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Endogenous as well as exogenous atrial natriuretic peptide (ANP) attenuates the development of chronic hypoxic pulmonary hypertension (CHPH) in rats. We built a recombinant adenovirus type 5 containing ANP cDNA under the control of the Rous sarcoma virus long terminal repeat (Ad.ANP). The efficiency of this vector in delivering the ANP gene was first examined in rat primary cultures of pulmonary vessel smooth muscle cells (SMCs) in comparison with Ad.βGAL. Conditioned medium collected from Ad.ANP-infected cells (1000 TCID50/cell) contained 5 × 109M immunoreactive ANP and elicited relaxation of isolated rat pulmonary arteries preconstricted with phenylepinephrine. To examine the effects of adenovirus-mediated ANP expression in the CHPH rat lung, Ad.ANP or Ad.βGAL was administered via the tracheal route. Immunoreactive ANP was detected in bronchoalveolar fluid as early as 4 days and until 10-17 days after Ad.ANP administration (5 × 108 TCID50). Lung ANP immunostaining was mainly localized in bronchial and alveolar epithelial cells. As compared with Ad.βGAL-treated controls, rats given Ad.ANP (5 × 108 TCID50) on the day before a 2-week exposure to hypoxia (10% O2) had lower values for pulmonary artery pressure (32.1 ± 1.93 vs. 35.5 ± 2 mmHg, p < 0.01) and Fulton's index (0.52 ± 0.089 vs. 0.67 ± 0.12, p < 0.001) and less severe right ventricular hypertrophy and distal vessel muscularization. These results suggest that induction of ANP expression in the lung may hold promise in the treatment of pulmonary hypertension.

To investigate the respective role of transduced cells in the induction of immune response following intramuscular inoculation of adenovirus-based vaccines, we generated several replication-defective adenoviruses expressing the glycoprotein D gene of pseudorabies virus under the control of four different promoters: major late promoter of adenovirus type 2, human cytomegalovirus immediate-early promoter/enhancer (CMV), Rous sarcoma virus-long terminal repeat promoter, and human desmin gene 5′ regulatory region (DES). All the adenovirus constructs were able to fully protect mice, in the contrary of direct DNA inoculation of plasmids harboring the same transcription units. The far most effective adenovirus constructs, on the criterion of protective doses and specific antibody response induction, were those in which the foreign gene was driven by the DES or CMV promoter. Wide variations in promoter strengthin vitrowere evidenced in several cell culture types representative of putative target cells following muscular inoculation (myoblasts, myotubes, fibroblasts, macrophages, and endothelial cells). The level of efficacyin vivo,was not correlated with the level of expressionin vitroin myotubes, but paralleled the level of expression in endothelial cells and in myoblasts. Together with previously published data, these results suggest that, following adenovirus injection, locally produced cytokines may induce myoblasts to act as local antigen presenting cells.

Atrial natriuretic factor (ANF) is a potent natriuretic, diuretic, and vasoactive hormone produced and released by atrial cardiomyocytes. We investigated whether adenovirus-mediated ANF gene delivery to dogs leads to a sustained increase in circulating ANF levels resulting in long-lasting biological effects. An adenoviral vector containing the canine ANF cDNA under the control of the Rous sarcoma virus 3' long terminal repeat (AdRSV-ANF) was injected via the intrahepatic route to nonvaccinated 2-month-old dogs. In the first group of four dogs injected with AdRSV-ANF (10 10.2 TCID50), a short-lived increase in plasma ANF concen- trations not associated with biological effects occurred 8-10 days after the injection, as compared with four control dogs injected with an adenovirus encoding a luciferase reporter gene (AdRSV-luc). In a second series of experiments, six dogs received AdRSV-ANF at a dose of 10 10 TCID50 and a replication-defective type 5 adenovirus harboring a modified VAI gene (Ad-VAr) at the same dose. Sustained increases in plasma ANF concentrations and urinary cGMP excretion starting on day 2 and persisting until day 20 were seen, as well as concomitant elevations in natriuresis and diuresis, a transient increase in cardiac output, and a delay in body weight gain, as compared with control dogs injected with AdRSV-luc/Ad-VAr. These results show that adenovirus-mediated ANF gene expression can lead to systemic biological effects in dogs, a finding of potential relevance for the treatment of cardiovascular diseases and sodium-retaining disorders.