Expression of Wild-Type and Noncleavable Fas Ligand by Tetracycline-Regulated Adenoviral Vectors to Limit Intimal Hyperplasia in Vascular Lesions

Abstract

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 provides 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 FasL and a noncleavable mutant of FasL (FasL-NC). Transduction of FasL and FasL-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 FasL or FasL-NC transduction was also dependent on cotransfection with the rtTA adenovirus and administration of DOX by subcutaneous injection. In contrast to wild-type FasL, transduction of FasL-NC did not result in the production of soluble (cleaved) FasL in the medium of infected cells in 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.

Heat Shock Protein 70 Participates in the Neuroprotective Response to Intracellularly Expressed β-Amyloid in Neurons

“…The TRE cassette was then subcloned into the multiple cloning site of pShuttle vector, and recombination with pAdEasy-1 vector was achieved following manufacturer's instructions (Quantum Biotechnologies, Montreal, Quebec, Canada). The AdTet-On and AdTRE-LacZ viruses were previously described (Mano et al, 2000). The cDNA of human ␤APP751 was digested from NSE-hAPP751 vector (provided by Dr. C. R. Abraham, Boston University, Boston, MA), and inserted into the HindIII site in the plasmid pAdTrack-CMV (Clontech), followed by recombination with pAdEasy-1, as described above.…”

Rheumatoid arthritis synovial fluid macrophages express decreased tumor necrosis factor–related apoptosis‐inducing ligand R2 and increased decoy receptor tumor necrosis factor–related apoptosis‐inducing ligand R3

“…One positive clone was amplified, purified through cesium chloride gradient, and dialyzed. Tetracycline-inducible gene expression was achieved with a binary adenovirus strategy, as previously described (17). Ad-Tet-␤-Gal and Ad-Tet-FasL are under the control of a minimally active CMV promoter downstream of 7 tetracycline operator sites.…”

Phosphatidylinositol 3-Kinase/Akt Signaling Controls Endothelial Cell Sensitivity to Fas-Mediated Apoptosis via Regulation of FLICE-Inhibitory Protein (FLIP)

“…The adenoviral vector AdTet-LacZ encoding ␤-galactosidase (␤-gal) was generated by using p⌬1ATRE, as described previously. 23 The adenoviral vector AdCMV-rtTA encodes a chimeric transcription factor composed of a mutant tetracycline repressor fused to the VP16 trans-activator under control of the CMV promoter/enhancer. Replication-defective adenovirus vectors expressing dominantnegative and constitutively active forms of murine Akt from the CMV promoter have been described previously.…”

“…The AdTet-LacZ vector, expressing ␤-gal from the tetracyclineregulated promoter, was used to evaluate inducible expression with this system by using X-Gal staining. 23 FLIP expression from the AdTet-FLIP vector was analyzed by Western blot analysis. As shown in Figure 4B, coinfection of HUVEC cultures with AdTet-FLIP and AdCMV-rtTA, each at an MOI of 2, for 12 hours in the presence of 300 ng/mL Dox increased the level of FLIP that was slightly above the level of endogenous expression.…”