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Preliminary Characterization of Angiogenic Activity in Media Conditioned by Cells from Luteinized Rat Ovaries^*

Departments ofGynecology and Obstetrics (J.D.R., A.L.G.) and Physiology (A.L.G.), Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Janice D. Rone, Department of Gynecology and Obstetrics, Park Building Room B2-232, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, Maryland 21205.

Abstract

Angiogenic activity was detected in media conditioned by ovarian cells from superovulated, pseudopregnant (PMSG/human CG treated) immature Holtzman rats. Media conditioned by cells from luteinized rat ovaries stimulated the directed migration of rabbit endothelial cells or mouse Balb/ c3T3 cells, but was not mitogenic to either cell type. That endotheliotropic activity was not associated with a steroid was indicated by the finding that chemoattractant activity was detected in fractions after reversed-phase Ci8 chromatography, which removes more than 95% of steroids present in the media, and that chemoattractant activity was precipitated by ammonium sulfate and by ethanol. Full chemoattractant activity was recovered after boiling (95 C for 30 min), lyophilization, dialysis, Sephadex G-25 desalting columns, and pH changes from 3–10. After Sephadex G-200 chromatography, chemoattractant activity emerged at elution volumes corresponding to 20,000–30,000 mol wt. Chemoattractant activity was not retained by Concanavalin A-Sepharose or gelatin-Sepharose, and was only partially retained by heparin-agarose. Chemoattractant activity was also partially retained on both cation and anion exchange columns. Our collective findings indicate the presence of a nonsteroidal, heat-stable, pronase-sensitive factor, nominal mol wt of 20,000–30,000, in media conditioned by cells from luteinized rat ovaries; this factor is chemoattractive but not mitogenic to endothelial cells. Ovarian-derived chemoattractant activity appears to be distinct from fibroblast growth factor because it lacked detectable mitogenic activity, and because fibroblast growth factor was not active in our cell migration bioassay. Because stimulation of endothelial cell migration is a key event during angiogenesis, demonstration of an ovarian endotheliotropic chemoattractant is consistent with our hypothesis that angiogenesis factors play a role in the paracrine regulation of ovarian function. (Endocrinology 127: 2821–2828,1990)

Footnotes

^* This work was supported in part by NIH Grants HD-22202 and SO7-RR 05378, and The Rockefeller Foundation. Presented in part at the 20th Annual Meeting of the Society for the Study of Reproduction, Urbana, IL, 1987 (Abstract 271) and at the 70th Annual Meeting of The Endocrine Society, New Orleans, LA, 1988 (Abstract #1122).

Received April 16, 1990.

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