DIFFERENTIAL HORMONAL-REGULATION OF VASCULAR ENDOTHELIAL GROWTH-FACTORS VEGF, VEGF-B, AND VEGF-C MESSENGER-RIBONUCLEIC-ACID LEVELS IN CULTURED HUMAN GRANULOSA-LUTEAL CELLS

The development of ovarian follicles and subsequent corpus luteum form ation is accompanied by very active angiogenesis. Ovarian granulosa ce lls produce vascular endothelial growth factor (VEGF), which is a pote nt endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regula tion in the ovary. We first studied the expression of the messenger RN As (mRNAs) of the three VEGF isotypes in freshly isolated human granul osa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertil ization. The hormonal regulation of these mRNAs was subsequently studi ed in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express th e alternatively spliced transcripts representing 121-, 145-, and 165-a mino acid VEGF isoforms. Northern blot hybridization analyses indicate d that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of cul ture. For studying the hormonal regulation of VEGF isotype. mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE(2), as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A-and protein kinase C-dependent signaling pathways, r espectively. All test agents stimulated the expression of VEGF mRNA le vels in a concentration-dependent manner. Time-course studies indicate d that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels w ere not regulated by any of the test agents. However, we found that hC G and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal respons e observed at 24 and 48 h after cellular treatment. We conclude that t he mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells a nd that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation o f VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VE GF isotypes may play different roles during the vascularization of the human ovarian follicle and corpus luteum.