(Verbatim from the application): Proliferation of vascular smooth muscle cells (VSMC) is a hallmark of arteriosclerosis. Because no genes have been identified that specifically inhibit VSMC proliferation, it has been difficult to develop an effective therapy for excessive VSMC growth. We have isolated the cDNA encoding APEG-l, a novel protein preferentially expressed in differentiated VSMC. During dedifferentiation of VSMC in vitro and in vivo, APEG-l mRNA underwent a rapid downregulation. Infecting VSMC with a recombinant adenovirus of constitutively active cGMP-responsive protein kinase (PKG) reversed this downregulation. Microinjection of an APEG-l expression plasmid into VSMC inhibited DNA synthesis, implicating APEG-l as a growth inhibitor. Thus, we hypothesize that APEG-l is a VSMC-specific growth inhibitor that functions downstream of the cGMP/PKG-signaling pathway. A 2.7-kb fragment of mouse APEG-l 5-flanking DNA directed a high level of VSMC-specific promoter activity, which could be repressed by a 0.8-kb APEG-l DNA fragment located 5' to the 2.7-kb fragment. The goals of our proposed work are to investigate the molecular mechanisms mediating APEG-l's VSMC-specific promoter activity, to test whether APEG-l is a VSMC-specific growth inhibitor, and to determine the molecular mechanisms by which APEG-l regulates VSMC growth and differentiation. To achieve these goals, we propose four specific aims.
AIM 1. Identify specific DNA sequences (cis-acting elements) and their cognate DNA-binding proteins (trans-acting proteins) important for APEG-l 's restricted expression in VSMC in vitro. In addition, we will identify the cGMP-responsive element(s) and their binding proteins in the APEG-l promoter.
AIM 2. Test the specificity and potency of the APEG-l VSMC-specific cis-acting elements identified in AIM 1 by using recombinant adenoviruses or transgenes containing reporters driven by these elements in mice.
AIM 3. Study the effect of APEG-l on growth and differentiation in VSMC.
AIM 4. Isolate APEG- l's interaction partners to elucidate the molecular mechanisms by which APEG- 1 regulates VSMC growth and differentiation. information obtained from these proposed experiments should provide fundamental knowledge about the biology of APEG-l and the differentiation of VSMC, and also critical insight into the treatment of atherosclerosis and restenosis after coronary artery bypass or angioplasty/stent placement. Furthermore, the identification of VSMC-specific promoters will be crucial for future use of gene therapy to direct foreign gene expression in the blood vessel wall.
