Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Genome-wide identification of human microRNAs located in leukemia-associated genomic alterations.

Cytogenetic alterations, such as amplifications, deletions, or translocations, contribute to myeloid malignancies. MicroRNAs (miRNAs) have emerged as critical regulators of hematopoiesis, and their aberrant expression has been associated with leukemia. Genomic regions containing sequence alterations and fragile sites in cancers are enriched with miRNAs; however, the relevant miRNAs within these regions have not been evaluated on a global basis. Here, we investigated miRNAs relevant to acute myeloid leukemia (AML) by (1) mapping miRNAs within leukemia-associated genomic alterations in human AML cell lines by high-resolution genome arrays and (2) evaluating absolute expression of these miRNAs by massively parallel small RNA sequencing. Seventy-seven percent (542 of 706) of miRNAs mapped to leukemia-associated copy-number alterations in the cell lines; however, only 18% (99 of 542) of these miRNAs are expressed above background levels. As evidence that this subset of miRNAs is relevant to leukemia, we show that loss of 2 miRNAs identified in our analysis, miR-145 and miR-146a, results in leukemia in a mouse model. Small RNA sequencing identified 28 putative novel miRNAs, 18 of which map to leukemia-associated copy-number alterations. This detailed genomic and small RNA analysis points to a subset of miRNAs that may play a role in myeloid malignancies.

Tyk2 tyrosine kinase expression is required for the maintenance of mitochondrial respiration in primary pro-B lymphocytes.

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-beta. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-beta. Finally, Tyk2(-/-) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.

miR-143/145 differentially regulate hematopoietic stem and progenitor activity through suppression of canonical TGFß signaling.

Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs), but activation of progenitor cells (HPCs). We identify components of the transforming growth factor ß (TGFß) pathway as key targets of miR-143/145. Enforced expression of the TGFß adaptor protein and miR-145 target, Disabled-2 (DAB2), recapitulates the HSC defect seen in miR-143/145-/- mice. Despite reduced HSC activity, older miR-143/145-/- and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy, associated with expansion of HPC. Thus, miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFß/DAB2 activation, and loss of these miRNAs creates a preleukemic state.

[Bacterial and fungal bloodstream infections in patients at the University Hospital of Cracow]. / Bakteryjne i grzybicze zakazenia krwi u chorych w Oddzialach Klinicznych Szpitala Uniwersyteckiego w Krakowie.

In presented material evaluation of changes in sepsis and types of bloodstream infections of hospitalized patients in Wards of the University Hospital in Cracow were examined. Results of 9,138 blood cultures studied in years 1989-1999 were analysed. All of the blood samples were recovered from 4,656 infected adults at Clinics of the University Hospital in Cracow. Microbiological blood examinations were held in system of constant monitoring of isolated cultures applying BacT/Alert--colorimetric system (Organon Teknika). Cultured micro--organisms were identified using commercial biochemical tests (bio-Merieux). During period of research changes of profile of isolated microorganisms was observed. Percentage of blood infections of Enterococcus spp. etiology increased from 2.2% in 1989 to 9.8% in 1997-98 (p = 0.014). Dynamic growth of non-fermentative S. maltophilia bacilli to 5.5% (p = 0.036) and Serratia marcescens to 13.8% (p = 0.042) in 1999 was revealed. Designed according to our research review of fungal flora in years 1989-1999 revealed tendency of systematic growth of invasive candidemia frequency, from 1.1% to 10.4%. Diagnostic and therapeutic profile of Departments was in a strict connection with increase of the number and meaning of the politiological bacteremias (p = 0.036) in total number of systemic infections cases.

[Infections with Van B phenotype Enterococcus faecium and E. faecalis in patients with immune deficiency during the course of hematologic neoplasms]. / Zakazenia fenotypem Van B Enterococcus faecium i E. faecalis u chorych z niedoborem immunologicznym w przebiegu chorób nowotworowych ukladu krwiotwórczego.

In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. The aim of this study was to characteristics of the types of resistance to glycopeptide antibiotic and to check the resistance patterns of these pathogens. A trial of monitoring the risk factors for colonization or infection with VRE as well as epidemiological investigation were undertaken. Genus identification of the isolates was performed according to the method of Facklam and Collins, and species were identified using the API 20 Strep test. MICs of different antimicrobial agents were determined by the E-test method. The isolates collected during the investigation demonstrated resistance to multiple antimicrobials, which is a common characteristic of VRE. Isolates were found to be uniformly resistant to penicillin, fluoroquinolones, tetracycline and to high concentrations of aminoglycosides. The only drugs with in vitro activity against the isolates were ampicillin (VRES), linezolid (VRES, VREM) and quinupristin-dalfopristin (VREM). Except for a single VREM isolate, they all revealed the typical VanB phenotype with resistance to vancomycin and susceptibility to teicoplanin. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene claster, including the regulatory genes vanRB and -SB, and the vancomycin--inducible promoter PYB. Our studies have found an association between colonization or infection with VRE and the mean duration of hospital stay, previously administration of glycopeptide, cephalosporins and imipenem. These organisms were a common cause of monoethiological bloodstream infections.

Accurate and safe mediastinal restaging by combined endobronchial and endoscopic ultrasound-guided needle aspiration performed by single ultrasound bronchoscope.

OBJECTIVES: The aim of this prospective trial was to assess the diagnostic utility of combined endobronchial (EBUS) and endoscopic (EUS) ultrasound-guided needle aspiration by use of a single ultrasound bronchoscope (CUSb-NA) in non-small-cell lung cancer (NSCLC) restaging in patients after induction therapy. METHODS: In a consecutive group of NSCLC patients with pathologically confirmed N2 disease (clinical stage IIIa and IIIb) who underwent induction chemotherapy, CUSb-NA was performed. All of the patients with negative or suspected for metastases (uncertain) diagnosed by endoscopy underwent subsequently transcervical extended mediastinal lymphadenectomy (TEMLA) as a confirmatory test. RESULTS: From January 2009 to December 2012, 106 patients met the inclusion criteria and underwent restaging CUSb-NA under mild sedation, in whom 286 (mean 2.7, range 2-5) lymph node stations were biopsied, 127 (mean 1.2, range 1-3) by EBUS-transbronchial needle aspiration (TBNA) and 159 (mean 1.5, range 1-4) by EUS-fine needle aspiration (FNA). The CUSb-NA revealed metastatic lymph node involvement in 37/106 patients (34.9%). In 69 (65.1%) patients with negative and uncertain CUSb-NA in 4 (3.8%) out of them, who underwent subsequent TEMLA metastatic nodes were found in 18 patients (17.0%) and there were single lymph nodes found only in one mediastinal station (minimal N2) in 10 (9.4%) out of them. False-positive results were found in 2 (1.9%) patients. In 9 (8.5%) patients CUSb-NA occurred to be false negative in Stations 2R and 4R (only accessible for EBUS), exclusively in small nodes and in 4 (3.8%) patients in Station 5-not accessible for CUSb-NA. The prevalence of mediastinal lymph node metastases in the present study was 51.9%. Diagnostic sensitivity, specificity, total accuracy, positive predictive value and negative predictive value (NPV) of the restaging CUSb-NA were 67.3% (95% CI [confidence interval]-53-79), 96.0% (95% CI-86-99), 81.0% (95% CI-73-87), 95.0% (95% CI-83-99) and 73.0% (95% CI-61-83), respectively. The sensitivity, accuracy and NPV of CUSb-NA were higher compared with EBUS-TBNA and EUS-FNA alone. No complications of CUSb-NA were observed. CONCLUSIONS: The CUSb-NA is a reasonable and safe technique in mediastinal restaging in NSCLC patients after induction therapy. Following our data, in patients with negative result of CUSb-NA, a surgical restaging of the mediastinum should be considered.

Mouse models of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic malignancies characterized by peripheral cytopenias in the face of normo- or hypercellular, dysplastic bone marrow that arise from mutations in the hematopoietic stem/progenitor cell (HSPC). The disease is characterized by multiple cytogenetic and molecular defects, which result in an extremely heterogeneous phenotype. Recently, significant efforts have been made to develop appropriate mouse models to study this complex disease. Because of the heterogeneity of MDS, no single model is able to capture the MDS phenotype in its entirety. In this review, we describe several MDS mouse models and discuss the advances made in our understanding of the different disease mechanisms within the malignant clone and the marrow microenvironment. In addition, we describe progress in xenotransplantation models of MDS and discuss questions that remain to be answered.

MicroRNA-146a disrupts hematopoietic differentiation and survival.

OBJECTIVE: MicroRNAs (miRNAs) are short noncoding RNAs capable of exerting dramatic effects by postranscriptionally regulating numerous messenger RNA targets. Toll-like receptor-4 (TLR-4) activation by lipopolysaccharide (LPS) induces the expression of three miRNAs in myeloid cells. The aim of this study was to investigate the in vivo consequences of expressing one of the LPS-induced miRNA, miR-146a, in bone marrow cells. MATERIAL AND METHODS: The role of miR-146a in hematopoiesis was investigated by using retroviral infection and overexpression of miR-146a in mouse hematopoietic stem/progenitor cells, followed by bone marrow transplantations. RESULTS: miR-146a is mainly expressed in primitive hematopoietic stem cells and T lymphocytes. Overexpression of miR-146a in hematopoietic stem cells, followed by bone marrow transplantation, resulted in a transient myeloid expansion, decreased erythropoiesis, and impaired lymphopoiesis in select anatomical locations. Enforced expression of miR-146a also impaired bone marrow reconstitution in recipient mice and reduced survival of hematopoietic stem cells. CONCLUSIONS: Our results indicate that miR-146a, an LPS-induced miRNA, regulates multiple aspects of hematopoietic differentiation and survival. Furthermore, the consequences of miR-146a expression in hematopoietic cells mimics some of the reported effects with acute LPS exposure.

TRAF6 is an amplified oncogene bridging the RAS and NF-κB pathways in human lung cancer.

Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer-associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor-associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.

Infectious complications in patients with acute myeloid leukemia treated according to the protocol with daunorubicin and cytarabine with or without addition of cladribine. A multicenter study by the Polish Adult Leukemia Group (PALG).

OBJECTIVES: The addition of cladribine to the standard regimen consisting of daunorubicin and cytarabine has been reported to increase the efficacy of induction therapy in acute myeloid leukemia (AML). The goal of this study was to determine the effect of this modification on the incidence and spectrum of infectious complications. METHODS: Case report forms of 309 patients with newly diagnosed AML who had been enrolled in the prospective, randomized 'DAC-7 vs. DA-7' trial were reviewed. The frequency, etiology, localization, severity, and outcome of infections were compared for patients receiving only daunorubicin and cytarabine (DA-7) and those additionally treated with cladribine (DAC-7). RESULTS: A total of 443 febrile episodes were reported with no significant difference between the treatment groups. A trend towards a higher frequency of bacteremias was observed among DA-7 patients compared to those in the DAC-7 group (31% vs. 21%; p=0.08). The treatment arms did not differ in terms of the distribution of the isolated Gram-positive, Gram-negative, fungal, and viral organisms. However, when bacteremias were considered, Gram-positive blood cultures tended to be more frequent in the DA-7 compared to the DAC-7 group (16% vs. 8.5%; p=0.07). This difference reached statistical significance when major blood bacteremias were analyzed separately (13% vs. 5%; p=0.02). Complete recovery from infections was observed in the majority of patients across both treatment arms and no significant difference was noted regarding infection-related mortality. CONCLUSIONS: The addition of cladribine to standard induction chemotherapy has no impact on the incidence and spectrum of infectious complications in newly diagnosed AML patients.

Mitochondrial STAT3 supports Ras-dependent oncogenic transformation.

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor responsive to cytokine signaling and tyrosine kinase oncoproteins by nuclear translocation when it is tyrosine-phosphorylated. We report that malignant transformation by activated Ras is impaired without STAT3, in spite of the inability of Ras to drive STAT3 tyrosine phosphorylation or nuclear translocation. Moreover, STAT3 mutants that cannot be tyrosine-phosphorylated, that are retained in the cytoplasm, or that cannot bind DNA nonetheless supported Ras-mediated transformation. Unexpectedly, STAT3 was detected within mitochondria, and exclusive targeting of STAT3 to mitochondria without nuclear accumulation facilitated Ras transformation. Mitochondrial STAT3 sustained altered glycolytic and oxidative phosphorylation activities characteristic of cancer cells. Thus, in addition to its nuclear transcriptional role, STAT3 regulates a metabolic function in mitochondria, supporting Ras-dependent malignant transformation.

HIF-1 attenuates Ref-1 expression in endothelial cells: reversal by siRNA and inhibition of geranylgeranylation.

Redox factor-1 (Ref-1), a multifunctional protein with DNA repairing activities, plays a cytoprotective function by post-translational redox modification of numerous transcription factors, including hypoxia inducible factor-1 (HIF-1). In the present study, activation of HIF-1 by hypoxia and dimethyloxaloylglycine (DMOG), a hypoxia mimic, diminished Ref-1 mRNA and protein expression in human microvascular endothelial cells (HMEC-1). Similarly, adenoviral delivery of the stabilized form of HIF-1alpha decreased Ref-1 mRNA and protein levels. Accordingly, HIF-1alpha siRNA abolished the hypoxia-induced inhibition of Ref-1 expression, indicating the role of HIF-1 in down-regulation of Ref-1. Also, translocation of Ref-1 from nucleus to cytoplasm after HIF-1 activation was noted. Interestingly, we observed the restoration of Ref-1 expression in hypoxia by pharmacologically relevant doses of atorvastatin. This effect was dependent on the inhibition of protein geranylgeranylation, but not farnesylation, as only the inhibitor of the former but not the latter prenylation step restored the Ref-1 expression. The regulation of Ref-1 by statins may be considered as a novel mechanism of their beneficial effects on endothelium.

HIF-1 induction attenuates Nrf2-dependent IL-8 expression in human endothelial cells.

Through hypoxia-inducible factor 1 (HIF-1), hypoxia regulates the expression of numerous genes and is a potent inducer of angiogenesis. However, interleukin-8 (IL-8), an important angiogenic mediator, has been reported to be downregulated by HIF-1, although the mechanisms have not been elucidated. HIF-1 was induced in human endothelial cells by hypoxia and dimethyloxaloylglycine (DMOG). Interestingly, both hypoxia and DMOG attenuated IL-8 expression, and a similar effect has been obtained by adenoviral overexpression of the stable form of HIF-1alpha. Heme oxygenase-1 (HO-1) expression was also downregulated by HIF-1 induction. This suggests similar mechanisms of regulation of IL-8 and HO-1, indicating the involvement of Nrf2, a transcription factor previously linked to hypoxia-mediated inhibition of HO-1. Indeed, HIF-1-mediated downregulation of both IL-8 and HO-1 was associated with both lowered Nrf2 expression and induction of Bach1, a repressor of Nrf2 transcriptional activity. Accordingly, overexpression of Nrf2 reversed the inhibitory effect of HIF-1 on IL-8 and HO-1 expression. However, neither overexpression of HO-1 nor HO-1 inhibition affected IL-8 synthesis. The data indicate that HIF-1-dependent inhibition of IL-8 expression is caused by downregulation of Nrf2. However, expression of IL-8 is independent of HO-1. Cross-talk between HIF-1 and Nrf2 may influence the outcome of anti-angiogenic therapies aimed at targeting HIF-1. Antioxid.

Function of mitochondrial Stat3 in cellular respiration.

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3(-/-) cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the protein's function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

Immunogenicity of influenza vaccination in patients with non-Hodgkin lymphoma.

PURPOSE: The purpose of this study was to assess humoral response to influenza vaccine in patients (pts) with non-Hodgkin lymphoma (NHL) as compared to healthy subjects (ctrl). PATIENTS AND METHODS: In two epidemic seasons, 2003/2004 and 2004/2005, 163 pts and 92 ctrl were vaccinated. Antibody titers to hemagglutinin (HA) and neuraminidase (NA) were measured in serum samples collected before vaccination, and 1 and 6 months apart. Changes in antibody titers were assessed by comparing geometric mean titers (GMT), mean fold increases (MFI), and seroprotection and seroresponse rates to baseline values. RESULTS: Pts vaccinated in 2003/2004 had, after 1 month, increase in GMT by a factor of 8.64-26.60 for antihemagglutinin antibodies (HI) and 6.93-12.66 for antineuraminidase antibodies (NI), as compared to factor of 9.12-24.41 for HI and 4.83-10.31 for NI in ctrl. At 1 month after vaccination, seroprotection and seroresponse rates were similar in both groups, ranging from 68.42 to 84.21% and 71.93 to 94.74% in NHL, and 66.67-82.22% and 62.22-86.67% in ctrl, respectively. Pts vaccinated in 2004/2005 had increase in the GMT by a factor of 38.76-41.49 for HI and 26.59-30.31 for NI, as compared to factor of 81.19-104.32 for HI and 52.16-54.52 for NI in ctrl. Seroprotection and seroresponse rates were lower in the former group, ranging from 62.11 to 65.26% and 74.47 to 77.66%, respectively. In both seasons, pts achieved titres of antibodies greater than the protective threshold, irrespective of the previous chemotherapy administration. CONCLUSIONS: The results indicate that influenza vaccination induces sufficient immune response in pts with NHL, irrespective of previous chemotherapy.

Activation of Tyk2 and Stat3 is required for the apoptotic actions of interferon-beta in primary pro-B cells.

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.

Opposite effects of prostaglandin-J2 on VEGF in normoxia and hypoxia: role of HIF-1.

The vascular endothelial growth factor (VEGF) is produced in response to hypoxia or inflammatory cytokines. In normoxia VEGF synthesis is upregulated by 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) via induction of heme oxygenase-1 (HO-1). Here we compared the influence of 15d-PGJ(2) on VEGF expression in human microvascular endothelial cells in normoxia (approximately 20% O(2)) and hypoxia ( approximately 2% O(2)). Regardless of the oxygen concentration, 15d-PGJ(2) inhibited activity of hypoxia inducible factor-1 (HIF-1), the major hypoxic regulator of VEGF. However, in normoxic conditions 15d-PGJ(2) (1-10microM) activated the VEGF promoter and increased synthesis of the VEGF protein. Concomitantly, it strongly induced expression of HO-1. In contrast, in hypoxia, 15d-PGJ(2) decreased VEGF promoter activity and reduced VEGF release by 50%. Inhibition of HO-1 activity additionally attenuated VEGF synthesis in hypoxia. We conclude that induction of HO-1 by 15d-PGJ(2) results in augmentation of VEGF synthesis in normoxia. In hypoxia, however, the stimulatory effect of HO-1 is outweighed by 15d-PGJ(2)-mediated inhibition of the HIF-1 pathway.

Extracellular superoxide dismutase (EC-SOD) binds to type i collagen and protects against oxidative fragmentation.

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is mainly found in the extracellular matrix of tissues. EC-SOD participates in the detoxification of reactive oxygen species by catalyzing the dismutation of superoxide radicals. The tissue distribution of the enzyme is particularly important because of the reactive nature of its substrate, and it is likely essential that EC-SOD is positioned at the site of superoxide production to prevent adventitious oxidation. EC-SOD contains a C-terminal heparin-binding region thought to be important for modulating its distribution in the extracellular matrix. This paper demonstrates that, in addition to binding heparin, EC-SOD specifically binds to type I collagen with a dissociation constant (K(d)) of 200 nm. The heparin-binding region was found to mediate the interaction with collagen. Notably, the bound EC-SOD significantly protects type I collagen from oxidative fragmentation. This expands the known repertoire of EC-SOD binding partners and may play an important physiological role in preventing oxidative fragmentation of collagen during oxidative stress.