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

Collagen Res. ReI. Vol. 6/1986, pp. 125-152

The Extracellular Matrix is an Integrated Unit: Ultrastructural Localization of Collagen Types I, III, IV, V, VI, Fibronectin, and Laminin in Human Term Placenta PETER S. AMENTN, STEFFEN GAy2, ANTTI VAHERI 3 and ANTONIO MARTINEZ-HERNANDEZ! ! Hahnemann University, Department of Pathology and Laboratory Medicine, Broad and Vine, Philadelphia, PA 19102, USA, 2 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA and 3 Department of Virology, University of Helsinki, Helsinki, Finland. Abstract 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 30-35 nm, cross-banded fibers, often in the form of large fiber bundles. Type III collagen was present as thin 10-15 nm, beaded fibers often forming a meshwork which encased type I collagen fibers. Types V and VI collagen were present as 6-10 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. Key words: basement membrane, collagen, extracellular matrix, fibronectin, laminin. Introduction The extracellular matrix (ECM) is a stable complex of macromolecules surrounding stromal cells and lying beneath all epithelial cells (Hay, 1981). While initially considered an inert support, it is now apparent that the ECM functions in cell-matrix interactions, in matrix-matrix interactions, in differentiation, and in development (Bernfield and Banerjee, 1978; Hay, 1981). 9 Collagen 6/2

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Four major molecular species are found in the ECM: the collagens, the noncollagenous structural glycoproteins, elastin, and the proteoglycans. The collagens are a heterogeneous, but closely related group of proteins, with some ten genetically distinct types described (Cheah, 1985). The structural glycoproteins represent a distinct group of proteins, with the two best characterized being fibronectin and laminin. Fibronectin (M, = 440,000) found in both plasma and tissue forms, is capable of interacting with other components of the ECM (for review see Furcht, 1983; Ruoslahti et aI., 1981; Ruoslahti et aI., 1982; Vaheri et aI., 1983; Vartio and Vaheri, 1983; Yamada and Olden, 1978). Laminin (M, = 1,000,000) is exclusive to and found in all basement membranes studied to date (for review see Martinez-Hernandez and Amenta, 1983). Basement membranes (BM) also contain type IV collagen and another glycoprotein entactin (Bender et aI., 1981; Carlin et aI., 1981; Martinez-Hernandez and Chung, 1984; Mynderse et aI., 1983). The proteoglycans are a heterogeneous group of macromolecules, characterized by a protein core linked to glycosaminoglycan side chains (Hascall and Hascall, 1981; Hascall and Kimura, 1982; Iozzo, 1985). A heparan sulfate proteoglycan is present in many BM (Kanwar and Farquhar, 1979; Mynderse et aI., 1983). The prevalent use of the placenta as a source of ECM, is due to its availability, accessibility, and to the wealth of ECM components it contains (Chung et aI., 1976; Furuto and Miller, 1980; Isemura, 1984; Sage and Bornstein, 1979). Despite its extensive use in biochemical studies, little is known of the distribution of specific ECM components in the placenta. Further, although there are numerous studies describing the electron immunohistochemical localization of one or two ECM antigens in a tissue, there are few studies localizing multiple components which could provide information on the interrelationship of ECM components in the same tissue. Therefore, we decided to localize collagen types I, III, IV, V, VI, fibronectin, and laminin in the placenta. The results obtained illustrate the supramolecular organization of seven different components of the ECM in the human term placenta. BM contained laminin and type IV collagen, but no fibronectin. Type I collagen was the basic structural unit of the human term placenta, present as 30-35 nm cross-banded fibers. Type III collagen often codistributed with type I collagen, as did other interstitial proteins, fibronectin, and types V and VI collagen. The pattern observed with the antibodies to collagen types III, V, and VI, and fibronectin, suggests that one function of these proteins is to link type I collagen fibers to one another and to other structures. The well-known binding properties of fibronectin would be well-suited to this type of activity (for review see Furcht, 1983; Ruoslahti et aI., 1981).

Materials and Methods Tissue Fixation. Human term placentae were collected 1 to 2 h following normal delivery or Cesarean section, cut into 5 x 5 x 2 mm blocks, to include decidua and chorionic villi and processed as previously described (Amenta et aI., 1983; Boselli et aI., 1981 ; Martinez-Hernandez et aI., 1981; Martinez-Hernandez et aI., 1974). For conventional light microscopy, blocks were fixed in 4% formaldeyhde for 24 h, dehydrated in graded ethanol solutions, and embedded in paraffin via xylene. Sections were stained with hematoxylin and eosin, periodic acid Schiff (PAS), trichrome, and reticulin stains (Lillie and Fullmer, 1976). For conventional electron microscopy, after 3 h fixation in 3 % glutaraldehyde in 0.1 M sodium phosphate buffer with 26 mM NaCI,

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tissues were postfixed in 1 % OS04 for 1 h, dehydrated in graded ethanol solutions, and embedded in Medcast-Araldite via propylene oxide. Ultrathin sections were mounted on uncoated grids, stained with lead citrate and uranyl acetate. For light microscopic immunohistochemistry, blocks, were embedded in OCT® compound and quick frozen in methyl butane, precooled to liquid nitrogen temperature. For electron immunohistochemistry, blocks were fixed by immersion in freshly made 4% formaldehyde in 0.2 M sodium phosphate buffer (pH = 7.4) for 3 h, followed by multiple washings in phosphate-buffered saline (PBS) with 4% sucrose, with constant agitation, at 4°C for 16h. Following a final wash in PBS with 7% glycerol and 4% sucrose, they were quickly frozen in methyl butane, precooled to liquid nitrogen temperature, as previously described (Amenta et aI., 1983; Martinez-Hernandez, 1984).

Antibodies Polyclonal antibodies. All primary polyclonal antibodies were made in rabbits. The IgG and Fab fragments were obtained as previously described (Amenta et aI., 1983; Boselli et aI., 1981). Anti-mouse plasma fibronectin, anti-rat ED-PYS laminin, anticollagen types I, Ill, IV, and V antibodies were characterized by ELISA and e1ectroimmunoblotting, as previously described (Engvall and Pearlman, 1972; Gay et aI., 1976 a). Polyclona.1 antibodies to type VI collagen were the gift of Dr. Eva Engvall. Normal rabbit serum was used as negative control. Goat anti-rabbit Fab and rabbit Fab peroxidase-anti-peroxidase complex (PAP) were prepared as previously described (Amenta et aI., 1983; Slemmon et aI., 1980). Monoclonal antibodies. Monoclonal antibodies to human plasma fibronectin (amino terminal domain) were prepared as previously described (Pierschbacher et aI., 1981; Vartio et aI., 1983). Monoclonal antibodies to the other fibronectin domains (gelatin-, heparin-, and cell attachment site) were the gift of Dr. Erkki Ruoslahti and Michael Piersbacher. Monoclonal antibodies to type VI collagen were the gift of Dr. Eva Engvall. Normal mouse serum (Cappel Laboratories) was used as negative control. Goat anti-mouse IgG and mouse PAP were obtained from Cappel Laboratories. Immunohistochemical staining. Four !tm (for light microscopy) and 8 !tm (for electron microscopy) cryostat sections were mounted on albumin-coated slides and reacted with the corresponding antibodies. The optimum antibody concentration was obtained by checkerboard titration as previously described (Amenta et aI., 1983). Prior to incubation with primary antibodies, tissues were treated with sodium borohydride and normal goat serum. Between antibody incubations the tissues were washed three times (10 min) with PBS at 4°C. Following incubation in PAP, the sections were post-fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 30 min and reacted with 0.1 M glycine in PBS, prior to incubation in diaminobenzidine and H 2 0 2 • Sections processed for electron microscopy were reacted with 1 % OS04 in 0.1 M sodium phosphate buffer for 1 h, dehydrated, and embedded in Medcast®-Araldite. Ultrathin sections were mounted on uncoated copper grids and photographed without further staining.

Unmasking Experiments The following treatments were used: Trypsin. Trypsin (2,750 units/mg; Calbiochem, ultrapure #1605) was dissolved in PBS and passed through a Millipore filter prior to application. Sections were reacted for 20 min at room temperature with trypsin at concentrations of 12,000, 6,000, 4,000, 1,000 and 100 units/m!. To insure that the trypsin-treated sections where

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actually digested by specific activity and not by contaminant enzymes, alternate sections were treated with soybean trypsin inhibitor (Sigma T9-003 ) at 2.5 X the concentration necessary to completely inhibit the trypsin. Glycosidases. Mixed glycosidases (Seigake ET 5803) in 0.2 M NaP0 4 buffer, pH = 5.5 at 5 mg/ml and 10 mg/ml, were used for 30 min at room temperature. The enzyme preparation was passed through a millipore filter prior to application. Periodic Acid. Sections were treated with 10 mM Periodic Acid in PBS for 15 min at room temperature. Acetic Acid. Sections were treated with 0.25 M acetic acid 30 min at room temperature. Fig. 1. Conventional and Immunohistochemical light Microscopy 1 a. Methacrylate-embedded section of human term chorionic villi. The villi are bathed in maternal plasma present in the intervillous space (IVS). Each villus is surrounded by a layer of trophoblast (T) encasing a core of extracellular matrix and interstitial cells. Fetal capillaries (Cl) are scattered in the interstitium (I). Hematoxylin-eosin. Original magnification = x 65. 1 b. Laminin distribution. laminin is confined to basement membrane zones of the trophoblast (TBMZ) and endothelium (EBMZ). The plasma spaces (IVS and Cl) and interstitium (I) are negative. Original magnification = x 65. 1 c. Type IV collagen distribution . The localization of type IV collagen is similar to that of laminin, being present in all basement membrane zones (TBMZ and EBMZ); however, in contrast to the former, the interstitium (I) has some occasional reactivity. By electron microscopy immunohistochemistry, this reactivity is found in lamellar remnants of basement membrane (see Fig. 3e). The plasma spaces (IVS and Cl) are negative. Original magnification = x 65. 1 d. Fibronectin distribution (anti-amino terminal domain). The interstitium (I) contains most of the reactivity. There is occasional staining, in or near, the basement membrane zones of the trophoblast (TBMZ) and endothelium (EBMZ). Maternal plasma (MP) is positive. The staining obtained with this monoclonal antibody was indistinguishable from that obtained with the other anti-fibronectin antibodies. Original magnification = x 65. 1 e .Type I collagen distribution. The interstitium (I) of each chorionic villus is positive, with a diffuse, homogeneous pattern. The plasma spaces (IVS and Cl) are negative. Original magnification = x 65. 1 f . Type III collagen distribution . The interstitium (I) of the chorionic villus is positive. The distribution of type III collagen, although similar to that of type I, has a more distinct linear pattern. Plasma spaces are negative (IVS and Cl) . Original magnification = x 65 . 1 g. Type V collagen distribution . The interstitium (I) is positive, the pattern of distribution being most like that of type VI collagen (see Fig. 1 h) in its preferential perivascular distribution. Trophoblastic basement membrane zones are occasionally positive (arrowheads). Vascular spaces (lVS and Cl are negative). Original magnification = x 65 . 1 h. Type VI collagen distribution. The chorionic villus interstitium (I) is positive in a linear pattern, similar to that observed with anti-type V collagen. Trophoblastic basement membrane zones are occasionally positive (arrowheads). The vascular spaces (IVS and Cl) are negative. Original magnification = x 65. Abbreviations Used in All Figures: C = Cytotrophoblast; Cl = Capillary lumen; E = Endothelial; EBM = Endothelial Basement Membrane; EMBZ = Endothelial Basement Membrane Zone; F = Fibrocytic Cell; FP = Fetal Plasma; I = Interstitium; IVS = Intervillous Space; MP = Maternal Plasma; P = Pericytic Cell; S = Syncytiotrophoblast; SBM = Syncytiotrophoblastic Basement Membrane; 5MBM = Smooth Muscle Basement Membrane; SMC = Smooth Muscle Cell; T = Trophoblast; TBMZ = Trophoblastic Basement Membrane Zone Vl = Vascular lumena.

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Results Conventional Microscopy In general, conventional light (Fig. 1 a) and electron microscopy (Fig. 2 and 8 a) were consistent with previous reports on the structure of human term placenta (Boyd and Hamilton, 1970; Padykula, 1983; Rhodin and Terzakis, 1962), but demonstrated some features not previously described. The trophoblast, at term was composed of the outer syncytiotrophoblast and an occasional cytotrophoblastic (Langhan's) cell (Fig. 2 a). The syncytiotrophoblast contained numerous cisternae of rough endoplasmic reticulum and mitochondria (Fig. 2 a). These organelles were less abundant in the cytotrophoblast, accounting for the clear staining of these cells seen by conventional electron microscopy (Rhodin and Terzakis, 1962). The vascular surface of the syncytiotrophoblast contained numerous microvilli whereas its basal surface had numerous infoldings (Fig. 2 a and 2 b). In contrast, the cytotrophoblast had few, if any, surface projections. The basal surface of the trophoblast was observed to be in contact with a BM characterized by numerous projections, interposed between the basal infoldings of the syncytiotrophoblast (Fig. 2 b). The interstitium of the chorionic villi contained fibroblasts and occasional Hofbauer cells encased by an ECM of amorphous lamellae, fibrillar material, and cross-banded collagen fibers (Fig. 2 b, 2 c, and 2 d). These cross-banded collagen fibers (30-35 nm) were often in discrete bundles, surrounded by and intertwined with thin (10-15 nm) fibers. The latter fibers were characterized by an irregular-beaded periodicity (Fig. 2 d), varying from 30-65 nm, depending on the tortuosity of the fiber, where the periodicity was measured. Fine (6 to 10nm), non-beaded filaments were often present in contact with and perpendicular to, one or more beaded or banded fibers (Fig. 2 d and 8 a). The

Fig. 2. Conventional electron microscopy 2 a. Term trophoblast. The trophoblast at term is composed of a complete layer of syncytiotrophoblast (S) and an occasional cytotrophoblastic (Langhan's) cell (C). The endothelium (E) and trophoblast are separated from the interstitium (I) by a basement membrane (EBM and TBM, respectively). The interstitium between the two basement membranes contains collagen, fibrillar, and granular material. Details of the interstitium from this area are shown in Figure 2 b. Magnification = x 6,000. 2 b. Detail of the villar basement membranes. Between the trophoblastic (TBM) and endothelial (EBM) basement membranes, there are thick (30-35 nm), banded collagen fibers (straight arrows). Some of these cross-banded collagen fibers insert into the trophoblastic basement membranes (arrowhead). The endothelial basement membrane is lamellated and poorly defined. Note the trophoblastic (T) basal infoldings (curved arrow). Magnification = x 26,300. 2 c. The villar stroma. The stroma consists of cells and extracellular matrix. Numerous projections from fibrocytic cells (F) are present. Lamellate remnants of basement membrane are free in the interstitium (arrows). A pericytic cell (P) is surrounded by lamellae of basement membrane (arrows). Magnification = x 4,200. 2 d. Detail of the villar extracellular matrix. The extracellular matrix consists of bundles of thick cross-banded collagen fibers (30-35) (large arrowheads). Thinner, beaded fibers, forming a meshwork, are closely associated with this bundle (small arrowheads). Similar beaded fibers are present within the bundle proper, between the thicker-banded fibers (large arrows) . Fine filamentous structures associated with the larger, beaded and banded fibers are present (small arrows) (Please see Figure 8). Magnification = x 32,400.

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endothelial BM of the fetal capillaries was often thinned, usually lamella ted, and occasionally absent (Fig. 2 b). Collagen fibers were present between the lamellae. Often, lamellae of BM-like material were found free within the interstitium (Fig. 2 a). Occasionally, the endothelial and trophoblastic BM were closely apposed to one another, with occasional contact between the two; although, usually, there was ECM separating the two BM (Fig. 2 b). Cross-banded collagen fibers and beaded fibers were often observed inserted into the trophoblastic and endothelial BM (Fig. 2 b) .

Immunohistochemistry Laminin

Laminin was consistently localized to the chorionic villus (Fig. 1 b, 3 a-3 d), those of the trophoblastic and vascular BM, with a preferential reaction in the lamina rarae (Fig. 3a and 3 b) . Such a localization was prominent at the basal surface of the trophoblast, where reaction product was consistently present in the BM around cell surface projections, with the intensity of the reaction greater in the trophoblastic than in the endothelial BM (Fig. 3b). The interstitium of the chorionic villi contained no laminin, except for collagen fibers adjacent to BM, which were often coated with reaction product (Fig. 3b). Occasional cytotrophoblastic cells contained laminin antigen in the cisternae of the rough endoplasmic reticulum (Fig. 3c). Endothelial cells had no demonstrable laminin in positive intracytoplasmic organelles; however, pinocytotic vesicles containing laminin antigen on the ablumenal surface, were often present. One intersti-

Fig. 3. Localization of Basement membrane components 3 a. Laminin localization. Tangential section of chorionic villus. Both trophoblastic (TBM) and endothelial (EBM) basement membranes are positive. There is a preferential reaction with the lamina rarae of both basement membranes (arrowheads). The intervillous space (lVS) and capillary lumena (CL) are negative. Magnification = x 8,200. 3 b. Detail of laminin localization . The trophoblastic basement membrane (TBM) is positive with preferential localization in the lamina rara (arrowheads). The cell surface of the basal trophoblastic infoldings is positive (curved arrows). The lamellated endothelial basement membrane (EBM) is also positive. The reaction of the antibodies was consistently more intense with the trophoblastic than with the endothelial basement membrane. Collagen fibers adjacent to either basement membrane have some laminin on their surfaces (small straight arrows), whereas those fibers further from the basement membranes are negative (large straight arrows). The capillary lumen (CL) is negative. Magnification = x 9,800. 3 c. Laminin localization. Detail of a cytotrophoblastic cell. A cytotrophoblastic cell (C) contains laminin antigen in the cisternae of the rough endoplasmic reticulum (arrowheads). Magnification = x 7,300. 3 d. Laminin localization. Detail of interstitial cell. An interstitial cell (Hofbauer cell?) contains reaction product in the cisternae of the rough endoplasmic reticulum (curved arrows). Collagen fibers (small, straight arrows ) in the interstitium (I) are negative. Magnificiation = X 12,700. 3 e . Type IV collagen localization. The basement membranes are positive with preferential staining of the lamina densa (arrowheads). Positive lamellae of basement membrane material (large straight arrows) are present adjacent to the fetal vessels (CL). Collagen fibers within the interstitium are negative (open arrow) . The capillary lumen (CL) is negative. Magnification = x 6,700.

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tial cell also contained reaction product in the cisternae of the rough endoplasmic reticulum (Fig. 3 d). Although a definitive identification could not be made, its appearance was most consistent with its identification as a Hofbauer cell. The contents of vascular lumena, syncytiotrophoblastic, and endothelial cell membranes, in contact with plasma, were negative. Type IV Collagen

Type IV collagen was found in all BM with a distribution similar, but not identical to laminin (Fig. 1 c and 3 e). Differences included preferential staining of the lamina densa with anti-type IV collagen, the lack of localization in the interdigitations of BM between the syncytiotrophoblastic projections, and equally intense staining of endothelial and trophoblastic BM. Further, remnants of BM lamellae, free in the interstitium, were intensely reactive with anti-type IV collagen antibodies but reacted only weakly with anti-laminin (Fig. 3b and 3 e). This staining of such material may explain the occasional, interstitial reaction noted with anti-type IV antibodies by light microscopy (Fig. 1 c) . Collagen fibers adjacent to BM were observed to be coated with reaction product, whereas fibers away from BM were negative (Fig. 3 e). Unlike laminin, type IV collagen antigen was not found within the cytoplasm of any cell, only occasional pinocytotic vesicles were positive on the ablumenal surface of endothelial cells. Vascular spaces and cell surfaces in contact with plasma failed to react with the antibody. Fibronectin

Antibodies to fibronectin, both mono- and polyclonal, had similar reactions with placental tissues (Fig. 1d, 4 a-4 e) . However, there was a greater intensity of staining with the monoclonal antibody directed against to amino terminal domain of fibronectin, as compared to that obtained with the other antibodies. Fibronectin was present in Fig. 4. localization of Fibronectin 4 a. Anti-human plasma, fibronectin, gelatin binding domain. Fetal capillary space (Cl) contains fibronectin (FP). Basement membranes (TBM and EBM) and the intervening interstitial (I) collagen fibers are negative. Magnification = x 8,400. 4 b. Anti-human plasma fibronectin, amino-terminal domain . Detail for endothelial surface. Reaction product (in fetal plasma (FP) is present coating the endothelial cell surface and free within the lumen (Cl). The basement membranes (TBM and EBM) are negative. Magnification = x 37,300. 4 c. Anti-human plasma fibronectin, amino terminal domain. The interstitium (I) is diffusely positive, with multiple collagen fibers (arrows) coated with fibronectin. The trophoblastic basement membrane (TBM) is negative. Magnification = x 16,900. 4 d. Anti-human plasma fibronectin, amino terminal domain. Detail fo interstitium. Collagen fibers with the characteristic 64 nm, cross-banding (small arrowheads) are coated on their surface. Thinner 15-20 nm fibers are also positive (large arrowhead). Magnification = x 26,600. 4 e . Anti-human plasma fibronectin , amino terminal domain. Localization after treatment with glycosidases. There is no significant change from untreated sections. The interstitium is positive, collagen fibers (arrowheads) are coated with antigen and the trophoblastic basement membrane (TBM) remains negative. Tissue preservation is acceptable. Magnification = x 10,700. 4f. Normal mouse serum. All structures are negative. Magnification = x 9,700.

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both fetal and maternal plasma and was observed to coat those cell surfaces (endothelium and trophoblast) in contact with plasma. Pinocytotic vesicles on both the lumenal and ablumenal surfaces of endothelial cells were often positive. Fibronectin was also present in the ECM of the chorinoic villi (Fig. 4 c and 4 d), with the most intense reaction subjacent to the trophoblastic and endothelial BM. By light microscopy this appeared as a continuous linear staining, difficult to resolve from the BM itself (Fig. 1 d) . Collagen fibers of varying diameter were observed to be coated with fibronectin, which enhanced their periodicity (Fig. 4d) . Fibronectin was also present as free filaments or globular aggregates within the interstitium (Fig. 4d). None of the cells contained positive stained organelles. The BM of the chorionic villi were negative (Fig. 4 a-4 c and 4 e) except for occasional, spotty traces. Sections treated with enzymes or acids to unmask antigens, had no obvious change in antigenic localization. Specifically, fibronectin antibodies remained localized to the interstitium, with no demonstrable reaction with BM (Fig. 4 e). However, the preservation of tissue structure was poor with all concentrations of trypsin. Nuclear, organellar, and cellular membranes were removed with only occasional collagen aggregates readily identifiable (data not shown). Glycosidases, acetic and periodic acid-treated sections had ultrastructural preservation comparable to that observed in untreated sections. Type I Collagen

Type I collagen was restricted to the interstitium of the chorionic villus (Fig. 1 e, 5 a and 5 b) . The pattern of staining obtained with anti-type I collagen antibodies was distinct from all other antibodies, both by light and electron microscopy. By light microscopy the interstitium was diffusely and homogeneously stained throughout (Fig. 1e). By electron microscopy, the staining was virtually restricted to the surface of

Fig. 5. Localization of Collagens I and III 5 a. Type I collagen localization. Cross-banded (D = 64 nm) collagen fibers (arrowheads) adjacent to the trophoblastic basement membrane (TBM) are positive, highlighting the characteristic banding pattern of this collagen type. The adjacent trophoblastic (TBM) basement membrane is negative. Magnification = x 11,500. 5 b. Type I collagen localization. Detail of individual fibers. Cross-banded, 30-35 nm diameter fibers are positive. Thinner fibers adjacent to thick collagen fibers are negative (arrows). Virtually no reaction product is found free between the thick, type I collagen fibers. Magnification = x 31,000. 5 c. Type III collagen localization. Type III collagen fibers are intensely stained. The type III fibers encase type I fibers, (see Fig. 5 d) as a consequence of this encasement, large type I collagen fibers appear in negative image (arrowheads). The trophoblastic basement membrane (TBM) is negative. Magnification = x 11,000. 5 d. Type III collagen localization. Detail of interstitium. Dense aggregates of type III collagen fibers surround type I fibers. Due to the density of these aggregates, it is difficult to resolve individual type III fibers . Because of their intimate association with thick type I collagen fibers (arrowheads), the latter appear in negative image with inconspicuous crossbanding. Magnification = x 17,300. 5 e. Type III collagen reaction. Detail of interstitium. Thin fibers, with a beaded periodicity, are stained with anti-type III antibody. Individual fibers can be resolved (arrowheads). Magnification = x 22,400.

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thick, cross-banded (30-35 nm) fibers, and bundles of these fibers. The staining often occurred at regular intervals which corresponded to the characteristic 64 nm crossbanded pattern of this collagen (Fig. 5 a and 5 b). The interstitial space between and surrounding these fibers was negative. Non-banded, smaller diameter (15-20nm) fibers, in contact with type I fibers, were occasionally positive, possibly representing thinner type I fibers. However, the majority of the thinner fibers did not react with type I antibody (Fig. 5 b). Type III Collagen

By light microscopy, type III collagen had a different distribution than type I collagen (Fig. 1 f and 5 c-5 e). By electron microscopy, anti-type III collagen anitbodies were localized on thin, non-banded, beaded fibers of 10-15 nm ciiameter and had a 40-60 nm beaded interval (Fig. 5 e) . These fibers were ubiquitous in the interstitium, usually present as dense bundles or aggregates in which resolution of individual fibers was difficult (Fig. 2 d, 5 c, 5 d and 8 a). Individual type III collagen fibers could be resolved occasionally at the edge of a bundle (Fig. 5 e). Type III collagen was abundant, encasing both fibers and bundles of type I collagen (Fig. 5 d), often resulting in a negative image of type I collagen fibers (Fig. 5 c and 5 d ). Further, the banding pattern obtained with anti-type I collagen antibodies on the thick, cross-banded fibers was obscured in sections where anti-type III collagen antibodies reacted intensely (Fig. 5 d). Often, the type I collagen fibers contained aggregates of reaction product along their surface, in a regular or irregular fashion, suggesting that type III collagen fibers may run both perpendicular and parallel to type I fibers. Occasionally BM, adjacent to the interstitium, were positive with anti-type III collagen antibody. However, BM of vascular smooth muscle and endothelial cells away from the interstitium, were consistently negative. Type V Collagen

Type V collagen (Fig. 1g, 6 a, 6 b ) was strictly limited to the interstitium of the chorionic villus in a distinct perivascular distribution. Non-beaded, filamentous, 6-10 nm structures on the surface of thick type I fibers and type III fibers reacted with the anti-type V collagen antibodies. Reaction product was so intense that individual filaments were often obscured. Type I collagen was often observed to be embedded within a dense matrix of type V collagen, producing a negative image of the large, cross-banded type I fibers. Aggregates of reaction product were observed on the surface of type I fibers in an irregular distribution (Fig. 6 a). Trophoblastic BM were consistently negative, as were BM of smooth muscle cells and endothelial BM of large vessels. Occasionally, vascular BM adjacent to the villus interstitium were positive with antitype V collagen antibody, as with the other collagens studied (Fig. 6 b ). Structures corresponding to the filaments demonstrated with anti-type V collagen, were identified on overstained sections, processed for conventional electron microscopy. Type VI Collagen

Type VI collagen was found to be localized exclusively in the interstitium of the chorionic villus, with a preferential distribution in perivascular regions and subjacent

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Fig. 6. Type V collagen localization 6 a. Type V collagen localization. The reaction product is restricted to the villar interstitium. Thick collagen fibers are encased by reaction product. An occasional filament can be resolved (arrows). The trophoblastic basement membrane (TBM) is negative. Magnification = x 22,100. 6 b. Type V localization. Detail of villar basement membranes. The endothelial basement membrane is difficult to identify but is apparently positive (large arrowheads). The basement membrane separating two pericytic cells (P) and an endothelial cell (E) is negative (small arrowhead), as is the trophoblastic basement membrane (TBM). Magnification = x 25,200.

to the trophoblastic BM (Fig. 1 hand 7 a-7 e). The monoclonal and polyclonal antibodies directed against type VI collagen showed identical localizations. By electron microscopy, type VI collagen appeared to coat the surfaces of collagen fibers of all diameters und reacted with 6 to 10 nm filaments in the interstitium (Fig. 7 d). Although types III, V, and VI collagens were all often associated with thick (30-35 nm), crossbanded type I collagen fibers and formed fine fibrils, type III and often type V collagen formed a meshwork encasing type I fibers, veiling their periodicity (Fig. 7 d). In contrast, staining with anti-type VI collagen often coated the surface of type I collagen fibers, enhancing the 64 nm banding pattern (Fig. 7 d). This marking of type I collagen periodicity and the presence of free interstitial aggregates was reminiscent of the pattern obtained with anti-fibronectin antibodies (Fig. 4 d and 7 d). BM did not contain type VI collagen, except for occasional BM adjacent to the interstitium. A summary of the relationships of the collagens studied and fibronectin in the ECM of the chorionic villus is depicted in Table I and in graphic form in Figure 8 b. Discussion The human placenta is a complex organ that undergoes rapid growth and remodeling during gestation. A brief review of its features (Boyd and Hamilton, 1970; Padykula, 1983; Rhodin and Terzakis, 1962) would facilitate discussion of our results.

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The human term placenta has two components: maternal decidua basalis and fetal chorion frondosum. At term, the decidua occupies the entire surface of the endometrium. The regions of the decidua are termed according to their topographical relation to the trophoblastic implantation site. That portion of the decidua underlying the fetal chorion frondosum is the decidua basalis and anchors the placenta on the uterine wall. Microscopically, the decidua is composed of large, maternal cells derived from endometrial stroma, containing occasional fetal cytotrophoblast cells encased in lipid, glycogen, and ECM (Padykula, 1983). At term, the chorionic villi form an intricate, "branching" network of vessels, surrounded by connective tissue and are lined externally by trophoblast. The development of the villi proceeds through a series of stages. Primary villi (third week of gestation) consist of a peripheral rim of syncytiotrophoblast cells encasing a core of cytotrophoblastic cells. The cytotrophoblast is eventually replaced by mesodermal tissue, leaving only a single layer of cytotrophoblast, forming the secondary villus. By the end of the fourth week of gestation, blood vessels begin to develop within the interstitium forming the tertiary villus. The continuous layer of cytotrophoblast disappears by the 12th to 16th week of gestation; therefore, at term only an occasional cytotrophoblastic (or Langhan's cell) is present, underlying a thinned syncytiotrophoblast. The chorion frondosum is best considered a tree-like network, where larger or stem villi contain more fibroconnective tissue than do the smaller, "free-floating" peripheral villi. The trophoblastic BM is often better defined than the endothelial BM. The former is characterized by numerous projections which interdigitate with basal infoldings of the syncytiotrophoblast. Occasional lamellae of BM material were found in the villar interstitium; probably representing residual BM left over from the continuous growth and remodeling of the placenta during gestation. The connective tissue core of the chorionic villus (exclusive of BM) contains fibrillar material and collagen fibers of

Fig. 7. Type VI collagen localization 7 a . Type VI collagen localization. Type VI collagen is diffusely distributed throughout the interstitium (I) . The trophoblast basement membrane (TBM) is negative. Polyclonal antibody. Magnification = x 6,400. 7 b. Type VI collagen localization. Detail of trophoblastic basement membrane. Type VI collagen filaments are intensely positive. The reaction product is so intense, as to obscure individual filaments. The trophoblastic basement membrane (TBM) is negative. The endothelial basement membrane (arrowheads) surrounding this vessel is poorly defined. Polyclonal antibody. Magnification = x 10,000. 7 c .Type VI collagen localization . Detail of the interstitium. Type VI collagen is prevalent in the interstitium. Numerous cross-banded, type I collagen fibers (arrowheads) are coated with type VI collagen. Poly clonal antibody. Magnification = x 6,000. 7 d. Type VI collagen localization . Detail of interstitium. Thick, banded fibers are positive (large arrowheads) revealing the characteristic periodicity associated with type I collagen. Thinner fibers are also coated. The reaction product is so prevalent as to often obscure individual fibers. Thin filaments are also positive (small arrows). This pattern of staining most resembles that seen with anti-fibronectin (see Figure 4 d) . Polyclonal antibody. Magnification = x 29,500. 7 e. Type VI collagen localization. Detail for an arteriole. The vascular lumena (VL) is negative as is the endothelial surface and smooth muscle basement membrane (SMBM) . The endothelial basement membrane is negative (EBM) . The interstitium (I) is positive with large collagen fibers (arrowhead) coated with type VI collagen. Monoclonal antibody. Magnification = x 14,000.

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various sizes (Fig. 2). Within this interstitial core there are two basic cell types, fibrocytic and Hofbauer cells. In the present study, seven ECM components were localized in the chorionic villi of the human term placenta. Laminin and type IV collagen, as expected, were localized in both the trophoblastic and endothelial BM. As previously shown (Gil and MartinezHernandez, 1984; Martinez-Hernandez, 1984; Martinez-Hernandez and Chung, 1984; Martinez-Hernandez et al., 1982 b), laminin was preferentially localized in the lamina rara of both trophoblastic and endothelial BM. This was best illustrated in the trophoblastic BM where the syncytiotrophoblastic cell surface, at the basal infoldings, was coated with reaction product and the BM projections were heavily stained. In contrast, anti-type IV collagen was localized preferentially in the lamina densa (Martinez-Hernandez, 1984; Mynderse et al., 1983) and absent in the trophoblastic associated BM projections or basal cell surface. This differential localization of type IV collagen and laminin demonstrates that under the short diaminobenzidine incubation times used in this study, diffusion of reaction product is minimal (Courtoy et al., 1983). If a diffusion artefact was of any significance, it would be expected that the reaction product of anti-type IV collagen would also coat the trophoblastic cell membrane, but this was not the case (Fig. 3 band 3 e) . The finding of remnants of BM in the villar interstitium probably reflects the continuous remodeling of the placenta. Of interest was the relative greater reactivity of these remnants with anti-type IV collagen than with anti-laminin. This may reflect the relative resistance of type IV collagen to protease digestion. The alternative is that these lamellae are remnants of endothelial BM, since they react more intensely with anti-type IV collagen than with anti-laminin antibody. The results obtained with the polyclonal, as well as four monoclonal antibodies to fibronectin, were virtually identical and consistent with previous studies (Alitalo et al., 1980; Alitalo and Vaheri, 1982; Amenta et al., 1983; Boselli et al., 1981; Courtoy and Boyles, 1983; Gil and Martinez-Hernandez, 1984; Hedman et al., 1982 a, b; Madri et al., 1980 b; Martinez-Hernandez, 1984; Martinez-Hernandez et al., 1981; Semoff et al., 1983; Vaheri et al., 1978). Fibronectin was not observed in BM, but in plasma and

Fig. 8. Conventional electron microscopy and graphic summary of relationships of interstitial collagens 8 a. Conventional electron microscopy. Cross-banded type I collagen fibers (large arrowheads) (30-35 nm) are enmeshed by numerous beaded, thin (10-15 nm) type III collagen fibers (small arrowheads). Type III collagen fibers are often interspersed between type I fibers within the bundles. This observation, along with immunohistochemical studies demonstrate that these bundles are heterogeneous. Thin (6-10 nm) filaments, some perpendicular to larger fibers are present (arrows). The immunohistochemical data suggests these filamentous structures represent types V and VI collagen as well as fibronectin. The dimensions of these fibers and filaments may not represent fiber dimensions in other organs or even in placenta at other gestational dates. Magnification = x 31,200. 8 b. Graphic summary. Thick, cross-banded (D = 64 nm) type I collagen fibers (gray, 30-35 nm) form the major support of the placental extracellular matrix. Thinner (10-15 nm) irregularly, beaded type III fibers often are closely associated or apposed to type I fibers. The irregularity of the beading (30-64 nm) may be the consequence of the tortuosity of these fibers, i. e. where fibers are straight D = 64 nrn. Thinner, filamentous structures (6-10 nm) represent types V and VI collagen as well as fibronectin. These filamentous proteins may act as connector proteins, interconnecting larger fibers.

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Table I. The tissue distribution, supra molecular organization and relationships of the extracellular matrix components studied. The Extracellular Matrix Is An Integrated Unit: Ultrastructural Localization of Collagen Types I, III, IV, V, VI, Fibronectin and Laminin in Human Term Placenta Summary of the Ultrastructural Distribution and Associations of the ECM Components Tissue Distribution

Supramolecular Organization

Collagen Type I

Interstitium

Basic structural component Cross-banded fibers; diameter = 30-35 nm; associated with collagen types III, V, VI and fibronectin banding interval =64nm

Collagen Type III

Interstitium

Beaded fibers; diameter Associated other interstitial col= 15-20 nm; beaded lagens and fibronectin interval = 40-64 nm

Collagen Type V

Interstitium

Thin filaments; diameter = 10 nm

Associated with types I and III often extending to this interstitial aspect of vascular basement membranes

Collagen Type VI

Interstitium

Thin filaments; diameter = 10 nm

Associated with types I and III often extending to the interstitial aspect of basement membranes

Fibronectin Interstitium and plasma

Associations

Thin filaments and glo- Associated with interstitial colbular aggregates diame- lagen ter = 10nm

Laminin

Basements membra- Not resolved nes both laminae but preferentially in the lamina rara

Associated with type IV collagen

Collagen Type IV

Basement membrane Not resolved both laminae but preferentially in the lamin a densa

Associated with laminin

stroma. Collagen fibers of all diameters were coated with reaction product. The crossbanding of type I collagen fibers was enhanced by all the anti-fibronectin antibodies (Fig. 4). The localization obtained with all the monoclonal anti-fibronectin antibodies, directed against determinants of different functional domains, was identical. This identical localization suggests that fibronectin is present in tissues in a globular or folded configuration. If fibronectin was present in tissues in an unfolded or linear configuration, a slightly different pattern of distribution might be expected for the respective monoclonal antibodies. It is interesting, that fibronectin was rarely localized in the trophoblastic BM, a BM with a prominent filtering function . This was in contrast to the renal glomerulus and murine parietal yolk sac, each of which consistently contain trace amounts of fibronec-

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tin. This may indicate differences in the structure and function of the trophoblastic BM when compared to the other two filtering BM. Functional differences among these BM are well known, for example, IgG crosses the placental BM in the last trimester of gestation (Dancis et al., 1961; Gitlin et al., 1964), but it is retained by the the glomerular basement membrane (Farquhar, 1981). The possibility that in multicomponent structures, such as BM, the antigenic determinants of one or more components may be "masked" by their interaction with other components, was considered (Finley and Petruz, 1982). To rule out this possibility, tissue sections were treated with different enzymes and acidic solutions. None of these treatments revealed fibronectin in BM. Since all known components of BM, laminin, entactin, type IV collagen, and heparan sulfate proteoglycan are demonstrable without prior treatment (Amenta et al., 1983; Mynderse et al., 1983), it would have been surprising if the absence of demonstrable fibronectin in BM was due to masking. Enzyme digestion of tissue sections is commonly used in immunohistochemical studies, particularly for diagnostic pathology. In this respect it is worth noting that, in our hands, trypsin digestion, at any concentration used, resulted in complete destruction of cellular architecture. With this degree of morphologic disruption, antigen displacement and relocation should be a major concern in studies using enzyme digestion. The localization of type I collagen in the chorionic villus was in keeping with that reported in other organs (Boselli et al., 1981; Clark et al., 1982; Fleischmajer et al., 1980; Fleischmajer et al., 1981; Martinez-Hernandez, 1984; Martinez-Hernandez et al., 1981). Type I collagen antigens were localized virtually exclusively on thick, crossbanded (D = 64 nm) fibers. Some studies using conventional eletron microscopy have used different diameters of cross-banded fibers as an indicator of different genetic collagen types (Fleischmajer et al., 1980; Fleischmajer et al., 1981). In contrast, our findings in the placenta and those reported in other organs (Boselli et al., 1981; Clark et al., 1982; Martinez-Hernandez, 1984; Martinez et al., 1981) indicated that type I collagen exists in tissues as thick, cross-banded fibers, arid that all cross-banded fibers represent type I collagen. Although there are reports localizing type III collagen by immunofluorescence (D'ardenne et al., 1983; Gay et al., 1976 a; Gay et al., 1976 b), there is a paucity of data regarding the ultrastructural localization of this collagen type (Fleischmajer et al., 1980; Fleischmajer et al., 1981; Gay et al., 1976 b). In the present study, antibodies against type III collagen localized exclusively on thin, (10-15 nm) beaded, non-crossbanded fibers. Anti-type III collagen antibody binding sites were prevalent throughout the interstitium. Often type III collagen was closely associated with type I collagen, encasing or forming a meshwork around type I fibers (Fig. 5 d). This intimate, relationship of type III with type I collagen was not surprising in view of the known occasional cross-linking of type III with type I collagen (Henkel, 1982). In addition to its close association with type I collagen fibers, type III was also found unassociated with type I, often as bundles or aggregates, or occasionally as individual fibers. The reason for the beaded appearance of type III fibers is not clear, but in view of the reported incorporation of the aminopropeptide into the mature type III fiber (Fleischmajer et al., 1981; Gay et al., 1976 a), it is tempting to postulate that the beaded region corresponds to the propeptide domains. It is worth noting that studies using ferritin-labelled antibodies (Fleischmajer et al., 1980; Fleischmajer et al., 1981) report the existence of crossbanded type III collagen fibers. In our studies, type III collagen antigens were found not to react with cross-banded fibers. However, we often found thin, beaded type III fibers closely associated with cross-banded type I fibers. It is conceivable that in these prior

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reports the identification of cross-banded fibers as type III was due to the close association of both collagen types. A particular silver impregnation, the "reticulin" stain, is used to demonstrate connective tissues. The patterm obtained with this stain is distinct from that obtained with the trichrome and PAS stains. The difference is useful in the histopathologic diagnosis of sarcomas, hemopoietic, and hepatic disorders (Lille and Fullmer, 1976). Attempts have been made to equate the pattern obtained with the reticulin stain with the distribution of specific ECM components, usually type III collagen and/or fibronectin (Fleischmajer et aI., 1980; Timpl et aI., 1977; Unsworth et aI., 1982). The present study, as well as others (D'Ardenne et aI., 1983; Martinez-Hernandez, 1984; Stenman and Vaheri, 1978), failed to demonstrate any correlation between "reticulin" and any specific ECM component. The silver impregnation apparently reacts with carbohydrate moieties rather than with the protein core (Stern, 1979). It would seem unlikely that this technique would recognize exclusively one molecular species. Therefore, the "reticulin" stain, although a useful diagnostic tool, should not be interpreted as demonstrating any specific ECM component. Type V collagen has been localized in several tissues by light and electron immunohistochemical techniques (Biempca et aI., 1980; Gay et aI., 1981; Gay and Miller, 1978; Linsenmayer et aI., 1983; Madri et aI., 1980 a; Madri and Furthmayr, 1979; Martinez-Hernandez et aI., 1982 a; Roll et aI., 1980). In the placenta its relationship with the other collagens was similar to that observed with type III collagen, rather than type VI collagen. For example type V collagen often formed an encasement around type I fibers, as did type III collagen, obscuring the banding pattern of the thicker type I fibers (Fig. 5 d) . Unlike type III collagen antibodies, type V collagen antibodies reacted with 6-10 nm, nonbeaded filaments representing true type V collagen fibers. The distribution of type V collagen in the present study was in agreement with some prior reports (Linsenmayer, 1983; Martinez-Hernandez et aI., 1982 a) . However, there has been some controversy concerning the presence of type V collagen in BM. Whereas some authors have reported type V in some BM (Bailey et aI., 1979; Roll et aI., 1980; Stenn et aI., 1979), more recent studies using both monoclonal and polyclonal antibodies have failed to demonstrate type V collagen in BM (Linsenmayer et aI., 1983). In the present study placental BM did not contain type V collagen, except occasionally the adventitial aspect of ill-defined BM of fetal capillaries. There have been few studies localizing type VI collagen (Fleischmajer et aI., 1985; Gibson and Cleary, 1983; Hessle and Engvall, 1984; von der Mark et aI., 1984). In the present study, type VI collagen was in the form of fine non-beaded filaments 6-10 nm in diameter. Prior studies have reported similar dimensions for type VI collagen. This genetically distinct collagen type has been called "intima" collagen, since it was originally isolated from aortic intima (Chung et aI., 1976) and because of its vascular distribution by immunofluorescence (Furuto and Miller, 1980; Furuto and Miller, 1981; Jander et aI., 1983; Odermatt et aI., 1983). The term "intima" is a misnomer; since in tissues, at least in the placenta, type VI collagen does not have a preferential association with endothelium (Fig. 7 e). Furthermore, although this collagen type has a clear perivascular distribution, it is more prevalent in the interstitium, outside of the vessel than within the vessel wall. Therefore, type VI collagen is, properly speaking, an interstitial collagen rather than an "intima" collagen. Interstitium is a term morphologists use to describe the intercellular matrix. With the discovery of collagen types V to X, it became obvious that the different collagens could be grouped according to their properties, such as degrees of hydroxylation and glycosylation, continuous or inter-

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rupted helices, etc. In this light, it was obvious that collagen types I, II, and III had common properties, and the term "interstitial" collagens was used to describe this group. However, collagen types V and VI are also present in the interstitium and therefore, they are, properly speaking, interstitial collagens. It seems that another term, without erroneous implications, should be coined to categorize collagen types according to their properties. Perhaps the fiber size can be used to classify these collagens; types I, II, and III can be called "fibrillar" collagens and types V and VI "filamentous" collagens. It should be noted here that with each antibody no type III, V, or VI collagen was identified within type I collagen fibers. Therefore, in the placenta collagen fibers are apparently homopolymers. Nevertheless, individual bundles of collagen fibers are heterogeneous, often containing variable ratios of type I, III, V, and VI. Similar data has been obtained in other organs (Karkavelas et ai., 1986), implicating that bundles in different tissues or regions of an organ may have different composition and properties. The ultrastructural localization of seven different components of the ECM in the same tissue, has not only provided new information on the morphology and distribution of each individual component, but also on the organization, relationships, and interactions among these ECM components. The morphology distribution, interactions and known physical properties of type I collagen indicate that this collagen is the basic unit of structural support. The other ECM components may act as connecting structures. For instance, the binding of fibronectin to collagens is well known (Engvall et ai., 1978) and would be well suited to interconnecting collagens with other structures. Similarly, the interaction of type I collagen with proteoglycans has been shown biochemically and morphologically Uunqueira et ai., 1980; Scott, 1980; Toole and Lowther, 1968). It seems likely that these other ECM components connect type I collagen bundles among themselves, to stromal cells and BM. These associations transform the ECM from a collection of heterogeneous components into an integrated, functional unit. It is possible to visualize type I collagen as the basic building block of the ECM, with the other components binding and possibly modifying the properties of type I collagen. In this manner, subtle variations in the ratio of the different components may result in significant changes in the properties of the ECM. For instance, a relative increase in type III collagen, may result in increased plasticity of the matrix, an increase in fibronectin may result in better coupling between cells and matrix, or an increase in type VI may result in tighter binding between the matrix and vascular basement membranes. These qualitative, rather than quantitative variations, may account for the unique properties of specific extracellular matrices such as, the hepatic, renal, or muscular.

Footnotes: Abbrevations Used in Text: BM = Basement Membrane(s) ECM = Extracellular Matrix PBS = Phosphate-buffered saline PAP = Perxidase-anti-peroxidase complex

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Acknowledgements The authors are grateful to Drs. E. Ruoslahti and M. D. Piersbacher for the generous gift to monoclonal antibodies against human fibronectin (cell attachment, heparin- and gelatin binding domains) and to Dr. Eva Engvall for monoclonal and polyclonal antibodies against human type VI collagen. The authors thank Dr. George Martin for his reviewing the manuscript. The excellent technical assistance of Frank Gigliotti, Vicki Sabo, Lynn Armor and Bridget MeGeever and Jacqueline Hankins for her typing of the manuscript. Supported in part by grants AM 25254, AM 28488 and AA 05662 from the National Institutes of Health.

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