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1.
Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots 总被引:2,自引:0,他引:2
Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM epsilon-aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen-depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis. 相似文献
2.
Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin 总被引:7,自引:0,他引:7
Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis. 相似文献
3.
C Y Cheng C G Leggett A C Reese 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1988,188(4):444-450
Fibronectin is a normal glycoprotein component of plasma, interstitial fluid, and extracellular matrix which has binding sites for collagen, gelatin, actin, glycosaminoglycans, fibrin, Staphylococcus aureus, and some cells. Since it is a dimer, it can crosslink these substances to each other or to extracellular components of basement membrane, thereby affecting many physiological processes. The level of circulating fibronectin is markedly reduced following even moderate blunt or operative trauma, thermal injury, starvation, advanced cancers, hemorrhage, etc. Replacement therapy has been tried with some success in patients who become septic following multiple injuries. The reduction in plasma fibronectin has been attributed to several causes including consumption by binding to cell debris at the site of injury, binding to circulating cell debris and its subsequent removal by elements of the phagocytic system, and degradation by proteolytic cleavage. However, the amount of fibronectin removed from circulation raises some question about this. In this paper, we used indomethacin, ibuprofen, imidazole, and essential fatty acid deprivation to inhibit the synthesis of prostaglandins in young adult rats. Thirty minutes after ip administration of one of the inhibitors, the rats were subjected to a midline laparotomy and mild intestinal manipulation. Blood samples were taken at intervals following closure of the incision and analyzed for fibronectin. In all cases, the normal decline in plasma fibronectin seen in untreated rats was abrogated by inhibiting prostaglandin synthesis. Since imidazole specifically inhibits thromboxane A synthesis, this strongly suggests that thromboxanes directly or indirectly control the trauma-induced reduction in circulating fibronectin. This was confirmed by ip injection of thromboxane into the rats which resulted in a decline in plasma fibronectin levels. 相似文献
4.
Role of fibronectin in primary mesenchyme cell migration in the sea urchin 总被引:6,自引:0,他引:6
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We studied the effect of fibronectin (FN) on the behavior of primary mesenchyme cells isolated from sea urchin mesenchyme blastulae in vitro using a time-lapse technique. The migration of isolated primary mesenchyme cells reconstituted in seawater and horse serum is dependent on the presence or absence of exogenous FN in the culture media. The cells in FN, 4 and 40 micrograms/ml, show a high percentage of migration and migrate long distances, whereas a higher concentration of FN at 400 micrograms/ml tends to inhibit migration. 相似文献
5.
Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholate-soluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KDiss was 3.6 X 10(-8) M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors. 相似文献
6.
Cultured neural crest cells which are freshly trypsinized require serum or purified fibronectin to attach to collagen substrates of types I–V. Furthermore, neural crest cells migrate in a Boyden chamber in response to fibronectin, and a “checkerboard” analysis demonstrates that fibronectin is both chemotactic and chemokinetic for these cells. It is proposed that collagen serves as a substrate for neural crest cells as they migrate in the early embryo. By mediating the cells' attachment to collagen, fibronectin may influence the movement of the cells. Local differences in fibronectin concentration or availability in the matrix could affect the degree of attachment of the cells to the collagen substrate and could also direct their migration by serving as a chemoattractant. 相似文献
7.
Role of newly synthesized fibronectin in vascular smooth muscle cell migration on matrix-metalloproteinase-degraded collagen 总被引:3,自引:0,他引:3
Stringa E White D Tuan RS Knauper V Gavrilovic J 《Biochemical Society transactions》2002,30(2):102-111
The migration of vascular smooth muscle cells (VSMC) is known to be a key process in the development of a number of vascular lesions, although the precise mechanisms involved have still to be elucidated. In the present study, the production of endogenous fibronectins by VSMC migrating across intact and matrix-metalloproteinase-degraded collagen type I has been explored. Cellular fibronectin seems to play a role in the enhanced migration seen when VSMC are exposed to degraded collagen and platelet-derived growth factor-BB. VSMC were found to synthesize both exon IIIA-containing fibronectin (which predominated) and exon IIIB-containing fibronectin. When these cells were exposed to substrates consisting of recombinant exon IIIA- or exon IIIB-containing fibronectin, rates of migration were not elevated above those seen with undegraded collagen. Endogenous fibronectin production may thus be necessary, but not sufficient, for VSMC migration over degraded collagenous substrates. 相似文献
8.
Zand L Qiang F Roskelley CD Leung PC Auersperg N 《In vitro cellular & developmental biology. Animal》2003,39(3-4):178-182
Summary The main form of fibronectin (FN) encountered by tumor cells in vivo is cellular FN (cFN), which differs structurally and
functionally from the commonly used plasma FN (pFN). We compared the effects of cFN and pFN on the ovarian carcinoma lines
OVCAR-3 and SKOV-3 and on cultures of normal ovarian surface epithelium, which is the precursor of the epithelial ovarian
carcinomas. Ovarian surface epithelial cells and SKOV-3 cells attached and spread faster on cFN than on pFN. On cFN, SKOV-3
migration was enhanced compared with pFN or plastic. In a matrigel transfilter assay, cFN strongly inhibited SKOV-3 invasion,
whereas pFN did not. In contrast to SKOV-3, OVCAR-3 cells adhered faster on FN than on plastic but did not discriminate between
cFN and pFN, and they did not migrate or invade matrigel either with or without FN. In both carcinoma lines, proliferation
was unaffected by either FN. The results show profound differences in the responses to cFN and pFN by two invasive ovarian
carcinoma lines. Because cFN is the main type that cancer cells encounter in vivo, extrapolations from culture data to in
vivo events should preferably be based on studies using this form of FN. 相似文献
9.
Incorporation of thrombospondin into fibrin clots 总被引:9,自引:0,他引:9
Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure. 相似文献
10.
Summary To study the role of phagocytosis in periodontal tissues, internalization of fibronectin-coated latex beads by Gin-1 fibroblast populations was investigated. Demonstration of phagocytosis by internalization of beads was confirmed by immunofluorescence microscopy, electron microscopy, and flow-cytometry. The percent of cells phagocytosing beads measured by flow-cytometry was negligible at 4° and 23°C, but increased to approximately 17% at 37°C. As measured by automated image analysis, the percentage of phagocytosing cells increased linearly from 8 to 22 with increasing fibronectin concentration of the incubation solution from 30 ng to 300 g/ml. Similar linear increases in the percentage of phagocytosing cells were observed when beads were incubated with cells for periods ranging from 2 h to 2 days. To examine the role of the Arg-Gly-Asp receptor in mediating phagocytosis, fibronectin-coated beads were first coated with either Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro peptides at concentrations of 0.125, 0.5, and 1 mg/ml, or with control vehicle, and then incubated with cells. Phagocytosis was completely blocked at 1 mg/ml of the Gly-Arg-Gly-Asp-Ser-Pro peptide, but the Gly-Arg-Gly-Glu-Ser-Pro peptide showed no significant inhibition compared to control values. Blocking antibodies to the cell attachment domain of the fibronectin molecule also reduced the percentage of phagocytosing cells significantly. The data show that these phagocytic assays are sensitive enough to detect the influence of incubation temperature and time, cellular heterogeneity, ligand type, and ligand concentration on the percentage of phagocytosing cells. Further, the mechanisms which determine internalization of fibronectin-coated beads rely in part on the initial binding of ligand to the Arg-Gly-Asp receptor present on fibroblasts. 相似文献
11.
Yeong-Hau Lien Mitchell J. Wong Mitchell S. Golbus Robert Stern 《Journal of cellular physiology》1984,120(1):103-107
The effect of hydrocortisone on fibronectin synthesis was investigated in cultured skin fibroblasis. Confluent cells were treated with hydrocortisone (10?7 M to 10?5 M) for 2 days and labeled with [3H]proline for 24 h. Fibronectin levels in both the culture medium and the cell layer were studied by gelatin-Sepharose affinity chromatography and SDS-polyacrylamide gel electrophoresis. In control cultures of human fetal skin fibroblasts, fibronectin constituted 8% of the total labeled proteins in the medium. The proportion of fibronectin increased to 13.1% at 10?7 M hydrocortisone, 15.5% at 10?6 M and to 19.4% at 10?5 M. The proportion of fibronectin associated with the cell layer remained at 2-3% of total [3H]prolne-labeled proteins and did not increase with hydrocortisone exposure. The stimulating effect of hydrocortisone on medium fibronectin was also demonstrated in cultured human newborn foreskin fibroblasts and in rabbit skin fibroblasts. 相似文献
12.
Characterization of fibronectin from fetal human plasma in comparison with adult plasma fibronectin 总被引:1,自引:0,他引:1
Y Yamaguchi M Isemura M Kosakai A Sato M Suzuki M Kan Z Yosizawa 《Biochimica et biophysica acta》1984,790(1):53-60
Peptides containing fewer than 50 amino acids show little ordered structure under physiological conditions. In this paper it is shown that in the receptor environment, secondary structure could be induced in small peptides that involves 87% of all the amino acid residues. The statistical methods of Chou and Fasman are used to predict the conformation of 41 peptide hormones or neuromodulators in the proteinaceous environment of the receptor, and four distinct conformational groupings are elucidated. beta-bend, beta-structure and alpha-helical conformation are possible for distinct groups of linear peptides, and disulfide bridge containing peptides show a common beta-bend beta-structure conformation at the receptor. In the predicted receptor conformation, the peptides show hydrophobic and hydrophilic domains that must reflect the distribution of corresponding regions in the ligand-binding site of the receptor. The predicted ligand conformation should allow a more rational approach to interpreting existing structure activity studies and the design of new analogs of pharmacological interest. 相似文献
13.
Mast cells enhance migration and proliferation of fibroblasts into an in vitro wound 总被引:4,自引:0,他引:4
The effects of mast cells (MC) in an in vitro wound model were studied. The model consisted of rat peritoneal MC cultured on confluent monolayers of 3T3 fibroblasts (MC/3T3). A linear wound was performed by cutting along the midline and scraping one half of the monolayer. After 42 h fibroblasts were counted in the scraped area of the wound. In the MC/3T3 cocultures 27.6 +/- 2.1 fibroblasts were found compared to 16.6 +/- 0.9 in the 3T3 cultures. The most significant increase in the number of fibroblasts was obtained upon activation of the MC with anti-IgE antibodies immediately after wound production (39.9 +/- 2.1). Stimulation with compound 48/80 had a weaker effect (32.7 +/- 1.5). Incubation of 3T3 wounded monolayers with supernatants of anti-IgE- or compound 48/80-activated MC, or with sonicated MC, induced an increase in fibroblast number similar to that found in unactivated MC/3T3. [3H]Thymidine incorporation followed by autoradiography was performed to assess fibroblast mitosis. The highest number of labeled fibroblasts beyond the wound line was found in immunologically activated MC/3T3 (29.7 +/- 4.4), followed by compound 48/80-activated MC/3T3 (18.4 +/- 1.5), MC/3T3 (15.1 +/- 3.6), and 3T3 (10.6 +/- 2.6). After addition of aphidicolin, which inhibited fibroblast mitosis, MC were still effective in enhancing fibroblast migration. In all the cocultures MC were observed to have migrated alongside fibroblasts. Thus merely the presence of MC adhering to wounded fibroblast monolayers significantly enhanced migration and proliferation of the fibroblasts. A further increase was achieved by immunological activation of the MC. We therefore suggest that MC have a facilitating role in this in vitro wound model. 相似文献
14.
Curli fibers of Escherichia coli mediate internalization of bacteria by eukaryotic cells. As curli fibers bind fibronectin with high affinity, the role of fibronectin in the uptake process was studied. The experiments presented here support the involvement of fibronectin in internalization of bacteria. Furthermore, a peptide containing the RGD motif, responsible for interaction of fibronectin with cellular integrins, can strongly inhibit curli-mediated internalization. The ability of curli fibers to bind fibronectin can therefore be linked to virulence. 相似文献
15.
Ramprasad OG Srinivas G Rao KS Joshi P Thiery JP Dufour S Pande G 《Cell motility and the cytoskeleton》2007,64(3):199-216
The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton. 相似文献
16.
The elastic modulus (G') of factor XIIIa induced fibrinogen gels was found to be substantially lower than the G' of fibrin gels that were formed by clotting fibrinogen with thrombin. The addition of fibronectin and/or the reducing reagent dithiothreitol (DTT) to the factor XIIIa coagulation mixture led to the formation of a weaker gel structure, while the rigidity of thrombin induced clots was not appreciably affected by the inclusion of the DTT but increased somewhat in the presence of fibronectin. The reasons for the differing clot rigidities are discussed in terms of biochemical mechanisms. 相似文献
17.
Millard CJ Ellis IR Pickford AR Schor AM Schor SL Campbell ID 《The Journal of biological chemistry》2007,282(49):35530-35535
The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN. 相似文献
18.
In addition to mediating cell adhesion, fibronectin (FN) also affects the migration of different cell types. However, the role of FN in lymphocyte migration is unclear. In this study, we examined the effects of FN on the in vitro migration of lymphocytes. Using the checkerboard analysis in a blind-well microchemotaxis assay, soluble FN was determined to have neither a chemotactic nor chemokinetic effect on spleen or thymus lymphocytes. However, when the nitrocellulose filter was coated unidirectionally with FN, the migration of both spleen and thymus lymphocytes into the filter was enhanced, indicating that FN is haptotactic for lymphocytes. When the filter was coated bidirectionally, no enhancement in migration was observed, indicating that FN is not haptokinetic for lymphocytes. When the FN cell-binding domain and the heparin-binding domain were tested, the cell-binding domain was haptotactic for both spleen and thymus lymphocytes, whereas the heparin-binding domain was only haptotactic for spleen lymphocytes. Because the heparin-binding domain can mediate strong adhesion of thymus lymphocytes, the lack of haptotactic activity is likely to be the result of excessive binding that prevents cell motility. 相似文献
19.
Subunit interactions in human plasma fibronectin 总被引:1,自引:0,他引:1
The fibronectin molecule was split chemically into its two constituent chains (mol. wt. 220,000) by mild reduction with dithiothreitol. However, physical properties (molecular weight and diffusion coefficient from light scattering, and elution in gel exclusion chromatography) remained those of intact fibronectin, except (reversibly) in the presence of denaturants which also change the conformation of non-reduced fibronectin to a more open form. Similarly, during digestion of fibronectin by plasmin to fragments of molecular weight less than 200,000, the light scattering intensity drops to roughly half in 30% glycerol but not in the absence of glycerol. These results suggest that the compact conformation of native fibronectin is stabilized by specific noncovalent contacts between constituent chains. 相似文献
20.
Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in a time- and temperature-dependent manner and were not removed from the cell surface by trypsin or serum. Thus, the gangliosides appeared to be stably incorporated into the lipid bilayer of the plasma membrane. Fluorescent photobleaching recovery measurements showed that the inserted gangliosides were free to diffuse in the plane of the membrane with a high diffusion coefficient of approximately 10(-8) cm2/s. When the ganglioside-treated cells were washed and incubated in fresh medium, the surface gangliosides became internalized with time, and localized in the perinuclear region of the fibroblasts. In dense cultures of fibroblasts, a large fraction of the fluorescent gangliosides were organized in a fibrillar network and were immobile on the time scale of fluorescent photobleaching recovery measurements. Using antifibronectin antibodies and indirect immunofluorescence, these gangliosides were found to co-distribute with fibrillar fibronectin. Thus, exogenous gangliosides appear to be stably inserted into the lipid bilayer of the plasma membrane and to diffuse freely in its plane as well as form a less mobile state with the fibrillar networks of fibronectin associated with the cells. 相似文献