Estrogen regulates vascular endothelial growth permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

Vascular endothelial growth/permeability factor (VEG/PF) is expressed in some normal tissues and at high levels in a wide range of tumors. This growth factor is believed to be a key mediator of angiogenesis. Recent reports have shown that VEG/PF mRNA in the normal rat uterus is stimulated by estradiol (E(2)). In this study, we investigated the expression of VEG/PF in the mammary gland and 7,12-dimethylbenz(a)anthracene (DMBA)-induced, hormone-dependent mammary tumor of the rat model, and also whether VEG/PF is regulated by E(2). VEG/PF mRNA from tumor extracts was amplified by RT-PCR with VEG/PF primers and generated two main products which corresponded in size to those expected for VEG/PF 164 and 120. In some cases, a third product corresponding in size to that expected for VEG/PF 188 was also generated. No such PCR products were generated from equal amount of RNA from normal mammary tissue, rat brain, or liver. Using immunocytochemistry, VEG/PF expression was detected in the epithelial cells of the tumors. We developed an ELISA assay to measure VEG/PF protein concentrations and found a 4-fold difference between normal mammary glands (1.3 +/- 0.11 ng/mg protein) and tumors (4.44 +/- 0.66) (P < 0.01). E(2) treatment (5 mu g/rat, sc) of rats 24 h after ovariectomy, greatly enhanced the expression of RT-PCR products in tumors within 2 h, which reached a maximum at 6-8 h but declined by 48 h. VEG/PF concentrations were also increased 8-12 h after E(2) injection. When rats were given two injections of aromatase inhibitor 4-hydroxyandrostenedione (4-OHA 10 mg/rat sc) 24 h apart, to reduce estrogen concentrations, a low level of RT-PCR products was maintained for at least 96 h. After a single injection of 4-OHA, RT-PCR products remained low until 36 h when an increase occurred corresponding with a rise in plasma E(2) levels. Injection of E(2) 2 h after 4-OHA treatment, caused a rise in RT-PCR products in 6-8 h. However, there was no significant change in VEG/PF concentrations. An increase in VEG/PF protein concentrations followed the increase in mRNA levels by 4-6 h. Thus, it appears that E(2) causes a rapid induction of VEG/PF expression in mammary tumors that is similar to that observed in the normal uterus. These findings suggest that one mechanism by which estrogen acts as a mammary tumor promotor is by stimulating VEG/PF, leading to increased tumor angiogenesis and/or permeability of the microvessels to allow tumor cell migration.