Gene transfer to blood vessels in vivo generally requires interruption of blood flow. Thus, gene transduction to cerebral blood vessels in vivo has not yet been achieved. In this study, we injected replication-deficient adenovirus into cerebrospinal fluid in an attempt to transduce genes to cerebral blood vessels. Recombinant adenovirus (1 x 10(9) infectious units) expressing nuclear-targeted bacterial beta-galactosidase driven by the cytomegalovirus promoter was injected into the cisterna magna of Sprague-Dawley rats. The brains were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 1 to 7 days after injection of adenovirus. Leptomeningeal cells overlying the major arteries were efficiently transduced, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. beta-Galactosidase was expressed on days 1 and 3 after injection but was undetectable by day 7. Expression of the gene was 'targeted' by altering the position of the head. When viral suspension was injected while the rat was in a nose-down position, the reporter gene was expressed extensively on the ventral surface of the brain, especially along the circle of Willis. When the position was changed to the nose-up or lateral position, the inferior or lateral region of the brain was stained primarily. Administration of the virus into the lateral ventricle provided extensive expression in ependymal cells and leptomeninges with some transduction to cerebral blood vessels. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, cisternal delivery may target specific brain regions by positioning of the head.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene transfer with replication-deficient adenovirus is a potentially useful tool to study vascular biology. We have constructed a replication-deficient adenovirus (AdRSVeNOS) that carries cDNA for endothelial nitric oxide synthase (eNOS). Transfection of COS-1 cells with AdRSVeNOS increased nitric oxide synthase activity (measured as production of L-citrulline from L-arginine) that was calcium dependent and inhibited by N omega-nitro-L-arginine methyl ester. To investigate effects of overexpression of eNOS on vascular function, we incubated common carotid arteries from rabbits in organ culture with AdRSVeNOS or AdRSV beta gal encoding beta-galactosidase. Transgene expression and responses to vasoactive agents were examined 1 day after transduction. Histochemical staining of beta-galactosidase and immunohistochemistry for eNOS indicated transgene expression in endothelium and adventitial cells. After precontraction with phenylephrine, vessels treated with AdRSVeNOS demonstrated greater relaxation to acetylcholine than vessels treated with vehicle or AdRSV beta gal. Relaxation to calcium ionophore A-23187 was much greater in vessels treated with AdRSVeNOS than in vessels treated with vehicle or AdRSV beta gal. Augmented relaxation in response to A-23187 was also observed after denudation of endothelium in vessels treated with AdRSVeNOS and was inhibited by N omega-nitro-L-arginine. Thus vasorelaxation in response to stimuli that release nitric oxide is augmented after adenovirus-mediated overexpression of eNOS. Transgene expression in adventitial cells appears to be sufficient to alter vasomotor function.

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Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O2.-) and oxidize LDL in vitro; however, the role of O2.- in endothelial cell-induced LDL oxidation is unclear. Incubation of human LDL (200 microg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5+/-1.1 versus 6.3+/-0.2 [mean+/-SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 microg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O2.- release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O2.- contributes to endothelial cell-induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.