Enhancement of intracellular glutathione protects endothelial cells against oxidant damage

We studied the role of glutathione in the endothelial cell defense against H2O2 damage. Treatment of endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, depleted the cells of GSH, while L-2-oxothiazolidine-4-carboxylate, an effective intracellular cysteine delivery agent, markedly enhanced endothelial cell GSH concentration. Depletion of intracellular GSH sensitized the endothelial cells to injury by H2O2 either preformed or generated by the glucose-glucose oxidase system. In contrast, an increase of intracellular GSH protected the cells from H2O2 damage. There was an inverse, linear relationship between the intracellular GSH concentrations and killing of endothelial cells by H2O2. Our results suggest that enhancement of endothelial cell GSH may be an alternative approach toward the prevention of oxidant-induced endothelial damage such as adult respiratory distress syndrome.     

Gpx4 protects mitochondrial ATP generation against oxidative damage

Mitochondrial ATP production can be impaired by oxidative stress. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme found in mitochondria as well as other subcellular organelles that directly detoxifies membrane lipid hydroperoxides. To determine if Gpx4 protects ATP production in vivo, we compared mitochondrial ATP production between wild-type mice and Gpx4 transgenic mice using a diquat model. Diquat (50 mg/kg) significantly decreased mitochondrial ATP synthesis in livers of wild-type mice; however, no decrease in mitochondrial ATP synthesis was detected in Gpx4 transgenic mice after diquat. We observed no differences in activities of mitochondrial respiratory chain complexes between Gpx4 transgenic mice and wild-type mice. However, compared to wild-type mice, diquat-induced loss of mitochondrial membrane potential was attenuated in Gpx4 transgenic mice. Therefore, our results indicate that decreased ATP production under oxidative stress is primarily due to reduced mitochondrial membrane potential and overexpression of Gpx4 maintains mitochondrial membrane potential under oxidative stress.     

Hydrogen sulfide protects against vascular remodeling from endothelial damage

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H₂S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT + NaHS (sodium hydrogen sulfide, precursor of H₂S) treatment (30 μmol/L in drinking water/6 weeks) and WTD + NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H₂S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H₂S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.     

Prion protein protects against DNA damage induced by paraquat in cultured cells

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.     

Cellular prion protein protects against reactive-oxygen-species-induced DNA damage

Although the cellular form of the prion protein (PrPC) is critical for the development of prion disease through its conformational conversion into the infectious form (PrPSc), the physiological role of PrPC is less clear. Using alkaline single-cell gel electrophoresis (the Comet assay), we show that expression of PrPC protects human neuroblastoma SH-SY5Y cells against DNA damage under basal conditions and following exposure to reactive oxygen species, either hydroxyl radicals following exposure to Cu2+ or Fe2+ or singlet oxygen following exposure to the photosensitizer methylene blue and white light. Cells expressing either PrPDeltaoct which lacks the octapeptide repeats or the prion-disease-associated mutants A116V or PG14 had increased levels of DNA damage compared to cells expressing PrPC. In PrPSc-infected mouse ScN2a cells there was a significant increase in DNA damage over noninfected N2a cells (median tail DNA 2.87 and 7.33%, respectively). Together, these data indicate that PrPC has a critical role to play in protecting cells against reactive-oxygen-species-mediated DNA damage; a function which requires the octapeptide repeats in the protein, is lost in disease-associated mutants of the protein or upon conversion to PrPSc, and thus provide further support for the neuroprotective role for PrPC.     

Hsa-miRNA-29a protects against high glucose-induced damage in human umbilical vein endothelial cells

Sustained exposure to high glucose (HG) results in dysfunction of vascular endothelial cells. Hence, diabetic patients often suffer from secondary vascular damages, such as vascular sclerosis and thrombogenesis, which may eventually cause cardiovascular problems. Thus, elucidating how HG results in vascular endothelial cell damage and finding an approach for prevention are important to prevent and treat vascular damages in diabetic patients. In the current study, we first showed that 72-hour exposure to HG-decreased hsa-miRNA-29a and increased the expression of Bcl-2 associated X protein (Bax), which subsequently inhibited Bcl-2 and promoted the expression of apoptotic protease activating factor-1 and activation of caspase-3, thus directly triggering the mitochondrial apoptotic pathway in human umbilical vein endothelial cells (HUVECs). Study of the underlying mechanism showed that hsa-miRNA-29a/Bax plays an essential role in the decreased proliferation and increased apoptosis of HUVECs induced by HG, and overexpression of hsa-miRNA-29a effectively inhibits HG-induced apoptosis and restores the proliferation and tube formation of HUVECs exposed to HG by inhibiting its target gene Bax. In short, our study demonstrates that hsa-miRNA-29a is a promising target for the prevention and treatment of vascular injury in diabetic patients.     

Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.     

AKR1B10 protects against UVC-induced DNA damage in breast cancer cells

The cellular response to DNA damage is crucial for maintaining the integrity and stability of molecular structure.To maintain genome stability,DNA-damaged cells...     

Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage.

Rat ventricular myocytes have been isolated and cultured by two separate procedures. Using phase-contrast and electron microscopies, we illustrate that (a) definitive cell damage is produced when myocytes are exposed to xanthine oxidase--hypoxanthine and (b) purpurogallin between 0.25 and 1.0 mM prolongs survival of both myocyte preparations in a dose-dependent manner. The cytoprotection produced by 1 mM purpurogallin exceeds that given by 2 mM each of ascorbate, Trolox, and mannitol, or 24,200 IU superoxide dismutase/L and (or) 92,000 IU catalase/L. Furthermore, we noted, for the first time, that purpurogallin markedly protects rat aortic endothelial cells, a key target of free radical generation and attack. In contrast, Trolox has a negligible effect here. Mechanistically, we showed that purpurogallin inhibits urate formation by xanthine oxidase more potently than allopurinol. Also, the compound diminishes formation of superoxide-reduced cytochrome c. Therefore, purpurogallin is a potent protector of ventricular myocytes and aortic endothelial cells, both of which are important cells in the cardiovascular system.     

Autophagy protects auditory hair cells against neomycin-induced damage

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.     

Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells

BackgroundApoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5′-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC.Methods and resultsTo simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po₂ < 4 mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/?0.4; P < 0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/?0.5; P < 0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αβ-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP.ConclusionThe data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.     

MMS1 protects against replication-dependent DNA damage in Saccharomyces cerevisiae

A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.     

Macelignan protects HepG2 cells against tert-butylhydroperoxide-induced oxidative damage

In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.     

XRCC1 protects against the lethality of induced oxidative DNA damage in nondividing neural cells

XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR). Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias—aprataxin and tyrosyl-DNA phosphodiesterase 1—implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. We demonstrate herein that shRNA lentiviral-mediated XRCC1 knockdown in human SH-SY5Y neuroblastoma cells results in a largely selective increase in sensitivity of the nondividing (i.e. terminally differentiated) cell population to the redox-cycling agents, menadione and paraquat; this reduced survival was accompanied by an accumulation of DNA strand breaks. Using hypoxanthine–xanthine oxidase as the oxidizing method, XRCC1 deficiency affected both dividing and nondividing SH-SY5Y cells, with a greater effect on survival seen in the former case, suggesting that the spectrum of oxidative DNA damage created dictates the specific contribution of XRCC1 to cellular resistance. Primary XRCC1 heterozygous mouse cerebellar granule cells exhibit increased strand break accumulation and reduced survival due to increased apoptosis following menadione treatment. Moreover, knockdown of XRCC1 in primary human fetal brain neurons leads to enhanced sensitivity to menadione, as indicated by increased levels of DNA strand breaks relative to control cells. The cumulative results implicate XRCC1, and more broadly SSBR, in the protection of nondividing neuronal cells from the genotoxic consequences of oxidative stress.     

Werner protein protects nonproliferating cells from oxidative DNA damage

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.     

Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages

Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.     

The DNA dioxygenase ALKBH2 protects Arabidopsis thaliana against methylation damage

The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro, but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo. The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function.     

Magnesium tanshinoate B protects endothelial cells against oxidized lipoprotein-induced apoptosis

The activation of c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in the induction of cell apoptosis. We previously reported that magnesium tanshinoate B (MTB), a compound purified from a Chinese herb danshen (Salvia miltiorrhiza), could inhibit ischemia/reperfusion-induced myocyte apoptosis in the heart. The objective of the present study was to investigate whether MTB can prevent oxidized lipoprotein-induced apoptosis in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated with copper-oxidized very low density lipoprotein (Cu-OxVLDL) or copper-oxidized low density lipoprotein (Cu-OxLDL). Treatment of cells with Cu-OxVLDL or Cu-OxLDL resulted in a 3-fold increase in the JNK activity. The amount of cytochrome c released and the activity of caspase-3 in cells treated with Cu-OxVLDL or Cu-OxLDL were significantly elevated, indicating the occurrence of apoptosis. The presence of MTB was able to abolish the JNK activation, cytochrome c release, and caspase-3 activation induced by Cu-OxVLDL or Cu-OxLDL, resulting in a marked reduction in apoptosis in endothelial cells. The data from this study indicate that oxidized lipoproteins induce apoptosis in endothelial cells. We postulate that the inhibition of the JNK signaling pathway by MTB is a key mechanism that protects these cells from oxidized lipoprotein-induced apoptosis.