ARTICLE |
Correspondence to: Nicolai Miosge, Abteilung Histologie, Kreuzbergring 36, 37075 Göttingen, Germany. E-mail: nmiosge@gwdg.de
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Summary |
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The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo. (J Histochem Cytochem 51:285296, 2003)
Key Words: collagen type XVIII, endostatin domain, ultrastructural tissue, localization, in situ hybridization, mouse development
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Introduction |
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ANGIOGENESIS is a fundamental process, especially during embryonic development and in many other instances, such as wound healing or tumorigenesis (
Collagen type XVIII protein was found in basement membrane zones of embryonic chick and adult murine blood vessels (1-chain of collagen type XVIII have been identified in families suffering from the Knobloch syndrome, a disease involving retinal detachment (
The 1-chain of collagen type XVIII is homologous with that of collagen type XV (
Endostatin is part of the larger C-terminal globular NC1 domain of collagen type XVIII (
The involvement of the endostatin domain of collagen type XVIII (endostatin-XVIII) in mouse development is still not quite clear. We localized endostatin-XVIII at the light and ultrastructural level and found it to be a true basement membrane component even early during development. To test binding partners of endostatin-XVIII demonstrated by solid-phase assays in vitro (
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Materials and Methods |
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Animals
NMRI mice were kept on a normal day/night cycle. Day 0 of gestation was defined as starting at 1100 hr on the day on which a vaginal plug was detected after a mating period of 3 hr. On the respective days of gestation, beginning with day 8, pregnant mice were anesthetized with ether and sacrificed by cervical dislocation. After dissection of the uterine horns, the embryos (days 8, 10, 12) or fetuses (days 14, 16, 18) were removed. Three embryos or fetuses of each developmental stage were investigated. For light microscopic analysis of adult tissues, three kidneys from different 3-month-old NMRI mice were taken.
Fixation and Preparation of Tissues
All specimens were fixed by immersion in 4% paraformaldehyde in PBS, pH 7.2, at 4C. They were then dehydrated in an ascending series of ethanol from 30% to 100% and embedded in paraffin. Serial sections of 5 µm were cut with a Reichert's microtome. Every fifth section was stained with hematoxylin for topological orientation and staging was achieved by comparison with the appropriate Theiler stages. Tissue pieces (1 mm3) of adult and fetal NMRI mouse kidneys, fetal skin, and lung were applied for immunogold staining and were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde for 15 min, dehydrated, and embedded in the acrylic resin LR Gold (London Resin Company; Reading, UK). Ultrathin sections were cut with a Reichert's ultramicrotome and collected on formvar-coated nickel grids for immunoelectron microscopy as described previously (
Antibodies
Affinity-purified rabbit antibody (2.26 mg/ml) against the endostatin domain from mouse collagen type XVIII (
Light Microscopic Immunohistochemistry
Sections were deparaffinized, rehydrated, and rinsed for 10 min in PBS. Endogenous peroxidase was blocked by incubation in 3% H2O2 in methanol for 45 min in the dark. Each of the reaction steps was followed by rinsing for 10 min in PBS. Sections were pretreated for 5 min with 10 µg/ml protease XXIV (Sigma; Deisenhofen, Germany). The anti-endostatin-XVIII antibody was used at a dilution of 1:100 for 1 hr at 37C. The anti-perlecan antibody was applied at a dilution of 1:100 for 24 hr at 37C and the anti-nidogen-1 and anti-fibulin-2 antibodies at a dilution of 1:100, all in PBS, for 1 hr at RT. A peroxidaseanti-peroxidase method followed the previously described procedures (
Electron Microscopic Immunohistochemistry
For single labeling, the tissue sections were incubated for 1 hr at 4C with the antibodies against endostatin-XVIII. Sections were rinsed in PBS and incubated with the 8-nm gold-coupled goat anti-rabbit IgG diluted 1:200 in PBS for 20 min at RT. Further sections for single labeling were incubated for 1 hr at 4C with the antibodies against perlecan (1:50), nidogen-1 (1:100), or fibulin-2 (1:50) directly coupled to 16-nm colloidal gold particles. Thereafter, all sections were rinsed with water and stained with uranyl acetate (10 min) and lead citrate (8 min). Sections were examined with a LEO 906E electron microscope. For double labeling, all sections were incubated for 5 min at RT with 1% bovine serum albumin (BSA) in PBS and then rinsed in PBS. Thereafter, anti-endostatin-XVIII antibody diluted 1:50 in PBS was applied for 1 hr at 4C. After being rinsed with PBS, the gold-coated (8 nm) goat anti-rabbit antibody diluted 1:200 was applied for 20 min at RT. The sections were thoroughly rinsed with PBS. The gold-coated (16 nm) anti-perlecan antibody diluted 1:50 in PBS or the anti-nidogen-1 antibody diluted 1:100 in PBS was then incubated. All sections were finally rinsed with water and stained with uranyl acetate (10 min) and lead citrate (8 min). As control for the double labeling experiments we also applied a monoclonal (rat) endostatin-XVIII antibody (
Statistical Analyses
The numbers of gold particles, shown as means ± SEM, were counted on 10 randomly chosen micrographs of identical size and magnification. Each micrograph included a basement membrane of approximately 120 nm in width and 6.5 µm in length. The number of gold particles representing individual labeling and co-localizations, defined as different gold particles less than 30 nm apart, was determined for proximal tubule basement membranes after double labeling. With the immunogold method, a correlation between the number of gold particles and the relative protein amount can be established (
In Situ Hybridization
A 205-bp sac/hinc II fragment corresponding to the C-terminal endostatin domain of collagen type XVIII was used for hybridization (
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Results |
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Light Microscopic Localization of Collagen Type XVIII mRNA During Mouse Development
ISH revealed the mRNA of collagen type XVIII on days 8, 10, 12, 14, 16, and 18 of mouse development. On day 8 the mRNA was detected only in the mesoderm, not in the ectoderm or endoderm. In addition, the ectoplacental conus was positive for the mRNA (data not shown). On day 10 the mRNA was located widespread in the cytoplasm of epithelial and mesenchymal cells, e.g., the structures of the developing head mesenchyme, the gut, the neural tube and ganglia, or the heart and blood vessels (Fig 1A). From day 12 onwards, the collagen type XVIII mRNA was seen in the mesenchymal and epithelial cells of all major organ anlagen, e.g., the kidney, liver, or, as shown here, the lung (Fig 1B). In the vascular system of all developmental stages investigated, all endothelial cells of the developing capillaries stained for the mRNA, as seen here in the lung mesenchyme on day 14 (Fig 1B). On day 16 the mRNA of collagen type XVIII was detected, e.g., in the epithelial cells of the developing skin, whereas the mesenchymal cells of the developing dermis remained unstained (Fig 1C). None of the control ISHs with the corresponding sense probes (Fig 1D1F) showed any staining.
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Light Microscopic Localization of Endostatin-XVIII During Mouse Development
Day 8 mouse embryos are still surrounded by Reichert's membrane, a thick and multilayered basement membrane, which stained positive for endostatin-XVIII. In the early embryo the three germ layers, endoderm, ectoderm, and mesoderm, are separated by basement membranes exhibiting staining for the protein (Fig 2A). From day 10 to day 18, staining for endostatin-XVIII was seen in almost all epithelial basement membrane zones in all consecutive stages of development. In addition, the endothelial basement membrane zone of capillaries and larger blood vessels revealed staining for the protein (Table 1). In the brain, the neuroectodermal cells themselves were not stained. In contrast, the basement membrane zones of the neuroectoderm and the adjacent leptomeningeal anlage were positive for endostatin-XVIII (Fig 2B). In the peripheral nervous system, the basement membrane zones of the ganglia exhibited staining, as did the endoneurium of peripheral nerves (Fig 2C). No staining was seen in any developmental stages of bone formation, either in the condensed mesenchyme or the chondrocytes or at the stage of onset of calcification. The same was true for the myocytes of the developing skeletal muscles (Fig 2D). The basement membrane zones of the skin stained positive for endostatin-XVIII, but neither the fibroblasts, nor the keratinocytes showed any staining (Fig 2E). In the lung anlage, neither the mesenchyme nor the developing epithelial cells were stained, but the protein was found in the basement membrane zones of the branching bronchi (Fig 2F). The cells of the developing myocardium, endocardium, and pericardium were never stained, but their corresponding basement membrane zones were positive for endostatin-XVIII. In the liver anlage, the protein was found in the sinusoids and underneath the mesothelium. The hepatocytes and the hematopoietic cells showed no staining (Fig 2G). The basement membrane zones of the developing exocrine glands in the pancreas anlage and the salivary glands in the head mesenchyme were positive for endostatin-XVIII. In the small and large intestine the epithelial cells were not stained, whereas the basement membrane zones underlying the developing epithelium were stained for endostatin-XVIII. In all stages of kidney organogenesis, only basement membrane zones of the consecutive stages of glomeruli development (comma, S-shaped, early glomeruli) and those of the tubules were positive for endostatin-XVIII. The surrounding mesenchyme was never stained (Fig 2H).
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Ultrastructural Localization of Endostatin-XVIII During Mouse Development
Immunogold histochemistry at the ultrastructural level revealed that the endostatin domain of collagen type XVIII is indeed a true component of developing basement membranes in the mouse embryo and fetus. Endostatin-XVIII was found in the basement membranes of the developing epithelial cells in the lung anlage (Fig 3A). In the kidney, endostatin-XVIII was detected in the basement membranes of developing tubules (Fig 3B). The same was true for the basement membranes underneath endothelial cells, as shown here for a capillary in the dermis. The extracellular matrix, adjacent to the capillary, also stained positive (Fig 3C).
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Light Microscopic Localization of Endostatin-XVIII, Perlecan, Nidogen-1, and Fibulin-2 in Adult Mouse Kidney
Endostatin-XVIII was found in adult mouse kidney, especially in the basement membrane zones of the Bowman's capsule and the proximal and distal tubules. The protein was also detected in connective tissue surrounding arterioles (Fig 4A). The basement membrane zones of the collecting ducts, loops of Henle (Fig 4B), and the ureter (Fig 4C) stained positive. The urothelium itself was free of any staining, but the lamina propria and lamina muscularis contained endostatin-XVIII next to elastic fibers. Perlecan (Fig 4D) and nidogen-1 (Fig 4E) were found in all basement membrane zones in the mouse kidney, including tubules and glomeruli as well as Bowman's capsule. Fibulin-2 was detected only in the walls of arterioles in connection with elastic fibers. In contrast, no tubular or glomerular basement membrane zones stained for fibulin-2 (Fig 4F).
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Ultrastructural Localization of Endostatin-XVIII, Nidogen-1, and Perlecan in Adult Mouse Kidney
We found endostatin-XVIII in the proximal (Fig 5A) and distal tubules as well as in the Bowman's capsule. Less labeling was seen in the basement membranes of the glomeruli and some intracellular labeling was seen. Free endostatin-XVIII was also detectable in the serum and urine (
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Immunogold Double Labeling of Endostatin-XVIII with Nidogen-1 and Perlecan
Because the endostatin domain of collagen type XVIII exhibits various binding activities in solid-phase assays (
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Statistical Analysis of the Immunogold Results
The amount of endostatin-XVIII detected in the two co-localization experiments was almost identical (Fig 7A and Fig 7B, Lanes a). Approximately 50% of the detectable endostatin-XVIII (Fig 7A, Lane a) was co-localized with perlecan (Fig 7A, Lane c). In contrast to the amount of nidogen-1 detected (Fig 7B, Lane b), the co-localization rate with nidogen-1 (Fig 7B, Lane c) was below the background level (Fig 7B, Lane d). The difference between the co-localization rates of nidogen-1 and perlecan with endostatin-XVIII was statistically significant with p values <0.001 (Fig 7A and Fig 7B, Lanes c, asterisks).
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Discussion |
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The C-terminal globular endostatin domain of collagen type XVIII is of special interest because of its putative value as an anti-angiogenic agent in human anti-tumor therapy (v and
5 integrin-dependent endothelial cell migration (
As one of the first steps to further elucidate the physiological role of collagen type XVIII during mouse development, we localized its mRNA. Expression is seen in the mesoderm, but not in the ecto- or endoderm, at early stages of development and is later found in the mesenchyme. From day 12 onwards, the epithelial cell compartments also express collagen type XVIII mRNA. Interestingly, we found the mRNA in both cell compartments, epithelial and mesenchymal, throughout kidney and lung development. Collagen type XVIII is implicated in kidney and lung patterning processes during epithelialmesenchymal tissue interaction in an in vitro culture system (
In agreement with the wide distribution of the collagen type XVIII mRNA early on during mouse development, the protein is also seen from day 8 onwards. The antibody applied detects the C-terminal domain of collagen type XVIII, which means that we cannot distinguish between free and bound endostatin. Therefore, it remains to be elucidated whether the whole molecule plays a role during development or whether the endostatin domain is cleaved from the molecule but remains in the same tissue location. Endostatin-XVIII was seen in all epithelial and endothelial basement membranes from day 8 onwards in all developing organs investigated, and might therefore be involved in early developmental processes. It appears to be a basement membrane component as abundant as collagen type IV. This was also recently demonstrated for adult human tissues (
As determined by immunogold histochemistry, endostatin-XVIII is a true basement membrane component even early during mouse development. Collagen type XVIII might be an integral basement membrane component involved in maintaining its structural network integrity. On the other hand, cleavage of the endostatin domain from the collagen type XVIII molecule is one of the main features of its anti-angiogenic function (
To investigate the association of endostatin-XVIII with the known basement membrane components in vivo, we performed immunogold double labeling of endostatin-XVIII together with nidogen-1 or perlecan. These were chosen because their binding affinities were demonstrated in vitro (
In summary, endostatin-XVIII is already found in the early mouse embryo and has a widespread distribution, mainly in basement membranes of epithelial and endothelial tissues. This supports a physiological role during organogenesis, not only for the regulation of angiogenesis. Expression of the mRNA is seen in epithelial as well as mesenchymal cells. In the adult mouse kidney, approximately 50% of the detectable endostatin domain is co-localized with perlecan in basement membranes. This seems to be at least one mechanism to connect collagen type XVIII to the basement membrane scaffold in vivo.
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Acknowledgments |
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Supported by a grant from the German Research Council (DGF) to N. Miosge (Mi 573/2-1). Parts of this work were taken from the doctoral thesis of O. Abdul-Malak.
We would like to thank Drs R. Timpl and T. Sasaki for providing antibodies and cDNA, and Cyrilla Maelicke, BSc, for editing the manuscript.
Received for publication July 29, 2002; accepted November 6, 2002.
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