Malignant Ascites Fluid (MAF), Including Ovarian-Cancer-Associated MAF, Contains Angiostatin and Other Factor(s) Which Inhibit Angiogenesis

OBJECTIVE: The aim was to determine whether human malignant ascites fluid (MAF) associated with abdominal cancer, including ovarian cancer, contained factors which inhibit angiogenesis as well as others which stimulate this process. METHODS: MAF was collected from six patients, four with ovarian cancer, one with gastric cancer, and one with liver metastases. Using the chick chorioallantoic membrane (CAM) the effect of MAF on 7-day-old CAM capillaries was examined for 48 h. Vascular endothelial growth factor (VEGF) was evaluated by ELISA. Five samples of MAF were fractionated by lysine-Sepharose chromatography and the lysine-bound and -unbound fractions were eluted by epsilon-amino-n-hexanoic acid. Whole MAF, the lysine-bound and -unbound fractions, and human angiostatin were subjected to SDS-PAGE/Western blot analysis and immunostained after exposure to anti-human plasminogen. Human plasminogen was exposed to conditioned medium from ovarian epithelial cancer (HEY) cells and subjected to similar Western blot analysis. RESULTS: Despite containing VEGF, each MAF sample examined caused a loss of capillaries from the CAM; a similar response was seen using purified human angiostatin. Whole MAF and the lysine-bound fraction contained plasminogen (90 kDa) and a 55-kDa protein which migrated in a similar manner to human angiostatin on Western blot. Both the lysine-bound and -unbound fractions caused a loss of capillaries in the CAM. Human plasminogen exposed to conditioned medium from HEY cells yielded a fragment which was similar in size to angiostatin. CONCLUSIONS: MAF from patients with various clinical presentations contains angiostatin and VEGF as well as other factors which are capable of inhibiting angiogenesis.