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
promoter region were inactivated, cloned into pGL3, and co-transfected with phRL-TK for dual-luciferase assays.
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
expression, whereas Keap1-specific RNAi increased it. Putative ARE sites (ARE
, ARE
) were identified in the
promoter region. ARE
mutagenesis significantly reduced
promoter activity, but ARE
excision did not. Functional ARE
site was essential for methysticin-mediated induction of
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.
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.
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.
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.
A new multiple trauma model of the mouse Fitschen-Oestern, Stefanie; Lippross, Sebastian; Klueter, Tim ...
BMC musculoskeletal disorders,
11/2017, Letnik:
18, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Blunt trauma is the most frequent mechanism of injury in multiple trauma, commonly resulting from road traffic collisions or falls. Two of the most frequent injuries in patients with multiple trauma ...are chest trauma and extremity fracture. Several trauma mouse models combine chest trauma and head injury, but no trauma mouse model to date includes the combination of long bone fractures and chest trauma. Outcome is essentially determined by the combination of these injuries. In this study, we attempted to establish a reproducible novel multiple trauma model in mice that combines blunt trauma, major injuries and simple practicability.
Ninety-six male C57BL/6 N mice (n = 8/group) were subjected to trauma for isolated femur fracture and a combination of femur fracture and chest injury. Serum samples of mice were obtained by heart puncture at defined time points of 0 h (hour), 6 h, 12 h, 24 h, 3 d (days), and 7 d.
A tendency toward reduced weight and temperature was observed at 24 h after chest trauma and femur fracture. Blood analyses revealed a decrease in hemoglobin during the first 24 h after trauma. Some animals were killed by heart puncture immediately after chest contusion; these animals showed the most severe lung contusion and hemorrhage. The extent of structural lung injury varied in different mice but was evident in all animals. Representative H&E-stained (Haematoxylin and Eosin-stained) paraffin lung sections of mice with multiple trauma revealed hemorrhage and an inflammatory immune response. Plasma samples of mice with chest trauma and femur fracture showed an up-regulation of IL-1β (Interleukin-1β), IL-6, IL-10, IL-12p70 and TNF-α (Tumor necrosis factor- α) compared with the control group. Mice with femur fracture and chest trauma showed a significant up-regulation of IL-6 compared to group with isolated femur fracture.
The multiple trauma mouse model comprising chest trauma and femur fracture enables many analogies to clinical cases of multiple trauma in humans and demonstrates associated characteristic clinical and pathophysiological changes. This model is easy to perform, is economical and can be used for further research examining specific immunological questions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract To improve skin flap healing, one promising strategy in reconstructive surgery might be to optimize platelet rich plasma (PRP) bioactivity and the ischemia-altered expression of genes. We ...studied both the effect of PRP on ischemic flaps, and whether in vivo bioluminescence imaging (BLI) is a suitable method for the longitudinal monitoring of angiogenesis in surgical wounds. Axial murine skin flaps were created in four experimental groups. In vivo measurements of VEGFR2 expression levels were made every other day until the 14th day. The local VEGF level and microvessel density were quantified on the 14th day via ELISA and immunohistochemistry, and flap survival rates were measured. We demonstrated that PRP and induced ischemia have a beneficial influence on angiogenesis and flap healing. Combining the two resulted in a significantly robust increase in angiogenesis and flap survival rate that was corroborated by bioluminescence imaging of VEGFR2 activity. This study shows that angiogenic effects of PRP may be potentialized by the stimulus of induced ischemia during free flap harvesting, and thus the two procedures appear to have a synergistic effect on flap healing. This study further demonstrates that BLI of modulated genes in reconstructive surgery is a valuable model for longitudinal in vivo evaluation of angiogenesis.
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.
The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus pneumoniae. By entering the brain, host cells will be activated and proinflammatory cytokines ...like interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are released. The goal of the current study was to examine the interaction between IL-6 and TNFR1 as receptor for TNF-α and the innate immune response in vivo in a model of Streptococcus pneumoniae-induced meningitis. For the experiments IL-6−/−, TNFR1−/−, and TNFR1-IL-6−/− KO mice were used. Our results revealed higher mortality rates and bacterial burden after infection in TNFR1−/−, IL-6−/−, and TNFR1-IL-6−/− mice and a decreased immune response including lower neutrophil infiltration in the meninges of TNFR1−/− and TNFR1-IL-6−/− mice in contrast to IL-6−/− and wild type mice. Furthermore, the increased mortality of TNFR1−/− and TNFR1-IL-6−/− mice correlated with decreased glial cell activation compared to IL-6−/− or wild type mice after pneumococcal meningitis. Altogether, the results show the importance of TNFR1 and IL-6 in the regulation of the innate immune response. The lack of TNFR1 and IL-6 results in higher mortality by weakened immune defence, whereas the lack of TNFR1 results in more severe impairment of the innate immune response than the lack of IL-6 alone.
Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. ...Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2–ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2–ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.
Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians’ ...focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.