Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks

Different biochemical and cytochemical techniques were applied to characterize the sites of localization of thrombospondin in cultured endothelial cells. The results obtained by [35S]methionine labeling, immunoblotting, immunoprecipitation, fluorescence microscopy, ultracytochemistry, immunogold labeling, and silver enhancement experiments revealed that thrombospondin secreted by endothelial cells is structurally organized together with proteoheparan sulfate in spherical granules at the cell surface. These granules are about 100 to 300 nm in size. Heparin or enzymatic degradation with heparitinase, but not with ABC lyase, release thrombospondin from the cell surface. Fibronectin is expressed in the extracellular matrix of endothelial cells in a fibrillar organization, clearly distinct from the punctate pattern of thrombospondin on the cell surface. Furthermore, secreted thrombospondin is highly enriched together with fibronectin and proteoheparan sulfate in cell attachment sites and in cell migration tracks. In cell migration tracks proteoheparan sulfate more clearly resembles the fibrillar distribution pattern of fibronectin, whereas thrombospondin reveals a rather monodisperse pattern. The obtained data suggest preferential sites of interaction between thrombospondin and heparan sulfate proteoglycans on the cell surface and a participation of thrombospondin in cell adhesion and cell migration.     

Thrombospondin: biosynthesis, distribution, and changes associated with wound repair in corneal endothelium

Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.     

Immunocytochemical localization of coated vesicle protein in rodent nervous system

Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.     

Thrombospondin is an osteoblast-derived component of mineralized extracellular matrix

Thrombospondin, the most abundant protein of platelet alpha granules, is a biosynthetic product of a variety of connective tissue cells and a component of many extracellular matrices. In this study, thrombospondin distribution in bone was investigated using a monoclonal antibody specific for the human protein. Thrombospondin was localized in osteoid of undemineralized, frozen sections of fetal subperiosteal bone, and identified as a component of mineralized bone matrix of neonatal and/or young (growing) bone of many animal species by Western blot analysis. Adult human bone cells were demonstrated to contain mRNA for thrombospondin by hybridization of a cDNA thrombospondin probe to a 6.1 kb mRNA. Pulse-chase experiments indicated that the protein was synthesized and the majority was secreted from osteoblastic cells. Treatment of the cells with TGF-beta (0.01-10 ng/ml) slightly decreased total thrombospondin synthesis, but caused an increase in the retention on newly synthesized thrombospondin in the cell layer/matrix fraction. In cell attachment assays, thrombospondin mediated adhesion, but not spreading of adult human bone cells.     

Binding and degradation of platelet thrombospondin by cultured fibroblasts

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I- thrombospondin. The second fraction was localized to a trypsin- sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I- thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.     

Studies on the assembly of the rat lens capsule. Biosynthesis and partial characterization of the collagenous components.

1. Isolated rat lens capsules synthesized hydroxy[3H]proline-containing polypeptides when incubated with [3H]proline. 2. The collagenous polypeptides synthesized during a 2 h incubation were analyzed by both gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and shown to have an apparent mol.wt. of approx. 180,000. 3. No evidence was obtained for conversion of these polypeptides into a lower-molecular-weight species in experiments where capsules were labelled for 2 h and chased with non-radioactive proline for up to 22 h. However, a time-dependent incorporation of the 180,000-mol.wt. species into a larger collagenous component was observed and this could be prevented by the inclusion of beta-aminopropionitrile in the incubation medium. 4. The radioactive components synthesized by the capsules correspond to subunits of the intact lens capsule and the direct incorporation of the polypeptide of mol.wt. 180,000 into deoxycholate-insoluble basement membrane was demonstrated.     

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.     

Two-dimensinal gel electrophoresis of rat liver nuclear washes,nuclear matrix,and hnRNA proteins

The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000- 75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA- bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.     

Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin

Type 1 plasminogen activator inhibitor (PAI-1) binds to the extracellular matrix of cultured bovine aortic endothelial cells. Bovine plasma and bovine lung extract contain protein(s) that bind to PAI-1 and prevent this interaction. One of these proteins was purified approximately 425-fold from ammonium sulfate-fractionated plasma using standard chromatographic procedures together with affinity chromatography on PAI-1-Sepharose. The final product consisted of a major polypeptide of Mr 65,000 and two minor polypeptides of Mr 80,000 and 57,000. NH2-terminal amino acid sequence analysis of the Mr 65,000 polypeptide revealed that it was homologous with vitronectin, and antiserum against this purified binding protein recognized vitronectin and vice versa. Immunological analysis using these antisera demonstrated that the three peptides were immunologically related, and that vitronectin was present in the extracellular matrix of bovine endothelial cells and also in bovine lung.     

A major heat-shock protein defined by a monoclonal antibody.

A monoclonal antibody reacts with a polypeptide of 68 000 mol. wt. (p68) that accumulates to high levels during heat shock. The intracellular distribution of this antigen in normal and heat-shocked cells has been studied. It is a major component of non-stressed cells, where it is located predominantly in the cytoplasm, but also occurs in the nucleus. The nuclear accumulation is growth regulated, in that exponentially growing cells have strong nuclear immunofluorescence and confluent cells little. It is concentrated at the leading edge of motile fibroblasts and co-distributes with actin-containing microfilaments. Heat shock causes cytoplasmic and nuclear accumulation and there is new deposition in the periphery of cells. In normal cells the antigen in the nucleus is located in the nuclear lamina and matrix which increases during heat shock. The distribution of this molecule and the structures with which it interacts suggests that it is important in mediating the effects of heat shock.     

Interactions of thrombospondin with endothelial cells: receptor- mediated binding and degradation

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.     

Comparison of pathways of copper metabolism in aorta and liver. A functional test of metallothionein.

Soluble fractions from chick liver and aorta were examined for copper-binding proteins. In liver a zinc-binding thionein appeared to be the major binding protein for copper. Aortic tissue contained only traces of this thionein protein. Unlike liver, moderate amounts of soluble copper in aorta showed no association with macromolecules. Chicks fed on copper-deficient diets for 8 days had one-third the liver copper concentrations of controls. Aortic copper concentration was decreased only slightly, but the activity of lysyl oxidase, a copper-dependent enzyme in aorta, was decreased significantly. Treating the deficient chicks with CuSO4 (1 mg/kg) restored liver copper rapidly. The increase correlated with the binding of copper to a 10 000-mol.wt. component in the soluble fraction. Aortic copper concentrations responded much less to the CuSO4 treatment, but lysyl oxidase activity was again measurable in the tissue. Radioactive isotopes of copper bound almost exclusively to the 10 000-mol.wt. component in liver and to components of mol.wt. 30 000 or above in aorta. Hardly any of the administered radioactivity appeared with the 10 000-mol.wt. components in aorta, and none was found with unbound copper. The 30 000-mol.wt. components in aorta showed superoxide dismutase activity that was sensitive to NaCN. They also showed the highest specific activity of copper of any other aorta component. A clear distinction was seen between the metabolism of copper in liver and aortic tissues. Whereas a copper thionein, metallothionein, was a major component in the liver pathway, it is doubtful that this protein plays a major role in the intracellular metabolism of copper in aortic tissue.     

Absence of two membrane proteins containing extracellular thiol groups in Rhnull human erythrocytes.

Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.     

Brain clathrin and clathrin-associated proteins.

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.     

Contraction of fibrillar type I collagen by endothelial cells: A study in vitro

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.     

Release of two thioesterase domains from fatty acid synthetase by limited digestion with trypsin.

Limited digestion, with trypsin, of the fatty acid synthetase from rat mammary gland releases an enzymically active thioesterase component that, under denaturing conditions, consists of two major species of mol.wts. 35000 and 17500 and a minor species, mol.wt. 15,000. The 17500- and 150000-mol.wt. species are shown to originate from the 35000-mol.wt. species as a result of nicking by trypsin. The nicked polypeptides are enzymically active. The fatty acid synthetase is inhibited by [1,3-14C]di-isopropyl phosphorofluoridate, which is shown to bind to, and inactivate, two thioesterase active sites. When the [1,3-14C]di-isopropyl phosphate-labelled fatty acid synthetase is subjected to limited digestion with trypsin, all of the radioactivity is recovered in the isolated thioesterase component, i.e. in the 35000-mol.wt. polypeptide and its nicked products. Since the isolated thioesterase is shown to bind only one di-isopropyl phosphate residue per 35000-mol.wt. polypeptide, we conclude that the fatty acid synthetase has two thioesterase domains, both of which are removed by limited trypsin treatment.     

Phosphoproteins: structural components of oncornaviruses.

Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.     

The build-up of the thrombospondin extracellular matrix. An apparent dependence on synthesis and on preformed fibrillar matrix

Thrombospondin (TSP) is a multifunctional protein synthesized by several cell types in culture, among them endothelial cells, and incorporated into the extracellular matrix (ECM) of these cells. In vitro it has been detected in most interstitial spaces, and its presence has been suggested to be developmentally controlled. With the aim of elucidating the role of TSP in the extracellular matrix, we studied the build-up of this protein in the matrix as a function of cell type density and age in culture. The development of the TSP matrix was compared to that of fibronectin (FN) and of von Willebrand Factor (VWF) in the same cultures using our monoclonal anti-TSP antibody B7-3 and monospecific anti-FN or anti-VWF antibodies. In cultures of bovine aortic endothelial cells (BAEC) we observed that both the pattern and distribution of TSP in the matrix changed with cell density and time in culture; it started as a completely amorphous dense layer of protein when the cells were sparse and changed to well organized fibrils when the cells had been confluent for a while. The first appearance of the fibrillar arrays of TSP coincides perfectly with that of the FN fibers; extracellular VWF also was first detected at this point. From this time in culture, there was an increasing coincidence of the TSP with the FN and the VWF matrices suggesting the formation of a complex multicomponent structure of the fibrillar network.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombospondin: synthesis and secretion by cells in culture

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.