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1.
Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel 总被引:7,自引:0,他引:7
Cornelia Mauch Atsushi Hatamochi Karin Scharffetter Thomas Krieg 《Experimental cell research》1988,178(2):493-503
Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix. 相似文献
2.
Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors 总被引:12,自引:0,他引:12
Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast-like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor-beta 1 (TGF-beta 1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1-1.0 ng/ml), higher doses (greater than or equal to 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF-beta 1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF-beta 1 activity in highly contractile cells. Transforming growth factor-alpha moderately enhanced contraction by cells from older granulation tissue. While both TGF-beta 1 and bFGF enhanced wound repair, their differential effects on the fibroblast-like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair. 相似文献
3.
The fibrillar collagen I gel induced the formation of numerous dendritic cell-like protrusions (cell spikes) from the cell body, whereas monomeric collagen I induced typical cell spreading with filopodia and lamellipodia in skin fibroblasts. Peripheral, not central stress fibers appeared upon adhesion to fibrillar collagen gel, whereas both types of fibers were evident upon adhesion to monomeric collagen. Microtubules and vimentin filaments were elongated inside stress fibers along the terminal tip of cell spikes. Spike formation was totally inhibited by nocodazole and severely delayed by cytochalasin D. This suggests that cell spike formation is dependent on microtubules rather than on F-actin. We then investigated the intracellular signaling responsible for cytoskeleton organization to identify the key factor that induces cell spike morphology. During cell spike formation, FAK and CAS were activated. More CAS was activated in cells on fibrillar collagen gel than on the monomeric form, whereas FAK was activated to the same level on either. At 90 min of culture, Rac1 was activated in cells on monomeric collagen I, whereas Cdc42, Rac1 and RhoA were activated in cells on fibrillar collagen gel. These results suggest that microtubule organization via CAS and small GTPases is important for the cell spike formation that is involved in collagen gel contraction and in wound retraction in skin. 相似文献
4.
The effect of a native collagen gel substratum on the synthesis of collagen by bovine brain capillary endothelial cells 总被引:2,自引:0,他引:2
Cultured capillary endothelial cells, derived from bovine brain, and maintained on a plastic substratum synthesized predominantly interstitial collagens of which approximately 75 per cent were secreted into the medium. When grown on a native hydrated collagen type I gel, although no marked alteration in the 'collagen synthetic pattern' was observed, the overall level of collagen synthesis was increased by approximately 100 per cent. More dramatic, however, was the alteration in the distribution of these molecules between medium and cell layer. Interstitial collagens produced by cells grown on collagen gels were almost exclusively associated with the cell layer or collagenous gel. These studies, thus, demonstrate that an extracellular matrix may exert a considerable influence on the cellular synthetic activities and possibly cellular polarity of capillary endothelial cells. 相似文献
5.
Sato K Hattori S Irie S Sorimachi H Inomata M Kawashima S 《Journal of biochemistry》2004,136(6):777-785
When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas micro-calpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, alpha-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel. 相似文献
6.
Mark Eastwood Rebecca Porter Umraz Khan Gus McGrouther Robert Brown 《Journal of cellular physiology》1996,166(1):33-42
The force generated in granulation tissue during wound contraction is thought to be cell mediated; however, it is unclear whether contractile forces are generated by fibroblast locomotion or contraction of myofibroblasts. To help clarify this question the force of this contraction can now be determined accurately in a human dermal fibroblast collagen lattice system using a novel instrument known as a Culture Force Monitor. Three distinct phases of contraction of such collagen gels could be identified over the first 24 hours. Most of the force generated by human dermal fibroblasts was produced during the first stage in parallel with cell attachment and associated changes in cell shape, and the appearance of cell processes. During this initial 24 hours no evidence could be found for the presence of myofibroblasts, but stereoscopic and electron microscopic analysis at a range of time points indicated that migratory fibroblasts were present in the system. Comparison of the contraction profiles of cells extracted from other tissues (tendon and articular cartilage), and extracted by different means from the same tissue specimen, indicated that different populations of fibroblasts can be distinguished on the basis of their pattern of contractions. It would seem that most of the force generated in this model is a result of fibroblast attachment and movement within the collagen lattice. Furthermore, different groups of fibroblasts, even within the same tissue, may vary in their contraction (hence locomotory) activity. © 1996 Wiley-Liss, Inc. 相似文献
7.
Heparin modulates the organization of hydrated collagen gels and inhibits gel contraction by fibroblasts 总被引:5,自引:4,他引:5
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We studied the effects of extracellular matrix components on fibroblast contraction of hydrated collagen gels. After 4-h incubations, heparin-containing collagen gels contracted only 10% compared with 50% contraction of control gels. Contraction was not affected by hyaluronic acid, dermatan sulfate, or fibronectin, implying that the activity of heparin was specific. The possibility that heparin inhibited attachment of the cells to the gels was ruled out. Also, addition of heparin to the incubation medium had no effect on contraction. Microscopic examination showed that control collagen gels were composed of a uniform network of interlocking fibrils of similar sizes. Heparin-containing gels, on the other hand, were highly variable with some collagen bundles containing 5-6 collagen fibrils and other regions containing amorphous material. Unlike the control gels, the fibrils of heparin-containing gels were not continuously interconnected. Based on the results, we propose that fibroblasts attach normally to the collagen fibrils of heparin-containing gels and attempt to contract the gels, but the mechanical forces exerted by fibroblasts on individual collagen fibrils cannot be propagated throughout the gels. 相似文献
8.
The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL. 相似文献
9.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2017,1861(9):2261-2273
BackgroundTensioned collagen gels with dermal fibroblasts (DFs) as a dermis model are usually utilized in a static culture (SC) that lacks medium flowing. To make the model closer to its in vivo state, we created a device to perfuse the model with media flowing at a physiological velocity and examined the effects of medium flow (MF) on the cultures.MethodsWe constructed a medium perfusion device for human DF-embedded stretched collagen gels (human dermis model), exposed the model to media that flows upwardly at ~ 1 mL/day, and examined water retention of the gels, cells' growth ability, metabolic activity, expression profiles of nine extracellular matrix (ECM)-related genes. The obtained data were compared with those from the model in SC.ResultsMF increases the gels' water retention and cells' growth potential but had little effect on their metabolic activities. MF robustly enhanced hyaluronan synthase 2 (HAS2) and matrix metalloprotease 1 (MMP1) gene expressions but not of the other genes (MMP2, HYAL1, HYAL2, HYAL3, COL1A1, COL3A1, and CD44). MF significantly increased the amounts of cellular hyaluronan and adenosine triphosphate.ConclusionsThe MF at a physiological speed significantly influences the nature of ECMs and their resident fibroblasts and remodels ECMs by regulating hyaluronan metabolism.General significanceFibroblasts in tensioned collagen gels altered their phenotypes in a MF rate-dependent manner. Collagen gel culture with tension and MF could be utilized as an appropriate in vitro model of interstitial connective tissues to evaluate the pathophysiological significance of mechanosignals generated by fluid flow and cellular/extracellular tension. 相似文献
10.
Yin Q Nan HY Zhang WH Yan LF Cui GB Huang XF Wei JG 《Journal of cellular physiology》2011,226(8):2091-2102
Accumulation and activation of myofibroblasts are the hallmark of progressive pulmonary fibrosis, and the resident fibroblasts are the major source of myofibroblasts. However, the key factors involved in the transformation of fibroblasts are unknown. Pulmonary microvascular endothelial cells (PMVECs), major effector cells against pathogenesis in early stages of the disease, can secrete cytokines to induce the differentiation of mesenchymal cells. We speculated that PMVECs could secrete pro-fibrotic cytokines and promote the transformation of fibroblasts into myofibroblasts. Accordingly, we established a co-culture system with PMVECs and fibroblasts to examine the specific transformation and collagen synthesis of the co-cultured fibroblasts by FACS and Western blot, prior to and after treatment with neutralizing antibodies against transforming growth factor-beta1 (TGF-β1) and connective tissue growth factor (CTGF). We also analyzed expression of TGF-β1 and CTGF in PMVECs. The synthesis and secretion of TGF-β1 and CTGF protein were up-regulated in PMVECs isolated from bleomycin (BLM)-treated rats, most prominently at 7 days post-instillation. We showed that the PMVECs isolated from BLM-induced rats could induce the transformation of normal fibroblasts and their secretion of collagen I, which was inhibited by both neutralizing anti-TGF-β1 and anti-CTGF antibodies. Therefore, up-regulation of TGF-β1 and CTGF in PMVECs plays an important role in activation, transformation, and collagen synthesis of fibroblasts; in particular, these effects in PMVECs are likely to be the key factors for activation and stimulation of static fibroblasts in lung interstitium in early stages of pulmonary fibrosis disease. 相似文献
11.
Lactoferrin promotes collagen gel contractile activity of fibroblasts mediated by lipoprotein receptors. 总被引:1,自引:0,他引:1
Lactoferrin is an iron-binding glycoprotein that belongs to the transferrin family. Recent studies in vitro and in vivo suggest that lactoferrin is a potential therapeutic agent for wound healing. We have shown that both bovine and human lactoferrin enhance the collagen gel contractile activity of WI-38 human fibroblasts. The collagen gel contraction is considered as an in vitro model for reorganization of the collagen matrix during the wound healing process. The elevation of collagen gel contractile activity induced by lactoferrin was accompanied by activation of extracellular-regulated kinase (ERK) 1/2 and myosin light chain kinase (MLCK), and subsequent elevation of myosin light chain (MLC) phosphorylation. The effects of lactoferrin on collagen gel contraction and the activation of the signaling pathway were dependent on the expression of low-density lipoprotein receptor - related protein (LRP) - 1 in the fibroblasts. LRP-1 is known as an endocytosis receptor and is involved in the cellular uptake of diverse ligands, including lactoferrin. In addition, LRP-1 acts as a signaling lactoferrin receptor in mammalian cells by converting the lactoferrin-binding signal into the activation of the intracellular signaling pathway. This property was found to be independent of the endocytic function of LRP-1, as seen in osteoblast-like cells. 相似文献
12.
Hydrostatic fluid pressure promotes cellularity and proliferation of human dermal fibroblasts in a three-dimensional collagen gel/sponge 总被引:2,自引:0,他引:2
Cell constructs and culture systems are essential components of tissue engineering. Cell constructs are usually composed of a dense population of cells, for which long-term culture is required in vitro. However, the denser construct suffers from the absence of passive nutrient supply, gas exchange, and removal of degraded debris. We have developed a novel hydrostatic pressure/perfusion (HP/P) culture system that improves the quality of neo-tissues, providing an automated affordable system for clinical applications. We evaluated the effects of HP/P on cellularity, viability, and proliferation of human dermal fibroblasts seeded in a gel/sponge construct. HP/P and perfusion promoted cell migration and significantly increased proliferation and DNA content after 4 days culture compared to the static culture. HP/P culture is beneficial for building a denser three-dimensional fibroblast construct. 相似文献
13.
Takayama Y Mizumachi K Takezawa T 《Biochemical and biophysical research communications》2002,299(5):813-817
We have reported that bovine lactoferrin (bLf) promotes the contractile activity of collagen gels by WI-38 human fibroblasts via the phosphorylation of myosin light chain (MLC). To identify the region of bLf that is responsible for this activity, we prepared bLf fragments by limited proteolysis using trypsin and investigated the effects of each fragment on gel contractile activity. Lf consists of a single polypeptide chain containing two lobes that are independent globular structures termed the N- and C-lobes.The fragment corresponding to the C-lobe of bLf (amino acids 341-689) had a more prominent effect on collagen gel contractile activity than did that of either native bLf or its N-lobe (1-284). Further hydrolysis of the C-lobe with either pepsin or trypsin resulted in a loss of this activity. The effect of the C-lobe on collagen gel contraction by fibroblasts was dose-dependent and was associated with the elevation of MLC phosphorylation. 相似文献
14.
With the help of a ribonucleoprotein it is possible to precipitate collagen in a layer of fibers with a 700 Å period. As collagen is a constituent of many membrane systems in the body, it seemed interesting to investigate the permeability of ions and water through a native collagen membrane.The experiments were carried out with the help of an acryl glass apparatus, where an osmotic pressure, a hydrostatic pressure difference or both can be maintained between the two bulk phases separated by the membrane. The diffusion coefficients for NaCl and KCl were found to be comparable with those in other biological membranes (Ds = 9 · 10−7cm2 · s−1) whereas there is difference of more than three orders of magnitude in the hydraulic permeability (Lp = 6 cm4 · J−1 · s−1).Volume flow measurements caused by an osmotic gradient indicated that the reflection coefficient for NaCl and KCl is very small. In hydrostatic pressure experiments, the membrane shows a preferred direction for volume flows which seems to have something to do with the mode of preparation of the membrane. 相似文献
15.
16.
Edward Bańkowski Wiktor Rzeczycki Henry K. F. Nowak Kazimierz J. Jodczyk 《Molecular and cellular biochemistry》1978,20(2):77-83
Summary Rat kidney fibroblasts transformed with SV-40 produce in vitro a significantly lower amount of hydroxyproline-containing material which is collagenase sensitive as compared to normal cells. In contrast to normal fibroblast cultures, no collagenous material was found by histochemical methods in intercellular spaces of transformed cultures. 相似文献
17.
18.
Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides 总被引:3,自引:3,他引:0
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《The Journal of cell biology》1982,95(3):752-756
Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo. 相似文献
19.
When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI-38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y-27632), myosin light chain kinase inhibitor (ML-7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf-enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf-enhanced MLC phosphorylation was inhibited by Y-27632 and ML-7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation. 相似文献