Citations

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 × 10−7 M), endothelium-dependent relaxations to A-23187 (10−9–10−6 M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10−8–10−5 M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10−9–10−5 M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N G-nitro-l-arginine methyl ester (l-NAME; 3 × 10−4 M), and a cyclooxygenase inhibitor, indomethacin (10−5 M), partially reduced A-23187-induced relaxation. However, in rings with valves,l-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1 H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3×10−6 M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37°C) to an adenoviral vector [107, 108, or 109plaque-forming units (pfu)/ml] encoding either the iNOS gene or the β-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (109pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10−9–10−3M) and diethylaminodiazen-1-ium-1,2-dioate (10−10–10−5M) were significantly shifted to the right in iNOS gene (109pfu/ml)-transduced rings compared with control and β-galactosidase-transduced rings ( P < 0.05, n = 5–6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings ( P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O[Formula: see text]·) production were elevated in iNOS-transduced rings ( P < 0.05, n = 7 for cGMP; P < 0.01, n = 6–9 for O[Formula: see text]· production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.

The current study was designed to determine the effect of recombinant heme oxygenase-1 (HO-1) gene expression on endothelial function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector (10 10 PFU/mL, total volume 300 μL) encoding either the HO-1 gene (AdCMVHO-1) or the β-galactosidase (β-Gal) reporter gene (AdCMVβ-Gal). Twenty-four hours after transduction, arterial rings were suspended in organ chamber for isometric force recording. Endothelium-dependent relaxations were obtained in response to bradykinin (10 −10 to 10 −6 mol/L) during contraction to uridine-5′-triphosphate (UTP; 3 × 10 −6 to 3 × 10 −5 mol/L). Certain rings were incubated with oxyhemoglobin (OxyHb; 10 −5 mol/L) overnight (16 to 18 hours of 24 hours). Expression and localization of recombinant protein were shown by Western blot analysis and immunohistochemistry. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to forskolin (10 −9 to 10 −5 mol/L) and DEA-NONOate (10 −10 to 10 −5 mol/L) were identical in β-Gal– and HO-1–transduced arteries. Exposure to OxyHb caused impairment of endothelium-dependent relaxation to bradykinin ( P < 0.01). In contrast, OxyHb did not affect endothelium-dependent relaxation in arteries expressing recombinant HO-1 ( P > 0.05). This protective effect of HO-1 was reversed by coincubation with tin protoporphyrin (SnPP9; 10 −5 mol/L), a selective inhibitor of HO-1 ( P < 0.01). Basal levels of 3′,5′-cyclic monophosphate (cGMP) in HO-1–transduced vessels were not significantly different from those in β-Gal–transduced vessels. Pretreatment with OxyHb significantly reduced cGMP level in β-Gal–transduced rings ( P < 0.01), whereas it had no effect in HO-1–transduced rings. These results demonstrate that HO-1 gene transfer does not affect endothelial and smooth muscle function of normal arteries, and that expression of recombinant HO-1 in cerebral arteries protects vasomotor function against OxyHb-induced injury.

Heat shock protein 90 (HSP90), an essential component of several signal transduction systems, participates in the activation of endothelial nitric oxide synthase (eNOS) in cells. The objective of the current study was to determine if HSP90 and eNOS were functionally interdependent and colocalized in the cerebral circulation. The authors used isometric force recording, cyclic 3′5′-guanosine monophosphate (cGMP) radioimmunoassay (RIA), and immunogold electron microscopy (EM) to study canine basilar artery. They found that geldanamycin (0.1 to 10 μg/mL), a selective HSP90 inhibitor, caused concentration-dependent contractions in arterial rings (n = 6 dogs). Contractions to geldanamycin were unaffected by a cyclooxygenase inhibitor, indomethacin (10 μmol/L; P < 0.05, n = 6). Functional evidence for interaction between HSP90 and nitric oxide (NO)-mediated signaling included observations that the contractile effect of geldanamycin was the following: (1) endothelium-dependent, (2) abolished by N g -nitro-l-arginine methylester (L-NAME; 0.3 mmol/L), and (3) nonadditive with the contractile effect of this NOS inhibitor ( P < 0.01, n = 6 for each). Furthermore, RIA showed significant reduction in cGMP levels in arteries treated with geldanamycin (3 μg/mL; P < 0.02, n = 8), whereas immunogold EM demonstrated areas of colocalization of HSP90 and eNOS selectively in the cytoplasm of endothelial cells. The current findings suggest that in cerebral arteries, endothelial HSP90 plays an important role in modulation of basal NO-mediated signaling. This interaction may be particularly important in stress-induced up-regulation of HSP90 with subsequent alteration of vasomotor function.

Background and Purpose — Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179DeNOS) increases nitric oxide (NO) production constitutively in the absence of agonist stimulus. The present study was designed to determine the effect of mutant S1179DeNOS gene expression on vasomotor function of canine cerebral arteries. Methods — Isolated basilar and middle cerebral arteries were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector (10 10 plaque-forming units per milliliter) encoding the S1179DeNOS gene (AdCMVS1179DeNOS), the wild-type eNOS gene (AdCMVeNOS), or the green fluorescent protein (GFP) reporter gene (AdCMVGFP). Twenty-four hours after transduction, arteries were suspended in an organ chamber for isometric force recording, and levels of cGMP were measured by radioimmunoassay. Results — Transgene protein expression was detected mainly in the vascular adventitia. In AdCMVS1179DeNOS-transduced arteries, basal levels of cGMP were significantly elevated compared with those in control (nontransduced), AdCMVGFP-, or AdCMVeNOS-transduced vessels (n=8; P <0.01). The elevation of cGMP was abolished by a NOS inhibitor, N G -nitro- l -arginine methyl ester (L-NAME), or by incubation in the calcium-free medium in the presence of calcium chelators. In AdCMVS1179DeNOS-transduced arteries, contractions to endothelin-1 (10 −10 to 10 −8 mol/L) were significantly reduced compared with those in control and AdCMVGFP-transduced arteries (n=7; P <0.05). The vasoconstrictor effect of endothelin-1 was restored in the presence of the NOS inhibitor L-NAME. Conclusions — Our results suggest that in cerebral arteries, expression of recombinant S1179DeNOS increases basal production of NO and inhibits the vasoconstrictor effect of endothelin-1. This effect may have therapeutic application in prevention and treatment of cerebrovascular diseases.

Background and Purpose — Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model. Methods — Recombinant adenoviral vectors (5×10 9 plaque-forming units/animal) encoding genes for eNOS (AdeNOS) and β-galactosidase (AdLacZ) or vehicle were injected into the cerebrospinal fluid (CSF) of dogs on day −1 (ie, 24 hours before the first intra-CSF injection of blood on day 0). Cerebral angiography was performed at day 0 (baseline) and day 7 (immediately before death), and tissues were harvested for additional studies. Results — Western analysis and immunohistochemistry detected recombinant eNOS exclusively in cerebral arteries isolated from AdeNOS-transduced dogs, and in this group of animals CSF NO concentrations were significantly elevated by day 2. Analysis of day 7 versus day 0 cerebral angiograms for each group revealed significant spasm at the basilar artery midpoint in AdLacZ-transduced and nontransduced dogs but not in AdeNOS-transduced dogs. Isometric force recording of basilar arteries isolated from AdeNOS-transduced dogs showed significantly augmented relaxations to bradykinin and reduced contractions to endothelin-1. Conclusions — Our results suggest that expression of recombinant eNOS in the adventitia of cerebral arteries may contribute toward protection against post-SAH vasospasm.