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.

Persistence of Collagen Type II Synthesis and Secretion in Rapidly Proliferating Human Articular Chondrocytes In Vitro

2008 ◽  
Vol 14 (12) ◽  
pp. 1999-2007 ◽  
Author(s):  
Aboulghassem Shahdadfar ◽  
Sverre Løken ◽  
John Arne Dahl ◽  
Siv H. Tunheim ◽  
Philippe Collas ◽  
...  
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Type Ii ◽  
Collagen Type Ii

scholarly journals 9.7 Persistent collagen type II synthesis and secretion in rapidly proliferating human articular chondrocytes in vitro

2007 ◽  
Vol 15 ◽  
pp. B67
Author(s):  
A. Shahdadfar ◽  
S. Løken ◽  
S.H. Tunheim ◽  
J.B. Eriksen ◽  
J.A. Dahl ◽  
...  
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Type Ii ◽  
Collagen Type Ii

Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity

2007 ◽  
Vol 20 (03) ◽  
pp. 185-191 ◽  
Author(s):  
A. O. Oshin ◽  
E. Caporali ◽  
C. R. Byron ◽  
A. A. Stewart ◽  
M. C. Stewart
Keyword(s):  
Articular Cartilage ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Soluble Form ◽  
Mrna Levels ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Endogenous Expression ◽  
Chondrocytic Phenotype

SummaryArticular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.

Androgen regulation and site specificity of angiotensinogen gene expression and secretion in rat adipocytes

2000 ◽  
Vol 279 (6) ◽  
pp. E1398-E1405 ◽  
Author(s):  
Valérie Serazin-Leroy ◽  
Mireille Morot ◽  
Philippe de Mazancourt ◽  
Yves Giudicelli
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Mrna Expression ◽  
Protein Secretion ◽  
Male Rats ◽  
Fat Cells ◽  
Intact Male ◽  
Rat Adipocytes ◽  
Fat Deposits

Adipose tissue is an important source of angiotensinogen (ATG), and hypertension is commonly associated with android obesity. Therefore, we tested the hypothesis that androgens may control ATG gene expression and secretion in rat fat cells. In intact male rats, ATG mRNA expression (Northern blot and co-reverse transcription-polymerase chain reaction analysis) and protein secretion were significantly higher in deep intra-abdominal (perirenal and epididymal) than in subcutaneous adipocytes. After castration, ATG mRNA was reduced almost 50% in the three fat deposits, with parallel changes in ATG protein secretion. Conversely, testosterone treatment fully restored the ATG mRNA decrease after castration, whatever the anatomical origin of the adipocytes. Finally, a 24-h in vitro exposure of perirenal fat cells or differentiated preadipocytes from castrated rats to testosterone or dihydrotestosterone (10 nM free hormone concentration) increased ATG mRNA expression by 50–100%, an effect that was prevented by the anti-androgen cyproterone acetate. These data, demonstrating both in vivo and in vitro androgen induction of ATG mRNA expression in rat adipocytes, add further weight to the hypothesis of a link between adipose tissue ATG production, androgens, and android obesity-related hypertension.

scholarly journals Maintenance of chondrocyte phenotype during expansion on PLLA microtopographies

2018 ◽  
Vol 9 ◽  
pp. 204173141878982 ◽  
Author(s):  
Elisa Costa ◽  
Cristina González-García ◽  
José Luis Gómez Ribelles ◽  
Manuel Salmerón-Sánchez
Keyword(s):  
Lactic Acid ◽  
Type I Collagen ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Cell Phenotype ◽  
Type I ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Chondrocyte Phenotype

Articular chondrocytes are difficult to grow, as they lose their characteristic phenotype following expansion on standard tissue culture plates. Here, we show that culturing them on surfaces of poly(L-lactic acid) of well-defined microtopography allows expansion and maintenance of characteristic chondrogenic markers. We investigated the dynamics of human chondrocyte dedifferentiation on the different poly(L-lactic acid) microtopographies by the expression of collagen type I, collagen type II and aggrecan at different culture times. When seeded on poly(L-lactic acid), chondrocytes maintained their characteristic hyaline phenotype up to 7 days, which allowed to expand the initial cell population approximately six times without cell dedifferentiation. Maintenance of cell phenotype was afterwards correlated to cell adhesion on the different substrates. Chondrocytes adhesion occurs via the α5 β1 integrin on poly(L-lactic acid), suggesting cell–fibronectin interactions. However, α2 β1 integrin is mainly expressed on the control substrate after 1 day of culture, and the characteristic chondrocytic markers are lost (collagen type II expression is overcome by the synthesis of collagen type I). Expanding chondrocytes on poly(L-lactic acid) might be an effective solution to prevent dedifferentiation and improving the number of cells needed for autologous chondrocyte transplantation.

scholarly journals Expression of the Heparan Sulfate Proteoglycan, Perlecan, during Mouse Embryogenesis and Perlecan Chondrogenic Activity In Vitro

1999 ◽  
Vol 145 (5) ◽  
pp. 1103-1115 ◽  
Author(s):  
M.M. French ◽  
S.E. Smith ◽  
K. Akanbi ◽  
T. Sanford ◽  
J. Hecht ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Collagen Type ◽  
Heparan Sulfate Proteoglycan ◽  
Surrounding Tissue ◽  
Collagen Type Iv ◽  
Type Ii ◽  
Sulfate Proteoglycan ◽  
Collagen Type Ii ◽  
Murine Embryonic Fibroblast

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

2013 ◽  
Vol 19 (1-2) ◽  
pp. 59-65 ◽  
Author(s):  
Marijn Rutgers ◽  
Daniel B. Saris ◽  
Lucienne A. Vonk ◽  
Mattie H. van Rijen ◽  
Vanessa Akrum ◽  
...  
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Human Articular Chondrocytes ◽  
Type I ◽  
Type Ii
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