scholarly journals Collagenolytic Potential of Rat Liver Myofibroblasts

2013 ◽  
pp. 15-25 ◽  
Author(s):  
A. JIROUTOVÁ ◽  
E. PETEROVÁ ◽  
L. BITTNEROVÁ ◽  
R. SLAVKOVSKÝ ◽  
P. ČEVELOVÁ ◽  
...  
Keyword(s):  
Rat Liver ◽  
Type I Collagen ◽  
Collagen Gel ◽  
Plastic Substrate ◽  
Type I ◽  
Collagen Gels ◽  
Rt Pcr ◽  
Fibrotic Liver ◽  
Fibrin Gels ◽  
Liver Myofibroblasts

Rat liver myofibroblasts (MFB) were isolated by repeated passaging of nonparenchymal liver cell fraction. They were cultured on polystyrene Petri dishes, on fibrin or on type I collagen gels for 5 days. Quantitative RT-PCR, Western blotting, zymography and immunocytochemistry were used to study differences in cell morphology and protein expression. MFB were large and spread on plastic substrate, with prominent α-smooth muscle (α-SMA) fibres. They turned much smaller and elongated on collagen which was accompanied by the rearrangement of the cytoskeleton and a decrease in α-SMA and β-actin content. Collagen gel induced the expression of a group of metalloproteinases (MMP-2, -3, -9, -13), on mRNA and protein level which resulted in the degradation of the gel. This response was accompanied by changes in the mRNA expression of cytokines of TGF-β family, CTGF and interleukin-6, as well as of osteopontin and thrombospondin-2 that are involved in metalloproteinases (MMPs) regulation. The expression of MMPs substrates, collagen types I, IV and XII did not change or decreased. The effects of fibrin gels on MFB were milder than those of collagen. MFB assumed to deposit collagen and other ECM components in fibrotic liver, besides hepatic stellate cells, also possess a great collagenolytic potential.

scholarly journals EFFECT OF COLLAGEN I GEL ON APOPTOSIS OF RAT HEPATIC STELLATE CELLS

2013 ◽  
Vol 56 (2) ◽  
pp. 73-79
Author(s):  
Lenka Bittnerová ◽  
Alena Jiroutová ◽  
Emil Rudolf ◽  
Martina Řezáčová ◽  
Jiří Kanta
Keyword(s):  
Hepatic Stellate Cells ◽  
Type I Collagen ◽  
Collagen Gel ◽  
Stellate Cells ◽  
Type I ◽  
Function Type ◽  
Fibrotic Liver ◽  
Normal Rat ◽  
And Function

Activated hepatic stellate cells (HSC) are a major source of fibrous proteins in cirrhotic liver. Inducing or accelerating their apoptosis is a potential way of liver fibrosis treatment. Extracellular matrix (ECM) surrounding cells in tissue affects their differentiation, migration, proliferation and function. Type I collagen is the main ECM component in fibrotic liver. We have examined how this protein modifies apoptosis of normal rat HSC induced by gliotoxin, cycloheximide and cytochalasin D in vitro and spontaneous apoptosis of HSC isolated from CCl4-damaged liver. We have found that type I collagen gel enhances HSC apoptosis regardless of the agent triggering this process.

Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 605-622 ◽  
Author(s):  
G. Greenburg ◽  
E.D. Hay
Keyword(s):  
Type I Collagen ◽  
Collagen Gel ◽  
Type I ◽  
Basal Cells ◽  
Collagen Gels ◽  
Expression Change ◽  
Cell Functions ◽  
The Matrix ◽  
3D Matrix ◽  
Mesenchymal Transformation

In considering the mechanism of transformation of epithelium to mesenchyme in the embryo, it is generally assumed that the ability to give rise to fibroblast-like cells is lost as epithelia mature. We reported previously that a definitive embryonic epithelium, that of the anterior lens, gives rise to freely migrating mesenchyme-like cells when suspended in type I collagen matrices. Here, we show that a highly differentiated epithelium that expresses cytokeratin changes to a vimentin cytoskeleton and loses thyroglobulin during epithelial-mesenchymal transformation induced by suspension in collagen gel. Using dispase and collagenase, we isolated adult thyroid follicles devoid of basal lamina and mesenchyme, and we suspended the follicles in 3D collagen gels. Cells bordering the follicle lumen retain epithelial polarity and thyroid phenotype, but basal cell surface organization is soon modified as a result of tissue multilayering and elongation of basal cells into the collagenous matrix. Cytodifferentiation, determined by thyroglobulin immunoreactivity, is lost as the basal epithelial cells move into the matrix after 3–4 days in collagen. By TEM, it can be seen that the elongating cells acquire pseudopodia, filopodia and mesenchyme-like nuclei and RER. Immunofluorescence examination of intermediate filaments showed that freshly isolated follicles and follicles cultured on planar substrata react only with anticytokeratin. However, all of the mesenchyme-like cells express vimentin and they gradually lose cytokeratin. These results suggest that vimentin may be necessary for cell functions associated with migration within a 3D matrix. The mesenchymal cells do not revert to epithelium when grown on planar substrata and the transformation of epithelium to mesenchyme-like cells does not occur within basement membrane gels. The results are relevant to our understanding of the initiation of epithelial-mesenchymal transformation in the embryo and the genetic mechanisms controlling cell shape, polarity and cytoskeletal phenotype.

Stress-Dependent Effects of Platelet-Derived Growth Factor-BB on Fibroblast Migration and Traction Differ in Collagen and Fibrin

2000 ◽  
Author(s):  
David I. Shreiber ◽  
Paul A. J. Enever ◽  
Robert T. Tranquillo
Keyword(s):  
Type I Collagen ◽  
Cell Behavior ◽  
Environmental Cues ◽  
Type I ◽  
Collagen Gels ◽  
Fibrin Gels ◽  
Fibroblast Migration ◽  
Tissue Equivalents ◽  
Stress Dependent ◽  
Statistical Conclusion

Abstract We used our novel assays of cell behavior in tissue equivalents to study the dose-response effects of PDGF-BB on RDF migration and traction in mechanically stressed and stress-free type I collagen and fibrin gels. PDGF-BB increased fibroblast migration significantly in all assays, but the effects on traction depended on the presence of stress and the nature of the ECM. PDGF-BB decreased fibroblast traction in stressed collagen gels, but increased traction in stress-free gels. No statistical conclusion could be inferred for stressed fibrin gels, and increasing PDGF-BB decreased traction in stress-free fibrin gels. These results demonstrate the complex response of fibroblasts to environmental cues, and point to opportunities to orchestrate cell behavior to affect the outcome of wound healing.

scholarly journals Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate

1985 ◽  
Vol 228 (2) ◽  
pp. 463-469 ◽  
Author(s):  
G K Hunter ◽  
B L Allen ◽  
M D Grynpas ◽  
P T Cheng
Keyword(s):  
Type I Collagen ◽  
Chondroitin Sulphate ◽  
Collagen Gel ◽  
Calcium Pyrophosphate ◽  
Molar Ratio ◽  
Crystal Formation ◽  
Type I ◽  
Collagen Gels ◽  
High Concentrations ◽  
Hydroxyapatite Formation

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.

Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway

2000 ◽  
Vol 278 (5) ◽  
pp. L1032-L1038 ◽  
Author(s):  
X. D. Liu ◽  
C. M. Skold ◽  
T. Umino ◽  
J. R. Spurzem ◽  
D. J. Romberger ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Human Lung ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Lung Fibroblast ◽  
Type I ◽  
Collagen Gels ◽  
Human Lung Fibroblast ◽  
Collagen Gel Contraction ◽  
Cgmp Pathway

Nitric oxide (NO) relaxes vascular smooth muscle in part through an accumulation of cGMP in the target cells. We hypothesized that a similar effect may also exist on collagen gel contraction mediated by human fetal lung (HFL1) fibroblasts, a model of wound contraction. To evaluate this, HFL1 cells were cultured in three-dimensional type I collagen gels and floated in serum-free DMEM with and without various NO donors. Gel size was measured with an image analyzer. Sodium nitroprusside (SNP, 100 μM) significantly augmented collagen gel contraction by HFL1 cells (78.5 ± 0.8 vs. 58.3 ± 2.1, P < 0.01), whereas S-nitroso- N-acetylpenicillamine, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride, NONOate, and N G-monomethyl-l-arginine did not affect the contraction. Sodium ferricyanide, sodium nitrate, or sodium nitrite was not active. The augmentory effect of SNP could not be blocked by 1 H-[1,2,4]-oxadiazolo-[4,3- a]-quinoxalin-1-one, whereas it was partially reversed by 8-(4-chlorophenylthio) (CPT)-cGMP. To further explore the mechanisms by which SNP acted, fibronectin and PGE2 production were measured by immunoassay after 2 days of gel contraction. SNP inhibited PGE2 production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and PGE2 production. Addition of exogenous PGE2 blocked SNP-augmented contraction and fibronectin production by HFL1 cells. Therefore, SNP was able to augment human lung fibroblast-mediated collagen gel contraction, an effect that appears to be independent of NO production and not mediated through cGMP. Decreased PGE2 production and augmented fibronectin production may have a role in this effect. These data suggest that human lung fibroblasts in three-dimensional type I collagen gels respond distinctly to SNP by mechanisms unrelated to the NO-cGMP pathway.

scholarly journals The Role of Cytokines TGF-β1 and FGF-1 in the Expression of Characteristic Markers of Rat Liver Myofibroblasts Cultured in Three-Dimensional Collagen Gel

2016 ◽  
pp. 661-672 ◽  
Author(s):  
E. PETEROVÁ ◽  
A. MRKVICOVÁ ◽  
L. PODMOLÍKOVÁ ◽  
M. ŘEZÁČOVÁ ◽  
J. KANTA
Keyword(s):  
Growth Factor ◽  
Rat Liver ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Parenchymal Cell ◽  
Collagen Gel ◽  
Fibrotic Liver ◽  
Liver Myofibroblasts ◽  
Cellular Environment ◽  
Tgf Β1

Rat liver myofibroblasts (MFB) are the key cells involved in the deposition of extracellular matrix in fibrotic liver. They were isolated by repeated passaging of non-parenchymal cell fraction and cultured in 3-dimensional (3D) collagen gel mimicking tissue. The transfer of MFB from plastic dishes to collagen resulted in the change in their shape from large and spread to slender with long extensions. The expression of transforming growth factor-β1 (TGF-β1) and of MFB markers, α-smooth muscle actin (α-SMA) and cellular fibronectin (EDA-FN), on protein level was significantly decreased in collagen gel. The gel did not change the expression of metalloproteinase MMP-2 but activated the proenzyme. The experiments with inhibitors of metabolic pathways showed that EDA-FN and α-SMA were differently regulated. The expression of EDA-FN required functional TGF-β1 receptors and was also dependent on the activity of protein kinases MEK1 and MEK2. α-SMA expression was primarily determined by the 3D environment. Fibroblast growth factor-1 (FGF-1) in combination with heparin decreased the expression of α-SMA and increased the expression of EDA-FN in the cells on plastic. The cellular environment may influence the cells per se and may modify the action of other agents.

scholarly journals Multiscale Model Predicts Tissue-Level Failure From Collagen Fiber-Level Damage

2012 ◽  
Vol 134 (9) ◽  
Author(s):  
Mohammad F. Hadi ◽  
Edward A. Sander ◽  
Victor H. Barocas
Keyword(s):  
Type I Collagen ◽  
Soft Tissues ◽  
Mechanical Response ◽  
Collagen Gel ◽  
Multiscale Model ◽  
Tissue Level ◽  
Type I ◽  
Collagen Gels ◽  
Damage And Failure ◽  
Failure Data

Excessive tissue-level forces communicated to the microstructure and extracellular matrix of soft tissues can lead to damage and failure through poorly understood physical processes that are multiscale in nature. In this work, we propose a multiscale mechanical model for the failure of collagenous soft tissues that incorporates spatial heterogeneity in the microstructure and links the failure of discrete collagen fibers to the material response of the tissue. The model, which is based on experimental failure data derived from different collagen gel geometries, was able to predict the mechanical response and failure of type I collagen gels, and it demonstrated that a fiber-based rule (at the micrometer scale) for discrete failure can strongly shape the macroscale failure response of the gel (at the millimeter scale). The model may be a useful tool in predicting the macroscale failure conditions for soft tissues and engineered tissue analogs. In addition, the multiscale model provides a framework for the study of failure in complex fiber-based mechanical systems in general.

Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells

2002 ◽  
Vol 282 (5) ◽  
pp. L1049-L1056 ◽  
Author(s):  
Xiangde Liu ◽  
Tadashi Kohyama ◽  
Hangjun Wang ◽  
Yun Kui Zhu ◽  
Fu-Qiang Wen ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Collagen Gel ◽  
Tissue Remodeling ◽  
Independent Effect ◽  
Airway Smooth Muscle Cells ◽  
Type I ◽  
Dependent Manner ◽  
Th2 Cytokines ◽  
Collagen Gels ◽  
Collagen Gel Contraction

Asthma is characterized by chronic inflammation of the airway wall with the presence of activated T helper 2 (Th2) lymphocytes. The current study assessed the ability of Th2 cytokines to modulate fibroblast-mediated contraction of collagen gels to determine if Th2 cytokines could contribute to tissue remodeling by altering mesenchymal cell contraction. Human fetal lung fibroblasts, human adult bronchial fibroblasts and human airway smooth muscle cells were cast into native type I collagen gels and allowed to contract in the presence or absence of IL (interleukin)-4, IL-5, IL-10, or IL-13. IL-4 and IL-13 but not IL-5 and IL-10 augmented collagen gel contraction in a concentration-dependent manner. Neither IL-4 nor IL-13 altered fibroblast production of transforming growth factor-β or fibronectin. Both, however, decreased fibroblast prostaglandin (PG) E2 release. Decreased PGE2 release was associated with a decreased expression of cyclooxygenase 1 and 2 protein and mRNA. Indomethacin completely inhibited PGE2release and also augmented contraction. IL-4 and IL-13, however, added together with indomethacin further augmented contraction suggesting both a PGE-dependent and a PGE-independent effect. These findings suggest that IL-4 and IL-13 may modulate airway tissue remodeling and, therefore, could play a role in the altered airway connective tissue which characterizes asthma.

scholarly journals Targeting lncRNA H19/miR-29b/COL1A1 Axis Impedes Myofibroblast Activities of Precancerous Oral Submucous Fibrosis

2021 ◽  
Vol 22 (4) ◽  
pp. 2216
Author(s):  
Cheng-Chia Yu ◽  
Yi-Wen Liao ◽  
Pei-Ling Hsieh ◽  
Yu-Chao Chang
Keyword(s):  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Oral Submucous Fibrosis ◽  
Ectopic Expression ◽  
Collagen Gel ◽  
Type I ◽  
Migration Ability ◽  
Potentially Malignant ◽  
Submucous Fibrosis

Oral submucous fibrosis (OSF) is known as a potentially malignant disorder, which may result from chemical irritation due to areca nuts (such as arecoline). Emerging evidence suggests that fibrogenesis and carcinogenesis are regulated by the interaction of long noncoding RNAs (lncRNAs) and microRNAs. Among these regulators, profibrotic lncRNA H19 has been found to be overexpressed in several fibrosis diseases. Here, we examined the expression of H19 in OSF specimens and its functional role in fibrotic buccal mucosal fibroblasts (fBMFs). Our results indicate that the aberrantly overexpressed H19 contributed to higher myofibroblast activities, such as collagen gel contractility and migration ability. We also demonstrated that H19 interacted with miR-29b, which suppressed the direct binding of miR-29b to the 3′-untranslated region of type I collagen (COL1A1). We showed that ectopic expression of miR-29b ameliorated various myofibroblast phenotypes and the expression of α-smooth muscle actin (α-SMA), COL1A1, and fibronectin (FN1) in fBMFs. In OSF tissues, we found that the expression of miR-29b was downregulated and there was a negative correlation between miR-29b and these fibrosis markers. Lastly, we demonstrate that arecoline stimulated the upregulation of H19 through the transforming growth factor (TGF)-β pathway. Altogether, this study suggests that increased TGF-β secretion following areca nut chewing may induce the upregulation of H19, which serves as a natural sponge for miR-29b and impedes its antifibrotic effects.

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