The candidate is committed the development of a career in academic cardiovascular medicine, focusing on basic research. He demonstrated his potential as a basic investigator during the two years of his cardiology fellowship that he spent in the lab of Dr. Coleen McNamara. He will use the KO8 award to learn advanced methods in vascular research and to develop the ability to function as an independent investigator. The environment in which the candidate will perform the proposed research is well- suited to support the candidate's career development. The co- sponsors are enthusiastic to participate in the candidate's academic development. Dr. McNamara was the mentor for the candidate during his cardiology fellowship, and is eager to continue the mentoring environment developed during that time. Dr. Owens is a leader in the field of smooth muscle cell (SMC) growth and differentiation, and will contribute significant experience to the candidate's development. The overall objective of the proposal is to determine the mechanisms through which Id2 enhances SMC growth and to identify the SMC factor(s) with which Id2 interacts to exert this effect. Aim 1 is to identify factors that interact with Id2 in SMC and determine the effect of cdk2 phosphorylation on the range of Id2 binding partners. Studies will use the yeast and mammalian two- hybrid assays, as well as immunoprecipitation, to determine SMC factors that interact with Id2, including both traditional HLH binding partners and potential non-HLH interactions. In addition, the effect of a consensus cdk2 phosphorylation site (Ser5) on binding interactions will be studied using Id2 proteins which contain mutations of this site. Aim 2 is to determine the mechanisms of Id2-enhanced SMC growth. Preliminary work by the candidate identified p21 as an important cell cycle control factor which is transcriptionally regulated by Id2. Studies will be performed to further define the control of p21 by Id2, including experiments to determine the effect of Id2 over- expression on levels of endogenous p21 protein, as well as experiments to determine the ability of over-expression of p21 to overcome Id2-induced SMC growth. Additionally, promoter-reporter studies using cell cycle gene promoters containing HLH binding sites (including cyclin B1, cyclin D1, and pRb) will be performed to determine non p21-dependent mechanisms of Id2-enhanced SMC growth. Aim 3 is to determine the effect of inactivation of Id2 on vascular lesion formation in vivo. Initial studies will examine the expression pattern of Id2 in wild-type 129/Sv mice following a ligation injury. Further studies will examine the effect of inactivation of Id2 on lesion morphometry, using the Id2 knockout mouse. Altered lesion formation in an Id2-/- background will be determined using histological assessment of proliferation, apoptosis, and expression of cell cycle proteins that were demonstrated to interact with or be regulated by Id2 in aims 1 and 2.