Relative distribution of fibronectin and type I, III, IV, V collagens in normal and atherosclerotic intima of human arteries

Spatial distribution of fibronectin and type I, III, IV and V collagen has been investigated in normal arterial intima, fatty streaks, and atherosclerotic plaques by indirect immunofluorescence on transverse sections. Two distinct types of extracellular matrix were revealed in atherosclerotic lesions. The fibrous plaques consisted mostly of interstitial collagen types I and III, contained moderate amounts of type V and none of type IV collagen or fibronectin. In the extracellular matrix of the fatty streaks and in some areas of the fibrous plaques containing large amounts of subendothelial cells, some interstitial collagen was revealed, an increased amount of type IV, some type V collagen and a lot of fibronectin. Similarities of the extracellular matrix in atherosclerotic lesions and granulation tissues are discussed.     

Collagen isotypes, laminin, and fibronectin in granulomas of the liver and intestines of Schistosoma mansoni-infected mice

Patterns of fibrosis within hepatic and intestinal granulomas of Schistosoma mansoni-infected mice were analyzed by indirect immunofluorescence. Deposition of collagen isotypes, laminin, and fibronectin was evaluated semiquantitatively between 8 and 20 weeks of the infection. Liver granulomas were the largest at 8 weeks and contained low amounts of type I and higher amounts of type III collagen and fibronectin. Collagen deposition became pronounced as infection progressed. The relative amounts of type I collagen deposits rose and equalled that of type III. In the smaller immunomodulated granulomas at 20 weeks both types I and III were high, and type IV collagen deposition was observed. Fibronectin and laminin deposits were also detected. The small ileal granulomas did not change their size during the course of the infection. At 8 weeks, connective tissue matrix deposition was barely detectable within these lesions. Gradually, small deposits of types I and III appeared in equal amounts and attained highest levels by 20 weeks of the infection. Fibronectin deposits at that time were very prominent but laminin and type IV collagen were absent. Colon granulomas at 8 weeks of the infection were only somewhat smaller than those of the liver, yet contained very sparse deposits of types I and III collagen. During the ensuing weeks collagen deposits rose only slightly. By 20 weeks the granulomas diminished in size and within those lesions type III collagen was predominant. Whereas the presence of fibronectin was pronounced, type IV collagen and laminin were detectable only in trace amounts. These observations indicate the existence of important organ-related differences in the intragranulomatous deposition of connective tissue matrix.     

The Extracellular Matrix is an Integrated Unit: Ultrastructural Localization of Collagen Types I,III, IV,V, VI,Fibronectin, and Laminin in Human Term Placenta

The human term placenta is used extensively as a source of extracellular matrix components. To elucidate the tissue distribution and interrelationships of seven of these components, monospecific antibodies directed against collagen types I, III, IV, V, VI, fibronectin, and laminin were reacted with human term placenta and studied by light and electron immunohistochemistry.Type I collagen was the basic structural unit of human term placenta, present as 30\2-35 nm, cross-banded fibers, often in the form of large fiber bundles. Type III collagen was present as thin 10\2-15 nm, beaded fibers often forming a meshwork which encased type I collagen fibers. Types V and VI collagen were present as 6\2-10 nm filaments, often closely associated with types I and III collagen. Type VI collagen also coated collagen fibers of all diameters, enhancing their periodicity, providing a staining pattern often similar to that observed with anti-fibronectin antibodies. Fibronectin was present in both maternal and fetal plasma and throughout the stroma of the chorionic villus, as both free filaments and coating collagen fibers. Basement membranes contained laminin and type IV collagen, but no fibronectin. In summary, the non-basement membrane proteins studied often codistributed with type I collagen, between and apparently attached to fibers, suggesting that they may act as binding proteins, linking type I fibers and bundles, to themselves and to other structures.     

Immunohistochemical study of extracellular matrix in acute galactosamine hepatitis in rats.

A single injection of D-galactosamine hydrochloride induces acute self-limiting liver disease in rats that morphologically resembles drug-induced hepatitis in human beings. In this immunohistochemical study we examined the localization and expression of the hepatic extracellular matrix components fibronectin, laminin, collagen type I, collagen type III and collagen type IV and of the cell surface receptors (integrins) for fibronectin and laminin. Sections of liver tissue obtained at intervals of 6, 12, 18, 24, 30, 36, 48 and 72 hr and 7 and 21 days after galactosamine administration were immunostained with a panel of polyclonal monospecific antibodies and studied independently by two of us. Fibronectin was the first extracellular matrix component found to be increased, 12 hr after galactosamine injection, followed by collagen type III, and, in a later phase, collagen type IV, type I and laminin. Increased deposition of extracellular matrix was found in areas with liver cell necrosis and along sinusoids. Extracellular matrix immunoreactivity reached a maximum at 36 to 48 hr and decreased thereafter to preinjury levels 3 wk after galactosamine. Immunostaining for the fibronectin and laminin receptors revealed tissue localization identical to that of their ligands. However, the intensity of staining was opposite of that for the extracellular matrix, with a decrease of immunoreactivity after 24 to 48 hr. The observed sequence of changes in hepatic extracellular matrix proteins after galactosamine injection resembles the repair reaction in other tissues and may reflect the particular function that each carries out during the process of liver healing after toxic injury.     

Cell types involved in collagen and fibronectin production in normal and fibrotic human liver

Three collagen types (I, III and IV) and fibronectin were localized in normal and alcoholic human liver by light and electron microscopy using the indirect immunoperoxidase technique. In normal liver, most of the bundles of collagen fibers stained for type pro-III collagen while only a few reacted for type I. Basement membranes stained for type IV collagen which formed discontinuous discrete deposits in sinusoids. Only fibronectin appeared as an almost continuous layer in the space of Disse. At the intracellular level, hepatocytes were found to contain little type I collagen and large amounts of fibronectin. Fat-storing cells strongly stained for type IV collagen and expressed low amounts of types I and III collagen and fibronectin. Endothelial cells contained low amounts of all the components. Alcoholic livers were studied at three stages: steatosis, fibrosis and cirrhosis. Qualitative and quantitative differences were observed in extracellular and intracellular distributions of matrix proteins. Increased amounts of all components were usually found in fibrotic and cirrhotic livers compared to normal liver. In two fibrotic livers which contained numerous bundles of collagen in the sinusoids, fat-storing cells stained more intensely for type III collagen. In a cryptogenic fibrotic liver, abundant type IV collagen was observed in hepatocytes. These results suggest that hepatocytes, fat-storing cells and endothelial cells are engaged in production of extracellular matrix components in normal human liver. In fibrosis, hepatocytes which normally did not synthesize types III and IV collagen may produce these collagens.     

Immunohistochemistry of the hepatic extracellular matrix in acute viral hepatitis

The distribution of several extracellular matrix components in the liver of patients with acute viral hepatitis was studied by light and electron microscopy using indirect immunoperoxidase methods. Light microscopy revealed type III and type V collagen and fibronectin in the portal tracts and the area of focal necrosis, showing cell infiltration. Type III and type V collagen were more strongly stained in the periphery of focal necrosis. Type IV collagen was seen around the vessels and hepatocytes near the focal necrosis. Electron microscopy showed many transitional Ito cells in the area of focal necrosis and fibroblasts were observed in the portal tracts, showing collagen fiber deposition. Numerous collagen fibrils were observed around fibroblasts, Ito cells and hepatocytes. Using immunoelectron microscopy, type III and type IV collagen and fibronectin were observed in the rough endoplasmic reticulum of Ito cells and hepatocytes localized near the area of focal necrosis or fiber deposition. In addition, type IV collagen was seen in the rough endoplasmic reticulum of endothelial cells forming capillary vessels. These results suggest that several extracellular matrix components such as types III, IV and V collagen and fibronectin, produced by Ito cells, hepatocytes or endothelial cells, play important roles in the healing of liver damage in acute viral hepatitis.     

Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components.

We have isolated and characterized collagen type VI from murine, canine, and nonhuman primate hearts. In the three species studied, collagen type I was the major collagenous component of the cardiac interstitium (80% of total collagen), whereas collagen type VI represented approximately 5% of total collagen. To define the exact distribution of collagen type VI and its possible interactions with other components of the cardiac extracellular matrix, collagen types I, III, IV, and VI, laminin, and fibronectin were localized in the rat myocardium by immunohistochemistry, using monospecific antibodies. In the rat myocardium, collagen type VI was prevalent in the media and adventitia of muscular arteries, in fine connective tissue septa, in the area surrounding capillaries, and in the delicate endomysium in proximity to myocardial cells. When compared with the immunohistochemical localization of collagen types I, III, and IV, laminin, and fibronectin, the continuity and hierarchical organization of the cardiac extracellular matrix became apparent. The matrix forms a continuous network extending from the pericardium to the endocardium. Furthermore, there is an arborescent hierarchy in the system such that collagen type I is more prevalent in the wider septa, collagen type III being more obvious in medium-sized branches, and fibronectin and collagen type VI prevailing in the terminal (pericellular) aspects of the network. In this pericellular location, fibronectin and collagen type VI, by means of specific interactions, may act as anchor components linking the myocardial cell basement membranes not only to the extracellular matrix but also to the cardiac interstitial cells. This continuity, organization, and coupling of the cardiac extracellular matrix appears well suited to integrate and distribute the physical stress generated by the continuous contraction and relaxation of the myocardium.     

Age-dependent variations of the biosyntheses of fibronectin and fibrous collagens in mouse skin

Skin explant cultures from hairless mice of increasing age were incubated with radioactive precursors in order to determine the age-dependent variations of the biosyntheses of fibronectin and fibrous collagens (types I and III). Total collagen synthesis expressed as a percentage of total protein synthesis did not vary with age but, if expressed as micrograms hydroxyproline per mg wet weight of skin, decreased by about 30% between 2 and 22 months of age. Hydroxylation of collagen, expressed as the ratio of 3H-hypro over 3H (pro + hypro) incorporated in freshly synthesized collagen, decreased with age by about 40% between 2 and 22 months of age. The proportion of type III collagen expressed as % of type I + type III collagens increased progressively with age by about 25% at 12 months to 60% at 22 months of age. Fibronectin biosynthesis, determined by immunoprecipitation of 35S-methionine labeled peptides in SDS-extracts of skin increased progressively with age from about 2% of total incorporated radioactivity in fibronectin at 2 months to 4% at 22 months. Plasma fibronectin, of hepatic origin, was shown already to increase with age in humans. It appears thus that the expression of genes coding for extracellular matrix macromolecules is under age-dependent regulation. This regulation appears to be different for the investigated macromolecules.     

Localisation of collagen types and fibronectin in cartilage by immunofluorescence.

Collagens type I, II, III, IV, and V and the minor cartilage collagens, 1 alpha 2 alpha 3 alpha, C-PS 1, and C-PS 2, were purified, antibodies raised, and then used in immunofluorescence studies on bovine nasal cartilage (BNC). Punctate localisation was seen with the type II antibody. However, pretreatment of sections with hyaluronidase to remove the proteoglycan resulted in diffuse staining over all the section with this antibody. Antibodies to 1 alpha 2 alpha 3 alpha, C-PS 1, and C-PS 2 collagens gave no staining on untreated BNC sections, but after treatment with hyaluronidase all 3 antibodies showed as a diffuse 'halo' round each chondrocyte lacuna. Anti-type I, anti-type III, and anti-type IV collagen antibodies did not stain untreated or enzyme treated BNC. Type V collagen antibodies gave a bright ring in the pericellular region of the lacunae of hyaluronidase-treated BNC. This was unexpected, as we could not detect type V collagen biochemically in the same cartilage. Anti-fibronectin antibodies stained areas distant from the chondrocytes, these areas being distinct from those stained by 1 alpha 2 alpha 3 alpha and C-PS antibodies, suggesting that fibronectin is not associated with these collagens in BNC. These results suggest that different collagen types may have different locations within the cartilage matrix, that proteoglycans may inhibit antibody association with collagen, and that fibronectin is normally not associated with all types of collagen.     

Distribution of Type I,III, IV and V Collagen in Normal and Atherosclerotic Human Arterial Wall: Immunomorphological Characteristics

35 autopsies — aged 30 to 75 years — were investigated in order to establish trends of collagen localization in various types of arteries depending on age, arterial size and degree of atherosclerosis. Cryostat sections stained with highly specific antibodies to human types I, III, IV or V collagen, or with the antiserum to smooth muscle myosin were examined by the indirect immunofluorescence technique.Localization of type III collagen was very similar to that of type I. Fibrous structures of both type I and type III were then major constituents of the intima, media and adventitia. Sparse fibrils of type I and type III collagens were revealed in the subendothelium of unaffected intima. They gradually became abundant in the deeper intimal layers contrasting with loose fibrillar formations of the media. The content of interstitial collagens was significantly increased in the subendothelium of local intimal thickenings and in a thickened intima of the aged. This fact, considering the thrombogenicity of interstitial collagens, may be relevant to the atherogenesis through the "response-to-injury" mechanism. Type IV and type V collagens are localized to the endothelial basement membrane and basement membranes of smooth muscle cells of the intima and media. Diffusely distributed type V collagen was also observed in the intercellular space of the intimaIn lipid streaks, parallel layers of condensed interstitial collagens separated groups of cells and extracellular lipid depositions. In fibrous plaques, types I and III became prevalent structural elements and their densely packed fibers occupied whole regions devoid of any type IV and type V collagen. Heavily thickened type IV collagen structures surrounding individual smooth muscle cells were found in fibrous plaques, but never, in unaffected intima.     

Matrix Composition in Opossum Esophagus

The esophagus of mammalian species is organized into mucosa, connective tissue, and muscle, but little is known about the matrix of these layers. We studied by immunohistochemistry the distribution of collagens, fibronectin, versican, and elastin in the smooth muscle segment of the American opossum. Cryosections were exposed to specific antibodies and fluorescent-stained using conjugates of rhodamine or isothiocyanate. Staining was scored by two observers. We found that collagen I was prominent in the submucosa and in the muscular septa; collagen III formed fibrillar meshes in the lamina propria and the submucosa but was virtually absent from the epithelial and muscular layers; collagen IV was restricted to the base of the epithelium; collagen V, in contrast to collagen III, was prominent in epithelium and muscularis mucosae and sparse in muscular septa and submucosa. Fibronectin distribution followed collagen III; it formed layers in lamina propria and submucosa and strands in muscle septa and between individual muscle cells. Versican distribution followed collagen V; it was prominent in large muscle septa and formed thick sheets at the boundaries of submucosa/circular muscle and of circular/longitudinal muscle. We also determined the tissue contents of protein, hexuronic acid, and fibronectin. The mucosal layers exceeded the muscular layers in their content of hexuronic acid and fibronectin but not protein. We conclude that individual layers of the smooth muscle esophagus each have their own characteristic matrix. Lamina propria and submucosa are similar with regard to fiber orientation but lamina propria contains relatively more collagen III (small fibril) and submucosa comparatively more collagen I (large fibril). Nonfibrillar collagen V and versican are particularly prominent specifically on the boundaries between contracting muscle tissue and connective tissue framework.     

Distribution of extracellular matrix proteins in pancreatic ductal adenocarcinoma and its influence on tumor cell proliferation in vitro

Affinity-purified antibodies to major components of the extracellular matrix (fibronectin and collagen type I) and basal lamina (laminin) were used in indirect immunofluorescence studies on frozen sections of 12 pancreatic ductal adenocarcinoma of the human and on sections of normal and inflamed pancreatic tissue of the same patients. Laminin-specific immunoreactivity was distributed in close correlation to the grade of differentiation of the tumor tissue. Intact basement membranes, also with some structural irregularities were found only in the highest grade of differentiation where tumor cells grew as tubular structures. With progressive dedifferentiation basal laminae were either absent or the laminin-positive material was focally distributed without spatial association with tumor cells. In all cases of pancreatic tumors a remarkable increase in interstitial connective tissue was observed, as demonstrated by antibodies specific for human collagen type I and for human serum fibronectin. Tumor extracts contained high amounts of collagen type I and V but no significant amount of collagen type III as visualized by analytical SDS gel electrophoresis. A similar distribution of collagen types was observed in lymph node and liver metastases, and in tumors xenografted into nude mice. Since previously a close correlation between grading and growth kinetics of primary tumors had been observed, in vitro experiments were performed analyzing the effect of purified extracellular matrix proteins on tumor cell proliferation. In vitro cultivation of two established cell lines of pancreatic carcinoma on collagen type I or on laminin resulted in an arrest of proliferation on laminin substrates, while normal proliferation comparable to growth on regular culture dishes was found using collagen type I and fibronectin as substrates. Fine structural studies demonstrated a higher degree of cell differentiation in the presence of laminin, as compared to collagen type I or fibronectin.     

Connective tissue growth factor induces extracellular matrix in asthmatic airway smooth muscle

Transforming growth factor (TGF)-beta and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-beta increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF-beta and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF-beta and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase-dependent in both cell types but phosphoinositide-3 kinase-dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF-beta induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.     

Pancreatic extracellular matrix alterations in chronic pancreatitis

The proliferation of pancreatic extracellular matrix, which characterizes chronic pancreatitis, has been analysed using immunohistochemistry. The relationship of matrix components to intraductal precipitates and the presence of serum proteins in precipitates were also studied to investigate the suggestion that ductal permeability increases in chronic pancreatitis. Pancreatic tissue from organ donors was compared with that from patients with chronic calcifying or chronic obstructive pancreatitis. Frozen sections were labeled with monospecific antibodies to collagen types I, III, pro-III and IV, laminin, fibronectin, IgG, IgA, and IgM and then visualized by indirect immunofluorescence. In chronic pancreatitis, interstitial collagens and fibronectin appeared increased and disorganized in both fibrous tissue and areas that appeared histologically normal. Type IV collagen distribution was abnormal and in some sites was present with interstitial collagen. In addition, intraductal precipitates were shown to contain immunoglobulins, and defects were identified in the duct basal lamina associated with precipitates. These results demonstrate that in chronic pancreatitis interstitial collagens are extensively disorganized, the fibrosis possibly being relatively labile. The presence of serum proteins in intraductal precipitates confirms an increase in ductal permeability, and associated defects in the basal lamina appear to define a route via which serum proteins may enter the intraluminal compartment.     

Distribution of Collagen Types I,III, and IV in Peptic Ulcer and Normal Gastric Mucosa in Man

The quality of peptic ulcer healing does not only mean complete epithelial restitution of the mucosal surface but also adequate repair of the underlying connective tissue. To obtain more information about the metabolism of extracellular matrix proteins in gastric mucosa and submucosa, we investigated biopsy specimens from six patients with antral peptic ulcers and six normal controls by staining of collagen types I, III, and IV with an immunofluorescence technique. In normal mucosa we found a certain amount of collagen types I and III in equal distribution and almost no collagen type IV. In contrast, there was a remarkable increase of collagen types I and III in peptic ulcers predominantly located at the ulcer edges. These results are compatible with the view that extracellular matrix proteins play some part in the ulcer healing process.     

Immunolocalization of collagen types, laminin and fibronectin in the normal human pancreas

In order to define the connective matrix organization of the normal human pancreas collagen types I, III, pro-III and IV, laminin and fibronectin were labeled using specific, antihuman antibodies. Visualization was by indirect immunofluorescence. Collagen types I, III and pro-III were present within lobules: around acini, ducts and small vessels. Their immunofluorescence reaction was particularly obvious in septa and it also outlined interlobular vessels and ducts. The type III and pro-III fractions possessed a characteristic, branched appearance in many situations, when compared to the more linear type I reaction. Collagen type IV, laminin and fibronectin were closely applied to acini, ducts and vessels, but in contrast to the other collagen types were absent from septa.     

Extracellular matrix structure after heart transplantation

Following heart transplantation remodeling of the donor heart causes changes in the extracellular myocardial matrix. We investigated 20 right ventricular endomyocardial biopsies taken 17+/-4 days (group I, n=9) and 63+/-13 days (group II, n=11) after heart transplantation from 16 patients transplanted for end-stage cardiomyopathy (15 dilated/1 ischemic). Immunohistochemical staining for collagen I, collagen III, collagen IV, and fibronectin was used. Evaluation was performed at a magnification of 400x using a computer-assisted image analyzing system measuring the relative area stained by the immunoperoxidase method, the number of cells in the given area, and the total area. Collagen I per cell was 13.9+/-5.9 microm2 in group I and increased significantly 66+/-13 days after heart transplantation in the perimysium around the myocardial cells as well as in the endocardium to 49.9+/-15.1 microm2 (P<0.05). No quantitative change in collagen III was noted (75.7+/-12.4 versus 75.5+/-16.0 microm2 n.s.). Collagen IV was found in the perimysial, in the capillary bed and in the vascular network. Significant quantitative change in the amount of collagen IV was not found (64.1+/-12.6 versus 61.0+/-8.9 microm2). Fibronectin was found in the entire perimysial extracellular matrix and in the endocardium in relationship with collagen I and III. An increased amount of fibronectin from 87.09+/-9.9 microm2 (group I) to 140.8+/-17.9 microm2 (group II, P<0.05) was found. The cell area and cell diameters were not significantly different (group I; cell area 772+/-227 microm2, diameter 31.3 microm; group II; cell area 776+/-224 microm2, diameter 31.4 microm). It is concluded that remodeling of the donor heart after transplantation is characterized by a specific increase in collagen I and fibronectin, whereas a change in other collagen subtypes was not observed.     

Angiotensin II stimulates collagen synthesis in cultured vascular smooth muscle cells

The aim of the present study was to investigate whether angiotensin II, by increasing extracellular matrix synthesis, contributed to the vascular wall thickening observed in hypertension. Thus, we examined the direct effects of angiotensin II on collagen and fibronectin synthesis in cultured rat vascular smooth muscle cells by measuring 3H-proline incorporation. Angiotensin II, in a concentration of 10 mumol/l, increased collagen synthesis in a dose-dependent manner up to 1.8-fold. This increase occurred within 24 h after the addition of angiotensin II and the time required to reach maximum stimulation was approximately 48 h. This increase was receptor-mediated and correlated with an increase in its specific messenger RNA. A closer study of the collagen increase demonstrated a relatively greater increase in type V collagen than type I or type III collagen. Fibronectin synthesis was also increased 1.5-fold with 10 mumol/l angiotensin II. These data suggest that angiotensin II induces vascular wall thickening by acting directly on smooth muscle cells and enhancing the production of extracellular matrix proteins.     

Contribution of lung fibroblast migration in the fibrotic process of airway remodeling in asthma.

BACKGROUND: The fibrotic process in airway remodeling of asthma may be characterized by an exaggerated deposition of extracellular matrix (ECM) components such as fibronectin and type I, III and IV collagen. In the present study, we established airway remodeling model mice and examined the mechanism of fibrotic change by measuring chemotactic activity of lung fibroblasts and quantifying collagen content in lung tissues. METHODS: Airway remodeling model mice were made by ovalbumin (OA) sensitization and inhalation. Bronchoalveolar lavage (BAL) and bronchial biopsy were performed. Cell migration was assessed by the Boyden's chamber technique. The collagen content of lung tissue was measured using ELISA. RESULTS: The chemotactic activity in lung fibroblasts toward the mouse BAL fluid (BALF) was significantly increased in OA-inhaled mice. Total soluble collagen content was significantly increased in OA-inhaled mice. We observed markedly increased collagen deposition around the airway wall in OA-inhaled mice, which was not shown in saline-inhaled mice. Furthermore, fibronectin in the BALF of OA-inhaled mice was significantly higher than that in the control mice. CONCLUSIONS: The total soluble collagen content increased during the fibrotic change of airway remodeling in asthma. Furthermore, migration of fibroblasts may play a key role in this remodeling process, and fibronectin and type I and IV collagen seem to be chemotactic factors for the fibroblasts.