Genetic Alterations in Oxidant and Anti-Oxidant Enzymes in the Vascular System

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.

The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4(-/-) mice. Nox4 was highly expressed in the endothelium and contributed to H(2)O(2) formation. Nox4(-/-) mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4(-/-) lung endothelial cells (LECs) was attenuated and restored by low concentrations of H(2)O(2,) whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.