scholarly journals Effect of PEP-1-GLRX-1 on catabolic gene expression in human articular chondrocytes and in mouse carrageenan-induced PAW edema model

2016 ◽  
Vol 24 ◽  
pp. S385
Author(s):  
I. Park ◽  
H. Hwang ◽  
S. Choi ◽  
D. Kim ◽  
H. Kim
Keyword(s):  
Gene Expression ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Catabolic Gene ◽  
Paw Edema ◽  
Edema Model

scholarly journals PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes

2016 ◽  
Vol 41 (1) ◽  
pp. 252-264 ◽  
Author(s):  
Hyun Sook Hwang ◽  
In Young Park ◽  
Hyun Ah Kim ◽  
Soo Young Choi
Keyword(s):  
Nitric Oxide ◽  
Western Blot ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
No Production ◽  
Paw Edema ◽  
Edema Model

Background: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. Methods: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. Results: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. Conclusion: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

scholarly journals The effect of interleukin-1 on cytokine gene expression in cultured human articular chondrocytes analyzed by messenger rna phenotyping

1993 ◽  
Vol 36 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Janet M. Seid ◽  
Shamim Rahman ◽  
Robert Graveley ◽  
Rowena A. D. Bunning ◽  
Rene Nordmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Articular Chondrocytes ◽  
Interleukin 1 ◽  
Human Articular Chondrocytes ◽  
Cytokine Gene Expression ◽  
Cytokine Gene

scholarly journals Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity

2019 ◽  
Vol 20 (4) ◽  
pp. 795 ◽  
Author(s):  
Ufuk Tan Timur ◽  
Marjolein Caron ◽  
Guus van den Akker ◽  
Anna van der Windt ◽  
Jenny Visser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Protein Secretion ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Reporter Assay

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

scholarly journals Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes

2002 ◽  
Vol 46 (8) ◽  
pp. 2095-2104 ◽  
Author(s):  
Michael Ulrich-Vinther ◽  
Michael D. Maloney ◽  
J. Jeffrey Goater ◽  
Kjeld Søballe ◽  
Mary B. Goldring ◽  
...  
Keyword(s):  
Gene Expression ◽  
Articular Chondrocytes ◽  
Virus Vector ◽  
Human Articular Chondrocytes ◽  
Gene Transduction ◽  
Adeno Associated Virus

scholarly journals Effect of Inflammatory Signaling on Human Articular Chondrocyte Hypertrophy: Potential Involvement of Tissue Repair Macrophages

Cartilage ◽  
2021 ◽  
pp. 194760352110219
Author(s):  
Mauricio N. Ferrao Blanco ◽  
Yvonne M. Bastiaansen-Jenniskens ◽  
Mark G. Chambers ◽  
Andrew A. Pitsillides ◽  
Roberto Narcisi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Cytokines ◽  
Tissue Repair ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Human Chondrocytes ◽  
Articular Chondrocyte ◽  
Inflammatory Signaling ◽  
Chondrocyte Hypertrophy ◽  
Pro Inflammatory Cytokines

Objective In osteoarthritis, chondrocytes tend to acquire a hypertrophic phenotype, which contributes to the modification of the extracellular matrix, resulting in permanent cartilage changes. In mouse chondrocytes, pro-inflammatory macrophages and pro-inflammatory cytokines have been shown to stimulate hypertrophy via the activation of the nuclear factor kappa B (NF-κB) pathway. Whether or not this also occurs in human chondrocytes remains unclear. We therefore aimed to investigate whether hypertrophy-like responses in human cartilage are driven mainly by intrinsic inflammatory signaling or shaped by specific macrophage populations. Design Human articular chondrocytes were cultured with pro-inflammatory cytokines or medium conditioned by defined macrophage subsets. Furthermore, the effect of inhibition of NF-κB-dependent gene expression was evaluated using the NF-κB inhibitor SC-514. Hypertrophy was assessed by measuring the transcription level of alkaline phosphatase ( ALPL), type X collagen ( COL10A1), Indian hedgehog ( IHH), and runt-related transcription factor 2 ( RUNX2). Results The expression of hypertrophic genes was not promoted in human chondrocytes by pro-inflammatory cytokines neither pro-inflammatory M(IFNγ + TNFα) macrophages. Inhibition of the NF-κB-dependent gene expression did not affect human articular chondrocyte hypertrophy. However, tissue repair M(IL4) macrophages induced hypertrophy by promoting the expression of COL10A1, RUNX2, and IHH. Conclusion Intrinsic inflammatory signaling activation is not involved in the hypertrophic shift observed in human articular chondrocytes cultured in vitro. However, tissue repair macrophages may contribute to the onset of this detrimental phenotype in human osteoarthritic cartilage, given the effect observed in our experimental models.

scholarly journals AB0036 APPA (APOCYNIN AND PAEONOL) REDUCES ROS PRODUCTION AND SENESCENCE IN HUMAN ARTICULAR CHONDROCYTES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1051.1-1051
Author(s):  
M. Fernandez-Moreno ◽  
N. Larkins ◽  
A. Reynolds ◽  
T. Hermida Gómez ◽  
F. J. Blanco
Keyword(s):  
Gene Expression ◽  
Research And Development ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Ros Production ◽  
Tnf Α ◽  
Inflammatory Stimuli ◽  
And Cartilage ◽  
Pro Inflammatory Cytokines ◽  
Inflammatory Effect

Background:Disease modification is not yet possible for osteoarthritis (OA). Mitochondrial ROS and pro-inflammatory cytokines are involved in the pathogenesis of OA and are potential therapeutic targets. APPA, a combination of apocynin (AP) and paeonol (PA), has the potential capacity to modulate synthesis of pro-inflammatory stimuli.Objectives:To investigate the anti-inflammatory effect of APPA in human articular chondrocytes and cartilage.Methods:Tissue and chondrocytes from human OA cartilage were isolated. The effect of APPA on chondrocyte viability was analyzed using MTT. IL-1β 10 ng/mL and LPS 10 ng/mL were used as pro-inflammatory stimuli. ROS production was evaluated by flow cytometry using DCFH-DA and MitoSoxRed. The percentage of senescent cells was evaluated through the quantification of Fluorescein di-β-D-galactopyranoside (FDG) by flow cytometry. The effect of APPA on gene expression of pro-inflammatory cytokines (IL-8 and TNF-α) and enzymes degrading cartilage (MMP-13 and MMP-3) were analyzed in chondrocyte and cartilage by RT-PCR. Quantification of Toluidine Blue (TB) staining in cartilage was performed to evaluate proteoglycans content using software ImageJ/Fiji. Release of Glycosaminoglycan (GAGs) into the supernatant was quantified using BlyscanTM Glycosaminoglycan assay. Statistical analyses were performed with GraphPad Prism v6.Results:Chondrocytes, incubated in presence of APPA 10 µg/mL for 24 h had viability >85%, reduced cytoplasmic ROS (p=0.028) and mitochondrial anion superoxide production induced by LPS 10 ng/mL (p=0.057). Chondrocytes incubated in presence of APPA 10 µg/mL for 2 hours contained significantly fewer senescent cells (p=0.0079). APPA significantly reduced the gene expression induced by IL-1β 10 ng/mL in chondrocytes of IL-8, TNF-α, MMP-13 and MMP-3. Cartilage incubated with APPA 60 and 100 µg/mL for 48 h showed decreased the MMP-3 gene expression induced by IL-1β (p=0.021 and p<0.0001 respectively). Quantification of TB showed that APPA 60 and 100 µg/mL during 48h increased the proteoglycans in intermedial layer, which had been decreased through the incubation with IL-1β (p=0.0018 and p=0.018 respectively). Quantification of release GAGs into the supernatant decreased significantly when the cartilage explants were incubated for 48h in presence of APPA 100 µg/mL (p=0.028).Conclusion:APPA has a clear anti-inflammatory effect on human articular chondrocytes, and could reduce extracellular matrix degradation of cartilage. This could be mediated by the capacity to modulate ROS production and reduce senescence.Disclosure of Interests:Mercedes Fernandez-Moreno: None declared, Nicholas Larkins Shareholder of: I am a shareholder in AKL Research and Development Ltd, Alan Reynolds Shareholder of: I have share options in AKL Research and Development Ltd, Speakers bureau: I have not been a paid speaker for a pharma company - at least not since 2008 whichI think is outside the scope of this, Consultant of: The last time I was a paid consultant was in 2017 when I acted as a consultant for Avillion and Norgine, Employee of: I am also an employee of AKL Research and Development Ltd, Tamara Hermida Gómez: None declared, Francisco J. Blanco Speakers bureau: LillyPfizerSanofiGalapagos, Consultant of: LillyPfizerSanofiGalapagos, Grant/research support from: LillyMSDMerck SeronoPfizerPierre-FabraRocheSanofiServierUCBAbbvieAmgenBioibericaBristol MayerCelgeneCelltrionCellerixGrunenthalGebro PharmaAKL Research and Development Ltd

SOX9 transduction increases chondroitin sulfate synthesis in cultured human articular chondrocytes without altering glycosyltransferase and sulfotransferase transcription

2008 ◽  
Vol 414 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Simon R. Tew ◽  
Peraphan Pothacharoen ◽  
Theoni Katopodi ◽  
Timothy E. Hardingham
Keyword(s):  
Gene Expression ◽  
Chondroitin Sulfate ◽  
Monolayer Culture ◽  
Articular Chondrocytes ◽  
High Mobility ◽  
Protein Translation ◽  
Dermal Fibroblasts ◽  
Human Articular Chondrocytes ◽  
Total Capacity ◽  
Synthetic Capacity

The transcription factor SOX9 (Sry-type high-mobility-group box 9) is expressed in all chondrocytes and is essential for the expression of aggrecan, which during biosynthesis is substituted with more than 10 times its weight of CS (chondroitin sulfate) and is secreted by chondrocytes to form the characteristic GAG (glycosaminoglycan)-rich ECM (extracellular matrix) of cartilage. SOX9 expression rapidly falls during monolayer culture of isolated chondrocytes and this turns off aggrecan and associated CS synthesis. We therefore investigated whether SOX9 transduction of cultured human articular chondrocytes had any effect on the gene expression of the glycosyltransferases and sulfotransferases necessary for GAG biosynthesis. Retroviral SOX9 transduction of passaged chondrocytes increased the endogenous rate of GAG synthesis and the total capacity for GAG synthesis assessed in monolayer culture with β-xyloside. Both the endogenous rate and the total capacity of GAG biosynthesis were increased further in chondrogenic cell aggregate cultures. The GAG synthesized was predominantly CS and the hydrodynamic size of the newly synthesized chains was unchanged by SOX9 transduction. Aggrecan gene expression was increased in the SOX9-transduced chondrocytes and increased further in chondrogenic culture, but no comparable effects were found in SOX9 transduced dermal fibroblasts. However, the expression of CS glycosyltransferase and sulfotransferase genes in chondrocytes was unaffected by SOX9 transduction. Therefore SOX9 transduction in chondrocytes increased their CS synthetic capacity, but this was not accompanied by changes in the transcription of the CS biosynthetic enzymes and must occur by indirect regulation of enzyme activity through control of enzyme protein translation or enzyme organization.

Sign in / Sign up

Export Citation Format

Share Document