A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer

Abstract

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of beta-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases beta-galactosidase expression 90-fold in SMC in vitro and approximately 40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.

Figure 1

Schematic representation of the transgene expression cassette within each of the vectors studied.

Figure 2

Expression of β-galactosidase in SMC after infection with recombinant adenoviral vectors. PASMC infected with (a) Ad5-lacZ, (b) RAd36, (c) Ad5-PP-lacZ, (d) Ad5–PMEP-lacZ, (e) Ad5–PREP-lacZ after cytochemical staining with X-gal. All cells were infected at MOI=10. (f) The proportion of β-galactosidase-positive cells 72 h post-infection is significantly greater after infection with Ad5–PREP-lacZ than with any other vector (all P<0.001).

Figure 3

β-galactosidase activity in lysates of cultured SMC and COS-7 cells. At MOI 10 (a), all MIEmCMV-based vectors give rise to greater activity than the MIEhCMV-based vector Ad5-lacZ (all P<0.001). Activity is significantly greater in Ad5-PREP-lacZ infected cells than all others (all P<0.0001). At MOI 500 (b), β-galactosidase activity is significantly greater in Ad5-PREP-lacZ-infected cells than all other groups (all P<0.001), almost five-fold greater than that induced by RAd36, and almost 90-fold greater than Ad5-lacZ. In fibroblasts (COS-7) infected at MOI 10 (c), there is no significant difference between the β-galactosidase activity induced by any of the MIEmCMV-based vectors containing WPRE (P=0.25).

Figure 4

Immunohistochemistry of an Ad5-PREP-lacZ infected vessel. Sections of porcine coronary artery underwent immunofluorescent staining 72 h after infection with Ad5-PREP-lacZ. (a) Vessel costained for SM α-actin (green) and β-galactosidase (red). Vessel histology is demonstrated. Red fluorescence is seen within the media. (b) Vessel at higher power showing colocalization of the cytoskeletal α-actin stain and the cytosolic β-galactosidase stain (bars=50 μm).

Figure 5

β-Galactosidase expression in infected coronary vessels. (a) The area of X-gal staining 72 h after exposure to each virus. Staining is significantly greater in vessels infected with Ad5–PREP-lacZ than with any other vector (all P<0.001). (b) β-galactosidase activity in lysates of coronary arteries 72 h after Infiltrator-mediated delivery of adenovirus vectors. Ad5-PREP-lacZ-infected vessels revealed ≈40-fold greater β-galactosidase activity than Ad5-lacZ-infected vessels (P<0.05) and ≈20-fold greater activity than RAd36-, Ad5-PP-lacZ- and Ad5-PMEP-lacZ-infected vessels (all P<0.01).

Figure 6

β-Galactosidase expression in infected coronary arteries. Sections of coronary artery stained with X-gal 72 h after Infiltrator-mediated infection with each of the vectors used. The greatest area of β-galactosidase staining is observed in the Ad5-PREP-lacZ-treated vessels (e) (bars=50 μm).