BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 235, 59–63 (1997) ARTICLE NO. RC976728 Purification of a Vascular Apoptosis-Inducing Factor from Hemorrhagic Snake Venom S. Masuda, S. Araki, T. Yamamoto, K. Kaji,* and H. Hayashi Sugashima Marine Biological Laboratory, School of Science, Nagoya University, Toba, Mie 517, Japan; and *Department of Life Health Science, Shizuoka University, Shizuoka, Shizuoka 422, Japan Received April 28, 1997 MATERIALS AND METHODS Hemorrhagic snake venom induces apoptosis in vas- cular endothelial cells specifically. We report here the Reagents. MCDB-107 medium for cell culture was purchased purification of an apoptosis-inducing factor from the from Kyokuto Pharmaceutical Industry (Tokyo, Japan). Fibroblast growth factor (FGF) was extracted from bovine brains by the method venom of the rattlesnake Crotalus atrox. The purified of Lobb and Fett (6). Fetal bovine serum (FBS) was purchased from factor was a homodimeric protein with a molecular GIBCO (Grand Island, NY, U.S.A.). Crude snake venom and crystal- mass of 110 kD and an isoelectric point of 8.5. When line bovine serum albumin were obtained from Sigma Chemical Com- exposed to the factor, vascular endothelial cells in cul- pany (St. Louis, MO, U.S.A.). Cycloheximide (CHX) was purchased ture died, with the characteristic features of apoptosis, from Wako Pure Chemical Industries (Osaka, Japan). All other such as fragmentation of cells and cleavage of DNA chemicals were of ultrapure grade. into fragments that yielded a characteristic ladder Cell culture. Human umbilical vein endothelial cells were ob- pattern upon electrophoresis. The activity seemed to tained by the method of Jaffe et al. (7). The cells were cultured on be specific to endothelial cells. This factor may prove collagen-coated plastic dishes in MCDB-107 culture medium that to be a useful tool for studies of the molecular mecha- had been supplemented with 10% FBS, 70 ng/ml FGF, and 100 mg/ ml heparin at 37 °C in an atmosphere of 5% carbon dioxide and 95% nisms of vascular apoptosis.  1997 Academic Press air. The cells were stained immunochemically by treatment with antibodies raised in rabbit against human factor VIII to confirm that they were endothelial cells. RSMC-3 (rat smooth muscle cells), TIG- 7 (human fibroblasts) and ECV-304 (transformed VEC) were cultured Vascular endothelial cells (VEC) are important in in a medium that consisted of MEM supplemented with 10% FBS. wound healing, in the progression of cancer, in the ho- Assay of viability. VEC were grown until cultures had reached meostasis of the circulatory system and in embryogene- confluence or sub-confluence. Then the medium was replaced with sis as a consequence of their capacity for angiogenesis the basal medium (without FGF and FBS) after cells had been washed once with phosphate-buffered saline without magnesium or (1,2). Vascular degradation is of equal importance in calcium (PBS 0 ). The cells in test media were kept in an incubator such phenomena. For example, degeneration of tumor with or without the fraction to be tested. In order to determine num- vessels induces tumor regression (3), and degradation bers of living cells, which adhered to the surface of the plastic dishes, of vessels during the healing of a wound is important trypsinized cells were counted in a Coulter counter (Coulter Elec- tronic, Inc., Hialeah, FL, U.S.A.) after an appropriate time interval. for the prevention of a hypertrophic scar (4). Further- Cells that had disintegrated and detached from dishes were washed more, many vascular degenerative disease are known, away with PBS 0 before treatment with trypsin. The cells that re- such as atherosclerosis and diabetic angiopathy, as mained attached to dishes after washing were not stained with eryth- well as hemorrhagic diseases or conditions caused by rosin B and were regarded as living cells. viruses, bacteria, snake venoms and autoantigens (5). Purification of the protein. Crude snake venom from Crotalus We have been investigating cell death in VEC be- atrox was fractionated by column chromatography on hydroxyapatite cause it seems possible that cell death might play an and isoelectric focusing in a polyacrylamide gel. The activity of frac- tions was assayed by monitoring the viability of cultured VEC and active role in the control of vascular degeneration (8,9). fractions with apoptotic activity were identified. Protein concentra- We showed previously that crude hemorrhagic snake tions were measured by the BCA assay (Pierce Chemical Co. Rock- venoms are effective for the induction of apoptosis in ford, IL, U.S.A.) on microtiter plates with bovine serum albumin as VEC. The activity is specific to VEC (10), and neuro- the standard. toxic snake venoms lack such activity. In this study, Gel electrophoresis. Cells were plated on dishes of 100 mm in we purified a vascular apoptosis-inducing factor from diameter. After treatment with test medium, DNA was extracted the hemorrhagic venom of the rattlesnake Crotarus from cells and loaded immediately onto a 2.0% agarose gel for conven- tional electrophoresis, as described previously (8). atrox. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 59