Components of subendothelial aorta basement membrane. Immunohistochemical localization and role in cell attachment

Cryosections of fetal and adult bovine aorta were stained with purified, cross-absorbed antibodies against various connective tissue components. The antibodies to the basement membrane components, laminin, heparan sulfate proteoglycan, and type IV collagen, gave a sharp reaction in the subendothelial layer. Antibodies against type III procollagen showed a broad endothelial staining, and staining was also seen in the media layer. A similar staining reaction was seen with antibodies against fibronectin. Bovine fetal aortic endothelial (BAE) cells were isolated and cultured in vitro. The cells became stained by the indirect immunofluorescence method with antibodies against laminin and heparan sulfate proteoglycan and also with antibodies against types III and IV collagen and type I procollagen, as in previously reported experiments. The attachment properties of endothelial cells to the same extracellular matrix components were also studied. BAE cells became attached most readily to surfaces coated with fibronectin or type III or type IV collagen. Laminin and collagen types I and V served as less effective substrates. Attachment to heparan sulfate proteoglycan was slowest of the tested components. The results of the study demonstrate that the BAE cells are associated with basement membranes in vivo. The BAE cells in culture produced interstitial connective tissue components in addition to basement membrane components and showed no clear specific preference in their attachment to any of these.     

Alterations in the extracellular matrix components in human glomerular diseases

Summary We investigated the distribution of extracellular matrix components such as fibronectin, laminin, type III, IV, V, and VI collagens and heparan sulfate proteoglycan (HSPG) in normal and diseased glomeruli using the indirect immunofluorescence method. This study included 96 renal biopsies: 7 controls, 3 minimal change nephrotic syndrome (MCNS), 47 mesangial proliferative glomerulonephritis (PGN), 25 membranous nephropathy (MN) and 14 membranoproliferative glomerulonephritis (MPGN) including 3 lupus nephritis. Fibronectin was detected predominantly in the mesangium and less prominently in the glomerular basement membrane (GBM) of normal glomeruli. Laminin and type IV collagen were present in the mesangium and GBM, type III collagen in the interstitium, and type V collagen in the mesangium, interstitium and a part of GBM. Type VI collagen was observed in the mesangium, interstitium and slightly in GBM. Anti-HSPG antibody reacted with the mesangium and GBM. MCNS showed a distribution of these antigens similar to that in normal controls. The finding that staining for HSPG was not decreased in the GBM and mesangium indicated that there was no change in the core protein of HSPG. Fibronectin, laminin, type IV collagen and HSPG were increased in the thickened GBM of MN and in the expanded mesangium of PGN. In MPGN, these matrix components were increased in the mesangium and GBM with remarkable increase of type V and VI collagens. While type III collagen was not found in normal glomeruli, it became detectable in the mesangium and a part of GBM in MPGN. No significant decrease in the intensity of fluorescence for HSPG was observed in the glomeruli from nephrotic patients.These findings suggest that proteinuria might be caused by the structural alteration in the glycosaminoglycan portion of HSPG, changes in any anionic material other than HSPG, or both, and also indicate that the glomerular mesangial sclerosis is closely related to the increase of type V and VI collagens.     

Distribution of extracellular matrix glycoproteins during normal development of human kidney. An immunohistochemical study

The distribution of types I, III, and IV collagens, fibronectin, laminin, and heparan sulfate proteoglycan during human fetal kidney development has been studied by indirect immunofluorescence. Fibronectin and interstitial collagens types I and III are present in undifferentiated mesenchyme, whereas the intrinsic basement membrane components, type IV collagen, laminin, and heparan sulfate proteoglycan are not detected. In differentiated cortex, types I and III collagens are detected only as interstitial connective fibers, whereas fibronectin is found in both interstitium and glomeruli where its distribution is dependent on the stage of maturation. It is found in both the mesangium and along capillary walls of immature glomeruli, and principally in the mesangium of mature ones. Basement membrane components delineated the branching ureteric bud. They also outlined the structures of the earliest stage of nephrogenesis (epithelial differentiation). In mature nephrons, they are found along glomerular, capsular, and tubular basement membranes.     

Light microscopic immunolocalization of type IV collagen,laminin, heparan sulfate proteoglycan,and fibronectin in the basement membranes of a variety of rat organs

Immunohistochemical methods were used to determine whether type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan were present in diverse basement membranes. Antisera or antibodies against each substance were prepared, tested by enzyme-linked immunosorbent assay, and exposed to frozen sections of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth from rats aged 20 days to 34 months. Bound antibodies were then localized by indirect or direct peroxidase methods for examination in the light microscope. Immunostaiing for type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan was observed in all of the basement membranes encountered. Fibronectin was also found in connective tissue. In general, the intensity of immunostaining was strong for type IV collagen and laminin, moderate for heparan sulfate proteoglycan, and weak for fibronectin. The pattern was similar in the age groups under study. Very recently the sulfated glycoprotein, entactin, was also detected in the basement membranes of the listed tissues in 20-day-old rats. It is accordingly proposed that, at least in the organs examined, type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan, and entactin are present together in basement membranes.     

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.     

Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions.

The immunohistochemical localization of the extracellular matrix was examined in 31 cases with different degrees of human diabetic nephropathy using antisera to human collagen types I, III, IV, V, fibronectin, laminin, and basement-membrane-associated heparan sulfate proteoglycan (HSPG). In normal glomeruli, HSPG was predominantly localized in the glomerular basement membrane and in the mesangium, and to minor extent in the basement membranes of tubules and Bowman's capsule. Collagen IV and laminin were distributed in glomerular basement membrane and mesangium in minor amounts. Interstitial collagens usually do not occur within glomeruli except for collagen V which has a light microscopic glomerular distribution similar to collagen IV. In diabetic diffuse glomerulosclerosis, the enlarged mesangial matrix showed an increased staining reaction for collagen IV, V, laminin, and fibronectin whereas the staining pattern of HSPG was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane components, whereas HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for all basement membrane components diminished, whereas interstitial collagens V and III, but not collagen I, were present in these noduli in substantial amounts. These initial studies provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphologic correlate to the impaired filter function of the glomerular basement membrane.     

The extracellular matrix in sarcomatoid carcinomas of the breast

The distribution of the main extracellular matrix components has been investigated immunohistochemically in four cases of breast sarcomatoid carcinomas. The histogenesis of these tumours is still unclear, but most evidence suggests that the sarcomatous component originates from mesenchymal conversion of carcinomatous cells. We found that carcinomatous portions of the tumours were associated with linear basement membrane-like deposition of laminin, type IV collagen and heparan sulphate proteoglycan that partially circumscribed the epithelial nests. The sarcomatous components produced an extracellular matrix immunoreactive to fibronectin, type I, III and VI collagens and tenascin. However, in two cases, in some sarcomatous areas, focal pericellular staining for antibodies to laminin and type IV collagen was seen. These results indicate that modifications observed in the double tissue component of these tumours involve not only the cell shape and the cytoskeleton, but also the components of the extracellular matrix. The significance of these findings and their relevance in the understanding of the phenotypic pattern changes of these biphasic tumours are discussed.     

AA glomerular amyloid. An ultrastructural immunogold study of the colocalization of heparan sulphate proteoglycan and P component with amyloid fibrils together with changes in distribution of type IV collagen and fibronectin

An ultrastructural investigation was undertaken on paraformaldehyde-fixed Lowicryl resin-embedded human kidneys of three patients with AA amyloidosis to investigate the association of various basement membrane components with amyloid fibrils. An immunogold technique was used and antibodies to serum amyloid A, heparan sulphate proteoglycan, type IV collagen, P component, and fibronectin were applied to human normal and amyloid glomeruli. The amyloid was identified as AA, and P component was shown to be intimately associated with the fibrils. In addition, heparan sulphate proteoglycan was associated with amyloid in all subendothelial, subepithelial and intramembranous glomerular basement membrane deposits, and those throughout the mesangial matrix. This contrasted with the distribution of the proteoglycan in the normal glomerulus where it was found predominantly on the epithelial aspect of the basement membrane and only in the more peripheral regions of the mesangium. The accumulation of heparan sulphate proteoglycan with amyloid resulted in a marked increase in its amount in the glomeruli. The amyloid deposits contained little or no type IV collagen or fibronectin. These findings demonstrate a strong association of heparan sulphate proteoglycan with amyloid and suggest different roles for the various glomerular basement membrane components in amyloidogenesis.     

Distribution of the extracellular matrix components in human glomerular lesions

Most glomerular pathologies are associated with alterations of the matrix compartment. Using reagents directed against the α/α2 and α3 chains of type IV collagen [α1/α2(IV), α3(IV)], laminin, heparan sulphate proteoglycan (HPG), fibronectin, collagen I, and collagen III, we investigated the modifications of the glomerular matrix components in several human glomerular lesions compared with normal kidney. In type I membranous glomerulo-nephritis (MGN) (nine cases), we did not observe alterations in the matrix component distribution. In MGN types II and III (five cases), the spikes and chainettes were made of the α3(IV) chain, laminin, and HPG, while the α1/α2(IV) chains were localized along the subendothelial side of the glomerular basement membrane (GBM). In focal and segmental glomerulosclerosis (six cases), fibronectin, α1/α2(IV) chains, laminin, and small amounts of interstitial collagens were detected within the collapsed capillary loops; the newly formed matrix material between the podocytes and the GBM contained the α1/α2(IV) chains, laminin, and HPG but not the α3(IV) chain. In crescentic glomerulo-nephritis (six cases), fibronectin was the most abundant and, in purely cellular crescents, the unique component. A basement membrane-like network containing laminin, HPG, α1/α2(IV) chains, and interstitial collagens developed in a second step between the crescent cells. Interstitial collagens were present in the crescent framework, even when the integrity of Bowman's capsule was preserved. In membranoproliferative glomerulonephritis (five cases), we observed strong accumulation of fibronectin in the thickened mesangial spaces together with accumulation of laminin, α1/α2(IV) chains, and HPG; type I collagen was also present in the central part of the mesangial areas. This study shows that each glomerular lesion is characterized by particular alterations of the matrix components.     

Participation of collagen types I, III, IV, V, and fibronectin in the formation of villi fibrosis in human term placenta.

The indirect immunofluorescence method was used to study the human term placenta in pathological pregnancy for the distribution of collagen types I, III, IV, V, and fibronectin in fibrosis stromatis villi. All collagen types and fibronectin were shown to participate in fibrosis villorum formation. Fibronectin was also detected in the fibrinoid that surrounded villi at stroma. The presence of free cytotrophoblast cells in the fibrinoid was accompanied by a noticeable increase in fibronectin fluorescence. A significant amount of collagen types IV and V and a less amount of collagen types I and III were identified.     

Extracellular matrix synthesis by undifferentiated childhood tumor cell lines.

The authors have examined extracellular matrix (ECM) biosynthesis by small round cell tumors of childhood. Basal lamina (laminin and Type IV collagen) and stroma (collagens I, III, and V and fibronectin) constituents were studied. It was found that these tumors synthesize ECM in characteristic patterns. Five Ewing's sarcomas variably synthesized small amounts of all ECM constituents except Type V collagen. All eight neural tumors (neuroblastoma and primitive neural tumors) synthesized fibronectin (unlike some Ewing's sarcomas), as well as laminin and Type IV collagen (2 cases lacked Type IV collagen synthesis). No stromal (I/III) collagen synthesis was observed by neural tumors. All soft tissue sarcomas except an embryonal rhabdomyosarcoma synthesized stromal collagens and often laminin or fibronectin as well. Lymphomas synthesized no ECM of any kind. The synthesis of stromal collagens by sarcomas but not neural tumors serves to distinguish these two tumor types, especially Ewing's sarcoma from neuroblastoma. The presence of any ECM synthesis excludes lymphoma from diagnostic consideration.     

Structure,composition, and assembly of basement membrane

Basement membranes are thin layers of matrix separating parenchymal cells from connective tissue. Their ultrastructure consists of a three-dimensional network of irregular, fuzzy strands referred to as “cords”; the cord thickness averages 3–4 nm. Immunostaining reveals that the cords are composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin. Collagen IV has been identified as a filament of variable thickness persisting after the other components have been removed by plas-min digestion or salt extraction. Heparan sulfate proteoglycan appears as sets of two parallel lines, referred to as “double tracks,” which run at the surface of the cords. Laminin is detected in the cords as diffuse material within which thin wavy lines may be distinguished. The entactin and fibronectin present within the cords have not been identified as visible structures. The ability of laminin, heparan sulfate proteoglycan, fibronectin, and entactin to bind to collagen IV has been demonstrated by visualization with rotary shadowing and/or biochemical studies. Incubation of three of these substances–collagen IV, laminin (with small entactin contamination), and proteoglycan–at 35°C for 1 hr resulted in a precipitate that was sectioned for electron microscopic examination and processed for gold im-munolabeling for each of the three incubated substances. Three structures are present in the precipitate: (1) a lacework, exclusively composed of heparan sulfate proteoglycan in the form of two parallel lines, similar to double tracks; (2) semi-solid, irregular accumulations, composed of the three initial substances distributed on a cord network; and (3) convoluted sheets, which are also composed of the three initial substances distributed on a cord network but which, in addition, have the uniform appearance and thickness of the lamina densa of basement membrane. Hence these sheets are closely similar to the main component of authentic basement membranes.     

Biosynthesis of basement membrane molecules by salivary adenoid cystic carcinoma cells: an immunofluorescence and confocal microscopic study

The biosynthesis of basement membrane molecules and fibronectin was studied in vitro in the two different human cell systems (ACC2 and ACC3) established from adenoid cystic carcinomas (ACC) of the salivary gland using immunofluorescence and confocal microscopy. When the cells were attached and spread on dishes, fine granular immunofluorescence for type IV collagen, laminin, heparan sulphate proteoglycan, entactin, and fibronectin first appeared diffusely in the cytoplasm, and then changed into aggregation of coarse granules in the perinuclear area. With formation of colonies, these signals were present in the extracellular space, initially in the basal aspect of attached cells and consequently in the lateral intercellular space. After the cells formed a confluent monolayer, extracellular signals started to decrease in inverse proportion to the reappearance of intracellular ones. The results indicate that the parenchymal cells of ACC synthesize these five extracellular matrix molecules, secrete them into the extracellular milieu and remodel the extracellular deposits. It is suggested that the characteristic stromal architecture of ACC, represented by stromal pseudocysts, results from their own secretion of the basement membrane molecules and fibronectin.     

Fibrosarcoma: immunohistochemical study of the extracellular matrix

Immunofluorescent examination of extracellular matrix of 5 fibrosarcomas showed the matrix to contain fibronectin and collagen type I, III, V. Fibrosarcoma matrix is characterized by the absence of collagen type IV and collagen type III predominance over type I differentiating this tumor from synovial sarcoma, neurogenic sarcoma, dermatofibrosarcoma protuberans, malignant fibrous histiocytoma. Their matrices showed the presence of type IV collagen, equal proportion of interstitial collagens or collagen type I predominance over type III typical for synovial sarcoma. The findings can be employed in practical morphologic diagnosis of soft tissue tumors.     

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.     

Histological pattern and changes in extracellular matrix in aortic dissections.

Samples from 34 patients were studied both histologically and immunocytochemically by the indirect biotin-avidin peroxidase technique to analyse the distribution of the extracellular matrix components (type IV collagen, fibronectin, types I and III collagens) in dissection of the aorta. Most showed defects in type IV collagen around medial smooth muscle cells. Defects in smooth muscle cell basement membrane were found throughout the media in cystic medial degeneration and in medionecrosis, whereas in atherosclerosis such unlabelled areas were found only above advanced atherosclerotic plaques. In aortitis other defects in the smooth muscle cell basement membrane were found in areas of inflammatory infiltrates. In all of these conditions similar defects in fibronectin expression were also found. No defects in the expression of interstitial collagens type I and III were seen in the dissecting aortas. Moreover, cystic medial degeneration, medionecrosis, and atherosclerosis were characterised by intense staining of these interstitial matrix components. In the pathogenesis of the aortic dissection local changes in the basement membranes of the medial layer may be important.     

Distribution of connective tissue proteins in chick muscle spindles as revealed by monoclonal antibodies: A unique distribution of brachionectin/tenascin

The distribution in chick muscle spindles of eight connective tissue proteins (collagen types I, IV, V, and VI, laminin, heparan sulfate, fibronectin, and brachionectin/tenascin) was examined by immunofluorescent histochemistry. Intrafusal fibers were surrounded by layers of collagen type VI and fibronectin, and by an external lamina containing collagen type IV, laminin, and heppran sulfate. Most of these layers displayed a different pattern of staining at the sensory region of the equator than at the polar region. The crescent-like sheath that caps each intrafusal fiber and sensory terminal at the equator was strongly positive for collagen type I and weakly positive for collagen type V. The outer spindle capsule contained laminin, heparan sulfate, collagen types IV and VI, brachionectin/tenascin, fibronectin, and to a lesser degree also collagen types I and V. Brachionectin/tenascin had the narrowest distribution of any of the connective tissue macromolecules studied. It was found only in the outer capsule and in the coverings of blood vessels and nerves associated with the outer capsule.     

Co-deposition of basement membrane components during the induction of murine splenic AA amyloid

Past studies have demonstrated that during murine AA amyloid induction there is co-deposition of the AA amyloid peptide and the basement membrane form of heparan sulfate proteoglycan. The synthesis and accumulation of heparan sulfate proteoglycan does not usually occur in the absence of other basement membrane components, such as type IV collagen, laminin, and fibronectin. Using immunohistochemical techniques, the present experiments have demonstrated that in addition to the heparan sulfate proteoglycan, there are other basement membrane components present in splenic AA amyloid deposits and these are present as soon as AA amyloid deposits are detectable. The results indicate that within the time constraints imposed by the experiments, the basement membrane components, fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan are co-deposited 36 to 48 hours after the AgNO3 and amyloid enhancing factor induction of amyloid, the period when amyloid is first detected. These observations raise the possibility that an abnormality in basement membrane metabolism is a very early event, and potentially plays an integral part in the process of AA amyloidogenesis.     

Immunomorphologic study of the extracellular matrix in neurofibroma and benign schwannoma

The distribution of type I, III, IV and V collagen and fibronectin in schwannomas and neurofibromas showed the similarity in the extracellular matrix structure of both tumours. Both the components of the interstitial connective tissue (collagen of I, III, V types and fibronectin) and component of basal membrane (the IV type collagen) are found in the fibrillar matrix of these two tumours. It is shown for the first time that matrix of rhythmical structures of schwannoma contains the collagen of type IV and fibronectin while the interstitial collagen of types I, III and V is absent. The application of the results obtained for the differential diagnosis of fibrous and neurogenic tumours is discussed.