ARTICLE |
Correspondence to: Ryoji Kobayashi, Dept. of Chemistry, Kagawa Medical University, Ikenobe, Miki-cho, Kagawa 761-0793, Japan.
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Summary |
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We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastinmicrofibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis. (J Histochem Cytochem 47:10491056, 1999)
Key Words: MAGP-36, tropoelastin, microfibrils, immunoelectron microscopy
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Introduction |
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MICROFIBRILS are small-diameter (818 nm) extracellular matrix fibrils found in close proximity to elastic fibers and the basement membrane (
In addition to its major component, fibrillin, many microfibril-associated proteins have been identified. These include MAGP-1 and 2 (
MAGP-36 has recently received much attention because its human homologue, AAAP-40 (MAGP-3), is immunoreactive with IgG purified from the serum and aortic wall of patients with abdominal aortic aneurysms (AAAs) (
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Materials and Methods |
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Antibodies
An antiserum was prepared from rabbits immunized with purified porcine MAGP-36 as previously described (
Sample Preparation
Specimens of hypertrophic scar tissue from a 19-year-old woman were obtained during surgery. Samples of normal human skin were taken from the marginal part of the surgical specimen after informed consent was obtained from the patient. Specimens of other tissues from adult cows were obtained from a local slaughterhouse. Tissues for postembedding immunoelectron microscopy were fixed in 4% formaldehyde0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 5 hr at 4C, washed extensively with 0.1 M phosphate buffer, dehydrated through a graded series of ethanolwater mixtures at -20C, and embedded in LR White. The resin was polymerized at 40C for 48 hr under vacuum. For pre-embedding immunolabeling, tissue samples were fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.4, for 5 hr at 4C, rinsed twice with 0.1 M phosphate buffer, cryoprotected in 20% sucrose at 4C, and frozen with OCT compound in dry iceethanol. Some samples were also fixed in 4% formaldehyde2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 hr at 4C and postfixed in cacodylate-buffered 1% OsO4 for 1 hr at 4C. The samples were washed in buffer, 50% ethanol and then stained en bloc with 2% uranyl acetate in 50% methanol for 1 hr at 4C. After dehydration through a graded series of ethanolwater mixture and propylene oxide, these samples were embedded in Epon 812.
Immunolabeling
Postembedding immunoelectron microscopy was conducted according to
Pre-embedding double labeling experiments were performed on cryosections (15 µm thick) that were mounted on slides. After washing several times in PBS, the sections were blocked with PMT for 30 min and incubated with a mixture of anti-MAGP-36 rabbit serum and anti-fibrillin-1 MAb overnight at 4C. The antibodies were diluted 1:100 for anti-MAGP-36 rabbit serum and to 40 µg/ml for anti-fibrillin-1 MAb in PMT. The sections were then washed several times in PBS and incubated overnight at 4C with a mixture of goat anti-rabbit IgG antibodies conjugated with 5-nm gold particles and goat anti-mouse IgG antibodies conjugated with 10-nm gold particles. The secondary antibodies were diluted 1:20 in PMT. After rinsing several times in PBS, sections were fixed in 2.5% glutaraldehyde in PBS for 30 min, dehydrated, and embedded in Epon 812.
All ultrathin sections for electron microscopy were stained for 1530 min with 2% alcoholic uranyl acetate and 5 min with Reynold's lead citrate before examination in a JEOL 1200EX electron microscope.
Immunohistochemistry was performed on cryosections of formaldehyde-fixed tissue using the avidinbiotin complex (ABC) method. Cryosections (15 µm thick) were saturated with PMT for 30 min and incubated with primary antibodies at 4C overnight. These antibodies were detected with an ABC kit (Vector Laboratories; Burlingame, CA). Alkaline phosphatase activity of tissue-bound ABC complex was visualized with 0.3 mg/ml nitroblue tetrazolium (NBT) and 0.1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) diluted in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1 M NaCl and 5 mM MgCl2.
Method specificity was confirmed by one or more of the following controls: (a) the sections were processed without primary antibody; (b) the primary antibody was replaced by non-immune serum; or (c) the primary antibody was replaced by antiserum adsorbed with purified MAGP-36 which was excised from the gel. For the double labeling, crossreactions between inappropriate antibodies were also checked by applying each primary antibody separately and linking them with the inappropriate secondary antibody.
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Results |
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Specificity of Antibodies
Antibody specificity was carefully assayed against crude tissue preparations. Anti-MAGP-36 antibody did not react with other protein species because it recognized only two bands corresponding to MAGP-36 and its dimeric form (68 kD) by Western blotting of crude extracts from human skin and bovine aorta (Figure 1) (
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Ultrastructural Localization of MAGP-36 in Normal Skin and Aorta
Strong labeling was observed on elastic fibers in dermis and all layers of aorta, intima, media, and adventitia by immunohistochemistry and postembedding immunoelectron microscopy (Figure 2, Figure 4, Figure 11, and Figure 12). In elastic fibers, labeling for MAGP-36 was associated with their surface and with electron-dense bundles at the outer periphery of their amorphous elastic cores. Although some microfibrils were observed in the interior of the elastic cores, labeling of these microfibrils was sparse.
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Electron-dense bundles varied in density in Epon sections (Figure 5). Bundles increased from moderate to high density between the outer periphery of the elastic cores and their centers. Tubular microfibrils and electron-dense amorphous material were present in bundles at the periphery of the elastic core. Specific structures could not be resolved at the center of these cores, where density of bundles was highest. Electron-dense amorphous materials were also observed at the surface of elastic cores, although these were not as clear as in bundles that occurred at moderate density around the core. Fibrillin was also detected by immunoelectron microscopy in the electron-dense bundles (Figure 6).
With the pre-embedding procedure, labeling for MAGP-36 was detected on the surface of elastic cores and at the elastinmicrofibril interface (Figure 3). Intense labeling covered regions of the surface of elastic core in which microfibrils had apparently been stripped off. The microfibrils away from the elastic core did not bind the antibody for MAGP-36.
Ultrastructural Localization of MAGP-36 in Hypertrophic Scars
MAGP-36 was localized in most of the immature elastic fibers observed in hypertrophic scar tissue. Some of these immature elastic fibers did not contain any amorphous elastic deposits, but tropoelastin was detected by immunolabeling in all fibers in which MAGP-36 was present. The amount of tropoelastin varied, depending on the development of the fibers. In early immature elastic fibers, labeling for tropoelastin was intermingled with labeling for MAGP-36. Some labeling for tropoelastin was closely associated with the labeling for MAGP-36 at this stage (Figure 7a). At later stages of development, some amorphous elastin deposits were seen. The majority of gold particles specific for MAGP-36 were located where microfibrils intersected with these elastin deposits (Figure 7b).
Ultrastructural Localization of MAGP-36 in Other Tissues
In spleen tissue, labeling of typical elastic fibers with anti-MAGP-36 and anti-tropoelastin antibodies was identical to that observed in normal skin (Figure 8). Labeling for MAGP-36 was localized where microfibrils intersect with the amorphous core of these fibers. Labeling for tropoelastin, by contrast, was localized within the amorphous core. In the red pulp region of spleen, labeling was limited for both MAGP-36 and tropoelastin. In kidney, the mesangial region of the glomerulus is known to contain microfibrils that are not associated with elastin. We did not observe labeling with either anti-MAGP-36 or anti-tropoelastin antibody in the mesangial region of the glomerulus (Figure 10). Labeling was also sparse in other regions of kidney for both MAGP-36 and tropoelastin. In the eye, the ocular zonule contained bundles of microfibrils in parallel alignment. These microfibrils did not react with either anti-MAGP-36 or anti-tropoelastin antibody (Figure 9 and Figure 13).
Gold deposits were not seen in control specimens prepared using normal rabbit serum or the serum preadsorbed with purified MAGP-36 instead of anti-MAGP-36 antibody. Completely negative results were also obtained when anti-tropoelastin antibody or anti-fibrillin-1 antibody, but not gold-labeled second antibody, was omitted from the immunolocalization protocol described in Materials and Methods. Crossreaction was not detected in control specimens that were stained with combinations of inappropriate antibodies.
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Discussion |
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The results reported here describe the ultrastructural distribution of MAGP-36 in different tissues and its localization during elastogenesis. Among several microfibril-associated proteins, such as fibrillin, emilin, MAGP-1, MAGP-2, fibulin-2, and amyloid P, MAGP-36 is unique in that its distribution is exactly matched by the distribution of tropoelastin (
There are some previous descriptions of electron-dense bundles in elastic fibers (Ross et al. 1969;
Immunoelectron microscopy with anti-fibrillin antibody also suggested the presence of microfibrils in the electron-dense bundles. It appears certain that these peripheral dense bundles contain microfibrils and electron-dense amorphous materials.
Two human homologues of MAGP-36, MFAP-4 and AAAP-40 (MAGP-3) have been reported (
In this study, our findings suggest that MAGP-36 may be involved in elastogenesis. By affecting elastogenesis, MAGP-36 might be related to the pathogenesis of several diseases, such as AAAs, SMS, and hypertrophic scar formation. Further studies are needed to determine the function of MAGP-36 and its relationship to the pathogenesis of these diseases.
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