analytical issues in commonly used MP assays and briefly discuss some novel approaches for the detection and characterisation of MPs. Ideally MP measurements should be performed in plasma, freshly prepared directly after blood withdrawal. As platelet contamination seems to be one of the major pre-analytical problems in processing plasma for MP measurement, the use of platelet-free plasma may be preferred. When frozen-thawed plasma is used, especially PMP and annexinV-positive MP counts should be interpreted with caution. When flow cytometry is chosen as a method for quantification of MPs, some analytical conditions should be standardised, e.g. settings of the flow cytometer, quality of the antibodies, and use of counting beads. Fluorescence-nanoparticle tracking analysis and atomic force microscopy can accurately count nanosized MPs, but unfortunately the operational procedures of both methods are still time consuming and they give no information on the functional properties of MPs. The MP-TF activity assay provides information on MPs carrying active TF, regardless of their parental origin. Ultimately, standardisation of pre-analytical procedures and the introduction of reliable and rapid methods for the measurement of MPs are urgently needed to facilitate their use as biomarker in the pathophysiology of diseases. Show less
Background: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in ... Show moreBackground: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio) vascular disorders and cancer. Most of these MPs are derived from platelets. Objectives: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. Methods: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG(1) isotype control was used as a negative control. Results: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation = 16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (d(sph)) ranged from 10 to 475 nm (mean: 67.5 +/- 26.5 nm) and from 5 to 204 nm (mean: 51.4 +/- 14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702 x 10(9) L-1 plasma vs. 11-626 x 10(6) L-1 plasma). After filtration of isolated MPs through a 0.22-mu m filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean d(sph): 37.2 +/- 11.6 nm), but not by flow cytometry. Conclusions: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry. Show less
