Cell-based gene transfer to the pulmonary vasculature - Endothelial nitricoxide synthase overexpression inhibits monocrotaline-induced pulmonary hypertension

To circumvent the problems of in vivo transfection and avoid the use of vir al vectors or proteins, we sought to establish whether smooth-muscle cells (SMCs) transfected ex vivo could be delivered via the systemic venous circu lation into the pulmonary bed to achieve local transgene expression in the lung. Primary cultures of pulmonary artery SMCs from Fisher 344 rats were l abeled with a fluorescent, membrane-impermeable dye chloromethyl trimethyl rhodamine or transfected with the beta-galactosidase (beta Gal) reporter ge ne under the control of the cytomegalovirus (CMV) enhancer/promoter (pCMV-b eta). Transfected or labeled SMCs (5 x 10(5) cells/animal) were delivered t o syngeneic recipient rats by injection into the jugular vein; the animals were killed at intervals between 15 min and 2 wk; and the lungs, spleens, k idneys, and skeletal muscle were excised and examined. At 15 min after tran splantation, injected cells were detected mainly in the lumen of small pulm onary arteries and arterioles, often in groups of three or more cells. Afte r 24 h, labeled SMCs were found incorporated into the vascular wall of pulm onary arterioles, and transgene expression persisted in situ for 14 d with no evidence of immune response. Using simple geometric assumptions, it was calculated that approximately 57 +/- 5% of the labeled cells reintroduced i nto the venous circulation could be identified in the lungs after 15 min, 3 4 +/- 7% at 48 h, 16 +/- 3% at 1 wk, and 15 +/- 5% at 2 wk. Similiar result s were observed using cells transfected with the reporter gene beta Gal. To determine whether this method of gene transfer could prove effective in in hibiting the development of pulmonary vascular disease, pulmonary artery SM Cs were transfected with either the full-length coding sequence of endothel ial nitric oxide synthase (NOS) under the control of the CMV enhancer/promo ter or with the control vector (pcDNA3.1) and injected simultaneously with the pulmonary endothelial toxin monocrotaline. At 28 d after injection the right ventricular systolic pressure was significantly decreased from 50 +/- 4 mm Hg in animals injected with the null-transfected cells to 33 +/- 3 mm Hg in animals injected with the NOS-transfected cells (P < 0.01). These re sults suggest that a cell-based strategy of ex vivo transfection may provid e an effective nonviral approach for the selective delivery of foreign tran sgenes to pulmonary microvessels in the treatment of pulmonary vascular dis ease.