Publications by authors named "Athanassios Fragoulis"

35 Publications

Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury.

Int J Mol Sci 2021 Jul 22;22(15). Epub 2021 Jul 22.

Department of Pharmacology and Toxicology, Uniklinik RWTH Aachen University, 52074 Aachen, Germany.

qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 () and C-X-C motif ligand 1 () as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of and gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.
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http://dx.doi.org/10.3390/ijms22157853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346155PMC
July 2021

Adverse Effects of Oxidative Stress on Bone and Vasculature in Corticosteroid-Associated Osteonecrosis: Potential Role of Nuclear Factor Erythroid 2-Related Factor 2 in Cytoprotection.

Antioxid Redox Signal 2021 Aug 5;35(5):357-376. Epub 2021 Apr 5.

Department of Anatomy and Cell Biology, RWTH Aachen University, Aachen, Germany.

Osteonecrosis (ON) is characterized by bone tissue death due to disturbance of the nutrient artery. The detailed process leading to the necrotic changes has not been fully elucidated. Clinically, high-dose corticosteroid therapy is one of the main culprits behind osteonecrosis of the femoral head (ONFH). Numerous studies have proposed that such ischemia concerns various intravascular mechanisms. Of all reported risk factors, the involvement of oxidative stress in the irreversible damage suffered by bone-related and vascular endothelial cells during ischemia simply cannot be overlooked. Several articles also have sought to elucidate oxidative stress in relation to ON using animal models or cell cultures. However, as far as we know, antioxidant monotherapy has still not succeeded in preventing ONFH in humans. To provide this desideratum, we herein summarize the current knowledge about the influence of oxidative stress on ON, together with data about the preventive effects of administering antioxidants in corticosteroid-induced ON animal models. Moreover, oxidative stress is counteracted by nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent cytoprotective network through regulating antioxidant expressions. Therefore, we also describe Nrf2 regulation and highlight its role in the pathology of ON. This is a review of all available literature to date aimed at developing a deeper understanding of the pathological mechanism behind ON from the perspective of oxidative stress. It may be hoped that this synthesis will spark the development of a prophylactic strategy to benefit corticosteroid-associated ONFH patients. 35, 357-376.
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http://dx.doi.org/10.1089/ars.2020.8163DOI Listing
August 2021

Effects of Strontium-Doped β-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

Int J Mol Sci 2020 May 1;21(9). Epub 2020 May 1.

Department of Anatomy and Cell Biology, RWTH Aachen University Hospital, 52074 Aachen, Germany.

It was hypothesized that strontium (Sr)-doped β-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed β-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the β-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the β-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the β-TCP group, whereas the percentage of osseous tissue formation in the β-TCP group was significantly higher than in the β-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.
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http://dx.doi.org/10.3390/ijms21093208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246816PMC
May 2020

Nrf2 expression driven by Foxp3 specific deletion of Keap1 results in loss of immune tolerance in mice.

Eur J Immunol 2020 04 14;50(4):515-524. Epub 2020 Jan 14.

Department of Pediatrics, Medical Faculty, RWTH Aachen, Aachen, Germany.

The transcription factor Nrf2 regulates oxidative stress responses. However, the specific function of Nrf2 in Tregs, the central regulators of immune homeostasis, is unclear. Here, we report an unexpected but important role of Nrf2 in Tregs. Nrf2 expression driven by Foxp3 specific deletion of Keap1 resulted in an autoinflammatory phenotype with enhanced effector T cell activation and immune cell infiltrates in the lung. While early postnatal death of mice with Foxp3 specific deletion of Keap1 was most probably due to ectopic Foxp3 expression and subsequent Keap1 deletion in epithelial cells, bone marrow chimeras suggest that Nrf2 activation intrinsically in Tregs contributes to a loss of Treg cells and diminished peripheral tolerance. Moreover, Nrf2 activation was associated with a loss of Foxp3 expression, but an enhanced glucose uptake and mTOR activity in Tregs, thus mimicking a metabolic phenotype that is associated with impaired lineage stability and cell functioning.
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http://dx.doi.org/10.1002/eji.201948285DOI Listing
April 2020

Kinetic modelling of quantitative proteome data predicts metabolic reprogramming of liver cancer.

Br J Cancer 2020 01 10;122(2):233-244. Epub 2019 Dec 10.

Molecular Tumor Biology, Department of General, Visceral and Transplantation Surgery, RWTH University Hospital, 52074, Aachen, Germany.

Background: Metabolic alterations can serve as targets for diagnosis and cancer therapy. Due to the highly complex regulation of cellular metabolism, definite identification of metabolic pathway alterations remains challenging and requires sophisticated experimentation.

Methods: We applied a comprehensive kinetic model of the central carbon metabolism (CCM) to characterise metabolic reprogramming in murine liver cancer.

Results: We show that relative differences of protein abundances of metabolic enzymes obtained by mass spectrometry can be used to assess their maximal velocity values. Model simulations predicted tumour-specific alterations of various components of the CCM, a selected number of which were subsequently verified by in vitro and in vivo experiments. Furthermore, we demonstrate the ability of the kinetic model to identify metabolic pathways whose inhibition results in selective tumour cell killing.

Conclusions: Our systems biology approach establishes that combining cellular experimentation with computer simulations of physiology-based metabolic models enables a comprehensive understanding of deregulated energetics in cancer. We propose that modelling proteomics data from human HCC with our approach will enable an individualised metabolic profiling of tumours and predictions of the efficacy of drug therapies targeting specific metabolic pathways.
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http://dx.doi.org/10.1038/s41416-019-0659-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052204PMC
January 2020

A Newly Established Murine Cell Line as a Model for Hepatocellular Cancer in Non-Alcoholic Steatohepatitis.

Int J Mol Sci 2019 Nov 12;20(22). Epub 2019 Nov 12.

Department of General, Visceral and Transplantation Surgery, Uniklinik RWTH Aachen, 52074 Aachen, Germany.

Non-alcoholic steatohepatitis (NASH) has become a major risk factor for hepatocellular cancer (HCC) due to the worldwide increasing prevalence of obesity. However, the pathophysiology of NASH and its progression to HCC is incompletely understood. Thus, the aim of this study was to generate a model specific NASH-derived HCC cell line. A murine NASH-HCC model was conducted and the obtained cancer cells (N-HCC25) were investigated towards chromosomal aberrations, the expression of cell type-specific markers, dependency on nutrients, and functional importance of mTOR. N-HCC25 exhibited several chromosomal aberrations as compared to healthy hepatocytes. Hepatocytic (HNF4), EMT (Twist, Snail), and cancer stem cell markers (CD44, EpCAM, CK19, Sox9) were simultaneously expressed in these cells. Proliferation highly depended on the supply of glucose and FBS, but not glutamine. Treatment with a second generation mTOR inhibitor (KU-0063794) resulted in a strong decrease of cell growth in a dose-dependent manner. In contrast, a first generation mTOR inhibitor (Everolimus) only slightly reduced cell proliferation. Cell cycle analyses revealed that the observed growth reduction was most likely due to G/G cell cycle arrest. These results indicate that N-HCC25 is a highly proliferative HCC cell line from a NASH background, which might serve as a suitable in vitro model for future investigations of NASH-derived HCC.
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http://dx.doi.org/10.3390/ijms20225658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888677PMC
November 2019

Nrf2 deficiency increases oligodendrocyte loss, demyelination, neuroinflammation and axonal damage in an MS animal model.

Metab Brain Dis 2020 02 16;35(2):353-362. Epub 2019 Sep 16.

Institute of Neuroanatomy, Uniklinik RWTH Aachen, Wendlingweg 2, 52074, Aachen, Germany.

Oxidative stress is a pathophysiological hallmark of many CNS diseases, among multiple sclerosis (MS). Accordingly, boosting the astrocytic transcription factor nuclear factor E2-related factor 2 (Nrf2) system in an MS mouse model efficiently ameliorates oligodendrocyte loss, neuroinflammation and axonal damage. Moreover, Dimethylfumarate, an efficient activator of Nrf2, has recently been approved as therapeutic option in MS treatment. Here, we use the cuprizone mouse model of MS to induce oxidative stress, selective oligodendrocyte loss, microglia and astrocyte activation as well as axonal damage in both wild type and Nrf2-deficient mice. We found increased oligodendrocyte apoptosis and loss, pronounced neuroinflammation and higher levels of axonal damage in cuprizone-fed Nrf2-deficient animals when compared to wild type controls. In addition, Nrf2-deficient animals showed a higher susceptibility towards cuprizone within the commissura anterior white matter tract, a structure that is relatively insensitive to cuprizone in wild type animals. Our data highlight the cuprizone model as a suitable tool to study the complex interplay of oxidative stress, neuroinflammation and axonal damage. Further studies will have to show whether distinct expression patterns of Nrf2 are involved in the variable susceptibility towards cuprizone in the mouse.
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http://dx.doi.org/10.1007/s11011-019-00488-zDOI Listing
February 2020

Macrophages protect against loss of adipose tissue during cancer cachexia.

J Cachexia Sarcopenia Muscle 2019 10 18;10(5):1128-1142. Epub 2019 Jul 18.

Department of General, Visceral and Transplantation Surgery, University Hospital RWTH Aachen, Aachen, Germany.

Background: Cancer cachexia represents a central obstacle in medical oncology as it is associated with poor therapy response and reduced overall survival. Systemic inflammation is considered to be a key driver of cancer cachexia; however, clinical studies with anti-inflammatory drugs failed to show distinct cachexia-inhibiting effects. To address this contradiction, we investigated the functional importance of innate immune cells for hepatocellular carcinoma (HCC)-associated cachexia.

Methods: A transgenic HCC mouse model was intercrossed with mice harbouring a defect in myeloid cell-mediated inflammation. Body composition of mice was analysed via nuclear magnetic resonance spectroscopy and microcomputed tomography. Quantitative PCR was used to determine adipose tissue browning and polarization of adipose tissue macrophages. The activation state of distinct areas of the hypothalamus was analysed via immunofluorescence. Multispectral immunofluorescence imaging and immunoblot were applied to characterize sympathetic neurons and macrophages in visceral adipose tissue. Quantification of pro-inflammatory cytokines in mouse serum was performed with a multiplex immunoassay. Visceral adipose tissue of HCC patients was quantified via the L3 index of computed tomography scans obtained during routine clinical care.

Results: We identified robust cachexia in the HCC mouse model as evidenced by a marked loss of visceral fat and lean mass. Computed tomography-based analyses demonstrated that a subgroup of human HCC patients displays reduced visceral fat mass, complementing the murine data. While the myeloid cell-mediated inflammation defect resulted in reduced expression of pro-inflammatory cytokines in the serum of HCC-bearing mice, this unexpectedly did not translate into diminished but rather enhanced cachexia-associated fat loss. Defective myeloid cell-mediated inflammation was associated with decreased macrophage abundance in visceral adipose tissue, suggesting a role for local macrophages in the regulation of cancer-induced fat loss.

Conclusions: Myeloid cell-mediated inflammation displays a rather unexpected beneficial function in a murine HCC model. These results demonstrate that immune cells are capable of protecting the host against cancer-induced tissue wasting, adding a further layer of complexity to the pathogenesis of cachexia and providing a potential explanation for the contradictory results of clinical studies with anti-inflammatory drugs.
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http://dx.doi.org/10.1002/jcsm.12450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818538PMC
October 2019

Role of Nrf2 in Fracture Healing: Clinical Aspects of Oxidative Stress.

Calcif Tissue Int 2019 10 24;105(4):341-352. Epub 2019 Jun 24.

Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany.

Fracture healing is a natural process that recapitulates embryonic skeletal development. In the early phase after fracture, reactive oxygen species (ROS) are produced under inflammatory and ischemic conditions due to vessel injury and soft tissue damage, leading to cell death. Usually, such damage during the course of fracture healing can be largely prevented by protective mechanisms and functions of antioxidant enzymes. However, intrinsic oxidative stress can cause excessive toxic radicals, resulting in irreversible damage to cells associated with bone repair during the fracture healing process. Clinically, patients with type-2 diabetes mellitus, osteoporosis, habitual drinkers, or heavy smokers are at risk of impaired fracture healing due to elevated oxidative stress. Although increased levels of oxidative stress markers upon fracture and effects of antioxidants on fracture healing have been reported, a detailed understanding of what causes impaired fracture healing under intrinsic conditions of oxidative stress is lacking. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been identified as a key transcriptional regulator of the expression of antioxidants and detoxifying enzymes. It further not only plays a crucial role in preventing degenerative diseases in multiple organs, but also during fracture healing. This narrative review evaluates the influence of intrinsic oxidative stress on fracture healing and sheds new light on the intriguing role of Nrf2 during bone regeneration in pathological fractures.
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http://dx.doi.org/10.1007/s00223-019-00576-3DOI Listing
October 2019

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

Oncogene 2019 07 1;38(28):5670-5685. Epub 2019 May 1.

Molecular Tumor Biology, Department of General Visceral and Transplantation Surgery, RWTH University Hospital, Pauwelsstraße 30, 52074, Aachen, Germany.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/β-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.
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http://dx.doi.org/10.1038/s41388-019-0816-4DOI Listing
July 2019

Nrf2 Ameliorates DDC-Induced Sclerosing Cholangitis and Biliary Fibrosis and Improves the Regenerative Capacity of the Liver.

Toxicol Sci 2019 06;169(2):485-498

Department of Anatomy and Cell Biology.

The Nrf2 pathway protects against oxidative stress and induces regeneration of various tissues. Here, we investigated whether Nrf2 protects from sclerosing cholangitis and biliary fibrosis and simultaneously induces liver regeneration. Diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was fed to Nrf2-KO mice (Nrf2-/-), mice with liver-specific hyperactivated Nrf2 (HKeap1-/-) and wild-type (WT) littermates to induce cholangitis, liver fibrosis, and oval cell expansion. HKeap1-/--mice were protected from almost all DDC-induced injury compared with WT and Nrf2-/-. Liver injury in Nrf2-/- and WT mice was mostly similar, albeit Nrf2-/- suffered more from DDC diet as seen for several parameters. Nrf2 activity was especially important for the expression of the hepatic efflux transporters Abcg2 and Abcc2-4, which are involved in hepatic toxin elimination. Surprisingly, cell proliferation was more enhanced in Nrf2-/-- and HKeap1-/--mice compared with WT. Interestingly, Nrf2-/--mice failed to sufficiently activate oval cell expansion after DDC treatment and showed almost no resident oval cell population under control conditions. The resident oval cell population of untreated HKeap1-/--mice was increased and DDC treatment resulted in a stronger oval cell expansion compared with WT. We provide evidence that Nrf2 activation protects from DDC-induced sclerosing cholangitis and biliary fibrosis. Moreover, our data establish a possible role of Nrf2 in oval cell expansion.
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http://dx.doi.org/10.1093/toxsci/kfz055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542338PMC
June 2019

Mitochondrial Impairment in Oligodendroglial Cells Induces Cytokine Expression and Signaling.

J Mol Neurosci 2019 Feb 13;67(2):265-275. Epub 2018 Dec 13.

Institute of Neuroanatomy, Faculty of Medicine, RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany.

Widespread inflammatory lesions within the central nervous system grey and white matter are major hallmarks of multiple sclerosis. The development of full-blown demyelinating multiple sclerosis lesions might be preceded by preactive lesions which are characterized by focal microglia activation in close spatial relation to apoptotic oligodendrocytes. In this study, we investigated the expression of signaling molecules of oligodendrocytes that might be involved in initial microglia activation during preactive lesion formation. Sodium azide was used to trigger mitochondrial impairment and cellular stress in oligodendroglial cells in vitro. Among various chemokines and cytokines, IL6 was identified as a possible oligodendroglial cell-derived signaling molecule in response to cellular stress. Relevance of this finding for lesion development was further explored in the cuprizone model by applying short-term cuprizone feeding (2-4 days) on male C57BL/6 mice and subsequent analysis of gene expression, in situ hybridization and histology. Additionally, we analyzed the possible signaling of stressed oligodendroglial cells in vitro as well as in the cuprizone mouse model. In vitro, conditioned medium of stressed oligodendroglial cells triggered the activation of microglia cells. In cuprizone-fed animals, IL6 expression in oligodendrocytes was found in close vicinity of activated microglia cells. Taken together, our data support the view that stressed oligodendrocytes have the potential to activate microglia cells through a specific cocktail of chemokines and cytokines among IL6. Further studies will have to identify the temporal activation pattern of these signaling molecules, their cellular sources, and impact on neuroinflammation.
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http://dx.doi.org/10.1007/s12031-018-1236-6DOI Listing
February 2019

Nrf2 Signaling in Sodium Azide-Treated Oligodendrocytes Restores Mitochondrial Functions.

J Mol Neurosci 2018 Oct 23;66(2):229-237. Epub 2018 Aug 23.

Department of Anatomy and Cell Biology, Medical Faculty, Uniklinik RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany.

Mitochondrial dysfunctions mark a critical step in many central nervous system (CNS) pathologies, including multiple sclerosis (MS). Such dysfunctions lead to depolarization of mitochondrial membranes and imbalanced redox homeostasis. In this context, reactive oxygen species (ROS) are potentially deleterious but can also act as an important signaling step for cellular maintenance. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the key regulator in the cellular oxidative stress-response, induces a battery of genes involved in repair and regeneration. Here, we investigated the relevance of Nrf2 signaling for the prevention of cellular damage caused by dysfunctional mitochondria. We employed sodium azide (SA) as mitochondrial inhibitor on oligodendroglial OliNeu cells in vitro, and the cuprizone model with wild type and GFAP-Cre::Keap1 mice to induce mitochondrial defects. The importance of Nrf2 for cellular functions and survival after SA treatment was elucidated by in vitro knockdown experiments with shRNA directed against Nrf2 and its inhibitor Keap1 as well as by methysticin treatment. Metabolic activity, cytotoxicity, and depolarization of the mitochondrial membrane were analyzed after SA treatment. The expression of Nrf2 target genes as well as endoplasmic reticulum stress response genes was additionally measured by real-time PCR (in vitro) and PCR gene arrays (in vivo). Treatment of OliNeu cells with SA resulted in significant depolarization of the mitochondrial membrane, decreased metabolic activity, and increased cytotoxicity. This was partly counteracted in Nrf2-hyperactivated cells and intensified in Nrf2-knockdown cells. Our studies demonstrate a key role of Nrf2 in maintaining cellular functions and survival in the context of mitochondrial dysfunction.
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http://dx.doi.org/10.1007/s12031-018-1159-2DOI Listing
October 2018

Nrf2 Is a Central Regulator of Metabolic Reprogramming of Myeloid-Derived Suppressor Cells in Steady State and Sepsis.

Front Immunol 2018 6;9:1552. Epub 2018 Jul 6.

Department of Pediatrics, Medical Faculty, RWTH Aachen, Aachen, Germany.

Arising in inflammatory conditions, myeloid-derived suppressor cells (MDSCs) are constantly confronted with intracellular and extracellular reactive oxygen species molecules and oxidative stress. Generating mice with a constitutive activation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) we show a pivotal role of the antioxidant stress defense for development of these immune-modulatory cells. These mice are characterized by a massive increase of splenic CD11bGr-1 cells, which exhibit typical suppressive characteristics of MDSCs. Whole transcriptome analysis revealed Nrf2-dependent activation of cell cycle and metabolic pathways, which resemble pathways in CD11bGr-1 MDSCs expanded by LPS exposure. Constitutive Nrf2 activation thereby regulates activation and balance between glycolysis and mitochondrial metabolism and hence expansion of highly suppressive MDSCs, which mediate protection in LPS-induced sepsis. Our study establishes Nrf2 as key regulator of MDSCs and acquired tolerance against LPS-induced sepsis.
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http://dx.doi.org/10.3389/fimmu.2018.01552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6043652PMC
July 2018

Platelet-Released Growth Factors Modulate the Secretion of Cytokines in Synoviocytes under Inflammatory Joint Disease.

Mediators Inflamm 2017 19;2017:1046438. Epub 2017 Nov 19.

Department of Traumatology, University Hospital of Schleswig-Holstein, Kiel, Germany.

The etiology and pathogenesis of rheumatoid arthritis (RA) are marked by a complex interplay of various cell populations and is mediated by different signaling pathways. Traditionally, therapies have primarily focused on pain relief, reducing inflammation and the recovery of joint function. More recently, however, researchers have discussed the therapeutic efficacy of autologous platelet-rich plasma (PRP). The main objective of this work is to examine the influences of platelet-released growth factor (PRGF) on human synoviocytes under inflammatory conditions. Additionally, it is checked to which extend treatment with platelet concentrate influences the release of cytokines form synoviocytes. For this purpose, an in vitro RA model was created by stimulating the cells with the TNF-. The release of cytokines was measured by ELISA. The cytokine gene expression was analyzed by real-time PCR. It has been observed that the stimulation concentration of 10 ng/ml TNF- resulted in a significantly increased endogenous secretion and gene expression of IL-6 and TNF-. The anti-inflammatory effect of PRGF could be confirmed through significant reduction of TNF- and IL-1. An induced inflammatory condition seems to cause PRGF to inhibit the release of proinflammatory cytokines. Further study is required to understand the exact effect mechanism of PRGF on synoviocytes.
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http://dx.doi.org/10.1155/2017/1046438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733972PMC
August 2018

Chemical hypoxia-induced integrated stress response activation in oligodendrocytes is mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2).

J Neurochem 2018 02 5;144(3):285-301. Epub 2018 Jan 5.

Department of Anatomy II, Ludwig-Maximilians-University of Munich, Munich, Germany.

The extent of remyelination in multiple sclerosis lesions is often incomplete. Injury to oligodendrocyte progenitor cells can be a contributing factor for such incomplete remyelination. The precise mechanisms underlying insufficient repair remain to be defined, but oxidative stress appears to be involved. Here, we used immortalized oligodendrocyte cell lines as model systems to investigate a causal relation of oxidative stress and endoplasmic reticulum stress signaling cascades. OLN93 and OliNeu cells were subjected to chemical hypoxia by blocking the respiratory chain at various levels. Mitochondrial membrane potential and oxidative stress levels were quantified by flow cytometry. Endoplasmic reticulum stress was monitored by the expression induction of activating transcription factor 3 and 4 (Atf3, Atf4), DNA damage-inducible transcript 3 protein (Ddit3), and glucose-regulated protein 94. Lentiviral silencing of nuclear factor (erythroid-derived 2)-like 2 or kelch-like ECH-associated protein 1 was applied to study the relevance of NRF2 for endoplasmic reticulum stress responses. We demonstrate that inhibition of the respiratory chain induces oxidative stress in cultured oligodendrocytes which is paralleled by the expression induction of distinct mediators of the endoplasmic reticulum stress response, namely Atf3, Atf4, and Ddit3. Atf3 and Ddit3 expression induction is potentiated in kelch-like ECH-associated protein 1-deficient cells and absent in cells lacking the oxidative stress-related transcription factor NRF2. This study provides strong evidence that oxidative stress in oligodendrocytes activates endoplasmic reticulum stress response in a NRF2-dependent manner and, in consequence, might regulate oligodendrocyte degeneration in multiple sclerosis and other neurological disorders.
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http://dx.doi.org/10.1111/jnc.14270DOI Listing
February 2018

Widespread Post-transcriptional Attenuation of Genomic Copy-Number Variation in Cancer.

Cell Syst 2017 10 11;5(4):386-398.e4. Epub 2017 Oct 11.

European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Cambridge CB10 1SD, UK. Electronic address:

Copy-number variations (CNVs) are ubiquitous in cancer and often act as driver events, but the effects of CNVs on the proteome of tumors are poorly understood. Here, we analyze recently published genomics, transcriptomics, and proteomics datasets made available by CPTAC and TCGA consortia on 282 breast, ovarian, and colorectal tumor samples to investigate the impact of CNVs in the proteomes of these cells. We found that CNVs are buffered by post-transcriptional regulation in 23%-33% of proteins that are significantly enriched in protein complex members. Our analyses show that complex subunits are highly co-regulated, and some act as rate-limiting steps of complex assembly, as their depletion induces decreased abundance of other complex members. We identified 48 such rate-limiting interactions and experimentally confirmed our predictions on the interactions of AP3B1 with AP3M1 and GTF2E2 with GTF2E1. This study highlights the importance of post-transcriptional mechanisms in cancer that allow cells to cope with their altered genomes.
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http://dx.doi.org/10.1016/j.cels.2017.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660600PMC
October 2017

Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH.

Oxid Med Cell Longev 2017 6;2017:3420286. Epub 2017 Jun 6.

Department of Medicine III, University Hospital RWTH Aachen, Aachen, Germany.

We have recently shown that hepatocyte-specific c-met deficiency accelerates the progression of nonalcoholic steatohepatitis in experimental murine models resulting in augmented production of reactive oxygen species and accelerated development of fibrosis. The aim of this study focuses on the elucidation of the underlying cellular mechanisms driven by Nrf2 overactivation in hepatocytes lacking c-met receptor characterized by a severe unbalance between pro-oxidant and antioxidant functions. Control mice (c-met), single c-met knockouts (c-met), and double c-met/Keap1 knockouts (met/Keap1) were then fed a chow or a methionine-choline-deficient (MCD) diet, respectively, for 4 weeks to reproduce the features of nonalcoholic steatohepatitis. Upon MCD feeding, met/Keap1 mice displayed increased liver mass albeit decreased triglyceride accumulation. The marked increase of oxidative stress observed in c-met was restored in the double mutants as assessed by 4-HNE immunostaining and by the expression of genes responsible for the generation of free radicals. Moreover, double knockout mice presented a reduced amount of liver-infiltrating cells and the exacerbation of fibrosis progression observed in c-met livers was significantly inhibited in met/Keap1. Therefore, genetic activation of the antioxidant transcription factor Nrf2 improves liver damage and repair in hepatocyte-specific c-met-deficient mice mainly through restoring a balance in the cellular redox homeostasis.
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http://dx.doi.org/10.1155/2017/3420286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476895PMC
January 2018

Oral administration of methysticin improves cognitive deficits in a mouse model of Alzheimer's disease.

Redox Biol 2017 08 19;12:843-853. Epub 2017 Apr 19.

Department of Anatomy and Cell Biology, Uniklinik RWTH Aachen University, Aachen, Germany. Electronic address:

Introduction: There is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid β (Aβ)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD.

Methods: The kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aβ burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze.

Results: Methysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aβ deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice.

Conclusion: In summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs.
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http://dx.doi.org/10.1016/j.redox.2017.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406548PMC
August 2017

Psoriasin has divergent effects on the innate immune responses of murine glial cells.

J Neurochem 2017 04 27;141(1):86-99. Epub 2017 Feb 27.

Department of Anatomy and Cell Biology, RWTH Aachen University, Aachen, Germany.

Antimicrobial peptides are an important part of the innate immune defense in the central nervous system (CNS). The expression of the antimicrobial peptides psoriasin (S100A7) is up-regulated during bacterial meningitis. However, the exact mechanisms induced by psoriasin to modulate glial cell activity are not yet fully understood. Our hypothesis is that psoriasin induced pro- and anti-inflammatory signaling pathways as well as regenerative factors to contribute in total to a balanced immune response. Therefore, we used psoriasin-stimulated glial cells and analyzed the translocation of the pro-inflammatory transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) in murine glial cells and the expression of pro- and anti-inflammatory mediators by real time RT-PCR, ELISA technique, and western blotting. Furthermore, the relationship between psoriasin and the antioxidative stress transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) was investigated. Stimulation with psoriasin not only enhanced NFκB translocation and increased the expression of the pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) but also neurotrophin expression. Evidence for functional interactions between psoriasin and Nrf2 were detected in the form of increased antioxidant response element (ARE) activity and induction of Nrf2/ARE-dependent heme oxygenase 1 (HO-1) expression in psoriasin-treated microglia and astrocytes. The results illustrate the ability of psoriasin to induce immunological functions in glia cells where psoriasin exerts divergent effects on the innate immune response.
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http://dx.doi.org/10.1111/jnc.13959DOI Listing
April 2017

In vivo imaging of antioxidant response element activity during liver regeneration after partial hepatectomy.

J Surg Res 2016 12 9;206(2):525-535. Epub 2016 Aug 9.

Department of General, Visceral, and Transplantation Surgery, RWTH Aachen University Hospital, Aachen, Germany.

Background: The nuclear factor-erythroid 2-related factor 2 (Nrf2) -antioxidant response element (ARE) pathway is important for the regulation of antioxidative stress response and detoxification. To activate the expression of its target genes, such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1), Nrf2 binds to the ARE within the promoter region of these genes. Partial hepatectomy and consecutive liver regeneration lead to oxidative stress with activation of the Nrf2-ARE pathway. The aim of this study was to investigate ARE activity in vivo during liver regeneration after partial hepatectomy.

Materials And Methods: Transgenic ARE-luc mice were used. In these mice, the luciferase reporter gene is under the control of an ARE promoter element. Following 2/3 partial hepatectomy (PHx), mice underwent in vivo bioluminescence imaging up until the ninth postoperative day. In addition, liver tissue was analyzed by immunohistochemistry (Nrf2 and HO-1), quantitative reverse transcription-PCR (HO-1 and NQO1) and in vitro luminescence assays.

Results: Bioluminescence imaging revealed a significant increase in Nrf2-ARE activity after PHx. The signal maximum was recorded on the third day after PHx. Seven days postoperatively, the signal almost reached baseline levels. In immunohistochemistry, significantly more hepatocytes were positive for Nrf2 and HO-1 on the third postoperative day compared with baseline levels. The mRNA expression of HO-1 and NQO1 were significantly increased on day 3 as measured by qRT-PCR.

Conclusions: This study demonstrated the time-dependent activation of the Nrf2-ARE system during liver regeneration in vivo. The transgenic ARE-luc mouse provided a convenient model for studying Nrf2-mediated gene expression noninvasively and may facilitate further experiments with therapeutic modulation of the antioxidative stress response.
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http://dx.doi.org/10.1016/j.jss.2016.08.008DOI Listing
December 2016

The effects of Nrf2 deletion on placental morphology and exchange capacity in the mouse.

J Matern Fetal Neonatal Med 2017 Sep 3;30(17):2068-2073. Epub 2016 Oct 3.

a Department of Anatomy and Cell Biology , RWTH Aachen University Hospital , Aachen , Germany.

Objectives: Intrauterine growth restriction (IUGR) is defined as a pathological decreased fetal growth. Oxidative stress has been connected to the restriction in the fetal growth. The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is a potent activator of the cellular antioxidant response. The effect Nrf2 on fetal-placental development has not yet been sufficiently investigated. Here, we evaluated the placental and fetal growth in Nrf2 knockout (Nrf2-KO) and Nrf2-wild type mice (Nrf2-WT) throughout pregnancy.

Methods: Heterozygote Nrf2 (Nrf2) mice were paired to get Nrf2-KO and Nrf2-WT in the litters. Placentae and embryos from both genotypes were collected and weighed on days 13.5, 15.5 and 18.5 post coitum. The absolute volumes of the labyrinth zone and the total volume of the placenta were determined using the Cavalieri principle.

Results: On E 18.5 the fetal weight in Nrf2-KO was significantly reduced versus Nrf2-WT indicating a decrease in placental efficiency. A significant reduction in both total and labyrinth-volume in the placenta of Nrf2-KO mice was observed.

Conclusion: This data points out the necessity of functional Nrf2 for fetal and placental growth. A deficiency in Nrf2 signaling may negatively affect nutrient transfer capacity which is then no longer able to meet fetal growth demands.
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http://dx.doi.org/10.1080/14767058.2016.1236251DOI Listing
September 2017

Hepatocyte-specific Keap1 deletion reduces liver steatosis but not inflammation during non-alcoholic steatohepatitis development.

Free Radic Biol Med 2016 Feb 15;91:114-26. Epub 2015 Dec 15.

Department of Medicine III, RWTH Aachen University Hospital, Aachen, Germany. Electronic address:

Generation of reactive oxygen species (ROS) in response to fatty acids accumulation has been classically proposed as a possible "second hit" triggering progression from simple steatosis to non-alcoholic steatohepatitis (NASH). In this study we challenged hepatocyte-specific Keap1 knockout mice (Keap1(Δhepa)) and littermate Cre- controls (Keap1(fx/fx)) with two different diet models of NASH in order to evaluate the effects of the anti-oxidant transcription factor Nrf2 over-activation on hepatic metabolism and disease progression. After 4 weeks of MCD diet the liver/body weight ratio of Keap1(Δhepa) mice was significantly higher compared to littermate controls with no differences in total body weight. Strikingly, liver histology revealed a dramatic reduction of lipid droplets confirmed by a decreased content of intra-hepatic triglycerides in Keap1(Δhepa) compared to controls. In parallel to reduced expression of genes involved in lipid droplet formation, protein expression of Liver X Receptor (LXRα/β) and Peroxisome proliferator-activated receptor α (PPARα) was significantly decreased. In contrast, genes involved in mitochondrial lipid catabolism were markedly up-regulated in Keap1(Δhepa) livers. A similar phenotype characterized by inhibition of lipogenesis in favor of increased mitochondrial catabolic activity was also observed after 13 weeks of western diet administration. MCD-induced apoptosis was significantly dampened in Keap1(Δhepa) compared to Keap1(fx/fx) as detected by TUNEL, cleaved caspase-3 and Bcl-2 protein expression analyses. However, no differences in inflammatory F4/80- and CD11b-positive cells and pro-fibrogenic genes were detected between the two groups. Although hepatic lack of Keap1 did not ameliorate inflammation, the resulting constitutive Nrf2 over-activation in hepatocytes strongly reduced hepatic steatosis via enhanced lipid catabolism and repressed de novo lipogenesis during murine NASH development.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.014DOI Listing
February 2016

Nrf2 in health and disease: current and future clinical implications.

Clin Sci (Lond) 2015 Dec;129(12):989-99

Department of Anatomy and Cell Biology, University Hospital, RWTH Aachen University, Aachen, Germany

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a major regulator of oxidative stress defence in the human body. As Nrf2 regulates the expression of a large battery of cytoprotective genes, it plays a crucial role in the prevention of degenerative disease in multiple organs. Thus it has been the focus of research as a pharmacological target that could be used for prevention and treatment of chronic diseases such as multiple sclerosis, chronic kidney disease or cardiovascular diseases. The present review summarizes promising findings from basic research and shows which Nrf2-targeting therapies are currently being investigated in clinical trials and which agents have already entered clinical practice.
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http://dx.doi.org/10.1042/CS20150436DOI Listing
December 2015

Corrigendum to "Enoxaparin Prevents Steroid-Related Avascular Necrosis of the Femoral Head".

ScientificWorldJournal 2015 26;2015:264241. Epub 2015 Aug 26.

Department of Orthopedic and Trauma Surgery, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany ; Department of Orthopaedic and Spine Surgery, AGAPLESION EV. BATHILDISKRANKENHAUS gemeinnützige GmbH, Maulbeerallee 4, 31812 Bad Pyrmont, Germany.

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http://dx.doi.org/10.1155/2015/264241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550074PMC
January 2016

Mechanical forces induce changes in VEGF and VEGFR-1/sFlt-1 expression in human chondrocytes.

Int J Mol Sci 2014 Sep 1;15(9):15456-74. Epub 2014 Sep 1.

Department of Orthopaedic Surgery, RWTH Aachen University, 52074 Aachen, Germany.

Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.
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http://dx.doi.org/10.3390/ijms150915456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200847PMC
September 2014

Nrf2 augments skeletal muscle regeneration after ischaemia-reperfusion injury.

J Pathol 2014 Dec 16;234(4):538-47. Epub 2014 Sep 16.

Department of Anatomy and Cell Biology, University Hospital, RWTH Aachen University, Germany.

Skeletal muscles harbour a resident population of stem cells, termed satellite cells (SCs). After trauma, SCs leave their quiescent state to enter the cell cycle and undergo multiple rounds of proliferation, a process regulated by MyoD. To initiate differentiation, fusion and maturation to new skeletal muscle fibres, SCs up-regulate myogenin. However, the regulation of these myogenic factors is not fully understood. In this study we demonstrate that Nrf2, a major regulator of oxidative stress defence, plays a role in the expression of these myogenic factors. In both promoter studies with myoblasts and a mouse model of muscle injury in Nrf2-deficient mice, we show that Nrf2 prolongs SC proliferation by up-regulating MyoD and suppresses SC differentiation by down-regulating myogenin. Moreover, we show that IL-6 and HGF, both factors that facilitate SC activation, induce Nrf2 activity in myoblasts. Thus, Nrf2 activity promotes muscle regeneration by modulating SC proliferation and differentiation and thereby provides implications for tissue regeneration.
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http://dx.doi.org/10.1002/path.4418DOI Listing
December 2014

Enoxaparin prevents steroid-related avascular necrosis of the femoral head.

ScientificWorldJournal 2014 2;2014:347813. Epub 2014 Jul 2.

Department of Orthopedic and Trauma Surgery, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany ; Department of Orthopaedic and Spine Surgery, AGAPLESION EV. BATHILDISKRANKENHAUS gemeinnützige GmbH, Maulbeerallee 4, 31812 Bad Pyrmont, Germany.

Nontraumatic osteonecrosis of the femoral head is still a challenging problem in orthopedic surgery. It is responsible for 10% of the 500,000 hip replacement surgeries in the USA and affects relatively young, active patients in particular. Main reasons for nontraumatic osteonecrosis are glucocorticoid use, alcoholism, thrombophilia, and hypofibrinolysis (Glueck et al., 1997; Orth and Anagnostakos, 2013). One pathomechanism of steroid-induced osteonecrosis is thought to be impaired blood flow to the femoral head caused by increased thrombus formation and vasoconstriction. To investigate the preventive effect of enoxaparin on steroid-related osteonecrosis, we used male New Zealand white rabbits. Osteonecrosis was induced by methylprednisolone-injection (1 × 20 mg/kg body weight). Control animals were treated with phosphate-buffered saline. Treatment consisted of an injection of 11.7 mg/kg body weight of enoxaparin per day (Clexane) in addition to methylprednisolone. Four weeks after methylprednisolone-injection the animals were sacrificed. Histology (hematoxylin-eosin and Ladewig staining) was performed, and empty lacunae and histological signs of osteonecrosis were quantified. Histomorphometry revealed a significant increase in empty lacunae and necrotic changed osteocytes in glucocorticoid-treated animals as compared with the glucocorticoid- and Clexane-treated animals and with the control group. No significant difference was detected between the glucocorticoid and Clexane group and the control group. This finding suggests that cotreatment with enoxaparin has the potential to prevent steroid-associated osteonecrosis.
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http://dx.doi.org/10.1155/2014/347813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106066PMC
April 2015

Interplay between nuclear factor erythroid 2-related factor 2 and amphiregulin during mechanical ventilation.

Am J Respir Cell Mol Biol 2014 Nov;51(5):668-77

1 Institute of Pharmacology and Toxicology and.

Mechanical ventilation (MV) elicits complex and clinically relevant cellular responses in the lungs. The current study was designed to define the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a major regulator of the cellular antioxidant defense system, in the pulmonary response to MV. Nrf2 activity was quantified in ventilated isolated perfused mouse lungs (IPL). Regulation of amphiregulin (AREG) was investigated in BEAS-2B cells with inactivated Nrf2 or Keap1, the inhibitor of Nrf2, using a luciferase vector with AREG promoter. AREG-dependent Nrf2 activity was examined in BEAS-2B cells, murine precision-cut lung slices (PCLS), and IPL. Finally, Nrf2 knockout and wild-type mice were ventilated to investigate the interplay between Nrf2 and AREG during MV in vivo. Lung functions and inflammatory parameters were measured. Nrf2 was activated in a ventilation-dependent manner. The knockdown of Nrf2 and Keap1 via short hairpin RNA in BEAS-2B cells and an EMSA with lung tissue revealed that AREG is regulated by Nrf2. Conversely, AREG application induced a significant Nrf2 activation in BEAS-2B cells, PCLS, and IPL. The signal transduction of ventilation-induced Nrf2 activation was shown to be p38 MAP kinase-dependent. In vivo ventilation experiments indicated that AREG is regulated by Nrf2 during MV. We conclude that Areg expression is regulated by Nrf2. During high-pressure ventilation, Nrf2 becomes activated and induces AREG, leading to a positive feedback loop between Nrf2 and AREG, which involves the p38 MAPK and results in the expression of cytoprotective genes.
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http://dx.doi.org/10.1165/rcmb.2013-0279OCDOI Listing
November 2014

Nrf2 protects against TWEAK-mediated skeletal muscle wasting.

Sci Rep 2014 Jan 10;4:3625. Epub 2014 Jan 10.

Department of Anatomy and Cell Biology, University Hospital, RWTH Aachen University, Aachen, Germany.

Skeletal muscle (SM) regeneration after injury is impaired by excessive inflammation. Particularly, the inflammatory cytokine tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a potent inducer of skeletal muscle wasting and fibrosis. In this study we investigated the role of Nrf2, a major regulator of oxidative stress defence, in SM ischemia/reperfusion (I/R) injury and TWEAK induced atrophy. We explored the time-dependent expression of TWEAK after I/R in SM of Nrf2-wildtype (WT) and knockout (KO) mice. Nrf2-KO mice expressed significant higher levels of TWEAK as compared to WT mice. Consequently, Nrf2-KO mice present an insufficient regeneration as compared to Nrf2-WT mice. Moreover, TWEAK stimulation activates Nrf2 in the mouse myoblast cell line C2C12. This Nrf2 activation inhibits TWEAK induced atrophy in C2C12 differentiated myotubes. In summary, we show that Nrf2 protects SM from TWEAK-induced cell death in vitro and that Nrf2-deficient mice therefore have poorer muscle regeneration.
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http://dx.doi.org/10.1038/srep03625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887379PMC
January 2014
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