Endothelial microparticles (EMP) for the assessment of endothelial function: an in vitro and in vivo study on possible interference of plasma lipids

Abstract

Background: Circulating endothelial microparticles (EMP) reflect the condition of the endothelium and are of increasing interest in cardiovascular and inflammatory diseases. Recently, increased numbers of EMP following oral fat intake, possibly due to acute endothelial injury, have been reported. On the other hand, the direct interference of lipids with the detection of EMP has been suggested. This study aimed to investigate the effect of lipid-rich solutions, commonly administered in clinical practice, on the detection, both in vitro and in vivo, of EMP.

Methods: For the in vitro assessment, several lipid-rich solutions were added to whole blood of healthy subjects (n = 8) and patients with coronary heart disease (n = 5). EMP (CD31+/CD42b-) were detected in platelet poor plasma by flow cytometry. For the in vivo study, healthy volunteers were evaluated on 3 different study-days: baseline evaluation, following lipid infusion and after a NaCl infusion. EMP quantification, lipid measurements and peripheral arterial tonometry were performed on each day.

Results: Both in vitro addition and in vivo administration of lipids significantly decreased EMP (from 198.6 to 53.0 and from 272.6 to 90.6/µl PPP, respectively, p = 0.001 and p = 0.012). The EMP number correlated inversely with the concentration of triglycerides, both in vitro and in vivo (r = -0.707 and -0.589, p<0.001 and p = 0.021, respectively). The validity of EMP as a marker of endothelial function is supported by their inverse relationship with the reactive hyperemia index (r = -0.758, p = 0.011). This inverse relation was confounded by the intravenous administration of lipids.

Conclusion: The confounding effect of high circulating levels of lipids, commonly found in patients that receive intravenous lipid-based solutions, should be taken into account when flow cytometry is used to quantify EMP.

Figure 1. Decrease in EMP after in vitro administration of lipid-rich solutions.

Whole blood was taken from 8 healthy volunteers (black) and 5 patients with coronary heart disease (red). PPP was prepared from different aliquots to which lipid-rich solutions were added in different concentrations. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b−. The figure shows EMP numbers/µl PPP from samples without lipid-rich solutions (PPP), and samples with added lipid-rich solutions in the lowest concentration (Lipid). The number of EMP detected by flow cytometry decreased significantly (all data taken together p = 0.001). EMP = endothelial microparticles, PPP = platelet poor plasma.

Figure 2. The inverse relation between EMP and TG after the in vitro administration of lipid-rich solutions to whole blood.

Whole blood was taken from 8 healthy volunteers (black) and 5 patients with coronary heart disease (red). PPP was prepared from different aliquots to which lipid-rich solutions were added in different concentrations. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b−, and TG concentration were determined in plasma. The figure shows EMP numbers/µl PPP from samples without lipid-rich solutions (black inversed triangles for the healthy volunteers and red diamonds for the cardiovascular patients) and samples with added lipid-rich solutions in different concentrations (black quadrangles for the healthy volunteers and red triangles for the cardiovascular patients). Taken all samples together a non-linear inverse relation exists between TG concentration and the number of EMP (r = −0.707, p<0.001). EMP = endothelial microparticles, PPP = platelet poor plasma, TG = triglycerides.

Figure 3. Evolution of EMP detected by flow cytometry in the in vivo study.

5 healthy volunteers were evaluated on 3 different study days (all 5 represented in a different color and symbol). Day A: blood was collected after an overnight fast. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b− in PPP. Day B: NaCl 0.9% was administered in fasting conditions and the same measurements as on day A were performed. Day C: the same protocol was used as on day B, but a pure lipid solution was infused instead of NaCl. A lower number of EMP was detected in all healthy volunteers after intravenous administration of the lipid emulsion (p = 0.012). EMP = endothelial microparticles, PPP = platelet poor plasma.

Figure 4. In vivo there is a non-linear inverse relation between TG concentration and EMP detected in plasma.

5 healthy volunteers were evaluated on 3 different study days. Day A: blood was collected after an overnight fast. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b− in PPP, and lipid profile was determined on frozen plasma samples. (Black dots) Day B: NaCl 0.9% was administered in fasting conditions and the same measurements as on day A were performed. (Black open triangles) Day C: the same protocol was used as on day B, but a pure lipid solution was infused instead of NaCl. (Gray diamonds) There is an inverse non-linear relation between the TG concentration in plasma and the number of EMP number/µl PPP detected (r = −0.589, p = 0.021). EMP = endothelial microparticles, PPP = platelet poor plasma, TG = triglycerides.

Figure 5. In healthy volunteers the inverse relation of EMP with RHI is confounded by the intravenous administration of a pure lipid solution.

5 healthy volunteers were evaluated on 3 different study days. Day A: blood was collected after an overnight fast and PAT was performed. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b− in PPP. Day B: a NaCl 0.9% infusion was administered in fasting conditions and the same measurements as on day A were performed. Day C: the same protocol was used as on day B, but a parenteral lipid emulsion was infused instead of NaCl. On day A and B (black dots) there was an inverse relation between EMP numbers/µl PPP and RHI (r = −0.758, p = 0.011 combining data from day A and day B). This relation was confounded by lipid infusion as on day C (gray quadrangles) (r = −0.254, p = 0.361 combining all data). EMP = endothelial microparticles, PAT = peripheral arterial tonometry, PPP = platelet poor plasma, RHI = reactive hyperemia index.

Figure 6. Gating strategy microparticle dimension criteria.

Fluoresbrite YG 1 µm calibration size beads (Polysciences, Eppelheim Germany) were used to set forward (FSC) and side scatter (SSC) criteria for the assessment of microparticles. Beads were measured at low flow rate and threshold was placed on both FSC and SSC. More in particular, the MP analysis region was defined as follows: at first, the bead population was selected on a histogram of the FSC signal (A), while the events within this gate were further selected on SSC signal (B); the upper detection limit of the microparticle gate was set on the peak of the selected 1 µm beads on histogram plots of FSC (C) and SSC (D). For illustrative purposes, the population hierarchy is shown in figure E. The lower dimension criterion was set just above the electronic noise of the cytometer, which corresponded with the upper boarder of the Fluoresbrite YG 0.5 µm calibration size beads on FSC as illustrated in figures F and G. In figure F, the threshold was placed on FITC, and in figure G, the threshold was placed on FSC and SSC. As an illustration our microparticle dimension criteria are shown as a rectangular gate in figure F and G. The position of the peak signal of the 1 µm beads on FSC and SSC was maintained at the same place by adjusting the FSC and SSC voltage when necessary. FITC = fluorescein, FSC-H = forwards scatter height, SSC-H = sight scatter height, MP = microparticles, PPP = platelet poor plasma.

Figure 7. Microparticle gating strategy.

For the detection of EMP, PPP was incubated at 4°C for 20 minutes with CD31-PE (BD Biosciences, Erembodegem Belgium) and CD42b-FITC (BD Bioscience, Erembodegem Belgium), both 0.22 µm filtered and azide free. Samples were diluted in 0.22 µm filtered FACSflow (BD Biosciences, Erembodegem Belgium), to allow sample acquisition at less than 1000 events/sec on low flow rate. Samples were analyzed on a FACSCantoII (BD Biosciences, Erembodegem Belgium). Antibodies were titrated, and isotype fluorescence-minus-one for FITC (C) and PE (D) and unstained samples (A) were run as controls. EMP were defined as particles smaller than 1 µm that were CD31-positive and CD42b-negative (B). For illustrative reasons the MP gate is shown as a rectangular gate. For the settings of the dimension criteria see figure 6. EMP = endothelial microparticle, FITC = fluorescein, FSC-H = forwards scatter height, SSC-H = sight scatter height, MP = microparticle, PE = phycoerythrin, PPP = platelet poor plasma.