Alpha-lipoic Acid modulates heat shock factor-1 expression in streptozotocin-induced diabetic rat kidney

Increased oxidative stress and impaired heat shock protein (HSP) synthesis may contribute to diabetic nephropathy. The question of whether 8-week thiol antioxidant alpha-lipoic acid (LA) supplementation modulates HSP response and oxidative stress was studied in the kidney of streptozotocin-induced diabetic (SID) and nondiabetic rats. SID caused a histological mesangial expansion, tubular dilatation, and increased levels of transforming growth factor-beta (TGF-beta), a mediator of glomerulosclerosis. SID increased 4-hydroxynonenal (4-HNE) protein adduct formation, a marker of lipid peroxidation, and heme oxygenase-1 (HO-1), also a marker of oxidative stress. Moreover, SID increased the DNA-binding activity of heat shock factor-1 (HSF-1) and expression of heat shock protein 60 (HSP60). In contrast, LA supplementation partially reversed histological findings of glomerulosclerosis and decreased TGF-beta. LA also increased HSF-1 and decreased HO-1 protein expression, without affecting 4-HNE protein adduct levels. At the mRNA level, LA increased expression of HSF-1, HSP90, and glucose-regulated protein (GRP75) in both control and diabetic animals and HSP72 in SID rats. However, LA supplementation did not affect these HSPs at the protein level. These findings suggest that in addition to its antiglomerulosclerotic effects, LA can induce cytoprotective response in SID.     

Glial heme oxygenase-1 expression in Alzheimer disease and mild cognitive impairment

We determined whether oxidative stress is an early event in the pathogenesis of sporadic Alzheimer disease (AD), and correlated oxidative stress with neuropsychological functions and neurofibrillary pathology in AD and mild cognitive impairment (MCI). Oxidative stress was measured as the percentage of astrocytes expressing heme oxygenase-1 (HO-1) in post mortem temporal cortex and hippocampus after dual HO-1/glial fibrillary acidic protein (GFAP) immunohistochemistry. Glial HO-1 expression in the MCI temporal cortex and hippocampus was significantly greater than in the non-demented group and did not differ from AD values. Astroglial HO-1 expression in the temporal cortex was associated with decreased scores for global cognition, episodic memory, semantic memory and working memory. Hippocampal astroglial HO-1 expression was associated with lower scores for global cognition, semantic memory and perceptual speed. Glial HO-1 immunoreactivity in the temporal cortex, but not hippocampus, correlated with the burden of neurofibrillary pathology. Cortical and hippocampal oxidative stress is a very early event in the pathogenesis of sporadic AD and correlates with the development of specific cognitive deficits in this condition.     

Chronically increased oxidative stress in fibroblasts from Alzheimer's disease patients causes early senescence and renders resistance to apoptosis by oxidative stress

It is well established that oxidative stress is involved in several neurodegenerative disorders, including Alzheimer's disease (AD). Study of the induction and consequences of oxidative stress in the peripheral tissues of the familial AD patients can help to elucidate the inherent abnormalities and the mechanism of pathogenesis of this disease. AD fibroblasts have been used as a model to investigate the underlying mechanisms of oxidative stress. In our study, we used AD fibroblasts from six different donors who are either at high risk of developing AD or have already been diagnosed with AD to study the effect of oxidative stress in comparison with the effect on non-AD normal human fibroblast. Oxidative stress was induced by a brief exposure of the cells to 250microM H(2)O(2) followed by incubation in normal conditions. Neuronal loss due to oxidative stress is a characteristic of Alzheimer's patients; however, our results showed that AD fibroblasts were more resistant to oxidative stress compared to non-AD fibroblasts. Measurement of reactive oxygen species (ROS) indicated that AD fibroblasts produced more ROS than did non-AD NHF cells either in basal conditions or after induction of oxidative stress. Furthermore, we found that expression of p21 was significantly higher in AD cells than in non-AD cells and expression of Bax, a pro-apoptotic protein was downregulated/absent in AD cells during normal or under conditions of external oxidative stress. Further experiments revealed that mitochondria in AD cells moved to the peri-nuclear region following induction of oxidative stress. Thus, these results suggest that AD fibroblasts are chronically exposed to oxidative stress that may trigger senescent phenotype, making AD cell resistant to apoptosis by external oxidative stress.     

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation,thermal or oxidative stress

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.     

Increased expression of Leydig cell haem oxygenase-1 preserves spermatogenesis in varicocele

BACKGROUND: Oxidative stress is involved in the pathogenesis of testicular disorders. Haem oxygenase-1 (HO-1) plays an important cytoprotective role against oxidative stress. We investigated the presence of oxidative stress, represented by generation of 4-hydroxy-2-nonenal (4-HNE)-modified proteins, and expression of HO-1 in varicocele testes of human. METHODS: Thirty testicular biopsies from patients with left varicocele and 10 from patients with normal spermatogenesis were included. Generation of 4-HNE-modified proteins was examined as a marker of oxidative stress. Expression of HO-1 was assessed by western blotting and immunohistochemistry. The expression was compared with clinico-pathological parameters. RESULTS: Increased generation of 4-HNE-modified proteins was observed in varicocele testes. HO-1 expression was significantly correlated with varicocele grade (P < 0.01 in grade 2 and 3 compared to control) and expression of 4-HNE-modified proteins (r = 0.508, P < 0.01). The immunoreactivity was increased in Leydig cells in varicocele testes. There were significant correlations between age, total motile sperm count, Johnsen's mean score and HO-1:4-HNE-modified protein ratio (r = 0.206, 0.405 and 0.408, P = 0.027, 0.027 and 0.025 respectively). CONCLUSIONS: In testes with varicocele, there are increases in 4-HNE-modified proteins, suggesting that oxidative stress is present. Increased HO-1 expression, mainly in Leydig cells, is considered to protect the cells against oxidative stresses in varicocele testes.     

Chronic oxidative stress causes increased tau phosphorylation in M17 neuroblastoma cells

Tau hyperphosphorylation appears to be a critical event leading to abnormal aggregation and disrupted function of tau in affected neurons in Alzheimer's disease (AD). As a prominent early event during AD pathogenesis, oxidative stress is believed to contribute to tau phosphorylation and the formation of neurofibrillary lesions. However, acute oxidative stress has disparate effects on tau phosphorylation. Given the chronic nature of AD, in this study, we aimed to determine the long-term effect of oxidative stress on tau phosphorylation. In this regard, we established a novel in vitro model of chronic oxidative stress through inhibition of glutathione (GSH) synthesis with BSO. We confirmed that these cells were under a chronic mild oxidative stress by looking at oxidative response, the induction of heme oxygenase 1 (HO-1) without neuronal death. Chronic oxidative stress increased levels of tau phosphorylated at PHF-1 epitope (serine 399/404) in a time-dependent manner. Our data further suggest that increased activity of JNK and p38 and decreased activity of PP2A are likely involved in chronic oxidative stress-induced tau phosphorylation. In conclusion we suggest that chronic oxidative stress contributes to increased tau phosphorylation in vitro and could play a critical role in neurofibrillary pathology in vivo.     

Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenic mouse model of Alzheimer's disease: a chronic oxidative paradigm for testing antioxidant therapies in vivo.

Increased expression of antioxidant enzymes and heat-shock proteins are key markers of oxidative stress. Such proteins are abnormally present within the neuropathological lesions of Alzheimer's disease (AD), suggesting that oxidative stress may play significant but yet undefined roles in this disorder. To gain further insight into the role of oxidative stress in AD, we studied the expression of CuZn superoxide dismutase (SOD) and hemoxygenase-1 (HO-1), two established markers of oxidative stress, in a transgenic mouse model of AD. Immunohistochemistry with anti-SOD and anti-HO-1 antibodies revealed a very pronounced increase of these proteins only in aged transgene-positive mice. Interestingly, the distribution of the oxidative burden was largely overlapping with dystrophic neuritic elements in the mice as highlighted with anti-ubiquitin antibodies. Because the most conspicuous alterations were identified around amyloid (Abeta) deposits, our results provide strong support for the hypothesis that Abeta is neurotoxic in vivo and that such toxicity is mediated by free radicals. To obtain additional experimental evidence for such an interpretation (ie, a cause-effect relationship between Abeta and oxidative neurotoxicity), PC12 cells were exposed to increasing concentrations of Abeta or to oxidative stress. In agreement with the in vivo findings, either treatment caused marked induction of SOD or HO-1 in a dose-dependent fashion. These results validate the transgenic approach for the study of oxidative stress in AD and for the evaluation of antioxidant therapies in vivo.     

Evidence for a novel heme-binding protein, HasAh, in Alzheimer disease

Multiple lines of evidence indicate that oxidative stress is an integral component of the pathogenesis of Alzheimer disease (AD). The precipitating cause of such oxidative stress may be misregulated iron homeostasis because there are profound alterations in heme oxygenase-1 (HO-1), redox-active iron, and iron regulatory proteins. In this regard, HasA, a recently characterized bacterial protein involved in heme acquisition and iron metabolism, may also be important in the generation of reactive oxygen species (ROS) given its ability to bind heme and render iron available for free radical generation through the Fenton reaction. To study further the role of heme binding and iron metabolism in AD, we show an abnormal localization of anti-HasA to the neurofibrillary pathology of AD, but not in normal-appearing neurons in the brains of cases of AD or in age-matched controls. These results suggest the increased presence in AD of a HasA homologue or protein sharing a common epitope with HasA, which we term HasAh. We conclude that heme binding of HasAh is a potential source of free soluble iron and therefore toxic free radicals in AD and in aging. This furthers the evidence that redox-active iron and subsequent Fenton reaction generating reactive oxygen are critical factors in the pathogenesis of AD.     

Redox regulation of heat shock protein expression by signaling involving nitric oxide and carbon monoxide: relevance to brain aging, neurodegenerative disorders, and longevity

Increased free radical generation and decreased efficiency of the reparative/degradative mechanisms both primarily contribute to age-related elevation in the level of oxidative stress and brain damage. Excess formation of reactive oxygen and nitrogen species can cause proteasomal dysfunction and protein overloading. The major neurodegenerative diseases are all associated with the presence of abnormal proteins. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed vitagenes, including the heat shock protein (HSP) system. Of the various HSPs, heme oxygenase-I (HO-1), by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. The HO-1 gene is redox regulated and its expression is modulated by redox active compounds, including nutritional antioxidants. Given the broad cytoprotective properties of the heat shock response, there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. These findings have opened up new neuroprotective strategies, as molecules inducing this defense mechanism can be a therapeutic target to minimize the deleterious consequences associated with accumulation of conformationally aberrant proteins to oxidative stress, such as in neurodegenerative disorders and brain aging, with resulting prolongation of a healthy life span.     

Gene expression of heme oxygenase-1 during glial activation by lipopolysaccharide

The effect of bacterial lipopolysaccharide (LPS) on the expression of mRNA encoding heme oxygenase-1 (HO-1), which is the rate limiting enzyme in heme catabolism and is also known as heat shock protein 32 (HSP32), was examined in primarily cultured rat glial cells. Treatment of cells with LPS elicited an increase in HO-1 mRNA, accompanying down regulation of delta-aminolevulinate synthase, in a dose-dependent fashion. HO-1 mRNA increased markedly at 12 h after LPS treatment (10 microg/ml) and reached a maximum at 24 h. In contrast, HSP70, a major heat shock protein, slightly increased only at 6 h after LPS treatment and returned to the control level by 12 h. These results suggest that HSPs are induced under separate regulation during glial activation by LPS through oxidative stress, a part of which is likely mediated by intracellular free heme.     

Opposite effect of oxidative stress on inducible nitric oxide synthase and haem oxygenase-1 expression in intestinal inflammation: anti-inflammatory effect of carbon monoxide

Inducible nitric oxide synthase (iNOS) is expressed in intestinal epithelial cells (IEC) of patients with active inflammatory bowel disease (IBD) and in IEC of endotoxaemic rats. The induction of iNOS in IEC is an element of the NF-kappaB-mediated survival pathway. Haem oxygenase-1 (HO-1) is an AP-1-regulated gene that is induced by oxidative stress. The enzyme produces carbon monoxide (CO), which may attenuate the inflammatory response. The aim of this study was to investigate the regulation and interaction of iNOS and HO-1 in response to inflammation and oxidative stress. Male Wistar rats were treated with the thiol-modifying agent diethylmaleate (DEM) to induce oxidative stress and rendered endotoxaemic by LPS injection. Human colonic biopsies and the human colon carcinoma cell line DLD-1 were treated with DEM and the lipid peroxidation end-product 4-hydroxynonenal to induce oxidative stress and exposed to cytokine mix (CM) to mimic inflammation. In some experiments, cells were incubated with 250-400 ppm CO prior to and during stimulation with CM. HO-1 and iNOS expression was evaluated by RT-PCR, western blotting, and immunohistology. NF-kappaB activation was evaluated by EMSA. LPS induced iNOS but not HO-1 in epithelial cells of the ileum and colon. Oxidative stress strongly induced HO-1 in epithelial and inflammatory cells. Combined oxidative stress and endotoxaemia decreased iNOS expression but strongly induced HO-1 expression. Similarly, CM induced iNOS but not HO-1 in colonic biopsies and DLD-1 cells. Oxidative stress prevented iNOS induction in an NF-kappaB-dependent manner but increased HO-1 expression in CM-exposed DLD-1 cells. CO inhibited iNOS mRNA induction in CM-stimulated DLD-1 cells. These data demonstrate opposite regulation of iNOS and HO-1 in intestinal epithelial cells in response to cytokine exposure and oxidative stress. These findings suggest that iNOS (NF-kappaB driven) and HO-1 (AP-1 driven) represent mutually exclusive survival mechanisms in intestinal epithelial cells.     

Proteomic identification of proteins specifically oxidized by intracerebral injection of amyloid beta-peptide (1-42) into rat brain: implications for Alzheimer's disease

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.     

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.     

Age-related changes in HSP25 expression in basal ganglia and cortex of F344/BN rats

Normal aging is associated with chronic oxidative stress. In the basal ganglia, oxidative stress may contribute to the increased risk of Parkinson's disease in the elderly. Neurons are thought to actively utilize compensatory defense mechanisms, such as heat shock proteins (HSPs), to protect from persisting stress. Despite their protective role, little is known about HSP expression in the aging basal ganglia. The purpose of this study was to examine HSP expression in striatum, substantia nigra, globus pallidus and cortex in 6-, 18- and 30-month-old Fischer 344/Brown Norway rats. We found robust age-related increases in phosphorylated and total HSP25 in each brain region studied. Conversely, HSP72 (the inducible form of HSP70) was reduced with age, but only in the striatum. p38 MAPK, a protein implicated in activating HSP25, did not change with age, nor did HSC70 (the constitutive form of HSP70), or HSP60. These results suggest that HSP25 is especially responsive to age-related stress in the basal ganglia.     

热休克蛋白90在硫化氢抗化学性缺氧诱导心肌细胞氧化应激损伤中的作用

目的： 探讨热休克蛋白90（HSP90）在硫化氢（H2S）对抗化学性低氧模拟剂氯化钴（CoCl2）诱导H9c2心肌细胞氧化应激损伤中的作用。方法： 应用CoCl2处理H9c2心肌细胞,建立化学性缺氧损伤心肌细胞的实验模型。在CoCl2处理H9c2心肌细胞前30 min,把硫氢化钠（NaHS,H2S的供体）加入培养基中,作为预处理。应用高效液相色谱法（HPLC）检测细胞内ATP的含量;罗丹明123（Rh123）染色荧光显微镜照相检测线粒体膜电位（MMP）;超氧化物歧化酶（SOD）检测试剂盒检测SOD活性;免疫印迹法（Western blotting）检测血红素氧合酶-1（HO-1）的表达。结果： 600 μmol/L CoCl2明显地降低H9c2心肌细胞内SOD活性、ATP水平及MMP,并增加HO-1表达。400 μmol/L NaHS 预处理可显著地抑制CoCl2诱导的细胞毒性及氧化应激反应,使SOD活性、ATP水平及MMP提高,HO-1表达减少。热休克蛋白90抑制剂17-丙烯胺基-17去甲氧基格尔德霉素（17AAG）能明显地阻断H2S对CoCl2诱导的细胞毒性和氧化应激反应的抑制作用,使细胞内ATP水平及MMP降低,HO-1表达增多,但对SOD活性的影响不明显。结论： 热休克蛋白90可通过抑制化学性缺氧引起的氧化应激反应来介导H2S的心肌保护作用。     

Blood-brain barrier disruption and oxidative stress in guinea pig after systemic exposure to modified cell-free hemoglobin

Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis.     

Amyloid beta-peptide(1-42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain

Oxidative stress is extensive in Alzheimer disease (AD) brain. Amyloid beta-peptide (1-42) has been shown to induce oxidative stress and neurotoxicity in vitro and in vivo. Genetic mutations that result in increased production of Abeta1-42 from amyloid precursor protein are associated with an early onset and accelerated pathology of AD. Consequently, Abeta1-42 has been proposed to play a central role in the pathogenesis of AD as a mediator of oxidative stress. In this review, we discuss the role of Abeta1-42 in the lipid peroxidation and protein oxidation evident in AD brain and the implications of such oxidative stress for the function of various proteins that we have identified as specifically oxidized in AD brain compared to control, using proteomics methods. Additionally, we discuss the critical role of methionine 35 in the oxidative stress and neurotoxic properties exhibited by Abeta1-42.