Nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency causes age-dependent progression of female osteoporosis.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice. METHODS: Eighteen female wild type (WT) and 16 Nrf2-knockout (KO) mice were sacrificed at different ages (12 weeks = young mature adult and 90 weeks = old) to analyze their femurs. The morphological properties (trabecular and cortical) were evaluated by micro-computed tomography (µCT) and compared to gold standard histochemistry analysis. The quasi-static compression tests were performed to calculate the mechanical properties of bones. Additionally, the population of bone resorbing cells and aromatase expression by osteocytes was immunohistochemically evaluated and empty osteocyte lacunae was counted in cortical bone. RESULTS: Old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness, cortical area, and bone fraction compared to old WT mice, regardless of no significant difference in skeletally mature young adult mice between WT and KO. Specifically, while all old WT mice showed thin metaphyseal trabeculae, trabecular bone was completely absent in 60% of old KO mice. Additionally, old KO mice showed significantly more osteoclast-like cells and fewer aromatase-positive osteocytes than WT mice, whereas the occurrence of empty osteocyte lacunae did not differ between both groups. Nrf2-KO mice further showed an age-dependently reduced fracture resilience compared to age-matched WT mice. CONCLUSION: Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.

[Ethical, Psychosocial and Legal Aspects of the Treatment of Pregnant Patients with Brain Death]. / Ethische, psychosoziale und rechtliche Aspekte der Behandlung hirntoter Schwangerer.

The therapy of brain-dead pregnant women is an extreme example not only of the possibilities in current critical care, but also of resulting ethical, social and legal controversies, an area not familiar to most clinicians. Based on the case of a patient with fatal traumatic brain injury, a previously unknown early pregnancy and stated will to donate organs, we will discuss several aspects using published case reports: therapeutic goals, especially palliative care vs. continuation; implications of brain death diagnosis; considerations on legal care; involvement of relatives, especially the child's father; dynamics within the care team; and finally the issue of putative organ donation. This complex case once more depicts that even facing such highly unfavourable framework and seemingly irreconcilable factors, pregnancy can prevail. The researched facts and considerations in this article are intended to give an overview of potential dilemmas and might serve as a starting point in similar situations.

Impaired Fracture Healing after Hemorrhagic Shock.

Impaired fracture healing can occur in severely injured patients with hemorrhagic shock due to decreased soft tissue perfusion after trauma. We investigated the effects of fracture healing in a standardized pressure controlled hemorrhagic shock model in mice, to test the hypothesis that bleeding is relevant in the bone healing response. Male C57/BL6 mice were subjected to a closed femoral shaft fracture stabilized by intramedullary nailing. One group was additionally subjected to pressure controlled hemorrhagic shock (HS, mean arterial pressure (MAP) of 35 mmHg for 90 minutes). Serum cytokines (IL-6, KC, MCP-1, and TNF-α) were analyzed 6 hours after shock. Fracture healing was assessed 21 days after fracture. Hemorrhagic shock is associated with a significant increase in serum inflammatory cytokines in the early phase. Histologic analysis demonstrated a significantly decreased number of osteoclasts, a decrease in bone quality, and more cartilage islands after hemorrhagic shock. µCT analysis showed a trend towards decreased bone tissue mineral density in the HS group. Mechanical testing revealed no difference in tensile failure. Our results suggest a delay in fracture healing after hemorrhagic shock. This may be due to significantly diminished osteoclast recruitment. The exact mechanisms should be studied further, particularly during earlier stages of fracture healing.

Abrasion arthroplasty increases mesenchymal stem cell content of postoperative joint effusions.

BACKGROUND: Abrasion arthroplasty (AAP) is a procedure by which intrinsic cartilage healing is believed to be stimulated. Although clinically accepted for degenerative and traumatic cartilage lesions scientific evidence at a molecular level that proves the effect of AAP is scarce. METHOD: Mononuclear cells were extracted from postoperative joint effusions 21.5 h post AAP and simple debridement of cartilage lesions. Luminex, ELISA and FACS experiments were performed. Immunohistochemical stainings of cell cultures for cartilage markers were used to confirm the findings. RESULTS: Postoperative joint effusions after AAP showed increased contents of Mononuclear cells compared to Arthroscopic Chondroplasty (ACP). BMP-4 and IGF were increased in AAP as complared to ACP. Mononuclear cells isolated after AAP express the MSC markers CD 73, CD 105, CD 90, CD 44 and are CD34 negative. Chondrogenic differentiation was demonstrated by positive staining for Sox9, collagen II, proteoglycan, chondroitin-4-sulfate. CONCLUSION: Our results support the clinical application of AAP as a procedure that enhances cartilage repair as an alternative to far more complex procedures that have gained popularity. Furthermore the data presented supports clinical investigations that recommend not to use suction drainage as by this procedure a considerable amount of the regeneratory potential of postoperative joint effusions might be extracted.

Nrf2 deficiency impairs fracture healing in mice.

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.

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

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

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

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

Nrf2/ARE Signaling Directly Regulates SOX9 to Potentially Alter Age-Dependent Cartilage Degeneration.

Oxidative stress is implicated in osteoarthritis, and nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway maintains redox homeostasis. We investigated whether Nrf2/ARE signaling controls SOX9. SOX9 expression in human C-28/I2 chondrocytes was measured by RT-qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap ''n'' collar homology-associated protein 1 (Keap1). To verify whether Nrf2 transcriptionally regulates SOX9, putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL-TK for dual-luciferase assays. SOX9 promoter activities without and with Nrf2-inducer methysticin were compared. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Nrf2-specific RNAi significantly decreased SOX9 expression, whereas Keap1-specific RNAi increased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, but ARE1 excision did not. Functional ARE2 site was essential for methysticin-mediated induction of SOX9 promoter activity. Young Nrf2-knockout mice revealed significantly lower Sox9-positive chondrocytes, and old Nrf2-knockout animals showed thinner cartilage and more cartilage degeneration. Our results suggest Nrf2 directly regulates SOX9 in articular cartilage, and Nrf2-loss can develop mild osteoarthritis at old age. Pharmacological Nrf2 induction may hold the potential to diminish age-dependent cartilage degeneration through improving SOX9 expression.

Expansion of functional personalized cells with specific transgene combinations.

Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.

In-vitro cell treatment with focused shockwaves-influence of the experimental setup on the sound field and biological reaction.

BACKGROUND: To improve understanding of shockwave therapy mechanisms, in vitro experiments are conducted and the correlation between cell reaction and shockwave parameters like the maximum pressure or energy density is studied. If the shockwave is not measured in the experimental setup used, it is usually assumed that the device's shockwave parameters (=manufacturer's free field measurements) are valid. But this applies only for in vitro setups which do not modify the shockwave, e.g., by reflection or refraction. We hypothesize that most setups used for in vitro shockwave experiments described in the literature influence the sound field significantly so that correlations between the physical parameters and the biological reaction are not valid. METHODS: To reveal the components of common shockwave in vitro setups which mainly influence the sound field, 32 publications with 37 setups used for focused shockwave experiments were reviewed and evaluated regarding cavitation, cell container material, focal sound field size relative to cell model size, and distance between treated cells and air. For further evaluation of the severity of those influences, experiments and calculations were conducted. RESULTS: In 37 setups, 17 different combinations of coupling, cell container, and cell model are described. The setup used mainly is a transducer coupled via water to a tube filled with a cell suspension. As changes of the shockwaves' maximum pressure of 11 % can already induce changes of the biological reaction, the sound field and biological reactions are mainly disturbed by use of standard cell containers, use of coupling gel, air within the 5 MPa focal zone, and cell model sizes which are bigger than half the -6 dB focal dimensions. CONCLUSIONS: Until now, correct and sufficient information about the shockwave influencing cells in vitro is only provided in 1 of 32 publications. Based on these findings, guidelines for improved in vitro setups are proposed which help minimize the influence of the setup on the sound field.

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

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.

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

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.

Nitrate patch prevents steroid-related bone necrosis.

Avascular necrosis of the femoral head is a common complication with disabling effect for young patients after high-dose corticosteroid treatment. We could show that steroids have a vasoconstrictive effect on lateral epiphyseal arteries of the femoral head which could lead to ischemia and subsequent necrosis. In this study we investigated the preventive effect of a nitrate patch on steroid-related bone necrosis in a rabbit model. New Zealand White rabbits (male; 3-4.5 kg bodyweight) were injected with 20 mg/kg bodyweight methylprednisolone (GC group; n = 6). Control animals (n = 6) were treated with phosphate-buffered saline. A third group (GC + N; n = 6) additionally received a nitrate patch (0.675 mg/day). Four weeks after i.m. methylprednisolone injection the animals were sacrificed. For histology and immunohistochemistry, tissue samples were fixed in 3% paraformaldehyde, embedded in paraffin, sectioned, dewaxed, and stained with Ladewig. For quantification of empty lacunae, a histologic sign of FHN, histomorphometry was performed. Histomorphometry revealed a significant increase of empty lacunae in glucocorticoid-treated animals compared to controls and GC + N-treated animals. No significant difference in empty lacunae count was detected between the GC + N group and controls. HE staining revealed the different osteocyte amount in the GC versus GC and nitrate patch-treated groups. This study demonstrates an increased number of empty osteocyte lacunae representing a pathologic feature of osteonecrosis, in the GC group. Less empty lacunae were counted in the GC animals after additional treatment with a nitrate patch. This finding suggests that nitrate co-treatment has the potential to prevent steroid-associated FHN.