Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice

Abstract

It has been shown that mice deficient in the gene coding for endothelial nitric-oxide synthase (eNOS) have increased pulmonary arterial pressure and pulmonary vascular resistance. In the present study, the effect of transfer to the lung of an adenoviral vector encoding the eNOS gene (AdCMVeNOS) on pulmonary arterial pressure and pulmonary vascular resistance was investigated in eNOS-deficient mice. One day after intratracheal administration of AdCMVeNOS to eNOS(-/-) mice, there was an increase in eNOS protein, cGMP levels, and calcium-dependent conversion of l-arginine to l-citrulline in the lung. The increase in eNOS protein and activity in eNOS(-/-) mice was associated with a reduction in mean pulmonary arterial pressure and pulmonary vascular resistance when compared with values in eNOS-deficient mice treated with vehicle or a control adenoviral vector coding for beta-galactosidase, AdCMVbetagal. These data suggest that in vivo gene transfer of eNOS to the lung in eNOS(-/-) mice can increase eNOS staining, eNOS protein, calcium-dependent NOS activity, and cGMP levels and partially restore pulmonary arterial pressure and pulmonary vascular resistance to near levels measured in eNOS(+/+) mice. Thus, the major finding in this study is that in vivo gene transfer of eNOS to the lung in large part corrects a genetic deficiency resulting from eNOS deletion and may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced.

Figure 1

Comparison of mean pulmonary arterial pressure (Left) and pulmonary vascular resistance (Right) in eNOS^+/+ mice and eNOS^−/− mice 1 day after administration of vehicle or 1 day after transfection with AdCMVβgal or AdCMVeNOS. n = 7. *, P < 0.05 when compared with eNOS^+/+; **, P < 0.05 when compared with eNOS^−/− + AdCMVβgal group.

Figure 2

Western blot analysis of eNOS protein in the lung of eNOS^+/+ mice (lane 1) and eNOS^−/− mice 1 day after intratracheal administration of vehicle (lane 2), transfection with AdCMVβgal (lane 3), or transfection with AdCMVeNOS (lane 4). eNOS protein levels are expressed as gel unit per mg protein. n = 6. *, P < 0.05 when compared with eNOS^+/+; **, P < 0.05 when compared with the eNOS^−/− + AdCMVβgal group.

Figure 3

Comparison of calcium-dependent (Left) and calcium-independent (Right) l-[H³]arginine conversion to l-[H³]citrulline in lung tissue from eNOS^+/+ mice and eNOS^−/− mice 1 day after administration of vehicle or transfection with AdCMVβgal or AdCMVeNOS. Values are pmol/mg protein/h citrulline formation. n = 6. *, P < 0.05 when compared with eNOS^+/+; **, P < 0.05 when compared with the eNOS^−/− + AdCMVβgal group.