Optimising adenoviral vascular gene transfer in vivo: Impact of early acute inflammation on transgene expression and vasomotor function
Channon KM., Youngblood SA., Olmez E., Shetty GA., Blazing MA., George SE.
Recombinant adenoviruses are attractive vectors for in-vivo vascular gene therapy, although chronic inflammation and transgene loss results from host immunity to viral proteins. However, early acute effects of adenovirus may also have important implications for in vivo vascular gene transfer. We aimed to assess the mechanism and functional significance of endothelial injury after adenoviral gene transfer. We performed β-Galactosidase (β-Gal) gene transfer to rabbit carotid arteries (CA), using increasing viral titers. Arteries were either left in place in-vivo, or removed and incubated ex-vivo. After 3 days, we determined: (1) β-Gal protein levels, by ELISA; (2) endothelial VCAM-1 and ICAM-1 expression, and inflammation, by immunohistochemistry; or (3) endothelial function, by isometric vasomotor studies. The importance of neutrophils (PMN) was examined by inducing neutropenia with vinblastine. Results: A total of 68 rabbit CA were studied. (1) β-Gal expression was reduced 4-fold in in-vivo CA compared with paired ex-vivo CA (n=18, p<0.001). Increasing viral titer > 5 × 109 pfu/ml had little effect on β-Gal expression in-vivo. (2) Endothelial PMN were not seen ex-vivo but increased with viral titer in-vivo (21.9 ± 4.2 PMN/section at 8 × 109 pfu/ml vs. 2.0 ± 2.4 at 1 × 109 pfu/ml; p<0.01). (3)Phenylephrine-induced contraction was unaffected by gene transfer. In contrast, endothelial dependent relaxation to acetylcholine (ACh) was greatly impaired (31 ± 6.0 % relaxation at 1 × 1011 pfu/ml vs. 53 ± 5.8 % in control CA, at 10..μM ACh; p<0.01). (4) Vinblastine-induced neutropenia virtually abolished PMN infiltration in CA, and improved ACh dependent relaxation. Conclusions: In vivo vascular adenoviral gene transfer can result in endothelial injury, impaired vasomotor function, and greatly reduced transgene expression. Acute endothelial toxicity is mediated largely by PMN. However, we identify a "window" of viral titer for optimal vascular adenoviral gene transfer, between 1 and 5 × 109 pfu/ml, when transgene expression is high but vessel injury is minimized.