Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. ...The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (
,
and terminal differentiation markers (e.g.,
) were affected by osmolarity. Particularly, the expression of minor collagens (e.g.,
,
) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.
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.
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.
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.
Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Nrf2 signaling pathway prevents cancer initiation, but genetic mutations that activate this pathway are found in various types of cancer. The molecular mechanisms underlying this Janus-headed ...character are still not understood. Here, we show that sustained Nrf2 activation induces proliferation and dedifferentiation of a Wnt-responsive perivenular hepatic progenitor cell population, transforming them into metastatic cancer cells. The neoplastic lesions display many histological features known from human hepatoblastoma. We describe an Nrf2-induced upregulation of β-catenin expression and its activation as the underlying mechanism for the observed malignant transformation. Thus, we have identified the Nrf2–β-catenin axis promoting proliferation of hepatic stem cells and triggering tumorigenesis. These findings support the concept that different functional levels of Nrf2 control both the protection against various toxins as well as liver regeneration by activating hepatic stem cells. Activation of the hepatic stem cell compartment confers the observation that unbridled Nrf2 activation may trigger tumorigenesis.
Platelet‐released growth factors (PRGF) and its related clinically used formulations e.g. Vivostat platelet‐rich fibrin (PRF®) are thrombocyte concentrate lysates that support healing of chronic, ...hard‐to‐heal and infected wounds. Human beta‐defensin‐2 (hBD‐2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound‐related bacteria. In this study, we analysed the influence of PRGF on hBD‐2 expression in human primary keratinocytes and the influence of Vivostat PRF® on hBD‐2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD‐2 gene and protein expressions in a concentration‐ and time‐dependent manner. The use of blocking antibodies revealed that the PRGF‐mediated hBD‐2 induction was partially mediated by the epidermal growth factor receptor and the interleukin‐6 receptor (IL‐6R). Luciferase gene reporter assays indicated that the hBD‐2 induction through PRGF required activation of the transcription factor activator protein 1 (AP‐1), but not of NF‐kappaB. In concordance with these cell culture data, Vivostat PRF® induced hBD‐2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD‐2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds.
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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The etiology and pathogenesis of rheumatoid arthritis (RA) are marked by a complex interplay of various cell populations and is mediated by different signaling pathways. Traditionally, therapies have ...primarily focused on pain relief, reducing inflammation and the recovery of joint function. More recently, however, researchers have discussed the therapeutic efficacy of autologous platelet-rich plasma (PRP). The main objective of this work is to examine the influences of platelet-released growth factor (PRGF) on human synoviocytes under inflammatory conditions. Additionally, it is checked to which extend treatment with platelet concentrate influences the release of cytokines form synoviocytes. For this purpose, an in vitro RA model was created by stimulating the cells with the TNF-α. The release of cytokines was measured by ELISA. The cytokine gene expression was analyzed by real-time PCR. It has been observed that the stimulation concentration of 10 ng/ml TNF-α resulted in a significantly increased endogenous secretion and gene expression of IL-6 and TNF-α. The anti-inflammatory effect of PRGF could be confirmed through significant reduction of TNF-α and IL-1β. An induced inflammatory condition seems to cause PRGF to inhibit the release of proinflammatory cytokines. Further study is required to understand the exact effect mechanism of PRGF on synoviocytes.
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.
Antimicrobial peptides (APs) are an important part of the innate immune system in epithelial and non-epithelial surfaces. So far, many different antimicrobial peptides from various families have been ...discovered in non-vertebrates and vertebrates. They are characterized by antibiotic, antifungal and antiviral activities against a variety of microorganisms. In addition to their role as endogenous antimicrobials, APs participate in multiple aspects of immunity. They are involved in septic and non-septic inflammation, wound repair, angiogenesis, regulation of the adaptive immune system and in maintaining homeostasis. Due to those characteristics AP could play an important role in many practical applications. Limited therapeutic efficiency of current antimicrobial agents and the emerging resistance of pathogens require alternate antimicrobial drugs. The purpose of this review is to highlight recent literature on functions and mechanisms of APs. It also shows their current practical applications as peptide therapeutics and bioactive polymers and discusses the possibilities of future clinical developments.
Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF®)) contain a variety of chemokines, cytokines, and growth factors and ...are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR). In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds.
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