Multiple and masked pools of fibronectin in murine fibroblast cell-substratum adhesion sites

Substrate-attached material (SAM) prepared from murine BALB/c 3T3 cells and various derivatives contains adhesion sites which pinch off from the cell surface during EGTA-mediated detachment but which remain bound to the serum-coated tissue culture substratum. SAM contains the related adhesive glycoproteins cold-insoluble globulin (CIG) (from serum in the medium) and fibronectin (synthesized by the cells) as detected by immune staining of electrophoretically separated proteins, using antibodies of defined specificity. Serum and SAM contain cross-linked multimers of serum-derived CIG (not disulfide-mediated) but not of cell-derived fibronectin; therefore, thiol-resistant cross-linking between CIG and fibronectin is not involved in adhesion of these cells. Immunofluorescence microscopy of SAM from sparse cultures reveals fibrillar pools containing cellular fibronectin, although most retraction fibers seen on EGTA-treated cells do not stain, even after treatment with non-ionic detergent. Very little specific staining can be detected in SAM prepared from dense cultures, although gel electrophoretic analysis reveals proportionately as much murine fibronectin as is found in SAM from sparse cultures. Hyaluronidase digestion of SAM has no effect on the immunofluorescent staining, while gentle trypsin digestion completely abolishes staining without removing all biochemically detectable fibronectin. We conclude that some of the fibronectin and CIG in adhesion sites is masked and unavailable for antibody binding and that multiple pools of fibronectin exist in this adhesive material.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.     

Biosynthesis of laminin and fibronectin by rat satellite cells during myogenesis in vitro

The biosynthesis of fibronectin and laminin was studied in satellite cells cultured from adult rat muscles before (day 4) and after fusion and formation of myotubes (day 14) using (35S) methionine as a tracer. The kinetics of incorporation into immunoprecipitable fibronectin and laminin were recorded at intervals from 1.5 to 24 hours of incubation with the tracer from the cells, the pericellular matrix and the culture medium. The rate of synthesis of fibronectin and laminin expressed as dpm/microgram DNA were constant from the mononucleated cell to the myotube state. Both glycoproteins were detected in the cells and in the pericellular matrix. When the results were expressed as the percentage of incorporation into total protein, major changes could be observed in the early phase of the kinetic studies in the cells and the pericellular matrix. Both showed an increase from the mononucleated myoblast to myotube, suggesting that an increasing fraction of total protein biosynthesis is directed towards these two extracellular matrix glycoproteins. At the same time, there was a decrease in the secretion into the medium of freshly synthesized radiolabeled fibronectin and laminin. Our results confirm the synthesis of varying ratios of both extracellular matrix macromolecules by undifferentiated mononucleated myogenic cells as well as myotubes.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Structure of the carbohydrate units of human amniotic fluid fibronectin

Human amniotic fluid fibronectin was found to contain three types of carbohydrates: complex-type N-glycosidic glycans, lactosaminoglycans, and O-glycosidic glycans. The structures of the complex-type glycans were established by carbohydrate and methylation analysis, Smith degradation, sequential exoglycosidase treatments, lectin chromatography, and DEAE-Sephadex chromatography. Lactosaminoglycans were analyzed by fast atom bombardment mass spectrometry, and the O-glycosidically-linked oligosaccharides by gas-liquid chromatography-mass spectrometry and high-pressure liquid chromatography. The results show that amniotic fluid fibronectin contains 2 mol of biantennary and 2-3 mol of triantennary, complex-type N-glycosidic glycans. Unlike the N-glycosidic glycans of human adult plasma fibronectin, which contain only traces of fucose and are completely sialylated, the glycans from amniotic fluid fibronectin are fucosylated and only partially sialylated. The complex-type N-glycosidic glycans present in amniotic fluid fibronectin also include a fractional amount (0.1 mol) of glycans with a polylactosaminyl structure. In addition, 4 mol of O-glycosidic oligosaccharides, which have not previously been described in fibronectins, were found in amniotic fluid fibronectin. The major oligosaccharides in this fraction have the structures Gal beta 1----3GalNAcol, NeuNAc alpha 2----3Gal beta 1----3GalNAcol and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcol. O-glycosidically linked oligosaccharides were also detected in human adult plasma fibronectin but in smaller amounts than in amniotic fluid fibronectin. These results show that amniotic fluid fibronectin differs from plasma fibronectin with regard to the number of glycans attached to the polypeptide and that the glycans present in these two fibronectins differ in structure.     

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

ED-A sequence containing fibronectin in human amniotic fluid and amnion epithelial cells

1. Human amniotic fluid fibronectin (aFn) was studied by using a monoclonal antibody 52DHl (DH) that recognizes the extra domain (ED-A) sequence of cellular Fn (cFn). 2. In immunoblotting the DH antibody reacted with a sharp polypeptide band at the top of the bulk of the diffuse aFn. Another monoclonal antibody 52BF12 (BF) against the cell binding site of Fn, recognized the whole aFn. 3. The ED-A sequence containing cFn (EcFn) formed a constant proportion in aFns from all amniotic fluid preparations studied. 4. In amniotic membranes the DH antibody revealed bright subepithelial immunofluorescence. 5. Also isolated and cultured human amnion epithelial cells were strongly positive in immunofluorescence and secreted EcFn into the culture medium as revealed by immunoblotting. 6. The results indicate that aFn is a composition of at least two different Fn subtypes of which the EcFn most probably originates from amnion epithelial cells.     

Characterization of a type IV procollagen synthesized by human amniotic fluid cells in culture.

Fetal epithelioid cells, isolated from human amniotic fluid, synthesize and secrete a type IV-like procollagen characterized by a unique pattern of cyanogen bromide (CNBr)-produced peptides. The procollagen is disulfide-bonded and, after reduction, migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a doublet between collagen beta components and pro-alpha 1(I) chains. No conversion of the procollagen to collagen or to procollagen intermediates is observed in cell culture. The procollagen was purified by salt fractionation and ion exchange chromatography; its amino acid composition resembles that of collagenous proteins extracted from basement membranes, with a high 3- and 4-hydroxyproline and hydroxylysine content and low levels of alanine and arginine. The major products obtained after limited proteolytic digestion of the protein retain interchain disulfide bonds and, after reduction, migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis near intact pro-alpha 1(I) chains. The procollagen is secreted efficiently by amniotic fluid cells despite almost complete inhibition of peptidyl hydroxylation but, unlike type I procollagen, the secreted underhydroxylated chains lack interchain disulfide bonds. Since these cells also secrete fibronectin and elaborate an extensive extracellular matrix, the system should prove useful in the study of cell-matrix interactions.     

Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

Regulation of growth and gene activity in euploid hybrids between human neonatal fibroblasts and epithelioid amniotic fluid cells.

Pure populations of proliferating synkaryons were obtained from polyethylene glycol-mediated crosses between diploid human foreskin fibroblasts and epithelioid amniotic fluid cells. These hybrids proved to be chromosomally stable tetraploids. They continuously produced heteropolymeric G6PD and showed strictly additive patterns of silver staining of both parental sets of nucleolar organizing chromosomes. Collagenous proteins characteristic of the fibroblast parent were synthesized, while fibronectin production appeared to be directed by the epithelioid portion of the genome. Even though these heterotypic hybrids proliferated at a reduced rate and achieved fewer population doublings relative to homotypic (fibroblast X fibroblast) crosses, they survived passage by trypsinization better than pure populations of epithelioid cells. These observations suggest a concerted action of both parental genomes with respect to proteins responsible for "household" functions, but complementation and possibly modulation of gene action with respect to "luxury" protein synthesis and cell growth.     

An irreversible tissue inhibitor of collagenase in human amniotic fluid: Characterization and separation from fibronectin

Soluble fibronectin isolated from human plasma and amniotic fluid by gelatin-Sepharose affinity chromatography was tested for inhibitory activity against specific collagenase secreted by human and rabbit fibroblasts. The fibronectin preparation derived from plasma showed little inhibition, but the one derived from amniotic fluid contained potent inhibitory activity against collagenase. This activity was separated from fibronectin on a DE-52 cellulose column and did not cross-react with antibodies to fibronectin. The inhitor was a glycoprotein that was partially purified from amniotic fluid by concanavalin A-Sepharose affinity chromatography. Inhibition was irreversible and enzyme activity was not recovered after reaction with latent or activated collagenase by either trypsin or organomercurial treatment.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

Developmental changes in the glycosylation and binding properties of human fibronectins. Characterization of the glycan structures and ligand binding of human fibronectins from adult plasma, cord blood and amniotic fluid

Fibronectins from human adult plasma, fetal plasma and from amniotic fluid obtained during early and late gestation were compared with respect to (i) their reactivity with lectins, (ii) their binding to the physiological ligands gelatin and heparin, and (iii) the role of the carbohydrate residues in the binding to these two ligands. The two fibronectin isoforms displayed distinct developmental differences in both glycosylation and binding properties: (i) Proportions of tri/tetraantennary complex glycans compared to the fraction of biantennary structures, as inferred from the reactivity with concanavalin A, were highest in amniotic fluid fibronectin from late pregnancy, lower in amniotic fluid fibronectin from early gestation, and even lower in fetal and adult plasma fibronectins. Likewise, fucose (alpha 1-6) linked to the innermost N-acetylglucosamine of the chitobiosyl core, defined by reactivity with Lens culinaris agglutinin (LCA), was present primarily in amniotic fluid fibronectin, and decreased in content during gestation from the 2nd. to the 3rd. trimenon. Both fetal and adult plasma fibronectins were only weakly reactive with LCA, indicating a low content of (alpha 1-6) linked fucose residues. After prior treatment with sialidase, both plasma and amniotic fluid fibronectins strongly reacted with erythrocyte phytohaemagglutinin (E-PHA), indicating that both fibronectin isoforms contain bisecting (beta 1-4) N-acetylglucosamine residues. Amniotic fluid fibronectins showed much greater reactivity than adult and fetal plasma fibronectins with wheat germ agglutinin; binding of this lectin to amnion fluid fibronectins was not decreased by desialylation indicating the presence of poly(N-acetyllactosamine) units. Whereas amniotic fluid fibronectins were strongly reactive with peanut agglutinin, neither adult nor fetal plasma fibronectins did bind to this lectin unless after prior desialylation. Hence, both fibronectin isoforms contain O-glycan residues that are fully sialylated in fetal and adult plasma fibronectins, but only partly sialylated in amniotic fluid fibronectins. According to these differences, glycosylation of plasma and amniotic fluid fibronectins is under developmental regulation. (ii) Amniotic fluid fibronectins had a significantly lower binding activity for both heparin and gelatin than plasma fibronectins. Moreover, amnion fibronectin from late gestation displayed a significantly lower binding to these two ligands than amnion fibronectin from early gestation. Fetal plasma fibronectins had a lower binding activity for gelatin than adult plasma fibronectin. (iii) Treatment of fibronectins with sialidase, fucosidase and removal of N-glycans with endoglycosidases H and F did not affect binding to gelatin and heparin, indicating that the interaction of plasma and amnion fibronectin with these two ligands is not influenced by their oligosaccharide moieties.     

Matrix metalloproteinases MMP-2 and MMP-9 of wound and burn exudates and their effect on extracellular matrix proteins

The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.     

Biosynthesis of fibronectin by rabbit aorta

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found. These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.     

Demonstration of astrocytes in cultured amniotic fluid cells of three cases with neural-tube defect

Summary We have investigated the origin of rapidly adhering (RA) cells in three cases of neural tube defects (two anencephali, one encephalocele). We were able to demonstrate the presence of glial fibrillary acidic (GFA) protein in variable percentages (4–80%) of RA cells cultured for 4–6 days by use of indirect immunofluorescence with GFA antiserum. Cells cultured from amniotic fluids of normal pregnancies and fetal fibroblasts were completely GFA protein negative. GFA protein is well established as a highly specific marker for astrocytes. Demonstration of astrocytes may prove to be a criterion of high diagnostic value for neural tube defects. The percentage of astrocytes decreased with increasing culture time, while the percentage of fibronectin positive cells increased both in amniotic fluid cell cultures from neural tube defects and normal pregnancies.     

Fibroblast receptor for cell-substratum adhesion: studies on the interaction of baby hamster kidney cells with latex beads coated by cold insoluble globulin (plasma fibronectin)

Studies were carried out on the interactions of uncharged latex beads (0.76 micrometer) with baby hamster kidney cells. Binding of beads to the cells occurred if the beads were coated by cold insoluble globulin (CIG) (plasma fibronectin) but not if the beads were coated by bovine albumin. Bovine albumin-coated beads did not bind to the cells even in the presence of excess CIG in the incubation medium. Binding of beads occurred randomly over the entire surfaces of cells in suspension. However, cell receptors for CIG beads were no longer detectable on the upper surface of cells spread onCIG-coated tissue culture dishes. Binding of CIG beads to cells occurred at all temperatures tested from 4 degrees to 37 degrees C but the rate was lowest at 4 degrees C. At 37 degrees C, binding was accompanied by endocytosis and the beads were found inside vesicles which appeared to be lysosomes. There was also release of radioactivity from radiolabeled CIG beads during incubation with the cells at 37 degrees C. Binding of CIG beads to cells did not require divalent cations. Finally, the cell receptor for CIG beads was lost after cell trypsinization. The data are discussed in terms of current ideas about the basis for cell adhesion.     

Fibrin-enhanced endothelial cell organization

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.