Over expression of gax, a novel homeobox protein induces apoptosis in serum stimulated vascular smooth muscle cells in vitro

The proliferation of the underlying vascular smooth muscle cells has been attributed to restenosis. Our laboratory has cloned a novel growth-inhibitory homeobox gene, gax, from vascular smooth muscle cell library. In this work we demonstrate that overexpression of gax will induce apoptosis. This phenomena has been shown in neuronal cells that overexpress gas3/PMP22. a protein that has inhibitory properties similar to gax. Primary rat vascular smooth muscle cells that had been serum deprived for 72 hours were infected with a nonreplicating adenoviral construct expressing either the gax cDNA fused to a hemagglutinin epitope tag (Ad-gax) or B-gal cDNA (Ad-B-gal). We demonstrate 100% infection for both Ad-β-gal and Ad-gax-infected cells. Ad-gax infected cells do not incorporate tritiated thymidine compared to Adβ-gal infected or non-infected control cells when restimulated with serum. Cell viability assays revealed that 80% of the Ad-gax infected cells died within 48 hours unlike the uninfected control or Ad-β-gal infected cells which doubled during the same period. Immunohistochemical analysis utiliizing the ApopTag system indicated that Ad-gax-infected cells were undergoing apoptosis by 24 hours post serum stimulation and exhibited condensed nuclei. In contrast Ad-β-gal-infected or uninfected control cells contained normal nuclei and exhibited no labeling with ApoTag. DNA extracted from the Ad-Gax-infected cells exhibited the nucleosomal 180-bp ladder characteristic of apotosis. Our in-vitro results provides a basis for future animal studies using the rat carotid balloon injury model of restenosis where preliminary results indicate that Ad-gax administration to the vessel wall prevents neointimal formation.