scholarly journals Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system.

1991 ◽  
Vol 112 (3) ◽  
pp. 501-513 ◽  
Author(s):  
L C Gerstenfeld ◽  
W J Landis
Keyword(s):  
Extracellular Matrix ◽  
Culture System ◽  
Time Course ◽  
Collagen Synthesis ◽  
Collagen Type ◽  
Fold Increase ◽  
Proteoglycan Synthesis ◽  
Type Ii ◽  
Collagen Types ◽  
Matrix Mineralization

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Time course of gene expression in rat experimental autoimmune myocarditis

2002 ◽  
Vol 103 (6) ◽  
pp. 623-632 ◽  
Author(s):  
Haruo HANAWA ◽  
Satoru ABE ◽  
Manabu HAYASHI ◽  
Tsuyoshi YOSHIDA ◽  
Kaori YOSHIDA ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Time Course ◽  
Collagen Type ◽  
Fold Increase ◽  
Cardiac Myosin ◽  
Autoimmune Myocarditis ◽  
Experimental Autoimmune Myocarditis ◽  
Experimental Autoimmune

Genetic responses that characterize experimental autoimmune myocarditis (EAM) have not yet been determined. To investigate gene expression in the myocardium of EAM, absolute copy numbers of 44 mRNA species [calcium-handling proteins, contractile proteins, natriuretic peptides (NPs), cytokines, chemokines, growth factors, renin–angiotensin–aldosterone (RAA) system, endothelins (ETs) and extracellular matrix] in synthesized cDNA from a fixed quantity of total heart RNA were assessed using real-time reverse-transcriptase PCR at days 0, 14, 21 and 28 after immunization. α-Cardiac myosin showed a 26.3-fold decrease and β-cardiac myosin a 3.75-fold increase at day 14. Atrial NP and brain NP increased 47.7- and 6.35-fold at days 21 and 14 respectively. Angiotensin II type 1 receptor, angiotensin-converting enzyme and ET1 increased 22.3-fold at day 21, 6.30-fold at day 21 and 16.8-fold at day 14 respectively. Aldosterone receptor decreased 2.15-fold at day 14, but aldosterone synthetase was detected only at days 14 and 21. Interleukin (IL)-2, IL-10, interferon-γ and monocyte chemo-attractant protein-1 increased 9.08-fold at day 14, 398-fold at day 21, 43.1-fold at day 14 and 142-fold at day 14 respectively. Collagen type 3, collagen type 1 and fibronectin increased 34.6-, 1.74- and 44.4-fold respectively at day 21. Interestingly, osteopontin showed a 4540-fold increase and it was the highest mRNA of all at day 14. An isoform of cardiac myosin and NP are dramatically changed in EAM. RAA system and ET expressions are changed differently during the EAM time course. Cytokine, chemokine and extracellular matrix greatly increase and, in particular, large numbers of osteopontin mRNA are expressed in early EAM.

Independent deposition of collagen types II and IX at epithelial-mesenchymal interfaces

Development ◽  
1989 ◽  
Vol 105 (1) ◽  
pp. 85-95 ◽  
Author(s):  
J.M. Fitch ◽  
A. Mentzer ◽  
R. Mayne ◽  
T.F. Linsenmayer
Keyword(s):  
Collagen Type ◽  
Immunohistochemical Study ◽  
Type Ii Collagen ◽  
The Other ◽  
Extracellular Matrices ◽  
Type Ii ◽  
Collagen Deposition ◽  
Collagen Types ◽  
Hind Limbs ◽  
The Matrix

Previous studies have demonstrated the presence of type II collagen (in mature chickens predominantly a ‘cartilage-specific’ collagen) in a variety of embryonic extracellular matrices that separate epithelia from mesenchyme. In an immunohistochemical study using collagen type-specific monoclonal antibodies, we asked whether type IX collagen, another ‘cartilage-specific’ collagen, is coexpressed along with type II at such interfaces. We confirmed that, in the matrix underlying a variety of cranial ectodermal derivatives and along the ventrolateral surfaces of neuroepithelia, type II collagen is codistributed with collagen types I and IV. Type IX collagen, however, was undetectable at those sites. We observed immunoreactivity for type IX collagen only within the notochordal sheath, where it first appeared at a later stage than did collagen types I and II. We also observed type II collagen (without type IX) beneath the dorsolateral ectoderm at stage 16; this correlates with the period during which limb ectoderm has been reported to induce the mesoderm to become chondrogenic. Finally, in older hind limbs we observed subepithelial type II collagen that was not associated with subsequent chondrogenesis, but appeared to parallel the formation of feathers and scales in the developing limb. These observations suggest that the deposition of collagen types II and IX into interfacial matrices is regulated independently, and that induction of mesenchymal chondrogenesis by such matrices does not involve type IX collagen. Subepithelial type IX collagen deposition, on the other hand, correlates with the assembly of a thick multilaminar fibrillar matrix, as present in the notochordal sheath and, as shown previously, in the corneal primary stroma.

scholarly journals Modeling appendicular skeletal cartilage development with modified high-density micromass cultures of adult human bone marrow-derived mesenchymal progenitor cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Alessandro Pirosa ◽  
Karen L. Clark ◽  
Jian Tan ◽  
Shuting Yu ◽  
Yuanheng Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Culture System ◽  
Collagen Type ◽  
Micromass Culture ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Mesenchymal Progenitor Cells ◽  
Environmental Toxicity ◽  
Gfp Reporter

Abstract Background Animal cell-based systems have been critical tools in understanding tissue development and physiology, but they are less successful in more practical tasks, such as predicting human toxicity to pharmacological or environmental factors, in which the congruence between in vitro and clinical outcomes lies on average between 50 and 60%. Emblematic of this problem is the high-density micromass culture of embryonic limb bud mesenchymal cells, derived from chick, mouse, or rat. While estimated predictive value of this model system in toxicological studies is relatively high, important failures prevent its use by international regulatory agencies for toxicity testing and policy development. A likely underlying reason for the poor predictive capacity of animal-based culture models is the small but significant physiological differences between species. This deficiency has inspired investigators to develop more organotypic, 3-dimensional culture system using human cells to model normal tissue development and physiology and assess pharmacological and environmental toxicity. Methods We have developed a modified, miniaturized micromass culture model using adult human bone marrow-derived mesenchymal progenitor cells (hBM-MPCs) that is amenable to moderate throughput and high content analysis to study chondrogenesis. The number of cells per culture was reduced, and a methacrylated gelatin (gelMA) overlay was incorporated to normalize the morphology of the cultures. Results These modified human cell-based micromass cultures demonstrated robust chondrogenesis, indicated by increased Alcian blue staining and immunodetectable production of collagen type II and aggrecan, and stage-specific chondrogenic gene expression. In addition, in cultures of hBM-MPCs transduced with a lentiviral collagen type II promoter-driven GFP reporter construct, levels of GFP reporter activity correlated well with changes in endogenous collagen type II transcript levels, indicating the feasibility of non-invasive monitoring of chondrogenesis. Conclusions The modified hBM-MPC micromass culture system described here represents a reproducible and controlled model for analyzing mechanisms of human skeletal development that may later be applied to pharmacological and environmental toxicity studies.

Antibodies to Collagen Types I–VI in Dupuytren’s Contracture

1986 ◽  
Vol 11 (1) ◽  
pp. 58-60
Author(s):  
R. S. PEREIRA ◽  
C. M. BLACK ◽  
S. M. TURNER ◽  
J. D. SPENCER
Keyword(s):  
Patient Group ◽  
Blood Donor ◽  
Collagen Type ◽  
Type Ii Collagen ◽  
Dupuytren’S Contracture ◽  
Type Ii ◽  
Collagen Types ◽  
Igg Antibody ◽  
Dupuytren's Contracture ◽  
Human Collagen

Sera from 16 patients with Dupuytren’s contracture were tested for IgG and IgM antibodies to native and denatured human collagen types I, II, III, IV, V and VI. IgG antibody to at least one collagen type was found in 11/16 (69%) of these patients, compared with 27/96 (28%) normal adult blood donor controls. The prevalence of antibody to denatured type II collagen was raised, and although there was no overall increase in HLA-DR4 compared with a control population, this antibody was associated with HLA-DR4 in this patient group.

scholarly journals Enterococcus faecalis Adhesin, Ace, Mediates Attachment to Extracellular Matrix Proteins Collagen Type IV and Laminin as well as Collagen Type I

2000 ◽  
Vol 68 (9) ◽  
pp. 5218-5224 ◽  
Author(s):  
Sreedhar R. Nallapareddy ◽  
Xiang Qin ◽  
George M. Weinstock ◽  
Magnus Höök ◽  
Barbara E. Murray
Keyword(s):  
Extracellular Matrix ◽  
Enterococcus Faecalis ◽  
Collagen Type ◽  
Collagen Type I ◽  
Immune Serum ◽  
Collagen Type Iv ◽  
Type I ◽  
Collagen Types ◽  
Type Iv ◽  
Ecm Proteins

ABSTRACT Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ∼105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF ace mutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 μg/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.

scholarly journals Comparison of the effects of triamcinolone acetonide or platelet-rich plasma on expression of extracellular matrix-related genes in equine healthy chondrocytes in vitro

2019 ◽  
Vol 49 (7) ◽  
Author(s):  
Heloisa Einloft Palma ◽  
Miguel Gallio ◽  
Gabriele Biavaschi da Silva ◽  
Camila Cantarelli ◽  
Kalyne Bertolin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Collagen Synthesis ◽  
Platelet Rich Plasma ◽  
Joint Damage ◽  
Type Ii Collagen ◽  
Control Group ◽  
Type Ii ◽  
Healthy Cartilage ◽  
Pro Inflammatory Cytokines

ABSTRACT: In healthy cartilage, chondrocytes maintain an expression of collagens and proteoglycans and are sensitive to growth factors and cytokines that either enhance or reduce type II collagen synthesis. In osteoarthritis, pro-inflammatory cytokines, such as IL-6, induce overexpression of metalloproteinases (MMP) and decreasing synthesis of aggrecan. Use of chondroprotectors agents, such as Platelet-Rich Plasma (PRP) and triamcinolone (TA) are alternatives to reduce the progression of joint damage. In this study, we used chondrocytes extracted from metacarpophalangeal joints of healthy horses as the experimental model. Cells were treated in vitro with PRP or TA. No differences were observed between these treatments in comparison to the control group when the expressions of MMP9, MMP13, IL-6 and ACAN genes were evaluated (P<0.05). With these results, we can suggest that the treatments were not deleterious to equine cultured chondrocyte, once they did not stimulate MMPs and IL-6 synthesis or caused changes in ACAN.

scholarly journals Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes

2010 ◽  
Vol 15 (4) ◽  
Author(s):  
Kristin Schram ◽  
Sabrina Girolamo ◽  
Siham Madani ◽  
Diana Munoz ◽  
Farah Thong ◽  
...  
Keyword(s):  
Heart Failure ◽  
Extracellular Matrix ◽  
P38 Mapk ◽  
Collagen Synthesis ◽  
Collagen Type ◽  
Mapk Signaling ◽  
Conditioned Media ◽  
Type I ◽  
Dependent Manner ◽  
Extracellular Matrix Components

AbstractA clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.

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