Nitric oxide and blood pressure in mice lacking extracellular-superoxide dismutase

Nitric oxide is a major vasorelaxant and regulator of the blood pressure. The blood vessels contain several active sources of the superoxide radical, which reacts avidly with nitric oxide to form noxious peroxynitrite. There are large amounts of extracellular-superoxide dismutase (EC-SOD) in the vascular wall. To evaluate the importance of EC-SOD for the physiology of nitric oxide, here we studied the blood pressure in mice lacking the enzyme. In chronically instrumented non-anaesthetized mice there was no difference in mean arterial blood pressure between wild-type controls and EC-SOD mutants. Extensive inhibition of nitric oxide synthases with N -monomethyl- l -arginine however resulted in a larger increase in blood pressure, and infusion of the nitric oxide donor nitrosoglutathione caused less reduction in blood pressure in the EC-SOD null mice. We interpret the alterations to be caused by a moderately increased consumption of nitric oxide by the superoxide radical in the EC-SOD null mice. One role of EC-SOD may be to preserve nitric oxide, a function that should be particularly important in vascular pathologies, in which large increases in superoxide formation have been documented.     

Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung and vascular tissue. Localization of EC-SOD to the matrix of the lung may protect against oxidative tissue damage that leads to pulmonary fibrosis. This study directly examines the protective role of EC-SOD in a bleomycin model of pulmonary fibrosis and the effect of this enzyme on oxidative protein fragmentation. Mice null for ec-sod display a marked increase in lung inflammation at 14 d post-bleomycin treatment as compared to their wild-type counterparts. Hydroxyproline analysis determined that both wild-type and ec-sod null mice display a marked increase in interstitial fibrosis at 14 d post-treatment, and the severity of fibrosis is significantly increased in ec-sod null mice compared to wild-type mice. To determine if the lack of EC-SOD promotes bleomycin-induced oxidative protein modification, 2-pyrrolidone content (as a measure of oxidative protein fragmentation at proline residues) was assessed in lung tissue from treated mice. 2-Pyrrolidone levels in the lung hydrolysates from ec-sod null mice were increased at both 7 and 14 d post-bleomycin treatment as compared to wild-type mice, indicating EC-SOD can inhibit oxidative fragmentation of proteins in this specific model of oxidative stress.     

Phenotypes of mice lacking extracellular superoxide dismutase and copper- and zinc-containing superoxide dismutase

Mice lacking the secreted extracellular superoxide dismutase (EC-SOD) or the cytosolic copper- and zinc-containing SOD (CuZn-SOD) show relatively mild phenotypes. To explore the possibility that the isoenzymes have partly overlapping functions, single and double knockout mice were examined. The absence of EC-SOD was found to be without effect on the lifespan of mice, and the reduced lifespan of CuZn-SOD knockouts was not further shortened by EC-SOD deficiency. The urinary excretion of isoprostanes was increased in CuZn-SOD knockout mice, and plasma thiobarbituric acid-reactive substances levels were elevated in EC-SOD knockout mice. These oxidant stress markers showed potentiated increases in the absence of both isoenzymes. Other alterations were mainly found in CuZn-SOD knockout mice, such as halved glutathione peroxidase activity in the tissues examined and increased glutathione and iron in the liver. There were no changes in tissue content of the alternative superoxide scavenger ascorbate, but there was a 25% reduction in ascorbate in blood plasma in mice lacking CuZn-SOD. No increase was found in the urinary excretion of the terminal metabolites of NO, nitrite, and nitrate in any of the genotypes. In conclusion, apart from the increases in the global urinary and plasma oxidant stress markers, our phenotype studies revealed no other evidence that the copper- and zinc-containing SOD isoenzymes have overlapping roles.     

Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase.

Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis.     

The site of nonenzymic glycation of human extracellular-superoxide dismutase in vitro.

The secretory enzyme extracellular-superoxide dismutase (EC-SOD) has affinity for heparin and some other sulfated glycosaminoglycans and is in vivo bound to heparan sulfate proteoglycan. Nonenzymic glycation of EC-SOD, both in vivo and in vitro, is associated with a reduction in heparin affinity, whereas the enzymic activity is not affected. The glycation sites in EC-SOD are further studied in the present article. It is shown that modification of a few of the five lysyl residues of the subunits of the enzyme with trinitrobenzene sulfonic acid nearly abolishes the in vitro glycation susceptibility. From a chymotryptic digest of in vitro glycated EC-SOD, two peptides with affinity for boronate could be isolated. Amino acid sequence analysis showed that both encompassed the carboxyterminal end. epsilon-Glucitol lysine was identified in both peptides at positions 211 and 212. The primary glycation sites in EC-SOD are thus lysine-211 and lysine-212 in the putative heparin-binding domain in the carboxyterminal end.     

In vitro photochemical cataract in mice lacking copper-zinc superoxide dismutase

We here evaluate cataract formation in mice lacking the cytosolic copper-zinc superoxide dismutase (CuZn-SOD) in an in vitro model using irradiation with visible light and riboflavin as a photosensitizing agent. Isolated, cultured lenses from wild-type and CuZn-SOD-null mice were irradiated for 1.5 h by a daylight fluorescent light after preincubation with 10 microM riboflavin for 24 h. Cataract formation was evaluated daily with digital image analysis and ocular staging, and after 5 d 86Rb uptake and water contents of the lenses were determined. Basal superoxide concentrations in freshly isolated lenses from wild-type and CuZn-SOD-null mice were determined with lucigenin-derived chemiluminescense, and enzymatic activities of all three SOD isoenzymes in the murine lens were determined with a direct spectrophotometric method. The cytosolic CuZn-SOD accounts for 90% of the total SOD activity of the murine lens. CuZn-SOD-null lenses showed a doubled basal superoxide concentration, and were more prone to develop photochemical cataract in the present model with more opacification, more hydration, and less 86Rb uptake than lenses from wild-type mice. We conclude that CuZn-SOD is an important superoxide scavenger in the lens, and that it may have a protective role against cataract formation.     

Enhanced recovery from ionizing radiation damage in a lepidopteran insect cell line

TN-368 lepidopteran insect cells display a pronounced resistance to the lethal effects of ionizing radiation and exhibit superior DNA repair capabilities. When a TN-368 cell population entering stationary growth phase is irradiated with 137Cs gamma rays and then incubated for several hours before cell dilution and plating for colony formation, the surviving fraction is increased several-fold over cells diluted and plated immediately after irradiation. Similarly, the survival of cells plated immediately following the second of two equivalent doses separated by several hours is greater than the survival of cells plated immediately following a single dose equal to the sum of the split doses. Both processes exhibit similar biphasic repair kinetics and reach maximal levels by 6 h. The phenomena appear initially to be analogous to confluent-holding and split-dose recovery as described for mammalian cells. However, the survival levels obtained for doses of 61-306 Gy after allowing for these recovery processes to occur are quite high and greatly exceed survival levels for all but relatively low doses less than 50 Gy. For example, while the survival of cells irradiated with 150 Gy is near 0.15, the survival of cells receiving 306 Gy in two equivalent split doses is approximately 0.77. Even if damage induced by the first of the split doses was completely repaired, it might be expected that the survival would be near the level of the second dose alone, or near 0.15. Instead the survival is approximately five times greater, suggesting that the first split dose stimulated a repair system not present in unirradiated cells. The situation for confluent-holding recovery is similar to that for split-dose recovery.     

Enhanced viral immunoinflammatory lesions in mice lacking IL-23 responses

Herpes simplex virus (HSV) infection of the cornea culminates in an immunopathological lesion (stromal keratitis--SK) that impairs vision. This report shows that HSV infection results in IL-23 up-regulation, but if this response fails to occur, as was noted in p19-/- mice, the severity of lesions, their incidence and the level of viral induced angiogenesis were significantly increased compared to wild-type (WT) animals (p<0.05). The higher disease severity in p19-/- mice appeared to be the consequence of an increased IL-12 response that in turn led to the induction of higher numbers of IFN-gamma producing CD4(+)T cells, the principal orchestrators of SK. Our results indicate that the severity of HSV induced immunopathological lesions may be mainly the consequence of IL-12 driven Th1 T cell reactions rather than the action of IL-17 producing cells controlled by IL-23.     

Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase

Asbestosis is a chronic form of interstitial lung disease characterized by inflammation and fibrosis that results from the inhalation of asbestos fibers. Although the pathogenesis of asbestosis is poorly understood, reactive oxygen species may mediate the progression of this disease. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) can protect the lung against a variety of insults; however, its role in asbestosis is unknown. To determine if EC-SOD plays a direct role in protecting the lung from asbestos-induced injury, intratracheal injections of crocidolite were given to wild-type and ec-sod-null mice. Bronchoalveolar lavage fluid (BALF) from asbestos-treated ec-sod-null mice at 24 h, 14 days, or 28 days posttreatment showed increased inflammation and total BALF protein content compared to that of wild-type mice. In addition, lungs from ec-sod-null mice showed increased hydroxyproline content compared to those of wild-type mice, indicating a greater fibrotic response. Finally, lungs from ec-sod-null mice showed greater oxidative damage, as assessed by nitrotyrosine content compared to those of their wild-type counterparts. These results indicate that depletion of EC-SOD from the lung increases oxidative stress and injury in response to asbestos.     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial infarct size in pigs.

The efficacy of human extracellular-superoxide dismutase type C (EC-SOD C) to limit infarct size after ischemia and reperfusion was explored and compared to that of EC-SOD C combined with catalase (CAT) and to that of CAT alone. EC-SOD C binds to heparan sulphate proteoglycan on the cell surfaces. Thirty-two pigs were subjected to 45 min of myocardial ischemia followed by 4 h of reperfusion. Control pigs (group A; n = 8) received 300 mL of saline into the great cardiac vein during a 30-min period started 5 min prior to reperfusion; pigs in group B (EC-SOD C; n = 8) got 16.6 mg of EC-SOD C; pigs in group C (EC-SOD C + CAT; n = 8) got 16.6 mg of EC-SOD C together with 150 mg of CAT. Pigs in group D (CAT; n = 8) received 150 mg of CAT. In groups B, C, and D, the drug was dissolved in saline and infused into the great cardiac. Infarct size expressed as percent of area at risk was smaller in groups B (14.5 +/- 16.7%) and C (40.8 +/- 13.3%) than in groups A (78.8 +/- 8.6%) and D (67.2 +/- 18.6%; p less than .05). Creatine kinase (CK) activity in ischemic myocardium was higher in groups B (1740 +/- 548 U/g) and C (1729 +/- 358 U/g) than in groups A (1184 +/- 237 U/g) and D (1251 +/- 434 U/g; p less than .05). There was an inverse relation (r = -.83) between infarct size and CK content. The EC-SOD C infusions resulted in only minimal increases in plasma SOD activities. In conclusion, the presence of SOD on the cell surfaces is of importance in the prevention of reperfusion injury rather than circulating SOD.     

Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various growth factors. Epidermal growth factor receptor (EGF-R) is expressed throughout the developing pancreas. We have analyzed here the pancreatic phenotype of EGF-R deficient (-/-) mice, which generally die from epithelial immaturity within the first postnatal week. The pancreata appeared macroscopically normal. The most striking feature of the EGF-R (-/-) islets was that instead of forming circular clusters, the islet cells were mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal EGF-R (-/-) beta-cells was significantly reduced (2.6+/-0.4 versus 5.8+/-0.9%, P<0.01) and the difference persisted even at 7-11 days of age. Analysis of embryonic pancreata revealed impaired branching morphogenesis and delayed islet cell differentiation in the EGF-R (-/-) mice. Islet development was analyzed further in organ cultures of E12.5 pancreata. The proportion of insulin-positive cells was significantly lower in the EGF-R (-/-) explants (27+/-6 versus 48+/-8%, P<0.01), indicating delayed differentiation of the beta cells. Branching of the epithelium into ducts was also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity was reduced 20% in EGF-R (-/-) late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-1 (PAI-1) were markedly higher, while no apparent differences were seen in the levels of active uPA and tPa between EGF-R (-/-) and wild-type pancreata. Our findings suggest that the perturbation of EGF-R-mediated signalling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-1 production and decreased gelatinolytic activity correlated to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development.     

Heparin-, dextran sulfate- and protamine-induced release of extracellular-superoxide dismutase to plasma in pigs

Intravenous heparin has previously been shown to release the high-heparin-affinity fraction C of extracellular-superoxide dismutase (EC-SOD, EC 1.15.1.1) to plasma in man and other mammals. This paper reports on further studies of the phenomena in the pig. A dose-response curve of the effect of heparin revealed that 1000 IU/kg body weight is needed for maximal release of EC-SOD C. This dose is an order of magnitude larger than that needed for the maximal release to plasma of factors such as lipoprotein lipase, hepatic lipase, and diamine oxidase, which are distributed between plasma and endothelium similarly to EC-SOD C. Thus EC-SOD C appears to have an unusually high affinity for endothelial cell-surface sulfated glycosaminoglycans relative to the affinity for heparin. There was no significant difference in releasing potency between unfractionated heparin and heparin subfractions with high or low affinity for antithrombin III. The heparin structure conferring high-affinity binding to antithrombin III is thus not specifically involved in binding to EC-SOD C. The non-biosynthetic compound dextran sulfate 5000 was an order of magnitude more efficient than heparin. Protamine displayed dual effects. Given alone in high dose it released EC-SOD to plasma, probably due to binding to endothelial cell-surface sulfated glycosaminoglycans displacing fraction C of the enzyme. When given after heparin, in a dose just below that expected to neutralize the heparin, protamine reversed the heparin-induced EC-SOD release.     

Enhanced activation of pro-inflammatory cytokines in mice lacking natriuretic peptide receptor-A

Natriuretic peptide receptor-A (NPRA) is the principal receptor for the cardiac hormones ANP and BNP. Mice lacking NPRA develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for hypertrophic growth in the absence of NPRA signaling are not yet known. In the present study, we determined whether deficiency of NPRA/cGMP signaling alters the cardiac pro-inflammatory cytokines gene expression in Npr1 (coding for NPRA) gene-knockout (Npr1(-/-)) mice exhibiting cardiac hypertrophy and fibrosis as compared with control wild-type (Npr1(+/+)) mice. A significant up-regulation of cytokine genes such as TNF-alpha (five-fold), IL-6 (three-fold) and TGF-beta1 (four-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-kappaB binding activity was almost five-fold greater in the nuclear extract of Npr1(-/-) mutant mice hearts as compared with wild-type Npr1(+/+) mice hearts. Guanylyl cyclase (GC) activity and cGMP levels were drastically reduced by 10- and 5-fold, respectively, in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory cytokines, probably via NF-kappaB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.     

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.     

Enhanced learning and memory in mice lacking Na+/Ca2+ exchanger 2

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) plays a role in regulation of intracellular Ca(2+) concentration via the forward mode (Ca(2+) efflux) or the reverse mode (Ca(2+) influx). To define the physiological function of the exchanger in vivo, we generated mice deficient for NCX2, the major isoform in the brain. Mutant hippocampal neurons exhibited a significantly delayed clearance of elevated Ca(2+) following depolarization. The frequency threshold for LTP and LTD in the hippocampal CA1 region was shifted to a lowered frequency in the mutant mice, thereby favoring LTP. Behaviorally, the mutant mice exhibited enhanced performance in several hippocampus-dependent learning and memory tasks. These results demonstrate that NCX2 can be a temporal regulator of Ca(2+) homeostasis and as such is essential for the control of synaptic plasticity and cognition.     

Mitochondrial reactive oxygen species in mice lacking superoxide dismutase 2: attenuation via antioxidant treatment

Mice that lack the mitochondrial form of superoxide dismutase (SOD2) incur severe pathologies and mitochondrial deficiencies, including major depletion of complex II, as a consequence of buildup of endogenous reactive oxygen species (Melov, S., Coskun, P., Patel, M., Tuinstra, R., Cottrell, B., Jun, A. S., Zastawny, T. H., Dizdaroglu, M., Goodman, S. I., Huang, T. T., Miziorko, H., Epstein, C. J., and Wallace, D. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 846-851 and Li, Y., Huang, T. T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Noble, L. J., Yoshimura, M. P., Berger, C., Chan, P. H., Wallace, D. C., and Epstein, C. J. (1995) Nat. Genet. 11, 376-381). These problems can be greatly attenuated or rescued by synthetic antioxidant treatment, such as with the catalytic antioxidant EUK189 (Hinerfeld, D., Traini, M. D., Weinberger, R. P., Cochran, B., Doctrow, S. R., Harry, J., and Melov, S. (2004) J. Neurochem. 88, 657-667). We have used heart mitochondria from sod2 null mice to better understand mitochondrial reactive oxygen species production both in the absence of SOD2 and following in vivo antioxidant treatment. Isolated heart mitochondria from 5-day-old sod2 null animals respiring on the complex II substrate succinate exhibited statistically significant higher levels of mitochondrial O2* (157%, p < 0.01) but significantly less H2O2 (33%, p < 0.001) than wild type littermates. Treatment of sod2 nullizygous mice with EUK189 proportionately increased the levels of complex II and H2O2. Increased production of O2* resulting from complex II normalization had no effect on steady state levels due to the rapid conversion to H2O2, a process presumably aided by the presence of the EUK189, an SOD mimetic.     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.     

Enhanced platelet activation mediates the accelerated angiogenic switch in mice lacking histidine-rich glycoprotein

Background

The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice.

Methodology/Principal Findings

To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG^−/− mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG^+/+ littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice.

Conclusions

Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.