Alipio O. Carmo

Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells

NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22phox, Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22phox, Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.

A new look at old agents for pleurodesis: nitrogen mustard, sodium hydroxide, and silver nitrate.

In this review we analyze the evolution of pleurodesis. In spite of the fact that this procedure started in the beginning of the 20th century, the ideal sclerosing agent is not yet known. Emphasis is placed on the current tendency toward minimally invasive procedures in which insertion of catheters is favored over surgical procedures such as placement of chest tubes or thoracoscopy. Among the sclerosing agents, talc is preferred throughout the world. However, the possible development of acute respiratory distress syndrome, which is sometimes fatal, caused the awakening of interest in other drugs. Nitrogen mustard induces pleurodesis but causes important side effects. Sodium hydroxide and silver nitrate are effective and may be used in humans beings.

Effectiveness of sodium hydroxide as a pleural sclerosing agent in rabbits: Influence of concomitant intrapleural lidocaine

The two agents that have been used most commonly to produce a pleurodesis are tetracycline and bleomycin. Tetracycline is no longer generally available because of more stringent requirements on the manufacturing process. Bleomycin is very expensive. Therefore, alternative agents are necessary particularly in developing countries. The objective of this project was to determine whether 0.5% sodium hydroxide is an effective sclerosant in an experimental model in rabbits. Sodium hydroxide (NaOH) (2 ml of 0.5%) was instilled intrapleurally in 24 anesthetized male rabbits. Half the rabbits received 1 ml of 2% lidocaine 3–5 min before the NaOH. Twenty-eight days after the instillation, the animals were sacrificed, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. The results indicated that the intrapleural injection of NaOH was effective in creating a pleurodesis only if the animals were not premedicated with lidocaine. The mean (±S.D.) degree of gross pleurodesis after NaOH alone 2.8 (1.0) was significantly (p < 0.001) greater than after that following the combination 1.3 (0.5). We conclude that NaOH is an effective pleural sclerosant but that it is ineffective if it is injected concomitantly with lidocaine.

Effectiveness of silver nitrate compared to talc slurry as pleural sclerosing agent in rabbits. Influence of concomitant intrapleural lidocaine

UNLABELLED The ideal agent for producing pleurodesis has not been identified. Talc, the most commonly used, poses several problems. Another possibility is silver nitrate, which was widely used in the past. PURPOSE To determine the influence of the intrapleural instillation of lidocaine in producing a pleurodesis with silver nitrate, to define the effect of lidocaine in the maturation of the collagen fibers, and to confirm that the pleurodesis after silver nitrate is stronger than after talc. METHODS We studied three groups of 8 rabbits. Two groups received 0.5% silver nitrate; in one we had previously injected 0.5 ml of 2% lidocaine. The third group received 400 mg/kg talc (2 ml). The animals were sacrificed 28 days after the injection, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of inflammation and fibrosis. The total amount of pleural collagen and the distribution of thick and thin collagen fibers were quantified. Collagen was identified using picrosirius red stain. RESULTS In the two groups that received silver nitrate (without lidocaine: 3.5 + 03 and with lidocaine: 3.2 + 0.3), the macroscopic pleurodesis (scale 0 - 4) was significantly (p = 0.001) better than that resulting from talc (1.6 + 0.2). The mean degree of pleural fibrosis induced by silver nitrate (3.5 + 0.2) was significantly (p = 0.004) higher than that induced by talc (1.9 + 0.1). The previous instillation of lidocaine resulted in a tendency for decreased amounts of fibrosis (3.1 + 0.4). The mean amount (10(3)mm2) of pleural collagen was significantly (p = 0.009) greater in the rabbits that received silver nitrate (116.9 + 22.7) than in those that received talc (10.7 + 3.4). The injection of lidocaine slightly reduced the collagen (80.1 + 30.3). The distribution of collagen fibers did not differ among the groups. CONCLUSION This rabbit model clearly confirms that intrapleural silver nitrate is more effective than talc for producing pleurodesis. The previous intrapleural instillation of lidocaine results in a decreasing trend in the amount of collagen, but does not change the effectiveness of the pleural fusion or modify the process of collagen maturation.

Effectiveness of Ethanolamine Oleate as a Pleural Sclerosing Agent in Rabbits

The ideal pleural sclerosing agent should be easily administered, without significant side effects, inexpensive, and widely available. None of the agents presently used meets all of these criteria. Ethanolamine oleate (ETH) is a sclerosing agent used in the sclerotherapy treatment of varicose veins of the legs and esophagus. The objective of the present study was to assess the efficacy of ETH as a pleural sclerosant in rabbits. An additional objective was to assess if better results were obtained when dextrose 50% (D50) as opposed to saline was used as the diluent. Each group of 10 rabbits received a total volume of 2 ml intrapleurally. The eight treatments were as follows: (1) 2 ml saline; (2) 2 ml D50; (3) 25 mg ETH plus 1.5 ml saline; (4) 25 mg ETH plus 1.5 ml D50; (5) 50 mg ETH plus 1.0 ml saline; (6) 50 mg ETH plus 1 ml D50; (7) 75 mg ETH plus 0.5 ml D50, and (8) 100 mg ETH. The rabbits were sacrificed 28 days after the injection. The intrapleural instillation of ETH resulted in evident pleurodesis, which was dose-dependent; 100 mg ETH induced significantly (p < 0.05) more adhesions than did any other treatment. The selection of the diluent had no effect on the pleurodesis. The microscopic examination of the right visceral pleura showed that the mean degree of fibrosis after 100 mg ETH was significantly (p < 0.05) greater than that after the other solutions. The mean degree of pleural inflammation, lung inflammation and lung fibrosis was minimal in all the groups. From this study we conclude that undiluted ETH produces pleurodesis in our experimental model. At the doses used, the pleurodesis was less than that produced after talc, tetracycline derivatives or silver nitrate in the same model.

Pleurodese: perspectivas futuras

This article addresses the evolution of pleurodesis since the beginning of the 20th century and defines the characteristics of the ideal sclerosing agent. Emphasis is placed on the current tendency towards minimally invasive procedures where insertion of catheters is usually given priority over certain surgical procedures such as placement of drains or thoracoscopy. Among the sclerosing drugs, talc is the one preferred throughout the world. However, the possible appearance of respiratory distress syndrome, which is sometimes fatal, caused the awakening of interest in other drugs. Anti neoplastic drugs do not induce a very efficient pleurodesis and still have the disadvantage of causing important side effects. Sodium hydroxide and silver nitrate produce effective pleurodesis. Both can be used in humans.