Expression of wild-type and noncleavable Fas ligand by tetracycline-regulated adenoviral vectors to limit intimal hyperplasia in vascular lesions

Proliferation of vascular smooth muscle cells (VSMCs) and the infiltration of T cells and macrophages into vessel wall are considered to be important for intimal lesion formation after balloon angioplasty. Previous studies have shown that Fas ligand (FasL) gene transfer to balloon-injured vessels inhibits lesion formation by killing both proliferating VSMCs and infiltrating inflammatory cells. Here, we describe the construction and utility of a binary, tetracycline-regulated adenovirus system that pro,ides controlled transgene expression in vitro and in vivo. In this system, optimal transgene expression required cotransfection with an adenovirus encoding the tetracycline-dependent trans-activator (rtTA) and induction with doxycycline hydrochloride (DOX), an analog of tetracycline. Using this system, adenovirus constructs were designed that allow regulated expression of wild-type Fast and a noncleavable mutant of Fast (Fast-NC). Transduction of Fast and Fast-NC induced similar extents of apoptosis in proliferating VSMCs in vitro in a manner that was dependent on the doses of the rtTA adenovirus and the presence of DOX in the medium. Furthermore, inhibition of intimal hyperplasia in injured carotid arteries by Fast or Fast-NC transduction was also dependent on cotransfection with the rtTA adenovirus and administration of DOX by subcutaneous injection. In contrast to wild-type Fast, transduction of Fast-NC did not result in the production of soluble (cleaved) Fast in the medium of infected cells lit vitro, or in the serum of rats after local gene transfer to carotid arteries, In conclusion, this binary tetracycline-inducible adenovirus system may allow for safer delivery of cytotoxic genes for therapeutic purposes.