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Biomedical subjects

Tak Yee Aw

Publications and source records attributed to Tak Yee Aw.

17 recordsLinked to original sources
PubMed2006

NRF2-dependent glutamate-L-cysteine ligase catalytic subunit expression mediates insulin protection against hyperglycemia- induced brain endothelial cell apoptosis.

Increased oxidative stress and susceptibility of brain endothelium are contributing factors in the development of central nervous system complications in neuro-degenerative disorders in diabetes, Alzheimer's and Parkinson's disease. The molecular mechanisms underpinning the vulnerability of brain endothelial cells to chronic oxidative challenge have not been elucidated. Here, we investigated the oxidative susceptibility of human brain endothelial cells (IHEC) to chronic hyperglycemic stress and insulin signaling and cytoprotection. Chronic hyperglycemia exacerbated IHEC apoptosis in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance, and actin/Keap-1 S-glutathionylation. Insulin attenuated hyperglycemia-induced apoptosis via restored cytosolic and mitochondrial redox. Insulin stimulated glutamate-L-cysteine ligase (GCL) activity by activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling, increased serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and upregulation of Nrf2-dependent GCL-catalytic (GCLc) subunit expression. Expression of the GCL-modulatory subunit (GCLm) was unchanged. Inhibitors of insulin receptor tyrosine kinase, PI3K, Akt and mTOR abrogated insulin-induced Nrf2-mediated GCLc expression, redox balance, and IHEC survival. Collectively, these results demonstrate that human brain endothelial cells exhibit vulnerability to hyperglycemic stress which is associated with marked cytosolic and mitochondrial redox shifts. Activation of insulin signaling through PI3K/Akt/mTOR/Nrf2/ GCLc pathway affords significant cell protection by maintaining cellular redox balance.

Actins↗
PubMed2005

Apolipoprotein A-IV attenuates oxidant-induced apoptosis in mitotic competent, undifferentiated cells by modulating intracellular glutathione redox balance.

Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity.

Animals↗
PubMed2005

Fatty acid-mediated intracellular iron translocation: a synergistic mechanism of oxidative injury.

Fatty acid has been reported to be associated with cardiovascular diseases and cancer, but the possible mechanism remains unclear. Here, we reported a novel mechanism for the permissive role of fatty acid on iron intracellular translocation and subsequent oxidative injury. In vitro study from endothelial cells showed that iron alone had little effect, whereas in combination with PA (palmitic acid), iron-mediated toxicity was markedly potentiated, as reflected in mitochondrial dysfunction, cell death, apoptosis, and DNA mutation. We also showed that PA not only facilitated iron translocation into cells through a transferrin-receptor (TfR)-independent mechanism, but also translocated iron into mitochondria; the subsequent intracellular iron overload resulted in reactive oxygen species (ROS) overgeneration and lipid oxidation. Further investigation revealed that PA-facilitated iron translocation is due to Fe/PA-mediated extracellular oxidative stress and the subsequent membrane damage with increased membrane permeability. Fe/PA-mediated toxic effects were reduced in rho0 cells lacking mitochondrial DNA or by antioxidant enzyme SOD, especially mitochondrially localized MnSOD, suggesting a permissive role of PA for iron deposition on the vascular wall and its subsequent toxicity via mitochondrial oxidative stress. This observation was confirmed in vivo in mice, wherein higher vascular iron deposition and accompanying superoxide release were observed in the presence of a high-fat diet with iron administration.

Animals↗
PubMed2005

Intestinal glutathione: determinant of mucosal peroxide transport, metabolism, and oxidative susceptibility.

The intestine is a primary site of nutrient absorption and a critical defense barrier against dietary-derived mutagens, carcinogens, and oxidants. Accumulation of oxidants like peroxidized lipids in the gut lumen can contribute to impairment of mucosal metabolic pathways, enterocyte dysfunction independent of cell injury, and development of gut pathologies, such as inflammation and cancer. Despite this recognition, we know little of the pathways of intestinal transport, metabolism, and luminal disposition of dietary peroxides in vivo or of the underlying mechanisms of lipid peroxide-induced genesis of intestinal disease processes. This chapter summarizes our current understanding of the determinants of intestinal absorption and metabolism of peroxidized lipids. I will review experimental evidence from our laboratory and others (Table 1) supporting the pivotal role that glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) play in mucosal transport and metabolism of lipid hydroperoxides and how reductant availability can be compromised under chronic stress such as hypoxia, and the influence of GSH on oxidative susceptibility, and redox contribution to genesis of gut disorders. The discussion is pertinent to understanding dietary lipid peroxides and GSH redox balance in intestinal physiology and pathophysiology and the significance of luminal GSH in preserving the integrity of the intestinal epithelium.

Glutathione↗
PubMed2005

Differential susceptibility of naive and differentiated PC-12 cells to methylglyoxal-induced apoptosis: influence of cellular redox.

Neuropathologies have been associated with neuronal de-differentiation and oxidative susceptibility. To address whether cellular states determines their oxidative vulnerability, we have challenged naive (undifferentiated) and nerve growth factor-induced differentiated pheochromocytoma (PC12) with methylglyoxal (MG), a model of carbonyl stress. MG dose-dependently induced greater apoptosis (24 h) in naive (nPC12) than differentiated (dPC12) cells. This enhanced nPC12 susceptibility was correlated with a high basal oxidized cellular glutathione-to-glutathione disulfide (GSH/GSSG) redox and an MG-induced GSH-to-Disulfide (GSSG plus protein-bound SSG) imbalance. The loss of redox balance occurred at 30 min post-MG exposure, and was prevented by N-acetylcysteine (NAC) that was unrelated to de novo GSH synthesis. NAC was ineffective when added at 1h post-MG, consistent with an early window of redox signaling. This redox shift was kinetically linked to decreased BcL-2, increased Bax, and release of mitochondrial cytochrome c which preceded caspase-9 and -3 activation and poly ADP-ribose polymerase (PARP) cleavage (1-2 h), consistent with mitochondrial apoptotic signaling. The blockade of apoptosis by cyclosporine A supported an involvement of the mitochondrial permeability transition pore. The enhanced vulnerability of nPC12 cells to MG and its relationship to cellular redox shifts will have important implications for understanding differential oxidative vulnerability in various cell types and their transition states.

Acetylcysteine↗
PubMed2005

Hyperglycemia potentiates carbonyl stress-induced apoptosis in naïve PC-12 cells: relationship to cellular redox and activator protease factor-1 expression.

The mechanism(s) of central nervous system complication associated with neurodegenerative disorders such as diabetes is unknown. Previous studies demonstrated that carbonyl stress induced by methylglyoxal (MG) mediates differential apoptosis of rat pheochromocytoma (PC12) cells in the naïve or differentiated transition states. Since chronic hyperglycemia is central to diabetic complications, and poorly differentiated cells are oxidatively more vulnerable, we currently investigated the effect of glycemic status on MG-induced apoptosis in naïve (nPC12) cells focusing on glutathione-to-glutathione disulfide (GSH/GSSG) redox signaling. nPC12 cells were exposed to 25 mM glucose acutely for 24h or chronically for 1 week. A role for glycemic fluctuation was tested in chronic high glucose-adapted cells subjected to acute reduction in glucose availability. Acute hyperglycemia potentiated MG-induced nPC12 apoptosis in accordance with cellular redox (GSH-to-Disulfide (GSSG plus protein-bound SSG)) imbalance. Chronic hyperglycemia exacerbated baseline and MG-induced apoptosis that corresponded to exaggerated loss of cytosolic and mitochondrial redox balance, impaired glucose 6-phosphate dehydrogenase (G6PD) activity, and enhanced basal expression of apoptosis protease activator factor-1 (Apaf-1). Reduced glucose availability in hyperglycemia-adapted nPC12 cells induced by acute lowering of glucose or by dehydroepiandrosterone (DHEA, G6PD inhibitor) further enhanced MG-induced apoptosis in association with greater cytosolic and mitochondrial redox and G6PD impairment and elevated basal Apaf-1 expression. These findings demonstrate that chronic hyperglycemia or acute glucose reduction from the chronic hyperglycemic state potentiates carbonyl stress, which collectively contribute to oxidative susceptibility of poorly differentiated cells such as that which occurs in brain neurons of neurodegenerative disorders like diabetes and Alzheimer's disease.

Animals↗
PubMed2004

Apoptosis in acute and chronic neurological disorders.

Programmed cell death or apoptosis is a physiologically important process in neurogenesis wherein approximately 50% of the neurons apoptose during maturation of the nervous system. However, premature apoptosis and/or aberrations in apoptosis control contribute to the pathogenesis of a variety of neurological disorders including acute brain injury such as trauma, spinal cord injury, ischemic stroke and ischemia/reperfusion as well as chronic disease states such as Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, spinal muscular atrophy, and diabetic neuropathy. The current review will focus on two major topics, namely, the general concepts of our current understanding of the apoptosis death machinery, its mediators and regulation, and the relationship between aberrant apoptosis and genesis of neurodegenerative disorders. This knowledge of apoptosis mechanisms will underpin the basis for development of novel therapeutic strategies and treatment modalities that are directed at control of the neuronal apoptotic death program.

Acute Disease↗
PubMed2004

Susceptibility of murine periportal hepatocytes to hypoxia-reoxygenation: role for NO and Kupffer cell-derived oxidants.

Ischemia/reperfusion (I/R) is an important problem in liver resection and transplantation that is associated with hepatocellular dysfunction and injury. This study was designed to investigate whether a difference in hepatocyte susceptibility occurs in the periportal (PP) and/or perivenous (PV) zones in response to hypoxia/reoxygenation (H/R), and to delineate the mechanisms underlying this susceptibility. H/R was induced in an in situ perfused mouse liver model with deoxygenated Krebs-Henseleit buffer followed by oxygenated buffer. Selective destruction of PP or PV sites was achieved by digitonin perfusion into the portal or inferior vena cava, and was confirmed by histological evaluations and zone-specific enzymes. Hepatocellular injury was assessed by alanine aminotransferase (ALT) release. In whole liver, H/R significantly increased perfusate ALT. H/R of PP-enriched zones caused ALT release that was similar to that of whole liver (80 + 10 vs. 70 + 12 U/mg protein), consistent with significant PP hepatocyte injury. Minimal ALT release occurred in PV zones (10 + 5 U/mg protein). Administration of N-acetyl L-cysteine or a chimeric superoxide dismutase (SOD)-SOD2/3, a genetically engineered SOD-abrogated ALT release in H/R-perfused PP zones, implicating a role for superoxide (O(2) (-)). This elevated ALT release was attenuated by gadolinium chloride pretreatment, indicating that Kupffer cells are the O(2) (-) source. Enzymatic inhibition of cellular nitric oxide synthase (NOS) or genetic depletion of endothelial nitric oxide synthase (eNOS) aggravated hypoxia injury while exogenous NO and inducible nitric oxide synthase (iNOS) deficiency abolished reoxygenation injury. In conclusion, PP hepatocytes are more vulnerable to H/R; this injury is mediated directly or indirectly by Kupffer cell derived O(2) (-) and is limited by eNOS-derived NO.

Animals↗
PubMed2004

Apoptosis: a key in neurodegenerative disorders.

Apoptosis is an important process in the development of the nervous system. Typically, approximately 50% of the neurons apoptose during neurogenesis before the nervous system matures. However, recent paradigms implicate premature apoptosis and/or aberrations in the fine control of neuronal apoptosis in the pathogenesis of a variety of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, stroke, brain trauma, spinal cord injury, and diabetic neuropathy. This review will focus on the current concepts salient to understanding the apoptosis death program, the mediators and control of cellular apoptosis, and the relationship between aberrant apoptosis and genesis of neurodegenerative disorders. The discussion will also highlight current advances in methodology, such as utilization of neuronal cell lines and mutant animal models, in investigations of neuronal apoptotic death. The knowledge of apoptosis mechanisms could underpin the basis for development of novel therapeutic strategies and treatment modalities that are directed at control of the neuronal apoptotic death program.

Animals↗
PubMed2003

Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells.

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.

Animals↗
PubMed2003

Chronic exposure to subtoxic levels of peroxidized lipids suppresses mucosal cell turnover in rat small intestine and reversal by glutathione.

Oxidative challenge can compromise intestinal growth and death responses. This studyexamines the effect of chronic consumption of subtoxic levels of peroxidized lipids on intestinal redox balance and turnover and the effect of glutathione (GSH) supplementation. Male Sprague-Dawley rats were fed standard rat chow or 4% peroxidized menhaden oil chow (2-8 weeks). Intestinal GSH and glutathione disulfide (GSSG), GSH synthetic and redox enzymes as well as proliferative (ornithine decarboxylase, ODC) and apoptotic activities were evaluated. Chronic peroxide intake did not affect overall animal growth, but decreased intestinal GSH/GSSG ratio that directly correlated with decreased GSH and increased GSSG, and suppressed peak circadian ODC activities and post-prandial mucosal apoptosis. Supplementation with GSH restored the mucosal GSH/GSSG ratio and abrogated the peroxide-induced suppression of intestinal cell turnover. Collectively, the results show that chronic lipid peroxide consumption induces intestinal GSH redox imbalance that interferes with regulation of enterocyte death and proliferation in vivo. These disruptive effects of lipid peroxides were reversed by GSH supplementation in accordance with the normalization of tissue GSH/GSSG redox balance.

Animals↗
PubMed2003

Chronic lipid hydroperoxide stress suppresses mucosal proliferation in rat intestine: potentiation of ornithine decarboxylase activity by epidermal growth factor.

We recently demonstrated that chronic exposure of rat intestine to sublethal levels of peroxidized lipids suppresses ornithine decarboxylase (ODC) activity, consistent with attenuated intestinal proliferation. The current study was designed to better understand the influence of exogenous epidermal growth factor (EGF) on intestinal proliferation in normal intestine and intestine that was challenged by oxidative stress induced by dietary consumption of peroxidized lipids. Male Sprague-Dawley rats (250-300 g) were fed standard chow (control) or peroxidized lipid chow for 4 weeks. EGF was injected intraperitoneally at a dose of 40 microg/kg. Intestinal proliferation was evaluated by ODC activity in fed or fasted states and at specified times during the circadian phase. Chronic peroxide consumption significantly attenuated ODC activities in association with increased tissue peroxide content. The suppressed ODC activities were restored to control values by EGF in the small intestine; in the colon, EGF increased ODC activity threefold over control rats given EGF. This elevated colonic ODC activity was correlated with decreased tissue GSSG and an increased GSH/GSSG ratio. These results show that EGF administration reverses the suppression of intestinal ODC activities induced by chronic peroxidized lipid intake. In contrast, EGF significantly elevates proliferative activity in the peroxide-stressed colon. This exaggerated proliferation may contribute to a better understanding of colonic susceptibility to oxidant-induced malignant transformation.

Animals↗
PubMed2002

Endothelial cells maintain a reduced redox environment even as mitochondrial function declines.

Human umbilical vein endothelial cells (HUVECs) are an endothelial model of replicative senescence. Oxidative stress, possibly due to dysfunctional mitochondria, is believed to play a key role in replicative senescence and atherosclerosis, an age-related vascular disease. In this study, we determined the effect of cell division on genomic instability, mitochondrial function, and redox status in HUVECs that were able to replicate for approximately 60 cumulative population doublings (CPD). After 20 CPD, the nuclear genome deteriorated and the protein content of the cell population increased. This indicated an increase in cell size, which was accompanied by an increase in oxygen consumption, ATP production, and mitochondrial genome copy number and approximately 10% increase in mitochondrial mass. The antioxidant capacity increased, as seen by an increase in reduced glutathione, glutathione peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase. However, by CPD 52, the latter two enzymes decreased, as well as the ratio of mitochondrial-to-nuclear genome copies, the mitochondrial mass, and the oxygen consumption per milligram of protein. Our results signify that HUVECs maintain a highly reducing (GSH) environment as they replicate despite genomic instability and loss of mitochondrial function.

Cells, Cultured↗
PubMed2002

The human immunodeficiency virus-1 Tat protein activates human umbilical vein endothelial cell E-selectin expression via an NF-kappa B-dependent mechanism.

Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-kappaB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin kappaB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIkappaBalphaSR) that constitutively sequesters IkappaB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated kappaB translocation and E-selectin up-regulation.

Cells, Cultured↗
PubMed2002

Lipid peroxide-induced redox imbalance differentially mediates CaCo-2 cell proliferation and growth arrest.

Dietary oxidants like lipid hydroperoxides (LOOH) can perturb cellular glutathione/glutathione disulphide (GSH/GSSG) status and disrupt mucosal turnover. This study examines the effect of LOOH on GSH/GSSG balance and phase transitions in the human colon cancer CaCo-2 cell. LOOH at 1 or 5 micro m were noncytotoxic, but disrupted cellular GSH/GSSG and stimulated proliferative activity at 6 h that paralleled increases in ornithine decarboxylase activity, thymidine incorporation, expression of cyclin D1/cyclin-dependent kinase 4, phosphorylation of retinoblastoma protein, and cell progression from G0/G1 to S. At 24 h, LOOH-induced sustained GSH/GSSG imbalance mediated growth arrest at G0/G1 that correlated with suppression of proliferative activity and enhanced oxidative DNA damage. LOOH-induced cell transitions were effectively blocked by N-acetylcysteine. Collectively, the study shows that subtoxic LOOH levels induce CaCo-2 GSH/GSSG imbalance that elicits time-dependent cell proliferation followed by growth arrest. These results provide insights into the mechanism of hydroperoxide-induced disruption of mucosal turnover with implications for understanding oxidant-mediated genesis of gut pathology.

Acetylcysteine↗
PubMed2002

Apoptosis in mitotic competent undifferentiated cells is induced by cellular redox imbalance independent of reactive oxygen species production.

Oxidants are known to induce cell apoptosis. Because oxidants also elicit redox imbalance, it is difficult to distinguish the direct effects of cellular redox from that of oxidants. This study tests the hypothesis that induction of redox imbalance independent of reactive oxygen species (ROS), can induce cell apoptosis in a mitotic competent, undifferentiated cell line, PC-12. Cells grown in standard DMEM containing 25 mM glucose were treated with diamide, a thiol oxidant, at a concentration that did not generate ROS. Diamide caused a rapid increase in oxidized glutathione (GSSG) and a loss of mitochondrial cytochrome c in 15-30 min, caspase-3 activation in 2 h, and apoptosis in 24 h. N-Acetyl cysteine attenuated GSSG elevation and diamide-induced apoptosis. Incubation of cells in 5 mM glucose or inhibition of the pentose phosphate pathway maintained GSSG elevation and accelerated cell apoptosis. Collectively, these results show that loss of redox balance is an upstream event that kinetically preceded mitochondrial apoptotic signaling. A sustained redox change was not critical or necessary for apoptotic progression, but its prolongation exacerbated apoptotic death. The potentiation of apoptosis by sustained redox imbalance was correlated with decreases in NADPH supply for GSSG reduction.

Acetylcysteine↗