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1.
The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype 总被引:3,自引:0,他引:3
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McDaniel DP Shaw GA Elliott JT Bhadriraju K Meuse C Chung KH Plant AL 《Biophysical journal》2007,92(5):1759-1769
Cells receive signals from the extracellular matrix through receptor-dependent interactions, but they are also influenced by the mechanical properties of the matrix. Although bulk properties of substrates have been shown to affect cell behavior, we show here that nanoscale properties of collagen fibrils also play a significant role in determining cell phenotype. Type I collagen fibrils assembled into thin films provide excellent viewing of cells interacting with individual fibrils. Cells can be observed to extensively manipulate the fibrils, and this behavior seems to result in an incompletely spread stellate morphology and a nonproliferative phenotype that is typical of these cells in collagen gels. We show here that thin films of collagen fibrils can be dehydrated, and when seeded on these dehydrated fibrils, smooth muscle cells spread and proliferate extensively. The dehydrated collagen fibrils appear to be similar to the fully hydrated collagen fibrils in topology and in presentation of β1 integrin ligation sites, but they are mechanically stiffer. This decrease in compliance of dehydrated fibrils is seen by a failure of cell movement of dehydrated fibrils compared to their ability to rearrange fully hydrated fibrils and from direct measurements by nanoindentation and quantitative atomic force measurements. We suggest that increase in the nanoscale rigidity of collagen fibrils can cause these cells to assume a proliferative phenotype. 相似文献
2.
The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator. 相似文献
3.
Macrophages (Mφs) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, Mφs reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by Mφs is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors. 相似文献
4.
Modulation of smooth muscle cell behaviour by platelet-derived factors and the extracellular matrix 总被引:2,自引:0,他引:2
We have studied the combined effects of platelet-derived soluble factors and three types of macromolecular substrata on the proliferation and migration of smooth muscle cells in vitro. Bovine aortic smooth muscle cells were plated onto three-dimensional gels of type I collagen or onto cell-free extracellular matrices deposited on such gels by either bovine aortic endothelial cells or smooth muscle cells. The cells were cultured in the presence of whole-blood serum (WBS) or platelet-poor plasma (PPP). Smooth muscle cell proliferation on type I collagen gels was dependent on the presence of platelet-derived factors, i.e. the cells proliferated in the presence of WBS but not in PPP. In contrast, cell proliferation on the extracellular matrices occurred at the same rate in PPP and WBS. Smooth muscle cells plated onto collagen gels rapidly migrated down into the gel matrix; the percentage of cells migrating was inversely proportional to cell density. The presence of extracellular matrices did not alter the rate of cell migration into the underlying gel matrix. Irrespective of the substratum used, smooth muscle cell migration was independent of platelet-derived or plasma factors and occurred in the absence of proliferation. These results indicate that possible chemotactic, chemokinetic, and/or mitogenic factors produced by the vascular cells and deposited within the extracellular matrix may play an important role in modulating smooth muscle cell behaviour in the vascular wall. 相似文献
5.
Douglas E. Coplen Pamela S. Howard John W. Duckett Howard M. Snyder Edward J. Macarak 《In vitro cellular & developmental biology. Animal》1994,30(9):604-608
Summary For the first time we report on the growth, culture, and matrix production characteristics of a cell type isolated from the
lamina propria of the urinary bladder wall. A fibroblastlike cell was identified as distinct from bladder detrusor smooth
muscle cells and urothelium based on morphology, growth characteristics, and immunohistochemical staining. Characterization
of extracellular matrix synthesis by this cell type using35S-methionine metabolic labeling demonstrated that these cells are capable of secreting components of the surrounding connective
tissue, including several fibrillar collagens, a basement membrane collagen, and fibronectin. 相似文献
6.
The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture 总被引:1,自引:0,他引:1
Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present. 相似文献
7.
8.
Enderling H Alexander NR Clark ES Branch KM Estrada L Crooke C Jourquin J Lobdell N Zaman MH Guelcher SA Anderson AR Weaver AM 《Biophysical journal》2008,95(5):2203-2218
Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells. 相似文献
9.
The formation of microvascular sprouts during angiogenesis requires that endothelial cells move through an extracellular matrix. Endothelial cells that migrate in vitro generate forces of traction that compress (i.e., contract) and reorganize vicinial extracellular matrix, a process that might be important for angiogenic invasion and morphogenesis in vivo. To study potential relationships between traction and angiogenesis, we have measured the contraction of fibrillar type I collagen gels by endothelial cells in vitro. We found that the capacity of bovine aortic endothelial (BAE) cells to remodel type I collagen was similar to that of human dermal fibroblasts—a cell type that generates high levels of traction. Contraction of collagen by BAE cells was stimulated by fetal bovine serum, human plasma-derived serum, bovine serum albumin, and the angiogenic factors phorbol myristate acetate and basic fibroblast growth factor (bFGF). In contrast, fibronectin and immunoglobulin from bovine serum, several nonserum proteins, and polyvinyl pyrrolidone (a nonproteinaceous substitute for albumin in artificial plasma) were not stimulatory. Contraction of collagen by BAE cells was diminished by an inhibitor of metalloproteinases (1, 10-phenanthroline) at concentrations that were not obviously cytotoxic. Zymography of proteins secreted by BAE cells that had contracted collagen gels revealed matrix metalloproteinase 2. Subconfluent BAE cells that were migratory and proliferating were more effective contractors of collagen than were quiescent, confluent cells of the same strain. Moreover, bovine capillary endothelial cells contracted collagen gels to a greater degree than was seen with BAE cells. Collectively, our observations indicate that traction-driven reorganization of fibrillar type I collagen by endothelial cells is sensitive to different mediators, some of which, e.g., bFGF, are known regulators of angiogenesis in vivo. © 1996 Wiley-Liss, Inc. 相似文献
10.
Biosynthesis of sulphated macromolecules by rabbit lens epithelium. II. Relationship to basement membrane formation
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Rabbit lens epithelial cells display a similar "cobblestone" morphology and produce the same complement of sulphated macromolecules (also see Heathcote, J.G., and R.W. Orkin, 1984, J. Cell Biol., 99:852-860) whether grown on plastic or glass, dried films of gelatin or type IV collagen with laminin, or on gels of type I collagen. There was no evidence of basement membrane formation by these cells when they were grown on plastic, glass, or dried films. In contrast, cultures that had been grown on gels deposited a discrete basement membrane that followed the contours of the basal surfaces of the cells and in addition, they secreted amorphous basement membrane-like material that diffused into the interstices of the gel and associated with the collagen fibrils of the gel. A significant proportion (approximately 70%) of the heparan sulphate proteoglycan fraction that was secreted into the culture medium (fraction MI) when the cells were grown on plastic became associated with the cell-gel layer in the gel cultures. Further, when basement membrane was isolated by detergent extraction, greater than 90% of the 35S-labeled material present was in this heparan sulphate proteoglycan. 相似文献
11.
Kirkpatrick ND Andreou S Hoying JB Utzinger U 《American journal of physiology. Heart and circulatory physiology》2007,292(6):H3198-H3206
To better understand interstitial matrix remodeling during angiogenesis, we probed endogenous optical signatures of collagen fibrils and cells with multiphoton microscopy to noninvasively visualize, in real-time, changes to fibril organization around angiogenic sprouts and growing neovessels. From analyses of the second-harmonic generation signal from fibrillar collagen and two-photon excited fluorescence, as well as coherent transmitted light from vascular cells, we found that microvessel fragments interacting with the collagen matrix exhibited two key features: a strong association of fibrillar collagen around the parent vessel fragment during vessel construct reconstitution and a substantial collagen fibril reorganization by sprout and neovessel tips. Results indicate that angiogenic sprouts and growing neovessels actively and differentially remodel existing collagen fibrils. This imaging approach to assess local changes in matrix organization may have a broader impact on tissue biology and mechanics during angiogenesis and allow for new insights in cardiovascular, diabetes, and cancer research. 相似文献
12.
A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X 总被引:2,自引:0,他引:2
G. P. Bates S. L. Schor M. E. Grant 《In vitro cellular & developmental biology. Plant》1987,23(5):374-380
Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen
or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that
cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic
or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than
the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated
that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes
cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen,
independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions
of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional
gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial
array could be an important aspect in the control of type IX and X collagen expression by chondrocytes.
The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged. 相似文献
13.
The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells. 相似文献
14.
Modulator protein as a component of the myosin light chain kinase from chicken gizzard. 总被引:30,自引:0,他引:30
The Ca2+-dependent regulation of smooth muscle actomyosin involves a myosin light chain kinase (ATP: myosin light chain phosphotransferase). It has been shown (Dabrowska, R., Aromatorio, D., Sherry, J.M.F., and Hartshorne, D.J. 1977, Biochem. Biophys. Res. Commun. 78, 1263) that the kinase is composed of two proteins of approximate molecular weights 105 000 and 17 000. In this communication it is demonstrated that the 17 000 component is the modulator protein. This conclusion is based on: (1) the identical behavior of the 17 000 kinase component and modulator protein in assays of actomyosin Mg2+-ATPase activity, phosphorylation of myosin, and phosphodiesterase activity, and, (2) the similarity of the 17 000 kinase component and the modulator protein with respect to amino acid composition, absorption spectrum, and electrophoresis in urea-polyacrylamide gels. It is shown also that the modulator protein from smooth muscle and troponin C are distinct proteins. 相似文献
15.
Fibronectin, hyaluronan, and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration. 总被引:7,自引:0,他引:7
"Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo. 相似文献
16.
Effect of heparin on synthesis of short chain collagen by chondrocytes and smooth muscle cells 总被引:1,自引:0,他引:1
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The treatment of embryonic chick chondrocyte cultures with heparin results in a decrease in collagen synthesis. One of the collagens synthesized by hypertrophic chondrocytes, specifically type X collagen, may play an important role in cartilage mineralization and endochondral ossification. Recently a new short chain collagenous component was found in cultures of rat vascular smooth muscle cells (Majack, R. A., and P. Bornstein, 1985, J. Cell Biol., 100: 613-619). The present study was initiated to investigate heparin's effect on type X collagen in embryonic chick chondrocytes and to further evaluate the nature of the short chain component synthesized by rat vascular smooth muscle cells. Different tissues may respond differently to the administration of heparin. In chondrocyte cultures heparin decreased both total collagen synthesis as well as the synthesis of type X collagen. There was an accumulation of collagen precursors, found principally in the cell layer compartment, which appeared to be the result of heparin's inhibition of the NH2-terminal protease. In cultures of rat vascular smooth muscle cells heparin was found to increase the synthesis of a short chain collagenous component as previously reported. However, comparison with a type X collagen standard showed this to be different from type X. In all cases, the effect of heparin on collagen chain precursors, chondrocyte type X synthesis, and synthesis of a vascular smooth muscle short chain collagen was shown to be reversible. Similar effects were obtained by adding chondroitin sulfate to chondrocytes, suggesting a role for extracellular matrix components in the modulation of collagen synthesis. These findings are consistent with the concept of a group of short chain collagens with type X collagen being unique to hypertrophic chondrocytes. 相似文献
17.
Jessica Trombetta-eSilva Erik P. Eadie Yuhua Zhang Russell A. Norris Thomas K. Borg Amy D. Bradshaw 《PloS one》2013,8(11)
Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts. 相似文献
18.
Fibroblasts retain their tissue phenotype when grown in three-dimensional collagen gels 总被引:4,自引:0,他引:4
Fibroblasts are responsible for the synthesis, assembly, deposition, and organization of extracellular matrix molecules, and thus determine the morphology of connective tissues. Deposition of matrix molecules occurs in extracellular compartments, where the sequential stages are under cellular control. Cell orientation/polarity is important in determining how the cell orients these extracytoplasmic compartments and therefore how the matrix is assembled and oriented. However, the control of cell orientation is not understood. Fibroblasts from three tissues with different morphologies were studied to determine whether cells maintained their characteristic phenotype. Fibroblasts from cornea, which in vivo are oriented in orthogonal layers along with their matrix; from tendon, a uniaxial connective tissue, where cells orient parallel to each other; and from dermis, a connective tissue with no apparent cellular orientation, were used to study cell morphology and orientation in three-dimensional collagen gels. The different cells were grown for 3 and 7 days in identical three-dimensional collagen gels with a nonoriented matrix. Confocal fluorescence microscopy demonstrated that corneal fibroblasts oriented perpendicular to one another at 3 days, and after 7 days in hydrated gels these cells formed orthogonal sheets. Tendon fibroblasts were shown by the same methods to orient parallel to one another in bundles at both 3 and 7 days, throughout the depth of the gel. Dermal fibroblasts showed no apparent orientation throughout the hydrated gels at either time point examined. The organization of these different cell types was consistent with their tissue of origin as was the cell structure and polarity. These studies imply that cellular and tissue phenotype is innate to differentiated fibroblasts and that these cells will orient in a tissue-specific manner regardless of the extracellular matrix present. 相似文献
19.
When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins. 相似文献
20.
Ascorbate-generated endogenous extracellular matrix affects cell protein synthesis in calf aortic smooth muscle cells 总被引:1,自引:0,他引:1
Mark A. Zern Elaine Schwartz Marie-Adele Giambrone Olga O. Blumenfeld 《Experimental cell research》1985,160(2):307-318
Ascorbate supplementation of cultured fetal calf aortic smooth muscle cells leads to increased deposition of extracellular matrix proteins and stimulation of cellular protein synthesis (E. Schwartz et al., J cell biol 92 (1983) 462) [7]. In the present study, we have investigated this phenomenon at the level of gene expression. Cells were grown for three weeks on tissue culture plastic with or without ascorbate (50 micrograms/ml). When compared to controls, cells grown in presence of ascorbate had twice as much poly(A+) RNA per microgram of total RNA, and ascorbate led to a 50% increase in [35S]methionine incorporation when the total RNA was translated in the reticulocyte lysate system. SDS-PAGE revealed no change in the protein pattern under the two conditions. "Northern" hybridization revealed a two- to fivefold increase in the sequence content of beta-actin, alpha-tubulin and type I pro alpha 1-collagen in total RNA of ascorbate-supplemented cells, but no difference was observed in the mRNA sequence content for the three specific proteins when equal amounts of poly(A+) RNA from ascorbate and control cells were hybridized with the three cloned cDNAs. To evaluate the effect of an exogenous matrix, cells were also plated on collagen gels. RNA isolated from cells grown on collagen without added ascorbate exhibited translational activity and mRNA sequence content similar to cells grown with ascorbate on tissue culture plastic. In contrast, no differences from controls were found in cells grown for one week in the presence of ascorbate, at which time no significant deposition of collagen occurs in the extracellular matrix. These results suggest that the stimulation in protein synthesis in fetal calf smooth muscle cells supplemented with ascorbate is associated with an increase in the proportion of poly(A+) RNA in the total RNA pool, and that the production of an endogenous collagen-rich matrix in the presence of ascorbate may be the basis for these pretranslational changes. 相似文献