Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries

Abstract

Nitric oxide (NO), synthesized from L-arginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or beta-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal fluid (CSF) via the cisterna magna (final viral titer in CSF, 10(9) pfu/ml). Adventitial transgene expression was demonstrated 24 h later by beta-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels (P < 0.05). The increased receptor-mediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood flow.

Figure 3

β-Gal levels in AdCMVLacZ- and AdCMVNOS-transduced cerebral arteries 24 h after gene transfer. (A) β-Gal levels in basilar and middle cerebral arteries of same dogs after in vivo gene transfer (final viral titer in CSF, 10⁹ pfu/ml). (B) β-Gal levels in basilar arteries of same dogs after ex vivo gene transfer (final viral titers in the incubation media, 10⁸, 10⁹, and 10¹⁰ pfu/ml). Data are expressed as means ± SEM (n = 4 from 4 dogs). ∗, P < 0.05, LacZ vs. eNOS (one-way factorial ANOVA).

Figure 1

Morphological demonstration of transgene expression on canine brain and cerebral arteries 24 h after intracisternal injection of viral vector (final viral titer in CSF, 10⁹ pfu/ml). (A) β-Gal staining on the dorsal surface of the brain. (B) β-Gal staining on the ventral surface of the same brain. Note increased β-gal staining in B compared with A. (C) Microscopic view (cross section) of β-gal staining in a basilar artery after being counterstained with nuclear fast red. (D) Microscopic view (cross section) of immunohistochemical staining of eNOS in a middle cerebral artery 24 h after in vivo eNOS gene transfer. (Bar = 0.1 mm.)

Figure 2

EM immunogold labeling showing recombinant eNOS cellular localization in adventitia fibroblast of a basilar artery. Gold particles (15 nm; also see Inset) indicating recombinant eNOS localized along plasma membranes and inside the cytoplasm. N, nucleus; M, mitochondria. (Bar = 1 μm.)

Figure 4

Western blot analysis demonstrating recombinant eNOS expression after in vivo gene transfer. Lanes: 1, homogenate of bovine aortic endothelial cells (positive control); 2 and 3, AdCMVLacZ-transduced basilar and middle cerebral arteries, respectively; 4 and 5, AdCMVNOS-transduced basilar and middle cerebral arteries, respectively. Similar results were seen in duplicate experiments of four dogs.

Figure 5

Effects of transgene expression on contractions to UTP, relaxations to diethylamine NONOate and bradykinin in middle cerebral arteries 24 h after in vivo gene transfer. Shown are concentration-response curves to (A) UTP with endothelium (P > 0.05); (B) diethylamine NONOate with endothelium (P > 0.05); (C) bradykinin with endothelium (P < 0.05); and (D) bradykinin without endothelium (P < 0.05) (two-way RM-ANOVA). Data are expressed as means ± SEM (n = 8 for A and D; n = 4 for B and C). Contractions are expressed in grams, and relaxations are expressed as percentage of the maximal relaxation to papaverine (3 × 10⁻⁴ M).

Figure 6

Basal cGMP production in AdCMVLacZ- and AdCMVNOS-transduced basilar arteries 24 h after gene transfer, and the effects of NOS inhibitor l-NMMA and calcium depletion. Data are expressed as means ± SEM (n = 7 from 7 dogs each). P < 0.05, eNOS vs. LacZ (one-way factorial ANOVA); eNOS vs. eNOS + l-NMMA (one-way RM-ANOVA); and eNOS vs. eNOS without calcium (one-way RM-ANOVA).