Savita Khanna

Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing.

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H2O2 potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp −194 to −50 of the VEGF promoter is responsible for the H2O2 response. It was established that at μm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.

Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system?

One of the many functions of liver peroxisomes is the beta-oxidation of long-chain fatty acids. It is essential for the continuation of peroxisomal beta-oxidation that a redox shuttle system exist across the peroxisomal membrane to reoxidize NADH. We propose that this redox shuttle system consists of a substrate cycle between lactate and pyruvate. Here we present evidence that purified peroxisomal membranes contain both monocarboxylate transporter 1 (MCT 1) and MCT 2 and that along with peroxisomal lactate dehydrogenase (pLDH) form a Peroxisomal Lactate Shuttle. Peroxisomal beta-oxidation was greatly stimulated by the addition of pyruvate and this increase was partially inhibited by the addition of the MCT blocker alpha-cyano-4-hydroxycinnamate (CINN). We also found that peroxisomes generated lactate in the presence of pyruvate. Together these data provide compelling that the Peroxisome Lactate Shuttle helps maintain organelle redox and the proper functioning of peroxisomal beta-oxidation.

Protective Effects of Anethole Dithiolethione against Oxidative Stress-induced Cytotoxicity in Human Jurkat T Cells

The protective effects of anethole dithiolethione (ADT) against H2O2- or 4-hydroxynonenal (HNE)-induced cytotoxicity in human Jurkat T cells were investigated. Jurkat T cells were pretreated with ADT (10-50 microM) for 18 hr and then challenged with H202 or HNE for up to 4 hr. Cytotoxicity was assessed by measuring: 1) leakage of lactate dehydrogenase from cells to medium; and 2) exclusion of the DNA intercalating fluorescent probe propidium iodide by viable cells. Pretreatment of cells with ADT (10 or 25 microM) for 18 hr significantly protected cells against H202- or HNE-induced cytotoxicity. Treatment of cells with ADT (10-50 microM) for 72 hr significantly increased the activities of catalase and glutathione reductase. The maximum effect of ADT treatment on the activity of these enzymes was observed when cells were treated with 25 microM of ADT for 72 hr. A significant increase in cellular GSH was observed in cells that were treated with ADT for 72 hr. Using monobromobimane as a thiol probe, we consistently observed that cells pretreated for 18 hr with ADT (25 or 50 microM) had also increased total thiol content. Exposure of Jurkat T cells to H202 or HNE resulted in a time-dependent decrease in cellular GSH. ADT (10-50 microM, 18 hr) pretreatment circumvented H202-dependent lowering of cellular GSH. In conclusion, ADT proved to be a potent cytoprotective thiol antioxidant with multifaceted mechanisms of action, suggesting that the drug has a remarkable therapeutic potential.

Skeletal muscle and liver lipoyllysine content in response to exercise, training and dietary α‐lipoic acid supplementation

In human cells, α‐lipoic acid (LA) is present in a bound lipoyllysine form in mitochondrial proteins that play a central role in oxidative metabolism. The possible effects of oral LA supplementation, a single bout of strenuous exercise and endurance exercise training on the lipoyllysine content in skeletal muscle and liver tissues of rat were examined. Incorporation of lipoyl moiety to tissue protein was not increased by enhanced abundance of LA in the diet. Endurance exercise training markedly increased lipoyllysine content in the liver at rest. A bout of exhaustive exercise also increased hepatic lipoyllysine content. A significant interaction of exhaustive exercise and training to increase tissue lipoyllysine content was evident. In vastus lateralis skeletal muscle, training did not influence tissue lipoyllysine content. A single bout of exhaustive exercise, however, clearly increased the level of lipoyllysine ill the muscle. Comparison of tissue lipoyllysine data with that of free or loosely‐bound LA results showed a clear lack of association between the two apparently related parameters. Tightly protein‐bound lipoyllysine pool in tissues appeared to be independent of the loosely‐bound or free LA status in the tissue.

Arachidonic Acid Metabolism and Lipid Peroxidation in Stroke: Alpha-Tocotrienol as a Unique Therapeutic Agent

Under normal physiological conditions, the human brain has one of the highest metabolic profiles of all organs, using 25% of glucose and 20% of all oxygen consumed by the body. When challenged by metabolic disruption as in ischemic or hemorrhagic stroke, brain tissue that is enriched with arachidonic acid (22:6n − 3 polyunsaturated fatty acid) is highly susceptible to oxidative stress. The consequence of increased generation of radical species in stroke-affected brain tissue is the uncontrolled oxidative metabolism of arachidonic acid, generating a host of secondary products that are culpable neuromodulators of the cell death cascade. In this chapter, preclinical models of ischemic and hemorrhagic stroke injury are explored. Subsequently, the arachidonic acid cascade is examined as a common pathological contributor of oxidative stress in both aforementioned stroke subtypes. Finally, the unique neuroprotective properties of the natural vitamin E alpha-tocotrienol are discussed as a potent intervention of the stroke-induced arachidonic acid cascade.