Publications by authors named "Christoph Jan Wruck"

33 Publications

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21093208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246816PMC
May 2020

Aggregates of RNA Binding Proteins and ER Chaperones Linked to Exosomes in Granulovacuolar Degeneration of the Alzheimer's Disease Brain.

J Alzheimers Dis 2020 ;75(1):139-156

Institute of Neuropathology, RWTH Aachen University Medical School, Aachen, Germany.

Granulovacuolar degeneration (GVD) occurs in Alzheimer's disease (AD) brain due to compromised autophagy. Endoplasmic reticulum (ER) function and RNA binding protein (RBP) homeostasis regulate autophagy. We observed that the ER chaperones Glucose - regulated protein, 78 KDa (GRP78/BiP), Sigma receptor 1 (SigR1), and Vesicle-associated membrane protein associated protein B (VAPB) were elevated in many AD patients' subicular neurons. However, those neurons which were affected by GVD showed lower chaperone levels, and there was only minor co-localization of chaperones with GVD bodies (GVBs), suggesting that neurons lacking sufficient chaperone-mediated proteostasis enter the GVD pathway. Consistent with this notion, granular, incipient pTau aggregates in human AD and pR5 tau transgenic mouse neurons were regularly co-localized with increased chaperone immunoreactivity, whereas neurons with mature neurofibrillary tangles lacked both the chaperone buildup and significant GVD. On the other hand, APP/PS1 (APPswe/PSEN1dE9) transgenic mouse hippocampal neurons that are devoid of pTau accumulation displayed only few GVBs-like vesicles, which were still accompanied by prominent chaperone buildup. Identifying a potential trigger for GVD, we found cytoplasmic accumulations of RBPs including Matrin 3 and FUS as well as stress granules in GVBs of AD patient and pR5 mouse neurons. Interestingly, we observed that GVBs containing aggregated pTau and pTDP-43 were consistently co-localized with the exosomal marker Flotillin 1 in both AD and pR5 mice. In contrast, intraneuronal 82E1-immunoreactive amyloid-β in human AD and APP/PS1 mice only rarely co-localized with Flotillin 1-positive exosomal vesicles. We conclude that altered chaperone-mediated ER protein homeostasis and impaired autophagy manifesting in GVD are linked to both pTau and RBP accumulation and that some GVBs might be targeted to exocytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JAD-190722DOI Listing
May 2021

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00223-019-00576-3DOI Listing
October 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kfz055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542338PMC
June 2019

The protective effect of platelet released growth factors and bone augmentation (Bio-Oss) on ethanol impaired osteoblasts.

Ann Anat 2017 Nov 31;214:36-42. Epub 2017 Jul 31.

Institute of Anatomy and Cell Biology, Medical Faculty, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany. Electronic address:

Background: Chronic alcohol consumption is a known limiting factor for bone healing. One promising strategy to improve bone augmentation techniques with Bio-Oss in oral and maxillofacial surgery might be the supportive application of platelet-concentrated biomaterials as platelet-released growth factor (PRGF). To address this matter, we performed an in vitro study investigating the protective effects of PRGF and Bio-Oss in ethanol (EtOH) treated osteoblasts.

Methods: The SAOS-2 osteosarcoma cell line, with and without EtOH pretreatment was used. The cell viability, proliferation and alkali phosphatase activity (ALP) after application of 0%, 5% and 10% PRGF and Bio-Oss were assessed.

Results: The application of PRGF and Bio-Oss in EtOH impaired osteoblasts showed a significant beneficial influence increasing the viability of the osteoblasts in cell culture. The synergistic effect of Bio-Oss and 5% PRGF on the proliferation of osteoblasts was also demonstrated. Bio-Oss only in combination with PRGF increases the alkaline phosphatase (ALP) activity in EtOH pretreated cells.

Conclusions: These results indicate that the simultaneous application of PRGF and Bio-Oss inhibits EtOH induced bone healing impairment. Furthermore, in the cells, PRGF induced a protective mechanism which might promote bone regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.aanat.2017.07.002DOI Listing
November 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2017.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406548PMC
August 2017

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/14767058.2016.1236251DOI Listing
September 2017

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/CS20150436DOI Listing
December 2015

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.4418DOI Listing
December 2014

Nrf2 deficiency impairs fracture healing in mice.

Calcif Tissue Int 2014 Oct 6;95(4):349-61. Epub 2014 Aug 6.

Department of Trauma Surgery, University Medical Center of Schleswig-Holstein, Campus Kiel, Kiel, Germany,

Oxidative stress plays an important role in wound healing but data relating oxidative stress to fracture healing are scarce. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that controls the cellular defence essential to combat oxidative stress by regulating the expression of antioxidative enzymes. This study examined the impact of Nrf2 on fracture healing using a standard closed femoral shaft fracture model in wild-type (WT) and Nrf2-knockout (Nrf2-KO)-mice. Healing was evaluated by histology, real-time RT-PCR, µCT and biomechanical measurements. We showed that Nrf2 expression is activated during fracture healing. Bone healing and remodelling were retarded in the Nrf2-KO compared to the WT-mice. Nrf2-KO-mice developed significantly less callus tissue compared to WT-mice. In addition, biomechanical testing demonstrated lower strength against shear stress in the Nrf2-KO-group compared to WT. The expression of vascular endothelial growth factor (VEGF) and osteocalcin is reduced during fracture healing in Nrf2-KO-mice. Taken together, our results demonstrate that Nrf2 deficiency in mice results in impaired fracture healing suggesting that Nrf2 plays an essential role in bone regeneration. Pharmacological activation of Nrf2 may have therapeutic potential for the enhancement of fracture healing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00223-014-9900-5DOI Listing
October 2014

A possible protective role of Nrf2 in preeclampsia.

Ann Anat 2014 Sep 27;196(5):268-77. Epub 2014 May 27.

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

Excess release of reactive oxygen species (ROS) is a major cause of oxidative stress. This disturbance has been implicated as a cause of preeclampsia, a pregnancy-related disorder characterized by hypertension and proteinuria. Increased oxidative stress leads to trophoblast apoptosis/necrosis and alters the balance between pro- and anti-angiogenic factors, resulting in generalized maternal endothelial dysfunction. Trials using antioxidants have significantly failed to improve the condition of, or in any way protect, the mother from the life-threatening complications of this syndrome. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a potent transcription activator that regulates the expression of a multitude of genes that encode detoxification enzymes and anti-oxidative proteins. Recent discussion on evidence of a link between Nrf2 and vascular angiogenic balance has focussed on the downstream target protein, heme oxygenase-1 (HO-1). HO-1 metabolizes heme to biliverdin, iron and carbon monoxide (CO). HO-1/CO protects against hypertensive cardiovascular disease and contributes to the sustained health of the vascular system. In one animal model, sFlt-1 (soluble fms-like tyrosine kinase-1) has induced blood pressure elevation, but the induction of HO-1 attenuated the hypertensive response in the pregnant animals. The special conditions under which Nrf2 participates in the pathogenesis of preeclampsia are still unclear, as is whether Nrf2 attenuates or stimulates the processes involved in this syndrome. In this review, we summarize recent theories about how Nrf2 is involved in the pathogenesis of preeclampsia and present the reasons for considering Nrf2 as a therapeutic target for the treatment of preeclampsia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.aanat.2014.04.002DOI Listing
September 2014

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1165/rcmb.2013-0279OCDOI Listing
November 2014

Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts.

Bone 2014 Aug 4;65:9-17. Epub 2014 May 4.

Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address:

Introduction: Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2-ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2).

Methods: Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors.

Results: The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells.

Conclusions: These results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2-ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2014.04.029DOI Listing
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep03625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887379PMC
January 2014

A novel laser-Doppler flowmetry assisted murine model of acute hindlimb ischemia-reperfusion for free flap research.

PLoS One 2013 20;8(6):e66498. Epub 2013 Jun 20.

Department of Oral and Maxillofacial Surgery, Medical Faculty, RWTH Aachen University, Aachen, Germany.

Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group) were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively) following 4 hours of complete hindlimb ischemia. Ischemia and reperfusion were monitored using Laser-Doppler Flowmetry. Hindlimb tissue components (skin and muscle) were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia-reperfusion such as occur during free flap procedures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066498PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688775PMC
February 2014

PP019. A new player in preeclampsia: The NF-E2-related factor 2 (NRF2).

Pregnancy Hypertens 2013 Apr 6;3(2):74. Epub 2013 Jun 6.

Introduction: Preeclampsia PE is characterized by diminished antioxidant capacity. These enzymes are mainly regulated via the transcription factor Nrf2.

Objectives: PE is associated with an increase in Nrf2 activity. Nrf2 involves also in the vascular homeostasis during PE. Respective hemodisturbances have been associated with impaired invasion of the extravillous trophoblast EVT in early onset IUGR associated with PE. To test this link, we studied in vitro the interaction between Nrf2 and VEGF, then their expression was determined in third trimester placental beds in cases of severe early onset IUGR/PE.

Methods: BeWo cells were used in the in vitro study.Western blot; ELISA and Dual Luciferase assay were applied. Full-thickness uterine tissues from 6 healthy and 6 women with severe early onset IUGR/PE were used to study the expression of VEGF, Nrf2 and 4-HNE in the EVT.

Results: Nrf2-activation and its downstream target protein HO-1 augmented CO production, which in turn up-regulated the expression of VEGF. EVT in cases with IUGR/PE showed increased expression of Nrf2 and decreased VEGF intensity.

Conclusion: In early onset IUGR/PE the EVT experience oxidative stress and try to counteract this by increased expression of Nrf2. However, since these cells fail to up-regulate VEGF, Nrf2-activation does not occur, leading to further trophoblast damage. At the same time, in vitro data show a protective role of the Nrf2/HO-1 pathway, which may have a therapeutic potential in PE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.preghy.2013.04.047DOI Listing
April 2013

PP021. The role of the transcription factor Nrf2 in the murine placental development.

Pregnancy Hypertens 2013 Apr 6;3(2):74-5. Epub 2013 Jun 6.

Introduction: The placenta is the key organ for successful pregnancy and fetal growth. Oxidative stress during early human placental development is associated with pregnancy-related disorders. The transcription of many antioxidative-genes is mediated mainly through the transcription factor Nrf2. Furthermore, a link between Nrf2, vascular homeostasis and extravillous trophoblast invasion has been discussed.

Objectives: Here, we investigated the placental phenotype, placental and fetal weight of the Nrf2 knockout (Nrf2(-/-)) and wild type (Nrf2(+/+)) mice and the vascular function of these placentas around embryonic day 18.5.

Methods: We performed H&E, Periodic Acid Schiff (PAS) and immunohistochemistry of paraffin-embedded mouse placenta samples.

Results: There is no significant difference in both placental and fetal weight of both geno types (Nrf2(-/-) and Nrf2(+/+)). Phenotypic analysis of ED 18.5 placentas showed presence of trophoblast clusters in the labyrinth and frequent enlarged maternal blood lacunae. Furthermore, Nr2(-/-) showed increased levels in the lipid peroxidation product 4-hydroxinonoeal (4-HNE), which is a sensitive marker of oxidative damage and lipid peroxidation.

Conclusion: This data point out the necessity of a functional Nrf2 for placental development, as it may interact with the differentiation of the trophoblast lineage from one side and to diminish the oxidative damage during pregnancy from the other side.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.preghy.2013.04.049DOI Listing
April 2013

PP020. Evidence of a preventive role of Nrf2 in preeclampsia.

Pregnancy Hypertens 2013 Apr 6;3(2):74. Epub 2013 Jun 6.

Introduction: Smoking during pregnancy is associated with lower preeclampsia risk. This has been mainly explained through the effect of carbon monoxide CO.

Objectives: Recent studies showed that the activation of heme oxygenase-1 HO-1 and consequently its metabolite CO in cultured cells mediated an inhibition of sFlt-1 and sEng release, and an up-regulation of the endogenous VEGF. The transcriptional regulation of the HO-1 gene is majorly regulated through the transcription factor Nrf2. The aim of this study was to investigate in vitro the effect of HO-1-activation via Nrf2 on the pro- and anti-angiogenic factors.

Methods: BeWo cells and HUVECs endothelial cells were used to study the angiogenic effect of Nrf2-activation. ELISA, scratch and tube formation assay were mainly applied.

Results: The activation of HO-1 via Nrf2 lead to an increase in the protein levels of VEGF (control 64.75pg/ml±4.3; Sulforaphane-treated cells 128.2pg/ml±6.5 p<0.005) and decrease in the augmented sFlt-1 in the supernatant of the treated cells (control 186.3pg/ml±28.7; H2O2-treatment 2026pg/ml±64, co-treatment with H2O2 and Sulforaphane 1200pg/ml±19.7 p<0.01). Up-regulation of HO-1/CO enhanced tube formation and migration of the endothelial cells.

Conclusion: The activation of HO-1/CO via Nrf2 inducer such as sulforaphane inhibited in vitro the release of sFlt-1, thus the activation of Nrf2 during the first trimester may improve the balance of the pro- and anti-angiogenic factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.preghy.2013.04.048DOI Listing
April 2013

Sulforaphane has opposing effects on TNF-alpha stimulated and unstimulated synoviocytes.

Arthritis Res Ther 2012 Oct 27;14(5):R220. Epub 2012 Oct 27.

Introduction: Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with rampantly proliferating synoviocytes and joint destruction due to oxidative stress. Recently, we described nuclear factor erythroid 2-related factor 2 (Nrf2) as a major requirement for limiting cartilage destruction. NF-κB and AP-1 are the main transcription factors triggering the inflammatory progression in RA. We used sulforaphane, an isothiocyanate, which is both an Nrf2 inducer and a NF-κB and AP-1 inhibitor.

Methods: Cultured synoviocytes were stimulated with sulforaphane (SFN) with or without TNF-α pre-treatment. NF-κB, AP-1, and Nrf2 activation was investigated via dual luciferase reporter gene assays. Matrix metalloproteinases (MMPs) were measured via zymography and luminex technique. Cytokine levels were detected using ELISA. Cell viability, apoptosis and caspase activity were studied. Cell proliferation was analysed by real-time cell analysis.

Results: SFN treatment decreased inflammation and proliferation dose-dependently in TNF-α-stimulated synoviocytes. SFN did not reduce MMP-3 and MMP-9 activity or expression significantly. Interestingly, we demonstrated that SFN has opposing effects on naïve and TNF-α-stimulated synoviocytes. In naïve cells, SFN activated the cytoprotective transcription factor Nrf2. In marked contrast to this, SFN induced apoptosis in TNF-α-pre-stimulated synoviocytes.

Conclusions: We were able to show that SFN treatment acts contrary on naïve and inflammatory synoviocytes. SFN induces the cytoprotective transcription factor Nrf2 in naïve synoviocytes, whereas it induces apoptosis in inflamed synoviocytes. These findings indicate that the use of sulforaphane might be considered as an adjunctive therapeutic strategy to combat inflammation, pannus formation, and cartilage destruction in RA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/ar4059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580531PMC
October 2012

A role for Nrf2 in redox signalling of the invasive extravillous trophoblast in severe early onset IUGR associated with preeclampsia.

PLoS One 2012 9;7(10):e47055. Epub 2012 Oct 9.

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

Background: Preeclampsia (PE) is characterized by increased lipid oxidation and diminished antioxidant capacity, while intrauterine growth restriction (IUGR) is characterized by impaired invasion of the extravillous trophoblast. Vascular endothelial growth factor (VEGF) has been reported to be altered in preeclampsia. A relationship between VEGF and nuclear factor erythroid 2-related factor-2 (Nrf2) has been shown in vitro, where VEGF prevents oxidative damage via activation of the Nrf2 pathway. In this study the expression of Nrf2, VEGF and 4-hydroxynonenal (4-HNE), was determined in interstitial and endovascular/intramural extravillous trophoblast (EVT) in normal pregnancies and those complicated by severe early onset IUGR associated with preeclampsia IUGR/PE.

Materials And Methods: Full-thickness uterine tissues derived from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and 6 women with severe early onset IUGR with preeclampsia (29-34 weeks gestation) were analyzed. Interstitial and endovascular extravillous trophoblast were quantified after immunohistochemical staining of paraffin sections using antibodies against Nrf2, 4-HNE, VEGF, and cytokeratin 7.

Results: Uterine tissues from women suffering from severe early onset IUGR/PE were characterized by reduced invasion of extravillous trophoblast into the endometrial and myometrial segments of spiral arteries in the placental bed. Extravillous trophoblast showed an increased cytoplasmic expression of Nrf2 and 4-HNE in IUGR/PE cases. The increased expression of Nrf2 in cases of IUGR/PE was associated with decreased expression of VEGF in these cells compared to controls.

Conclusion: Our data suggests that besides villous cytotrophoblast, also the extravillous trophoblast is a source of Nrf2-dependent genes. VEGF deficiency may cause higher oxidative stress in extravillous trophoblast in cases with IUGR/PE. The resulting reduced basal defence against oxidative stress and the higher vulnerability to oxidative damage may play a role in the limited trophoblast invasion into spiral arteries in cases suffering from severe early onset IUGR/PE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047055PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467234PMC
May 2013

Interplay between vascular endothelial growth factor (VEGF) and nuclear factor erythroid 2-related factor-2 (Nrf2): implications for preeclampsia.

J Biol Chem 2011 Dec 27;286(50):42863-72. Epub 2011 Oct 27.

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

Several recently published studies have suggested that decreasing VEGF levels result in placental oxidative stress in preeclampsia, although the question as to how decreased VEGF concentrations increase oxidative stress still remains unanswered. Here, we show that VEGF activated Nrf2, the main regulating factor of the intracellular redox balance, in the cytotrophic cell line BeWo. In turn, this activated the production of antioxidative enzymes thioredoxin, thioredoxin reductase, and heme oxygenase-1, which showed a decrease in their expression in the placentas of preeclamptic women. Nevertheless, this activation occurred without oxidative stress stimulus. As a consequence, the activation of Nrf2 protected BeWo cells against H(2)O(2)/Fe(2+)-induced oxidative damage. We further show that VEGF up-regulated the expression of itself. A positive feedback loop was described in which VEGF activated Nrf2 in an ERK1/2-dependent manner; the up-regulation of HO-1 expression by Nrf2 augmented the production of carbon monoxide, which in turn up-regulated VEGF expression. In conclusion, VEGF induces the Nrf2 pathway to protect against oxidative stress and, via a positive feedback loop, to elevate VEGF expression. Therefore, decreased VEGF bioavailability during preeclampsia may result in higher vulnerability to placental oxidative cell damage and a further reduction of VEGF bioavailability, a vicious circle that may end up in preeclampsia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.286880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234879PMC
December 2011

Nrf2 expression by neurons, astroglia, and microglia in the cerebral cortical penumbra of ischemic rats.

J Mol Neurosci 2012 Mar 7;46(3):578-84. Epub 2011 Sep 7.

Institute of Neuroanatomy, Medical Faculty, RWTH Aachen University, Aachen, Germany.

Experimental animal studies have demonstrated that oxidative stress plays an essential role during ischemic stroke. In addition to oxidizing macromolecules leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Nuclear factor erythroid 2-related factor 2 (Nrf2) represents one of the major regulators implicated in the endogenous defense system against oxidative stress. We have studied the expression and activation status of Nrf2 under stroke-like conditions using the temporary middle cerebral artery occlusion rat model. Inactive Nrf2 is proteasomal degraded within minutes but stabilized during activation. We analyzed Nrf2 activation and the resulting accumulation in post-ischemic rat brain cells using double immunofluorescence staining with antibodies directed against Nrf2 and cell type-specific markers. The core infarct region showed no obvious positive staining signal for Nrf2 24 h after the initiation of artery occlusion. However, Nrf2 immunoreactivity was detectable in the ipsilateral penumbra where microglia, astrocytes, and neurons contained Nrf2. Interestingly, Nrf2 was also significantly upregulated in neurons but not in other cell types of the unaffected contralateral site. These results provide strong evidence that Nrf2 is involved in acute stroke-dependent neurodegeneration in the penumbra but not core region and indicate the presence of a systemic Nrf2 activator independent from oxidative stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12031-011-9645-9DOI Listing
March 2012

Platelets display potent antimicrobial activity and release human beta-defensin 2.

Platelets 2012 13;23(3):217-23. Epub 2011 Sep 13.

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

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/10(9) platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/09537104.2011.610908DOI Listing
August 2012

Intraarticular injection of platelet-rich plasma reduces inflammation in a pig model of rheumatoid arthritis of the knee joint.

Arthritis Rheum 2011 Nov;63(11):3344-53

University Medical Center Schleswig Holstein, Kiel Campus, Kiel, Germany.

Objective: Treatment options for rheumatoid arthritis range from symptomatic approaches to modern molecular interventions such as inhibition of inflammatory mediators. Inhibition of inflammation by platelet-rich plasma (PRP) has been proposed as a treatment for tendinitis and osteoarthritis. The present study was undertaken to investigate the effect of PRP on antigen-induced arthritis (AIA) of the knee joint in a large animal model.

Methods: Six-month-old pigs (n = 10) were systemically immunized by bovine serum albumin (BSA) injection, and arthritis was induced by intraarticular BSA injection. PRP was injected into the knee joints of 5 of the animals after 2 weeks. An additional 5 animals received no systemic immunization (controls). Signs of arthritis were documented by plain histologic analysis, Safranin O staining, and immunohistochemistry analysis for type II collagen (CII), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF). Interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNFα), VEGF, and insulin-like growth factor 1 (IGF-1) protein content was measured by Luminex assay.

Results: In the pigs with AIA, plain histologic analysis revealed severe arthritic changes in the synovium. Safranin O and CII staining showed decreased proteoglycan and CII content in cartilage. Immunohistochemistry analysis revealed increased levels of IL-6 and VEGF in synovium and cartilage, and protein concentrations of IL-6, VEGF, IL-1β, and IGF-1 in synovium and cartilage were elevated as well; in addition, TNFα protein was increased in cartilage. Treatment with PRP led to attenuation of these arthritic changes in the synovium and cartilage.

Conclusion: We have described a porcine model of AIA. Experiments using this model demonstrated that PRP can attenuate arthritic changes as assessed histologically and based on protein synthesis of typical inflammatory mediators in the synovial membrane and cartilage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/art.30547DOI Listing
November 2011

Thrombocytes are effectors of the innate immune system releasing human beta defensin-3.

Injury 2011 Jul 10;42(7):682-6. Epub 2011 Feb 10.

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

Background: Thrombocyte concentrate i.e. platelet-rich plasma (PRP) has become a popular adjunct for many surgical procedures. It is believed to improve bone and soft tissue healing. Recently antimicrobial effects of the autologous preparation were reported by several groups. In this study we investigated the antimicrobial effect of PRP against gram-negative microbes which frequently cause severe complications in orthopaedic trauma surgery.

Methods: Platelet-rich plasma was produced from liquid preserved thrombocyte concentrates. ELISA, Western blot and immunohistochemistry were preformed to investigate the release and content of platelet concentrates. A radial diffusion assay was used to detect antimicrobial effects of PRP.

Results: We detected the human beta defensin-3 in bactericidal concentrations in platelet preparations by ELISA, Western blot and immunohistochemistry. In antimicrobial testing we demonstrated effective inhibition of Escherichia coli (ATCC 11303), Bacterium megaterium (ATCC 14581), Klebsiella pneumoniae (ATCC 13883), Enterococcus faecalis (ATCC 29212) and Proteus mirabilis (ATCC21100).

Conclusion: With this study we demonstrate antimicrobial action of a popular adjunct for orthopaedic and trauma surgery against gram-positive and gram-negative bacteria. We have identified a possible mechanism of action via the secretion of HBD-3 as a first line defence in contaminated wounds and in elective application of PRP. This finding supports a broader spectrum of clinical indications for an autologous platelet preparation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.injury.2010.12.010DOI Listing
July 2011

Role of oxidative stress in rheumatoid arthritis: insights from the Nrf2-knockout mice.

Ann Rheum Dis 2011 May 20;70(5):844-50. Epub 2010 Dec 20.

Correspondence to Christoph J Wruck, Department of Anatomy and Cell Biology, Medical Faculty, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.

Objectives: Increasing evidence suggests that oxidative stress may play a key role in joint destruction due to rheumatoid arthritis (RA). The aim of this study was to elucidate the role of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that maintains the cellular defence against oxidative stress, in RA.

Methods: The activation status of Nrf2 was assessed in synovial tissue from patients with RA using immunohistochemistry. Antibody-induced arthritis (AIA) was induced in Nrf2-knockout and Nrf2-wild-type control mice. The severity of cartilage destruction was evaluated using a damage score. The extent of oxidative stress, the activation state of Nrf2 and the expression level of Nrf2 target genes were analysed by immunhistological staining. The expression of vascular endothelial growth factor (VEGF)-A was examined on mRNA and protein using the Luminex technique. A Xenogen imaging system was used to measure Nrf2 activity in an antioxidant response element-luciferase transgenic mouse during AIA.

Results: Nrf2 was activated in the joints of arthritic mice and of patients with RA. Nrf2-knockout mice had more severe cartilage injuries and more oxidative damage, and the expression of Nrf2 target genes was enhanced in Nrf2-wild-type but not in knockout mice during AIA. Both VEGF-A mRNA and protein expression was upregulated in Nrf2-knockout mice during AIA. An unexpected finding was the number of spontaneously fractured bones in Nrf2-knockout mice with AIA.

Conclusion: These results provide strong evidence that oxidative stress is significantly involved in cartilage degradation in experimental arthritis, and indicate that the presence of a functional Nrf2 gene is a major requirement for limiting cartilage destruction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/ard.2010.132720DOI Listing
May 2011

Nrf2 induces interleukin-6 (IL-6) expression via an antioxidant response element within the IL-6 promoter.

J Biol Chem 2011 Feb 2;286(6):4493-9. Epub 2010 Dec 2.

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

IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-κB, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.162008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039356PMC
February 2011

Local treatment of meniscal lesions with vascular endothelial growth factor.

J Bone Joint Surg Am 2010 Nov;92(16):2682-91

Department of Orthopaedic Surgery, Otto-von-Guericke-University, Magdeburg, Germany.

Background: The healing potential in the avascular regions of the meniscus is very limited, and improving the vascularity might be a reasonable way to improve healing. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenetic factors. We hypothesized that the local application of VEGF(165) would (1) improve the healing of a lesion in the avascular region of the meniscus, (2) induce angiogenesis in both the avascular and vascular regions, and (3) increase the amounts of VEGF mRNA and VEGF.

Methods: In eighteen sheep, the medial menisci were cut longitudinally in the avascular region and were sutured. Three groups were established depending on the suture material: (1) uncoated Ethibond, (2) Ethibond coated with VEGF(165) and its carrier Poly(D,L-Lactide) (PDLLA), and (3) Ethibond coated with PDLLA. The contralateral medial menisci served as a control group. Each of the three suture type groups included six animals. After eight weeks, the sheep were killed, and the menisci were examined macroscopically. Immunohistochemistry of Factor VIII and VEGF and real-time reverse-transcription polymerase chain reaction (RT-PCR) of VEGF mRNA were performed. Additionally, the VEGF release kinetics from the VEGF/PDLLA-coated suture were evaluated in vitro.

Results: In this model, VEGF did not improve meniscal healing. It did not increase angiogenesis in the avascular or vascular region, the VEGF concentration, or the amount of VEGF mRNA. VEGF release from the coated suture peaked on Day 3 and was nearly zero on Day 9.

Conclusions: The local application of VEGF(165) as eluted from suture did not increase meniscal angiogenesis or improve meniscal healing. In addition, there was no effect on the amount of VEGF mRNA and VEGF. The VEGF carrier (PDLLA) may have been inadequate because of the short duration of VEGF supply.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2106/JBJS.I.01481DOI Listing
November 2010
-->