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Localization of collagen types I, III, IV and V, fibronectin and laminin in human arteries by the indirect immunofluorescence method
Path. Res. Pract. 181,568-575 (1986)
Localization of Collagen Types I, III, IV and V, Fibronectin and Laminin in Human Arteries by the Indirect Immunofluorescence Method Bruno Voss and Jurgen Rauterberg Institut tilr Arterioskleroseforschung an der Universitat MOnster, FRG
SUMMARY
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.
Introduction Collagen is a major constituent of the ar'terial walp8. Recent investitions have demonstrated that collagen occurs in a variety of genetically distinct molecules 2 • So far, ten types of collagen have been well characterized. The various molecules differ from each other in their amino acid composition, carbohydrate content, macromolecular organization, localization and function. The collagen types I and III are the major collagenous components of the arterial wa1l 18, 20. Chemical as well as immunohistological studies suggest a close association of collagens type I and III to fibronectin, a structural glycoprotein of the extracellular matrix. A second structural glycoprotein, laminin, is present in basement membranes where it is colocalized with type IV collagen27 • 0344-0338/86/0181-0568$3.50/0
The possible involvement of these matrix proteins in the pathogenesis of vascular diseases has been inferred from the reactions of platelets exposed to collagens and laminin in vitro l ,2,3,31. In vivo, platelets may perhaps react with matrix proteins after the first lesions of the arterial wAll develop. Therefore, it is necessary to elucidate the location of the different collagens and structural glycoproteins in normal and diseased human arterial walls. This is especially important, because there are only a few, generally controversial contributions dealing with the amount and distribution of types I, III and V collagen4, 15, 16. The present communication is concerned with the localization of the collagen types I, III, IV and V and the glycoproteins fibronectin and laminin in human arterial walls using indirect immunofluorescence microscopy.
© 1986 by Gustav Fischer Verlag, Stuttgart
Collagens and Glycoproteins in Arteries . 569
Material and Methods
Material Tissues of human arteries (aorta thoracica, aorta abdominalis, arteria renalis, arteria carotis, arteria basilaris from 50 cases aged 0-76 years,S arteriae iliaca) were obtained after autopsy from the Gerhard-Domagk Institute of Pathology, Munster, FRG, and stored immediately at - 70°C.
Preparation of antigens Type I and III collagen were prepared from human skin according to Miller et al. 17• Type IV collagen was isolated from bovine or human placenta and purified as described by Dixits. 7 S-collagen (short form) was obtained by bacterial collagenase digestion of purified type IV collagen at 37°C. The digests were purified by chromato£raphy on Sephacryl S-200 (Pharmacia) as described elsewhere 3. Collagen type V [(X 1(Vh (xz (V)1was isolated from bovine or human placenta. It was isolated following the method of Rhodes and Miller22. The glycoprotein fibronectin was isolated from human plasma by affinity chromatography on immobilized collagen and subsequently on arginine-sepharose6 . The basement membrane glycoprotein laminin was purified according to Foidart et aI8 ., a gift of Dr. R. Timpl and the laminin fragment PI was isolated from proteolytic digests of human placenta24 .
phosphate buffered saline (PBS). After washing, the sections were incubated with a second antibody conjugated to fluoresceine-iso-thiocyanate (Behringwerke, Marburg, or Cappel) and directed against the first antibody. Nonreacted IgG-conjugate was removed by washing with PBS. The sections were embedded with Entellan (Merck, Darmstadt). Fluorescence microscopy was carried out with an Orthoplan microscope equipped with epi-illumination (Leitz, Wetzlar). Microphotographs were taken with the camera system Orthomat (Leitz, Wetzlar).
Results
Distribution of collagens and structural glycoproteins in the normal aortic wall Fluoresrescence microscopical studies of the aortic tissue revealed an autofluorescence most of which was attributable to the elastic fibers. Such autofluorescence was sometimes more pronounced in aortic tissues of aged people, while elastic fibers of the aortas of newborns exhi-
Preparation of antibodies Goats were immunized with 20 mg of type I or III collagen dissolved in 5 ml of 0.1 % (v/v) acetic acid. The collagen solution was mixed with 5 ml of complete Freund's adjuvant (CFA). The homogenate was injected subcutaneously into different parts of the animals's lumbal region. Booster injections were made with the same quantity of collagen as above on days 21 and 42. For the last booster injection incomplete Freund's adjuvant (IFA) was used instead of CF A. Blood was withdrawn from the arteria carotis on day 60. Rabbits were immunized with 5 mg of type IV or type V collagen dissolved in 2 ml of 0.1 % acetic acid and 2 ml CFA. Booster injections were made with the same protein concentration on day 21 and 42. The last booster contained IFA instead of CFA. For immunization with 7S-collagen, 2,5 mg of the antigen were dissolved in 1 ml of 0.1 % acetic acid and 1 ml CFA. The following booster scheme was the same as described above. Immunization with fibronectin followed the procedure described for 7S-collagen. Serum was collected by heart puncture on day 60. To obtain antisera against laminin, goats were immunized with 2.5 mg of laminin PI peptide dissolved in 2 ml 0.1 % acetic acid and 2 ml CFA. For booster injections on day 21 and 42 the same quantity of antigen was used, and the immunization procedure was as described for collagen type I and III. Monospecific antibodies against the different collagens and glycoproteins were prepared by affinity chromatography according to the method of Timpl et a1. 26 • The specificity of monospecific antibodies type I and type III collagen was tested by passive hemaglutination. Antibodies against type IV, 7S, and type V collagen and fibronectin and laminin were tested with a radioimmunoassay28.
Immunohistochemistry Deep-frozen arteries were cut into 6-8 [tm thick sections at - 20°C, Air-dried tissue sections were first incubated with specific antibodies for 30 min and washed with
a
b Fig. 1. Immunofluorescence microscopy of type I collagen (a) and type III collagen (b) in the wall of an adult human aorta. The specific fluorescence is exhibited by the white areas. (From right to left): Type I collagen (a) as well as type III collagen are present in the intimal layer. Type III collagen-specific fluorescence is somewhat more intense than the type I collagen-specific fluorescence. Magnification 1 : 1000.
570 . B. Voss and]. Rauterberg bited only a very weak fluorescence. The specific fluorescence of collagen types I and III closely accompanied elastic fibers. The codistribution of these proteins was visible in tissues of the donors of all ages. The intenstiy of the specific fluorescence of type III collagen seemed to be higher than that of type I collagen in the subendothelial space of the aortic wall of children. In the aortic tissue of adults no great differences among the fluorescence intensities of the two interstitial collagens were observed (Fig. 1). In the medial layers of aortic walls type I and type III collagen showed a similar distribution. No age-dependent alteration of the distribution was detected. This was also true for the location of basement membrane collagens and of the structural glycoproteins: Type IV collagen was visible directly beneath the endothelial cells. The intensity decreased from the endothelial layer to the internal elastic fiber. In the medial layer of the aorta, type IV collagen was observed as a membraneous structure surrounding the smooth muscle cells. It was also present in thin filamentous structures between the cells. The extracellular matrix of the adventitia was generally free of type IV collagen. This collagen was localized only in the basement membranes of capillaries which invaded the adventitia. The localization of 75-collagen was coincident with that of type IV collagen. However, the intensity of the specific fluorescence of 75-collagen was sometimes lower than that of the complete protein molecule. Collagen type V was present in basement membrane areas as well. An accumulation of this collagen was not always observed in the intimal space of the aorta. The indirect fluorescence specific for collagen type V was intensive in the medial layer. Type V collagen was found along the elastic fibers and in the interstitial spaces of this part of the aortic wall. In the adventitia, type V was restricted to the basement membranes of the capillaries. The glycoprotein laminin was distributed as described for type IV collagen. Only basement membranes showed a positive reaction with the antibody against lamimin. Fibronectin was another structural glycoprotein of the arterial wall. This protein was found to be co-distributed with collagen types I and III.
Distribution of collagens and structural glycoproteins in normal small arteries The distributions of the collagen types and fibronectin in the walls of small arteries (arteria renalis, arteria carotis, arteria basilaris, arteria iliaca) were different from those for the aortic wall, because large elastic lamellae were absent. The deposition of the interstitial collagen types I and III and of fibronectin appeared to be at the surfaces of smooth muscle cells or along filaments of elastin. There were some differences between the distributions of type IV and type V collagen. The intensity of the specific fluorescence for type IV collagen was higher in subendothelial space than in the medial layer of the small artery. However, the positive reaction with antibodies against 75-collagen was more intense in the medial layer of these arteries. Type V collagen exhibited, like 75 collagen, a more intense fluorescence in the media than in the intima.
b Fig. 2. Indirect immunofluorescence staining for type IV. Collagen (a) and types V collagen (b) in arteries of a human kidney. Type IV collagen is deposited linearily along the smooth muscle cells. The same distribution was observed after specific staining for laminin (not shown). Type V collagen is coarsely deposited along the smooth muscle cells. Magnification 1 : 1000. Collagen type IV and its fragment, 75-collagen, as well as laminin, were located close to the endothelial cells and the internal elastic membrane. The smooth muscle cells were surrounded by a structure containing type IV, type V (Fig. 2) and laminin.
Distribution of collagens and structural glycoproteins in atherosclerotic vessel walls of arteries In those parts of the atherosclerotic vessel walls without plaque formations the localization of the intersitial collagen types I and III and the basement membrane collagens types IV and V as well as the structural glycoproteins fibronectin and laminin, were not different from those in normal blood vessel walls. An elevated intensity of fluorescence specific for collagen types I and III was observed in the thickened intima (Fig. 3 a, b), while fluorescence of fibronectin or laminin was not increased. The composition of atherosclerotic plaques varied. Two major types of plaques were observed. The first type of plaque contained small areas of connective tissue and many lipid-rich cells. Collagen types I and III were
Collagens and Glycoproteins in Arteries . 571
a
Fibrous plaques with marked specific fluorescence of collagen types I or of type III exhibited a weak reaction with antibodies against collagen types IV and V (Fig. 5) or against the basement membrane component laminin. In this respect these fibrous plaques resembled a typical scar formation. The prominent component of areas in fibrous plaques lacking collagen showed an emission of fluorescence light like the characteristic autofluorescence of elastin. In calcified parts of an atherosclerotic aorta the collagen and structural glycoproteins were totally masked. At the medial layer dense elastin depositions were observed. The lateral areas of the calcification often were occupied by lipid filled foam cells, probably macrophages. Between the subendothelial basement membrane and the calcified area fibers containing collagen types I or III were arranged vertical to the normal horizontal layer (Fig. 6). A surprising phenomenon was the occurrence of capillaries in the medial layer of a calcified aorta. The capillaries could be demonstrated by the indirect staining with antibodies against collagen type IV or laminin (Fig. 7). No difference was observed between the composition of plaques of the thoracic and abdominal part of aortas and of plaques of small arteries. Discussion
b Fig. 3. Localization of type I collagen (a) and type III collagen (b) in the thickened intima of a human aorta. The white areas represent collagen. The darker ligament (right side) is the non-stained basal lamina of the endothelium. The white spots on the ligament are due to non-specific reflecting light. Magnification 1 : 1000.
detected in the lipid-rich plaques. The basement membrane collagens type IV and type V as well as laminin were mostly restricted to the subendothelial basement membrane (Fig. 4). Fibronectin was obviously co distributed with the collagen type I and III. The second type of plaque, the fibrous plaque, could be subdivided into fibrous plaques consisting mostly of collagen type I and type III and into fibrous plaques containing areas with low collagen type I and type III content (Fig. 5).
The collagens and elastin are probably the most important proteins of blood vessel walls. The collagens are classified into genetically distinct macromolecules. Types I and III confer tensile strength to blood vessel walls. Type IV collagen and laminin are characteristic basement membrane components, while type V collagen and fibronectin may occur in both basement membranes and in the interstitial connective tissue 12, 18. In the present study the distribution of these various antigens was evaluated by immunohistochemical technique using mono-specific antibodies to identify the different collagens and the structural glycoproteins 29,30. The . monospecificity of the antibodies was achieved by affinity chromatography. Type I and type III collagen were generally found codistributed. The presence of both collagen types in the subendothelial layer is in afcreement with the biochemical studies of Rauterberg et al. and of Morton and Barnes 18 , However, this result contradicts earlier immunofluorescence microscopical studies which reported the presence of only one of either type m9 or type I collagen 14 in the subendothelial layer. The results obtained by McCullagh et a1. 15 concerning the distribution of collagen types I and III are difficult to interpret because these authors used antibodies raised in rabbits against pepsinextracted collagens (for a review of the immunologically relevant domains of the collagens see ref. 13). When native collagen type I or type III molecules are injected into rabbits, the resulting antibodies are predominantly directed against the extension peptides and not against the triple-helical region. However the pepsinextracted collagen molecules have no extension peptides.
°
572 . B. Voss and]. Rauterberg
a
b
c
d
Fig. 4. Localization of type I collagen (a), type V collagen (b), type IV collagen (c), and laminin (d) in intimal lipid-rich plaques. Type III collagen and fibronectin are co-localized with type I collagen (not shown). Type V collagen and type IV collagen and laminin are almost completely restricted to a subendothelial region (right side). Magnification 1: 1000.
In the medial layer of arterial walls types I and III collagen were essentially co-localized with elastic fibers. This was most obvious in the media of the aorta. The specific fluorescent labeling of type III collagen gave the impression that this protein is more intimately related to elastic fibers as type I collagen, although intermolecular crosslinks have been found between these two molecules ll . The distribution of the glycoprotein fibronectin was similar to that of the interstitial collagens. Fibronectin was
one of the predominant functional proteins of the blood vessel wall. The basement membranes in the intimal layer or around smooth muscle cells were composed of the collagen types IV and V and of the glycoprotein laminin as judged by indirect immunofluorescence microscopy. While in the aortic wall these basement membrane proteins were visible mainly along the elastic fibers, they were found in close apposition to smooth muscle cells in small arteries. Similar
Collagens and Glycoproteins in Arteries . 573
a
b
c
d
e
g
Fig. 5. Compostion of plaques in human aortas. The content of type I collagen (a) and type III collagen (b) is low in some fibrous plaques as shown by indirect immunofluorescence microscopy. In contrast, type I collagen (c) and type III collagen (d) are present in obviously higher amounts in other fibrous plaques as judged by the distribution of specific fluorescence. Fibronectin (e) is one of the components of fibrous plaques. Type V collagen (f) is visible in focal accumulations, especially below the endothelial layer (right side). Type IV collagen (g) is present in the basal lamina of the endothelial cells and in various scattered depositions in the fibrous plaque. Magnification 1 : 1000.
574 . B. Voss and J. Rauterberg
a
Fig. 7. Medial layer of an atherosclerotic wall of a human aorta stained specially for type IV collagen. The staining visualize basement membranes of capillaries which have invaded this part of the blood vessel wall. A similar staining was found for laminin (not shown). Magnification 1 : 1000.
labeling for the basement membrane components laminin and type IV collagen. Type V appeared to be poorly represented within the fibrous plaque as well. However, Morton and Barnes 18 as well as Ooshima 19 found by biochemical means a higher amount of type V collagen in fibrous b plaques as compared to the normal arterial wall. This Fig. 6. Collagen fibers in human aortic plaques. In some fibrous apparent contradiction may reflect tissue-specific differplaques the collagen fibers have changed their orientation from ences in the accessibility of type V antigenic determinants, horizontal to longitudinal with respect to the length of the aorta. as they have been reported by Linsenmeyer et al. 13. Type I collagen (a) and type III collagen occur in thin fibers. The The elasticity of the aortas and arteries is decreased due intense white areas arise from autofluorescent material. Magnifi- to scar formations and to progress of the calcification procation 1: 1000. cess of the blood vessel wall matrix. The disruption of the normal structural composition of the blood vessel wall provokes an undersupply of the smooth muscle cells in the observations have been made concerning the localization medial layer. This may be a reason for capillarization of of type VI collagen21 • The integration of various collagens, the media, observed by the indirect immunofluorescence structural glycoproteins, and elastin in the blood vessel of collagen type IV or laminin. A similar capillarization wall reflects the functional role of these proteins: They has been visualized in aortic coarctions (results unpubmodulate the elasticity, contractability and permeability of lished). the vessel. Any structural disorder of the vessel wall will be answered by a physiologic dysfunction. Atherosclerosis is the most common disease of blood vessels resulting from early events leading to a structural disorder of the vessel wall components. The aberrant composition of the Acknowledgements atherogsclerotic vessel wall matrix was observed in the present study in the form of increased deposition of types I The authors want to thank Miss Birgit Herrenpoth and Miss and III collagen in the thickened intima deriving from Manuela Kriiger for their excellent technical assistance, and Dr. smooth muscle cell infiltrations. On the ultrastructural level fibrogenesis is accompained by irregular formation of D. Troyer, Institut fiir Arterioskleroseforschung an der Univercollagen fibers 7, 10,25. The most impressive observation sitat Miinster, for reading the manuscript. We also thank Dr. D. B. v. Bassewitz and Dr. A. Roessner, Institute of Pathology, Uniusing immunofluorescence microscopy was that fibrous versity of Miinster, for careful evaluation of the autopsies. plaque formations, which coptained higher amounts of This study was supported by Deutsche Forschungsgesellschaft interstitial types I and III collagen as estimated by the grant Vo 33911-1 Dedicated to Professor Dr. U. Gerlach marked specific fluorescence, showed a weaker specific
Collagens and Glycoproteins in Arteries . 575
References 1 Balleisen L, Gay S, Marx R, Kiihn K (1975) Comparative investigations on the influence of human bovine collagen types I, II and III on the aggregation of human platelets. Klin Wschr 53: 903-905 I Balleisen L, Rauterberg J (1980) Platelet activation by basement membrane collagens. Thromb Res 18: 725-732 3 Balleisen L, Voss B, Rauterberg J, Miiller KM, Timpl R (1981) Subendotheliale Kollagene und Thrombozyten In: Lunge, Blutgerinnung und Hamostase Marx R. Thies KA (Eds), Edition Roche Basel, 25-40 4 Barnes MJ (1983) Collagen polymorphism in the normal and diseased blood vessel wall. Atherosclerosis 46: 249-251 5 Dixit SN (1978) Isolation and characterization of two collagenous components from anterior lens capsule. FEBS Lett 85: 153-157 6 Engvall E, Ruoslahti E, Miller EJ (1978) Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med 147: 1584-1595 7 Fischer N, Staubesand J (1982) Zur Ultrastruktur kollagener Fibrillen in normal en und veranderten BlutgefaSen. Acta nat 114: 125-145 8 FoidartJ-M, Timpl R, Furtmayr H, Martin GR (1982) Laminin, a glycoprotein from basement membranes. In: Immunochemistry of the extracellular matrix Vol 1, Methods Furthmayr H (Ed.) CRC Press Inc. Boca Raton, Florida, 125-134 9 Gay S, Balleisen L, Remberger K, Fietzek PP, Adelmann BC, Kiihn K (1975) Immunohistochemical evidence for the presence of collagen type III in human arterial wall, arterial thrombi, and in leucocytes, incubated with collagen in vitro. Klin Wschr 53: 899-902 10 Haust MD (1965) Fine fibrils of extracellular space (microfibrils). Their structure and role in connective tissue organization. Am J Pathol47: 1113-1137 11 Henkel W, Glanville RW (1982) Covalent crosslinking between molecules of type I and type III collagen. Eur J Biochem 122: 205-213 12 Leushner JRA, Haust MD (1984) Characterization of basement membrane collagens of bovine aortae. Atherosclerosis 50: 11-27 13 Linsenmayer TF (1981) Collagen. In: Cell biology of extracellular matrix. Hay ED (Ed) Plenum Press, New York, London, 5-37 14 Madri JA, Dreyer B, Pitlich FA, Furthmayr H (1980) The collagenous components of the sub endothelium. Correlation of structure and function. Lab Invest 43: 303-315 15 McCullagh KG, Duance VC, Bishop KA (1979) The distribution of collagen types I, III and V (AB) in normal and atherosclerotic human aorta. J Path 130: 45-55 16 McCullagh KG (1983) Increased type I collagen in human atherosclerotic plaque. Atherosclerosis 46: 247-248
17 Miller EJ, Epstein EH, Piez KA (1971) Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage collagen. Biochim Biophys Res Comm 42: 1029-1049 18 Morton LF, Barnes MJ (1982) Collagen polymorphism in the normal and diseased blood vessel wall. Investigations of collagens types I, III and V. Atherosclerosis 48: 41-51 19 Ooshima, A (1981) Collagen B chain: Increased proportion in human Atherosclerosis. Science 213: 666-668 20 Rauterberg J, Allam S, Brehmer U, Wirth W, Hauss WH (1979) Das Kollagen der Arterienwand. Charakterisierung in Gewebeproben und Untersuchungen zur Biosynthese in kultivierten Aortenwandzellen. Therapiewoche 29: 2717-2737 21 Rauterberg J, Jander R, Voss B, Furthmayr H, Glanville RW (1982) Characteristics and occurrence of short chain collagen. In: New Trends in Basement Membrane Research. Kiihn K, Schone H, Timpl R (Eds) Raven Press, New York, 107-119 22 Rhodes, R-K-P, Miller, E-J (1978) Physiochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 17: 3442-3448 23 Risteli J, Bachinger HP, Engel J. Furthmayr H, Timpl R (1980) 7S-collagen: characterization of an unusual basement membrane structure. Eur J Biochem 108: 239-250 24 Risteli L, Timpl R (1981) Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes. Biochem J 193: 749-755 25 Staubesand J, Fischer N (1979) Collagen dysplasia and matrix vesicles. Researches with the electron microscope into the problem of so-called 'weakness of the vessel wall'. Path Res Pract 165: 374-391 26 Timpl R, Wick G, Gay S (1977) Antibodies to distinct types of collagens and procollagens and their application in immunohistology. J Immunol Meth 18: 165-182 27 Timpl R, Rohde H, Robey PG, Rennard SI, Foidart J-M, Martin GR (1979) Laminin - a glycoprotein from basement membranes. J Bioi Chern 254: 9933-9937 28 Timpl R, Risteli L (1982) Radioimmunoassays in studies of connective tissue proteins. In: Furthmayr H (Ed) Immunochemistry of the extracellular matrix. Vol 1, Methods. CRC Press Inc Boca Raton, Florida, 199-235 29 Voss B, Allam S, Rauterberg J, Ullrich K, Gieselmann V, von Figura K (1979) Primary cultures of rat hepatocytes synthesize fibronectin. Biochem Biophys Res Comm 90: 1348-1354 30 Voss B, Rauterberg J, Allam S, Pott G (1980) Distribution of collagen type I and type III and of two collagenous components of basement membranes in the human liver. Path Res Pract
170: 50-60
31 Zucker MB, Borelli J (1962) Platelet clumping produced by connective tissue suspensions and by collagen. Proc Soc Exp Bioi Med 109: 779
Received May 13, 1985 . Accepted in revised form March 11, 1986
Key words: Human arteries - Collagens - Glycoproteins - Atherosclerosis - Fibronectin Dr. Bruno Voss, Institut fiir Arterioskleroseforschung an der Universitat Miinster, Domagkstr. 3, D-4400 Munster, FRG
Localization of Collagen Types I, III, IV and V, Fibronectin and Laminin in Human Arteries by the Indirect Immunofluorescence Method Bruno Voss and Jurgen Rauterberg Institut tilr Arterioskleroseforschung an der Universitat MOnster, FRG
SUMMARY
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.
Introduction Collagen is a major constituent of the ar'terial walp8. Recent investitions have demonstrated that collagen occurs in a variety of genetically distinct molecules 2 • So far, ten types of collagen have been well characterized. The various molecules differ from each other in their amino acid composition, carbohydrate content, macromolecular organization, localization and function. The collagen types I and III are the major collagenous components of the arterial wa1l 18, 20. Chemical as well as immunohistological studies suggest a close association of collagens type I and III to fibronectin, a structural glycoprotein of the extracellular matrix. A second structural glycoprotein, laminin, is present in basement membranes where it is colocalized with type IV collagen27 • 0344-0338/86/0181-0568$3.50/0
The possible involvement of these matrix proteins in the pathogenesis of vascular diseases has been inferred from the reactions of platelets exposed to collagens and laminin in vitro l ,2,3,31. In vivo, platelets may perhaps react with matrix proteins after the first lesions of the arterial wAll develop. Therefore, it is necessary to elucidate the location of the different collagens and structural glycoproteins in normal and diseased human arterial walls. This is especially important, because there are only a few, generally controversial contributions dealing with the amount and distribution of types I, III and V collagen4, 15, 16. The present communication is concerned with the localization of the collagen types I, III, IV and V and the glycoproteins fibronectin and laminin in human arterial walls using indirect immunofluorescence microscopy.
© 1986 by Gustav Fischer Verlag, Stuttgart
Collagens and Glycoproteins in Arteries . 569
Material and Methods
Material Tissues of human arteries (aorta thoracica, aorta abdominalis, arteria renalis, arteria carotis, arteria basilaris from 50 cases aged 0-76 years,S arteriae iliaca) were obtained after autopsy from the Gerhard-Domagk Institute of Pathology, Munster, FRG, and stored immediately at - 70°C.
Preparation of antigens Type I and III collagen were prepared from human skin according to Miller et al. 17• Type IV collagen was isolated from bovine or human placenta and purified as described by Dixits. 7 S-collagen (short form) was obtained by bacterial collagenase digestion of purified type IV collagen at 37°C. The digests were purified by chromato£raphy on Sephacryl S-200 (Pharmacia) as described elsewhere 3. Collagen type V [(X 1(Vh (xz (V)1was isolated from bovine or human placenta. It was isolated following the method of Rhodes and Miller22. The glycoprotein fibronectin was isolated from human plasma by affinity chromatography on immobilized collagen and subsequently on arginine-sepharose6 . The basement membrane glycoprotein laminin was purified according to Foidart et aI8 ., a gift of Dr. R. Timpl and the laminin fragment PI was isolated from proteolytic digests of human placenta24 .
phosphate buffered saline (PBS). After washing, the sections were incubated with a second antibody conjugated to fluoresceine-iso-thiocyanate (Behringwerke, Marburg, or Cappel) and directed against the first antibody. Nonreacted IgG-conjugate was removed by washing with PBS. The sections were embedded with Entellan (Merck, Darmstadt). Fluorescence microscopy was carried out with an Orthoplan microscope equipped with epi-illumination (Leitz, Wetzlar). Microphotographs were taken with the camera system Orthomat (Leitz, Wetzlar).
Results
Distribution of collagens and structural glycoproteins in the normal aortic wall Fluoresrescence microscopical studies of the aortic tissue revealed an autofluorescence most of which was attributable to the elastic fibers. Such autofluorescence was sometimes more pronounced in aortic tissues of aged people, while elastic fibers of the aortas of newborns exhi-
Preparation of antibodies Goats were immunized with 20 mg of type I or III collagen dissolved in 5 ml of 0.1 % (v/v) acetic acid. The collagen solution was mixed with 5 ml of complete Freund's adjuvant (CFA). The homogenate was injected subcutaneously into different parts of the animals's lumbal region. Booster injections were made with the same quantity of collagen as above on days 21 and 42. For the last booster injection incomplete Freund's adjuvant (IFA) was used instead of CF A. Blood was withdrawn from the arteria carotis on day 60. Rabbits were immunized with 5 mg of type IV or type V collagen dissolved in 2 ml of 0.1 % acetic acid and 2 ml CFA. Booster injections were made with the same protein concentration on day 21 and 42. The last booster contained IFA instead of CFA. For immunization with 7S-collagen, 2,5 mg of the antigen were dissolved in 1 ml of 0.1 % acetic acid and 1 ml CFA. The following booster scheme was the same as described above. Immunization with fibronectin followed the procedure described for 7S-collagen. Serum was collected by heart puncture on day 60. To obtain antisera against laminin, goats were immunized with 2.5 mg of laminin PI peptide dissolved in 2 ml 0.1 % acetic acid and 2 ml CFA. For booster injections on day 21 and 42 the same quantity of antigen was used, and the immunization procedure was as described for collagen type I and III. Monospecific antibodies against the different collagens and glycoproteins were prepared by affinity chromatography according to the method of Timpl et a1. 26 • The specificity of monospecific antibodies type I and type III collagen was tested by passive hemaglutination. Antibodies against type IV, 7S, and type V collagen and fibronectin and laminin were tested with a radioimmunoassay28.
Immunohistochemistry Deep-frozen arteries were cut into 6-8 [tm thick sections at - 20°C, Air-dried tissue sections were first incubated with specific antibodies for 30 min and washed with
a
b Fig. 1. Immunofluorescence microscopy of type I collagen (a) and type III collagen (b) in the wall of an adult human aorta. The specific fluorescence is exhibited by the white areas. (From right to left): Type I collagen (a) as well as type III collagen are present in the intimal layer. Type III collagen-specific fluorescence is somewhat more intense than the type I collagen-specific fluorescence. Magnification 1 : 1000.
570 . B. Voss and]. Rauterberg bited only a very weak fluorescence. The specific fluorescence of collagen types I and III closely accompanied elastic fibers. The codistribution of these proteins was visible in tissues of the donors of all ages. The intenstiy of the specific fluorescence of type III collagen seemed to be higher than that of type I collagen in the subendothelial space of the aortic wall of children. In the aortic tissue of adults no great differences among the fluorescence intensities of the two interstitial collagens were observed (Fig. 1). In the medial layers of aortic walls type I and type III collagen showed a similar distribution. No age-dependent alteration of the distribution was detected. This was also true for the location of basement membrane collagens and of the structural glycoproteins: Type IV collagen was visible directly beneath the endothelial cells. The intensity decreased from the endothelial layer to the internal elastic fiber. In the medial layer of the aorta, type IV collagen was observed as a membraneous structure surrounding the smooth muscle cells. It was also present in thin filamentous structures between the cells. The extracellular matrix of the adventitia was generally free of type IV collagen. This collagen was localized only in the basement membranes of capillaries which invaded the adventitia. The localization of 75-collagen was coincident with that of type IV collagen. However, the intensity of the specific fluorescence of 75-collagen was sometimes lower than that of the complete protein molecule. Collagen type V was present in basement membrane areas as well. An accumulation of this collagen was not always observed in the intimal space of the aorta. The indirect fluorescence specific for collagen type V was intensive in the medial layer. Type V collagen was found along the elastic fibers and in the interstitial spaces of this part of the aortic wall. In the adventitia, type V was restricted to the basement membranes of the capillaries. The glycoprotein laminin was distributed as described for type IV collagen. Only basement membranes showed a positive reaction with the antibody against lamimin. Fibronectin was another structural glycoprotein of the arterial wall. This protein was found to be co-distributed with collagen types I and III.
Distribution of collagens and structural glycoproteins in normal small arteries The distributions of the collagen types and fibronectin in the walls of small arteries (arteria renalis, arteria carotis, arteria basilaris, arteria iliaca) were different from those for the aortic wall, because large elastic lamellae were absent. The deposition of the interstitial collagen types I and III and of fibronectin appeared to be at the surfaces of smooth muscle cells or along filaments of elastin. There were some differences between the distributions of type IV and type V collagen. The intensity of the specific fluorescence for type IV collagen was higher in subendothelial space than in the medial layer of the small artery. However, the positive reaction with antibodies against 75-collagen was more intense in the medial layer of these arteries. Type V collagen exhibited, like 75 collagen, a more intense fluorescence in the media than in the intima.
b Fig. 2. Indirect immunofluorescence staining for type IV. Collagen (a) and types V collagen (b) in arteries of a human kidney. Type IV collagen is deposited linearily along the smooth muscle cells. The same distribution was observed after specific staining for laminin (not shown). Type V collagen is coarsely deposited along the smooth muscle cells. Magnification 1 : 1000. Collagen type IV and its fragment, 75-collagen, as well as laminin, were located close to the endothelial cells and the internal elastic membrane. The smooth muscle cells were surrounded by a structure containing type IV, type V (Fig. 2) and laminin.
Distribution of collagens and structural glycoproteins in atherosclerotic vessel walls of arteries In those parts of the atherosclerotic vessel walls without plaque formations the localization of the intersitial collagen types I and III and the basement membrane collagens types IV and V as well as the structural glycoproteins fibronectin and laminin, were not different from those in normal blood vessel walls. An elevated intensity of fluorescence specific for collagen types I and III was observed in the thickened intima (Fig. 3 a, b), while fluorescence of fibronectin or laminin was not increased. The composition of atherosclerotic plaques varied. Two major types of plaques were observed. The first type of plaque contained small areas of connective tissue and many lipid-rich cells. Collagen types I and III were
Collagens and Glycoproteins in Arteries . 571
a
Fibrous plaques with marked specific fluorescence of collagen types I or of type III exhibited a weak reaction with antibodies against collagen types IV and V (Fig. 5) or against the basement membrane component laminin. In this respect these fibrous plaques resembled a typical scar formation. The prominent component of areas in fibrous plaques lacking collagen showed an emission of fluorescence light like the characteristic autofluorescence of elastin. In calcified parts of an atherosclerotic aorta the collagen and structural glycoproteins were totally masked. At the medial layer dense elastin depositions were observed. The lateral areas of the calcification often were occupied by lipid filled foam cells, probably macrophages. Between the subendothelial basement membrane and the calcified area fibers containing collagen types I or III were arranged vertical to the normal horizontal layer (Fig. 6). A surprising phenomenon was the occurrence of capillaries in the medial layer of a calcified aorta. The capillaries could be demonstrated by the indirect staining with antibodies against collagen type IV or laminin (Fig. 7). No difference was observed between the composition of plaques of the thoracic and abdominal part of aortas and of plaques of small arteries. Discussion
b Fig. 3. Localization of type I collagen (a) and type III collagen (b) in the thickened intima of a human aorta. The white areas represent collagen. The darker ligament (right side) is the non-stained basal lamina of the endothelium. The white spots on the ligament are due to non-specific reflecting light. Magnification 1 : 1000.
detected in the lipid-rich plaques. The basement membrane collagens type IV and type V as well as laminin were mostly restricted to the subendothelial basement membrane (Fig. 4). Fibronectin was obviously co distributed with the collagen type I and III. The second type of plaque, the fibrous plaque, could be subdivided into fibrous plaques consisting mostly of collagen type I and type III and into fibrous plaques containing areas with low collagen type I and type III content (Fig. 5).
The collagens and elastin are probably the most important proteins of blood vessel walls. The collagens are classified into genetically distinct macromolecules. Types I and III confer tensile strength to blood vessel walls. Type IV collagen and laminin are characteristic basement membrane components, while type V collagen and fibronectin may occur in both basement membranes and in the interstitial connective tissue 12, 18. In the present study the distribution of these various antigens was evaluated by immunohistochemical technique using mono-specific antibodies to identify the different collagens and the structural glycoproteins 29,30. The . monospecificity of the antibodies was achieved by affinity chromatography. Type I and type III collagen were generally found codistributed. The presence of both collagen types in the subendothelial layer is in afcreement with the biochemical studies of Rauterberg et al. and of Morton and Barnes 18 , However, this result contradicts earlier immunofluorescence microscopical studies which reported the presence of only one of either type m9 or type I collagen 14 in the subendothelial layer. The results obtained by McCullagh et a1. 15 concerning the distribution of collagen types I and III are difficult to interpret because these authors used antibodies raised in rabbits against pepsinextracted collagens (for a review of the immunologically relevant domains of the collagens see ref. 13). When native collagen type I or type III molecules are injected into rabbits, the resulting antibodies are predominantly directed against the extension peptides and not against the triple-helical region. However the pepsinextracted collagen molecules have no extension peptides.
°
572 . B. Voss and]. Rauterberg
a
b
c
d
Fig. 4. Localization of type I collagen (a), type V collagen (b), type IV collagen (c), and laminin (d) in intimal lipid-rich plaques. Type III collagen and fibronectin are co-localized with type I collagen (not shown). Type V collagen and type IV collagen and laminin are almost completely restricted to a subendothelial region (right side). Magnification 1: 1000.
In the medial layer of arterial walls types I and III collagen were essentially co-localized with elastic fibers. This was most obvious in the media of the aorta. The specific fluorescent labeling of type III collagen gave the impression that this protein is more intimately related to elastic fibers as type I collagen, although intermolecular crosslinks have been found between these two molecules ll . The distribution of the glycoprotein fibronectin was similar to that of the interstitial collagens. Fibronectin was
one of the predominant functional proteins of the blood vessel wall. The basement membranes in the intimal layer or around smooth muscle cells were composed of the collagen types IV and V and of the glycoprotein laminin as judged by indirect immunofluorescence microscopy. While in the aortic wall these basement membrane proteins were visible mainly along the elastic fibers, they were found in close apposition to smooth muscle cells in small arteries. Similar
Collagens and Glycoproteins in Arteries . 573
a
b
c
d
e
g
Fig. 5. Compostion of plaques in human aortas. The content of type I collagen (a) and type III collagen (b) is low in some fibrous plaques as shown by indirect immunofluorescence microscopy. In contrast, type I collagen (c) and type III collagen (d) are present in obviously higher amounts in other fibrous plaques as judged by the distribution of specific fluorescence. Fibronectin (e) is one of the components of fibrous plaques. Type V collagen (f) is visible in focal accumulations, especially below the endothelial layer (right side). Type IV collagen (g) is present in the basal lamina of the endothelial cells and in various scattered depositions in the fibrous plaque. Magnification 1 : 1000.
574 . B. Voss and J. Rauterberg
a
Fig. 7. Medial layer of an atherosclerotic wall of a human aorta stained specially for type IV collagen. The staining visualize basement membranes of capillaries which have invaded this part of the blood vessel wall. A similar staining was found for laminin (not shown). Magnification 1 : 1000.
labeling for the basement membrane components laminin and type IV collagen. Type V appeared to be poorly represented within the fibrous plaque as well. However, Morton and Barnes 18 as well as Ooshima 19 found by biochemical means a higher amount of type V collagen in fibrous b plaques as compared to the normal arterial wall. This Fig. 6. Collagen fibers in human aortic plaques. In some fibrous apparent contradiction may reflect tissue-specific differplaques the collagen fibers have changed their orientation from ences in the accessibility of type V antigenic determinants, horizontal to longitudinal with respect to the length of the aorta. as they have been reported by Linsenmeyer et al. 13. Type I collagen (a) and type III collagen occur in thin fibers. The The elasticity of the aortas and arteries is decreased due intense white areas arise from autofluorescent material. Magnifi- to scar formations and to progress of the calcification procation 1: 1000. cess of the blood vessel wall matrix. The disruption of the normal structural composition of the blood vessel wall provokes an undersupply of the smooth muscle cells in the observations have been made concerning the localization medial layer. This may be a reason for capillarization of of type VI collagen21 • The integration of various collagens, the media, observed by the indirect immunofluorescence structural glycoproteins, and elastin in the blood vessel of collagen type IV or laminin. A similar capillarization wall reflects the functional role of these proteins: They has been visualized in aortic coarctions (results unpubmodulate the elasticity, contractability and permeability of lished). the vessel. Any structural disorder of the vessel wall will be answered by a physiologic dysfunction. Atherosclerosis is the most common disease of blood vessels resulting from early events leading to a structural disorder of the vessel wall components. The aberrant composition of the Acknowledgements atherogsclerotic vessel wall matrix was observed in the present study in the form of increased deposition of types I The authors want to thank Miss Birgit Herrenpoth and Miss and III collagen in the thickened intima deriving from Manuela Kriiger for their excellent technical assistance, and Dr. smooth muscle cell infiltrations. On the ultrastructural level fibrogenesis is accompained by irregular formation of D. Troyer, Institut fiir Arterioskleroseforschung an der Univercollagen fibers 7, 10,25. The most impressive observation sitat Miinster, for reading the manuscript. We also thank Dr. D. B. v. Bassewitz and Dr. A. Roessner, Institute of Pathology, Uniusing immunofluorescence microscopy was that fibrous versity of Miinster, for careful evaluation of the autopsies. plaque formations, which coptained higher amounts of This study was supported by Deutsche Forschungsgesellschaft interstitial types I and III collagen as estimated by the grant Vo 33911-1 Dedicated to Professor Dr. U. Gerlach marked specific fluorescence, showed a weaker specific
Collagens and Glycoproteins in Arteries . 575
References 1 Balleisen L, Gay S, Marx R, Kiihn K (1975) Comparative investigations on the influence of human bovine collagen types I, II and III on the aggregation of human platelets. Klin Wschr 53: 903-905 I Balleisen L, Rauterberg J (1980) Platelet activation by basement membrane collagens. Thromb Res 18: 725-732 3 Balleisen L, Voss B, Rauterberg J, Miiller KM, Timpl R (1981) Subendotheliale Kollagene und Thrombozyten In: Lunge, Blutgerinnung und Hamostase Marx R. Thies KA (Eds), Edition Roche Basel, 25-40 4 Barnes MJ (1983) Collagen polymorphism in the normal and diseased blood vessel wall. Atherosclerosis 46: 249-251 5 Dixit SN (1978) Isolation and characterization of two collagenous components from anterior lens capsule. FEBS Lett 85: 153-157 6 Engvall E, Ruoslahti E, Miller EJ (1978) Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med 147: 1584-1595 7 Fischer N, Staubesand J (1982) Zur Ultrastruktur kollagener Fibrillen in normal en und veranderten BlutgefaSen. Acta nat 114: 125-145 8 FoidartJ-M, Timpl R, Furtmayr H, Martin GR (1982) Laminin, a glycoprotein from basement membranes. In: Immunochemistry of the extracellular matrix Vol 1, Methods Furthmayr H (Ed.) CRC Press Inc. Boca Raton, Florida, 125-134 9 Gay S, Balleisen L, Remberger K, Fietzek PP, Adelmann BC, Kiihn K (1975) Immunohistochemical evidence for the presence of collagen type III in human arterial wall, arterial thrombi, and in leucocytes, incubated with collagen in vitro. Klin Wschr 53: 899-902 10 Haust MD (1965) Fine fibrils of extracellular space (microfibrils). Their structure and role in connective tissue organization. Am J Pathol47: 1113-1137 11 Henkel W, Glanville RW (1982) Covalent crosslinking between molecules of type I and type III collagen. Eur J Biochem 122: 205-213 12 Leushner JRA, Haust MD (1984) Characterization of basement membrane collagens of bovine aortae. Atherosclerosis 50: 11-27 13 Linsenmayer TF (1981) Collagen. In: Cell biology of extracellular matrix. Hay ED (Ed) Plenum Press, New York, London, 5-37 14 Madri JA, Dreyer B, Pitlich FA, Furthmayr H (1980) The collagenous components of the sub endothelium. Correlation of structure and function. Lab Invest 43: 303-315 15 McCullagh KG, Duance VC, Bishop KA (1979) The distribution of collagen types I, III and V (AB) in normal and atherosclerotic human aorta. J Path 130: 45-55 16 McCullagh KG (1983) Increased type I collagen in human atherosclerotic plaque. Atherosclerosis 46: 247-248
17 Miller EJ, Epstein EH, Piez KA (1971) Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage collagen. Biochim Biophys Res Comm 42: 1029-1049 18 Morton LF, Barnes MJ (1982) Collagen polymorphism in the normal and diseased blood vessel wall. Investigations of collagens types I, III and V. Atherosclerosis 48: 41-51 19 Ooshima, A (1981) Collagen B chain: Increased proportion in human Atherosclerosis. Science 213: 666-668 20 Rauterberg J, Allam S, Brehmer U, Wirth W, Hauss WH (1979) Das Kollagen der Arterienwand. Charakterisierung in Gewebeproben und Untersuchungen zur Biosynthese in kultivierten Aortenwandzellen. Therapiewoche 29: 2717-2737 21 Rauterberg J, Jander R, Voss B, Furthmayr H, Glanville RW (1982) Characteristics and occurrence of short chain collagen. In: New Trends in Basement Membrane Research. Kiihn K, Schone H, Timpl R (Eds) Raven Press, New York, 107-119 22 Rhodes, R-K-P, Miller, E-J (1978) Physiochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 17: 3442-3448 23 Risteli J, Bachinger HP, Engel J. Furthmayr H, Timpl R (1980) 7S-collagen: characterization of an unusual basement membrane structure. Eur J Biochem 108: 239-250 24 Risteli L, Timpl R (1981) Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes. Biochem J 193: 749-755 25 Staubesand J, Fischer N (1979) Collagen dysplasia and matrix vesicles. Researches with the electron microscope into the problem of so-called 'weakness of the vessel wall'. Path Res Pract 165: 374-391 26 Timpl R, Wick G, Gay S (1977) Antibodies to distinct types of collagens and procollagens and their application in immunohistology. J Immunol Meth 18: 165-182 27 Timpl R, Rohde H, Robey PG, Rennard SI, Foidart J-M, Martin GR (1979) Laminin - a glycoprotein from basement membranes. J Bioi Chern 254: 9933-9937 28 Timpl R, Risteli L (1982) Radioimmunoassays in studies of connective tissue proteins. In: Furthmayr H (Ed) Immunochemistry of the extracellular matrix. Vol 1, Methods. CRC Press Inc Boca Raton, Florida, 199-235 29 Voss B, Allam S, Rauterberg J, Ullrich K, Gieselmann V, von Figura K (1979) Primary cultures of rat hepatocytes synthesize fibronectin. Biochem Biophys Res Comm 90: 1348-1354 30 Voss B, Rauterberg J, Allam S, Pott G (1980) Distribution of collagen type I and type III and of two collagenous components of basement membranes in the human liver. Path Res Pract
170: 50-60
31 Zucker MB, Borelli J (1962) Platelet clumping produced by connective tissue suspensions and by collagen. Proc Soc Exp Bioi Med 109: 779
Received May 13, 1985 . Accepted in revised form March 11, 1986
Key words: Human arteries - Collagens - Glycoproteins - Atherosclerosis - Fibronectin Dr. Bruno Voss, Institut fiir Arterioskleroseforschung an der Universitat Miinster, Domagkstr. 3, D-4400 Munster, FRG