Localization of type I and III collagen and fibronectin production in injured gastrocnemius muscle.

The healing of muscle rupture consists of two simultaneous processes, regeneration of disrupted muscle fibers and production of connective tissue scar. These two processes are at the same time supportive to and competitive with each other. Their balanced progression is necessary for optimal healing. Synthesis of three connective tissue proteins, collagen types I and III and fibronectin, was analyzed during the regeneration process from 2 days to 3 weeks by Northern blot and in situ hybridization and immunohistochemistry. For this purpose a partial standard rupture of the gastrocnemius muscle was induced in 56 rats by a strike with a blunt spring-loaded hammer. Northern blot analysis of the specific mRNAs during the healing process revealed distinctly different expression patterns for fibronection and type I and III collagens. During the early stages (days 2 and 3) fibronectin, derived mainly from plasma, was abundant in the traumatized area, but local production of fibronectin mRNA by fibroblasts had also already started by day 2, closely followed by that of type III collagen. This early active synthesis of type III collagen and fibronectin was followed by a decrease after 1 week. The production of type I collagen mRNAs was activated somewhat later and remained elevated for at least 3 weeks. The muscle cells did not contain procollagen mRNAs. The observed sequence of connective tissue proteins reflects the particular function that each carries out during muscle wound healing (e.g., fibronectin in fibroblast trapping, type III collagen in plasticity/flexibility, and type I collagen in tensile strength).     

Localization of collagen types I, III, IV and V, fibronectin and laminin in human arteries by the indirect immunofluorescence method

The distribution of types I, III, IV and V collagen and of the glycoproteins fibronectin and laminin in sections of human aortas, arteries and atherosclerotic plaques were studied using monospecific antibodies and indirect fluorescence microscopy. Types IV and V collagen and laminin were present in a narrow zone, representing the basement membrane, apposed to the endothelial layers of all these tissues. Types I and III collagen and fibronectin were located in the interstitial spaces of the intima and the media of blood vessels walls, whereas types IV and V collagen and laminin were found in the basement membranes underlying smooth muscle cells in these areas. Two types of atherosclerotic plaques were observed. Lipid-rich plaques contained less collagen and reduced amounts of the glycoproteins. Fibrous plaques consisted of regions deficient in types I and III collagen and collagen-rich regions with elevated levels of these two collagens as well as more fibronectin. The collagen-rich regions of fibrous plaques contained, however, little type IV and type V collagen and little of the glycoproteins laminin and fibronectin. This may be due to the reduced number of cells involved in the biosynthesis of these basement membrane proteins.     

Attachment,spreading, and matrix formation by human gingival fibroblasts on porous-structured titanium alloy and calcium polyphosphate substrates

Porous calcium polyphosphate (CPP) structures represent promising resorbable implant systems that can promote anchorage to connective tissues. Previous studies focused on chondrocyte interactions with CPP, but there are limited data on interactions of soft connective tissue cells with these materials. We studied attachment, spreading, and matrix formation by human gingival fibroblasts when cultured on amorphous and crystalline CPP. Comparison with porous Ti6Al4V substrates of similar volume percent, porosity, and pore size distribution provided evaluations of fibroblast interactions with rapid, moderate, and nonbiodegradable systems, respectively. Cells were incubated on substrates in medium containing ascorbic acid and evaluated at 3, 24, 48, 72, and 96 h after plating. Attached cell counts, cytoplasmic actin filament area, and immunostained extracellular type 1 collagen or fibronectin were quantified by morphometric analyses using epifluorescence microscopy. Cell morphology and substrate interactions were evaluated by scanning electron microscopy. Spreading, attachment, and matrix production were similar for both CPP substrates. In contrast, titanium alloy substrates exhibited threefold more attachment and twofold more spreading than CPP substrates. The area per cell of immunostained extracellular collagen and fibronectin was similar for the three different substrates. The results indicate that the crystallinity and, hence, degradation rate of CPP substrates does not substantially affect the interactions of fibroblasts with CPP materials but that compared with titanium alloy substrates, spreading and attachment are inhibited.     

Collagen type I, III, IV and V and fibronectin in skin biopsies of patients with Ehlers-Danlos syndrome and cutis laxa

Immunofluorescence was employed to study distribution of collagen type I, III, IV and V, and fibronectin in skin biopsy specimens from patients with Ehlers-Danlos syndrome (EDS) and cutis laxa. Abnormal distribution of collagen type V in the skin biopsies was found out in EDS type VII. A high level of dermal fibronectin occurred in EDS type III. Tissue fibronectin was absent in the skin extracellular matrix in EDS type X. Defective distribution of collagen, predominantly of type III and V and fibronectin absence were registered in the skin connective tissue in cutis laxa.     

Modification of Ti6Al4V surfaces using collagen I, III, and fibronectin. I. Biochemical and morphological characteristics of the adsorbed matrix

Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). Aiming at load-bearing applications in bone contact, in this study the modification of titanium surfaces with the collagen types I and III in combination with fibronectin was undertaken; fibrillogenesis, fibril morphology and adsorption of type I, III and I/III-cofibrils onto titanium were assessed. Increasing the collagen type III amount resulted in a decrease of fibril diameter, while no significant changes in adsorption could be detected. The amount of fibronectin bound to the heterotypic fibrils depended on fibrillogenesis parameters such as ionic strength or concentration of phosphate, and varied with the percentage of integrated type III collagen.     

Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. III. Effects of chronic exposure to epidermal growth factor on growth, differentiation, and collagen biosynthesis

Studies have been performed to evaluate the effects of chronic exposure to epidermal growth factor (EGF) on a cell line (FRLE cells) established from the fetal rat lung type II alveolar epithelial cell. Chronic exposure to EGF enhanced proliferation and altered the culture morphology at the light microscopic level. At the ultrastructural level, chronic exposure to EGF inhibited the expression of lamellar body-like structures that occurs in the absence of EGF. Estimations of total protein and collagen production indicated that chronic exposure to EGF suppressed collagen production without significantly altering total protein synthesis. Quantitative evaluation of the genetic types of collagen (types I, III, IV, and V) produced by FRLE cells revealed decreased production of each collagen type in cultures chronically exposed to EGF. However, the magnitude of the decrease differed for each collagen type, with type III collagen synthesis being suppressed to the greatest extent. Additionally, chronic exposure to EGF resulted in enhanced secretion of types I, III, and IV collagen molecules and an increase in the ratios of type I-homotrimers to type I-heterotrimers and of type V-homotrimers to type V-heterotrimers. These findings demonstrate that chronic exposure to EGF selectively alters collagen expression in a cell line established from the fetal type II pneumocyte.     

Effects of transforming growth factor-beta on collagen synthesis by normal rat kidney epithelial cells.

The effects of transforming growth factor-beta (TGF-beta) on the growth of and collagen production by NRK52E cells, a clonal line established from normal rat kidney epithelial cells, have been characterized. NRK52E cells were grown in the absence or presence of TGF-beta for 4 days followed by incubation for 24 hours in serum-free medium containing [3H]proline. The secreted and cell-associated collagens produced by control and experimental cultures were isolated by limited pepsin digestion and differential salt fractionation. TGF-beta inhibited proliferation by about 50% but did not affect overall culture morphology. Both protein and collagen synthesis were increased in experimental cultures, but the increase in total collagen production exceeded that of total protein synthesis. Although NRK52E cells grown in the presence of TGF-beta continued to produce the same types of collagen (types I, III, IV, and V), their relative amounts were changed. In the experimental cultures, type I collagen production was increased eightfold, types III and V collagen levels were increased two-fold, but type IV production was only slightly enhanced. In addition to increasing total collagen production by about fivefold, TGF-beta increased the ratio of type I to type III about threefold but minimally affected the ratio of secreted to cell-associated molecules. These findings establish that TGF-beta specifically affects collagen production in NRK52E cells and that these alterations differ in many ways from the affects of epidermal growth factor. Because TGF-beta increased total collagen expression, these results provide additional evidence implicating this growth factor as a positive mediator of matrix accumulation in renal disease.     

Morphologic study and syntheses of type I collagen and fibronectin of human periodontal ligament cells cultured on poly(ethylene-co-vinyl alcohol) (EVA) with collagen immobilization

The purpose of this study was to regenerate the human periodontal ligament (PDL) around dental implants by using a hybrid structure of cells with materials, such as PDL cells + collagen + poly(ethylene-co-vinyl alcohol) (EVA) on titanium implant. Human PDL cells were cultured on the EVA surface coated with type I collagen and the cell adhesion and extension were investigated. Furthermore, collagen type I and fibronectin syntheses were analyzed. The serum free culture was also tried, to investigate the role of collagen in detail. The results showed that: 1. Satisfactory adhesion, extension, and proliferation of the PDL cells on the EVA films coated with collagen were observed, but were not good without collagen. 2. Immunostaining of cultured PDL cells revealed the syntheses of type I collagen, when cultured on the EVA coated with collagen or conventional culture dish, though fibronectin synthesis was observed even in the EVA without collagen. 3. Only PDL cells on the EVA coated with collagen proliferated well in the absence of serum. These results indicate that our novel implant material (EVA coated with collagen) provides a possibility of PDL regeneration on dental implants.     

Detection of fibronectin and collagen types I, III, IV and V in semithin sections of the human aorta

Localization of fibronectin and types I, III, IV and V collagen was investigated in semithin sections of fibrous atherosclerotic plaques and apparently normal intima of human aorta. The effect of different techniques of fixation and processing of the sections on immunostaining under peroxidase-antiperoxidase techniques have been examined. The tissue fixation in paraformaldehyde solution, removing the resin with sodium ethoxide and enzymatic pronase digestion of the sections resulted in successful specific staining of the antigens. It was found that some cells in fibrous plaques formed a cap of multiple layers of dense connective tissue containing fibronectin and type III, IV and V collagen in the absence of collagen type I.     

Type V collagen represses the attachment, spread, and growth of porcine vascular smooth muscle cells in vitro.

We attempted to clarify the effects of various purified extracellular components, including types I, III, IV, and V collagen and fibronectin on attachment, spread, growth, and DNA synthesis of porcine aortic smooth muscle cells (SMCs) in vitro. The number, area and shape index (SI = 4 pi S/L2) of cells attached to different substrates were determined at various intervals of incubation. The cell number and [3H]thymidine incorporation into DNA were measured on the 1st and 6th days of culture. SMCs showed the largest number of attached cells on fibronectin, but the smallest number of attached cells on type V collagen. There was no evidence of effects of the serum in media on the attachment of SMCs to the substrates. The areas of attached SMCs were the largest on fibronectin and the smallest on type V collagen. The shape index of SMCs on fibronectin decreased relative to those on other substrates. On the 6th day in culture, the number and population doubling of SMCs on type V collagen were significantly fewer than those on other substrates. Both the incorporation rate of [3H]thymidine into DNA and the percentage of nuclei labeled with [3H]thymidine were significantly less in the SMCs on type V collagen on the 1st day than those on other substrates. SMCs on types I, III, and IV collagen showed intermediate levels of cell attachment, spread, and growth. These results suggest that attachment, spread, and growth of SMCs are affected mainly by solid phase purified extracellular components and are most strongly suppressed by type V collagen. When DNA synthesis of growth-arrested SMCs was reinitiated by the addition of serum, type V collagen most intensively inhibited the rate and amount of [3H]thymidine incorporation. Flow cytometric analysis demonstrated an increased in the proportion of cells in G0/G1 phase on type V collagen in comparison with that on other substrates. Thus, the antiproliferative effect of type V collagen may relate to inhibition of transition of SMCs from the G0/G1 into the S phase.     

The extracellular matrix in large-cell lymphomas: a histopathologic, immunohistochemical, and ultrastructural study

We studied the histopathologic, immunohistochemical, and ultrastructural features of the extracellular matrix (ECM) in 20 immunologically defined large-cell lymphomas, including immunoblastic sarcoma of B cells (three patients), peripheral T cell lymphoma (five patients), large non-cleaved follicular center cell (FCC) lymphoma (nine patients), and non-marking large-cell lymphoma (three patients). Immunohistochemical studies were performed with antibodies to laminin, fibronectin, and collagen types I, III, IV, and V. The immunologically defined subgroups demonstrated characteristic differences in ECM in light microscopic appearance, composition, and ultrastructural features. Immunoblastic sarcomas of B cells showed delicate intercellular bands that were apparent only at high power but were distinct in exhibiting focal staining for basement membrane elements (laminin and type IV collagen) in addition to type I collagen and fibronectin. Electron microscopically, no basal lamina were apparent, although the collagen fibers were embedded in a dense matrix not seen in the other lymphomas. All peripheral T cell lymphomas exhibited a packeting pattern of intercellular bands and were distinguished by the frequent presence of intense pericellular staining for type V collagen as well as by focal pericellular staining for types I and III collagen. The latter finding corresponded to extensive areas of direct contact between tumor cells and the ECM by electron microscopy. The large non-cleaved FCC lymphomas and the non-marking large-cell lymphomas demonstrated both diffuse and compartmentalizing arrangements of intercellular bands that frequently coexisted and stained predominantly for fibronectin and types I and III collagen. All groups demonstrated myofibroblasts and fibroblasts partially or completely separating the ECM from tumor cells, suggesting that most of the ECM is part of a reaction to these lymphomas. These studies show more variation in light microscopic appearance, composition, and ultrastructural relationships of the intercellular and pericellular ECM than was apparent in earlier studies of cleaved FCC lymphomas.     

The microvascular extracellular matrix. Developmental changes during angiogenesis in the aortic ring-plasma clot model.

The composition of the extracellular matrix of developing microvessels in plasma clot cultures of rat aorta was studied with light and electron immunohistochemical techniques using affinity-purified antibodies against fibronectin, laminin, and collagen Types I, III, IV, and V. The extracellular matrix of solid endothelial sprouts in young cultures consisted of a delicate fibrillary network of fibronectin and Type V collagen and of patchy amorphous deposits of laminin and Type IV collagen. Rare fibrils of collagen Types I and III were also observed. Fibronectin stained intensely and appeared to be the predominant component of the provisional subendothelial matrix during vascular sprouting. As the cultures aged, laminin and Type IV collagen accumulated in the subendothelial space, forming a continuous feltwork around the newly formed microvessels. Patent microvessels were also surrounded by discontinuous deposits of fibronectin and by increased amounts of collagen Types I, III, and V. Ultrastructural studies revealed positive immunostaining for fibronectin, laminin, and collagen Types IV and V in the endoplasmic reticulum and in putative secretory vesicles, indicating active synthesis and secretion of these molecules by the endothelial cells. These observations indicate that the microvascular extracellular matrix undergoes significant dynamic changes during capillary development. The different composition and structural organization of the extracellular matrix at various stages of angiogenesis may have important effects on endothelial behavior and capillary morphogenesis.     

Culture of fibroblast-like cells derived from normal human liver. Identification by morphologic and immunologic criteria

Fibroblast-like cells were isolated from liver biopsies of normal adult donors. The cells were grown in tissue culture first as a heterogeneous population, afterwards as homogeneous cultures of fibroblast-like cells. Phase contrast microscopy demonstrated that cultured human liver fibroblast-like cells grew as monolayers of slender, spindle-shaped cells in parallel arrays. By transmission electron microscopy (TEM), cultured human liver fibroblasts were seen to have morphological characteristics of in vitro fibroblasts. By immunoelectronmicroscopy, cultured fibroblast-like cells were seen to produce components of connective tissue, such as fibronectin, collagen type I, type III, and small amounts of collagen type IV. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human liver fibroblasts from normal human liver.     

Differential localization of type I and type III procollagen messenger ribonucleic acids in inflamed periodontal and periapical connective tissues by in situ hybridization

Inflammatory lesions of periodontal and periapical connective tissue were studied by in situ hybridization to detect cells responsible for type I and type III collagen production. Formalin-fixed and paraffin-embedded tissue specimens from patients with oral lesions of various stages of inflammation were hybridized with cDNA probes specific for human pro alpha 1(I) and pro alpha 1(III) collagen mRNAs, and with bacteriophage lambda DNA as a control probe. This technique permitted us to localize fibroblasts active in type I collagen synthesis in the vicinity of inflammatory infiltrates in all the samples studied. Cells containing high levels of type III collagen mRNA were seen in early abscess formation and they were particularly abundant in pyogenic granuloma and irritation fibroma. Type I collagen mRNA was prominent in gingival fibrosis. In the infrabony lesions with active inflammatory infiltrations the production of collagen was confined mostly to the periphery of the lesions. These findings give indirect evidence that cytokines liberated during the early stages of the inflammatory process stimulate expression of the type III collagen gene by fibroblasts. In chronic lesions a gradual switch from type III to type I collagen gene expression occurs. The change in collagen types appears to underlie the observed isolation of the inflammation by a collagenous capsule. In all the samples studied fibroblasts exhibited marked variation in their levels of procollagen mRNAs, supporting previous views about their heterogeneity in connective tissues. The approach presented here offers new possibilities to study cellular interactions and metabolic activities in inflammatory lesions.     

Modification of Ti6AL4V surfaces using collagen I, III, and fibronectin. II. Influence on osteoblast responses

Responses of osteoblastic cells are influenced by morphology and composition of the extracellular matrix, and this fact has been used to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). In this study, the effect of the collagen types I and III on adhesion, proliferation, and differentiation was studied, using primary osteoblastic cells from rat calvariae. Differences in alkaline phosphatase activity (ALP) and collagen synthesis were observed between differently composed collagen coatings. An increase in collagen type III resulted in an increase in collagen synthesis and a concomitant decrease in ALP activity and Ca deposition. Initial adhesion mechanism of the cells depended on the substrate (titanium, collagen, fibronectin).     

A histologic study of the extracellular matrix during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The levels of four ECM components, i.e., collagens III, IV, VI, and laminin were markedly decreased in the sclerotic glomeruli as compared with week 8. In contrast, the amount of fibronectin in the sclerotic glomeruli increased dramatically. Immunoelectron microscopic examination showed fibronectin in the sclerotic lesions, in contrast to laminin, collagen type I, and collagen type IV. It is concluded that the sclerotic lesions in murine chronic GvHD contain fibronectin. The small amounts of the ECM components laminin, as well as collagens III, IV, and VI in the sclerotic glomeruli in GvHD, might represent remnants of mesangial material and collapsed capillary walls. These components are probably replaced by increased production and/or accumulation of collagen type I and fibronectin.     

Concomitant immunohistochemical localization of fibronectin and collagen in schistosome granulomata

Affinity purified antibodies to human collagen type I and III and antihuman fibronectin, were employed immunohistochemically on formalin-fixed, paraffin-embedded human tissue containing schistosome granulomata. The distribution of the three connective tissue proteins was studied in 45 biopsies in which there was Schistosoma haematobium (30) and Schistosoma mansoni (15) infestation. The granulomata were divided into early, intermediate and late according to the proportion of inflammatory cell types and the degree of fibrous tissue deposition. Only fibronectin was detected in the early granuloma where macrophages are the main cell type. In the intermediate granuloma both fibronectin and collagen type III were localized along reticulin positive fibres; spindle cells, (presumptive fibroblasts) and macrophages were the cell types present at this stage. Collagen type I was mainly localized in the late granuloma. Hyalinized connective tissue was negative for the three proteins. The loose connective tissue and walls of blood vessels were constantly stained for fibronectin and collagen type III in normal areas distant from granulomata. These results raise the possibility of using localization of fibronectin and collagen as a means to stage the duration of chronic fibrotic diseases.     

Culture and co-culture of DU145 prostate carcinoma,osteoblasts and HT-29 colon carcinoma cells on a fabricated type III collagen matrix

DU145 prostate carcinoma cells cultured on type III collagen possessed a highly migratory potential which was twice as much as HT-29 colon carcinoma cells. Prior to attachment to collagen, DU145 cells were highly reactive for fibronectin and after attachment clear zones between cells and collagen suggested protease activity. HT-29 cells attached to type III collagen forming dome-like polyps, however, tight and/or gap junctions were not observed. hFob osteoblasts were co-cultured with DU145 to establish a prostate cancer-collagen matrix barrier-bone cell metastasis model. Osteoblasts maintained their differentiated osteoblastic characteristics on one side of the collagen barrier, demonstrating high alkaline phosphatase, osteocalcin and insulin growth factor (IGF) activities. hFob cell growth was prominent adjacent to demineralized bone matrix particles (BMPs) embedded in type III collagen. The collagen matrix was deteriorated on the DU145 side of the collagen barrier. The DU145-collagen III-hFob model will allow an evaluation of the influence of the matrix on prostate cancer-bone cell interaction and regulation by growth factors.     

Comparative distribution patterns of type I and III collagens and fibronectin in human arteriosclerotic aorta

The distribution patterns of type I and III collagens and fibronectin were studied in arteriosclerotic lesions of human aortas as well as in the regions devoid of any macroscopic lesions of the same aortas. Frozen sections were analyzed for fibronectin and collagens by indirect immunofluorescence or immunoperoxidase analysis with specific antisera prepared in rabbits against human plasma fibronectin and against human type I and III collagens. Tissue samples were also stained for type I and III collagen distribution using Picro Sirius red according to the method described by Junqueira et al. (1979). In the regions devoid of any visible macroscopic lesions, there is a co-distribution in the media of the aortic wall of type I and III collagens, with a predominance of type I collagen. Type I collagen is also visible in the intima, as a continuous layer. This is attributed to an age-dependent change of the aorta (arteriosclerosis). Fibronectin can be demonstrated as a thin layer in the subendothelium and as a weak immunofluorescence over the media. In recent or evolutive arteriosclerotic plaques, fibronectin and type III collagen appear to be strongly increased and superimposed. There is also an increase of type I collagen in the intimal region of the plaque. The co-distribution of type III collagen and fibronectin in the arteriosclerotic plaques may suggest a tissue-repair-type of process, analogous to that which can be observed during wound healing. Our results suggest that in arteriosclerotic lesions, type III collagen and fibronectin are predominantly synthesized.(ABSTRACT TRUNCATED AT 250 WORDS)