Chandan K. Sen

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.

Neuroprotective effects of alpha-lipoic acid and its positively charged amide analogue.

Elevated levels of extracellular glutamate have been linked to reactive oxygen species mediated neuronal damage and brain disorders. Lipoic acid is a potent antioxidant that has previously been shown to exhibit neuroprotection in clinical studies. A new positively charged water soluble lipoic acid amide analog, 2-(N,N-dimethylamine) ethylamido lipoate HCl (LA-plus), with a better cellular reduction and retention of the reduced form was developed. This novel antioxidant was tested for protection against glutamate induced cytotoxicity in a HT4 neuronal cell line. Glutamate treatment for 12 h resulted in significant release of LDH from cells to the medium suggesting cytotoxicity. Measurement of intracellular peroxides showed marked (up to 200%) increase after 6 h of glutamate treatment. Compared to lipoic acid, LA-plus was more effective in (1) protecting cells against glutamate induced cytotoxicity, (2) preventing glutamate induced loss of intracellular GSH, and (3) disallowing increase of intracellular peroxide level following the glutamate challenge. The protective effect of LA-plus was found to be independent of its stereochemistry. The protective function of this antioxidant was synergistically enhanced by selenium. These results demonstrate that LA-plus is a potent protector of neuronal cells against glutamate-induced cytotoxicity and associated oxidative stress.

Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death

Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca(2+), progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants alpha-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for alpha-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca(2+). Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca(2+) chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acid did not protect the cells against glutamate treatment. The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.

Pine bark extract pycnogenol downregulates IFN-γ-induced adhesion of T cells to human keratinocytes by inhibiting inducible ICAM-1 expression

Expression of intercellular adhesion molecule-1 (ICAM-1) is necessary for leukocyte/keratinocyte interactions. Upregulation of ICAM-1 expression in keratinocytes has been observed in several inflammatory dermatoses, such as psoriasis, atopic dermatitis, and lupus erythematosus. Inflammatory cytokines, such as interferon-gamma (IFN-gamma), upregulate ICAM-1 expression in keratinocytes. Because of potent antioxidant and anti-inflammatory properties of the French maritime pine bark extract, Pycnogenol (Horphag Research, Geneva, Switzerland), its effects were investigated on the interaction of T cells with keratinocytes after activation with IFN-gamma and the molecular mechanisms involved in such interactions. Studies were performed using a human keratinocyte cell line, HaCaT. Cell adhesion in the presence of IFN-gamma was studied using a coculture assay. Treatment of HaCaT cells with 20 U/ml IFN-gamma for 24 h markedly induced adherence of Jurkat T cells to HaCaT cells. PYC pretreatment (50 microg/ml, 12 h) significantly inhibited IFN-gamma induced adherence of T cells to HaCaT cells (p < .01). ICAM-1 plays a major role in the IFN-gamma-induced adherence of T cells to keratinocytes. Thus, the effect of PYC on IFN-gamma-induced ICAM-1 expression was investigated as well. Pretreatment of HaCaT cells with PYC significantly inhibited IFN-gamma-induced expression of ICAM-1 expression in HaCaT cells. The downregulation of inducible ICAM-1 expression by PYC was both dose and time dependent. A 50 microg/ml dose of PYC and a 12 h pretreatment time (i.e., before activation with IFN-gamma) provided maximal (approximately 70%) inhibition of inducible ICAM-1 expression in HaCaT cells. Gamma-activated sequence present on the ICAM-1 gene confers IFN-gamma responsiveness in selected cells of epithelial origin (e.g., keratinocytes) that are known to express ICAM-1 on activation with IFN-gamma. Gel-shift assays revealed that PYC inhibits IFN-gamma-mediated activation of Stat1, thus suggesting a transcriptional regulation of inducible ICAM-1 expression by PYC. These results indicate the therapeutic potential of PYC in patients with inflammatory skin disorders.

Cytokine-induced glucose uptake in skeletal muscle: redox regulation and the role of α-lipoic acid

In L6 myotubes, glucose uptake stimulated by interferon (IFN)-γ or lipopolysaccharides (LPS) and a combination of LPS, IFN-γ, and tumor necrosis factor (TNF)-α was inhibited by the antioxidant pyrrolidinedithiocarbamate and potentiated in reduced glutathione (GSH)-deficient cells. Also, the stimulatory effect of LPS and IFN-γ individually, and of a combination of LPS, IFN-γ, and TNF-α, on glucose uptake was associated with an increased level of intracellular oxidants (dichlorofluorescein assay) and loss of intracellular GSH. Study of the individual effects of LPS, IFN-γ, and TNF-α as well as of a combination of the three activators provided evidence against a role of nitric oxide in mediating the stimulatory effect of the above-mentioned agents on glucose uptake. We also observed that the insulin-mimetic nutrient α-lipoic acid (LA; R-enantiomer) is able to stimulate glucose uptake in cytokine-treated cells that are insulin resistant. This study shows that cytokine-induced glucose uptake in skeletal muscle cells is redox sensitive and that, under conditions of acute infection that is accompanied with insulin resistance, LA may have therapeutic implications in restoring glucose availability in tissues such as the skeletal muscle.

Physical Exercise and Antioxidant Defenses in the Hearta

Abstract: Cardiac muscle relies highly on aerobic metabolism. Heart muscle has a high oxygen uptake at resting conditions, which increases many fold during exhaustive physical exercise. Such a high rate of oxidative metabolism is often associated with enhanced production of reactive oxygen metabolites. A single bout of strenuous exercise has been demonstrated to induce oxidative damage in heart. Such oxidant insult may lead to adaptive responses and strengthen antioxidant defenses in the heart tissue. Endurance exercise training has indeed been shown to upregulate heart tissue antioxidant defenses. Recently, we have observed that even predominantly anaerobic sprint training regimens may enhance cardiac antioxidant defenses. Regular physical exercise may beneficially influence cardiac antioxidant defenses and promote overall cardiac function.

Monochloramine Inhibits Phorbol Ester-inducible Neutrophil Respiratory Burst Activation and T Cell Interleukin-2 Receptor Expression by Inhibiting Inducible Protein Kinase C Activity

Monochloramine derivatives are long lived physiological oxidants produced by neutrophils during the respiratory burst. The effects of chemically prepared monochloramine (NH2Cl) on protein kinase C (PKC) and PKC-mediated cellular responses were studied in elicited rat peritoneal neutrophils and human Jurkat T cells. Neutrophils pretreated with NH2Cl (30–50 μm) showed a marked decrease in the respiratory burst activity induced by phorbol 12-myristate 13-acetate (PMA), which is a potent PKC activator. These cells, however, were viable and showed a complete respiratory burst upon arachidonic acid stimulation, which induces the respiratory burst by a PKC-independent mechanism. The NH2Cl-treated neutrophils showed a decrease in both PKC activity and PMA-induced phosphorylation of a 47-kDa protein, which corresponds to the cytosolic factor of NADPH oxidase, p47 phox . Jurkat T cells pretreated with NH2Cl (20–70 μm) showed a decrease in the expression of the interleukin-2 receptor α chain following PMA stimulation. This was also accompanied by a decrease in both PKC activity and nuclear transcription factor-κB activation, also without loss of cell viability. These results show that NH2Cl inhibits PKC-mediated cellular responses through inhibition of the inducible PKC activity.

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.

10 – Oxidants and Antioxidants in Apoptosis: Role of Bcl-2

The aim of this chapter is to provide a brief overview of the role of reactive species and antioxidants in regulating apoptosis. The chapter mainly focuses on the role of the Bcl-2 family proteins in the redox regulation of apoptosis. In some cases of apoptosis, oxidation-reduction, or redox-dependent mechanisms play a central role. This opens up the opportunity to modulate apoptosis by agents that may alter the intracellular redox status, including antioxidants. At present, the exact molecular mechanisms involved in the redox regulation of apoptosis remain elusive. Investigations exhibit that antioxidants may have antiapoptotic or proapoptotic properties, depending on the type of cells and the apoptosis-inducing signal involved. A trend showing that antioxidants may prevent apoptosis of healthy cells and may promote the apoptosis of diseased cells appears to be emerging. Such an observation, however, is not yet supported by reasonable mechanism-based details. A major function of the Bcl-2 family proteins is to regulate apoptosis. A strategic localization of the Bcl-2 protein inside the cell seems to be necessary to render its antiapoptotic property functional.

12 – Redox Regulation of Cell Adhesion Processes

This chapter discusses the role of oxidants and antioxidants in the regulation of cell adhesion processes as well as the molecular mechanisms of such regulation. Cell adhesion processes play a major role in the regulation of immune functions as well as of other vital biological processes, such as embryogenesis, cell growth, differentiation, and wound repair. Cell adhesion, which is a multistep process including rolling, firm attachment, and transmigration of leukocytes, is mediated by several classes of cell adhesion molecules. Cell adhesion molecule expression and adhesive properties of cells are modified greatly in several diseased conditions involving redox imbalances, such as cancer, atherosclerosis, diabetes, chronic inflammation, and ischemiareperfusion injury. Several stimuli such as cytokines, chemokines, and phorbol ester are known to activate the expression and function of cell adhesion molecules. Both these evidences indicate that the overall cell adhesion process is redox regulated—direct activation of cell adhesion processes by oxidants and inhibitory action of antioxidants on cell adhesion molecule expression and function.