Endothelium-derived microparticles have recently been described as a new marker of endothelial cell dysfunction. Increased levels of circulating microparticles have been documented in inflammatory disorders, diabetes mellitus, and many cardiovascular diseases. Perturbations of angiogenesis play an important role in the pathogenesis of these disorders. We demonstrated previously that isolated endothelial microparticles (EMPs) impair endothelial function in vitro, diminishing acetylcholine-induced vasorelaxation and nitric oxide production by rat aortic rings and simultaneously increasing superoxide production. Herein, using the Matrigel assay of angiogenesis in vitro and a topological analysis of the capillary-like network by human umbilical vein endothelial cells (HUVECs), we investigated the effects of EMPs on formation of the vascular network. All parameters of angiogenesis were affected by treatment for 48 h with isolated EMPs in a concentration of 105 but not 103 or 104 EMPs/ml. The effects included decreases in total capillary length (24%), number of meshes (45%), and branching points (36%) and an increase in mesh area (38%). The positional and topological order indicated that EMPs affect angiogenic parameters uniformly over the capillary network. Treatment with the cell-permeable SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (Mn-TBAP) partially or completely restored all parameters of angiogenesis affected by EMPs. EMPs reduced cell proliferation rate and increased apoptosis rate in time- and dose-dependent manners, and this phenomenon was also prevented by Mn-TBAP treatment. Our data demonstrate that EMPs have considerable impact on angiogenesis in vitro and may be an important contributor to the pathogenesis of diseases that are accompanied by impaired angiogenesis.
- human umbilical vein
- cardiovascular disease
microparticles are small membrane vesicles released from the plasma membrane into the extracellular space (13, 22). Many different cell types including B cells, T cells, monocytes, and endothelial cells (ECs) release microparticles both in vivo and in vitro (29, 32). Formation of microparticles is a part of normal cellular function (22) but is increased in conditions that cause cell stress, apoptosis, or altered cellular viability (25, 29).
Augmented release of microparticles by ECs has been documented in patients with many vascular diseases including lupus and acute coronary syndromes (23, 24, 29). Significantly elevated numbers of circulating endothelium- and platelet-derived microparticles have been found in patients with severe hypertension, and these levels correlated with blood pressure; this suggests a possible pathogenic role for these microparticles in mediating organ injury in severe hypertension (33). Furthermore, increased levels of endothelial microparticles (EMPs) correlate with high-risk angiographic lesions in acute coronary syndromes (3).
We have previously demonstrated that EMPs impair acetylcholine-induced vasorelaxation and nitric oxide production by rat aortic rings and simultaneously increase superoxide production. Superoxide is also produced by isolated EMPs, which express the p22phox subunit of NADPH oxidase (9). Similar impairment of endothelium-dependent relaxation of rat aortic rings by circulating microparticles obtained from patients with myocardial infarction has been reported by other investigators (5).
Angiogenesis plays a key role in a broad array of physiological and pathological processes (7, 10, 16). Normal development and maturation of tissues and organs are critically dependent on the establishment and proper function of the vascular system. The initial step in angiogenesis is the formation of a network-like pattern, the primary capillary plexus (10). However, formation of new vessels occurs not only during physiological processes (embryonic development, wound healing, menstrual cycle), but in many diseases as well, where it may interact with the disease process in a variable manner. Angiogenesis may be either suppressed (as in cardiac failure and late stages of diabetic nephropathy) or excessive (as during cancer, chronic inflammation, diabetic retinopathy, psoriasis, endometriosis, and adiposity; Refs. 10, 18). All of these conditions are also characterized by impaired endothelial function and an increased number of circulating EMPs (3, 14, 17, 21, 29, 33, 41). Our previous studies (12) showed that early transcriptional activation of the plasminogen activator inhibitor-1 gene, which is a marker of EC dysfunction, was associated with delayed branching of endothelial capillary cords. Plasminogen activator inhibitor-1 also increased mobilization of EMPs from the endothelium in vitro and in vivo (8).
Using the Matrigel assay of angiogenesis in vitro and a topological analysis of microtubule formation by human umbilical vein ECs (HUVECs), we investigated the possibility that EMPs may directly affect vascular network formation.
MATERIALS AND METHODS
HUVECs were obtained from Clonetics (Walkersville, MD) and cultured in EBM-2 medium supplemented with 2% fetal bovine serum (FBS) and growth factors (Clonetics) at 37°C in a 95% air-5% CO2 atmosphere. HUVEC passages 3–5 were used for the experiments.
Matrigel basement membrane matrix (growth-factor reduced and phenol red free) and Trucount tubes were purchased from BD Bioscience (San Jose, CA), Hoechst trihydrochloride trihydrate 33258 was from Molecular Probes (Eugene, OR), and the in situ cell death detection kit [terminal deoxytransferase-mediated dUTP-biotin nick-end labeling (TUNEL assay)] and cell proliferation ELISA 5-bromodeoxyuridine (BrdU) immunoassay were from Roche Applied Science (Penzberg, Germany). Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (Mn-TBAP) was from Alexis Biochemical (San Diego, CA).
Microparticle isolation from culture medium conditioned by ECs.
Microparticles were isolated from HUVECs cultured on 150-mm dishes. Confluent cells (100%) were incubated for 4 h in serum-free EBM-2 medium as previously described (8). Culture medium was collected and cleared of cells and cell debris by centrifugation at 2,000 g for 10 min. The supernatant was then subjected to ultracentrifugation at 100,000 g at 4°C for 2 h (13, 14), and the sediment EMPs were resuspended in EBM-2 medium that contained 2% FBS and were used immediately. The number of resulting microparticles was measured using FACS cytometer analysis with Trucount tubes (4, 26). The high-speed supernatant was used in the control experiments after its serum concentration was equalized with the original EBM-2 (2% FBS).
Matrigel angiogenesis assay.
To study angiogenesis, 250 μl of Matrigel substrate diluted with EMB-2 (1:1 dilution) was pipetted into each well of a 24-well plate and allowed to solidify for 30 min in the incubator at 37°C. Thereafter, 150,000 HUVECs in 0.5 ml of EBM-2 with 2% FBS were seeded into the wells (27). Cells were allowed to adhere for 20 min, after which an additional 0.5 ml of EBM-2 with 2% FBS that contained either different concentrations of microparticles or the high-speed supernatant were added to each well. The Matrigel assay for study of angiogenesis in vitro is widely used. However, there are several pitfalls associated with the use of this assay such as the subjective choice of field of view and variability of the network (reviewed in Ref. 11). To eliminate this problem, several solutions have been developed including miniaturizing the assay to monitor the entire well (34) and randomly selecting the fields for analysis (20, 27). However, with the use of a random field selection, it is difficult to analyze time-dependent changes in the network formation. To monitor the same field at different time points, we randomly selected four or five spots on the bottom of the culture wells with a fine-point marker and used these marks as a guide to help us find the same spots and further the analysis. Cells were photographed at 0, 6, 12, 24, and 48 h using a Nikon TE2000-U microscope equipped with a charge-couple device camera (Diagnostic Instruments).
Morphological and topological image analysis.
The topology of the capillary-like networks was measured using a modified analysis of the network (Fig. 1; Refs. 19, 31). To transform the dark-field microscopic photographs to black-and-white images, uneven backlighting was corrected with a closing top-hat transform on the negative image with a disk of radius 10 pixels (15) and subsequent binary thresholding using the equation threshold = min + (max − min)/20, with min and max denoting the minimum and maximum gray values in the filtered image (Fig. 1, B and C; Ref. 31). Parameters of angiogenesis (capillary length, number and area of meshes, branching points; Fig. 1C; Refs. 19, 37) were analyzed using the SDC Morphology Toolbox for C++ and MetaMorph (Universal Imaging) software.
A graph representing the positions of the branching points and their interconnections for further processing was constructed with Mathematica software (Fig. 1D; branching points shown as black dots; connections to neighboring points are not shown). This graph was used for further processing with Mathematica to calculate the mean (L̄) and standard deviation (σL) of the Euclidean distances between connected branching points (20, 37). Also, we determined the Voronoi diagrams of the branching points. The Voronoi diagram partitions the plane into regions, one belonging to each branching point, where each point is closer to its branching point than to any of the other branching points (see black polygons in Fig. 1D). The variation of the areas of the Voronoi regions indicates how evenly the branching points are distributed over the vascular network and is expressed by the positional order where Ā and σA are the mean and standard deviation of the Voronoi areas, respectively (19). The Voronoi regions intersecting with the boundaries were omitted. The positional order varies between 0 and 1, where 0 represents a perfectly even distribution of branching points (σA = 0) and larger values indicate more clustered branching points. The topological order where L̄ and σL are the mean and standard deviation of the lengths of the graph segments, respectively, is a measure of the variation in branch length, which also ranges between 0 and 1 and increases with branch-length heterogeneity.
Cell proliferation was determined using a colorimetric ELISA based on the measurement of 5-bromodeoxyuridine (BrdU) incorporation during DNA synthesis (Roche) according to the manufacturer's protocol. HUVECs were cultured in flat-bottomed, 96-well plates (3,000 cells/well) and allowed to adhere for 20 min at 37°C, after which EBM-2 with 2% FBS containing different numbers of EMPs was added to the wells. The high-speed supernatant was used as a control. Cells were cultured for 24 h at 37°C, BrdU was added to the wells, and cells were incubated at 37°C for an additional 4 h. The medium was then removed, cells were fixed, DNA was denatured, and the reaction product was quantified by measuring the absorbance at 450 nm with 690-nm reference wavelength using an ELx800 multiwell plate reader (Bio-Tek Instruments; Winooski, VT).
To detect apoptosis, HUVECs were cultured into eight-well glass chambers (Nalge Nunc International; Naperville, IL). Cells were allowed to adhere, and the culture medium was changed to EBM-2 medium that contained different numbers of EMPs. The high-speed supernatant was used as a control. Cells were fixed with 4% paraformaldehyde for 10 min at 6, 12, 24, and 48 h. Apoptotic cells were detected by TUNEL assay using an in situ apoptosis detection kit (Roche) according to the manufacturer's protocol. Additionally, cells were costained with Hoechst 33258 and analyzed using an inverted fluorescent microscope at 480/535 and 360/400 nm for TUNEL and Hoechst, respectively (35). Apoptotic cells were assessed as a green fluorescent (TUNEL-positive) cell with coexisting changes in the nucleus (revealed by the Hoechst staining) specific for apoptosis (dense, irregularly shaped, fragmented nucleus; Ref. 35). The TUNEL-positive cells were counted and analyzed using an inverted fluorescent microscope at 480- and 535-nm excitation and emission wavelengths, respectively, according to the manufacturer's protocol. With the use of two wells of an eight-well cluster per group, the effects of EMPs on the apoptotic rate in HUVECs were compared with those of the high-speed supernatant. The rate of apoptosis was calculated from four or five randomly selected fields (to eliminate a subjective selection of fields) from each well as the percentage of the TUNEL-positive cells to the total number of cells (revealed by the Hoechst staining) in the field. All experiments were repeated separately three times.
All experiments were repeated at least three times. All observations were completed by two independent observers who were blinded to the origin of the data. All data are presented as means ± SE unless otherwise specified. The means of two groups were compared using the two-tailed Student's t-test. For multiple comparisons, two-way ANOVA (factors were time and treatment) was used followed by the Bonferroni post hoc test. Pearson correlation analysis was used to determine the strength of association between the apoptosis rate and parameters of angiogenesis. Differences were considered statistically significant at P < 0.05.
EMPs impair HUVEC angiogenesis on Matrigel substrate.
HUVECs seeded on Matrigel substrate were initially dispersed evenly throughout the culture. Thereafter, cells spontaneously aligned to form a network of capillary-like structures within 6–12 h (Fig. 2). These structures were characterized by dense meshes with a high number of branching points. Once the network had formed, the number of branching points and meshes initially dropped quickly (50% of the initial values at 12 h of culture) and then stabilized (Fig. 3, A and C). The total capillary length gradually decreased during this time (Fig. 3D). The positional order remained constant, which indicates that although the mean distance between branching points increased, they did not cluster or disperse over time. The topological order significantly increased linearly at all time points (and without a drop at intermediate time points) from 0.41 ± 0.06 to 0.48 ± 0.02 (P < 0.005), which indicates a slightly broader distribution of branch lengths. Simultaneously, the mesh area increased and reached a plateau phase at 24 h (Fig. 3B), which represents the shrinkage of small meshes and dissolution of the borders between neighboring meshes (see Fig. 2).
Addition of microparticles to the medium resulted in dose-dependent changes to all topological characteristics of the capillary-like network. Thus pathophysiological concentrations of EMPs such as those found in the circulation of patients with cardiovascular diseases (105 EMPs/ml; Refs. 33, 38, 39) resulted in a significant decrease in the number of meshes and branching points. The number of branching points at 12 h of culture formed by HUVECs treated with 105 EMPs/ml was 11 ± 0.9 points/field compared with 17 ± 0.7 points/field (P = 0.001) in controls. At the same time, the number of meshes decreased from 9.7 ± 0.9 meshes/field in control cultures to 5.4 ± 0.6 meshes/field (P = 0.003) in EMP-treated cells. These significant differences persisted at 24 and 48 h of culture, when the numbers of meshes were 3.4 ± 0.6 vs. 6.1 ± 0.6 (P = 0.001) and 1.8 ± 0.3 vs. 3.1 ± 0.4 meshes/field (P = 0.01), respectively. The capillary length shrank more rapidly compared with control cultures, as the rate of mesh-area increase accelerated (Figs. 2 and 3). The physiological concentrations of 103 and 104 EMPs/ml that are found in the circulation of healthy persons (6, 24) did not change the parameters of angiogenesis significantly (Fig. 3). These results are congruent with our previous findings that 105 but not 104 EMPs/ml impair endothelium-dependent relaxation of artic rings (9). The positional and topological order of the EMP-treated cultures did not differ significantly from the control cultures, which indicates that EMP treatment reduced total capillary length and increased mesh area uniformly over the capillary network.
Mn-TBAP, a SOD mimetic, restores angiogenic pattern impaired by EMPs.
Based on the data obtained in the dose-response studies and the previously published data (9), we hypothesized that EMPs impair angiogenesis via oxidative stress. Previously we demonstrated (9) that isolated EMPs impaired endothelial function in vitro, which was accompanied by increased production of superoxide in the endothelium. Indeed, addition of the cell-permeable SOD mimetic Mn-TBAP (30 μM) to the cultures from the beginning of the experiments significantly improved angiogenesis affected by 105 EMPs/ml. Hence, Mn-TBAP cotreatment restored the number of branching points at 12 and 48 h [19 ± 1.4 vs. 11 ± 0.9 (P = 0.001) and 9.8 ± 0.8 vs. 4.9 ± 0.6 points/field (P = 0.02), respectively; Fig. 4C]. At the same time, other characteristics of angiogenesis such as the number of meshes, the mesh area, and the total capillary length were partially or completely restored (Fig. 4). Mn-TBAP alone did not only affect the angiogenic pattern (Fig. 2), but even stabilized the capillary-like network thereby preventing a decrease in total capillary length and number of branching points at 48 h (Fig. 4, C and D). The positional and topological order remained unchanged, which indicates that Mn-TBAP restores angiogenesis uniformly over the cell cultures.
EMPs affect cell division rate.
It has been demonstrated that parameters of angiogenesis are affected by the cell division rate (16, 28). We used the BrdU-incorporation assay to study the effects of EMPs on the cell proliferation rate. When HUVECs were cultured in the presence of EMPs for 24 h, the cell division rate was significantly reduced by 105 EMPs/ml (27% less than the high-speed supernatant). This effect was dose dependent; hence, 103 and 104 EMPs/ml reduced the cell proliferation rate by 8.5 and 13.2%, respectively, compared with control cells treated with the high-speed supernatant (Fig. 5).
EMPs increase apoptosis of HUVECs.
Apoptosis is an important factor affecting angiogenesis (16, 35). We investigated the possibility that EMPs may induce apoptosis in ECs and thus affect angiogenesis. It has been demonstrated that as many as 50% of cells became apoptotic in the Matrigel assay of angiogenesis after 24 h of culture (35). Therefore, we studied the effects of EMPs on the apoptosis rate with a different experimental method. HUVECs were cultured in complete EBM-2 medium that contained varying quantities of isolated EMPs. The high-speed supernatant served as a control. Apoptosis was detected by double staining of HUVECs with TUNEL assay and Hoechst stain (Fig. 6A). EMPs increased apoptosis in a dose-dependent manner with the maximum effect seen with 105 EMPs/ml. Thus at 24 and 48 h, the number of apoptotic cells in cultures treated with 105 EMPs/ml was increased by 7.7- and 5.2-fold, respectively (Fig. 6B). A less dramatic but significant increase in the number of apoptotic ECs was induced by 104 EMPs/ml at 24 and 48 h (increases of 3.1- and 2.1-fold, respectively).
Treatment with Mn-TBAP suggested that oxidative stress may be the key mechanism for the phenomena we observed. HUVECs cotreated with 30 μM Mn-TBAP and 105 EMPs/ml had significantly reduced numbers of apoptotic cells than cells treated with EMPs alone at both 24 and 48 h. However, Mn-TBAP did not totally prevent apoptosis in HUVECs, which suggests that EMPs have multiple actions on ECs, and oxidative stress was not the only mechanism by which apoptosis was induced. Mn-TBAP alone did not affect the apoptosis rate in HUVECs (Fig. 6C).
These data correlate well with the effects of EMPs on angiogenesis. Thus we found a strong correlation between both the EMP-induced apoptosis rate and the mean area of meshes (r = 0.83; P = 0.0009) and the EMP-induced apoptosis rate and the capillary length (r = −0.71; P = 0.009). However, the EMP-induced apoptosis rate and the number of meshes or branching points were not correlated (Fig. 7). Neither did the topological and positional orders correlate with the EMP-induced rate of apoptosis. This may reflect a relative stability of the capillary-like network formed by ECs (19, 20, 31, 37). The relative stability of the network was confirmed by the positional order, which remained constant; this indicated that although the mean distance between branching points increased, the branching points did not cluster or disperse over time. However, in the Matrigel assay, ECs rapidly undergo apoptosis (35), which results in a reduced number of cells forming capillaries, decreased capillary length, dissolving of borders between different meshes, and increased mesh area (see Fig. 2). These parameters gradually changed over time (see Fig. 3, B and D). The number of meshes and branching points changed rapidly during the first 12 h of culture (see Fig. 3, A and C) and then stabilized. The EMP-induced apoptosis rate also changed gradually over time (see Fig. 6B) and correlated well with capillary length and mesh area.
In the present study, we report that isolated EMPs in pathophysiological concentrations impair angiogenesis in vitro by affecting all parameters of the capillary-like network formation. This is the first detailed analysis of the direct action of EMPs on angiogenesis.
Despite the significant growth in microparticle research recently, very little is known about the role of endothelium-derived microparticles in the pathogenesis of vascular diseases and EC dysfunction (2, 21). Endothelium-derived microparticles represent a relatively small (5–15%; Refs. 1, 13, 30, 33) but very important subset of all circulating microparticles. This number may vary in different cardiovascular and inflammatory diseases (14, 21, 30), reflecting the interaction of pathological process with endothelium. Indeed, it has been demonstrated that circulating EMPs are a marker of preeclampsia (6) and are elevated in patients with acute coronary syndromes (29) and severe hypertension (33). Moreover, endothelium-derived microparticles may directly affect the endothelium and diminish its function (5, 9, 40).
However, the role of microparticles in angiogenesis has not been rigorously investigated. It has been demonstrated that platelet-derived microparticles induce angiogenesis in vitro by increasing the cell proliferation rate and decreasing apoptosis (28). Taraboletti et al. (36) reported that microparticles isolated from HUVECs promote angiogenesis in vitro in low concentrations, whereas high concentrations of EMPs suppress angiogenesis. These findings are consistent with our results. Herein, using a topological analysis with modified time-lapse microscopy, we provide detailed analysis of the formation and degradation of the capillary-like network and how this changes over time in response to EMPs. Furthermore, our study used direct measurements of EMP numbers, thereby demonstrating the effect to be dose dependent.
The use of randomly placed reference points on the bottom of the culture dishes enabled us to monitor the same spots over time and thus minimized the common problem of using the Matrigel angiogenesis assay, namely, the variability of the capillary network formation (11). Our technique also permitted the completion of the experiments in a cell culture incubator, which differs from conventional time-lapse microscopy, where the cells are kept under a microscope in room air. The modified technique also allowed us to run multiwell experiments to simultaneously study several experimental groups, unify experimental conditions, and reduce the possible effects of any systematic error.
We found that isolated EMPs in pathological concentrations affect all parameters of angiogenesis by uniformly decreasing total capillary length and numbers of meshes and branching points and increasing mesh area. Indeed, detailed analysis of individual experiments revealed that in the capillary networks formed by HUVECs treated with 105 EMPs/ml, the borders between meshes dissolved faster and at a greater rate than in cells treated with lower concentrations of EMPs or in control cultures (see Fig. 2). One of the possible mechanisms of this phenomenon may be increased oxidative stress in cells treated with EMPs as reported previously (9). This hypothesis is supported by the finding that treatment with the cell-permeable SOD mimetic Mn-TBAP partially or completely restored all parameters of angiogenesis affected by EMPs. Apoptosis may be the chief contributor to the processes of mesh-border dissolution and decreased total capillary length. In fact, the EMP-induced apoptosis rate correlated strongly with these parameters of angiogenesis (see Fig. 7). Isolated EMPs increased the apoptosis rate in a dose-response manner, which suggests an important role for this mechanism in the altered angiogenic pattern. The cell proliferation rate was reduced by EMPs, which may also contribute to diminished angiogenesis (see Fig. 5).
We would like to emphasize that “physiological” concentrations of EMPs such as those found in the circulation of healthy persons (103 and 104 EMPs/ml) did not affect any of the parameters of angiogenesis or apoptosis in vitro, which reinforces the importance of endothelium-derived microparticles as a marker of a disease. In low concentrations, microparticles did not affect the endothelium as others and we have demonstrated previously (9, 36) and in the present study. However, when the number of circulating EMPs exceeds a certain threshold, the EMPs became an important factor in the pathophysiology of the disease, directly affecting the endothelium and other circulating cells. Indeed, it has been demonstrated that EMPs can bind to and activate monocytes and facilitate their transmigration through endothelium (21, 26). Microparticles obtained from the blood of patients with acute myocardial infarction (5), from preeclamptic women (40), or isolated from cultured ECs (9) have been shown to significantly impair the endothelium-dependent relaxation of macro- and microvessels in vitro.
Our study and the recently published clinical data suggest that circulating EMPs are an important contributor to the pathogenesis of different cardiovascular diseases, all of which are accompanied by impaired angiogenesis. These key findings suggest the possibility that future therapeutic strategies aimed at reducing the number of circulating endothelium-derived microparticles or blocking their effects may be reasonable in different pathological processes where abnormalities of neovascularization and angiogenesis prevail.
These studies were supported in a part by National Institutes of Health Grants DK-064863 (to S. V. Brodsky), DK-45462, DK-45695, and DK-54602 (to M. S. Goligorsky), the Westchester Artificial Kidney Foundation (to M. S. Goligorsky), American Heart Association Grant 0430255N (to J. Chen), and a Kevin J. and Gloria B. Keily National Kidney Foundation fellowship (to E. O'Riordan).
The authors express great gratitude to Nathan O'Connor (Universal Imaging) for excellent technical expertise and support.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 by the American Physiological Society