scholarly journals Reduction of Matrix Metallopeptidase 13 and Promotion of Chondrogenesis by Zeel T in Primary Human Osteoarthritic Chondrocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Christelle Sanchez ◽  
Kathrin Hemmer ◽  
Natascha Krömmelbein ◽  
Bernd Seilheimer ◽  
Jean-Emile Dubuc ◽  
...  
Keyword(s):  
Differential Expression Analysis ◽  
Type Ii Collagen ◽  
Cartilage Degradation ◽  
Alginate Beads ◽  
Protein Quantification ◽  
Type Ii ◽  
Osteoarthritic Chondrocytes ◽  
Matrix Metallopeptidase ◽  
Oa Chondrocytes

Objectives: Zeel T (Ze14) is a multicomponent medicinal product. Initial preclinical data suggested a preventive effect on cartilage degradation. Clinical observational studies demonstrated that Ze14 reduced symptoms of osteoarthritis (OA), including stiffness and pain. This study aimed to explore these effects further to better understand the mode of action of Ze14 on human OA chondrocytes in vitro.Methods: Primary chondrocytes were obtained from the knees of 19 OA patients and cultured either as monolayers or in alginate beads. The cultures were treated with 20% or 10% (v/v) Ze14 or placebo. For RNA-seq, reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19). Differential expression analysis between Ze14 and placebo was performed in R using the DESeq2 package. Protein quantification by ELISA was performed on selected genes from the culture medium and/or the cellular fractions of primary human OA chondrocyte cultures.Results: In monolayer cultures, Ze14 20% (v/v) significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1, FOS, NR4A1, DUSP1, ZFP36, ZFP36L1, NFKBIZ, and CCN1 were upregulated and ATF7IP, TXNIP, DEPP1, CLEC3A, and MMP13 were downregulated after 24 h Ze14 treatment. Ze14 significantly increased (mean 2.3-fold after 24 h, p = 0.0444 and 72 h, p = 0.0239) the CCN1 protein production in human OA chondrocytes. After 72 h, Ze14 significantly increased type II collagen pro-peptide production by mean 27% (p = 0.0147). For both time points CCN1 production by OA chondrocytes was correlated with aggrecan (r = 0.66, p = 0.0004) and type II collagen pro-peptide (r = 0.64, p = 0.0008) production. In alginate beads cultures, pro-MMP-13 was decreased by Ze14 from day 7–14 (from −16 to −25%, p < 0.05) and from day 17–21 (−22%, p = 0.0331) in comparison to controls.Conclusion: Ze14 significantly modified the expression of DUSP1, DEPP1, ZFP36/ZFP36L1, and CLEC3A, which may reduce MMP13 expression and activation. Protein analysis confirmed that Ze14 significantly reduced the production of pro-MMP-13. As MMP-13 is involved in type II collagen degradation, Ze14 may limit cartilage degradation. Ze14 also promoted extracellular matrix formation arguably through CCN1 production, a growth factor well correlated with type II collagen and aggrecan production.

scholarly journals AB0072 A MULTICOMPONENT MEDICATION PROMOTES CHONDROGENESIS AND REDUCES MMP-13 IN PRIMARY ARTICULAR CHONDROCYTES FROM KNEE OSTEOARTHRITIS PATIENTS IN VITRO

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1336.1-1336
Author(s):  
C. Sanchez ◽  
K. Hemmer ◽  
N. Kroemmelbein ◽  
B. Seilheimer ◽  
J. E. Dubuc ◽  
...  
Keyword(s):  
Knee Osteoarthritis ◽  
Protein Production ◽  
Differential Expression Analysis ◽  
Rna Extraction ◽  
Type Ii Collagen ◽  
P Value ◽  
Type Ii ◽  
Regulatory Pathways ◽  
Oa Chondrocytes

Background:HE-1100 is a multicomponent medicinal product. Initial preclinical data potentially suggest a preventive effect on cartilage degradation.Objectives:This study aims to understand the mode of action of HE-1100 on OA chondrocytesin vitro.Methods:Primary chondrocytes were obtained from 10 knee osteoarthritis (OA) patients undergoing knee replacement surgery. The cultures were treated with 20% (v/v) HE-1100 or placebo. Samples were collected for subsequent RNA extraction using standard methods. The reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19) to generate the transcriptome. Differential expression analysis between HE-1100 and placebo was made in R using the DESeq2 package to identify the differentially expressed genes in the OA-associated regulatory pathways. The protein production of the selected genes was quantified by ELISA in 10 independent human OA chondrocytes cultures.Results:According to the DESeq2 analysis, HE-1100 significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1 (+93%), FOS (+87%), NR4A1 (+43%), DUSP1 (+18%), ZFP36 (+18%), ZFP36L1 (+14%), NFKBIZ (+16%) and CYR61 (+14%) were upregulated and ATF7IP (-10%), TXNIP (-11%), C10orf10 (-12%), CLEC3A (-12%) and MMP13 (-18%) were downregulated after 24h HE-1100 treatment. HE-1100 significantly increased (2.3 fold +/-1.2 after 24h, p=0.0444 and 2.3-fold +/-1.0 after 72h, p=0.0239) the CYR61 protein production by human OA chondrocytes. After 72h, HE-1100 slightly but not significantly increased aggrecan production by 14 ± 19 % (p=0.1117) and significantly increased type II collagen pro-peptide production by 27 ± 20 % (p=0.0147). For both time points CYR61 production by OA chondrocytes was positively and significantly correlated with aggrecan (r=0.66, p=0.0004) and type II collagen pro-peptide (r=0.64, p=0.0008) production. In alginate beads culture, pro-MMP-13 was significantly decreased by HE-1100 treated cultures from day 7 to day 14 (from -16 to -25 %, p<0.05) and from day 17 to 21 (-22 %, p=0.0331) in comparison to controls.Conclusion:HE-1100 significantly modified the expression of DUSP1, C10orf10, ZFP36/L1 and CLEC3A, which are pathway mediators involved in MMP-13 expression and activation. Further, long-term (28 days) treatment with HE-1100 significantly reduced the production of pro-MMP-13, the inactive precursor of the metalloproteinase MMP-13 involved in type II collagen degradation. HE-1100 also promoted extracellular matrix formation probably through CYR61 production, a growth factor well correlated with type II collagen and aggrecan production.References:/Acknowledgments:We would like to thank the staff of the GIGA ULiège Genomic Next Generation Sequencing platform for performing the RNA sequencing and Benoit Charloteaux for his help in RNAseq data analysis.Disclosure of Interests:christelle sanchez: None declared, Kathrin Hemmer Employee of: Heel, Natascha Kroemmelbein Employee of: Heel, Bernd Seilheimer Employee of: Heel, Jean-Emile Dubuc: None declared, Christophe Antoine Employee of: Artialis, Yves Henrotin Grant/research support from: HEEL, TILMAN

scholarly journals Irisin Recovers Osteoarthritic Chondrocytes In Vitro

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1478 ◽  
Author(s):  
Gianluca Vadalà ◽  
Giuseppina Di Giacomo ◽  
Luca Ambrosio ◽  
Francesca Cannata ◽  
Claudia Cicione ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Protein Quantification ◽  
Saline Control ◽  
Collagen Gene ◽  
Type Ii ◽  
Protein Levels ◽  
Collagen Gene Expression ◽  
Osteoarthritic Chondrocytes

Physical exercise favors weight loss and ameliorates articular pain and function in patients suffering from osteoarthritis. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types. This study aimed to investigate the effect of irisin on human osteoarthritic chondrocytes (hOAC) in vitro. Our hypothesis was that irisin would improve hOAC metabolism and proliferation. Cells were cultured in growing media and then exposed to either phosphate-buffered saline (control group) or human recombinant irisin (experimental group). Cell proliferation, glycosaminoglycan content, type II/X collagen gene expression and protein quantification as well as p38/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK), protein kinase B (Akt), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) involvement were evaluated. Furthermore, gene expression of interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 were investigated following irisin exposure. Irisin increased hOAC cell content and both type II collagen gene expression and protein levels, while decreased type X collagen gene expression and protein levels. Moreover, irisin decreased IL-1, IL-6, MMP-1, MMP-13 and iNOS gene expression, while increased TIMP-1 and TIMP-3 levels. These effects seemed to be mediated by inhibition of p38, Akt, JNK and NFκB signaling pathways. The present study suggested that irisin may stimulate hOAC proliferation and anabolism inhibiting catabolism through p38, Akt, JNK, and NFκB inactivation in vitro, demonstrating the existence of a cross-talk between muscle and cartilage.

scholarly journals The Role of MicroRNA-381 in Chondrogenesis and Interleukin-1-β Induced Chondrocyte Responses

2015 ◽  
Vol 36 (5) ◽  
pp. 1753-1766 ◽  
Author(s):  
Changhe Hou ◽  
Fangang Meng ◽  
Zhiqi Zhang ◽  
Yan Kang ◽  
Weishen Chen ◽  
...  
Keyword(s):  
Interleukin 1 ◽  
Type Ii Collagen ◽  
Cartilage Degeneration ◽  
Cartilage Degradation ◽  
Luciferase Assay ◽  
Type Ii ◽  
Osteoarthritic Cartilage ◽  
Enhanced Expression

Aim: The molecular pathways regulating cartilage degradation are unclear. miR-381 was identified as a putative regulator of chondrogenesis related genes. Here, we examined its role in chondrogenesis and osteoarthritic cartilage degeneration. Methods: miR-381 expression was assessed in vitro in response to IL-1β stimulation in primary human (PHC) and mouse (PMC) chondrocytes, and ATDC5 derived chondrocytes; and in vivo in mouse embryos and human osteoarthritic cartilage. The effects of miR-381 on chondrogenesis and NF-kB signaling were assessed using a synthetic RNA mimic or inhibitor and luciferase assay, respectively. Upstream regulators of miR381 were probed using siRNA or overexpression plasmids for Sox9 and Runx2. Results: miR-381 expression was elevated in chondrogenic and hypertrophic ATDC5 cells. miR-381 was induced in vitro by IL-1β in ATDC5 cells, PMCs, and PHCs, and was expressed in areas of cartilage degradation or absorption in vivo. Overexpression of Runx2 or Sox9 increased miR-381 expression in ATDC5 cells. miR-381 suppressed expression of collagen, type II, alpha 1, and enhanced expression of metalloproteinase-13 (MMP-13), but did not regulate NFKBIA and NKRF activity. Conclusion: miR-381 was highly expressed during chondrogenesis and in arthritic cartilage. It may contribute to absorption of the cartilage matrix by repressing type II collagen and inducing MMP-13.

scholarly journals GPDPLQ1237—A Type II Collagen Neo-Epitope Biomarker of Osteoclast- and Inflammation-Derived Cartilage Degradation in vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Henrik Löfvall ◽  
Anna Katri ◽  
Aneta Dąbrowska ◽  
Morten A. Karsdal ◽  
Yunyun Luo ◽  
...  
Keyword(s):  
Type Ii Collagen ◽  
Cartilage Degradation ◽  
Type Ii

scholarly journals Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ke Zhang ◽  
Zhuoying Li ◽  
Yunyang Lu ◽  
Linyi Xiang ◽  
Jiadong Sun ◽  
...  
Keyword(s):  
Western Blotting ◽  
Planar Cell Polarity ◽  
Type Ii Collagen ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Inflammatory Microenvironment ◽  
Functional Roles ◽  
Protein Levels ◽  
Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

Human Umbilical Cord Mesenchymal Stem Cells Induce into Chondrocytes in 3D Cultured System

2013 ◽  
Vol 749 ◽  
pp. 198-205
Author(s):  
Li Yu ◽  
Jing Liu ◽  
Chao Xu ◽  
Er Mei Luo ◽  
Ming Qiao Tang
Keyword(s):  
Real Time ◽  
Alcian Blue ◽  
Culture System ◽  
Type Ii Collagen ◽  
Human Umbilical Cord ◽  
Type Ii ◽  
Pellet Culture ◽  
Hla Dr

Objective: To investigate a better method of inducing hUC-MSCs into chondrocytes in different culture system in vitro. Method: hUC-MSCs were isolated and cultured by tissue block culture, and the cells surface antigens were identified by flow cytometry, hUC-MSCs were cultured with chondrogenic media and stained with Alcian Blue. The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method. Expressions of glycosaminoglycan (GAG), type II collagen and Sox-9 were assayed by real-time fluorescence quantitative PCR. Results: The cultured hUC-MSCs phenotype was CD105+/CD29+/CD44+/ CD31-/CD34-/ CD40-/CD45-/HLA-DR-. hUC-MSCs weakly expressed chondrocyte marker, which strongly expressed GAG and type II collagen after chondrogenic induction, and the cells were incubated in pellet culture with higher expression. Real-time PCR results demonstrated that chondrogenic induction cells were expressed GAG, type II collagen and Sox-9, and the cells were incubated in pellet culture with higher expression. Conclusion: hUC-MSCs incubated in pellet culture is more conducive to differentiate into chondrocytes than those cultured in monolayer culture system.

Type VI collagen expression is upregulated in the early events of chondrocyte differentiation

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 245-251
Author(s):  
R. Quarto ◽  
B. Dozin ◽  
P. Bonaldo ◽  
R. Cancedda ◽  
A. Colombatti
Keyword(s):  
Suspension Culture ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Type Vi Collagen ◽  
Full Spectrum ◽  
Collagen Expression ◽  
Hypertrophic Chondrocyte

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: From in vitro to in vivo

2007 ◽  
Vol 361 (1) ◽  
pp. 93-101 ◽  
Author(s):  
O.V. Nemirovskiy ◽  
D.R. Dufield ◽  
T. Sunyer ◽  
P. Aggarwal ◽  
D.J. Welsch ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Metalloproteinase Activity

scholarly journals Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

2021 ◽  
Vol 22 (21) ◽  
pp. 12012
Author(s):  
Manuela Minguzzi ◽  
Veronica Panichi ◽  
Stefania D’Adamo ◽  
Silvia Cetrullo ◽  
Luca Cattini ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Terminal Differentiation ◽  
Loss Of Function ◽  
Notch Receptors ◽  
Osteoarthritic Chondrocytes ◽  
Gene And Protein Expression ◽  
Luminescence Assay ◽  
Culture Models ◽  
Oa Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

scholarly journals Extracellular Nucleotides Selectively Induce Migration of Chondrocytes and Expression of Type II Collagen

2020 ◽  
Vol 21 (15) ◽  
pp. 5227
Author(s):  
Marcin Szustak ◽  
Edyta Gendaszewska-Darmach
Keyword(s):  
Migration Rate ◽  
Monolayer Culture ◽  
Type Ii Collagen ◽  
Extracellular Nucleotides ◽  
Nonbridging Oxygen ◽  
Nucleotide Receptors ◽  
Type Ii ◽  
Mrna Transcripts ◽  
Chondrogenic Phenotype

The migration of chondrocytes from healthy to injured tissues is one of the most important challenges during cartilage repair. Additionally, maintenance of the chondrogenic phenotype remains another limitation, especially during monolayer culture in vitro. Using both the differentiated and undifferentiated chondrogenic ATDC5 cell line, we showed that extracellular nucleotides are able to increase the migration rate of chondrocytes without affecting their chondrogenic phenotype. We checked the potency of natural nucleotides (ATP, ADP, UTP, and UDP) as well as their stable phosphorothioate analogs, containing a sulfur atom in the place of one nonbridging oxygen atom in a phosphate group. We also detected P2y1, P2y2, P2y4, P2y6, P2y12, P2y13, and P2y14 mRNA transcripts for nucleotide receptors, demonstrating that P2y1 and P2y13 are highly upregulated in differentiated ATDC5 cells. We showed that ADPβS, UDPβS, and ADP are the best stimulators of migration of differentiated chondrocytes. Additionally, ADP and ADPβS positively affected the expression of type II collagen, a structural component of the cartilage matrix.

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