Cell adhesion by aqueous extract of human placenta used as wound healer

Aqueous extract of human placenta, used as wound healer, has shown significant cell adhesion property on mouse peritoneal macrophages and P388D1 cultured macrophage cell line. This property was offered primarily by fibronectin type III like peptide present in the extract and is comparable to fibronectin on a molar basis. The peptide induce adhesion of cell through cell surface receptors having K(d) = 2.8 +/- 0.9 x 10(-5) M suggesting weak binding. This is in support of integrins receptors that typically exhibit low affinities. Cell adhesion was partially inhibited by Arg-Gly-Asp (RGD) peptide, anti-beta1 integrin suggesting that integrin beta1 receptors have roles to play in the process.     

Regulation of trypsin activity by peptide fraction of an aqueous extract of human placenta used as wound healer

An aqueous extract of human placenta, used as wound healer, shows stabilization of trypsin against autodigestion as one of the peptides of the extract binds very strongly with the protease. Trypsin retains 40% of activity at constant level between 20 and 26 days in presence of the extract against complete inactivation in its absence. Inhibition of esterolytic activity and inability to react with p-nitrophenyl-p'-guanidinobenzoate, HCl, an active site directed reagent, by trypsin in presence of a peptide fraction of the extract indicated blocking of the catalytic site of the enzyme. Rayleigh scattering, size-exclusion HPLC, fluorescence resonance energy transfer, and surface plasmon resonance show that fibronectin type III-like peptide present in the extract interacts with trypsin. The peptide-trypsin complex is dissociated in presence of high concentration of substrates. Thus, regulation of trypsin activity by the placental extract is evident.     

Atropa belladonna L. water extract: modulator of extracellular matrix formation in vitro and in vivo

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.     

The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded beta-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel beta-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin alpha4beta1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.     

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5β1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.     

Fibronectin (cold-insoluble globulin), VI. Influence of heparin and hyaluronic acid on the binding of native collagen.

Fibronectin of human plasma associated readily with denatured collagen but gave only a weak reaction with the native protein. In the presence of heparin, however, solutions of native collagen type III, and fibronectin produced precipitates at an ionic strength of 0.2. In the presence of fibronectin and optimal additions of heparin, up to 60% of soluble native 125I-collagen type III, but only about 10% of native 125I-collagen type I, were insolubilized. Heparin also enhanced the formation of insoluble complexes from fibronectin and denatured collagen type I and type III. In the absence of collagen 125I-fibronectin was partially precipitated by heparin. Electron micrographs showed filamentous structures. Collagen did not increase the amount of 125I-fibronectin precipitated by heparin unless a critical collagen concentration was exceeded. It is suggested that heparin induced the transition of fibronectin from a globular to an elongated form, capable of forming filamentous precipitates which adsorb native collagen. Hyaluronic acid and putrescine prevented the insolubilization of native collagen type III, by fibronectin and heparin.     

Mapping the recognition domains of pneumococcal fibronectin‐binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin‐binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non‐human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both β‐sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII¹, FnIII⁵ or FnIII¹⁵ bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.     

Degradation of fibrin-β amyloid co-aggregate: A novel function attributed to ubiquitin

Human placental extract contains numerous bioactive components that are effective wound healing, antimicrobial and anti-inflammatory agents. During our investigation on the therapeutic potency of human placental extract, we have purified ubiquitin-like molecules that showed strong fibrino(geno)lytic activity. Further investigation confirmed similar potency of ubiquitin purified from adult human erythrocyte. Additionally, ubiquitin efficiently degraded disordered amyloid β 42 peptide (Aβ42) aggregate and fibrin-Aβ42 co-aggregate in vitro and reduced co-aggregate induced cytotoxicity in SH-SY5Y human neuroblastoma cells as compared to plasmin. Ubiquitin also degraded abnormal co-aggregates of fibrin with other plasma proteins such as fibronectin, albumin, lysozyme, tranthyretin and α-synuclein. To elucidate the mechanism of degradation, synthetic peptides (ADG, GKT, DQQ, QRL, LIF, AGK, HLVL) derived from ubiquitin template as well as synthetic ubiquitin (8565.32?Da) were employed. Synthetic ubiquitin completely degraded preformed Aβ 42 aggregate and fibrin-Aβ42 co-aggregate, whereas, the smaller synthetic peptides showed varying degrees of degradation. These observations attribute a novel function of ubiquitin that may be used for degrading abnormal fibrin clots in human body. Thorough investigation might unfold a novel molecular mechanism of ubiquitin in protein homeostasis.     

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECM components.     

Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain

Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.     

Isolation and characteristics of ligand specificity of VLA-1 integrin from human smooth muscles]

Using affinity chromatography with immobilized monoclonal antibodies to the beta 1-subunit of human integrin, a total integrin fraction (subfamily beta 1) was isolated from the detergent extract of human smooth muscle (uterus). Immunoprecipitation and immunoblotting with specific antibodies revealed integrins VLA-1 and VLA-5. The former was isolated in a homogeneous state by chromatography on immobilized type I collagen in the presence of 1 mM Mn2+. The pure receptor yield was 2-4 mg per 400 g of smooth muscle tissue. Analysis of substrate specificity of VLA-1 in the liposome test revealed that this integrin possesses a broad spectrum of ligand specificity and can interact via a Ca2+, Mg(2+)-dependent mechanism with interstitial collagens of I, II and III types and with basal membrane proteins (type IV collagen and laminin). VLA-1 does not interact with fibronectin, thrombospondin or albumin. Denaturation of type I collagen decreases the liposome binding 5-7-fold. The peptide Gly-Arg-Gly-Asp-Ser-Pro added to the incubation mixture does not inhibit the liposome interaction with incorporated VLA-1 integrin, type I collagen and laminin.     

Evolution of the fibronectin gene. Exon structure of cell attachment domain

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.     

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).     

Collagen composition of normal and myxomatous human mitral heart valves.

The collagens were studied in 13 normal and 19 myxomatous human mitral valves. The collagens of the valve were completely solubilized by using a method consisting of guanidinium chloride extraction, limited pepsin digestions and CNBr cleavage of the residue. The normal valves contained 74% type I, 24% type III and 2% type V collagen. The type I and type III collagens had similar solubility patterns, although only type I collagen was detected in the guanidinium chloride extract. Type V collagen was only detected in the first pepsin extract. The type I and III collagens had higher contents of hydroxylysine than did the same collagens from age-matched dermis. The two-dimensional electrophoretic 'maps' of CNBr-cleavage peptides showed low recoveries of the C-terminal alpha 1(I) CB6 and alpha 1(III) CB9 peptides, which are involved in forming intermolecular cross-linkages. Most of the reducible cross-linkages were present in large-Mr peptide complexes, and these complexes were shown by labelling with 125I to include the tyrosine-containing alpha 1(I) CB6 peptide. The myxomatous valves contained 67% type I, 31% type III and 2% type V collagens. There was a significant increase in the concentration of each type of collagen, which consisted of a 9% increase of type I collagen, a 53% increase of type III collagen and a 25% increase of type V collagen. The contents of hydroxylysine in type I and III collagens and the electrophoretic 'maps' of the CNBr-cleavage peptides involved in cross-linkages did not differ significantly from the results obtained from the normal valves. The biochemical findings suggest that there is an increased production of collagen, in particular type III collagen, and glycosaminoglycan as well as a proliferation of cells as part of a repair process in the myxomatous valves.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

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.     

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.     

Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti

Endogenous chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. The chitinase gene was cloned from B. thuringiensis serovar alesti strain HD-16, and the deduced 676 amino acid sequence showed a high degree of similarity with other Bacillus chitinases. Additionally, the deduced amino acid sequence showed that the protein contained an amino terminus signal peptide and consisted of a catalytic domain, a fibronectin type III domain and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase or cellulase sequences.     

A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices.

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.     

Fibronectin involvement in granulation tissue and wound healing in rabbits

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.