Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries

We determined the expression and functional correlate of in vitro transfect ion with a recombinant adenoviral vector encoding the gene for bovine endot helial nitric oxide synthase (AdCMVeNOS) or Escherichia coli beta-galactosi dase (AdcMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscl e cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by dou ble-cesium gradient ultracentrifugation. Cell cultures and PA were incubate d with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37 degrees C. LacZ expression was assessed by his tochemical staining; eNOS expression was evaluated by Western blot analysis . Functional eNOS expression was determined by measurement of cGMP and quan tification of the relaxation response to bradykinin (BK). In PA, LacZ trans gene expression was preferentially localized to the adventitia and endothel ium. Increased eNOS protein expression was observed in EC and VSMC transfec ted with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in func tional expression and altered vasoactive responses in the neonatal pulmonar y vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.