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Significance: Reduction/oxidation (redox) balance could be defined as an even distribution of reduction and oxidation complementary processes and their reaction end products. There is a consensus that aberrant levels of reactive oxygen species (ROS), commonly observed in cancer, stimulate primary cell immortalization and progression of carcinogenesis. However, the mechanism how different ROS regulate redox balance is not completely understood. Recent Advances: In the current review, we have summarized the main signaling cascades inducing NADPH oxidase NOX1–5 and superoxide dismutase (SOD) 1–3 expression and their connection to cell proliferation, immortalization, transformation, and CD34+ cell differentiation in thyroid, colon, lung, breast, and hematological cancers. Critical Issues: Interestingly, many of the signaling pathways activating redox enzymes or mediating the effect of ROS are common, such as pathways initiated from G protein-coupled receptors and tyrosine kinase receptors involving protein kinase A, phospholipase C, calcium, and small GTPase signaling molecules. Future Directions: The clarification of interaction of signal transduction pathways could explain how cells regulate redox balance and may even provide means to inhibit the accumulation of harmful levels of ROS in human pathologies.

Background: Phosphorylation of the intracellular domain of the EPHA2 receptor tyrosine kinase (RTK) on serine 897 (S897) has been demonstrated to mediate EPHA2 oncogenic activity. Here, we show that in thyroid cancer cells harboring driver oncogenes that signal through the extracellular regulated kinase (ERK1/2) signaling pathway [rearranged RET RTK (RET/PTC), KRAS(G12R), or BRAFV600E oncogenes], EPHA2 is robustly phosphorylated on S897. EPHA2 S897 is embedded in a consensus sequence for phosphorylation by the AGC family kinases, including p90RSK (ribosomal protein S6 kinase), a direct ERK1/2 target. Methods: We show that recombinant p90RSK phosphorylates in vitro EPHA2 S897 and that treatment with chemical inhibitors targeting p90RSK or other components of the ERK1/2 pathway blunts S897 phosphorylation. Results: RNA interference-mediated knockdown combined with rescue experiments demonstrated that EPHA2 S897 phosphorylation mediates thyroid cancer cell proliferation and motility. Conclusions: These findings point to EPHA2 S897 as a crucial mediator of the oncogenic activity of the ERK1/2 signaling cascade in thyroid cancer.

Growth factors of the bombesin/gastrin releasing peptide (BN/GRP) family play a critical role in proliferation and progression of malignancies. Inhibitors targeting GRP signalling have been developed and tested as anticancer compounds showing promising preclinical and early phase clinical results. In this review, we will discuss the molecular signaling, expression and the functional role of BN/GRP-GRPR in different cancer models and will focus on the available strategies to target BN/GRP-GRPR in cancer treatment as well as in tumour diagnosis and follow up.

Sunitinib and sorafenib are broad-spectrum tyrosine kinase inhibitors (TKI) targeting, for example, VEGF1-3, PDGFRb, RET, FLT3, CD117 (c-KIT) and CSF-1R cell membrane receptors thus suppressing tumor angiogenesis and cancer cell growth. Recently it has been suggested that the kinases targeted by Sunitinib and/or Sorafenib regulate leukocyte transmigration, which might in part be responsible for the often-observed reduction in tumor-associated myeloid derived suppressor cells and regulatory T cells. The aim of the current study is to determine whether sunitinib or sorafenib inhibit leukocyte extravasation. Sunitinib, sorafenib, or vehicle treated animals did not show any difference in leukocyte trafficking either in peritonitis or in vivo homing experiments, although sunitinib treatment effectively inhibited growth of B16 melanoma tumors in WT, SCID and SCID beige mice. Inhibition of tumor growth was associated with an increased number of infiltrating CD11b+ cells in the tumor, while the numbers of CD8, Gr-1 and F4/80 expressing cells were unchanged. In conclusion, the findings suggest that despite multiple targets with a potential role in leukocyte extravasation, neither sunitinib nor sorafenib effectively inhibits this process in vivo. Thus, the observed specific effect on CD11b cells among tumor infiltrating leukocytes is most likely an indirect effect.

The Timeless (Tim) gene, originally identified in Drosophila melanogaster and subsequently in mammals, is involved in the molecular clockwork that drives 24h periodicity in physiology and behavior. The Tim protein is involved not only in circadian rhythmicity but also in embryonic development, cell cycle progression, DNA replication, and the DNA damage response (DDR). It is thus a multifaceted factor implicated in the maintenance of many cellular processes, tissue functions, and ultimately homeostasis of various organisms, from insects to humans. This review highlights the current knowledge of Tim functions, especially the most recent achievements, and illustrates the possible roles that this factor plays in the physiological preservation of health, as well as in the pathogenic mechanisms of related diseases.

Extracellular superoxide dismutase (SOD3) is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reaction that results in the formation of hydrogen peroxide. Both of these reactive oxygen species affect growth signaling in cells. Although SOD3 has growth-supporting characteristics, the expression ofSOD3is downregulated in epithelial cancer cells. In the current work, we studied the mechanisms regulatingSOD3expressionin vitrousing thyroid cell models representing different stages of thyroid cancer. We demonstrate that a low level of RAS activation increasesSOD3mRNA synthesis that then gradually decreases with increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells. Our data indicate thatSOD3regulation can be divided into two classes. The first class involves RAS–driven reversible regulation ofSOD3expression that can be mediated by the following mechanisms: RAS GTPase regulatory genes that are responsible forSOD3self-regulation; RAS-stimulated p38 MAPK activation; and RAS-activated increased expression of themir21microRNA, which inversely correlates withsod3mRNA expression. The second class involves permanent silencing ofSOD3mediated by epigenetic DNA methylation in cells that represent more advanced cancers. Therefore, the work suggests thatSOD3belongs to the group ofrasoncogene-silenced genes.

The early atmosphere of the earth contained low concentration of oxygen, which then slowly initiated to increase approximately 2.5 billion years ago after development of photosynthetic organisms and reached life-supporting levels 500–1000 million years ago. According to the oxygenic theory of evolution even the most primitive organisms used oxidative-antioxidative mechanisms in energy production, hence underlining the importance of reactive oxygen species (ROS) in normal cellular functions [1]. In reduction-oxidation (redox) biology the balance of oxidants and antioxidants determines the normal cellular homeostasis that is altered in tissue injuries and pathological conditions. Therefore, ROS, or unbalanced production of ROS, are often a consequence of injury or pathological condition with corresponding impact on cellular signal transduction and damage to macromolecules. The focus of this special issue is to characterize signaling pathways involved in tissue pathogenesis. Such studies potentially result in drug targets that supplement targets observed in currently used screenings [2].

// Giancarlo Vecchio 1, 2, * , Alessia Parascandolo 3, * , Chiara Allocca 1 , Clara Ugolini 4 , Fulvio Basolo 5 , Marco Moracci 6, 7 , Andrea Strazzulli 6, 7 , Beatrice Cobucci-Ponzano 6 , Mikko O. Laukkanen 3 , Maria Domenica Castellone 8 , Nobuo Tsuchida 9 1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli Federico II, Via Sergio Pansini, 5, Naples, Italy 2 Istituto Superiore di Oncologia, Via Sergio Pansini, 5, Naples and Via Balbi 5, Genoa, Italy 3 IRCCS SDN, Naples, Italy 4 Dipartimento di Medicina di Laboratorio, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy 5 Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell’Area Critica, University of Pisa, Italy 6 Institute of Biosciences and Bioresources (IBBR), National Research Council of Italy (CNR), Via P. Castellino, 111, Naples, Italy 7 Department of Biology, University of Naples “Federico II”, Complesso Universitario di Monte S. Angelo, Naples, Italy 8 Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” (IEOS), CNR, Naples, Italy 9 Graduate School of Medical and Dental Sciences Tokyo Medical and Dental University, Tokyo, Japan * These authors have contributed equally to this work Correspondence to: Maria Domenica Castellone, email: mcastell@unina.it Nobuo Tsuchida, email: ntutid.eme@tmd.ac.jp Keywords: glycosylation, fucosidase, thyroid carcinoma, metastasis Received: November 24, 2016 Accepted: January 24, 2017 Published: February 23, 2017 ABSTRACT Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.

Extracellular superoxide dismutase (SOD3) gene transfer to tissue damage results in increased healing, increased cell proliferation, decreased apoptosis, and decreased inflammatory cell infiltration. At molecular level,in vivoSOD3 overexpression reduces superoxide anion (O2-) concentration and increases mitogen kinase activation suggesting that SOD3 could have life-supporting characteristics. The hypothesis is further strengthened by the observations showing significantly increased mortality in conditional knockout mice. However, in cancer SOD3 has been shown to either increase or decrease cell proliferation and survival depending on the model system used, indicating that SOD3-derived growth mechanisms are not completely understood. In this paper, the author reviews the main discoveries in SOD3-dependent growth regulation and signal transduction.

Extracellular superoxide dismutase (SOD3) gene therapy has been shown to attenuate tissue damages and to improve the recovery of the tissue injuries, but the cellular events delivering the therapeutic response of the enzyme are not well defined. In the current work, we overexpressed SOD3 in rat hindlimb ischemia model to study the signal transduction and injury healing following the sod3 gene transfer. The data suggest a novel sod3 gene transfer-derived signal transduction cascade through Ras-Mek-Erk mitogenic pathway leading to activation of AP1 and CRE transcription factors, increased vascular endothelial growth factor (VEGF)-A and cyclin D1 expression, increased cell proliferation, and consequently improved metabolic functionality of the injured tissue. Increased cell proliferation could explain the improved metabolic performance and the healing of the tissue damages after the sod3 gene transfer. The present data is a novel description of the molecular mechanism of SOD3-mediated recovery of tissue injury and suggests a new physiological role for SOD3 as a Ras regulatory molecule in signal transduction.

ObjectivesThis study examined whether local gene therapy with extracellular superoxide dismutase (EC-SOD) could inhibit in-stent restenosis in atherosclerotic Watanabe heritable hyperlipidemic rabbits.BackgroundStenting causes an acute increase in superoxide anion production and oxidative stress; EC-SOD is a major component of antioxidative defense in blood vessels and has powerful cardioprotective effects in ischemic myocardium.MethodsEndothelial denudation and stenting were done in 36 adult (15 to 18 months old) rabbits. Catheter-mediated intramural delivery of clinical good manufacturing practice-grade adenoviruses encoding rabbit EC-SOD were done simultaneously with stenting. Control animals received adenovirus-encoding nuclear-targeted β-galactosidase (AdLacZ). Circulating markers for oxidative stress (nonesterified 8-iso-prostaglandin F2alpha) were measured. Analysis of 6-day, 28-day, and 90-day vessel histology, radical production, oxidation-specific epitopes, and expression studies were performed.ResultsThe EC-SOD treatment reduced oxidant production in stented vessels compared with control vessels. Early systemic recovery of total SOD activity was observed in the treated rabbits. The EC-SOD significantly accelerated endothelial recovery (67.4% ± 10.8% vs. 24.2.1% ± 4.6% at 6 days, p < 0.05; 89.3% ± 3.7% vs. 45.1% ± 9.6% at 28 days, p < 0.05), and the beneficial effect involved increased proliferation of regenerating endothelium. The EC-SOD group showed a 61.3% lower (p < 0.05) neointimal formation at 28 days, with a similar, albeit nonsignificant trend at 90 days (1.20 ± 0.32 mm2vs. 1.88 ± 0.24 mm2, p = 0.06).ConclusionsThe results suggest a central pathogenetic role of oxidation sensitive signaling processes in endothelial recovery and developing in-stent restenosis in atherosclerotic vessels. Local therapy against oxidative stress represents a promising therapeutic strategy in stent-induced vascular injury.

Mesenchymal stromal cells (MSCs) are able to influence the growth abilities of transformed cells. Here, we show that papillary thyroid cancer TPC1 and HEK 293T cells interact physically with human primary bone marrow-derived MSCs followed by evanescence of MSC cytoplasm. Interestingly, transformed cells were able to connect only to apoptotic MSCs that had lost their migration ability, whereas naïve MSCs avoided the direct contact. The interaction stimulated the proliferation of the cocultured transformed cells, activated mitogen and stress signaling, and increased resistance to cytotoxins. Consistent with in vitro data, the MSC interaction stimulated transformed cells had enhanced ability to grow and metastasize in vivo. The parental control cells showed mild tumorigenicity as compared to MSC interaction stimulated cells yielding measurable tumors in 31 days and 7 days, respectively. Our coculture model system describes how adjacent transformed cells absorb stromal cells thereby leading to the stroma-driven evolution of moderately carcinogenic cells to highly aggressive metastatic cells.

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream G?q and G?12/13 GTPases, and consistently, other G?q and G?13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active G?qQL and G?12/13QL mutants stimulated Gli. By using cells null for G?q and G?12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, G?q- and G?12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/G?q-12/13/Rho mediated activation of nuclear factor ?B (NF?B), which can stimulate a NF-?B response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.Oncogene advance online publication, 21 April 2014; doi:10.1038/onc.2014.104.

Recent reports linking insertional activation of LMO2 following gene therapy for X-linked severe combined immunodeficiency (X-SCID) have led to a re-evaluation of risks following gene therapy with retroviral vectors. In our analysis of 702 integration sites in rhesus macaques that underwent transplantation up to 7 years earlier with autologous CD34+ cells transduced with amphotropic murine leukemia virus (MLV)-derived retroviral vectors containing marker genes, we detected insertion into one locus, the Mds1/Evi1 region, a total of 14 times in 9 animals. Mds1/Evi1 integrations were observed stably long term, primarily in myeloid cells. We hypothesize that this over-representation likely results from an impact on the self-renewal and engraftment potential of CD34+ progenitor cells via insertional mutagenesis at this specific locus. There is no evidence of ongoing in vivo clonal expansion of the Mds1/Evi1 populations, and all animals are hematologically normal without evidence for leukemia. Characterization of integration sites in this relevant preclinical model provides critical information for gene therapy risk assessment as well as identification of genes controlling hematopoiesis. (Blood. 2005;106:2530-2533)

Due to high frequency of side effects caused by high-dose total body irradiation (TBI) the nonmyeloablative regimen together with cytotoxic agents is currently used especially for elderly patients. However, immediate and long-term effects of low-dose irradiation used in allogeneic transplantation on stem cells is less well known. We have studied the effect of low-dose 3 Gy TBI on the number of hematopoietic stem cell (HSC) clones contributing simultaneously to granulocyte production in rhesus macaque. The number of clones after 3 Gy TBI decreased markedly by 2 to 3 weeks after 3 Gy TBI, followed by a period of clonal instability, and recovery to almost pre–3 Gy TBI clonal diversity. The clones accounting for this recovery contributed before 3 Gy TBI, suggesting the profound initial impact of TBI was on a pool of progenitor cells, whereas most of the more primitive HSCs remained unaffected and were able to again contribute to hematopoiesis after recovery. Clonal fluctuation may indirectly suggest the presence of short-term/long-term HSC populations in rhesus macaque bone marrow as reported in a mouse model. The results indicate that even low-dose irradiation affects hematopoietic clonal dynamics and have implications for design of conditioning regimens for transplantation purposes.

Adenovirus is a widely used vector in gene transfer experiments because it produces high transduction efficiency in vitro and in vivo by means of the coxsackie-adenovirus receptor (CAR) and major histocompatibility complex (MHC) class I alpha-2 domain. Adenoviral gene transfer efficiency has been reported to correlate with cellular CAR expression. We report here a simple method to increase adenoviral gene transfer efficiency in cells that do not express high levels of CAR: preincubation of adenovirus for 30-40 minutes at +37 degrees C significantly increased the transduction efficiency in vitro in CHO and BALB/3T3 cells, in which CAR is expressed at very low levels. Increased transduction efficiency of preincubated adenovirus was also detected in vivo in rat brain tissue. In addition, we found that adenoviruses were rapidly inactivated in human serum in a complement-independent manner, whereas fetal bovine serum (FBS) had hardly any effects on the viral infectivity. We conclude that preincubation of adenoviral vectors at +37 degrees C may substantially increase gene transfer efficiency in applications in which target cells do not express high levels of CAR.

Clinical and epidemiological studies have provided circumstantial evidence that oxidized low-density lipoprotein (LDL) and antioxidants are involved in the pathogenesis of atherosclerosis. Superoxide dismutases (SODs) have been shown in vitro to protect LDL from deleterious effects of superoxide anions. In the present study, we have used adenoviral gene transfer to determine effect of extracellular SOD (EC-SOD) on atherogenesis in LDL receptor -/- mice. Intravenous administration of EC-SOD adenovirus (2 x 10(9) plaque forming units) into tail vein targeted transgene mainly to liver and induced a 3.5- to sevenfold increase in plasma total SOD activity. EC-SOD was secreted into circulation for 2-3 weeks mostly in a truncated B-form, suggesting that endogenous proteolytic mechanisms control the level and distribution of the enzyme. Therapeutic potential was determined by measuring plasma resistance against copper oxidation and analyzing atherosclerotic lesion areas in aortas of LDL receptor -/- mice. Mice were kept on a cholesterol diet for 10 weeks before gene transfer and 3 or 6 weeks after the gene transfer. Results showed a tendency for a reduction in the overall lesion area after EC-SOD gene transfer as compared with LacZ transduced control mice, but the difference did not reach statistical significance. It is concluded that short-term overexpression of EC-SOD in vivo does not affect atherogenesis in LDL receptor -/- mice.

Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidative enzyme with an abundant mRNA expression in kidney and arterial wall. In order to study expression and antioxidative function of EC-SOD, we cloned the rabbit ec-sod cDNA and produced the recombinant protein in cell culture. In vitro studies did not show a direct relationship between the amounts of synthesized mRNA and secreted protein activity, suggesting post-transcriptional regulation. The antiatherogenic role of EC-SOD was studied by determining the effect of EC-SOD on the oxidation (ox) of low density lipoprotein (LDL), and subsequent degradation of oxLDL in RAW 264 macrophages in vitro. It was found that recombinant EC-SOD reduced both the degradation of LDL in RAW 264 macrophages by 28-36% and its electrophoretic mobility caused by endothelial cell-mediated oxidation. It is therefore suggested that EC-SOD can act as a protective enzyme against the development of atherosclerosis.

BackgroundExtracellular superoxide dismutase (SOD3), which dismutates superoxide anion to hydrogen peroxide, has been shown to reduce the free radical stress derived apoptosis in tissue injuries. Since both superoxide anion and hydrogen peroxide have a marked impact on signal transduction pathways and could potentially explain a number of apoptosis and survival -related phenomena in different pathological conditions, we clarified the impact of SOD3 on Akt and Erk1/2 cell survival pathways in rat hind limb injury model.Methodology and principal findingsBased on our data, the hind limb ischemic rats treated with virally delivered sod3 have milder injury and less apoptosis than control animals that could be due to parallel activation of pro-proliferative and anti-apoptotic Erk1/2 and Akt pathways. The common downstream factor of both signaling pathways, the apoptosis related forkhead box protein O3a (FoxO3a), was phosphorylated and translocated to the cytoplasm in sod3 treated tissues and cell line. Additionally, we obtained increased mRNA production of elk-1, ets-1, and microRNA 21 (miR-21), whereas synthesis of bim mRNA was decreased in sod3 overexpressing tissues. We further showed that overexpression of sod3 modulated redox related gene expression by downregulating nox2 and inos when compared to injured control animals.Conclusions and significanceThe study shows the complexity of SOD3-derived effects on tissue injury recovery that are not limited to the reduction of superoxide anion caused cellular stress but highlights the impact of SOD3 related signal transduction on tissue functions and suggests an important role for SOD3 in attenuating cell stress effects in different pathological conditions.

Reactive oxygen species, specifically hydrogen peroxide (H2O2), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H2O2 synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H2O2-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C–Ca2+ and cAMP–protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.

Inflammatory cell migration characteristic of ischemic damages has a dual role providing the tissue with factors needed for tissue injury recovery simultaneously causing deleterious development depending on the quality and the quantity of infiltrated cells. Extracellular superoxide dismutase (SOD3) has been shown to have an anti-inflammatory role in ischemic injuries where it increases the recovery process by activating mitogen signal transduction and increasing cell proliferation. However, SOD3 derived effects on inflammatory cytokine and adhesion molecule expression, which would explain reduced inflammation in vascular lesions, has not been properly characterized. In the present work the effect of SOD3 on the inflammatory cell extravasation was studied in vivo in rat hind limb ischemia and mouse peritonitis models by identifying the migrated cells and analyzing SOD3-derived response on inflammatory cytokine and adhesion molecule expression. SOD3 overexpression significantly reduced TNF alpha, IL1 alpha, IL6, MIP2, and MCP-1 cytokine and VCAM, ICAM, P-selectin, and E-selectin adhesion molecule expressions in injured tissues. Consequently the mononuclear cell, especially CD68+ monocyte and CD3+ T cell infiltration were significantly decreased whereas granulocyte migration was less affected. According to our data SOD3 has a selective anti-inflammatory role in ischemic damages preventing the migration of reactive oxygen producing monocyte/macrophages, which in excessive amounts could potentially further intensify the tissue injuries therefore suggesting potential for SOD3 in treatment of inflammatory disorders.

Background aims Mesenchymal stromal cells (MSC) have been used in a wide variety of pre-clinical experiments and in an increasing number of human clinical trials. Although many of these studies have shown different levels of engraftment, the exact fate of MSC after transplantation and the tissue response to their engraftment have not been investigated in detail. In the present work we studied the distribution of human MSC in a rat hind limb ischemic injury model immediately after transplantation and also analyzed the recipient tissue response to transplanted cells. Methods We tracked the in vivo fate of the transplanted MSC utilizing bioluminescence imaging, fluorescence microscopy and gene/protein expression analysis in a rat hind limb ischemia model. We also monitored the viability of transplanted cells by graft versus recipient expression analysis and determined the angiogenic and proliferative effect of transplantation by histologic staining. Results According to imaging analysis only a small portion of cells persisted for an extended period of time at the site of injury. Interestingly, recipient versus graft expression studies showed increased synthesis of rat-origin angiogenic factors and no human-origin mRNA or protein synthesis in transplanted tissues. More importantly, despite the lack of robust engraftment or growth factor secretion the transplantation procedure exerted a significant pro-angiogenic and pro-proliferative effect, which was mediated by angiogenic and mitogenic signaling pathways. Conclusions Our results show an immediate temporal tissue effect in response to MSC transplantation that may represent a novel indirect paracrine mechanism for the beneficial effects of cell transplantation observed in injured tissues. © 2009 ISCT.

An understanding of the number and contribution of individual pluripotent hematopoietic stem cells (HSCs) to the formation of blood lineages has important clinical implications for gene therapy and stem cell transplantation. We have been able to efficiently mark rhesus macaque long-term repopulating stem and progenitor cells with retroviral vectors, and track their in vivo contributions to hematopoiesis using the linear amplification mediated–polymerase chain reaction (LAM-PCR) technique of insertion site analysis. We assessed the impact of busulfan on contributions of individual retrovirally marked clones to hematopoiesis. There were 2 macaques that received transplants of retrovirally transduced CD34+ cells 2 years previously that were then treated with 4 mg/kg busulfan. Despite only transient and mild suppression of peripheral blood counts, the numbers of individual stem/progenitor clones contributing to granulocyte production decreased dramatically, by 80% in the first monkey and by 60% in the second monkey. A similar impact was seen on clones contributing to T cells. The clone numbers recovered gradually back toward baseline by 5 months following busulfan in the first monkey and by 3 months in the second monkey, and have remained stable for more than one year in both animals. Tracking of individual clones with insertion-site–specific primers suggested that clones contributing to hematopoiesis prior to busulfan accounted for the majority of this recovery, but that some previously undetected clones began to contribute during this recovery phase. These results indicate that even low-dose busulfan significantly affects stem and progenitor cell dynamics. The clonal diversity of hematopoiesis was significantly decreased after even a single, clinically well-tolerated dose of busulfan, with slow but almost complete recovery over the next several months, suggesting that true long-term repopulating stem cells were not permanently deleted. However, the prolonged period of suppression of many clones suggests that transplanted HSCs may have a marked competitive advantage if they can engraft and proliferate during this time period, and supports the use of this agent in nonmyeloablative regimens

Tracking transplanted stem cells using magnetic resonance imaging (MRI) could offer biologic insight into homing and engraftment. Ultrasmall dextran-coated iron oxide particles have previously been developed for uptake into cells to allow MRI tracking. We describe a new application of much larger, micron-scale, iron oxide magnetic particles with enhanced MR susceptibility, which enables detection of single cells at resolutions that can be achieved in vivo. In addition, these larger particles possess a fluorophore for histologic confirmation of cell distribution. We demonstrate highly efficient, nontoxic, endosomal uptake of these particles into hematopoietic CD34+ cells and mesenchymal stem cells documented by confocal and electron microscopy. Labeled cells retain biologic activity with preservation of colony-forming ability and differentiation capacity. MRI studies could detect labeled CD34+ cells and mesenchymal stem cells (MSCs) at single cell resolution. This appears to be a promising tool for serial noninvasive monitoring of in vivo cell homing and localization using MRI.

Background— Restenosis is a frequent problem after invasive treatment of atherosclerotic vessels and is associated with intimal hyperplasia, which is primarily a result of proliferation and migration of smooth muscle cells, leading to the formation of neointima. Because there is no effective conventional medication for restenosis, gene therapy is a potential new treatment to prevent neointima formation. Methods and Results— In the present study, we analyzed the effects of adenovirus-mediated extracellular superoxide dismutase (EC-SOD) gene transfer (3×10 9 pfu/kg AdEC-SOD versus AdLacZ control virus) on neointima formation in balloon-denuded rabbit aortas. Local catheter-mediated gene transfer to the arterial wall reduced restenosis ( P P P Conclusions— The results suggest that EC-SOD gene transfer reduces restenosis and may be useful for the prevention of intimal hyperplasia after vascular manipulations.

Lipoprotein peroxidation in the arterial wall has been implicated in atherogenesis. The superoxide radical is formed in arteries and can induce such oxidation. Extracellular superoxide dismutase (EC-SOD) occurs in high concentration in the vascular wall interstitium, and in this study, we examined the importance of the enzyme in atherogenesis. On an apolipoprotein E–null background, the limited aortic lesions induced by a 1-month atherogenic diet were larger in EC-SOD wild-type mice than in EC-SOD–null mice, whereas there were no differences between the EC-SOD genotypes in the larger lesions seen after 3 months on the diet or after 8 months on normal chow. Despite smaller or equal lesions in the EC-SOD–null mice, their cholesterol levels were somewhat higher. Also, on a wild-type background, there were no effects produced by the absence or presence of EC-SOD on atherogenic diet–induced aortic root lesions. The urinary excretion of the lipid peroxidation biomarker 8-isoprostaglandin F2αwas related to the rates of atherogenesis in the mice but was not influenced by the EC-SOD genotype. Likewise, the EC-SOD status had no effect on the staining for oxidized low density lipoprotein epitopes in aortic root sections. Our findings suggest that EC-SOD has little influence on atherogenesis in mice.

Background Paracetamol overdose causes acute liver damage which leads to severe centrilobular hepatic necrosis. The hepatotoxic effect is caused by reactive metabolites and oxidative stress. Since extracellular superoxide dismutase (EC-SOD) protects tissues against the harmful effects of superoxide anion, the hypothesis that systemic adenovirus-mediated EC-SOD gene transfer could reduce liver damage was tested. Methods Mice were given paracetamol (600 mg/kg) enterally 2 days after adenovirus-mediated gene transfer of EC-SOD (2×109 pfu). Five days after gene transfer, plasma and tissue samples were collected for clinical chemistry analyses and tissue pathology evaluation. Results EC-SOD was expressed in a dose-dependent manner with the highest enzyme activity occurring 3 days after the gene transfer. Clinical chemistry and tissue pathology analyses showed that adenoviral EC-SOD gene transfer significantly attenuated release of liver enzymes and inhibited necrosis and apoptosis caused by paracetamol overdose. Conclusion The results indicate the involvement of superoxide anion in paracetamol-mediated liver damage and suggest a possible protective role for EC-SOD gene transfer in paracetamol-induced liver damage. Copyright © 2001 John Wiley & Sons, Ltd.

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1–5, avrl-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88–92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68–78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.

Extracellular superoxide dismutase (SOD3), an enzyme mediating dismutation of superoxide into hydrogen peroxide, has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues. In inflamed tissues, superoxide is produced by the phagocytic NOX2 complex, which consists of the catalytic subunit NOX2 and several regulatory subunits (e.g., NCF1). To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, we injected an adenoviral vector overexpressing SOD3 directly into the arthritic paws ofNcf1*/*mice with collagen-induced arthritis. SOD3 reduced arthritis severity in both oxidative burst-deficientNcf1*/*mice and also in wild-type mice. The NOX2 complex independent anti-inflammatory effect of SOD3 was further characterized in peritonitis, and SOD3 was found to reduce macrophage infiltration independently of NOX2 complex functionality. We conclude that the SOD3-mediated anti-inflammatory effect on arthritis and peritonitis operates independently of NOX2 complex derived oxidative burst.

Abstract —Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.