Verocytotoxin-Induced Apoptosis of Human Microvascular Endothelial Cells

Materials

Purified VT-1, VT-2, and VT-2c (1.0 to 1.2 mg protein/ml) were kindly provided by Dr. M. A. Karmali (Department of Microbiology, The Hospital for Sick Children, Toronto, Canada). The endotoxin content of the VT preparations was <0.05 EU/ml, as assessed using a Limulus amebocyte lysate assay with a detection level of 0.05 to 0.10 EU/ml. All experiments were performed with VT-2c (unless otherwise mentioned), which is structurally and functionally related to VT-1 and VT-2 and is associated with human disease (22) (Table 1).

Endothelial Cell Cultures

FMVEC were isolated and purified according to methods previously described by Davison et al. (23) and Voyta et al. (24). FMVEC were seeded on gelatin (1%; Fluka Biochemicals, Buchs, Switzerland)-coated, six-well plates (Costar, Cambridge, MA) and cultured in M199 (BioWhittaker, Walkersville, MD) supplemented with 10% (vol/vol) human serum (local blood bank), 10% (vol/vol) newborn calf serum (NBCS) (Life Technologies, Grand Island, NY), 2 mM L-glutamine (ICN Biomedicals, Zoetermeer, The Netherlands), 5 U/ml heparin (Leo Pharmaceutical, Weesp, The Netherlands), 100 IU/ml penicillin/0.1 mg/ml streptomycin (Yamanouchi Pharma B.V., Leiderdorp, The Netherlands), and 150 mg/liter crude endothelial cell growth factor [extracted from bovine brains as described by Maciag et al. (25)]. Cells were subcultured with mild trypsinization (3 to 5 min), using trypsin/ethylenediaminetetraacetate (EDTA) (0.5 g/liter trypsin, 1:250, and 0.2 g/liter EDTA; Life Technologies), after which the cells were replated with a split ratio of 1:3. For experiments, FMVEC were used after nine to 11 passages.

GMVEC were isolated and purified as previously described by van Setten et al. (18). HUVEC were isolated from umbilical cords according to the method described by Jaffe et al. (26). GMVEC and HUVEC were used after eight to 10 and two to four passages, respectively.

All endothelial cells used displayed the presence of von Willebrand factor, platelet endothelial cell adhesion molecule-1, and V- and E-cadherin at all passages used. No immunoreactivity to the anticytokeratin 20 antibody or the anti-α-smooth muscle actin antibody was observed, excluding the possibility of contaminating epithelial and mesangial cells, respectively (18).

Cytotoxicity Assays

Microvascular endothelial cells were cultured in complete medium on gelatin-coated, 24-well plates and were grown until confluence. Five days after reaching confluence, cells were preincubated with or without TNF-α (10 ng/ml) for 24 h. The next day, the medium was aspirated and fresh medium with different concentrations of VT was added. VT was diluted in medium with 20% fetal calf serum instead of 10% NBCS and 10% human serum, because previous studies indicated that NBCS and human serum may have neutralizing activity against VT. After 24 h, the cells were washed with phosphate-buffered saline (PBS) and released with trypsin/EDTA. Viable, trypan blue-excluding cells were then counted with a hemocytometer.

Protein Synthesis

Protein synthesis was assessed by assaying the incorporation of ³⁵S-labeled methionine (0.25 μCi/ml complete medium) into ³⁵S-proteins during 24-h incubations with different concentrations of VT. After incubation, the cells were washed and ³⁵S-labeled cellular proteins and ³⁵S-labeled proteins present in the medium were precipitated with the addition of TCA (10 and 20%, respectively). Precipitated proteins were dissolved in 0.3 ml of 0.3 M NaOH, 60 μl of 1.5 M HCl was added, and precipitated radioactivity was counted in a liquid scintillation counter (18,27).

Glycolipid Extraction and Thin Layer Chromatography

FMVEC were cultured in complete medium on gelatin-coated, six-well plates (Costar) and were grown to confluence. Highly confluent cells were exposed to complete medium alone or complete medium supplemented with TNF-α (10 ng/ml). After 24 h, the cells were trypsinized and washed three times with ice-cold PBS. Subsequently, glycolipids were extracted, separated, and assayed for ¹²⁵I-VT binding, as described previously (18,27). Thin layer chromatograms were analyzed with a Fuji BAS 1000 PhosphorImager (Fuji Photo Film Co., Tokyo, Japan).

Apoptosis Assays

Fluorescence-Activated Cell Sorting Analyses. FMVEC, HUVEC, and GMVEC were grown on gelatin-coated, 12-well plates (Costar) until they reached confluence. Highly confluent HUVEC and GMVEC were prestimulated with TNF-α (10 ng/ml) for 24 h, to upregulate VT receptors and induce VT susceptibility (18,27). Because FMVEC display VT susceptibility under basal conditions, these cells were not pretreated with TNF-α. Subsequently, unstimulated FMVEC and TNF-α-stimulated HUVEC and GMVEC were incubated for 4 or 24 h with complete medium, with complete medium supplemented with various concentrations of VT (0.1 pM to 10 nM), B-subunit (5 to 130 nM), or cycloheximide (CHX) (0.01 to 1 μg/ml), or with M199 alone. After the incubation period, detached cells were collected and pooled with trypsinized adherent cells. Cells were centrifuged at 200 × g for 5 min at 4°C, and the supernatant was removed. The cells were then washed with ice-cold binding buffer (10 mM Hepes, [pH 7.4], 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂), as previously described by Koopman et al. (28), supplemented with 0.1% bovine serum albumin (BSA) (ICN Biomedicals, Zoetermeer, The Netherlands). Cells were resuspended in 500 μl of binding buffer with 0.1% BSA, of which 445 μl was transferred into a tube suitable for fluorescence-activated cell sorting (FACS) analysis. Five microliters of FITC-conjugated annexin V (2 μg/ml, diluted in binding buffer with 0.1% BSA; Bender Medsystems, Vienna, Austria) and 50 μl of propidium iodide [100 μg/ml, diluted in RPMI DM (Flow Inc.) supplemented with 5% fetal calf serum and 2 mM CaCl₂; Sigma] were added, after which the cell suspension was gently mixed and incubated in the dark on ice for 10 min (28,29). Samples were assayed for viable, apoptotic, and necrotic cells by FACS analysis (Coulter 7 Epics 7 XL-MCL, Beckman Coulter Inc., Mijdrecht, The Netherlands). Necrotic cells were defined as cells demonstrating positive staining for both FITC-conjugated annexin V and propidium iodide. Viable cells were not positive for either FITC-conjugated annexin V or propidium iodide. Apoptotic cells were defined as cells exhibiting positive staining for FITC-conjugated annexin V and negative staining for propidium iodide. Fluorescence was measured on a double-parameter histogram, using logarithmic scales. For each tube, 5000 events were analyzed.

To assess the inhibition of the apoptosis-inducing effect by reversible inhibitors, FMVEC, cultured under identical conditions, were preincubated for 1 or 24 h with various concentrations of ICE inhibitor I [Ac-Tyr-Val-Ala-Asp-CHO (YVAD-CHO); Calbiochem, San Diego, CA] or CPP32/apopain inhibitor [Ac-Asp-Glu-Val-Asp-CHO (DEVD-CHO); Calbiochem] (30,31). After preincubation with these inhibitors, cells were rinsed once with M199, after which the cells were exposed for 16 h to complete medium or complete medium supplemented with VT (1 nM), in the presence of the same reversible inhibitor. Apoptosis was evaluated by FACS analysis as described previously. To detect the presence of caspase 3, caspase 6, or caspase 7 in FMVEC and GMVEC, Western blot analysis was performed according to established procedures (31), using primary antibodies against human CPP32/p20 (caspase 3) (N-19), Mhc2/p20 (caspase 6) (K-20), or ICE-LAP3 (caspase 7) (C-18) (Santa Cruz Biotechnology, Santa Cruz, CA) and a horseradish peroxidase-conjugated anti-goat IgG antibody (diluted 1:5000 in PBS with 0.5% BSA and 0.05% Tween-20; Nordic Immunology, Tilburg, The Netherlands) as a secondary antibody.

Fluorescence Microscopy Using Triple Staining of Cell Monolayers. Cells were seeded onto gelatin-coated glass coverslips, as described previously (18), and were grown to confluence. Highly confluent FMVEC were exposed to complete medium alone or complete medium supplemented with various concentrations of VT. After an incubation period of 16 h, adherent cells were stained with 500 μl of Hoechst 33342 (1 μg/ml in complete medium; Sigma) for 15 min at 37°C. Subsequently, cells were rinsed once with M 199 and incubated, on ice, with 500 μl of complete medium containing FITC-conjugated annexin V (1 μg/ml) and propidium iodide (1 μg/ml). Cells were analyzed by fluorescence microscopy within 15 min, using filters of 365, 490, and 560 nm for Hoechst 33342, FITC-conjugated annexin V, and propidium iodide staining, respectively. Definitions of viable, apoptotic, and necrotic cells were similar to those described for the FACS analyses.

DNA Fragmentation Analyses. FMVEC were grown in complete medium on gelatin-coated, six-well plates until they reached confluence. Highly confluent cells were exposed to complete medium or complete medium supplemented with various concentrations of VT or B-subunit. After 16 h of incubation, detached cells were collected on ice and pooled with trypsinized adherent cells. Cells were subjected to centrifugation at 200 × g for 5 min at 4°C, and the pellets were washed twice with ice-cold PBS. Subsequently, cells were lysed with 10 mM Tris (Boehringer Mannheim, Mannheim, Germany), pH 8.0, 10 mM EDTA (Life Technologies), 0.5% Triton X-100 (Boehringer Mannheim), as previously described by Bissonnette et al. (32). To separate fragmented DNA from intact chromatin, cells were centrifuged at 15,000 × g for 20 min at 4°C. Soluble fragmented DNA was transferred into a new tube and treated with RNase A (100 μg/ml; Sigma) for 1 h at 37°C, followed by treatment with proteinase K (200 μg/ml; Sigma) in 1% sodium dodecyl sulfate (Merck, Darmstadt, Germany) for 2 h at 50°C. DNA was precipitated with 0.1 volume of 3 M sodium acetate and 2 volumes of 96% ethanol for 16 h, after which the samples were centrifuged at 15,000 × g for 10 min at 4°C. Subsequently, DNA pellets were air-dried and dissolved in Tris-EDTA solution, pH 8.0 (10 mM Tris-HCl and 0.1 mM EDTA, both at pH 8.0). Loading buffer was added to each sample, and electrophoresis was performed at 80 V for 1.5 to 2 h on a 1.5% agarose gel containing 0.4 μg/ml ethidium bromide (Merck), with 1 × Tris/borate/EDTA running buffer (22.5 mM Tris-borate, 0.5 mM EDTA, pH 8.0). DNA was observed under ultraviolet light (350 nm) and photographed. DNA size calibration was performed using a 100-bp marker (Pharmacia, Roosendaal, The Netherlands).

Transmission Electron Microscopy. To evaluate the apoptosis-inducing effect of VT on adherent endothelial cells by transmission electron microscopy, FMVEC were cultured in complete medium on gelatin-coated, 12-well plates. After they reached confluence, the cells were exposed to control medium or control medium supplemented with various concentrations (0.1 pM to 10 nM) of VT. After 16 h of incubation, detached cells were pooled with trypsinized adherent cells. Cells were pelleted and fixed for 2 h at room temperature with 2.5% glutaraldehyde in cacodylate buffer. The cells were then carefully rinsed with 0.1 M cacodylate buffer for 30 min, after which the cells were immersed and pelleted in 15% gelatin. After immersion, alternate fixation with 1% OsO₄ in cacodylate buffer was performed for 30 min. Cells were then selectively dehydrated in graded ethanol and embedded in Epon 812. Thin sections were cut with an ultramicrotome and stained with uranyl acetate and lead citrate. Sections were examined with a Jeol 1200 EX electron microscope (Jeol Europe bv., Schilphol-Rijk, The Netherlands).

Additional Analytic Procedures

To determine whether the cultured cells exhibited signs of endogenous activation, the expression of monocyte chemoattractant protein-1 (MCP-1) (33), urokinase-type plasminogen activator (u-PA) (34), and vascular cell adhesion molecule-1 (VCAM-1) (18) in culture supernatants (u-PA and MCP-1) or on the cell surface (VCAM-1) was assessed according to established procedures.

Statistical Analyses

The statistical significance of differences between groups was analyzed by ANOVA followed by Bonferroni's modified t test. Differences were considered significant at P < 0.05 (two-sided).

Effects of VT on Unstimulated FMVEC

Under basal conditions, human FMVEC were very sensitive to VT, in contrast to GMVEC and HUVEC, which required preexposure to TNF-α or IL-1 to become sensitive to VT. Exposure of unstimulated FMVEC to various concentrations of VT (0.01 fM to 1 nM) for 24 h produced concentration-dependent cell toxicity and inhibition of overall protein synthesis (Figure 1). This high sensitivity to VT was related to high levels of specific VT binding to these cells, as assayed in binding experiments with ¹²⁵I-VT. Thin layer chromatographic analyses of cellular neutral glycolipids extracted from unstimulated FMVEC revealed strong binding of ¹²⁵I-VT to glycolipid species in the Gb3 region (Figure 2), with an additional increase after stimulation with TNF-α.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Cytotoxic effects of verocytotoxin-2c (VT-2c) on foreskin microvascular endothelial cells (FMVEC) and glomerular microvascular endothelial cells (GMVEC). Human FMVEC and GMVEC were grown to confluence and maintained confluent for 5 d. Subsequently, FMVEC (○), control GMVEC (□), and tumor necrosis factor-α (TNF-α) (10 ng/ml)-treated GMVEC (▪) were incubated with different concentrations of VT (0.01 fM to 10 nM). After 24 h, the number of viable cells (A) and the incorporation of [³⁵S]methionine into proteins (B) were determined as described in Materials and Methods. Data are expressed as the mean ± SD of two independent experiments, with two different donors [percentage of control (% of C)].

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

¹²⁵I-VT binding to glycolipids extracted from human GMVEC and FMVEC. Glycolipids were extracted from highly confluent endothelial cells, separated by thin layer chromatography, assayed for ¹²⁵I-VT binding, and observed by PhosphorImaging. Lanes A and B, glycolipid extracts of 20 cm² of human GMVEC from one representative donor; lane A, control GMVEC; lane B, TNF-α (10 ng/ml)-treated GMVEC. Lanes E and F, glycolipid extracts of 20 cm² of human FMVEC from one representative donor; lane E, control FMVEC; lane F, TNF-α (10 ng/ml)-treated FMVEC. Lanes C and G, standard neutral glycolipids (2 μg of each glycolipid). Lanes D and H, orcinol staining of standard neutral glycolipids (Gb1, galactosylceramide; Gb2, lactosylceramide; Gb3, globotriaosylceramide; Gb4, globotetraosylceramide; Gb5, Forssman pentasaccharide).

To investigate whether FMVEC had spontaneously acquired the activation phenotype that is induced by TNF-α or IL-1, we measured the levels of several compounds that are induced in endothelial cells by TNF-α and IL-1. Values for VCAM-1 (not detectable), MCP-1 (<0.3 ng/ml), and u-PA (0.2 ng/ml) in unstimulated FMVEC were essentially identical to those found in unstimulated GMVEC and HUVEC, and values increased during 24-h exposure to TNF-α (VCAM-1 clearly expressed; MCP-1, >2000 ng/ml; u-PA, 1.7 ng/ml; average of three independent determinations).

VT Induction of Apoptosis in FMVEC

To evaluate whether apoptosis is involved in VT-mediated FMVEC cell death, we initially performed FACS analyses using a dual-staining method with FITC-conjugated annexin V and propidium iodide. This method discriminates among viable, apoptotic, and necrotic cells (28,29) (see the Materials and Methods section for details). Figure 3 presents representative cytograms for control (Figure 3A) and VT-treated (Figure 3B) FMVEC. Whereas in control cells the numbers of apoptotic and necrotic cells were rather small (3 and 5%, respectively), VT-treated (0.1 nM for 24 h) cells demonstrated an impressive increase in apoptotic cells (40.7 ± 4.5%, n = 3) and a slight increase in necrotic cells (12.3 ± 2.9%, n = 3).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Flow cytometric analysis of apoptosis in control and VT-treated FMVEC. FMVEC were grown to confluence and maintained confluent for 5 d. Subsequently, cells were exposed to control medium alone (A) or control medium supplemented with 1 nM VT (B). After 16 h of incubation, detached cells were pooled with trypsinized adherent cells and assayed for the occurrence of apoptosis by fluorescence-activated cell sorting (FACS) analysis, as described in Materials and Methods. The lower left quadrant of each panel represents viable cells [annexin V (ANNEX)-negative/propidium iodide (PI)-negative]. The upper right quadrant represents nonviable necrotic cells (annexin V-positive/propidium iodide-positive). The lower right quadrant represents apoptotic cells (annexin V-positive/propidium iodide-negative). Data for a representative donor are presented; similar results were obtained in three other experiments with cells from two different donors.

The apoptosis-inducing effect of VT in FMVEC was time-and concentration-dependent (Figure 4). The apoptosis-inducing effect of VT was observed as early as 4 to 6 h after the beginning of the VT incubation, with an increase in the number of apoptotic cells after 24 h of VT exposure. The threshold dose for VT to induce apoptosis was 0.1 pM. Identical results were obtained when VT-1, VT-2, and VT-2c were compared (Table 1). The B-subunit of VT induced apoptosis only at very high concentrations. Exposure of the cells to 50 and 130 nM B-subunit for 24 h resulted in 29 ± 8% and 40 ± 8% apoptotic cells (mean ± SD of two independent experiments with two different donors), respectively. The percentages of necrotic cells were 9 ± 0.2% and 11 ± 2%, respectively.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Dose-response curves for VT-mediated apoptosis in human FMVEC. Human FMVEC were grown to confluence and maintained confluent for 5 d. Subsequently, cells were exposed to various concentrations of VT (0 to 10 nM) during a 5-h (○) or 24-h () incubation period. Detached and adherent cells were obtained and assayed for the occurrence of apoptosis by FACS analysis, as described in Materials and Methods. Data are expressed as the mean ± SD of three independent experiments with cells from two different donors.

VT-induced apoptosis in FMVEC was confirmed by direct observation, using a triple-staining method. As shown in Figure 5, highly confluent control and VT-exposed cells were stained with Hoechst 33342 (Figure 5, A and B) or FITC-conjugated annexin V and propidium iodide (Figure 5, C and D). Staining of control cells with Hoechst 33342 resulted in homogeneous diffuse nuclear staining (Figure 5A). After incubation of FMVEC with VT (10 nM) for 16 h, some of the adherent cells displayed condensed and fragmented nuclear chromatin, a characteristic feature of apoptosis (Figure 5B, arrows). Whereas almost all (>99%) control cells were negatively stained with annexin V and propidium iodide (Figure 5C), indicating that they were viable, a major fraction of VT-treated FMVEC demonstrated positive staining with annexin V and negative staining with propidium iodide, indicating apoptosis (Figure 5D, arrows). A minority of VT-exposed cells exhibited intracellular staining with annexin V and propidium iodide, which is characteristic of necrotic cells (Figure 5D, star).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Analysis of apoptosis in control- and VT-treated FMVEC by fluorescence microscopy, using triple-staining. FMVEC were grown to confluence and maintained confluent for 5 d. Subsequently, the cells were incubated for 16 h with control medium (A and C) or control medium supplemented with VT (10 nM) (B and D). After the incubation period, cells were stained with Hoechst 33342 (A and B) or FITC-conjugated annexin V and propidium iodide (C and D), as described in Materials and Methods. Magnification, ×200 in A to D. FMVEC indicated by arrows and stars represent apoptotic (annexin V-positive/propidium iodide-negative, approximately 24%) and necrotic (annexin V-positive/propidium iodide-positive) cells, respectively.

DNA Fragmentation Analysis and Ultrastructural Morphologic Features of VT-Exposed FMVEC

Observations made using FACS analysis and fluorescence microscopy were extended by DNA fragmentation analyses. Endonuclease-induced cleavage of nuclear DNA into mono-and oligonucleosomal fragments with approximate molecular sizes of multiples of 180 nucleotides is a characteristic feature of programmed cell death (35). Figure 6 demonstrates the electrophoretic patterns of DNA extracted from control and VT holotoxin-treated FMVEC. Whereas DNA from control cells remained intact throughout the 16-h incubation period (Figure 6, lane C), DNA from VT holotoxin (0.1 fM to 1 nM)-exposed cells clearly exhibited the DNA “ladder” pattern of multiples of 180-bp fragments. Exposure of the cells to 130 nM VT B-subunit induced a similar DNA ladder pattern (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

DNA fragmentation in VT-treated FMVEC, as determined by agarose gel electrophoresis. FMVEC were grown to confluence and maintained confluent for 5 d. Subsequently, FMVEC were incubated for 16 h with control medium alone or control medium supplemented with VT (0.1 fM to 1 nM), after which DNA was extracted, centrifuged, and analyzed for mono-/oligonucleosomal fragments by agarose gel electrophoresis, as described in Materials and Methods. Note that DNA fragmentation with the characteristic ladder pattern was observed only in VT-treated cells, whereas DNA from control cells remained intact. Shown is one representative experiment of two. M, marker; C, control untreated FMVEC.

In addition, the apoptosis-inducing effect of VT on FMVEC was studied by transmission electron microscopy. At the ultrastructural level, apoptotic cells exhibit distinctive alterations, primarily of the nucleus, which can be discriminated from events of necrosis (35). Representative micrographs of control and VT-treated FMVEC are presented in Figure 7. The morphologic features of control FMVEC in culture were maintained; cells exhibited nuclei with normal heterogeneous chromatin and a nuclear envelope, normal intact organelles, and normal plasma membranes (Figure 7A). In contrast, FMVEC exposed to VT (1 nM) for 16 h exhibited varying degrees of nuclear condensation (Figure 7, B and C), and even cellular fragmentation was noted (Figure 7D). Furthermore, we observed abundant cytoplasmic vacuolization and the presence of apoptotic bodies; however, cellular organelles of VT-treated FMVEC appeared normal. Although the integrity of the plasma membrane remained intact, blebbing of the plasma membrane occurred in VT-exposed FMVEC. These morphologic changes are indicative of apoptosis and are clearly different from those exhibited by cells undergoing necrosis.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7.

Ultrastructural morphologic features of control and VT-treated FMVEC, as assessed by transmission electron microscopy (TEM). FMVEC were grown to confluence and maintained confluent for 5 d. Subsequently, FMVEC were exposed for 16 h to control medium alone or control medium supplemented with VT (1 nM), after which the cells were prepared for TEM and subsequently examined as described in Materials and Methods. (A) Ultrastructural morphologic features of a representative normal control cell. (B, C, and D) Ultrastructural morphologic features of VT-treated cells, including features characteristic of apoptosis. Magnification, ×5800 in A; ×7500 in B through D.

Comparison of Apoptosis Induced by VT and CHX

To evaluate whether the induction of apoptosis by VT merely reflects inhibition of protein synthesis, we compared the effect of VT with that of the protein synthesis inhibitor CHX. For proper comparisons, we determined which CHX concentration inhibited protein synthesis to a similar extent, compared with VT (0.1 pM), in wells cultured in parallel. Because 0.1 μg/ml CHX and 0.1 pM VT both inhibited protein synthesis by 30%, their effects on apoptosis induction were compared. Only VT induced apoptosis (14 ± 3% for VT versus 2 ± 1% for CHX; mean of two independent experiments). These data indicate that VT is a more potent inducer of apoptosis than is CHX at comparable potencies for protein synthesis inhibition.

Apoptosis of TNF-α-Pretreated GMVEC and HUVEC after Exposure to VT

The effect of VT on the induction of apoptosis among GMVEC and HUVEC was subsequently investigated by FACS analysis, using a dual-staining method with FITC-conjugated annexin V and propidium iodide. VT did not induce significant cell death among untreated GMVEC or HUVEC (data not shown). However, when GMVEC or HUVEC were preincubated with TNF-α for 24 h, 4- to 6-h exposure of the cells to VT (0.1 to 10 nM) induced a dose-dependent increase in the number of apoptotic cells (Figure 8). Although no further increase in the percentage of apoptotic cells was observed after 24 h of VT exposure, the percentage of necrotic cells increased significantly.

• Download figure
• Open in new tab
• Download powerpoint

Figure 8.

Apoptosis of TNF-α-pretreated human umbilical vein endothelial cells (HUVEC) and GMVEC. Cells were grown to confluence. After 5 d of confluence, HUVEC and GMVEC were prestimulated with TNF-α (10 ng/ml) for 24 h. After pretreatment with TNF-α, the endothelial cells were incubated with VT (□, 0 nM; ▪, 0.1 nM; , 1 nM; , 10 nM) for 5 h. Subsequently, detached cells and trypsinized adherent cells were pooled and assayed for the presence of apoptotic cells by FACS analysis, as described in Materials and Methods. (A and C) Data are expressed as the mean ± SD of four (HUVEC) (A) or three (GMVEC) (C) independent experiments with cell cultures from different donors. (B and D) Representative flow cytometric analyses of control and VT (10 nM)-treated, TNF-α-stimulated HUVEC (B) and GMVEC (D) are presented.

Effects of Caspase Inhibitors on VT-Induced Apoptosis of FMVEC and GMVEC

Caspases such as caspase 1 (ICE) and caspase 3 (CPP32) have been implicated in the complex cascade of events that results in apoptosis (15). To determine whether these cysteine proteases play a role in VT-mediated apoptosis, DEVD-CHO and YVAD-CHO, specific inhibitors that discriminate between different caspases (inhibiting caspase 3 and caspase 1-like proteases, respectively), were used. FMVEC incubated with DEVD-CHO (3 to 300 μM) demonstrated dose-dependent inhibition of VT-induced apoptosis. The threshold dose to decrease the percentage of apoptotic cells was 30 μM; a maximal response of 60 to 70% inhibition of VT-mediated apoptosis was obtained with 300 μM DEVD-CHO (Tables 2 and 3). Similarly, DEVD-CHO partly inhibited VT-induced apoptosis in TNF-α-exposed GMVEC (Table 2). Administration of YVAD-CHO (3 to 300 μM) did not result in significant inhibition of VT-induced apoptosis in FMVEC (Table 2). Western blotting of cell extracts demonstrated that both FMVEC and unstimulated and TNF-α-stimulated GMVEC contained caspase 3, whereas caspases 6 (Mhc-2) and 7 (ICE-LAP, Mhc-3) could not be detected (data not shown). These data are consistent with a possible involvement of caspase 3 in VT-induced apoptosis in human microvascular endothelial cells.