Peptide fraction from B. jararaca snake venom protects against oxidative stress-induced changes in neuronal PC12 cell but not in astrocyte-like C6 cell.

Venom-derived proteins and peptides have prevented neuronal cell loss, damage, and death in the study of neurodegenerative disorders. The cytoprotective effects of the peptide fraction (PF) from Bothrops jararaca snake venom were evaluated against oxidative stress changes in neuronal PC12 cells and astrocyte-like C6 cells. PC12 and C6 cells were pre-treated for 4 h with different concentrations of PF, and then H2O2 was added (0.5 mM in PC12 cells; 0.4 mM in C6 cells) and incubated for 20 h more. In PC12 cells, PF at 0.78 µg mL-1 increased viability (113.6 ± 6.3%) and metabolism (96.3 ± 10.3%) cell against H2O2-induced neurotoxicity (75.6 ± 5.8%; 66.5 ± 3.3%, respectively), reducing oxidative stress markers such as ROS generation, NO production, and arginase indirect activity through urea synthesis. Despite that, PF showed no cytoprotective effects in C6 cells, but potentiated the H2O2-induced damage at a concentration lower than 0.07 µg mL-1. Furthermore, the role of metabolites derived from L-arginine metabolism was verified in PF-mediated neuroprotection in PC12 cells, using specific inhibitors of two of the key enzymes in the L-arginine metabolic pathway: the α-Methyl-DL-aspartic acid (MDLA) to argininosuccinate synthetase (AsS), responsible for the recycling of L-citrulline to L-arginine; and, L-NΩ-Nitroarginine methyl ester (L-Name) to nitric oxide synthase (NOS), which catalyzes the synthesis of NO from L-arginine. The inhibition of AsS and NOS suppressed PF-mediated cytoprotection against oxidative stress, indicating that its mechanism is dependent on the production pathway of L-arginine metabolites such as NO and, more importantly, polyamines from ornithine metabolism, which are involved in the neuroprotection mechanism described in the literature. Overall, this work provides novel opportunities for evaluating whether the neuroprotective properties of PF shown in particular neuronal cells are sustained and for exploring potential drug development pathways for the treatment of neurodegenerative diseases.

Renal protective effects of vicenin-2 and scolymoside in a mouse model of sepsis

This study was initiated to determine whether 2 structurally related flavonoids found in Cyclopia subternata-vicenin-2 (VCN) and scolymoside (SCL)-could modulate renal functional damage in a mouse model of sepsis, and to elucidate the relevant underlying mechanisms. The potential of VCN and SCL treatment to reduce renal damage induced by cecal ligation and puncture (CLP) surgery in mice was measured via assessment of serum creatinine, blood urea nitrogen (BUN), lipid peroxidation, total glutathione, glutathione peroxidase activity, catalase activity, and superoxide dismutase activity. Treatment with either VCN or SCL resulted in elevated plasma levels of BUN and creatinine, and of protein in the urine of mice with CLP-induced renal damage. Moreover, both VCN and SCL inhibited nuclear factor κB activation and reduced the induction of nitric oxide synthase and excessive production of nitric acid. VCN and SCL treatment also reduced the plasma levels of interleukin-6, and tumor necrosis factor-α, reduced lethality due to CLP-induced sepsis, increased lipid peroxidation, and markedly enhanced the antioxidant defense system by restoring the levels of superoxide dismutase, glutathione peroxidase, and catalase in kidney tissues. The present results suggest that VCN and SCL protect mice from sepsis-triggered renal injury

Efeito da talidomida sobre o processo inflamatório e desmielinização na encefalomielite auto-imune experimental: estudo histopatológico e expressão de TNF-α e iNOS / Effect of thalidomide on the inflammatory process and demyelination in experimental autoimmune encephalomyelitis: histopathologic study and expression of TNF-α e iN

A encefalomielite autoimune experimental (EAE) é uma doença inflamatória e desmielinizante do sistema nervoso central (SNC) caracterizada por incapacidades temporárias ou permanentes. A patogênese envolve a reação auto-imune associada com a produção de citocinas pró inflamatórias, tais como o fator de necrose tumoral alfa (TNF-α). Esta citocina está associada com o aumento de radicais livres de oxigênio, como o óxido nítrico, liberados pelas células imunes ativadas. Além de aumentar a inflamação, tanto o fator de necrose tumoral, como o óxido nítrico causam lesão tecidual direta. Este estudo avaliou o efeito da talidomida na progressão clínica da doença, desenvolvimento da reação inflamatória e desmielinização. A expressão tecidual "in situ" do TNF-α e iNOS, uma enzima associada com a produção de óxido nítrico, foi investigada em amostras do SNC obtidos durante o desenvolvimento do modelo de EAE em ratos Lewis. Métodos: Ratos Lewis(n = 30) foram divididos em grupo de controle saudável (I), grupo experimental de encefalomielite autoimune (II) e o grupo tratado com talidomida (III). Os ratos foram monitorizados durante 15 dias para determinação da condição clínica, após este período, os animais foram eutanasiados e as amostras do sistema nervoso central foram obtidas para a realização de estudo histopatológico e imuno-histoquímico Resultados: Todos os animais do grupo II tiveram sintomas relacionados a EAE, enquanto apenas um do grupo tratado talidomida apresentaram alterações clínicas. O estudo histopatológico revelou que as amostras de SNC do grupo II apresentaram áreas de intenso infiltrado inflamatório mononuclear difuso e presença de áreas de desmielinização. No entanto, os animais tratados com talidomida apresentaram ocasionalmente um leve infiltrado inflamatório e bainhas de mielina bem organizadas. Além disso, a expressão de TNF-α e iNOS foram significativamente maiores no grupo II, quando comparado com o grupo tratado com a talidomida. Conclusões: Os resultados considerados em conjunto sustentam a hipótese de que a talidomida inibe a intensidade do processo inflamatório e desmielinização, assim como reduz a produção de mediadores inflamatórios modulando o desenvolvimento da encefalomielite auto-imune experimental em ratos Lewis.

Experimental autoimmune encephalomyelitis is a inflammatory and demyelinating disease of central nervous system (CNS) characterized by permanents or temporary disabilities. Its pathogenesis involves autoimmune reaction associated with the production of pro inflammatory cytokines such as tumor necrosis factor alpha (TNF-α). This cytokine is associated with increase of reactive oxygen free radicals, such as nitric oxide, released by activated immune cells. Besides enhancing inflammation, both tumor necrosis factor as nitric oxide cause pathologically direct destruction of proteins and enzyme oxidation. This study focuses on clinical disease progression, development of the inflammatory reaction and evaluation axonal myelination . The " in situ" tissue expression of the TNF-α and inducible nitric oxide synthase iNOS ,an enzyme associated with the production of nitric oxide , were also investigated in CNS samples obtained during the development of experimental autoimmune encephalomyelitis model in Lewis rats. Methods: Lewis rats were used to perform the classical model of EAE. The rats ( n=30) were divided into the healthy control group (I), experimental autoimmune encephalomyelitis group (II) and thalidomide treated group (III). The rats were monitored for 15 days for determination of clinical score , after this period , the animals were euthanized and samples were obtained from the central nervous system in which histopatological study and immunohistochemistry for SNC in situ detection of TNF-α and inducible nitric oxide synthase (iNOS) were performed. Results: All animals of group II had symptoms related to experimental encephalomyelitis , while only one of the thalidomide treated group showed clinical changes. The histopatological study revealead that SNC samples of group II presented areas of intense focal and diffuse mononuclear inflammation and the myelin sheaths were scarce and poorly stained. However, thalidomide treated rats presented occasionally a mild perivascular inflammatory infiltrate and myelin sheaths were organized and well evidenced. In addition, the expression of TNF-α and iNOS were significantly higher in the group II when compared with thalidomide treated group. Conclusions: The results taken together support the hypothesis that thalidomide inhibits the intensity of the inflammation and demyelination process and as well as reduces the production of inflammatory mediators influencing the development of experimental autoimmune encephalomyelitis in Lewis rats

Contrasting effects of nitric oxide and corticotropin- releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice

The anxiogenic and antinociceptive effects produced by glutamate N-methyl-D-aspartate receptor activation within the dorsal periaqueductal gray (dPAG) matter have been related to nitric oxide (NO) production, since injection of NO synthase (NOS) inhibitors reverses these effects. dPAG corticotropin-releasing factor receptor (CRFr) activation also induces anxiety-like behavior and antinociception, which, in turn, are selectively blocked by local infusion of the CRF type 1 receptor (CRFr1) antagonist, NBI 27914 [5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)aminopyridine]. Here, we determined whether i) the blockade of the dPAG by CRFr1 attenuates the anxiogenic/antinociceptive effects induced by local infusion of the NO donor, NOC-9 [6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine], and ii) the anxiogenic/antinociceptive effects induced by intra-dPAG CRF are prevented by local infusion of Nω-propyl-L-arginine (NPLA), a neuronal NOS inhibitor, in mice. Male Swiss mice (12 weeks old, 25-35 g, N = 8-14/group) were stereotaxically implanted with a 7-mm cannula aimed at the dPAG. Intra-dPAG NOC-9 (75 nmol) produced defensive-like behavior (jumping and running) and antinociception (assessed by the formalin test). Both effects were reversed by prior local infusion of NBI 27914 (2 nmol). Conversely, intra-dPAG NPLA (0.4 nmol) did not modify the anxiogenic/antinociceptive effects of CRF (150 pmol). These results suggest that CRFr1 plays an important role in the defensive behavior and antinociception produced by NO within the dPAG. In contrast, the anxiogenic and antinociceptive effects produced by intra-dPAG CRF are not related to NO synthesis in this limbic midbrain structure.

Contrasting effects of nitric oxide and corticotropin- releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice.

The anxiogenic and antinociceptive effects produced by glutamate N-methyl-D-aspartate receptor activation within the dorsal periaqueductal gray (dPAG) matter have been related to nitric oxide (NO) production, since injection of NO synthase (NOS) inhibitors reverses these effects. dPAG corticotropin-releasing factor receptor (CRFr) activation also induces anxiety-like behavior and antinociception, which, in turn, are selectively blocked by local infusion of the CRF type 1 receptor (CRFr1) antagonist, NBI 27914 [5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)aminopyridine]. Here, we determined whether i) the blockade of the dPAG by CRFr1 attenuates the anxiogenic/antinociceptive effects induced by local infusion of the NO donor, NOC-9 [6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine], and ii) the anxiogenic/antinociceptive effects induced by intra-dPAG CRF are prevented by local infusion of N(ω)-propyl-L-arginine (NPLA), a neuronal NOS inhibitor, in mice. Male Swiss mice (12 weeks old, 25-35 g, N = 8-14/group) were stereotaxically implanted with a 7-mm cannula aimed at the dPAG. Intra-dPAG NOC-9 (75 nmol) produced defensive-like behavior (jumping and running) and antinociception (assessed by the formalin test). Both effects were reversed by prior local infusion of NBI 27914 (2 nmol). Conversely, intra-dPAG NPLA (0.4 nmol) did not modify the anxiogenic/antinociceptive effects of CRF (150 pmol). These results suggest that CRFr1 plays an important role in the defensive behavior and antinociception produced by NO within the dPAG. In contrast, the anxiogenic and antinociceptive effects produced by intra-dPAG CRF are not related to NO synthesis in this limbic midbrain structure.

Participation of nitric oxide synthase and cyclooxygenase-2 in the salivary secretion of hypothyroid endotoxemic rats / Participação da sintase do óxido nítrico e da ciclooxigenase-2 na secreção salivar de ratos hipotireoidianos endotoxêmicos

Purpose: In the present study the participation of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) on salivary secretion in endotoxemic hypothyroid rats was investigated. Methods: Male Wistar rats with an initial weight of 180 g were distributed into two groups, normal (N) or treated with propylthiouracil, 0.05 g/100 mL, administered orally for 5 weeks to induce hypothyroidism. Both groups were treated with lypopolysaccharide (LPS) (2.5 mg/kg; i.p.) to induce endotoxemia, or saline solution (SL), 90 min before salivary stimulation with pilocarpine (5 mg/kg; i.p.). Normal and PTU rats were divided into two groups each (n=07/09), receiving either L-NAME (10 mg/kg; i.p.), NOS inhibitor, or meloxicam (MLX) (0.5 mg/kg; i.p.), preferential COX-2 inhibitor, 30 min before endotoxemia challenge. Saliva was collected over a 15 min period (µL/min/100 g body wt.) from the time of the first drop of saliva. Results: Hypothyroidism decreased salivary flow rate in both groups of rats (LPS and SL). Endotoxemia and NOS inhibition by L-NAME reduced salivary flow in N rats. Meloxicam stimulated salivary secretion in the physiological state and systemic inflammation, induced by LPS, in N and PTU rats (Mann-Whitney Test; P < 0.05). Conclusion: In hypothyroid endotoxemic rats, it is COX-2 that modulates salivary secretion, not NOS.

Objetivo: Investigou-se a participação da sintase do óxido nítrico (NOS) e da ciclooxigenase-2 (COX-2) na secreção salivar de ratos hipotireoidianos endotoxêmicos. Metodologia: Ratos Wistar com peso inicial de 180 g foram distribuídos em dois grupos, normais (N) ou tratados com propiltiouracil (PTU) 0,05 g/100 mL, via oral, durante 5 semanas, para induzir hipotireoidismo. Ambos os grupos foram tratados com lipopolissacarídeo (LPS), 2,5 mg/kg i.p., para indução de endotoxemia ou salina (SL), 90 min antes da estimulação salivar com pilocarpina (5 mg/kg; i.p.). Os ratos N e PTU foram divididos em dois grupos cada (n = 07/09) e receberam injeções de L-NAME (10 mg/kg; i.p.), inibidor da NOS, ou meloxicam (MLX) (0,5 mg/kg;i.p.), inibidor preferencial da COX-2, 30 min antes da indução da endotoxemia. O fluxo salivar (µl/min/100 g de p.c.) foi avaliado durante um período de 15 min a partir da primeira gota de saliva. Resultados: O hipotireoidismo diminuiu o fluxo salivar em ratos tratados ou não com LPS. A endotoxemia e a inibição da NOS, através do L-NAME reduziu o fluxo salivar em ratos N. O MLX estimulou a salivação em situações fisiológicas e inflamatórias nos ratos N e PTU (Mann-Whitney; P < 0,05). Conclusão: A COX-2, mas não a NOS, modula a secreção salivar em ratos hipotireoidianos endotoxêmicos.

Participation of nitric oxide synthase and cyclooxygenase-2 in the salivary secretion of hypothyroid endotoxemic rats / Participação da sintase do óxido nítrico e da ciclooxigenase-2 na secreção salivar de ratos hipotireoidianos endotoxêmicos

Purpose: In the present study the participation of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) on salivary secretion in endotoxemic hypothyroid rats was investigated. Methods: Male Wistar rats with an initial weight of 180 g were distributed into two groups, normal (N) or treated with propylthiouracil, 0.05 g/100 mL, administered orally for 5 weeks to induce hypothyroidism. Both groups were treated with lypopolysaccharide (LPS) (2.5 mg/kg; i.p.) to induce endotoxemia, or saline solution (SL), 90 min before salivary stimulation with pilocarpine (5 mg/kg; i.p.). Normal and PTU rats were divided into two groups each (n=07/09), receiving either L-NAME (10 mg/kg; i.p.), NOS inhibitor, or meloxicam (MLX) (0.5 mg/kg; i.p.), preferential COX-2 inhibitor, 30 min before endotoxemia challenge. Saliva was collected over a 15 min period (µL/min/100 g body wt.) from the time of the first drop of saliva. Results: Hypothyroidism decreased salivary flow rate in both groups of rats (LPS and SL). Endotoxemia and NOS inhibition by L-NAME reduced salivary flow in N rats. Meloxicam stimulated salivary secretion in the physiological state and systemic inflammation, induced by LPS, in N and PTU rats (Mann-Whitney Test; P < 0.05). Conclusion: In hypothyroid endotoxemic rats, it is COX-2 that modulates salivary secretion, not NOS.

Objetivo: Investigou-se a participação da sintase do óxido nítrico (NOS) e da ciclooxigenase-2 (COX-2) na secreção salivar de ratos hipotireoidianos endotoxêmicos. Metodologia: Ratos Wistar com peso inicial de 180 g foram distribuídos em dois grupos, normais (N) ou tratados com propiltiouracil (PTU) 0,05 g/100 mL, via oral, durante 5 semanas, para induzir hipotireoidismo. Ambos os grupos foram tratados com lipopolissacarídeo (LPS), 2,5 mg/kg i.p., para indução de endotoxemia ou salina (SL), 90 min antes da estimulação salivar com pilocarpina (5 mg/kg; i.p.). Os ratos N e PTU foram divididos em dois grupos cada (n = 07/09) e receberam injeções de L-NAME (10 mg/kg; i.p.), inibidor da NOS, ou meloxicam (MLX) (0,5 mg/kg;i.p.), inibidor preferencial da COX-2, 30 min antes da indução da endotoxemia. O fluxo salivar (µl/min/100 g de p.c.) foi avaliado durante um período de 15 min a partir da primeira gota de saliva. Resultados: O hipotireoidismo diminuiu o fluxo salivar em ratos tratados ou não com LPS. A endotoxemia e a inibição da NOS, através do L-NAME reduziu o fluxo salivar em ratos N. O MLX estimulou a salivação em situações fisiológicas e inflamatórias nos ratos N e PTU (Mann-Whitney; P < 0,05). Conclusão: A COX-2, mas não a NOS, modula a secreção salivar em ratos hipotireoidianos endotoxêmicos.

Inhibition of bacillus Calmette-Guérin-induced nitric oxide in bladder tumor cells may improve BCG treatment.

Bacillus Calmette-Guérin (BCG) is considered to be one of the most effective treatments for superficial and in situ bladder cancer. However, either failure to respond initially or relapse within the first 5 years of treatment has been observed in some patients. As nitric oxide (NO) has been detected in the bladder of BCG-treated patients, we analyzed the role of endogenous NO generated after BCG treatments on human (T24) and murine (MB49 and MBT2) bladder tumor cells in the viability of tumor and immune cells, both in vitro and in vivo. In vitro inhibition of cancer cells after BCG treatment was evaluated by cell titer assay. NO production was determined as nitrite by Griess reagent. The death of immunocytes was evaluated by 51Cr release. Tumor histology with hematoxylin and eosin and Masson's trichrome staining was performed. BCG induced a direct inhibition of tumor cell growth in vitro, independently of NO levels. Besides, BCG-mediated NO production by tumor cells induced the death of spleen and peritoneal cells in syngeneic mice. The in vivo inhibition of NO synthase (NOS) activity by NG-nitro-L-arginine methyl ester in combination with BCG, improved tumor regression by generating a healing tissue. The increase of NO generated after BCG administration may induce the death of immunocytes. The in vivo inhibition of NO ameliorated immunotherapy with BCG by additional tumor growth inhibition. Our results suggested the possibility that the final outcome of patients with bladder tumors may improve by modulating NOS activity concomitantly with BCG therapy.

Role of nitric oxide increase on induced programmed cell death during early stages of rat liver regeneration.

We analysed the possible cellular mechanism involved in the NO action in the balance between apoptosis and cell proliferation in liver regeneration process. We determined p53, proapoptotic protein Bax, antiapoptotic Bcl-xL, proliferating cell nuclear antigen (PCNA) and apoptotic index at the early stages of regenerative process after NO increase by lipopolysaccharide-induction (LPS) of inducible-type nitric oxide synthase (iNOS) and by direct NO donor (sodium nitroprusside, SNP). Male Wistar rats were randomised in four experimental groups: sham operated control (Sh), partial hepatectomised control (PH-C), partial hepatectomised pretreated with LPS (2 mg/kg body weight, i.p.) (PH-LPS), and partial hepatectomised pretreated with SNP (2.5 mg/kg body weight, i.v. at a rate of 1 ml/h) (PH-SNP). Animals were killed 5 h post-surgery. Hepatic cytosolic iNOS showed an increase of 34% in PH-C animals with respect to Sh, and LPS-treatment increased iNOS protein levels 30% compared with PH-C. Bax and p53 protein levels showed significant increases in LPS- and SNP-treated hepatectomised rats with respect to PH-C. The apoptotic indexes were increased 75% in both, PH-LPS and PH-SNP rats versus PH-C. The increase of NO did not show any change in the proliferation process. These results suggest that NO is involved in apoptosis via p53 and Bax proteins after PH, showing a tightly regulated growth process in liver regeneration.

Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan-induced arthritis.

1. The contribution of nitric oxide (NO) and peroxynitrite (PN) to inflammation in a zymosan-induced (1 mg, intra-articular, i.art.) rat model of arthritis was assessed by histopathology and by measuring the glycosaminoglycan (GAG) content of the articular cartilage. 2. Progression of the chronic synovitis in zymosan-induced arthritis (ZYA) was associated with increased nitrite and nitrotyrosine (3-NT) levels in the joint exudates that paralleled a progressive loss of the GAG content. An increase in 3-NT was also observed after i.art. PN. 3. The nonselective nitric oxide synthase (NOS) inhibitor l-N(G)-nitroarginine methyl ester (25-75 mg x kg(-1)day(-1)) or the selective inducible NOS inhibitor aminoguanidine (50-100 mg x kg(-1)day(-1)) given 1 h before (prophylactic) or 3 days after (therapeutic) injection of the zymosan ameliorated the synovitis, but worsened the GAG loss, as measured at the end of the experiment (day 7). 4. The PN scavenger uric acid (100-250 mg x kg(-1) i.p. four times daily) given prophylactically until the end of the experiment (day 14), in a dose compatible with its PN scavenging activity, significantly decreased both the synovitis and the GAG loss. 5. In conclusion, PN formation is associated with cartilage damage in addition to proinflammatory activity in ZYA. NOS inhibitors and a PN scavenger were able to reduce the cellular infiltration, while displaying opposite effects on cartilage homeostasis either by enhancing or ameliorating the damage, respectively.

Nonadrenergic, noncholinergic relaxation of human isolated corpus cavernosum induced by scorpion venom.

OBJECTIVES: To examine the effects of Tityus serrulatus scorpion venom (TSV) on human corpus cavernosum (HCC) using a bioassay cascade. Priapism is occasionally observed in scorpion envenomation, mostly in children. METHODS: HCC strips were suspended in a cascade system and superfused with aerated and warmed Krebs' solution at 5 mL/min. Noradrenaline (3 micromol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclooxygenase products was prevented by infusing indomethacin (6 micromol/L). RESULTS: N(omega)-nitro-L-arginine methyl ester (10 micromol/L; n = 10) increased the tone of the preparations and significantly reduced (P <0.01) the acetylcholine (ACh) and TSV-induced relaxations. Subsequent infusion of L-arginine (300 micromol/L) partially reversed the increased tone and significantly restored the relaxations induced by TSV and ACh (P <0.01). The soluble guanylyl cyclase inhibitor ODQ (10 micromol/L; n = 8) markedly reduced (P <0.01) the relaxations induced by TSV, ACh, glyceryl trinitrate, and bradykinin. 7-Nitroindazole (10 micromol/L; n = 8) inhibited the relaxations induced by TSV by 84% (P <0.01) and also caused small, but significant, reductions in the ACh and bradykinin-induced HCC relaxations (P <0.05). Atropine (1 micromol/L; n = 6) abolished the relaxations evoked by ACh (P <0.01), but had no effect on those elicited by TSV. Tetrodotoxin (1 micromol/L; n = 6) abolished the relaxations induced by TSV (P <0.01) and also reversed the established TSV-induced relaxation (n = 4). CONCLUSIONS: Our results indicate that TSV relaxes HCC through the release of nitric oxide from nonadrenergic, noncholinergic (NANC) nerves. The elucidation of the mechanism responsible for the TSV-induced relaxations might be useful for a better understanding of the development of priapism in cases of scorpion envenomation.

Coronary endothelial dysfunction after ischemia and reperfusion: a new therapeutic target?

Although cardiac ischemia is usually characterized as a disease of the myocyte, it is clear that the vasculature, and especially endothelial cells, is also a major target of this pathology. Indeed, using a rat model of ischemia/reperfusion, we were able to detect severe endothelial dysfunction (assessed as a decreased response to acetylcholine) after acute or chronic reperfusion. Given the essential role of the endothelium in the regulation of vascular tone, as well as platelet and leukocyte function, such a severe dysfunction could lead to an increased risk of vasospasm, thrombosis and accelerated atherosclerosis. This dysfunction can be prevented by free radical scavengers and by exogenous nitric oxide. Endothelial dysfunction can also be prevented by preconditioning with brief periods of intermittent ischemia, thus extending to coronary endothelial cells the concept of endogenous protection previously described at the myocyte level. Experiments performed on cultured cells showed that the endothelial protection induced by free radical scavengers or by preconditioning was due to a lesser expression of endothelial adhesion molecules such as intercellular adhesion molecule-1, leading to a lesser adhesion of neutrophils to endothelial cells. Identification of the mechanisms of this protection may lead to the development of new strategies aimed at protecting the vasculature in ischemic heart diseases.

Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity.

Available evidence for oxidative stress after angioplasty is indirect or ambiguous. We sought to characterize the pattern, time course, and possible sources of free radical generation early after arterial balloon injury. Ex vivo injury performed in arterial rings in buffer with lucigenin yielded a massive oxygen-dependent peak of luminescence that decayed exponentially and was proportional to the degree of injury. Signals for injured vs. control arteries were 207. 1 +/- 17.9 (n = 13) vs 4.1 +/- 0.7 (n = 22) cpm x 10(3)/mg/min (p <. 001). Data obtained with 0.25 mmol/l lucigenin were validated with 0. 005-0.05 mmol/l lucigenin or the novel superoxide-sensitive probe coelenterazine (5 micromol/l). Gentle removal of endothelium prior to injury scarcely affected the amount of luminescence. Lucigenin signals were amplified 5- to 20-fold by exogenous NAD(P)H, and were >85% inhibited by diphenyliodonium (DPI, a flavoenzyme inhibitor). Antagonists of several other potential free radical sources, including xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport, were without effect. Overdistension of intact rabbit iliac arteries in vivo (n = 7) induced 72% fall in intracellular reduced glutathione and 68% increase in oxidized glutathione, so that GSH/GSSG ratio changed from 7.93 +/- 2.14 to 0. 81 +/- 0.16 (p <.005). There was also 28.7% loss of the glutathione pool. Further studies were performed with electron paramagnetic resonance spectroscopy. Rabbit aortas submitted to ex vivo overdistension in the presence of the spin trap DEPMPO (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide, 100 mmol/l, n = 5) showed formation of radical adduct spectra, abolished by DPI or superoxide dismutase. Computer simulation indicated a mixture of hydroxyl and carbon-centered radical adducts, likely due to decay of superoxide adduct. Electrical mobility shift assays for NF-kappaB activation were performed in nuclear protein extracts from intact or previously injured rabbit aortas. Balloon injury induced early NF-kappaB activation, which was decreased by DPI. In conclusion, our data show unambiguously that arterial injury induces an immediate profound vascular oxidative stress. Such redox imbalance is likely accounted for by activation of vessel wall NAD(P)H oxidoreductase(s), generating radical species potentially involved in tissue repair.