Physiomimetic biocompatibility evaluation of directly printed degradable porous iron implants using various cell types.

Additively manufactured (AM) degradable porous metallic biomaterials offer unique opportunities for satisfying the design requirements of an ideal bone substitute. Among the currently available biodegradable metals, iron has the highest elastic modulus, meaning that it would benefit the most from porous design. Given the successful preclinical applications of such biomaterials for the treatment of cardiovascular diseases, the moderate compatibility of AM porous iron with osteoblast-like cells, reported in earlier studies, has been surprising. This may be because, as opposed to static in vitro conditions, the biodegradation products of iron in vivo are transported away and excreted. To better mimic the in situ situations of biodegradable biomaterials after implantation, we compared the biodegradation behavior and cytocompatibility of AM porous iron under static conditions to the conditions with dynamic in situ-like fluid flow perfusion in a bioreactor. Furthermore, the compatibility of these scaffolds with four different cell types was evaluated to better understand the implications of these implants for the complex process of natural wound healing. These included endothelial cells, L929 fibroblasts, RAW264.7 macrophage-like cells, and osteoblastic MG-63 cells. The biodegradation rate of the scaffolds was significantly increased in the perfusion bioreactor as compared to static immersion. Under either condition, the compatibility with L929 cells was the best. Moreover, the compatibility with all the cell types was much enhanced under physiomimetic dynamic flow conditions as compared to static biodegradation. Our study highlights the importance of physiomimetic culture conditions and cell type selection when evaluating the cytocompatibility of degradable biomaterials in vitro. STATEMENT OF SIGNIFICANCE: Additively manufactured (AM) degradable porous metals offer unique opportunities for the treatment of large bony defects. Despite the successful preclinical applications of biodegradable iron in the cardiovascular field, the moderate compatibility of AM porous iron with osteoblast-like cells was reported. To better mimic the in vivo condition, we compared the biodegradation behavior and cytocompatibility of AM porous iron under static condition to dynamic perfusion. Furthermore, the compatibility of these scaffolds with various cell types was evaluated to better simulate the process of natural wound healing. Our study suggests that AM porous iron holds great promise for orthopedic applications, while also highlighting the importance of physio-mimetic culture conditions and cell type selection when evaluating the cytocompatibility of degradable biomaterials in vitro.

In vivo characterisation of the inflammatory reaction following mesh implantation in transgenic mice models.

INTRODUCTION: Hernia repair with prosthetic meshes represents one of the most common surgical procedures in the field of surgery. This intervention is always associated with an ensuing inflammatory response, angiogenesis and fibrotic encapsulation forming a foreign body granuloma (FBG) around the mesh fibres. Several studies have described this inflammatory reaction by characterising inflammatory cell infiltrate around the FBG after mesh explantation. However, very little is known about the real-time progression of such an inflammatory response. The aim of this study was to investigate the feasibility of monitoring the ongoing inflammatory response to mesh implantation using bioluminescence in vivo. MATERIALS AND METHODS: Three luciferase transgenic mice strains (FVB/N-Tg(Vegfr2-luc)-Xen, BALB/C-Tg(NFκB-RE-luc)-Xen and Tg(INS/EpRE-Luc)T20Rbl) were used. Mice were anaesthetized with 2 % isoflurane, and two incisions were made on the left and right sides of the abdomen of the mice. A 1-cm(2) propylene mesh was implanted subcutaneously in the right incision wound of each mouse, and the left wound served as control. Two hundred microliters of D-luciferin was injected into the mice, and bioluminescence measurements were done prior to the surgical intervention and subsequently every 3 days. After mesh explantation, histological analysis was done. Statistical analysis was done using prism GraphPad software. RESULTS: Bioluminescence results revealed different time points of maximum signal for the different mice strains. VEGFR2 gene expression peaked on day 6, NFkB on day 12 and ARE on day 3 post mesh implantation. We also observed much higher bioluminescent signal around the FBG surrounding the mesh as compared to the control wound, with p < 0.05 for all the different mice strains. CONCLUSION: Our results prove the possibility of monitoring the inflammatory reaction after mesh implantation in vivo using bioluminescence signal release. This provides a novel method of accessing and accurately describing the ongoing inflammatory response over a given period of time.

[New from old : relevant factors for fracture healing in aging bone]. / Aus alt mach neu : Relevante Faktoren bei der Frakturheilung im Alter.

BACKGROUND: Fracture healing is a complex biological process with specific temporal expression patterns. During this process new bone tissue is formed, which is similar to the original bone in quality and structure. This occurs in four phases: inflammation, formation of a soft tissue callus, formation of a bony callus and remodelling of the bony callus. This needs the precise orchestration of each cell type involved. OBJECTIVES: This article presents details of the fracture healing phases and the relevant factors. During the aging process there is an increase of reactive oxygen species and a change in expression pattern of growth factors that have a negative effect on the fracture healing process. METHODS: A selective review of the literature was carried out in PubMed concerning the influence of aging on fracture healing. CONCLUSION: The healing process is regulated by systemic and local factors. An understanding of these processes and the changes during aging is necessary in order to improve the knowledge of delayed or lack of fracture healing during aging to decide when an intervention is needed.

[Maternal-placental interactions and fetal programming]. / Mütterlich-plazentare Interaktionen und fetale Programmierung.

Pregnancy-related complications not only represent a risk for maternal and fetal morbidity and mortality, but are also a risk for several diseases later in life. Many epidemiological studies have shown clear associations between an adverse intrauterine environment and an increased risk of diabetes, hypertension, cardiovascular disease, depression, obesity, and other chronic diseases in the adult. Some of these syndromes could be prevented by avoiding adverse stimuli or insults including psychological stress during pregnancy, intake of drugs, insufficient diet and substandard working conditions. Hence, all of these stimuli have the potential to alter health later in life. The placenta plays a key role in regulating the nutrient supply to the fetus and producing hormones that control the fetal as well as the maternal metabolism. Thus, any factor or stimulus that alters the function of the hormone producing placental trophoblast will provoke critical alterations of placental function and hence could induce programming of the fetus. The factors that change placental development may interfere with nutrient and oxygen supply to the fetus. This may be achieved by a direct disturbance of the placental barrier or more indirectly by, e. g., disturbing trophoblast invasion. For both path-ways, the respective pathologies are known: while preeclampsia is caused by alterations of the villous trophoblast, intra-uterine growth restriction is caused by insufficient invasion of the extravillous trophoblast. In both cases the effect can be undernutrition and/or fetal hypoxia, both of which adversely affect organ development, especially of brain and heart. However, the mechanisms responsible for disturbances of trophoblast differentiation and function remain elusive.

Argon reduces neurohistopathological damage and preserves functional recovery after cardiac arrest in rats.

BACKGROUND: Xenon has profound neuroprotective effects after neurological injury and is currently undergoing phase 2 clinical trials in cardiac arrest patients. However, xenon is very costly, which might preclude its widespread use. We hypothesized argon, which is more available, might also protect central nervous tissues and allow better functional recovery in a rodent model of global cerebral ischaemia. METHODS: Fourteen male Sprague-Dawley rats were subjected to 7 min of cardiac arrest and 3 min of cardiopulmonary resuscitation (CPR). One hour after successful CPR, animals were randomized to either ventilation with 70% argon in oxygen (n = 7) for 1 h or 70% nitrogen (controls, n=7). A neurological deficit score (NDS) was calculated daily for the following 7 days, then the animals were killed and the brains harvested for histopathological analyses. RESULTS: All animals survived. Control rats had severe neurological dysfunction, while argon-treated animals showed significant improvements in the NDS at all time points. This was paralleled by a significant reduction in the neuronal damage index in the neocortex and the hippocampal CA 3/4 region. CONCLUSIONS: Our study demonstrates that a single 1 h application of 70% argon significantly reduced histopathological damage of the neocortex and hippocampus, associated with a marked improvement in functional neurological recovery.

Detection of vascular endothelial growth factor (VEGF) in moderate osteoarthritis in a rabbit model.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is detectable in later stages of human osteoarthritis (OA), but not in the healthy articular cartilage. Due to its capacity to increase matrix metalloproteinases and to decrease their inhibitors (tissue inhibitors of metalloproteinases or TIMPs) VEGF seems to play an important role in the development of osteoarthrosis. In late stages of osteoarthritis, invasion of blood vessels from the subchondral growth plate, synovitis with angiogenesis and osteophyte growth is observable. Several studies have revealed a central role for VEGF in all these phenomena. In order to investigate whether VEGF participates in early changes of OA or may even possess characteristics of a marker of OA, we developed an experimental posttraumatic OA New Zealand White rabbit animal model. MATERIALS AND METHODS: In four skeletally mature New Zealand White rabbits, OA was induced by joint instability after transsection of the anterior cruciate ligament in both knees. After eight weeks the animals were killed. OA was verified histologically using the Mankin scale. Expression of VEGF was detected by immunohistochemistry and RT-PCR. Proteoglycans were evaluated by using HE and safranin-O staining. Four non-surgically treated animals acted as a control. RESULTS: The mean Mankin score was 5.11 (±2.14), corresponding to a moderate OA. VEGF and VEGF transcripts were detectable in the cartilage of early experimental posttraumatic OA rabbits. Control samples remained negative for VEGF mRNA and protein. DISCUSSION: The results of this study are promising concerning the role of VEGF as a diagnostic marker. VEGF could further be participated in early changes of OA. A therapeutic approach by modulation of VEGF production could be a possibility for the future.

PP015. Differential expression of Nrf2 and VEGF in human placental beds from normal and pregnancies complicated with preeclampsia and IUGR.

INTRODUCTION: Impaired trophoblast invasion into the uteroplacental arteries is accompanied with an evidence of oxidative stress in the extravillous trophoblast in preeclampsia complicated with IUGR. OBJECTIVES: Preeclampsia is characterised by increased lipid oxidation and diminished antioxidant capacity; recently, we have shown that PE is associated with an increased expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) in villous cytotrophoblast. A possible relationship between the vascular endothelial growth factor (VEGF) and Nrf2 was established in vitro and the activation of Nrf2 pathway could lead to upregulation of VEGF synthesis through the induction of Nrf2-dependent Heme oxygenase-1 (HO-1). In this study the expression of Nrf2 and VEGF was determined in the interstitial and intramural extravillous trophoblast in normal pregnancies and those complicated by preeclampsia and intra-uterine growth restriction (IUGR). METHODS: Full-thickness uterine tissues were obtained from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and from 5 women with severe early-onset preeclampsia and IUGR (29-34 week's gestation). The interstitial and intramural trophoblasts were studied by immunohistochemical analysis of paraffin sections stained with anti VEGF and anti Nrf2. RESULTS: Cases suffering from preeclampsia with IUGR were characterised by reduced invasion of extravillous trophoblast into uteroplacental arteries in the endometrial and myometrial segments. In addition, these cells showed an increased expression of Nrf2 in the pathological sections. The overexpression of Nrf2 in cases with preeclampsia was associated with restricted expression of VEGF in these cells compared to controls. CONCLUSION: Our data suggest 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 preeclampsia with IUGR. 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 uteroplacental arteries in cases suffering from early onset preeclampsia and IUGR.

PP119. The role of the nuclear factor erythroid 2-related factor 2 (Nrf2) in preeclampsia.

INTRODUCTION: Preeclampsia is a multi-organ syndrome characterized by maternal endothelial damage, is an independent long-term risk factor for hypertension and cardiovascular disease. OBJECTIVES: In animal models the administration of the Vascular Endothelial Growth Factor (VEGF) could reverse the hypertensive signs accompanying this disease. In addition VEGF is implicated in placental oxidative stress during preeclampsia. One of the major cellular defence mechanisms against oxidative stress is the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the activation of Nrf2 up regulates the HO-1/CO system. The principal aim of this work is to investigate whether the activation of Nrf2 raises VEGF levels by up regulation of CO release. METHODS: This study took place in vitro, the choriocarcinoma cell line BeWo cells and the primary human umbilical vein endothelial cells (HUVECs) were used to study the relationship between VEGF and an Nrf2 inducer Sulforaphane, a naturally occurring compound derived from broccoli. ELISA, Western blot assay and the Dual Luciferase Assay were both mainly applied for protein and VEGF activity analysis. RESULTS: It was found that activation of HO-1 expression via Nrf2/ARE pathway augmented the production of CO, which in turn up-regulated the gene expression of VEGF, and down regulated the production of the antiangiogenic protein, the VEGF antagonist sFlt-1. CONCLUSION: Nrf2 driven HO-1 expression elevates the levels of VEGF via CO production. In particular, activating of Nrf2 via sulforaphane, may have therapeutic potential in preeclampsia.

[Avascular necrosis of the hip - diagnosis and treatment]. / Hüftkopfnekrose - Diagnostik und Differenzialtherapie.

Femoral head necrosis is an ischaemic bone necrosis of traumatic or nontraumatic pathogenesis which can lead to hip joint destruction in young age. It is today the indication for 10 % of all the total hip joint replacements. Known aetiologies of nontraumatic femoral head necrosis are alcoholism, steroids, sickle cell anaemia, caisson, and Gaucher's disease. Further risk factors are chemotherapy, chronic inflammatory bowel disease, systemic lupus erythematosus, and multiple sclerosis, in which also steroids are involved. Gravidity is another risk factor, but still idiopathic pathogenesis is found. In diagnosis, the ARCO-classification of the Association for the Research of Osseous Circulation is essential. While stage 0 can only be found histologically, the reversible early stage 1 shows MR signal changes. In the irreversible early stage 2, first native x-ray changes are seen as lower radiolucency reflects new bone apposition on dead trabeculae. In stage 3, subchondral fracture follows, and in stage 4 secondary arthritis of the hip. Established therapy in stage 1 is core decompression, physiotherapy, and more and more also bisphosphonates. Sufficient data to support extracorporeal shock wave therapy are still lacking. Stem cell therapy seems to be a promising new therapy method in stage 2. In stage 2 and 3 mainly proximal femoral osteotomies and (non)vascularised bone transplantation are performed. In stage 4, depending on size and location of the necrotic zone and pathology of the adjacent bone, resurfacing or short stem hip arthroplasty can be performed. However, conventional THA is still golden standard. The problem and challenge, however, is the often young patient age in femoral head necrosis. Especially chemotherapy-associated osteonecrosis in leukaemia is found in patients in their second decade of life. Therefore, the hip should be preserved as long as possible.

Scaffolds for bone healing: concepts, materials and evidence.

Critical sized bone defects have to be filled with material to allow bone healing. The golden standard for this treatment is autogenous bone grafting. Because of donor size morbidity, equivalent synthetic bone scaffolds should be developed. Different materials, especially ceramics and polymers are in the focus of research. Calcium phosphate ceramics show similar properties to bone and are degradable. Different modifications can improve the bioactive features. This article gives an overview about the current materials and their evidence of clinical use.

Complex patterns of ADAM12 mRNA and protein splice variants in the human placenta.

AIMS: Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. METHODS AND RESULTS: We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n=8) and 3rd (n=8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. CONCLUSION: The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell-cell and cell-matrix interactions between the placental syncytium and the maternal interface.

The role of pro-inflammatory and immunoregulatory cytokines in tendon healing and rupture: new insights.

Owing to limited self-healing capacity, tendon ruptures and healing remain major orthopedic challenges. Increasing evidence suggests that post-traumatic inflammatory responses, and hence, cytokines are involved in both cases, and also in tendon exercise and homeostasis. This review summarizes interrelations known between the cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF)α, IL-6 and vascular endothelial growth factor (VEGF) in tendon to assess their role in tendon damage and healing. Exogenic cytokine sources are blood-derived leukocytes that immigrate in damaged tendon. Endogenous expression of IL-1ß, TNFα, IL-6, IL-10 and VEGF was demonstrated in tendon-derived cells. As tendon is a highly mechanosensitive tissue, cytokine homeostasis and cell survival underlie an intimate balance between adequate biomechanical stimuli and disturbance through load deprivation and overload. Multiple interrelations between cytokines and tendon extracellular matrix (ECM) synthesis, catabolic mediators e.g. matrix-degrading enzymes, inflammatory and angiogenic factors (COX-2, PGE2, VEGF, NO) and cytoskeleton assembly are evident. Pro-inflammatory cytokines affect ECM homeostasis, accelerate remodeling, amplify biomechanical adaptiveness and promote tenocyte apoptosis. This multifaceted interplay might both contribute to and interfere with healing. Much work must be undertaken to understand the particular interrelation of these inflammatory and regulatory mediators in ruptured tendon and healing, which has relevance for the development of novel immunoregulatory therapeutic strategies.

The antimicrobial peptide HBD-2 and the Toll-like receptors-2 and -4 are induced in synovial membranes in case of septic arthritis.

Septic arthritis is frequently observed especially in immune-compromised or chronically diseased patients and leads to functional impairment due to tissue destruction. Recently, production of antimicrobial peptides (AMP) was observed in articular cartilage after exposure to bacteria. This report examines the role of synoviocyte-derived AMPs in innate defense mechanisms of articular joints. Samples of healthy, low-grade synovialitis and septic synovial membranes were assessed for the expression of human beta-defensin-2 (HBD-2) and Toll-like receptor-2 and -4 (TLR) by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). A stable synoviocyte line (K4IM) was used for in vitro experiments and assayed for endogenous HBD-2 and TLR production after exposure to inflammatory cytokines or bacterial supernatants by reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, ELISA, and dual luciferase assay. Healthy human synovial membranes and cultured synoviocytes are able to produce HBD-2 and TLR-1-5 at basal expression levels. Samples of bacteria-colonized synovial membranes produce higher levels of HBD-2 when compared with samples of healthy tissues. K4IM synoviocytes exposed to Staphylococcus aureus, Pseudomonas aeruginosa, or proinflammatory cytokines demonstrated a clear HBD-2 transcription and protein induction. TLR-2 and -4 are known to have a critical role in the recognition of gram-positive and gram-negative bacteria in epithelia and are induced in mesenchymal synoviocytes after bacterial exposure on transcription and on protein level. This report demonstrates an unappreciated role of synovial membranes: samples of septic synovial membranes and cultured synoviocytes exposed to bacteria produce increased amounts of the AMP HBD-2 and the bacteria recognition receptors TLR-2 and -4. The induction of anti-inflammatory pathways in infected synoviocytes suggests involvement in intra-articular defense mechanisms.

Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3.

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

The role of human beta-defensin-2 in bone.

Osteomyelitis often causes functional impairment due to tissue destruction. This report demonstrates a novel previously unappreciated role of osteoblasts. Samples of osteomyelitic bone and bacterially challenged osteoblasts produce increased amounts of antimicrobial peptides in order to combat bacterial bone infection. An osteomyelitis mouse model confirmed the osseous induction of the murine homologue of human beta-defensin-2, suggesting a central role in the prevention of bacterial bone infection. Antimicrobial peptides are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Some of them are produced constitutively, whereas others are induced during infection. Human beta-defensins represent a major subclass of antimicrobial peptides and act as a first line of defence through their broad spectrum of potent antimicrobial activity. The aim of the present in-vitro and in-vivo investigations was to study the expression and regulation of human beta-defensin-2 in the case of bacterial bone infection and to analyse the effects of immunosuppressive drugs on bone-derived antimicrobial peptide expression. Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (hFOB cells) were assessed for the expression of human beta-defensin-2. Regulation of human beta-defensin-2 was studied in hFOB cells after exposure to bacterial supernatants, proinflammatory cytokines and immunosuppressive drugs (glucocorticoids and methotrexate) and was assayed by enzyme-linked immunosorbent assay. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologue of human beta-defensin-2, named murine beta-defensin-3, by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Healthy human bone and cultured osteoblasts are able to produce human beta-defensin-2 under standard conditions. Samples of infected bone produce higher levels of endogenous antibiotics, such as human beta-defensin-2, when compared with samples of healthy bone. A clear induction of human beta-defensin-2 was observed after exposure of cultured osteoblasts to gram-positive bacteria or proinflammatory cytokines. Additional treatment with glucocorticoids or methotrexate prevented bacteria-mediated antimicrobial peptide induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional upregulation of the murine homologue of human beta-defensin-2, namely murine beta-defensin-3, in bone after intraosseous contamination of the tibia. Human and murine bone have the ability to produce broad-spectrum endogenous antibiotics when challenged by micro-organisms in vitro and in vivo. Immunosuppressive drugs, such as glucocorticoids or methotrexate, may increase the susceptibility to bone infection by decreasing antimicrobial peptide expression levels in case of microbial challenge. The induction of human beta-defensin-2 following bacterial contact suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection.

Involvement of formyl-peptide-receptor-like-1 and phospholipase D in the internalization and signal transduction of amyloid beta 1-42 in glial cells.

Recent studies suggest that the formyl-peptide-receptor-like-1 (FPRL1) plays an essential role in the inflammatory responses of host defense mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). We therefore analyzed whether amyloid beta1-42 (Abeta1-42) increased the activity of phospholipase D (PLD) via FPRL1, which is an enzyme involved in the secretion, endocytosis and receptor signaling. PLD activity was determined using a transphosphatidylation assay. The internalization of Abeta1-42 via FPRL1 was visualized using fluorescence microscopy and quantified by ELISA (Enzyme Linked Immunosorbent Assay). Determining receptor activity by extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement verified the Abeta1-42-induced activation of FPRL1. We were able to show that Abeta1-42 is rapidly internalized via FPRL1 in astrocytes and microglia. PLD was additionally activated by Abeta1-42 and via FPRL1 in rat glial cells. Furthermore, the ERK1/2 phosphorylation by FPRL1 agonists was dependent on the PLD product phosphatidic acid (PA). Together, these data suggest that PLD plays an important role in the regulation of Abeta1-42-induced endocytosis and FPRL1 receptor signaling.

Luteolin protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway.

Oxidative stress is central to neuronal damage in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In consequence, activation of the cerebral oxidative stress defence is considered as a promising strategy of therapeutic intervention. Here we demonstrate that the flavone luteolin confers neuroprotection against oxidative stress via activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor central to the maintenance of the cellular redox homeostasis. Luteolin protects rat neural PC12 and glial C6 cells from N-methyl-4-phenyl-pyridinium (MPP+) induced toxicity in vitro and effectively activates Nrf2 as shown by ARE-reporter gene assays. This protection critically depends on the activation of Nrf2 since downregulation of Nrf2 by shRNA completely abrogates the protection of luteolin in vitro. Furthermore, the neuroprotective effect of luteolin is abolished by the inhibition of the luteolin-induced ERK1/2-activation. Our results highlight the relevance of Nrf2 for neural cell survival conferred by flavones. In particular, we identified luteolin as a promising lead for the search of orally available, blood brain barrier permeable compounds to support the therapy of neurodegenerative disorders.

Influence of estradiol on vascular endothelial growth factor expression in bone: a study in Göttingen miniature pigs and human osteoblasts.

Ovariectomy (OVX) in animal models is an accepted method to simulate postmenopausal osteoprosis. Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation, hypertrophic cartilage remodeling, ossification, and angiogenesis. We hypothesized that reduced VEGF expression in bone contributes to OVX-induced bone loss and tested it in a miniature pig model and in vitro using human osteoblasts. Seventeen primiparous sows (Göttingen miniature pigs) were allocated to two experimental groups when they were 30 months old: a control group (n = 9) and an OVX group (n = 8). After 15 months, VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay and verified by Western blot analysis. VEGF and its receptor (VEGFR) were localized by immunohistochemistry. Expression of VEGF mRNA was analyzed by real-time reverse-transcription polymerase chain reaction. Differently sulfated glycosaminoglycans were localized in subchondral bone histochemically. Osteoblasts were immunopositive for VEGF. VEGF concentration in the vertebra was 27% lower in OVX miniature pigs. VEGFR-2 could be immunostained on osteoblasts. VEGF mRNA and protein were detectable in the lumbar vertebrae of all animals. In subchondral trabecular bone of OVX animals, significantly more islands of mineralized cartilage containing chondroitin 4- and 6-sulfate or keratan sulfate occurred compared to the control group. The occurrence of remnants of mineralized cartilage in subchondral bone of the OVX group may be caused by a delayed bone turnover due to low VEGF levels. In vitro experiments revealed an increase of VEGF in the supernatant of osteoblasts after incubation with estradiol. In conclusion, estrogen seems to be a key factor for regulation of VEGF expression in bone. Loss of VEGF due to menopause may be a reason for reduction of bone density.

Effects of pleiotrophin, a heparin-binding growth factor, on human primary and immortalized chondrocytes.

OBJECTIVE: Pleiotrophin (PTN) is a secreted heparin-binding peptide expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. In fetal and juvenile, but not in mature cartilage, PTN is abundant. Furthermore, PTN is re-expressed in chondrocytes in early stages of osteoarthritis (OA). Since little is known about the functions of PTN in cartilage, we investigated the occurrence of PTN receptors in human articular cartilage in situ and PTN effects on human primary and immortalized chondrocytes in vitro. METHODS: Receptor expression and regulation was monitored by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. PTN effects and signal transduction were studied by electrophoretic mobility shift, Boyden chamber cell migration and proliferation assays, effects on gene expression by real time RT-PCR and that on nitric oxide (NO) by the Griess reaction. RESULTS: Of the putative PTN signaling receptors, immortalized and primary chondrocytes (pc) expressed the anaplastic lymphoma kinase (ALK), less the receptor-type protein tyrosine phosphatase zeta/beta (PTPzeta). ALK expression was upregulated upon ligand exposure. PTN stimulation activated the AP-1 (activator protein-1) transcription factor and altered gene expression. Prolonged stimulation induced PTN mRNA expression slightly, reduced vascular endothelial growth factor (VEGF) mRNA as well as NO production. Whereas mRNA expression of matrix metalloproteinases (MMPs) MMP-1 and MMP-13 was reduced, their inhibitors TIMP-1 and TIMP-2 were induced. Furthermore, PTN stimulated chondrocyte migration and proliferation. CONCLUSIONS: These results show that PTN is an autocrine growth factor in cartilage. We suggest that PTN may be involved in the clustering and proliferation of chondrocytes observed in the early stages of OA.

TLR-2-mediated induction of vascular endothelial growth factor (VEGF) in cartilage in septic joint disease.

Bacterial arthritis is a progressive joint disease which includes rapid destruction of articular cartilage even after clearance of the causal factor. The resulting post-infectious arthropathy is mainly characterized by self-perpetuating joint destruction and extensive angiogenesis in the emerging pannus-like synovial membrane, but the underlying molecular mechanisms of the bacteria-initiated process remain incompletely understood. This study was conducted to elucidate the expression and regulation of angiogenic and cartilage-destructive vascular endothelial growth factor (VEGF) in septic arthritis. For that purpose, aspirates of synovial fluid from patients with pyogenic arthritis were examined for VEGF levels by ELISA. In vitro studies with primary and immortalized chondrocytes were performed to determine whether Gram-positive and Gram-negative bacteria induce VEGF expression, by using real-time RT-PCR, ELISA, and immunohistochemistry. Activation of the transcription factor AP-1 was assessed by EMSA experiments. The necessity of the Toll-like receptor-2 (TLR-2), ERK-1/-2, and AP-1 pathway for infectious VEGF induction in chondrocytes was examined by using specific blocking reagents. ELISA experiments revealed that aspirates of synovial fluid from patients with pyogenic arthritis contain elevated levels of VEGF. The in vitro results confirmed the transcriptional induction of VEGF in chondrocytes after bacterial challenge by real-time RT-PCR, ELISA, and immunohistochemistry. This activation was mediated by a TLR-2-, ERK-1/-2-, and AP-1-dependent pathway. The findings demonstrate the expression of Toll-like receptors on mesenchymal articular chondrocytes and reveal TLR-2-mediated VEGF induction in human chondrocytes after Gram-positive bacterial sensing. Since VEGF is a potent angiogenic and tissue remodelling factor, evidence that Toll-like receptors contribute to destructive arthropathy after microbial joint infection is provided. VEGF may be a therapeutic target in the future for the prevention of post-infectious cartilage degradation in articular joints.