Measured Superoxide Production Research Articles

Pravastatin decreases superoxide production by thick ascending limbs

Hydroxy‐methyl‐glutaryl‐CoA reductase inhibitors (statins) are used in the treatment of hyperlipidemia. Studies suggest that statins have anti‐inflammatory effects. Many of these may be mediated by nitric oxide (NO). Thick ascending limbs (TAL) express NO synthase and we have shown that statins increase NO production in TAL. In separate experiments we have shown that NO can reduce superoxide production. Thus, we hypothesized that pravastatin decreases superoxide production in TAL via NO. We measured superoxide production in 1‐minute periods in medullary TAL suspensions from Sprague‐Dawley rats using the lucigenin assay. The difference in luminescence in the absence and presence of tiron was taken as superoxide production. Tubules were treated with 10 uM pravastatin for 15 minutes. To study the role of NO, tubules were treated with 1 mM L‐NAME. When L‐NAME was used, it was added with the pravastatin. Control TAL produced superoxide at a rate of 1274 +/− 305 AU/min. TAL that had been treated with pravastatin produced superoxide at a rate of 697 +/− 225 AU/min (p < 0.01), 45% less. In the presence of L‐NAME alone, TAL produced superoxide at 1307 +/− 305 AU/min. Tubules treated with pravastatin plus L‐NAME produced superoxide at a rate of 595 ± 277 AU/min, 54% less (p< 0.01 vs. L‐NAME alone). We conclude that: 1) pravastatin inhibits superoxide production by TAL; and 2) NO does not mediate this effect.

Mitochondrial electron transport and glycolysis are coupled in articular cartilage

Although the majority of the adenosine triphosphate (ATP) in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport (ET) help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits ET and blocks oxidant production inhibits glycolytic ATP synthesis. Bovine osteochondral explants were treated with rotenone, an ET inhibitor; or oligomycin an ATP synthase inhibitor; or 2-fluoro-2-deoxy-D-glucose, a glycolysis inhibiter; or peroxide, an exogenous oxidant; or mitoquinone (MitoQ), a mitochondria-targeted anti-oxidant. Cartilage extracts were assayed for ATP, nicotine adenine dinucleotide (NAD+/H), and culture medium was assayed for pyruvate and lactate after 24 h of treatment. Imaging studies were used to measure superoxide production in cartilage. Rotenone and 2-FG caused a significant decline in cartilage ATP (P < 0.001). In contrast, ATP levels were not affected by oligomycin. Peroxide treatment blocked rotenone effects on ATP, while treatment with MitoQ significantly suppressed ATP levels. Rotenone and 2-FG caused a significant decline in pyruvate, but not in lactate production. NADH:NAD+ ratios decreased significantly in both rotenone and 2-FG-treated explants (P < 0.05). Rotenone also significantly reduced superoxide production. These findings showing a link between glycolysis and ET are consistent with previous reports on the critical need for oxidants to support normal chondrocyte metabolism. They suggest a novel role for mitochondria in cartilage homeostasis that is independent of oxidative phosphorylation.

Response to Tetrahydrobiopterin and Endothelial Nitric Oxide Synthase Uncoupling

Although we certainly recognize the excellent work done by Tsikas et al1 using purified endothelial NO synthase (eNOS) and incubation with tetrahydrobiopterin (H4B) at much higher concentrations (see Reference 4 and Figure in Reference 1) than what is found in vivo by many independent groups including us,2–4 we respectfully disagree based on the following grounds. First, accurate assessment of the uncoupling state of eNOS and determination of endogenous H4B content to the level of picomoles per milligram of protein in vivo can be successfully achieved. A large body of published work from many independent groups including ours has confirmed this consistently.2–4 In our case, we used the highly sensitive electron spin resonance method to measure superoxide production and high-performance liquid chromatography …

Trans Fatty Acids Induce Vascular Inflammation and Reduce Vascular Nitric Oxide Production in Endothelial Cells

Intake of trans fatty acids (TFA), which are consumed by eating foods made from partially hydrogenated vegetable oils, is associated with a higher risk of cardiovascular disease. This relation can be explained by many factors including TFA's negative effect on endothelial function and reduced nitric oxide (NO) bioavailability. In this study we investigated the effects of three different TFA (2 common isomers of C18 found in partially hydrogenated vegetable oil and a C18 isomer found from ruminant-derived—dairy products and meat) on endothelial NF-κB activation and nitric oxide (NO) production. Human endothelial cells were treated with increasing concentrations of Elaidic (trans-C18:1 (9 trans)), Linoelaidic (trans-C18:2 (9 trans, 12 trans)), and Transvaccenic (trans-C18:1 (11 trans)) for 3 h. Both Elaidic and Linoelaidic acids were associated with increasing NF-κB activation as measured by IL-6 levels and phosphorylation of IκBα, and impairment of endothelial insulin signaling and NO production, whereas Transvaccenic acid was not associated with these responses. We also measured superoxide production, which has been hypothesized to be necessary in fatty acid-dependent activation of NF-κB. Both Elaidic acid and Linoelaidic acid are associated with increased superoxide production, whereas Transvaccenic acid (which did not induce inflammatory responses) did not increase superoxide production. We observed differential activation of endothelial superoxide production, NF-κB activation, and reduction in NO production by different C18 isomers suggesting that the location and number of trans double bonds effect endothelial NF-κB activation.

Abstract P032: Nanoparticle Mediated Nox2-siRNA Therapy for Preventing Cardiac Dysfunction Following Myocardial Infarction

Myocardial infarction (MI) is the most common cause of heart failure in the developed world. Following MI, reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling leading to impaired ventricular function. Oxidative stress at high levels lead to many of the injury associated changes: proinflammatory cytokine release, myocyte apoptosis, cardiac fibrosis and hypertrophy. Nicotinamide adenine denucleotide phosphate (NADPH) oxidase with Nox2 as the catalytic subunit, is a major source for cardiac ROS production. After MI, Nox2 expression is significantly increased in the infarcted myocardium. Moreover, mice lacking the Nox2 gene are protected from ischemic injury. We used polyketals, a new class of acid-degradable polymers, as delivery vehicles for Nox2-siRNA to the post-MI environment. When engaged by macrophages, present in high quantities during MI, these particles have been shown to be taken up by macrophages and contents released within cells in active form. Nox2-siRNA was ion-paired to the cationic lipid DOTAP, and spherical particles averaging 500nm diameter were made by a single emulsion procedure with polyketal PK3 using PVA as surfactant. While a commercially available transfection reagent yielded 7% uptake as measured by flow cytometery, 81% of macrophages were positive for fluorescently-labeled siRNA in PK3-siNOX2 treated cells. These data were confirmed with confocal microscopy. Macrophages treated with PK3-siNOX2 demonstrated a significant 43% knockdown in Nox2 gene expression at 24 hours, while no reduction was seen with scrambled siRNA or empty particles. Functional activity was assessed by a fluorescent dihyroethidium dye based HPLC quantification after phorbol 12-myristate 13-acetate stimulation following 72 hours of particle treatment to measure superoxide production. PK3-siNox2 treated cells exhibited a 41% reduction in activity, with no significant changes seen with scrambled siRNA or empty particle treatment. Currently, the therapeutic potential of the Nox2-siRNA particles is being evaluated in a mouse model of ischemia-reperfusion. Successful completion of these studies could lead to a novel treatment for post-infarction injury.

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.

The Long Life of Birds: The Rat-Pigeon Comparison Revisited

The most studied comparison of aging and maximum lifespan potential (MLSP) among endotherms involves the 7-fold longevity difference between rats (MLSP 5y) and pigeons (MLSP 35y). A widely accepted theory explaining MLSP differences between species is the oxidative stress theory, which purports that reactive oxygen species (ROS) produced during mitochondrial respiration damage bio-molecules and eventually lead to the breakdown of regulatory systems and consequent death. Previous rat-pigeon studies compared only aspects of the oxidative stress theory and most concluded that the lower mitochondrial superoxide production of pigeons compared to rats was responsible for their much greater longevity. This conclusion is based mainly on data from one tissue (the heart) using one mitochondrial substrate (succinate). Studies on heart mitochondria using pyruvate as a mitochondrial substrate gave contradictory results. We believe the conclusion that birds produce less mitochondrial superoxide than mammals is unwarranted.We have revisited the rat-pigeon comparison in the most comprehensive manner to date. We have measured superoxide production (by heart, skeletal muscle and liver mitochondria), five different antioxidants in plasma, three tissues and mitochondria, membrane fatty acid composition (in seven tissues and three mitochondria), and biomarkers of oxidative damage. The only substantial and consistent difference that we have observed between rats and pigeons is their membrane fatty acid composition, with rats having membranes that are more susceptible to damage. This suggests that, although there was no difference in superoxide production, there is likely a much greater production of lipid-based ROS in the rat.We conclude that the differences in superoxide production reported previously were due to the arbitrary selection of heart muscle to source mitochondria and the provision of succinate. Had mitochondria been harvested from other tissues or other relevant mitochondrial metabolic substrates been used, then very different conclusions regarding differences in oxidative stress would have been reached.

Reduced endothelial dependent vasodilation in vessels from TLR4−/− mice is associated with increased superoxide generation

Toll like receptor (TLR)4 is a pattern recognition receptor expressed in endothelial and other cells, responsible for the sensing of endotoxin and host derived ligands. Our group has shown previously that the absence of TLR4 is associated with reduced endothelial dependent vasodilator responses and left heart hypertrophy in animal models. However, the mechanism behind reduced endothelial cell function in TLR4−/− mice is not known.We have used en face confocal imaging of mesenteric arteries from mice deficient in the TLR4 receptor stained with dihydroethidium (DHE) to measure superoxide production. Using the isometric wire myograph, mesenteric artery vasodilator responses to acetylcholine and MnCl2 (a superoxide dismutase mimetic) were measured. Mesenteric arteries from TLR4−/− mice had a reduced endothelial dependent relaxant response and increased superoxide levels when stimulated with acetylcholine. Increased levels of superoxide, as detected by DHE staining, were seen in vessels from TLR4−/− mice, which were reduced to control levels in the presence of MnCl2.Our observations suggest that loss of TLR4 increases superoxide generation which reduces the biological activity of endothelial derived nitric oxide and thereby explains the endothelial dysfunction and associated cardiovascular phenotype in TLR4−/− mice. These data implicate a novel cardio-protective role for TLR4 in vascular homeostasis.

Spatial and temporal patterns of oxidative stress in the brain of gerbils submitted to different duration of global cerebral ischemia

The present study was undertaken to examine spatial and temporal patterns of oxidative stress rate in the brain of Mongolian gerbils submitted to different duration of global ischemia/reperfusion. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. We followed the temporal ischemia-induced oxidative stress rate, the most important factor that exacerbates brain damage by reperfusion, starting from 24 h up to 28 days after reperfusion. The spatial ischemia-induced oxidative stress distribution was measured parallely in different brain regions: forebrain cortex, striatum, hippocampus and cerebellum. Post-ischemic effects were followed in vivo by monitoring the neurological status of whole animals and at the intracellular level by standard biochemical assays in different brain regions. We measured superoxide production, superoxide dismutase activity, nitric oxide production, index of lipid peroxidation, and reduced glutathione. Our results revealed a pattern of dynamic changes in each oxidative stress parameter that corresponded with ischemia duration in all tested brain structures. The highest levels were obtained in the first 24 h after the insult. After that, they slowly returned to nearly control values 28 days after reperfusion (with the exception of SOD activity that returned to control values at fourth day after reperfusion). The most sensitive oxidative stress parameter was index of lipid peroxidation. Our study confirmed spatial distribution of ischemia-induced oxidative stress. Tested brain structures showed different sensitivity to each oxidative stress parameter, although their basal levels were similar. These new findings could be valuable for creation and strategy of post-ischemic therapy.

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1: Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc1 can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQo) formed at the quinone oxidation/reduction Qo site (Qo) as a result of single-electron oxidation of quinol by the iron–sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme bL (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc1 that severely impeded the oxidation of FeS by cytochrome c1, changed density of FeS around Qo by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Qo and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQo that present a risk of generation of superoxide by cytochrome bc1. Isolation of this reaction sequence from multiplicity of possible reactions at Qo helps to better understand conditions under which complex III might contribute to ROS generation in vivo.

ICAM-1 cytoplasmic tail regulates endothelial glutathione synthesis through a NOX4/PI3-kinase-dependent pathway

We previously reported that ICAM-1 expression modulates endothelial intracellular glutathione (GSH) metabolism through unknown mechanisms. Here we report that the cytoplasmic tail of ICAM-1 is critically involved in governing intracellular GSH production. Peptides containing the antennapedia cell-permeative sequence (AP) or an AP peptide linked to the transmembrane and cytosolic tail of ICAM-1 (AP-ICAM) were synthesized and used to measure alterations in redox status in cultured endothelial cells and determine their biological effect. Treatment with AP-ICAM significantly increased GSH concentrations and glutamate–cysteine ligase (GCL) activity over time. Measuring reactive oxygen species (ROS) production with DCF revealed a rapid increase in ROS generation after AP-ICAM treatment. Measurement of superoxide production with hydroethidium revealed biphasic production at 30 min and 6 h after treatment with AP-ICAM. Apocynin, DPI, catalase, or SOD attenuated AP-ICAM-dependent ROS production, GCL activity, and GSH production, implicating superoxide production and dismutation to peroxide. Consistent with these findings, NOX4 siRNA knockdown blocked AP-ICAM peptide increases in GSH or GCL activity, demonstrating the importance of NADPH oxidase. Last, inhibition of PI3-kinase activity with LY 294002 or wortmannin blocked AP-ICAM GSH induction and ROS production. These data reveal that the ICAM-1 cytoplasmic tail regulates production of endothelial GSH through a NOX4/PI3-kinase-dependent redox-sensitive pathway.

Ketone pretreatment reduces superoxide production in human U87 glioblastoma cells exposed to normoxia and hyperoxia

Unlike normal brain cells, cancer cells lack the metabolic flexibility to use ketones as fuel. Brain cancer cells have defective mitochondria and derive energy primarily through aerobic glycolysis (the Warburg effect). Cancer growth is intimately linked to glucose‐stimulated reactive oxygen species (ROS) production. We hypothesized that ketones would reduce mitochondrial ROS production in cultured human U87 glioblastoma cells. Fluorescence microscopy and confocal spectral imaging were used to detect real‐time intracellular superoxide production with dihydroethidium (DHE). Cultured U87 cells were maintained in media containing 1mM glucose in the presence or absence of 2‐10mM D‐beta‐hydroxybutyrate (BHB) for 4‐6 days. Results demonstrate that superoxide production (measured as DHE fluorescence intensity units; FIU) was approximately 300% higher in untreated cells versus BHB‐treated cells (2 mM). Measurement of superoxide production during normoxia (60 minutes) increased FIUs 49 ± 3, 14 ± 2 and 38 ± 2 in untreated, 2mM and 10mM BHB, respectively. Hyperoxia‐induced mitochondrial superoxide production was abolished in U87 cells pretreated with 2mM BHB. We conclude that supplemental ketones significantly reduce superoxide production in U87 cells. These results suggest that supplemental ketones may confer protection against malignant brain cancer.

Systemic and microvascular oxidative stress induced by light chain amyloidosis

Light chain amyloidosis (AL) is a plasma cell dyscrasia associated with production of amyloidogenic immunoglobulin light chains (LC). Despite its often fatal course, the mechanism of injury remains unknown. We tested the hypothesis that AL is associated with oxidative stress by comparing serum protein carbonyl (a marker of protein oxidation and oxidative stress) in AL subjects (n=23, 60 ± 11 years) vs. controls (n=9, 54 ± 2 years); we also measured superoxide production (n=11) and dilator response to sodium nitroprusside (SNP, n=6) in isolated non-AL human adipose arterioles exposed to LC (20 μg/mL) purified from AL subjects for 1 h vs. control. Protein carbonyl was higher in AL patients (0.19 ± 0.04 vs. 0.003 ± 0.003 nmol/mg control, p=0.002). Post-exposure to LC proteins, arteriole superoxide was higher (1.89 ± 0.36 times control, p=0.03) with impaired dilation to SNP (10(-4) M, 54 ± 6 vs. 86 ± 4%, p=0.01, logEC50 -3.7 ± 0.2 vs. -6.7 ± 0.6, p=0.002). AL is associated with systemic oxidative stress and brief acute exposure to AL light chain proteins induces oxidative stress and microvascular dysfunction in human adipose arterioles. This novel mechanism of injury may be important in AL pathophysiology.

ROLE OF ENDOTHELIN IN IMPAIRED RESPONSES OF CEREBRAL ARTERIOLES DURING TYPE 1 DIABETES MELLITUS

Previous studies have suggested that endothelin‐1 may contribute to vascular abnormalities during a variety of disease states, including Type 1 diabetes mellitus. Endothelin‐1 may contribute to structural and functional abnormalities of the blood vessels, and may also regulate the expression of other growth factors and cytokines to influence vascular function. However, there is a lack of information regarding the precise role of endothelin‐1 in altered NOS‐dependent responses of cerebral arterioles during Type 1 diabetes. Thus, our goal was to determine whether acute inhibition of endothelin‐1 receptors (BQ‐123) could influence NOS‐dependent responses of cerebral arterioles in diabetic rats. We measured diameter of pial arterioles in nondiabetic and diabetic (STZ; 50 mg/kg) rats in response to eNOS‐ and nNOS‐dependent (ADP and NMDA) and ‐independent (nitroglycerin) agonists before and during treatment with BQ‐123. In addition, we measured superoxide production by cerebral cortex tissue obtained from nondiabetic and diabetic rats. We found that eNOS‐ and nNOS‐dependent dilation of pial arterioles was impaired in diabetic compared to nondiabetic rats. In addition, treatment with BQ‐123 restored impaired responses of cerebral arterioles in diabetic rats towards that observed in nondiabetic rats. Further the production of superoxide anion by cortex tissue was increased in diabetic rats when compared to nondiabetic rats. We suggest that endothelin‐1 may contribute to impaired responses of cerebral arterioles during Type 1 diabetes. We speculate that endothelin receptor antagonism may be a potential therapeutic tool for the treatment of cerebrovascular dysfunction observed in diabetic subjects.

Age‐Related Alterations in Reactivity of Cerebral Arterioles: Role of Oxidative Stress

Our goal was to identify the role of oxidative stress via activation of NAD(P)H oxidase in cerebrovascular dysfunction in aged rats. We examined the reactivity of cerebral arterioles in adult and aged Fisher-344 rats to endothelial nitric oxide synthase (eNOS)-dependent (acetylcholine and adenosine diphosphate [ADP]) and-independent (nitroglycerin) agonists before and during application of tempol, apocynin, and diphenyleneiodonium chloride (DPI). We used Western blot to examine subunits of NAD(P)H oxidase, eNOS, and superoxide dismutase (SOD-1) in cerebral microvessels and parietal cortex. Finally, we measured superoxide production by cortex tissue in adult and aged rats. Acetylcholine-and ADP-induced, but not nitroglycerin-induced, dilatation of cerebral arterioles was impaired in aged compared to adult rats. While tempol, apocynin, and DPI did not alter responses in adults, they alleviated impaired eNOS-dependent vasodilatation in aged rats, without influencing responses to nitroglycerin. eNOS and p67phox proteins were increased in cerebral microvessels from aged compared to adult rats. Further, p67phox and gp91phox proteins were increased, but SOD-1 protein was decreased, in cortex tissue of aged rats. Basal and agonist-induced production of superoxide was elevated in aged rats. Aging impairs eNOS-dependent reactivity of cerebral arterioles via an increase in superoxide produced by activation of NAD(P)H oxidase.

Glutathione Improves Impaired Responses of Cerebral Arterioles in Type 1 Diabetes

Our goal was to examine whether glutathione improves impaired nitric oxide synthase‐dependent responses of cerebral arterioles during Type 1 diabetes. We measured diameter of cerebral arterioles in nondiabetic and diabetic (streptozotocin; 50 mg/kg) rats to nitric oxide synthase‐dependent, acetylcholine and adenosine 5’‐diphosphate (ADP) and a nitric oxide synthase‐independent agonists, nitroglycerin. We measured responses before and during topical application of glutathione (5 mM). In addition, we measured superoxide production by brain tissue in nondiabetic and diabetic rats. In nondiabetic rats, acetylcholine and ADP produced dilatation of cerebral arterioles. However, there was significant impairment in reactivity of arterioles to acetylcholine and ADP in diabetic rats. Vasodilatation to nitroglycerin was similar in nondiabetic and diabetic rats. Glutathione did not alter baseline diameter of cerebral arterioles, but significantly improved impaired nitric oxide synthase‐dependent vasodilatation in diabetic rats. Superoxide production was increased under basal states in diabetic rats and glutathione restored superoxide levels to that observed in nondiabetic rats. Our findings suggest that glutathione selectively improves endothelial dysfunction of cerebral arterioles during Type 1 diabetes.

Nicotine Impairs eNOS‐Dependent Responses of the Basilar Artery

Our goals were to determine whether acute exposure to nicotine alters eNOS‐dependent responses of the basilar artery and to identify a potential role for activation of NAD(P)H oxidase. We measured diameter of the basilar artery to eNOS‐dependent (acetylcholine) and ‐independent (nitroglycerin) agonists before and during acute infusion of nicotine in the absence or presence of apocynin. In addition, we measure superoxide production by brain tissue to nicotine. We found that eNOS‐dependent vasodilatation was impaired during treatment with nicotine and that apocynin prevented this impairment. Further, the production of superoxide was increased by nicotine and this increase could be inhibited by apocynin. We suggest that nicotine impairs eNOS‐dependent dilatation of the basilar artery by activation of NAD(P)H oxidase.

Colchicine suppresses neutrophil superoxide production in a murine model of gouty arthritis: a rationale for use of low‐dose colchicine

When used to treat gouty arthritis, colchicine is believed to work by inhibiting microtubule-dependent cell infiltration. However, in vitro, colchicine also reduces monosodium urate (MSU)-induced superoxide production by neutrophils. Our study aimed to compare the effects of colchicine on neutrophil superoxide production and infiltration in an in vivo model of acute gouty inflammation. In vitro: Human and murine peritoneal neutrophils were incubated with MSU with and without colchicine, and superoxide production was measured. In vivo: Mice were treated with colchicine followed by an intraperitoneal injection of MSU to induce acute inflammation. After 4h, the peritoneal cells were recovered to measure superoxide production and neutrophil infiltration. Sera were tested for liver and renal toxicity. Colchicine dose-dependently inhibited MSU-induced superoxide production by both human and murine neutrophils in vitro. Oral colchicine inhibited MSU-induced superoxide production by neutrophils in vivo at doses 100 times lower than those required to inhibit neutrophil infiltration and without acute liver or renal toxicity. Neutrophils treated with colchicine in vivo still produced superoxide in response to another stimulus, 4-beta-phorbol-12-myristate-13-acetate. These results show a beneficial effect of colchicine for the treatment of MSU-induced superoxide production in vivo at sub-toxic doses without compromising superoxide production by other physiological processes. This is the first in vivo data to provide a biological rationale that supports the implementation of low dose, non-toxic, colchicine therapy for the treatment of gouty arthritis.

Prolonged survival of mice with established intracerebral glioma receiving combined treatment with peroxisome proliferator-activated receptor–γ thiazolidinedione agonists and interleukin-2–secreting syngeneic/allogeneic fibroblasts

In this study the authors explored the benefits of treating C57B1/6 mice with an established intracerebral glioma by combining immunotherapy with interleukin (IL)-2-secreting syngeneic/allogeneic fibroblasts administered into the tumor bed along with the chemotherapeutic agent pioglitazone, a thiazolidinedione (TZD). The TZDs are agonists of the peroxisome proliferator-activated receptor-gamma. They have been found to exert antiproliferative effects on several transformed cell lines. Data from prior studies by these authors have revealed the immunotherapeutic properties of the IL-2-secreting fibroblasts in treating intracerebral gliomas in mice. The sensitivity of GL261 glioma cells and primary astrocytes to pioglitazone was determined in vitro by incubating the cells with increasing amounts of the drug. Viability was assessed by measuring lactate dehydrogenase release, and effects on metabolism were determined by measuring superoxide production and levels of superoxide dismutase. The GL261 cells were injected intracerebrally into C57B1/6 mice, followed by treatment with pioglitazone either orally or intracerebrally into the tumor bed. The effect of the combined therapy was determined by injecting C57B1/6 mice with an established intracerebral GL261 glioma with IL-2-secreting allogeneic fibroblasts and pioglitazone directly into the tumor bed through a unique cannula system. Pioglitazone was found to induce cell death in GL261 glioma cells grown in vitro while causing only modest damage to astrocytes. The application of pioglitazone also resulted in a significantly greater induction of cellular superoxide in glioma cells than in astrocytes, which can activate apoptotic pathways. Pioglitazone administered intracerebrally (p < 0.05) but not orally was found to prolong survival in mice harboring an intracerebral glioma. Synergistic effects of combination therapy on prolonging survival were found in mice receiving both pioglitazone and IL-2-secreting fibroblasts (p < 0.005, compared with untreated animals). Pioglitazone induces metabolic and oxidative stresses that are tolerated by astrocytes but not glioma cells, which could account for selective vulnerability and increased sensitivity to IL-2, suggesting potential for the use of this Food and Drug Administration-approved drug in the treatment of brain tumors. The data indicate the beneficial effects of combination therapy using pioglitazone and immunotherapy in mice harboring intracerebral glioma.

CALORIE RESTRICTION REDUCES SUPEROXIDE PRODUCTION OF AGING MOUSE HYPOTHALAMUS

Previous investigations suggested that calorie restriction in mice significantly reduces reactive oxygen species (ROS), decreasing oxidative damage and extending lifespan. Calorie-restricted mice also increase expression of mitochondrial uncoupling proteins (UCPs), which have been suggested to reduce ROS generation. Thus, we hypothesized that the age-related increase in ROS would be attenuated in transgenic mice that overexpress UCP2 (TG-UCP2). To test this, we measured superoxide production in hypothalami from 8-, 19- and 26-month old female mice in the following ad libitum fed groups: TG-UCP2 mice, UCP2 knockout mice, and C57BL/6 wild type (WT) mice. Measurements were also made in hypothalami from C57BL/6 mice calorie-restricted to 70% of WT animals. Superoxide production in the fixed hypothalami was determined by measuring oxidation of fluorescent-labeled dihydroethidium to ethidium. We found a significant age-related increase in superoxide between 8 and 19 month WT mice without further increases at 26 months. Calorie restriction significantly reduced superoxide concentration compared to all other groups; and preliminary data suggest that increased UCP2 expression may contribute to this attenuation. (NIH grant AG19984; Genentech Support for Undergraduate Research)