Recent data suggests that for correct embryonic development multiple peptides must be expressed in a proper temporal and quantitative fashion. It has also been proposed that some of the mechanisms and proteins involved in embryogenesis may be reiterated in the adult, functioning in tissue repair and angiogenesis. Recent data from our laboratory would suggest that Ang II and the AT2 receptor may be such proteins. We have recently shown that AT2 receptors are expressed in the mesenchyme of the developing rat fetus. They are highly abundant but only transiently expressed during fetal development, reaching maximal abundance by embryonic day 19-21, and rapidly decreasing after birth. Our hypothesis is that Ang II, acting through its AT2 receptor, plays an important role in mesenchymal proliferation and/or differentiation in the fetus. The purpose of this proposal is to determine if the growth related actions of Ang II are mediated through the AT2 receptor and determine what is the physiologic function of Ang II in normal rat fetal development. Three major questions will be addressed. 1) Is Ang II necessary for normal embryonic/fetal development and, if so, is the presence of this peptide critical at a specific stage of differentiation/maturation? 2) Does Ang II function in mesenchymal cell proliferation and/or differentiation? 3) How does Ang II mediate its effect(s)? What is the second messenger system? Does Ang II function by potentiating and/or inducing the synthesis of other growth factors? Specifically, we will determine when AT2 receptors are first expressed during embryogenesis using an in situ receptor assay and determine the relative abundance of Ang I, II, and III peptides bound to these receptors. Using both in vivo and in vitro animal models, we will examine the effects AT2 receptor antagonists (PD123177 & CGP 42112A) have on embryogenesis. These antagonists will be administered to pregnant rats at varying times throughout the pregnancy or added directly to embryos growing in vitro. This second paradigm removes the confounding effects the antagonists may have on placental circulation. The fetuses will be evaluated for growth and development by measuring size, weight, total DNA, and protein content and gross morphologic defects. To directly evaluate the effects of Ang II and AT2 receptor blockade has on mesenchymal cell proliferation/differentiation, we will use both primary mesenchymal cultures and teratocarcinoma cell lines analyzing proliferation, DNA synthesis, protein synthesis and onset of senescence in the primary cultures. Finally, we will try to determine how Ang II mediates its effects by examining the most likely signaling mechanism(s) to be linked to the AT2 receptor.
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