Proliferation of vascular smooth muscle cells (VSMC) is a hallmark of arteriosclerosis. Despite its importance, no genes have been identified that inhibit VSMC growth in a cell type-specific manner.In addition, no effective therapy has been developed to effectively suppress VSMC growth in vivo. Several viral vectors have been used to deliver genes that inhibit VSMC growth in animals, however, these vectors lack cell type- specificity and thus represent an obstacle for gene therapy. The overall goal of this proposal is to identify the most potent VSMC-specific promoter for gene therapy. We have recently isolated the cDNA encoding APEG-1, a novel nuclear protein preferentially expressed in VSMC in vivo. Its expression in VSMC appears to be more restrict than SM22alpha or other smooth muscle-specific genes. Microinjection of APEG-1 expression plasmids into VSMC inhibits their 5-bromodeoxyuridine labeling, implicating APEG-1 as a growth inhibitor. In addition, a 2.7 kb mouse APEG-1 5' flanking DNA fragment directs high level of promoter activity in VSMC but not in other cell types. In this application, we propose to further test the effect of a retrovira1 construct (that can be induced to express APEG-1 by tetracyclin) on growth of VSMC and other cell types. We will identify the VSMC-specific positive regulatory elements present in the 2.7 kb of the APEG-1 5' DNA fragment and the specificity of these positive elements will be confirmed in transgenic mice. Furthermore, we will compare the VSMC-specific positive regulatory elements between APEG-1 and SM22alpha. Using these information, we plan to construct the most potent VSMC-specific artificial promoter. The potency and specificity of this promoter will also be tested in vitro and in transgenic mice in vivo. Finally, we will employ the most potent VSMC-specific promoter in conjunction with retroviral vectors to express APEG-1 (if it turns out to be a VSMC-specific growth inhibitor) or other growth inhibitor.to suppress proliferation of VSMC in mice and rabbits. The proposed experiments will allow us to determine the fundamental importance of APEG-1 in regulating growth and differentiation in VSMC. Furthermore, identification of VSMC-specific cis--acting elements will be crucial to the future success of efforts to restrict the expression of foreign genes to the blood vessel wall for gene therapy of vascular disease.
