application) The Ang II type 2 (AT2) receptor is expressed in the rat embryonic blood vessel at late gestation (embryonic days 16-21) and in the early neonatal period, but declines to very low levels in the adult. Cell culture studies and in vivo gene transfer experiments suggest that this receptor can inhibit DNA synthesis and induce apoptosis. Pharmacological blockade of the AT2 receptor using the specific antagonist PD123319 during embryonic days 16-21 results in decreased aortic DNA synthesis and subsequently in increased systemic blood pressure when measured at 3-5 weeks of age. Recently, the applicant's group and Inagami's group have obtained, using homologous recombination, AT2 receptor knockout mouse strains which exhibit enhanced pressor response to acute angiotensin infusion as compared to the wild type strain. In preliminary experiments, the applicant has observed that the knockout mouse has higher systemic vascular resistance, especially in response to Ang II infusion, and increased vascular thickening, especially of the small mesenteric arteries. These data have led the applicant to hypothesize that this receptor plays an important role in vascular development in late gestation. This proposal will employ homologous recombination and gene exchange technology to define further the role of the AT2 receptor in vascular remodeling in mouse development. 1) The applicant will characterize the relationship of receptor expression to vascular smooth muscle cell DNA synthesis, vascular smooth muscle cell differentiation and/or apoptosis. The structural influence of AT2 receptor expression will be studied by a systemic analysis of histology and morphometry of the blood vessels in knockout and wild type animals. The physiological consequence will be defined by in vivo hemodynamic and in vitro vascular contractility experiments. To amplify the phenotype, the applicant proposes to examine the effects of in utero Ang II infusion on vascular remodeling and development. 2) The investigators will perform AT1 vs AT2 gene exchange studies by generating recombinant animals using gene replacement technology. Thus, the coding region for the AT2 receptor gene will be replaced by that of the AT1a receptor, with the 5' and 3' flanking regions of the AT2 receptor gene remaining intact. This will enhance AT1 receptor expression at the time when the AT2 receptor would be expressed, and will amplify the AT1 receptor expression without AT2 receptor effect. Conversely, the reverse exchange of AT1 for AT2 will generate animals with """"""""unopposed"""""""" vascular AT2 receptor expression. Further modifications will involve the generation of mutations within the AT1a or AT2 receptors to obtain constitutively active receptors that are signaling in an angiotensin independent manner. Analysis of the effects of the gene exchanges on vascular remodeling and functional (phenotypic) consequences will be performed. Taken together, the above studies should define the role of the AT2 receptor in vascular development.
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