This proposal will test the hypothesis that A-II (A-II) plays an important role in control of smooth muscle cell (SMC) growth and differentiation/maturation during vascular development, as well as in mediating adaptive changes in the contractile mass of SMC in adult animals in response to increases in work load. This hypothesis will be tested by addressing the following three specific aims.
Aim 1 will be to determine the role of A-II in differentiation and maturation of vascular SMC during development. Studies will include determining whether knockout of the angiotensinogen, the angiotensin AT2 receptor, or the AT1 receptors (1a and 1b alone and in combination) is associated with alterations in expression of SMC differentiation markers during vascular development, changes in contractile properties of embryonic blood vessels (with A. Somlyo), and/or transcription of SMC differentiation marker genes (assessed by crossing knockout animals with a number of novel SM alpha-actin, SM22alpha, and SM MHC LacZ founder lines previously characterized in our lab). Studies will also include analysis of chimeric mice to determine cells lacking AT1 or AT2 receptors show defective SMC differentiation/maturation.
Aim 2 will be to determine the molecular mechanisms whereby contractile agonists such as A-II stimulate expression of SMC differentiation marker genes in vivo. Studies will include determination of the role of the homeodomain protein MHOX in cis elements of the SM alpha-actin promoter previously shown to be required for A-II responsiveness in cultured SMC.
Aim 3 also will be to determine mechanisms whereby A-II stimulates increased rRNA transcription, and how this contributes to regulation of contractile mass. Studies will include: a) determining phosphorylation sites that regulate the activity of the rRNA transcription factor upstream binding factor (UBF); b) identifying kinases that regulated increased phosphorylation of UBF in intact vessel preparations in organ culture (with A. Somlyo). The mechanisms whereby A-II and other contractile agonists regulate SMC growth, differentiation, and maturation in vivo during vascular development, as well as in physiological and pathological responses of blood vessels to high blood pressure.
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