PRODUCTION OF HEPARIN-BINDING ANGIOGENIC FACTOR(S) BY BOVINE CORPORA-LUTEA DURING PREGNANCY

Effects of luteal-conditioned media (LCM) on proliferation and migration of endothelial cells were used to assess angiogenic activity of corpora lutea (CL) obtained from cows on d 100 (n = 5), 150 (n = 6), 200 (n = 6), and 250 (n = 6) of gestation. Explants of CL (200 mg) were incubated for 6 h in 3 mL of serum-free media containing no hormone, LH (1-mu-g/mL), prostaglandin F2-alpha (PGF2-alpha; 3-mu-M), or both hormones. Media from the four stages of gestation were subjected to the following procedures: 1) ultrafiltration, 2) high-salt (3.0 M NaCl) treatment and then ultrafiltration, 3) heat treatment, 4) heparin-Sepharose affinity chromatography, 5) immuno-neutralization with specific antibodies against heparin-binding growth factor (HBGF)-1 and against HBGF-2, and 6) dot immunoblot assay for HBGF-2. Fractions from the first five procedures were evaluated in the endothelial cell proliferation bioassay. In addition, progesterone concentration of LCM was determined by RIA. Across all days of gestation and hormone treatments, LCM stimulated (P < .05) proliferation and migration of endothelial cells, but activities did not differ among stages of gestation or hormone treatments. Both mitogenic and migration-stimulating fractions seemed to have M(r) > 100,000. The mitogenic activity fraction had an apparent M(r) > 100,000 even after treatment with high salt and was heat-labile. This endothelial mitogen was retained on heparin-Sepharose columns and was eluted with 2.0 M NaCl. Mitogenic activity was partly neutralized (P < .05) by antibodies against HBGF-2 but not HBGF-1. Presence of HBGF-2 in LCM was detected by dot immunoblot assay. Secretion of progesterone by luteal tissue was stimulated by LH and PGF2-alpha on all days of gestation, indicating that progesterone secretion is regulated independently from production of angiogenic factor(s). These data demonstrate that bovine CL secrete angiogenic factor(s) throughout gestation. Bovine angiogenic factor(s) seems to belong to the family of heparin-binding (fibroblast) growth factors.