Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Vascular alterations upon activation of TGFβ signaling in fibroblasts - implications for systemic sclerosis

Tissue fibrosis and vascular disease are hallmarks of systemic sclerosis (SSc). Transforming growth factor β (TGFβ) is a key-player in fibroblast activation and tissue fibrosis in SSc. In contrast to fibrosis, evidence for a role of TGFβ in vascular disease of SSc is scarce. Using a transgenic mouse model with fibroblast-specific expression of a kinase-deficient TGFβ receptor type II, Derrett-Smith and colleagues demonstrate that aberrant TGFβ signaling in fibroblasts might result in activation of vascular smooth muscle cells and architectural changes of the vessel wall of the aorta.     

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.     

Toll-like receptor 3 upregulation by type I interferon in healthy and scleroderma dermal fibroblasts

Introduction  

Increased levels of genes in the type I interferon (IFN) pathway have been observed in patients with systemic sclerosis (SSc), or scleroderma. How type I IFN regulates the dermal fibroblast and its participation in the development of dermal fibrosis is not known. We hypothesized that one mechanism by which type I IFN may contribute to dermal fibrosis is through upregulation of specific Toll-like receptors (TLRs) on dermal fibroblasts. Therefore, we investigated the regulation of TLR expression on dermal fibroblasts by IFN.     

Hypoxia-generated superoxide induces the development of the adhesion phenotype

Adhesion fibroblasts exhibit higher TGF-beta1 and type I collagen expression as compared to normal peritoneal fibroblasts. Furthermore, exposure of normal peritoneal fibroblasts to hypoxia results in an irreversible increase in TGF-beta1 and type I collagen. We postulated that the mechanism by which hypoxia induced the adhesion phenotype is through the production of superoxide either directly or through the formation of peroxynitrite. To test this hypothesis, normal peritoneal and adhesion fibroblasts were treated with superoxide dismutase (SOD), a superoxide scavenger, and xanthine/xanthine oxidase, a superoxide-generating system, under normoxic and hypoxic conditions. Also, cells were treated with peroxynitrite. TGF-beta1 and type I collagen expression was determined before and after all treatments using real-time RT/PCR. Hypoxia treatment resulted in a time-dependent increase in TGF-beta1 and type I collagen mRNA levels in both normal peritoneal and adhesion fibroblasts. Similarly, treatment with xanthine oxidase, to endogenously generate superoxide, resulted in higher mRNA levels of TGF-beta1 and type I collagen in both normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD, to scavenge endogenous superoxide, resulted in a decrease in TGF-beta1 and type I collagen expression in adhesion fibroblasts to levels seen in normal peritoneal fibroblasts; no effect on the expression of these molecules was seen in normal peritoneal fibroblasts. Exposure to hypoxia in the presence of SOD had no effect on mRNA levels of TGF-beta1 and type I collagen in either normal peritoneal or adhesion fibroblasts. Peroxynitrite treatment alone significantly induced both adhesion phenotype markers. In conclusion, hypoxia, through the production of superoxide, causes normal peritoneal fibroblasts to acquire the adhesion phenotype. Scavenging superoxide, even in the presence of hypoxia, prevented the development of the adhesion phenotype. These findings further support the central role of free radicals in the development of adhesions.     

Inhibition of fibroblast chemotaxis by superoxide dismutase

Superoxide radicals are known to be important mediators in chronic inflammatory and fibrotic processes, in which accumulation of fibroblasts is thought to play a major role in the pathogenetic events. The enzyme superoxide dismutase removes these radicals by a catalytic reaction. Chemotactic response of human fibroblasts and fibrosarcoma-derived cells (HT-1080) to fibroblast conditioned medium, fibronectin and platelet-derived growth factor was inhibited in a dose-dependent manner in the presence of superoxide dismutase, while random migration, cell proliferation, cell viability and synthesis of collagen and non-collagenous proteins was not altered. In contrast, phorbol myristate acetate, an inducer of superoxide generation, stimulated the chemotactic movement of fibroblasts to the attractants. Evidence for the formation of superoxide is provided by the reduction of tetrazolium salt by activated fibroblasts which could be inhibited by superoxide dismutase. Thus, it is concluded that superoxide in small amounts is involved in the mechanism of fibroblast chemotaxis. Superoxide dismutase may, therefore, reduce fibroblast migration into sites of injury or inflammation.     

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions

Summary Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with⁵¹Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H₂O₂ became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress. Research supported by American Lung Association Fellowship Training Grant and Research Training Grant, the R. J. Reynolds Corporation, and National Institutes of Health Grants HL29784 and 1 HL 23805.     

Autoantibodies to fibrillin-1 activate normal human fibroblasts in culture through the TGF-beta pathway to recapitulate the "scleroderma phenotype"

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-beta1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-beta1 with anti-TGF-beta1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-beta1 from fibrillin-1-containing microfibrils in the ECM.     

An unexpected functional link between lysosomal thiol reductase and mitochondrial manganese superoxide dismutase

Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.     

CD109, a TGF-�� co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

Introduction

Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts.

Methods

CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot.

Results

The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-β₁-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.

Conclusion

The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.     

Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt₂)₂ was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728 nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5 min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.     

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts

Introduction

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote type I collagen (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants and are produced by lipoic acid synthetase (LIAS). Our goals in this study were to examine whether LA and LIAS were deficient in SSc patients and to determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison.

Methods

Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor 1 (PAI-1) and LIAS were measured by enzyme-linked immunosorbent assay. The expression of Col I was measured by immunofluorescence, hydroxyproline assay and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (αSMA) were measured by Western blotting. Student’s t-tests were performed for statistical analysis, and P-values less than 0.05 with two-tailed analysis were considered statistically significant.

Results

The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts; however, LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress and decreased PDGFR phosphorylation, Col I, PAI-1 and αSMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and MMP-3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc, but DHLA had a minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases.

Conclusions

DHLA acts not only as an antioxidant but also as an antifibrotic because it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC, LA, or DHLA, could be beneficial for patients with SSc.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0411-6) contains supplementary material, which is available to authorized users.     

Aggregate formation in Cu,Zn superoxide dismutase-related proteins

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.     

Levels of adiponectin,a marker for PPAR-gamma activity,correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker?

Introduction

Progressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.

Methods

Adiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.

Results

Patients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.

Conclusions

Levels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.     

Alarin alleviated cardiac fibrosis via attenuating oxidative stress in heart failure rats

The present study was to explore whether alarin could alleviate heart failure (HF) and attenuate cardia fibrosis via inhibiting oxidative stress. The fibrosis of cardiac fibroblasts (CFs) was induced by angiotensin (Ang) II. HF models were induced by ligation of the left anterior descending artery to cause ischemia myocardial infarction (MI) in Sprague–Dawley rats. Alarin (1.0 nM/kg/d) was administrated by intraperitoneal injection for 28 days. The decreases of left ventricular (LV) ejection fraction (EF), fractional shortening (FS), the maximum of the first differentiation of LV pressure (LV ± dp/dt_max) and LV systolic pressure (LVSP), and the increases of LV volume in systole (LVVS), LV volume in diastole (LVVD), LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) in MI rats were improved by alarin treatment. The increases in the expression levels of collagen I, collagen III, and transforming growth factor (TGF)-β were inhibited by alarin treatment in CFs and in the hearts of MI rats. The levels of NADPH oxidase (Nox) activity, superoxide anions and malondialdehyde (MDA) levels were increased, and the level of superoxide dismutase (SOD) activity was reduced in Ang II-treated CFs, which were reversed by alarin. Nox1 overexpression reversed the effects of alarin on attenuating the increases of collagen I, collagen III and TGF-β expression levels induced by Ang II in CFs. These results indicated that alarin improved HF and cardiac fibrosis via inhibiting oxidative stress in HF rats. Nox1 played important roles in the regulation of alarin effects on attenuating CFs fibrosis induced by Ang II.