The long term goal of this study is to elucidate the mechanisms controlling angiogenesis in the follicle and corpus luteum of the ovary. This will advance our knowledge not only of these key reproductive tissues, but also of the angiogenesis that is an intrinsic part of so many human diseases, particularly cancers. In the ovary, unlike other tissues and organs, blood vessels repeatedly and rapidly grow and regress in conjunction with follicular growth and corpus luteum formation. Substantial evidence indicates that these changes are controlled by angiogenic factors from the granulosa cells of the follicle and developing corpus luteum- a mechanism comparable to that operating in tumor angiogenesis. Progress to date shows that ovarian granulosa cells secrete several distinct factors that can affect endothelial cell (EC) growth or differentiation, and which could, therefore, participate in the angiogenic process. These include: (1) an EC mitogen that binds strongly to heparin and, therefore, is probably a member of the fibroblast growth factor (FGF) family; and (2) a heat stable, trysin sensitive, high molecular weight factor that induces EC elongation, migration, and angiogenesis in vitro. FGFs are prime candidates as regulators of angiogenesis, as well as numerous other vital developmental processes, since they are potent mitogens for ECs and several other cell types. Genes for six different FGFs have now been cloned. To determine which of these FGF genes are expressed by granulosa cells, the expression of the mRNA's for these factors will be examined using the new method of RT-PCR. This powerful procedure makes possible the study of expression of low copy number mRNAs or mRNA from very small numbers of cells. With RT-PCR, it was possible for the first time to show that both basic FGF (bFGF) and acidic FGF (aFGF) are produced in the rat ovary. Based on these findings, I propose to carry out experiments with the following two Specific Aims: I) To determine by RT-PCR whether the genes for the putative angiogenic proteins aFGF, bFGF, int-2, hst/KS, FGF-5, or FGF-6 are expressed in the various compartments of the rat ovary, whether expression varies during follicle growth and corpus luteum formation, and whether expression is hormonally regulated. II) To purify and identify the factor in GCCM that induces angiogenesis in vitro (IVAF). Identification of ovarian angiogenic factors and the elucidation of the mechanisms controlling their production will lead to a better understanding of normal and abnormal ovarian function, as well as the angiogenesis that contributes to tumor growth and several other major human diseases.
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