Endostatin Induces Endothelial Cell Apoptosis

Citation
Mohanraj Dhanabal, Ramani Ramchandran, Matthew J. F. Waterman, Hua Lu,
Bertrand Knebelmann, Mark Segal, and Vikas P. Sukhatme
From the Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School,
Boston, Massachusetts 02215
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 274, No. 17, Issue of April 23, pp. 11721–11726, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Received for publication, October 14, 1998, and in revised form, December 17, 1998

Summary
In this study researchers studied the apoptotic effects of endostatin, in endothelial cells and non endothelial cells. Endostatin is a fragment of Collagen XVIII that had been shown to reduce tumor proliferation and migration in mice. Because the process by which this happens was unknown, researchers at Harvard Medical School used four methods to try and track the mechanism by which endostatin increases apoptosis in cells and why it seemed to be endothelial cell specific. They studied cow pulmonary endothelial cells, bovine aortic endothelial cells, bovine adrenal capillary endothelial cells, and several non-endothelial cells including, HUVE, HMVE-L, several fibroblast cells and renal carcinoma cells. In order to study apoptosis, the authors used Annexin V, caspase 3, TUNEL staining, and Western Blot assays with different concentrations of recombinant mouse endostatin added to the various cells and added no endostatin to each cell line as a negative control and added human recombinant tumor necrosis factor as a positive control. In the Annexin assay, apoptosis was detected using a protein that has a high affinity for phosphatidylserine. The authors then used flow cytometry to analyze the percentage of apoptotic cells after various amounts of time had passed. The caspase 3 assay was used to track the activity of the caspase 3, a protein associated with cell apoptosis, in the cells that were introduced to endostatin. Finally, TUNEL staining and Western Blotting were used to detect DNA fragmentation and Bcl-2 and Bax proteins. Using these methods, the authors concluded that endothelial cells treated with endostatin had a rounder morphology associated with apoptosis, had PS moved from the inner membrane to the outside, had increased caspase activity, had increased DNA fragmentation (fig 4), and that Bcl-2 and Bax expression was unchanged (fig 5). From this the authors speculated that endostatin increased apoptosis by reducing the amount of Bcl-2, which is a pro-apoptotic protein that may also interfere with anti-apoptotic proteins such as Bax. Although several cell lines were studied, “the Cow Pulmonary Endothelial cells showed the most dramatic effect” when endostatin was introduced and non-endothelial cells showed none of these signs of apoptosis.

FIG. 4. A, detection of fragmented DNA in adherent C-PAE cells by the TUNEL assay. Adherent cells were fixed with fresh 4% formaldehyde/PBS at 4 °C for 25 min. The cells were permeabilized with prechilled 0.2% Triton X-100/PBS for 5 min on ice. After permeabilization, the TUNEL assay was performed along with propidium iodide dye staining. On staining, pictures were taken immediately using a fluorescent microscope attached to a digital camera and analyzed using the SPOT software. Panels a, c, e, and g are fields of cells stained with propidium iodide (1 mg/ml). Panels b, d, f, and h are the same fields under green fluorescent light. Control cells were incubated in DMEM with 2% FCS. Endostatin concentration in the terminal deoxynucleotidyltransferase1 and terminal deoxynucleotidyltransferase2 samples was 10 mg/ml. TNF-a was added at a final concentration of 20 ng/ml. B, quantitative determination of apoptosis. After TUNEL and propidium iodide staining, the cells were counted in 15 fields for control and endostatin-treated attached cells. For TNFa-treated cells, five fields were counted. The percentage of green divided by the number of red cells in a given field was determined and an average (mean 6 S.E.) of the different fields is presented.

FIG. 5. A, Western blot analysis of C-PAE cell lysate for Bcl-2 protein levels. C-PAE cells were treated with endostatin (10 mg/ml) for the indicated period of time. Total cell lysate from control (2) and treated (1) cultures was analyzed as described earlier. 30 mg of total protein was subjected to SDS polyacrylamide gel electrophoresis analysis followed by Western blot transfer. Immunoblot analysis was performed with antibodies directed against Bcl-2. Reprobing of the same blot with actin antibody was performed to normalize for protein loading. B, immunoblot detection of total cell lysate for Bax expression levels. 30 mg of total cell lysate was separated on 4–15% gradient gel. SDS-polyacrylamide gel electrophoresis analysis was followed by membrane transfer and probing with Bax antibody. Actin probing is shown, as well.

Criticism
The paper is thorough and easy to follow because the descriptions of assays used and procedures followed the same order as in the results. The experiments seem to be well thought out and executed. One thing that stood out to me was that for every negative control, the cells were not only unexposed to endostatin but also all the other buffers used for the assay. Instead, only PBS was added to the controls. Because of this, it can’t be certain that the endostatin is causing the cell apoptosis and not the other buffers used in the experiments. For example, in the caspase 3 assay they added bFGF along with the endostatin to the samples but didn’t add bFGF to the negative control. In the end, the authors were able to use four different assays that detected several different signs of apoptosis to show that endostatin causes apoptosis in cow pulmonary endothelial cells and not in non-endothelial cells.