In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant (original) (raw)
Release of MPs by HUVECs upon stimulation by TNF. To view the process of MP blebbing and shedding on HUVEC surface, cells were stimulated with TNF for 6 hours and prepared for scanning electron microscopic analysis. Unstimulated HUVECs displayed a cobblestone shape and a smooth surface, with a relatively low number of vesicles, presenting a sparse distribution (Figure 2a and b). After stimulation (Figure 2c), the cells exhibited a more fusiform shape and a blebby surface — due to a marked increase in the number of vesicles, which nearly covered the cell surface. At a higher magnification (Figure 2d), these blebs showed different diameters ranging from 0.5 to 2.5 μm (arrows). Some of the blebs were almost detached from the surface (Figure 2d, arrowhead). Analysis of culture supernatants from TNF-stimulated cells by scanning electron microscopy showed MPs with diameters ranging from 0.1 to 1.5 μm (Figure 2, e and f).
Morphology of HUVECs and EMPs. First-passage monolayers of resting (a and b) and TNF-stimulated (c and d) HUVECs were analyzed by scanning electron microscopy. (a and c) ×1,500; (b and d) ×3,000. (e and f) High-power magnification (×24,000 and ×36,000) of EMPs shed from TNF-stimulated HUVECs. Both resting and stimulated ECs showed surface blebs (arrows) or detached vesicles (arrowhead in d). Scale bars: 10 μm (a–d), 1 μm (e and f). P, filter pore.
We therefore quantitated the effect of TNF on vesiculation (Figure 3). In resting conditions, HUVECs released 90 ± 8 MPs per 103 cells in the supernatant over a 24-hour period, as determined by annexin V binding. TNF stimulation induced, in a dose-dependent manner, an increase in the number of MPs released from HUVECs by a maximum of 2.5-fold for 100 ng/mL TNF stimulation. This effect was abrogated when neutralizing anti-TNF antibody was added 1 hour prior to TNF. The antibody pretreatment alone had no effect. Apoptosis was not detectable in ECs in our conditions of TNF activation, as shown by PI and annexin V staining (data not shown). Other EC agonists were investigated for their capacity to induce vesiculation, including IL-1β (10 U/mL, 24 hours at 37°C), PMA (100 ng/mL, 24 hours at 37°C), thrombin (0.1 U/mL, 24 hours at 37°C), and calcium ionophore (100 μmol/L, 10 minutes at 37°C). MP numbers released in these conditions were similar to those obtained after TNF stimulation.
Effect of TNF on EMP shedding. HUVECs were incubated with either medium alone or varying concentrations of recombinant human TNF (a). A neutralizing anti-TNF antibody was added 1 hour before stimulation with the highest TNF concentration or was tested alone (b). Results are expressed in numbers of MPs labeled with annexin V-FITC, extracted from culture supernatants of 103 ECs. Bars represent SD of 3 determinations in 4 experiments.
Expression and modulation of surface molecules on ECs and EMPs. We determined whether MPs displayed the same phenotype as the cells from which they came. The presence of endothelial membrane antigens involved in coagulation (TF and TM) and adhesion (E selectin, ICAM-1, αvβ3, and PECAM-1) were assessed on both HUVECs and their shed MPs using flow cytometry (Figure 4).
Distribution of endothelial antigens on resting or TNF-stimulated HUVECs and their derived MPs. HUVECs were cultured for 24 hours in the presence or absence of TNF (100 ng/mL), detached and analyzed for mAb binding by flow cytometry (top). MPs derived from these ECs were labeled with the same mAb (bottom). For each antigen studied, the antibody binding was expressed as mean fluorescence intensity (MFI) of the positive population for HUVECs and as number of positive events for MPs, in view of the low intensity of labeling of the latter. For each mAb, MFI of cells and number of their derived MPs are shown under resting (left) and stimulated (right) conditions. Irrelevant mAb’s (both IgG1 and IgG2a) led to identical background staining.
Constitutive antigens of ECs, such as PECAM-1, αvβ3, ICAM-1, and TM, were present on both unstimulated cells and derived MPs. In response to a 24-hour TNF stimulation, the expression of PECAM-1 was not significantly modified on cells, whereas a 1.5-fold increase in the number of MPs expressing the corresponding antigen was observed. TNF enhanced the surface expression of αvβ3 (1.5-fold) and ICAM-1 (50-fold) on ECs concomitantly with an increase in the number of αvβ3- and ICAM-1–positive MP (3- and 4-fold, respectively). As expected, TM expression was decreased on TNF-stimulated HUVECs, and this decrease was accompanied by a slight diminution of TM-positive MPs. Inducible antigens, such as E-selectin and TF, were not detectable on resting ECs, whereas both peaked 4–6 hours after stimulation (data not shown) and presented a residual level of expression at 24 hours. It is noteworthy that TF- and E-selectin–positive EMPs were detectable after 24 hours of stimulation, compared with resting conditions (Figure 5, b versus a).
Flow cytometric analysis of EMPs under resting and stimulated conditions. MPs were obtained from EC supernatants and stained with mAb’s, as described in Methods. The cytograms (FL1/SSC) shown here are representative graphs of mAb binding on MPs, counted using 3-μm latex beads (gate B of Figure 1a) as an internal standard. In resting conditions (a), constitutive antigens such as PECAM-1, αvβ3, TM, and ICAM-1 were present on MPs, whereas inducible antigens such as TF and E-selectin were detectable only upon TNF stimulation (b). The horizontal bars represent the level of irrelevant mAb binding, used as control.
Representative staining patterns of MPs shed from HUVECs — stimulated by TNF or not — are illustrated in Figure 5, a and b, respectively. No direct comparison between detection by annexin V and mAb was made because surface distribution, affinity for the respective ligands, and incubation times were different. Nevertheless, these experiments indicate that particles found in the supernatant bore a substantial proportion of the antigens found on the corresponding cell.
TNF-generated procoagulant activity is associated with EMPs. In view of the EC changes suggesting a switch toward a procoagulant activity, the functional consequences of TF induction and TM downregulation were investigated. Accordingly, the procoagulant activity of MPs shed from 24-hour TNF-stimulated HUVECs was assessed by a clotting assay. A dose-response curve was established by incubating dilutions of the MP suspensions (5, 10, 15, and 25 μL of MP in 25 μL [final volume] of Owren-Koller buffer). A 35–70% shortening of the control plasma clotting time was observed after addition of increasing numbers of TNF-stimulated, HUVEC-derived MPs (Figure 6). MPs shed from unstimulated HUVECs or MPs derived from the different control groups (TNF-neutralizing antibody with or without TNF) resulted in a slight shortening of the clotting time (5–20%).
Plasma clotting time in the presence of EMPs. MPs were extracted by ultracentrifugation from the culture supernatant of unstimulated HUVECs (open circles), HUVECs incubated with anti-TNF antibody alone (filled triangles), anti-TNF antibody before TNF (100 ng/mL) (filled squares), and TNF (100 ng/mL) alone (filled diamonds). Various numbers of EMPs in suspension were added (10 μL corresponded to the number of MPs derived from 105 cells) to a chronometric test of normal (a) and factor VII–deficient (b) plasma clotting (Howell time). Data are expressed in absolute clotting times (measured in seconds).
Induction of TF on EMPs suggests that their procoagulant activity can be accounted for by activation of the extrinsic pathway of the coagulation system. To test this hypothesis, a factor VII–deficient plasma was used, allowing only intrinsic pathway involvement. In these conditions, a small decrease of the clotting time was observed after addition of MPs derived from TNF-stimulated HUVECs (a decrease of 5–30%). MPs obtained from control HUVECs or from HUVECs stimulated in the presence of anti-TNF antibody had a similar effect on the clotting time (5–15% decrease).
Identification of EMPs in peripheral blood. The demonstration that HUVECs and shed EMPs shared the same antigens was used to select a marker, or a combination of markers, that could allow the detection of EMPs in human plasma. We selected constitutive antigens that are highly expressed by ECs to ensure the most discriminative labeling of MPs. Accordingly, αvβ3 was used in combination with PECAM-1 to label MPs derived from HUVECs. As illustrated in Figure 7a, the whole MP population analyzed was double labeled. Biochemical evidence that EMPs express αvβ3 and PECAM-1 was provided by Western blot analysis on HUVEC and EMP lysates. Similar bands, at approximately 150 kDa for αvβ3 and 130 kDa for PECAM-1, were revealed on both HUVECs and EMPs (Figure 7b). Moreover, confocal laser microscopy showed αvβ3- and E-selectin–positive elements of less than 1.0 μm in diameter (Figure 8, c and d). Using transmission electron microscopy, TNF-induced EMPs appear as rounded vesicular structures with diameters ranging from 0.1 to 0.05 μm (Figure 8a).
Use of PECAM-1 and αvβ3 coexpression to delineate the endothelial origin of MPs: in vitro setup and ex vivo detection. For in vitro studies, the release of MPs in EC supernatants was induced by TNF, as described. (a) Setup of PECAM-1 and αvβ3 double labeling of MPs generated in vitro. (b) SDS-PAGE and Western blot analysis of HUVECs (E) and MP lysates (M) under nonreducing conditions, revealed with control isotype mAb, PECAM-1, and αvβ3. Ex vivo, double labeling of MPs was performed in normal human plasma (c–f). (c) Labeling with isotype control mAb’s, allowing the setting of the background noise position on the subsequent cytograms. (d) Positive double labeling of plasma MPs with FITC–anti-αvβ3 and either PE–anti-PECAM-1, PE–anti-CD41 (e), or PE–anti-CD14 (f).
Transmission electron and confocal laser microscopy of MPs. Transmission electron microscopy appearance of MPs generated in vitro (a) or in vivo (e), i.e., isolated from normal plasma (final magnification: ×25,000). In confocal laser microscopy, MPs appeared as elements with a diameter less than 1 μm and stained for αvβ3 (c) and E-selectin (d) in vitro. In vivo–generated MPs expressed αvβ3 (g) and CD41/GPIIb-IIIa (h). In this case, the majority of the elements were positive for CD41 (h), whereas only about 10% were positive for αvβ3 (g), confirming that they originate from ECs (final magnification: ×1,000). (b and f) Negative staining using isotope control IgG1.
In vivo–generated MPs revealed vesicular structures with diameters ranging from 0.1 to 1.0 μm (Figure 8e). Compared with those generated in vitro, these MPs appeared larger, more electron dense, and presented a more regular shape. The feasibility of MP detection using PECAM-1/αvβ3 double labeling was then investigated in normal human blood. Based on the control staining defined by irrelevant antibodies (Figure 7c), approximately 10% of MPs analyzed bound PECAM-1 and αvβ3 (Figure 7d), whereas about 90% were found to be positive only for PECAM-1, which is known to be expressed on blood cells. Because low levels of αvβ3 have been detected on platelets and monocytes, double-labeling experiments using anti-αvβ3 in combination with anti-CD41 or anti-CD14 were also performed. In both cases, no double-positive events were detectable; the proportion of αvβ3-labeled events was approximately 10%, whereas those positive for CD41 and CD14 represented 80% and 5%, respectively (Figure 7, e and f). These proportions of CD41-positive MPs versus αvβ3-positive elements were confirmed by confocal laser microscopy (Figure 8, g and h). Altogether, these experiments show that αvβ3 is not detectable on platelet- and monocyte-derived MPs and can be used to selectively discriminate EMPs in blood. Red blood cell, lymphocyte, and other leukocyte markers (glycophorin A, CD4, and CD45, respectively) were also found to be negative on αvβ3-labeled MPs (data not shown).
Quantitation of EMPs in blood samples from LA-positive patients. Because part of the MPs present in peripheral blood are of endothelial origin, we used αvβ3 to evaluate their number in 30 healthy individuals in comparison with a population presenting a thrombotic risk, such as LA-positive patients. Mean values of 37,000 ± 9,000 MPs/mL of plasma (± SD) were measured in healthy individuals, with no difference in EMP number linked to age and sex. The MP count was significantly higher in patients with LA, with mean values of 76,500 ± 64,000/mL of plasma (P = 0.001; Figure 9). Moreover, EMP count was significantly higher in patients who developed a thrombotic complication than in those who did not (102,000 ± 92,000 vs. 55,000 ± 19,000, respectively); results of the Mann-Whitney U test were P = 0.0074, even when the patient with the highest level of endothelial EMPs was excluded from the analysis. Interestingly, in the patients with thrombotic complications who received oral anticoagulants, the level of EMPs was not reduced when compared with untreated patients. Conversely, the highest EMP counts were found in 4 out of the 5 patients treated with coagulants.
Flow cytometric analysis of EMPs in the plasma from healthy donors (n = 30; open triangles) and LA patients (n = 30; all circles) was performed using FITC–anti-αvβ3 labeling. EMPs were quantitated in plasma as described in Methods. Among the LA patients 15 had a history of thrombosis (filled circles). The difference between groups was analyzed by the Mann-Whitney U test.