RESUMEN

BACKGROUND: Increased intrahepatic vascular tone in cirrhosis has been attributed to a decrease of hepatic nitric oxide (NO) secondary to disturbances in the post-translational regulation of the enzyme eNOS. NO scavenging by superoxide (O(2)(-)) further contributes to a reduction of NO bioavailability in cirrhotic livers. AIM: To investigate whether removing increased O(2)(-) levels could be a new therapeutic strategy to increase intrahepatic NO, improve endothelial dysfunction and reduce portal pressure in cirrhotic rats with portal hypertension. METHODS: Adenoviral vectors expressing extracellular superoxide dismutase (SOD) (AdECSOD) or beta-galactosidase (Adbetagal) were injected intravenously in control and CCl(4)-induced cirrhotic rats. After 3 days, liver O(2)(-) levels were determined by dihydroethidium staining, NO bioavailability by hepatic cGMP levels, nitrotyrosinated proteins by immunohistochemistry and western blot, and endothelial function by responses to acetylcholine in perfused rat livers. Mean arterial pressure (MAP) and portal pressure were evaluated in vivo. RESULTS: Transfection of cirrhotic livers with AdECSOD produced a significant reduction in O(2)(-) levels, a significant increase in hepatic cGMP, and a decrease in liver nitrotyrosinated proteins which were associated with a significant improvement in the endothelium-dependent vasodilatation to acetylcholine. In addition, in cirrhotic livers AdECSOD transfection produced a significant reduction in portal pressure (17.3 (SD 2) mm Hg vs 15 (SD 1.6) mm Hg; p<0.05) without significant changes in MAP. In control rats, AdECSOD transfection prevents the increase in portal perfusion pressure promoted by an ROS-generating system. CONCLUSIONS: In cirrhotic rats, reduction of O(2)(-) by AdECSOD increases NO bioavailability, improves intrahepatic endothelial function and reduces portal pressure. These findings suggest that scavenging of O(2)(-) might be a new therapeutic strategy in the management of portal hypertension.

Asunto(s)

Terapia Genética/métodos , Hipertensión Portal/terapia , Cirrosis Hepática Experimental/complicaciones , Presión Portal , Superóxido Dismutasa/genética , Adenoviridae/genética , Animales , Tetracloruro de Carbono , Endotelio Vascular/fisiopatología , Técnicas de Transferencia de Gen , Vectores Genéticos , Hipertensión Portal/etiología , Hipertensión Portal/metabolismo , Hipertensión Portal/fisiopatología , Circulación Hepática , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Masculino , Óxido Nítrico/metabolismo , Consumo de Oxígeno , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo

RESUMEN

This mini-review describes steps towards gene therapy to prevent vasospasm after subarachnoid hemorrhage, and summarizes some remaining obstacles. With recombinant adenoviruses, it is now possible to prevent vasospasm in experimental animals. If an adenoviral or other effective vector is demonstrated to be safe, it is likely that gene therapy will be used in patients to prevent vasospasm.

Asunto(s)

Transferencia de Gen Horizontal , Hemorragia Subaracnoidea/genética , Vasoespasmo Intracraneal/prevención & control , Regulación de la Expresión Génica , Humanos , Vasoespasmo Intracraneal/genética

RESUMEN

OBJECTIVE: Inducible nitric oxide synthase (iNOS) is expressed in arteries during inflammation and may contribute to vascular dysfunction. Effects of gene transfer of iNOS to carotid arteries were examined in vitro in the absence of systemic inflammation to allow examination of mechanisms by which iNOS impairs contraction and relaxation. METHODS AND RESULTS: After gene transfer of iNOS with an adenovirus (AdiNOS), constrictor responses to phenylephrine (PE) and U46619 were impaired. After AdiNOS, inhibition of soluble guanylate cyclase (sGC) with 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ) reduced the EC50 for PE from 4.33+/-0.78 micromol/L to 1.15+/-0.43 micromol/L (mean+/-SEM). These results imply that iNOS impairs contraction by activation of the NO/cGMP pathway. Relaxation to acetylcholine (ACh) also was impaired after AdiNOS. Sepiapterin (300 micromol/L), the precursor for tetrahydrobiopterin (BH4), improved relaxation to Ach. Because BH4 is an essential cofactor for production of NO by both iNOS and endothelial nitric oxide synthase (eNOS), these results suggest that iNOS may reduce production of NO by eNOS by limiting availability of BH4. Next, we examined effects of expression of iNOS in endothelium and adventitia. Selective expression of iNOS in endothelium, but not adventitia, impaired contraction to phenylephrine and relaxation to acetylcholine. CONCLUSIONS: We conclude that: (1) iNOS may impair contraction in part by activation of sGC; (2) iNOS impairs relaxation, at least in part, by limiting availability of BH4; and (3) expression of iNOS in endothelium may be a more important mediator of vascular dysfunction than expression of iNOS in adventitia.

Asunto(s)

Enfermedades de las Arterias Carótidas/fisiopatología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Vasculitis/fisiopatología , Adenoviridae/genética , Animales , Arterias Carótidas/fisiología , Enfermedades de las Arterias Carótidas/metabolismo , Endotelio Vascular/fisiología , Regulación Enzimológica de la Expresión Génica , Técnicas de Transferencia de Gen , Masculino , Conejos , Vasculitis/metabolismo , Vasoconstricción/fisiología , Vasodilatación/fisiología

RESUMEN

BACKGROUND AND PURPOSE: These studies evaluated whether gene transfer of inducible nitric oxide synthase (iNOS) is a sufficient stimulus to produce vascular dysfunction in cerebral arteries. METHODS: Intracranial (pial) arteries were dissected from human brain tissue obtained during elective surgery. Isolated human arteries were incubated in vitro with adenovirus containing iNOS (AdiNOS) or a nonexpressive transgene (control, AdBglII) (500 micro L, 3x10(9) plaque-forming units per milliliter), and vascular function was examined 24 hours later. In anesthetized rabbits, AdiNOS or AdBglII (300 microL 1x10(10)) was injected into the cisterna magna. Three days later, the basilar artery was removed, and reactivity was examined ex vivo. RESULTS: In submaximally precontracted vessels, we observed impairment of NO-dependent relaxation in human cerebral arteries after gene transfer of iNOS. Maximum relaxation to bradykinin (1 micromol/L, an endothelium-dependent agonist) was 77+/-11% (mean+/-SE) after AdBglII and 31+/-22% (P<0.05) after AdiNOS. After AdiNOS, responses to nitroprusside (an endothelium-independent NO donor) also were impaired. Responses to both nitroprusside and bradykinin were improved by aminoguanidine (300 micromol/L), an inhibitor of iNOS. AdiNOS produced no change in vasoconstrictor responses to U46619. In basilar arteries from rabbits examined in vitro after gene transfer in vivo, responses to histamine, serotonin, and nitroprusside all were similar after AdiNOS or AdBglII. In contrast, relaxation to acetylcholine was significantly depressed after AdiNOS. Maximum relaxation to acetylcholine (10 micromol/L) was 90+/-3% after AdBglII and 68+/-5% (P<0.05) after AdiNOS. Relaxation of arteries after AdiNOS was improved by aminoguanidine. CONCLUSIONS: These studies suggest that expression of iNOS may impair NO-dependent relaxation in both human and rabbit cerebral arteries.

Asunto(s)

Arterias Cerebrales/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Vasodilatación/efectos de los fármacos , Sistema Vasomotor/efectos de los fármacos , Animales , Bradiquinina/farmacología , Arterias Cerebrales/citología , Arterias Cerebrales/fisiología , Técnicas de Transferencia de Gen , Humanos , Inmunohistoquímica , Técnicas In Vitro , Masculino , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa/farmacología , Óxido Nítrico Sintasa de Tipo II , Conejos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Vasodilatación/fisiología , Vasodilatadores/farmacología , Sistema Vasomotor/fisiología

RESUMEN

Gene transfer may be appropriate for therapeutic protocols targeted at the vascular endothelium. Endothelial dysfunction is the principal phenotype associated with atherosclerosis and hypertension. Oxidative stress has been implicated in the development of endothelial dysfunction. We have explored the ability of overexpressing anti-oxidant genes (superoxide dismutases; SODs) in vitro and in vivo to assess their potential for reversing endothelial dysfunction in a rat model, the stroke-prone spontaneously hypertensive rat (SHRSP). Western blotting and immunofluorescence assays in vitro showed efficient overexpression of MnSOD and ECSOD with respect to localisation to the mitochondria and extracellular surface, respectively. Transgene functional activity was quantified with SOD activity assays. MnSOD and ECSOD overexpression in intact SHRSP vessels in vivo led to endothelial and adventitial overexpression. Pharmacological assessment of transduced vessels following in vivo delivery by basal NO availability quantification demonstrated that the "null" adenovirus and MnSOD adenovirus did not significantly increase NO availability. However, AdECSOD-treated carotid arteries showed a significant increase in NO availability (1.91 +/- 0.04 versus 0.75 +/- 0.08 g/g, n = 6, P = 0.029). In summary, efficient overexpression of ECSOD, but not MnSOD in vivo, results in improved endothelial function in a rat model of hypertension and has important implications for the development of endothelial-based vascular gene therapy.

Asunto(s)

Endotelio Vascular/fisiopatología , Depuradores de Radicales Libres/metabolismo , Terapia Genética/métodos , Hipertensión/terapia , Superóxido Dismutasa/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Células Cultivadas , Técnicas de Transferencia de Gen , Vectores Genéticos/uso terapéutico , Hipertensión/enzimología , Hipertensión/fisiopatología , Masculino , Ratas , Ratas Endogámicas SHR , Superóxido Dismutasa/genética

RESUMEN

Adenovirus-mediated gene transfer to blood vessels is relatively inefficient because binding of adenovirus to vessels is limited. The authors have reported that incorporation of cationic polymer and lipids with adenovirus augments gene transfer to blood vessels ex vivo. In this study, the authors determined whether complexes of adenovirus and cations improve efficiency of gene transfer in vivo. Poly-L-lysine, lipofectamine, or lipofectin was complexed with adenovirus encoding beta-galactosidase. Optimum ratios of the cations per adenovirus were determined by gene transfer to fibroblasts. After injection of the adenovirus into the cisterna magna of anesthetized rabbits, transgene activity was greater in the adventitia of intracranial arteries and meninges after injection of the complexes than adenovirus alone. Thirty minutes after application of adenovirus with the cations, binding of adenovirus to fibroblast cells in vitro or the basilar artery in vivo (by Southern blot analysis) was augmented, which suggests that enhanced binding of virus contributes to augmentation of transgene expression. Thus, cationic polymer and lipids improve transgene expression in intracranial arteries, primarily in the adventitia, after adenovirus-mediated gene transfer in vivo. This strategy may be applicable to studies of gene transfer and eventually for gene therapy.

Asunto(s)

Adenoviridae/genética , Arteria Basilar/fisiología , Técnicas de Transferencia de Gen , Glicerofosfolípidos/farmacocinética , Fosfatidiletanolaminas , Espermina/farmacocinética , Células 3T3 , Adenoviridae/metabolismo , Animales , Southern Blotting , Cisterna Magna , Expresión Génica , Masculino , Ratones , Conejos , Espermina/análogos & derivados , Transgenes/genética , beta-Galactosidasa/genética

RESUMEN

Proinflammatory stimuli produce expression of inducible NO-synthase (iNOS) within blood vessels and are associated with impaired endothelium-dependent relaxation. Gene transfer of iNOS was used to test the hypothesis that expression of iNOS in blood vessels produces impairment of NO-dependent relaxation as well as contraction. An adenoviral vector containing cDNA for murine iNOS, AdCMViNOS, and a control virus, AdCMVBglII, were used for gene transfer to rabbit carotid arteries in vitro and in vivo. After gene transfer of iNOS in vitro, contractile responses to KCl, phenylephrine, and U46619 were impaired. Relaxation in response to acetylcholine, ADP, A23187, and nitroprusside was also impaired. For example, maximum relaxation of vessels to acetylcholine (10 micromol/L) was 78+/-4% (mean+/-SE) after AdBglII (10(10.5) plaque-forming units) and 34+/-5% after AdiNOS (10(10.5) plaque-forming units, P<0.05). NO-independent relaxation in response to 8-bromo-cGMP and papaverine was not impaired after AdiNOS. Contraction and relaxation were improved in carotid arteries expressing iNOS by aminoguanidine and L-N-iminoethyl lysine, inhibitors of iNOS. After intraluminal gene transfer of iNOS in vivo, contraction of vessels in vitro was normal, but responses to acetylcholine were impaired. In summary, the major finding is that NO-dependent relaxation is impaired in arteries after gene transfer of iNOS in vitro and in vivo. Thus, expression of iNOS per se impairs NO-dependent relaxation.

Asunto(s)

Arterias Carótidas/fisiología , Técnicas de Transferencia de Gen , Óxido Nítrico Sintasa/genética , Óxido Nítrico/metabolismo , Vasodilatación/fisiología , Adenoviridae , Animales , Arterias Carótidas/enzimología , Arterias Carótidas/patología , ADN Complementario , Vectores Genéticos , Inmunohistoquímica , Técnicas In Vitro , Masculino , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Conejos , Superóxidos , Vasoconstrictores/farmacología , Vasodilatadores/farmacología

RESUMEN

Hyperhomocysteinemia is associated with increased risk for cardiovascular events, but it is not certain whether it is a mediator of vascular dysfunction or a marker for another risk factor. Homocysteine levels are regulated by folate bioavailability and also by the methyl donor S-adenosylmethionine (SAM) and its metabolite S-adenosylhomocysteine (SAH). We tested the hypotheses that endothelial dysfunction occurs in hyperhomocysteinemic mice in the absence of folate deficiency and that levels of SAM and SAH are altered in mice with dysfunction. Heterozygous cystathionine beta-synthase-deficient (CBS(+/-)) and wild-type (CBS(+/+)) mice were fed a folate-replete, methionine-enriched diet. Plasma levels of total homocysteine were elevated in CBS(+/-) mice compared with CBS(+/+) mice after 7 weeks (27.1+/-5.2 versus 8.8+/-1.1 micromol/L; P<0.001) and 15 weeks (23.9+/-3.0 versus 13.0+/-2.3 micromol/L; P<0.01). After 15 weeks, but not 7 weeks, relaxation of aortic rings to acetylcholine was selectively impaired by 35% (P<0.05) and thrombomodulin anticoagulant activity was decreased by 20% (P<0.05) in CBS(+/-) mice. Plasma levels of folate did not differ between groups. Levels of SAH were elevated approximately 2-fold in liver and brain of CBS(+/-) mice, and correlations were observed between plasma total homocysteine and SAH in liver (r=0.54; P<0.001) and brain (r=0.67; P<0.001). These results indicate that endothelial dysfunction occurs in hyperhomocysteinemic mice even in the absence of folate deficiency. Endothelial dysfunction in CBS(+/-) mice was associated with increased tissue levels of SAH, which suggests that altered SAM-dependent methylation may contribute to vascular dysfunction in hyperhomocysteinemia.

Asunto(s)

Cistationina betasintasa/deficiencia , Endotelio Vascular/fisiopatología , Hiperhomocisteinemia/fisiopatología , S-Adenosilhomocisteína/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiopatología , Encéfalo/metabolismo , Enfermedad Crónica , Cistationina betasintasa/genética , Modelos Animales de Enfermedad , Ácido Fólico/sangre , Alimentos Fortificados , Heterocigoto , Homocisteína/sangre , Hiperhomocisteinemia/sangre , Técnicas In Vitro , Hígado/metabolismo , Metionina/sangre , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , S-Adenosilmetionina/metabolismo , Trombomodulina/metabolismo , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Sistema Vasomotor/efectos de los fármacos , Sistema Vasomotor/fisiopatología

RESUMEN

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.

Asunto(s)

Ácido Araquidónico/farmacología , Arteria Basilar/fisiología , Flavanonas , Lipooxigenasa/metabolismo , Canales de Potasio/metabolismo , Vasodilatación/fisiología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animales , Arteria Basilar/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Indometacina/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacología , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Tetraetilamonio/farmacología , Tritio , Vasodilatación/efectos de los fármacos

RESUMEN

Gene transfer to blood vessels is remarkably effective in altering vasomotor function, and has proven to be a useful tool for studying vascular biology. Gene therapy for cardiovascular diseases is an attractive new approach, which will be used initially for diseases in which pharmacologic approaches are not effective. Preliminary data suggest that two possible targets for gene therapy are prevention of cerebral vasospasm after subarachnoid hemorrhage and treatment of pulmonary hypertension. Perhaps, as better vectors are developed, common clinical problems such as hypertension and hypercholesterolemia also may become targets for gene therapy.

Asunto(s)

Vasos Sanguíneos/fisiología , Técnicas de Transferencia de Gen , Animales , Terapia Genética , Humanos , Proyectos de Investigación , Sistema Vasomotor/fisiología

RESUMEN

BACKGROUND AND PURPOSE: The first goal of the present study was to examine the hypothesis that relaxation of cerebral arteries to nitric oxide in primates is dependent on activation of soluble guanylate cyclase (sGC). The second goal was to determine whether the role of sGC in mediating responses to nitric oxide is altered in atherosclerosis. METHODS: Basilar arteries from normal and atherosclerotic monkeys were studied in vitro. After precontraction with prostaglandin F(2alpha) (0.1 to 1 micromol/L), concentration-response curves to authentic nitric oxide (1 nmol/L to 1 micromol/L), sodium nitroprusside (10 nmol/L to 10 micromol/L; a nitric oxide donor), and papaverine (10 nmol/L to 10 micromol/L; a non-nitric oxide, non-sGC-dependent stimulus) were generated in the presence and absence of 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 and 10 micromol/L; an inhibitor of sGC). The effect of ODQ on basal tone of basilar arteries from normal and atherosclerotic monkeys was also examined. RESULTS: Nitric oxide, sodium nitroprusside, and papaverine produced relaxation that was similar (P:>0.05) in normal and atherosclerotic monkeys. ODQ produced marked inhibition (P:<0.05) of vasorelaxation in response to nitric oxide and nitroprusside but not papaverine. For example, relaxation of the basilar artery in response to nitric oxide (0.1 micromol/L) was inhibited by approximately 85% and 73% by ODQ (1 micromol/L) in normal and atherosclerotic monkeys, respectively. ODQ produced contraction of the basilar arteries, and the increase in tension to ODQ was greater in normal (2.7+/-0.3 g; mean+/-SE) than in atherosclerotic monkeys (1.4+/-0.4 g; P:<0.05). In contrast, contraction to prostaglandin F(2alpha) was similar in the basilar artery from normal and atherosclerotic monkeys. CONCLUSIONS: These findings suggest that (1) relaxation of cerebral arteries in primates in response to nitric oxide is normally dependent, in large part, on activation of sGC and (2) the influence of sGC (via reduced production and/or activity of basal nitric oxide) on cerebral vascular tone is reduced in atherosclerosis.

Asunto(s)

Arterias Cerebrales/metabolismo , Arteriosclerosis Intracraneal/metabolismo , Óxido Nítrico/metabolismo , Vasodilatación , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/metabolismo , Arteria Basilar/patología , Arteria Basilar/fisiopatología , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/patología , Arterias Cerebrales/fisiopatología , Colesterol/sangre , Dieta Aterogénica , Dinoprost/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/metabolismo , Técnicas In Vitro , Arteriosclerosis Intracraneal/fisiopatología , Macaca fascicularis , Masculino , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/antagonistas & inhibidores , Donantes de Óxido Nítrico/farmacología , Oxadiazoles/farmacología , Papaverina/farmacología , Quinoxalinas/farmacología , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología

RESUMEN

New diagnostic and treatment strategies are being developed for stroke. Gene therapy has several potential advantages over classical pharmacologic therapy. Direct administration of DNA into the brain offers the advantage of producing high concentrations of therapeutic agents in a relatively localized environment. Gene transfer also provides longer duration of effect than traditional drug therapy. Recent studies indicate that gene transfer can produce functional proteins in brain parenchyma and cerebral blood vessels after stroke. In animal models, gene transfer may reduce effects of cerebral ischemia or subarachnoid hemorrhage. This review summarizes some current methods of gene transfer to the brain and recent progress that may lead to gene therapy for stroke.

Asunto(s)

Terapia Genética/tendencias , Accidente Cerebrovascular/terapia , Predicción , Humanos

RESUMEN

BACKGROUND: Hyperhomocysteinemia is associated with increased risk of atherosclerotic and thrombotic vascular disease. In many patients, hyperhomocysteinemia can be treated or prevented by dietary supplementation with B vitamins, but the clinical benefit of B vitamins for the prevention of vascular disease has not been proven. METHODS AND RESULTS: Using an atherogenic diet that produces both hyperhomocysteinemia and hypercholesterolemia, we tested the hypothesis that dietary supplementation with B vitamins (folic acid, vitamin B(12), and vitamin B(6)) would prevent hyperhomocysteinemia, vascular dysfunction, and atherosclerotic lesions in monkeys. After 17 months, plasma total homocysteine increased from 3.6+/-0.3 to 11.8+/-1.7 micromol/L in monkeys fed an unsupplemented atherogenic diet (P<0.01) but did not increase in monkeys fed an atherogenic diet supplemented with B vitamins (3.8+/-0.3 micromol/L). Serum cholesterol increased from 122+/-7 to 550+/-59 mg/dL in the unsupplemented group (P<0.001) and from 118+/-5 to 492+/-55 mg/dL in the supplemented group (P<0.001). Responses to endothelium-dependent vasodilators, both in resistance vessels in vivo and in the carotid artery ex vivo, were impaired to a similar extent in groups that did and did not receive vitamin supplements. Anticoagulant responses to the infusion of thrombin were also impaired to a similar extent in both groups. Vitamin supplementation failed to prevent intimal thickening in the carotid or iliac arteries. CONCLUSIONS: These findings demonstrate that supplementation with B vitamins prevents hyperhomocysteinemia but is not sufficient to prevent the development of vascular dysfunction or atherosclerotic lesions in monkeys with marked hypercholesterolemia, even in the absence of preexisting atherosclerosis.

Asunto(s)

Dieta Aterogénica , Ácido Fólico/administración & dosificación , Piridoxina/administración & dosificación , Enfermedades Vasculares/prevención & control , Vitamina B 12/administración & dosificación , Animales , Arteriosclerosis/sangre , Arteriosclerosis/fisiopatología , Arteriosclerosis/prevención & control , Coagulación Sanguínea/efectos de los fármacos , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Colesterol/sangre , Suplementos Dietéticos , Modelos Animales de Enfermedad , Hipercolesterolemia/sangre , Hipercolesterolemia/complicaciones , Hipercolesterolemia/fisiopatología , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/complicaciones , Hiperhomocisteinemia/fisiopatología , Hiperhomocisteinemia/prevención & control , Técnicas In Vitro , Macaca fascicularis , Tiempo de Tromboplastina Parcial , Trombina/farmacología , Resultado del Tratamiento , Enfermedades Vasculares/sangre , Enfermedades Vasculares/etiología , Enfermedades Vasculares/fisiopatología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología

RESUMEN

BACKGROUND AND PURPOSE: Copper-zinc superoxide dismutase (CuZnSOD) is expressed intracellularly, while extracellular SOD (EC-SOD) is released from cells. The purpose of this study was to determine whether gene transfer of CuZnSOD increases SOD activity predominantly in tissues, and gene transfer of EC-SOD increases SOD activity in cerebrospinal fluid (CSF). We also determined whether heparin or dextran sulfate releases EC-SOD into CSF. METHODS: We injected recombinant adenoviruses expressing EC-SOD (AdEC-SOD), CuZnSOD (AdCuZnSOD), or beta-galactosidase (Adbeta-gal) into the cisterna magna of rabbits. RESULTS: Total SOD activity in CSF was 39+/-11 U/mL (mean+/-SE) before virus injection. Three days later, total SOD activity in CSF increased to 148+/-22 U/mL after AdEC-SOD and 92+/-10 U/mL after AdCuZnSOD (P:<0.05 versus AdEC-SOD), with no change after Adbeta-gal (49+/-5 U/mL). EC-SOD protein was detected in CSF after AdEC-SOD but not AdCuZnSOD or Adbeta-gal. Injection of heparin or dextran sulfate into the cisterna magna increased total SOD activity 27-fold and 32-fold over basal values, respectively, in CSF of rabbits that received AdEC-SOD. In contrast to effects in CSF, total SOD activity in basilar artery and meninges was significantly higher after AdCuZnSOD and tended to be higher after AdEC-SOD than after Adbeta-gal. CONCLUSIONS: -We have developed a method for intracranial gene transfer of CuZnSOD and EC-SOD. After gene transfer, CuZnSOD was expressed mainly in tissues, and EC-SOD was released into the CSF, especially after injection of heparin or dextran sulfate. Gene transfer of different isoforms of SOD may be useful in studies of cerebral vascular physiology and pathophysiology.

Asunto(s)

Líquido Cefalorraquídeo/enzimología , Técnicas de Transferencia de Gen , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Adenoviridae/genética , Animales , Arteria Basilar/química , Arteria Basilar/enzimología , Arteria Basilar/metabolismo , Western Blotting , Cisterna Magna , Sulfato de Dextran/administración & dosificación , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Genes Reporteros , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Heparina/administración & dosificación , Inyecciones Intravenosas , Inyecciones Intraventriculares , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Meninges/química , Meninges/enzimología , Meninges/metabolismo , Conejos , beta-Galactosidasa/genética

RESUMEN

Brain infarction is one of the most important age-associated diseases. We have developed aged animal models for brain ischemia, and found the age-related neuronal vulnerability to brain ischemia. Investigation of that mechanism would lead to the effective treatment of brain infarction in the elder population. Recent advancement of gene transfer technique has provided strong tools for the neuronal and vascular biology. We described our recent approaches of gene transfer to blood vessels, including cerebral circulation, using adenoviral vectors. Cerebral blood vessels, atherosclerotic endothelium, and ischemic brain tissue are good targets of gene transfer. Development of these techniques would offer new therapeutic strategies for the age-related neuronal vulnerability and other age-associated diseases.

RESUMEN

The first part of this paper focuses on unusual aspects of the cerebral circulation. Cerebral vessels have less smooth muscle and adventitia than other vessels, and the endothelial blood-brain barrier is unique. Because the wall of the arteries is thin, one might expect that the vessels are especially vulnerable to rupture. Pressure in intracranial arteries, however, is lower than in other arteries, because resistance of larger cerebral arteries is remarkably high. The low pressure in cerebral arteries presumably protects against rupture of the vessels. The second part of the paper summarizes some new insights into regulation of cerebral circulation. One concept is that "breakthrough" of autoregulation, with dilatation of cerebral vessels at high levels of pressure, is an active process, rather than a passive phenomenon. This conclusion is based on the finding that inhibitors of calcium-dependent potassium channels greatly attenuate the cerebral vasodilator response during acute hypertension. The third part of the paper focuses on effects of gene transfer to cerebral blood vessels. Gene transfer to intracranial and extracranial vessels is feasible and vasomotor function can be altered. Gene transfer has proven to be useful to study vascular biology, and we are optimistic that the approach will ultimately lead to gene therapy.

Asunto(s)

Distinciones y Premios , Circulación Cerebrovascular/fisiología , Trastornos Cerebrovasculares/fisiopatología , Animales , Trastornos Cerebrovasculares/historia , Trastornos Cerebrovasculares/terapia , Terapia Genética , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Microcirculación , Estados Unidos

RESUMEN

We sought to determine whether adenovirus-mediated gene transfer in vivo of calcitonin gene-related peptide (CGRP), a potent vasodilator, ameliorates cerebral vasoconstriction after experimental subarachnoid hemorrhage (SAH). Arterial blood was injected into the cisterna magna of rabbits to mimic SAH 5 days after injection of AdRSVCGRP (8x10(8) pfu), AdRSVbetagal (control virus), or vehicle. After injection of AdRSVCGRP, there was a 400-fold increase in CGRP in cerebrospinal fluid. Contraction of the basilar artery to serotonin in vitro was greater in rabbits after SAH than after injection of artificial cerebrospinal fluid (P<0.001). Contraction to serotonin was less in rabbits with SAH after AdRSVCGRP than after AdRSVbetagal or vehicle (P:<0.02). Basal diameter of the basilar artery before SAH (measured with digital subtraction angiogram) was 13% greater in rabbits treated with AdRSVCGRP than in rabbits treated with vehicle or AdRSVbetagal (P:<0.005). In rabbits treated with vehicle or AdRSVbetagal, arterial diameter after SAH was 25+/-3% smaller than before SAH (P<0.0005). In rabbits treated with AdRSVCGRP, arterial diameter was similar before and after SAH and was reduced by 19+/-3% (P<0.01) after intracisternal injection of CGRP-(8-37) (0.5 nmol/kg), a CGRP(1) receptor antagonist. To determine whether gene transfer of CGRP after SAH may prevent cerebral vasoconstriction, we constructed a virus with a cytomegalovirus (CMV) promoter, which results in rapid expression of the transgene product. Treatment of rabbits with AdCMVCGRP after experimental SAH prevented constriction of the basilar artery 2 days after SAH. Thus, gene transfer of CGRP prevents cerebral vasoconstriction in vivo after experimental SAH.

Asunto(s)

Arteria Basilar/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/uso terapéutico , Terapia Genética/métodos , Hemorragia Subaracnoidea/terapia , Vasodilatadores/uso terapéutico , Adenoviridae/genética , Angiografía , Animales , Arteria Basilar/patología , Péptido Relacionado con Gen de Calcitonina/líquido cefalorraquídeo , Péptido Relacionado con Gen de Calcitonina/genética , Técnicas de Transferencia de Gen , Histamina , Inyecciones Intraventriculares , Conejos , Serotonina , Hemorragia Subaracnoidea/líquido cefalorraquídeo , Factores de Tiempo , Vasoespasmo Intracraneal/inducido químicamente , Vasoespasmo Intracraneal/prevención & control

RESUMEN

We used mice deficient in expression of inducible NO synthase (iNOS -/-) to directly examine the role of iNOS in impaired vasoconstrictor responses following tumor necrosis factor-alpha (TNF-alpha). In iNOS +/+ mice, contraction of carotid arteries in response to prostaglandin F(2alpha) (PGF(2alpha)) was impaired following TNF-alpha (100 microg/kg ip)(n = 10, P < 0.01). In contrast to responses in wild-type mice, contraction to low concentrations of PGF(2alpha) were normal, but maximum contraction to PGF(2alpha) was impaired in arteries from iNOS -/- mice treated with TNF-alpha [0.35 +/-.0.02 g (n = 8) following vehicle and 0.25 +/- 0.02 g (n = 7) following TNF-alpha (P < 0.05)]. Aminoguanidine, a relatively selective inhibitor of iNOS, partially restored contraction to PGF(2alpha) in vessels from iNOS +/+ mice but had no effect in iNOS -/- mice injected with TNF-alpha, suggesting that a mechanism(s) other than iNOS contributes to impaired responses. In contrast to contractile responses, relaxation of the carotid artery in response to acetylcholine and nitroprusside was not altered following TNF-alpha in iNOS +/+ or iNOS -/-mice. Responses of carotid arteries from iNOS -/- mice and effects of aminoguanidine suggest that both iNOS-dependent and iNOS-independent mechanisms contribute to impaired contractile responses following TNF-alpha.

Asunto(s)

Arterias Carótidas/fisiología , Contracción Muscular , Relajación Muscular , Óxido Nítrico Sintasa/deficiencia , Factor de Necrosis Tumoral alfa/farmacología , Acetilcolina/farmacología , Animales , Arterias Carótidas/enzimología , Dinoprost/farmacología , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Cloruro de Potasio/farmacología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Little is known about the role of interleukin-10 (IL-10), an anti-inflammatory cytokine, in blood vessels. We used IL-10-deficient mice (IL-10 -/-) to examine the hypothesis that IL-10 protects endothelial function after lipopolysaccharide (LPS) treatment. The responses of carotid arteries were studied in vitro 6 h after injection of a relatively low dose of LPS (10 microgram ip). In IL-10 -/- mice, the maximum relaxation to ACh (3 microM) was 56 +/- 6% (means +/- SE) after LPS injection and 84 +/- 4% after vehicle injection (P < 0.05). Thus endothelium-dependent relaxation was impaired in carotid arteries from IL-10 -/- mice after LPS injection. In contrast, this dose of LPS did not alter relaxation to ACh in vessels from wild-type (IL-10 +/+) mice. Relaxation to nitroprusside and papaverine was similar in arteries from both IL-10 -/- and IL-10 +/+ mice after vehicle or LPS injection. Because inflammation is associated with increased levels of reactive oxygen species, we also tested the hypothesis that superoxide contributes to the impairment of endothelial function by LPS in the absence of IL-10. Results using confocal microscopy and hydroethidine indicated that levels of superoxide are elevated in carotid arteries from IL-10 -/- mice compared with IL-10 +/+ mice after LPS injection. The impaired relaxation of arteries from IL-10 -/- mice after LPS injection was restored to normal by polyethylene glycol-suspended superoxide dismutase (50 U/ml) or allopurinol (1 mM), an inhibitor of xanthine oxidase. These data provide direct evidence that IL-10 protects endothelial function after an acute inflammatory stimulus by limiting local increases in superoxide. The source of superoxide in this model may be xanthine oxidase.

Asunto(s)

Enfermedades de las Arterias Carótidas/fisiopatología , Endotelio Vascular/fisiopatología , Interleucina-10/deficiencia , Superóxidos/metabolismo , Vasculitis/fisiopatología , Animales , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Colorantes Fluorescentes , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Fenantridinas , Valores de Referencia , Vasculitis/metabolismo , Vasculitis/patología

RESUMEN

Hypertension and vascular injury usually require prolonged treatment, and compliance is a key to efficacy for pharmacologically-based antihypertensive therapy. Gene therapy has the potential to be long lasting, with few side effects. Recent studies have provided promising results, in which hypertension can be treated by either augmentation of vasodilation or inhibition of vasoconstriction through gene transfer in experimental models. Gene transfer is also becoming useful for the study of mechanisms of physiologic and pathophysiologic conditions, including hypertension. In this mini-review, we summarize some recent studies in this area of research, and suggest some areas where progress is needed to advance the research toward gene therapy.

Asunto(s)

Terapia Genética , Hipertensión/terapia , Técnicas de Transferencia de Gen , Humanos , Hipertensión/patología , Vasoconstricción/efectos de los fármacos , Vasodilatadores/uso terapéutico , Sistema Vasomotor/efectos de los fármacos , Sistema Vasomotor/fisiología