DEVELOPMENTAL REGULATION OF ANGIOTENSIN II VASCULAR MITOGENESIS

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Angiotensin II is a potent vasoconstrictor which also induces growth in vascular smooth muscle cells (VSMC). Evidence supports an essential role for AngII in fetal/neonatal vasculogenesis. We have shown in adult porcine aortic VSMC the AngII mitogenic action is incomplete, confined to late cell-cycle events: AngII induces completion of cell division by cells reversibly arrested in G2 Phase but has no capacity to stimulate or protein DNA synthesis. In contrast, based on our preliminary results, AngII potently stimulated DNA and protein synthesis and increased cell proliferation rate in neonatal porcine VSMC. This, together with published evidence that the full mitogenic response to AngII in human fetal mesangial cells is lost with aging, prompts our HYPOTHESIS that AngII mitogenic capacity is developmentally regulated. Using our established porcine VSMC model, we will test this in inbreed microswine, permitting control of precisely-timed fetal and neonatal tissues, reducing nonspecific genetic variability, and reducing number of passages in culture by pooling of VSMC from sex-matched littermates. Our three SPECIFIC AIMS each address VSMC obtained from fetal, neonatal, vs. post- pubertal microswine and studied at matched subconfluent density in 3rd- 5th passage. In SA#1, we will determine for each developmental stage the early cell-cycle vs. late cell-cycle loci of AngII mitogenic actions in (respectively) G0- or G2-synchronized VSMC using a newly validated synchronization protocol. In SA#2, we will assess developmental regulation of AngII receptor number, subtype, and AT-1:AT-2 ratio and determine the functional role of each subtype in the mitogenic actions of AngII defined at each developmental age. In SA#3, based on the importance of the Epidermal Growth Factor (EGF) receptor in fetal development and its capacity to increase AT and decrease AT-2 AngII receptors, we will determine the role of constitutive EGF-R number and activation status in the developmental regulation of AngII mitogenic capacity and will and compare effects of chronic "priming" with Transforming Growth Factor (TGF) alpha, the dominant fetal ligand for EGF receptor activation, on basal growth, on mitogenic responses to AngII, and on both Ang II receptors (AT- 1 and AT-2) and EGF-R in VSMC from each developmental stage. Because a fetal-like phenotype can be re-induced in mature VSMC in response to injury, understanding the developmental physiology of vascular growth regulation has relevance not only to normal growth, but also to preventive/therapeutic interventions in both children and adults at risk for vascular disease.