Article Figures & Data
Figures
- FIG. 1.
Kinetics of cell death. HUVEC were either treated with LT (100 ng of LF + 500 ng of protective antigen per ml) at the start of the experiment only (diamonds) or treated with toxin at the start of the experiment and then daily thereafter with fresh toxin and medium (squares). At the indicated time points, the viability of toxin-treated cells was determined with a formazan-based substrate as described in the text. Percent viability was determined by normalizing results to otherwise identically treated control assays in which LT was not added. Shown are mean and standard deviations of assays performed in octuplicate.
- FIG. 2.
LT induces apoptosis. (A) After 18 h of treatment with LT, 20 μM PD98059, or LT and 50 μM Z-VAD-FMK, HUVEC were stained with annexin V, Alexa Fluor 488 conjugate, and propidium iodide (PI). Following flow cytometry, histograms were generated by gating on propidium iodide-negative cells (annexin) and observing green fluorescence (FL-1) or by gating on propidium iodide-positive cells (propidium iodide) and observing red fluorescence (FL-3). Alternatively, HUVEC were stained with 10 μM fluorescein isothiocyanate-ZVAD-FMK, a fluorescent probe that binds to activated caspases. Following flow cytometry, histograms (FITC-ZVAD) were generated by observing green fluorescence (FL-1) for the entire cell population. Percentages are the number of cells within the marked histogram region expressed as a fraction of all cells analyzed. Results are representative of at least three different experiments. (B) After 18 h of treatment with anthrax factors, HUVEC were fixed, permeabilized, stained with DAPI, and examined by fluorescent microscopy. Shown is a representative field from LF- and protective antigen (PA)-treated and untreated controls. Apoptotic nuclei were scored based on characteristic apoptotic features of increased fluorescence (indicative of chromatin condensation) and nuclear fragmentation. A large number of apoptotic nuclear fragments are present in the LT-treated sample. (C) Data presented are the mean and standard deviation of one randomly chosen 100× field from four parallel assays with apoptotic nuclei expressed as a percentage of total HUVEC per field. LT-treated cells showed a significantly increased level of apoptotic nuclei (P < 0.01; Student's t test) compared to all other conditions.
- FIG. 3.
LT inhibits tubule formation. HUVEC were embedded in type I collagen gel containing 16 nM phorbol myristate acetate and treated with either LT or 20 μM PD98059 (an inhibitor of MEK1/2) or left untreated. After 2 days, cells were fixed, stained with DAPI, and photographed with phase contrast (P/C, 200×) and fluorescent (DAPI, 200×) microscopy. Alternatively, cultures were fixed on day 3, paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E, 100×). Note the presence of tubules in untreated controls and their total disruption in LT- and PD98059-treated cultures. In addition, DAPI images of LT- and PD98059-treated cultures show endothelial cells with morphological hallmarks of apoptosis, i.e., condensed chromatin and nuclear fragmentation. Examples of apoptotic nuclei within the plane of focus in this three-dimensional assay are indicated by arrows.
- FIG. 4.
LT induces endothelial death in two-dimensional cord assays. Confluent monolayers of HUVEC were overlaid with 0.5 mg of type I collagen per ml and treated with16 nM phorbol myristate acetate (PMA), LT, ET, and/or 20 μM PD98059. In the absence of PMA, typical cords form 6 h after collagen overlay (upper left panel, phase contrast, 100×); however, by 24 h (upper right panel), most of the endothelial cells are no longer viable. In contrast, in the presence of PMA, HUVEC remained viable as confluent monolayers even after 48 h (middle left panel). However, despite the presence of PMA, a 48-h treatment with LT (middle right panel) or PD98059 (lower left panel) led to significant cell death, leaving HUVEC in atypical cord-like arrangements. In contrast, ET did not have a deleterious effect on endothelial survival (lower right panel).
- FIG. 5.
Time course of ERK inhibition pathway by LT. HUVEC were serum starved and then treated with LT for the indicated number of hours. They were then treated with 30 ng of bFGF per ml, an activator of the ERK pathway, for 5 min, and lysed in protein sample buffer. Western blots were probed with antibody against phosphorylated ERK (phospho-Thr202/Tyr204), then stripped and probed with antibody to total ERK. Alternatively, blots were probed with an antibody against the N terminus (NH2) of MEK1, then stripped and probed with an antibody (COOH) against a sequence C-terminal to the LT cleavage site in MEK1/2.
- FIG. 6.
LT inhibits phosphorylation of ERK, p38, and JNK/SAPK. HUVEC were serum starved and then treated with LT for 7 h or 20 μM PD98059 for 2 h. Cells were then exposed to 30 ng of bFGF per ml or 10% serum for 5 min and immediately harvested. Western blots were probed with phosphorylation-specific antibodies for ERK, p38 and JNK/SAPK. Blots were then stripped and probed with antibodies against total ERK, p38, and JNK/SAPK.
- FIG. 7.
LT cleaves multiple MAP kinase kinases that signal through the ERK, p38, and JNK/SAPK pathways. Serum-starved HUVEC were treated with LT for 7 h and 30 ng of FGF per ml or 10% fetal bovine serum for 5 min prior to preparation of cell lysates. Western blots were probed with antibodies against epitopes N-terminal (NH2) or C-terminal (COOH) to previously described LT cleavage sites within the indicated MKKs. As expected, a short treatment with bFGF or serum did not alter MKK cleavage patterns. The MEK1/2 COOH blot was also probed with an antibody to α-tubulin (short arrow). This protein serves as a reference point to appreciate the slightly reduced molecular weight (dashed arrow) of MEK1/2 observed after LT treatment. Solid arrows point to the untreated MKKs. The band marked by the arrow in the MKK-4 COOH blot is exactly superimposable on the band marked in the NH2 blot. For unknown reasons, a cleaved form was not observed with the COOH antibody.
- FIG. 8.
ERK pathway inhibition leads to decreased endothelial viability. HUVEC were treated for 24 h with inhibitors of the ERK (20 μM PD98059), p38 (3 μM SB202190), and JNK/SAPK (5 μM SP600125) pathways. Values shown are mean and standard deviations for sextuplicate cell viability assays normalized to controls incubated in the absence of inhibitors.
