Dismutase Knockout Mice Research Articles

Senolytic treatment does not mitigate oxidative stress-induced muscle atrophy but improves muscle force generation in CuZn superoxide dismutase knockout mice.

Oxidative stress is associated with tissue dysfunctions that can lead to reduced health. Prior work has shown that oxidative stress contributes to both muscle atrophy and cellular senescence, which is a hallmark of aging that may drive in muscle atrophy and muscle contractile dysfunction. The purpose of the study was to test the hypothesis that cellular senescence contributes to muscle atrophy or weakness. To increase potential senescence in skeletal muscle, we used a model of oxidative stress-induced muscle frailty, the CuZn superoxide dismutase knockout (Sod1KO) mouse. We treated 6-month-old wildtype (WT) and Sod1KO mice with either vehicle or a senolytic treatment of combined dasatinib (5 mg/kg) + quercetin (50 mg/kg) (D + Q) for 3 consecutive days every 15 days. We continued treatment for 7 months and sacrificed the mice at 13 months of age. Treatment with D + Q did not preserve muscle mass, reduce NMJ fragmentation, or alter muscle protein synthesis in Sod1KO mice when compared to the vehicle-treated group. However, we observed an improvement in muscle-specific force generation in Sod1KO mice treated with D + Q when compared to Sod1KO-vehicle mice. Overall, these data suggest that reducing cellular senescence via D + Q is not sufficient to mitigate loss of muscle mass in a mouse model of oxidative stress-induced muscle frailty but may mitigate some aspects of oxidative stress-induced muscle dysfunction.

Neonatal Extracellular Superoxide Dismutase Knockout Mice Increase Total Superoxide Dismutase Activity and VEGF Expression after Chronic Hyperoxia.

Bronchopulmonary dysplasia (BPD) is a common lung disease affecting premature infants that develops after exposure to supplemental oxygen and reactive oxygen intermediates. Extracellular superoxide dismutase (SOD3) is an enzyme that processes superoxide radicals and has been shown to facilitate vascular endothelial growth factor (VEGF) and nitric oxide (NO) signaling in vascular endothelium. We utilized a mouse model of neonatal hyperoxic lung injury and SOD3 knockout (KO) mice to evaluate its function during chronic hyperoxia exposure. Wild-type age-matched neonatal C57Bl/6 (WT) and SOD3−/− (KO) mice were placed in normoxia (21% FiO2, RA) or chronic hyperoxia (75% FiO2, O2) within 24 h of birth for 14 days continuously and then euthanized. Lungs were harvested for histologic evaluation, as well as comparison of antioxidant enzyme expression, SOD activity, VEGF expression, and portions of the NO signaling pathway. Surprisingly, KO-O2 mice survived without additional alveolar simplification, microvascular remodeling, or nuclear oxidation when compared to WT-O2 mice. KO-O2 mice had increased total SOD activity and increased VEGF expression when compared to WT-O2 mice. No genotype differences were noted in intracellular antioxidant enzyme expression or the NO signaling pathway. These results demonstrate that SOD3 KO mice can survive prolonged hyperoxia without exacerbation of alveolar or vascular phenotype.

Age-related NMDA signaling alterations in SOD2 deficient mice

Oxidative stress affects the survival and function of neurons. Hence, they have a complex and highly regulated machinery to handle oxidative changes. The dysregulation of this antioxidant machinery is associated with a wide range of neurodegenerative conditions. Therefore, we evaluated signaling alterations, synaptic properties and behavioral performance in 2 and 6-month-old heterozygous manganese superoxide dismutase knockout mice (SOD2+/− mice). We found that their low antioxidant capacity generated direct oxidative damage in proteins, lipids, and DNA. However, only 6-month-old heterozygous knockout mice presented behavioral impairments. On the other hand, synaptic plasticity, synaptic strength and NMDA receptor (NMDAR) dependent postsynaptic potentials were decreased in an age-dependent manner. We also analyzed the phosphorylation state of the NMDAR subunit GluN2B. We found that while the levels of GluN2B phosphorylated on tyrosine 1472 (synaptic form) remain unchanged, we detected increased levels of GluN2B phosphorylated on tyrosine 1336 (extrasynaptic form), establishing alterations in the synaptic/extrasynaptic ratio of GluN2B. Additionally, we found increased levels of two phosphatases associated with dephosphorylation of p-1472: striatal-enriched protein tyrosine phosphatase (STEP) and phosphatase and tensin homolog deleted on chromosome Ten (PTEN). Moreover, we found decreased levels of p-CREB, a master transcription factor activated by synaptic stimulation. In summary, we describe mechanisms by which glutamatergic synapses are altered under oxidative stress conditions. Our results uncovered new putative therapeutic targets for conditions where NMDAR downstream signaling is altered. This work also contributes to our understanding of processes such as synapse formation, learning, and memory in neuropathological conditions.

A new mouse model of frailty: the Cu/Zn superoxide dismutase knockout mouse

Frailty is a geriatric syndrome that is an important public health problem for the older adults living in the USA. Although several methods have been developed to measure frailty in humans, we have very little understanding of its etiology. Because the molecular basis of frailty is poorly understood, mouse models would be of great value in determining which pathways contribute to the development of frailty. More importantly, mouse models would be critical in testing potential therapies to treat and possibly prevent frailty. In this article, we present data showing that Sod1KO mice, which lack the antioxidant enzyme, Cu/Zn superoxide dismutase, are an excellent model of frailty, and we compare the Sod1KO mice to the only other mouse model of frailty, mice with the deletion of the IL-10 gene. Sod1KO mice exhibit four characteristics that have been used to define human frailty: weight loss, weakness, low physical activity, and exhaustion. In addition, Sod1KO mice show increased inflammation and sarcopenia, which are strongly associated with human frailty. The Sod1KO mice also show alterations in pathways that have been proposed to play a role in the etiology of frailty: oxidative stress, mitochondrial dysfunction, and cell senescence. Using Sod1KO mice, we show that dietary restriction can delay/prevent characteristics of frailty in mice.

Superoxide dismutase deficiency impairs olfactory sexual signaling and alters bioenergetic function in mice.

Oxidative stress (an overproduction of reactive oxygen species in relation to defense mechanisms) may restrict investment in life history traits, such as growth, reproduction, lifespan, and the production of sexual signals to attract mates. The constraint on sexual signaling by oxidative stress is of particular interest because it has been proposed as a mechanism ensuring that only good-quality males produce the most attractive sexual signals. Despite these predictions, evidence supporting this theory is, at best, equivocal. We used a superoxide dismutase knockout mouse to demonstrate that oxidative stress directly impairs investment in morphological (preputial glands) and molecular (major urinary proteins) components of olfactory signaling essential for mate attraction. By maintaining males in a much more competitive environment than usual for mouse laboratory experiments, we also revealed a range of phenotypes of superoxide dismutase deficiency not observed in previous studies of this mouse model. This range included impaired bioenergetic function, which was undetectable in the control environment of this study. We urge further examination of model organisms in seminatural conditions and more competitive laboratory environments, as important phenotypes can be exposed under these more demanding conditions.

Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression

To understand the role of mitochondrial-produced superoxide (O2 (•-)) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver. For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression (Sod2 (+/-)) and age-matched littermate control mice (LMC), expressing Sod2 gene on both alleles, were exposed to either 10% (w/v) ethanol in the drinking water or plain water (control) for 7 d. Total cellular O2 (•-) levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O2 (•-)-sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O2 (•-) in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. Sod2 (+/-) mice expressed 40% less MnSOD protein (SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 (+/-) and LMC mice. O2 (•-) levels in hepatocytes of untreated Sod2 (+/-) mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O2 (•-) levels in mitochondria of Sod2 (+/) mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O2 (•-) levels in hepatocytes of LMC mice than in Sod2 (+/-) mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O2 (•-) levels in both Sod2 (+/-) and control mice. Mitochondrial O2 (•-) is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O2 (•-) levels in hepatocytes, which appears not to originate from mitochondria.

Complement component 3 inhibition by an antioxidant is neuroprotective after cerebral ischemia and reperfusion in mice

Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that pro-inflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up-regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen-glucose deprivation in brain cells. Persistent up-regulation of complement component 3 expression was reduced in copper/zinc-superoxide dismutase transgenic mice, and manganese-superoxide dismutase knock-out mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N-tert-butyl-α-phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N-tert-butyl-α-phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen-glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke.

Effect of dietary copper concentrations on dyslipidemia in SOD1−/− mice

We previously observed dyslipidemia in Cu,Zn‐superoxide dismutase knockout mice (SOD1−/−). This study was conducted to test if the dyslipidemic phenotype in the SOD1−/− mice was ameliorated by feeding a high level of dietary Cu. Weanling male SOD1−/− mice and their wild‐type (WT) controls (3‐wk old, n = 7–10 per genotype by treatment) were fed a Torula‐yeast based, Cudeficient (< 1 mg/kg) diet (BD) or BD plus Cu (as CuSO4·5H2O) at 10 and 100 mg/kg for 6 wk. Compared with the 10 mg Cu/kg diet, the BD elevated (P < 0.05) plasma cholesterol and non‐esterifed fatty acid concentrations. However, neither the BD nor the high Cu diet (100 mg/kg) removed the genotype differences in body weight, plasma lipid profiles, or hepatic lipid profiles, although the high Cu diet decreased the relative difference in hepatic triglyceride concentration between the SOD1−/− and WT mice. Meanwhile, the BD aggravated (P < 0.05) the genotype differences in hepatic cholesterol and triglyceride concentrations. Hepatic nuclear sterol regulatory element‐binding protein‐1 and ‐2 were affected (P < 0.05) by the high Cu diet in an opposite way, but the genotype difference in these two key lipogenesis regulatory proteins remained. In conclusion, dyslipidemia in the SOD1−/− mice was not likely attributed to Cu deficiency, and could not be ameliorated by a high dietary Cu supplementation (supported in part by NIH 53018).

Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse

Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.

Characterization of novel kidney specific manganese superoxide dismutase knockout mice

Inactivation of manganese superoxide dismutase (MnSOD), the major mitochondrial antioxidant, is a key issue in renal failure following transplantation or ischemia/reperfusion (IR) injury. Homozygous MnSOD knock out (KO) mice die shortly after birth, and heterozygous MnSOD KO mice have decreased MnSOD activity in all tissues/organs. To address the role MnSOD inactivation has on renal injury, we generated bi‐transgenic kidney specific MnSOD KO (50% and 100%) mice by breeding KidneyCre mice1 and floxed MnSOD mice2. Characterization of these KOs revealed that phenotypically the mice are smaller in size compared to littermate controls, but show no survival differences. Immunohistochemistry data showed that the Cre‐recombinase mediated MnSOD deletion occurred specifically in the kidney and was located predominantly in the medullary region. The kidney specific KOs exhibited histologic evidence of mild renal damage, increased tyrosine nitration, and renal cell death. KO mice renal function was not altered; however, more renal damage was observed following IR injury compared to littermate controls. In summary, we have developed a novel kidney specific MnSOD KO mouse model that will help define the molecular pathways that occur downstream of MnSOD inactivation during renal injury.Supported by NIH grant 1RO1DK0789361.

Oxidative Stress Increases Phosphorylation of IκB Kinase-α by Enhancing NF-κB-Inducing Kinase after Transient Focal Cerebral Ischemia

The IkappaB kinase (IKK) complex is a central component in the classic activation of the nuclear factor-kappaB (NF-kappaB) pathway. It has been reported to function in physiologic responses, including cell death and inflammation. We have shown that IKK is regulated by oxidative status after transient focal cerebral ischemia (tFCI) in mice. However, the mechanism by which oxidative stress influences IKKs after tFCI is largely unknown. Nuclear accumulation and phosphorylation of IKKalpha (pIKKalpha) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knockout mice, levels of NF-kappaB-inducing kinase (NIK) (an upstream kinase of IKKalpha), pIKKalpha, and phosphorylation of histone H3 (pH3) on Ser10 were increased after tFCI and were higher than in wild-type mice. Immunohistochemistry showed nuclear accumulation and pIKKalpha in mouse brain endothelial cells after tFCI. Nuclear factor-kappaB-inducing kinase was increased, and it enhanced pH3 by inducing pIKKalpha after oxygen-glucose deprivation (OGD) in mouse brain endothelial cells. Both NIK and pH3 interactions with IKKalpha were confirmed by coimmunoprecipitation. Treatment with IKKalpha small interfering RNA significantly reduced cell death after OGD. These results suggest that augmentation of NIK, IKKalpha, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke).

Protective role of glutathione S-transferase A4 induced in copper/zinc-superoxide dismutase knockout mice

Copper/zinc-superoxide dismutase (SOD1) plays a protective role in cells by catalyzing the conversion of the superoxide anion into molecular oxygen and hydrogen peroxide. Although SOD1 knockout (KO) mice exhibit a reduced life span and an elevated incidence of dysfunctions in old age, young SOD1 KO mice grow normally and exhibit no abnormalities. This fact leads to the hypothesis that other antioxidative proteins prevent oxidative stress, compensating for SOD1. Differently expressed genes in 3-week-old SOD1 KO and littermate wild-type mice were explored. A gene remarkably elevated in SOD1 KO mouse kidneys was identified as the glutathione S-transferase Alpha 4 gene ( Gsta4), which encodes the GSTA4 subunit. The GSTA4 protein level and activity were also significantly increased in SOD1 KO mouse kidneys. The administration of an iron complex, a free radical generator, induced GSTA4 expression in wild-type mouse kidneys. Iron deposition detected in SOD1 KO mouse kidney is thought to be an inducer of GSTA4. In addition, overexpression of mouse GSTA4 cDNA in human embryonic kidney cells decreased cell death caused by both 4-hydroxynonenal and hydrogen peroxide. These findings suggest that compensatory induced GSTA4 plays a protective role against oxidative stress in young SOD1 KO mouse kidneys.

Increased mitochondrial capacity due to chronic oxidative stress is reduced with age

Oxidative stress has been proposed to modulate mitochondrial function by reducing the coupling of oxidative phosphorylation (P/O) and inducing mitochondrial biogenesis. Here we test whether the effects of a chronic oxidative stress are age‐dependent. We compare in vivo mitochondrial metabolism and mitochondrial protein expression in wild‐type and Cu, Zn‐superoxide dismutase knockout mice (SOD1) at multiple ages. We used 31P NMR and optical spectroscopies to measure ATP and O2 fluxes in vivo. The 7‐month old SOD1 mice exhibited a significantly reduced mitochondrial P/O (P=0.05) and an increase in both maximal mitochondrial ATP production (ATPmax) (P=0.02) and O2 consumption (VO2max) (P=0.01). The greater in vivo mitochondrial capacity in these mice was accompanied by increased expression of electron transport chain proteins. In contrast, in the 20‐month old mice there was no difference in P/O, ATPmax, and VO2max between the wild‐type and SOD1 genotypes. The effect of the SOD1 knockout on electron transport chain protein expression was also reduced in the 20‐month old mice. These data suggest that in young adults the chronic oxidative stress results in changes in mitochondrial metabolism that lead to increased mitochondrial capacity, whereas this response is reduced by 20 months. Supported by NIH grants AG028455, AG022385, and the Ellison Medical Foundation.

Increased mitochondrial content in response to mitochondrial dysfunction in skeletal muscle of Cu, Zn superoxide dismutase knockout mice

Mitochondrial biogenesis is an important mechanism allowing cells to adapt to energetic stress. Here we test whether skeletal muscle compensates for mitochondrial dysfunction induced by oxidative stress by increasing mitochondrial content in vivo. We used 31P NMR and optical spectroscopies to measure ATP and O2 fluxes in vivo in wild‐type and Cu, Zn‐superoxide dismutase knockout mice (SOD1). Adult SOD1 mice demonstrated significant mitochondrial dysfunction as indicated by a 36% lower energy coupling ratio (P/O) (P=0.03) and a 13% lower resting ATP/PCr (P=0.01). Mitochondrial dysfunction in the SOD1 mice was associated with an increased capacity for mitochondrial ATP production (1.08 ± 0.04 vs. 0.72 ± 0.10 mM ATP s−1, P=0.01) and O2 uptake (O2max) (0.30 ± 0.04 vs. 0.13 ± 0.02 mM O2 s−1, P<0.01). Cytochrome oxidase protein and gene transcript and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PGC1a) transcript levels were positively correlated with O2max and negatively correlated with P/O across treatment groups (P<0.05), supporting a relationship between reduced energy coupling and increased mitochondrial content. These results indicate that skeletal muscle compensates for mitochondrial dysfunction in vivo by activating mitochondrial biogenesis and increasing mitochondrial content. Supported by NIH grants AG028455, AG022385, and the Ellison Medical Foundation.

Contractile Properties of Whole Skeletal Muscle and Single Muscle Fibers from Young and Old Superoxide Dismutase Knockout Mice

CuZn‐SOD knockout mice (Sod1−/−) have elevated oxidative stress, decreased body mass, and decreased lifespan compared to wild‐type mice (Sod1+/+). Sod1−/− exhibit age‐related decreases in whole muscle mass, absolute maximum isometric force (F0) and specific F0 (sF0), F0 normalized by cross‐sectional area (CSA). Reduced sF0 in whole muscle of Sod1−/− could be due to decreased sF0 of individual fibers or to non‐functional fibers that contribute to CSA but not force. We tested the hypothesis that sF0 of single fibers from Sod1−/− is lower than that of Sod1+/+ mice. Single fibers were obtained from the gastrocnemius muscle of Sod1+/+ and Sod1−/− mice at 8 and 20 mo of age. Single fiber F0 and CSA were measured and sF0 was calculated. The F0 of single fibers decreased with age (P=0.037) in both Sod1+/+ and Sod1−/− groups, but no difference was observed between the groups (P=0.825). The CSA of the fibers decreased with age (P=0.027) for both groups, but no differences were observed between groups (P=0.971). No differences in sF0 were observed among age or genotype groups (P=0.368, 0.906). Therefore, the decrease in sF0 of whole muscles in Sod1−/− mice may be explained by an increase in a population of non‐functional fibers that contribute to the CSA of the whole muscle, but not to force production. Supported by P30 AG13283.

Acceleration of diabetic renal injury in the superoxide dismutase knockout mouse: effects of tempol

Indices of renal injury and oxidative stress were examined in mice with deficiency of cytosolic Cu 2+/Zn 2+ superoxide dismutase (SOD1−/−, KO) and their wild-type (WT) littermates with streptozotocin-induced diabetes. After 5 weeks of diabetes, KO diabetic (D) but not WT-D mice developed marked albuminuria, increases in glomerular content of transforming growth factor β, collagen α1(IV), and nitrotyrosine, and higher glomerular superoxide compared with corresponding values in nondiabetics. After 5 months of diabetes, increases in these parameters, mesangial matrix expansion, renal cortical malondialdehyde content, and severity of tubulointerstitial injury were all significantly greater, whereas cortical glutathione was lower, in KO-D than in WT-D. In contrast to WT-D, after 4 weeks of diabetes, KO-D mice did not develop the increase in inulin clearance ( C In) characteristic of early diabetes. The nitric oxide synthase inhibitor N ω-nitro- l-arginine methylester suppressed C In in WT-D, but had no effect on C In in KO-D. Treatment of KO-D with the SOD mimetic tempol for 4 weeks suppressed albuminuria, increases in glomerular transforming growth factor β, collagen α1(IV), nitrotyrosine, and glomerular superoxide, and concurrently increased C In. The latter action of tempol in KO-D was blocked by the N ω-nitro- l-arginine methylester. The findings provide support for a role for superoxide and its metabolism by SOD1 in the pathogenesis of renal injury in diabetes in vivo, and implicate increased interaction of superoxide with nitric oxide as a pathogenetic factor.

Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of Nitrate Tolerance

Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.

Skeletal muscle reperfusion injury is enhanced in extracellular superoxide dismutase knockout mouse

This study investigates the role of extracellular SOD (EC-SOD), the major extracellular antioxidant enzyme, in skeletal muscle ischemia and reperfusion (I/R) injury. Pedicled cremaster muscle flaps from homozygous EC-SOD knockout (EC-SOD-/-) and wild-type (WT) mice were subjected to 4.5-h ischemia and 90-min reperfusion followed by functional and molecular analyses. Our results revealed that EC-SOD-/- mice showed significantly profound I/R injury compared with WT littermates. In particular, there was a delayed and incomplete recovery of arterial spasm and blood flow during reperfusion, and more severe acute inflammatory reaction and muscle damage were noted in EC-SOD-/- mice. After 90-min reperfusion, intracellular SOD [copper- and zinc-containing SOD (CuZn-SOD) and manganese-containing (Mn-SOD)] mRNA levels decreased similarly in both groups. EC-SOD mRNA levels increased in WT mice, whereas EC-SOD mRNA was undetectable, as expected, in EC-SOD-/- mice. In both groups of animals, CuZn-SOD protein levels decreased and Mn-SOD protein levels remained unchanged. EC-SOD protein levels decreased in WT mice. Histological analysis showed diffuse edema and inflammation around muscle fibers, which was more pronounced in EC-SOD-/- mice. In conclusion, our data suggest that EC-SOD plays an important role in the protection from skeletal muscle I/R injury caused by excessive generation of reactive oxygen species.

Inhibition of iNOS attenuates skeletal muscle reperfusion injury in extracellular superoxide dismutase knockout mice

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely involved in the mechanism of skeletal muscle ischemia/reperfusion (I/R) injury. This study was designed to determine the effects of inducible nitric oxide synthase (iNOS) inhibitor 1400 W on the reperfused cremaster muscle in extracellular super-oxide dismutase knockout (EC-SOD(-/-)) mice. The muscle was exposed to 4.5 h of ischemia, followed by 90 min of reperfusion. Mice received either 3 mg/kg of 1400 W or the same amount of phosphate-buffered saline (PBS, as a control) subcutaneously at 10 min before the start of reperfusion. 1400 W treatment markedly improved the recovery speed of vessel diameter and blood flow in the reperfused cremaster muscle of EC-SOD(-/-) mice compared to controls. Histological examination showed reduced edema in the interstitial space and muscle fiber, and reduced density of nitrotyrosine (a marker of total peroxi-nitrate (ONOO(-)) level) in 1400 W-treated muscles compared to controls. Our results suggest that iNOS and ONOO(-) products are involved in skeletal muscle I/R injury. Reduced I/R injury by using selective inhibition of iNOS perhaps works by limiting cytotoxic ONOO(-) generation, a reaction product of nitric oxide (NO) and super-oxide anion (O(2) (-)). Thus, inhibition of iNOS appears to be a treatment strategy for reducing clinical I/R injury.

Endothelial Progenitor Cells at Work

Tissue replacement in the adult organism by cell-specific differentiation of autologous stem/progenitor cells has evolved as a fascinating concept in stem cell biology. After organ damage, bone marrow–derived circulating or tissue-resident progenitor cells are thought to differentiate toward the type of cell needed for repair. According to this concept, maturation of these cells would be expected to result at best in a perfect morphological and functional replacement of the injured tissue. However, in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , He et al show that endothelial progenitor cells (EPCs) are more than just as capable of in vitro angiogenic tube formation as mature endothelial cells (ECs), but are truly advantageous when it comes to stress tolerance.1 See page 2021 EPCs were originally characterized as cells that are mobilized from the bone marrow and circulate in the peripheral blood and express certain surface membrane markers including the vascular endothelial growth factor receptor (VEGF-R2) KDR and the hematopoietic progenitor cell markers CD34 and CD133.2–4 During ex vivo expansion, these cells develop morphological and functional characteristics typical for ECs, including formation of vascular-like structures in matrigel and other in vitro angiogenesis assays. Most importantly, however, transplanted EPCs exhibit an extraordinary potent in vivo capacity to improve the neovascularization of ischemic tissue in the adult organism.5 In this regard, EPCs were shown to be more effective than mature ECs in animal models of hind limb ischemia,6–8 although mature ECs are well established to exert a potent in vitro angiogenic activity. Thus, the …