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Intracellular formation of collagen microfibrils in granulation tissue
Experimental and Molecular Pathology 79 (2005) 244 – 248 www.elsevier.com/locate/yexmp
Intracellular formation of collagen microfibrils in granulation tissue Keisuke Ina *, Hirokazu Kitamura, Shuji Tatsukawa, Takashi Miyazaki, Hirokazu Abe, Yoshihisa Fujikura Division of Morphological Analysis, Department of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, 1-1, Idaigaoka, Hasama-machi, Oita-gun, Oita, Japan Received 10 August 2005 Available online 10 October 2005
Abstract It is important to determine the biosynthesis process of collagen fibers to elucidate the mechanism by which granulation tissue is induced after injury. The purpose of this study is to investigate whether collagen microfibrils can be formed not only outside but also inside a cell. Fibroblastlike cells in granulation tissue resulting from incision and ligation were examined. The cells possessed vesicles containing collagen microfibrils. The vesicles were present in connection with Golgi apparatus or the rough endoplasmic reticulum. Furthermore, the vesicles were exhibited to be secretory granules with the secretory granule marker Rab3A. The fibroblast-like cells were also indicated to be myofibroblasts, using conventional transmission electron microscopy and immunoelectron microscopy for the myofibroblast marker a smooth muscle actin. In conclusion, it was demonstrated that collagen microfibrils could be formed in the cell in the case of collagen fiber overproduction. D 2005 Elsevier Inc. All rights reserved. Keywords: Collagen fiber; Granulation tissue; Myofibroblast; Rab3A; a smooth muscle actin
Introduction It appears to be a common concept that collagen microfibrils are formed by fibroblasts after secreting out of them (Trelstad and Hayashi, 1979; Davidson and Berg, 1981), while they are never biosynthesized inside a cell. The process of collagen microfibril biosynthesis is considered to be as follows: in the rough endoplasmic reticulum (RER), a chain of procollagen is synthesized, is in turn hydroxylated on proline and lysine residues, and is added to galactoses. Furthermore, three chains of procollagen are bound through disulfide binding (Engel and Prockop, 1991), and the triple-strand structure of procollagens is formed. The procollagens are then transferred to the Golgi apparatus, where they are added to glucoses. They are exocytosed by transferring via the transport vesicle. Procollagens are secreted out of the cell, the propeptides are removed in the extracellular space, and tropocollagens are formed. Finally, regular tropocollagen cross-linking occurs (Eyre et al., 1984), and the collagen microfibril is formed. Collagen microfibrils
* Corresponding author. Fax: +81 97 586 5630. E-mail address: [email protected] (K. Ina). 0014-4800/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2005.08.003
are characterized by a regular cross-striation structure, with a 67 nm duration and 80 nm width in transmission electron microscopy (TEM). On the other hand, regular cross-striated constituents with a collagen microfibril-like period have been demonstrated in intracellular vesicles (Weinstock and Leblond, 1974; Kajikawa and Kakihara, 1974; Eyden, 1989, 1991). It is discussed whether these constituents are present in the synthesis – secretion system or in the endocytosis –digestion system. On the whole, it has been concluded by findings with conventional TEM that cross-striated constituents with the same duration as collagen microfibrils are found in the endocytosis – digestion system; however, in many cases, it has been impossible to confirm the identity of the intracellular vesicle (e.g. the secretory granule, endocytotic vesicle, and lysosome) with conventional TEM. In this study, we attempted to determine the identity of the vesicle, including cross-striated constituents, using the antibody for the secretory granule marker Rab3A. It is well known that Rab3A is present on the membrane of secretory granules and in the cytoplasm of the cell secreting substances (Castillo et al., 1997; Chaudhuri et al., 2001). Active Rab3A binding to GTP is exclusively present on the membrane of the secretory
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granule, while inactive Rab3A binding to GDP is found in the cytoplasm. Active Rab3A plays a crucial role in the fusion of the secretory granule with the cell membrane (Carroll et al., 2001; Yi et al., 2002). Therefore, Rab3A can act as a marker of the secretory granule. It is demonstrated that vesicles with the same cross-striated constituents as collagen microfibrils are, at least in part, secretory granules, and the intracellular formation of collagen microfibrils occurs in granulation tissue. Materials and methods Experimental design Male Sprague – Dawley rats, weighing 180 to 220 g, were used in these experiments. Our experiments were subjected to the Guidelines for Animal
Fig. 2. Conventional TEM of collagen microfibrils in close proximity to the Golgi area. (a) Collagen microfibrils (arrow) are shown as contained in the vesicle between the RERs (R) and the Golgi area (G). Scale bar: 200 nm (b) The vesicle (V) containing collagen microfibrils (arrow) is shown to connect with the Golgi vesicle (G). Collagen microfibrils (arrowhead) are also seen in the Golgi vesicle. Scale bar: 200 nm. Experimentation of Oita University. Animals underwent incision of the abdominal wall and opening of the abdominal cavity under pentobarbital anesthesia. The abdominal cavity was subsequently closed by ligation. Rats were sacrificed under anesthesia for histologic evaluation 7 days post-operation.
Tissue preparation for TEM
Fig. 1. Conventional TEM of a cell containing collagen microfibrils in the granulation tissue. (a) Vesicles including cross-striation constituents (arrows) are seen in the cell. The period and width of the cross-striation are identical to those of collagen microfibrils in the extracellular space. Scale bar: 500 nm. (b) Some intracellular collagen microfibrils (arrow) exhibit a hair root-like appearance (arrowhead). Scale bar: 200 nm. (c) The vesicle containing the collagen microfibril is open to the extracellular space. Scale bar: 200 nm R, RER.
Animals were anesthetized with pentobarbital and perfused with two-fold diluted Karnovsky’s fixative (Karnovsky, 1965) for 10 min. The whole layer of the abdominal wall at the ligation site was removed. Pieces of removed tissue were additionally immersion-fixed in the same fixative for 2 h at 4-C and then washed in 0.1 M cacodyrate buffer and postfixed in 2% osmium tetroxide – 0.05% potassium ferrocyanide for 2 h at 4-C. The tissues were dehydrated in an ascending series of ethanol and embedded in epoxy resin. Ultrathin sections were cut on an ultramicrotome (LKB 2088 Ultratom V, Bromma, Sweden), mounted on copper grids, and stained with methanolic uranyl acetate (Stempak and Ward, 1964) and lead citrate (Reynolds, 1963). The sections were observed and photographed under a transmission electron microscope (TEM-1200 EX II, JEOL, Tokyo, Japan) at 80 kV.
Tissue preparation for immunoelectron microscopy Animals were perfused with 4% paraformaldehyde – 0.5% glutaraldehyde. Pieces of the operated site tissue were additionally immersion-fixed
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Fig. 5. Immunoelectron microscopy for Rab3A. Immunolabeling (arrowheads) for Rab3A is seen on the membrane of the containing collagen microfibrils (arrows). Furthermore, immunolabeling is shown in the cytoplasm. Scale bar: 200 nm. UK) for 36 h at 60-C. Ultrathin sections were then cut and mounted on nickel grids.
Immunoelectron microscopy procedure for Rab3A, propeptide, and a smooth muscle actin
Fig. 3. Conventional TEM of collagen microfibrils in close proximity to the RER. (a) The vesicle (V) including collagen microfibrils (arrow) is seen to connect with the RER (R) at the site marked with . Scale bar: 200 nm. (b) The vesicle (V) containing collagen microfibrils (arrow) is shown to connect with the RER (R), which also includes the collagen microfibril (arrowhead). Scale bar: 200 nm. in the same fixative for 2 h at 4-C. After rinsing in 0.1 M cacodylate buffer, the pieces were dehydrated in a graded ethanol series and embedded in LR white resin (London Resin Company Ltd., London,
Fig. 4. Conventional TEM of actin microfilaments and dense bodies in the cell with intracellular collagen microfibrils. Actin microfilament bundles (A) and dense bodies (D) are seen in the periphery of the cell containing the vesicle with collagen microfibrils (arrow). Scale bar: 200 nm.
Immunolabeling was performed to detect Rab3A as the secretory granule marker, the propeptide, and a smooth muscle actin (aSMA) as the myofibroblast marker, using the indirect immunogold – silver method (Kitamura et al., 1991). In brief, ultrathin sections were first reacted with the primary antibody, rabbit anti-Rab3A antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-procollagen type I antibody (Santa Cruz Biotechnology) or mouse anti-aSMA monoclonal antibody (Sigma, St. Louis, MO), for 36 h at 4-C. The epitope against anti-procollagen type I antibody is present in the propeptides. The sections were then rinsed in phosphate buffer (pH 7.4) and further incubated for 1.5 h at room temperature with gold-labeled secondary antibody, goat anti-rabbit IgG antibody for Rab3A or rabbit anti-goat IgG antibody for procollagen type I or goat anti-mouse IgG antibody for aSMA (Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK). Sections were finally developed (Kitamura et al., 1991) for 10 min at 20-C. The developer contained 0.02% 18-Crown-6 (Aldrich Chem. Co., WI), 0.17% hydroquinone, 0.17% silver nitrate in 0.1 M citrate buffer (pH 3.45). Immunolabeled sections were observed and photographed under a transmission electron microscope at 60 kV. The specificity was checked by omitting the primary antibodies and using nonimmune serum.
Fig. 6. Immunoelectron microscopy for procollagen type I. Immunolabeling for procollagen type I is seen in the vesicle containing collagen microfibrils in the process of the cell. Scale bar: 100 nm.
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Results Conventional TEM The granulation tissue formed 1 week after incision and ligation of the abdominal wall. Fibroblast-like cells and collagen fibers were abundantly included in the granulation tissue. Vesicles containing cross-striated constituents were occasionally seen in these cells. Some exhibited a hair rootlike appearance (Fig. 1). The duration and width of the crossstriation were very similar to those of collagen microfibrils in the extracellular space. Thus, the cross-striated constituents were considered to be collagen microfibrils. The vesicles containing collagen microfibrils were occasionally seen in close proximity to the Golgi apparatus (Fig. 2). Fig. 2b demonstrates that the vesicle including collagen microfibrils is linked to the Golgi vesicle. The collagen microfibrils are also present in the Golgi vesicle. The vesicles including collagen microfibrils were shown in close proximity to the RER, too (Fig. 3). Figs. 3a and b exhibit that the vesicle containing collagen microfibrils connects with the RER. In Fig. 3b, the collagen microfibril is also seen in the RER. Abundant actin microfilaments and dense bodies were frequently noticed in the cytoplasm of the cell with vesicles containing collagen microfibrils (Fig. 4). Immunoelectron microscopy Immunolabeling for Rab3A was found on the membrane of the vesicle containing collagen microfibrils as well as in the cytoplasm of the cell (Fig. 5). The anti-Rab3A antibody used in this study bound to both. Thus, the vesicle containing collagen microfibrils was revealed to be a secretory granule. Immunolabeling for procollagen type I was seen in the vesicle, including collagen microfibrils (Fig. 6). The antiprocollagen type I antibody used bound to the epitope in the propeptide. The collagen microfibril is completed through the
Fig. 7. Immunoelectron microscopy for aSMA. Immunolabeling (arrowheads) for aSMA is seen on actin microfilaments in the cell containing the vesicle with collagen microfibrils (arrow). This cell is suggested as the myofibroblast. Scale bar: 500 nm.
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cross-linking of tropocollagens. Tropocollagen is formed by removing propeptides from procollagen. Fig. 6 shows that removed propeptides were colocalized with collagen microfibrils in the vesicle. Immunolabeling for aSMA was observed in the cell containing collagen microfibrils (Fig. 7). No immunolabeling was detected when the slides were processed either in the absence of the primary antibody or in the presence of non-immune serum. Discussion It is generally accepted that collagen microfibrils form in the extracellular space (Trelstad and Hayashi, 1979; Davidson and Berg, 1981). Namely, procollagens are secreted out of fibroblasts, and then tropocollagens are formed by removing propeptides. Collagen microfibrils are formed by the regular cross-linking of tropocollagens in the extracellular space (Eyre et al., 1984). In this study, cross-striated constituents were shown in the vesicles of cells. Since the duration and width of the crossstriation were identical to those of collagen microfibrils in the extracellular space, the cross-striated constituents were confirmed as collagen microfibrils. Using the common concept, if collagen microfibrils are found in a cell, they will be absent from the biosynthesis – secretion system and present in the endocytosis – digestion system. Eyden demonstrated cross-striated constituents (collagen microfibrils) identical to those of this study in myofibroblasts of human spindle cell tumors (Eyden, 1989, 1991). He discussed whether vesicles containing collagen microfibrils were lysosomes because they had a distinct submembrane space of uniform width and microvesicles. He also noted that, unless vesicles with collagen microfibrils had these findings, they might be secretory granules in the terminal stage. On the other hand, there are reports that odontoblasts in the incisor teeth of young rats possessed secretory granules containing cross-striated constituents resembling those in this study, but they did not exhibit the complete form of the collagen microfibril (Weinstock and Leblond, 1974; Kajikawa and Kakihara, 1974). In this study, vesicles containing collagen microfibrils connected with the Golgi vesicle and the RER. These findings suggest that the vesicle with collagen microfibrils is present in the synthesis – secretion system. In addition, since these vesicles had no distinct submembrane space of uniform width and microvesicles, they were not considered to be lysosomes. To determine the identity of vesicles containing collagen microfibrils, the vesicle marker was investigated. IEM were performed using the antibody against Rab3A present exclusively on the membrane of secretory granules except for the cytoplasm. Immunolabeling for Rab3A was positive on the membrane of vesicles with collagen microfibrils. It has been demonstrated in various tissues (Castillo et al., 1997; Chaudhuri et al., 2001; Carroll et al., 2001; Yi et al., 2002) that the Rab3A molecule plays an important role in the fusion of secretory granules with the cell membrane in exocytosis.
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Taken together, collagen microfibrils were indicated to be, at least in part, present in secretory granules. The cells were considered to be myofibroblasts since they were revealed to possess abundant actin microfilaments and dense bodies in TEM (Gabbiani, 1977; Eyden, 2001; Ru et al., 2003), and these microfilaments were exhibited to be aSMA-positive in IEM (Hirschel et al., 1971; Komuro, 1990; Gabbiani, 1992; Ina et al., 2002). Myofibroblasts are known as Fthe active fibroblast_ (Gabbiani, 1992) and are well known to overproduce extracellular matrices, including collagen fibers, and to induce fibrosis (Schmitt-Gra¨ff et al., 1993; Essawy et al., 1997). In conclusion, it was indicated that collagen microfibrils were already formed in the cell before collagens were secreted when the cell overproduced collagen. The cells were considered to be myofibroblasts. Acknowledgment We are grateful for the excellent secretarial assistance of Ms. Yukari Goto. References Carroll, K., Ray, K., Helm, B., Carey, E., 2001. Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines. Eur. J. Cell Biol. 80, 295 – 302. Castillo, P.E., Janz, R., Su¨dhof, T.C., Tzounopoulos, T., Malenka, R.C., Nicoll, R.A., 1997. Rab3A is essential for mossy fibre long-term potentiation in the hippocampus. Nature 388, 590 – 593. Chaudhuri, S., Kumar, A., Berger, M., 2001. Association of ARF and Rabs with complement receptor type-1 storage vesicles in human neutrophils. J. Leukocyte Biol. 70, 669 – 676. Davidson, J.M., Berg, R.A., 1981. Posttranslational events in collagen synthesis. Methods Cell Biol. 23, 136 – 199. Engel, J., Prockop, D.J., 1991. The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper. Annu. Rev. Biophys. Chem. 20, 137 – 152. Essawy, M., Soylemezoglu, O., Muchaneta-Kubara, E.C., Shortland, J., Brown, C.B., El Nahas, A.M., 1997. Myofibroblasts and the progression of diabetic nephropathy. Nephrol. Dial. Transplant. 12, 43 – 50. Eyden, B., 1989. Collagen secretion granules in reactive stromal myofibroblasts, with preliminary observations on their occurrence in spindle cell tumours. Virchows Arch., A Pathol. Anat. 415, 437 – 445.
Eyden, B., 1991. Collagen secretion granules: intracellular collagen. In: Eyden, B.P. (Ed.), Organelles in Tumor Diagnosis: An Ultrastructural Atlas. New York-Igaku-Shoin, New York, pp. 69 – 71. Eyden, B., 2001. The myofibroblast: an assessment of controversial issues and a definition useful in diagnosis and research. Ultrastruct. Pathol. 25, 39 – 50. Eyre, D.R., Paz, M.A., Gallop, P.M., 1984. Cross-linking in collagen and elastin. 53, 717 – 748. Gabbiani, G., 1977. Reparative processes in mammalian wound healing: the role of contractile phenomena. Int. Rev. Cytol. 48, 187 – 219. Gabbiani, G., 1992. The biology of the myofibroblast. Kidney Int. 41, 530 – 532. Hirschel, B.J., Gabbiani, G., Ryan, G.B., Majno, G., 1971. Fibroblasts of granulation tissue: immunofluorescent staining with anti-smooth muscle serum. Proc. Soc. Exp. Biol. Med. 138, 466 – 469. Ina, K., Kitamura, H., Tatsukawa, S., Takayama, T., Fujikura, Y., Shimada, T., 2002. Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy. Med. Electron Microsc. 35, 87 – 95. Kajikawa, K., Kakihara, S., 1974. Odontoblasts and collagen formation: an ultrastructural and autoradiographic study. J. Electron Microsc. 23, 9 – 17. Karnovsky, M.J., 1965. A formaldehyde – glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A. Kitamura, H., Nagai-Takita, K., Nakamura, M., 1991. Improvement of a physical developer devoid of gum Arabic for immunohistochemical use. Acta Histochem. Cytochem. 24, 521. Komuro, T., 1990. Re-evaluation of fibroblasts and fibroblast-like cells. Anat. Embryol. 182, 103 – 112. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 – 212. Ru, Y., Eyden, B., Curry, A., McWilliam, L.J., Coyne, J.D., 2003. Actin filaments in human renal tubulo-interstitial fibrosis: significance for the concept of epithelial – myofibroblast transformation. J. Submicrosc. Cytol. Pathol. 35, 221 – 233. Schmitt-Gra¨ff, A., Chakroun, G., Gabbiani, G., 1993. Modulation of perisinusoidal cell cytoskeletal features during experimental hepatic fibrosis. Virchows Arch., A Pathol. Anat. 422, 99 – 107. Stempak, J.H., Ward, R.T., 1964. An improved staining method for electron microscopy. J. Cell Biol. 22, 697 – 701. Trelstad, R.L., Hayashi, K., 1979. Tendon collagen fibrillogenesis: intracellular subassemblies and cell surface changes associated with fibril growth. Dev. Biol. 71, 228 – 242. Weinstock, M., Leblond, C.P., 1974. Synthesis, migration, and release of precursor collagen by odontoblasts as visualized by radioautography after [3H]proline administration. J. Cell Biol. 60, 92 – 127. Yi, Z., Yokota, H., Torii, S., Aoki, T., Hosaka, M., Zhao, S., Takata, K., Takeuchi, T., Izumi, T., 2002. The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules. Mol. Cell. Biol. 22, 1858 – 1867.
Intracellular formation of collagen microfibrils in granulation tissue Keisuke Ina *, Hirokazu Kitamura, Shuji Tatsukawa, Takashi Miyazaki, Hirokazu Abe, Yoshihisa Fujikura Division of Morphological Analysis, Department of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, 1-1, Idaigaoka, Hasama-machi, Oita-gun, Oita, Japan Received 10 August 2005 Available online 10 October 2005
Abstract It is important to determine the biosynthesis process of collagen fibers to elucidate the mechanism by which granulation tissue is induced after injury. The purpose of this study is to investigate whether collagen microfibrils can be formed not only outside but also inside a cell. Fibroblastlike cells in granulation tissue resulting from incision and ligation were examined. The cells possessed vesicles containing collagen microfibrils. The vesicles were present in connection with Golgi apparatus or the rough endoplasmic reticulum. Furthermore, the vesicles were exhibited to be secretory granules with the secretory granule marker Rab3A. The fibroblast-like cells were also indicated to be myofibroblasts, using conventional transmission electron microscopy and immunoelectron microscopy for the myofibroblast marker a smooth muscle actin. In conclusion, it was demonstrated that collagen microfibrils could be formed in the cell in the case of collagen fiber overproduction. D 2005 Elsevier Inc. All rights reserved. Keywords: Collagen fiber; Granulation tissue; Myofibroblast; Rab3A; a smooth muscle actin
Introduction It appears to be a common concept that collagen microfibrils are formed by fibroblasts after secreting out of them (Trelstad and Hayashi, 1979; Davidson and Berg, 1981), while they are never biosynthesized inside a cell. The process of collagen microfibril biosynthesis is considered to be as follows: in the rough endoplasmic reticulum (RER), a chain of procollagen is synthesized, is in turn hydroxylated on proline and lysine residues, and is added to galactoses. Furthermore, three chains of procollagen are bound through disulfide binding (Engel and Prockop, 1991), and the triple-strand structure of procollagens is formed. The procollagens are then transferred to the Golgi apparatus, where they are added to glucoses. They are exocytosed by transferring via the transport vesicle. Procollagens are secreted out of the cell, the propeptides are removed in the extracellular space, and tropocollagens are formed. Finally, regular tropocollagen cross-linking occurs (Eyre et al., 1984), and the collagen microfibril is formed. Collagen microfibrils
* Corresponding author. Fax: +81 97 586 5630. E-mail address: [email protected] (K. Ina). 0014-4800/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2005.08.003
are characterized by a regular cross-striation structure, with a 67 nm duration and 80 nm width in transmission electron microscopy (TEM). On the other hand, regular cross-striated constituents with a collagen microfibril-like period have been demonstrated in intracellular vesicles (Weinstock and Leblond, 1974; Kajikawa and Kakihara, 1974; Eyden, 1989, 1991). It is discussed whether these constituents are present in the synthesis – secretion system or in the endocytosis –digestion system. On the whole, it has been concluded by findings with conventional TEM that cross-striated constituents with the same duration as collagen microfibrils are found in the endocytosis – digestion system; however, in many cases, it has been impossible to confirm the identity of the intracellular vesicle (e.g. the secretory granule, endocytotic vesicle, and lysosome) with conventional TEM. In this study, we attempted to determine the identity of the vesicle, including cross-striated constituents, using the antibody for the secretory granule marker Rab3A. It is well known that Rab3A is present on the membrane of secretory granules and in the cytoplasm of the cell secreting substances (Castillo et al., 1997; Chaudhuri et al., 2001). Active Rab3A binding to GTP is exclusively present on the membrane of the secretory
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granule, while inactive Rab3A binding to GDP is found in the cytoplasm. Active Rab3A plays a crucial role in the fusion of the secretory granule with the cell membrane (Carroll et al., 2001; Yi et al., 2002). Therefore, Rab3A can act as a marker of the secretory granule. It is demonstrated that vesicles with the same cross-striated constituents as collagen microfibrils are, at least in part, secretory granules, and the intracellular formation of collagen microfibrils occurs in granulation tissue. Materials and methods Experimental design Male Sprague – Dawley rats, weighing 180 to 220 g, were used in these experiments. Our experiments were subjected to the Guidelines for Animal
Fig. 2. Conventional TEM of collagen microfibrils in close proximity to the Golgi area. (a) Collagen microfibrils (arrow) are shown as contained in the vesicle between the RERs (R) and the Golgi area (G). Scale bar: 200 nm (b) The vesicle (V) containing collagen microfibrils (arrow) is shown to connect with the Golgi vesicle (G). Collagen microfibrils (arrowhead) are also seen in the Golgi vesicle. Scale bar: 200 nm. Experimentation of Oita University. Animals underwent incision of the abdominal wall and opening of the abdominal cavity under pentobarbital anesthesia. The abdominal cavity was subsequently closed by ligation. Rats were sacrificed under anesthesia for histologic evaluation 7 days post-operation.
Tissue preparation for TEM
Fig. 1. Conventional TEM of a cell containing collagen microfibrils in the granulation tissue. (a) Vesicles including cross-striation constituents (arrows) are seen in the cell. The period and width of the cross-striation are identical to those of collagen microfibrils in the extracellular space. Scale bar: 500 nm. (b) Some intracellular collagen microfibrils (arrow) exhibit a hair root-like appearance (arrowhead). Scale bar: 200 nm. (c) The vesicle containing the collagen microfibril is open to the extracellular space. Scale bar: 200 nm R, RER.
Animals were anesthetized with pentobarbital and perfused with two-fold diluted Karnovsky’s fixative (Karnovsky, 1965) for 10 min. The whole layer of the abdominal wall at the ligation site was removed. Pieces of removed tissue were additionally immersion-fixed in the same fixative for 2 h at 4-C and then washed in 0.1 M cacodyrate buffer and postfixed in 2% osmium tetroxide – 0.05% potassium ferrocyanide for 2 h at 4-C. The tissues were dehydrated in an ascending series of ethanol and embedded in epoxy resin. Ultrathin sections were cut on an ultramicrotome (LKB 2088 Ultratom V, Bromma, Sweden), mounted on copper grids, and stained with methanolic uranyl acetate (Stempak and Ward, 1964) and lead citrate (Reynolds, 1963). The sections were observed and photographed under a transmission electron microscope (TEM-1200 EX II, JEOL, Tokyo, Japan) at 80 kV.
Tissue preparation for immunoelectron microscopy Animals were perfused with 4% paraformaldehyde – 0.5% glutaraldehyde. Pieces of the operated site tissue were additionally immersion-fixed
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Fig. 5. Immunoelectron microscopy for Rab3A. Immunolabeling (arrowheads) for Rab3A is seen on the membrane of the containing collagen microfibrils (arrows). Furthermore, immunolabeling is shown in the cytoplasm. Scale bar: 200 nm. UK) for 36 h at 60-C. Ultrathin sections were then cut and mounted on nickel grids.
Immunoelectron microscopy procedure for Rab3A, propeptide, and a smooth muscle actin
Fig. 3. Conventional TEM of collagen microfibrils in close proximity to the RER. (a) The vesicle (V) including collagen microfibrils (arrow) is seen to connect with the RER (R) at the site marked with . Scale bar: 200 nm. (b) The vesicle (V) containing collagen microfibrils (arrow) is shown to connect with the RER (R), which also includes the collagen microfibril (arrowhead). Scale bar: 200 nm. in the same fixative for 2 h at 4-C. After rinsing in 0.1 M cacodylate buffer, the pieces were dehydrated in a graded ethanol series and embedded in LR white resin (London Resin Company Ltd., London,
Fig. 4. Conventional TEM of actin microfilaments and dense bodies in the cell with intracellular collagen microfibrils. Actin microfilament bundles (A) and dense bodies (D) are seen in the periphery of the cell containing the vesicle with collagen microfibrils (arrow). Scale bar: 200 nm.
Immunolabeling was performed to detect Rab3A as the secretory granule marker, the propeptide, and a smooth muscle actin (aSMA) as the myofibroblast marker, using the indirect immunogold – silver method (Kitamura et al., 1991). In brief, ultrathin sections were first reacted with the primary antibody, rabbit anti-Rab3A antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-procollagen type I antibody (Santa Cruz Biotechnology) or mouse anti-aSMA monoclonal antibody (Sigma, St. Louis, MO), for 36 h at 4-C. The epitope against anti-procollagen type I antibody is present in the propeptides. The sections were then rinsed in phosphate buffer (pH 7.4) and further incubated for 1.5 h at room temperature with gold-labeled secondary antibody, goat anti-rabbit IgG antibody for Rab3A or rabbit anti-goat IgG antibody for procollagen type I or goat anti-mouse IgG antibody for aSMA (Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK). Sections were finally developed (Kitamura et al., 1991) for 10 min at 20-C. The developer contained 0.02% 18-Crown-6 (Aldrich Chem. Co., WI), 0.17% hydroquinone, 0.17% silver nitrate in 0.1 M citrate buffer (pH 3.45). Immunolabeled sections were observed and photographed under a transmission electron microscope at 60 kV. The specificity was checked by omitting the primary antibodies and using nonimmune serum.
Fig. 6. Immunoelectron microscopy for procollagen type I. Immunolabeling for procollagen type I is seen in the vesicle containing collagen microfibrils in the process of the cell. Scale bar: 100 nm.
K. Ina et al. / Experimental and Molecular Pathology 79 (2005) 244 – 248
Results Conventional TEM The granulation tissue formed 1 week after incision and ligation of the abdominal wall. Fibroblast-like cells and collagen fibers were abundantly included in the granulation tissue. Vesicles containing cross-striated constituents were occasionally seen in these cells. Some exhibited a hair rootlike appearance (Fig. 1). The duration and width of the crossstriation were very similar to those of collagen microfibrils in the extracellular space. Thus, the cross-striated constituents were considered to be collagen microfibrils. The vesicles containing collagen microfibrils were occasionally seen in close proximity to the Golgi apparatus (Fig. 2). Fig. 2b demonstrates that the vesicle including collagen microfibrils is linked to the Golgi vesicle. The collagen microfibrils are also present in the Golgi vesicle. The vesicles including collagen microfibrils were shown in close proximity to the RER, too (Fig. 3). Figs. 3a and b exhibit that the vesicle containing collagen microfibrils connects with the RER. In Fig. 3b, the collagen microfibril is also seen in the RER. Abundant actin microfilaments and dense bodies were frequently noticed in the cytoplasm of the cell with vesicles containing collagen microfibrils (Fig. 4). Immunoelectron microscopy Immunolabeling for Rab3A was found on the membrane of the vesicle containing collagen microfibrils as well as in the cytoplasm of the cell (Fig. 5). The anti-Rab3A antibody used in this study bound to both. Thus, the vesicle containing collagen microfibrils was revealed to be a secretory granule. Immunolabeling for procollagen type I was seen in the vesicle, including collagen microfibrils (Fig. 6). The antiprocollagen type I antibody used bound to the epitope in the propeptide. The collagen microfibril is completed through the
Fig. 7. Immunoelectron microscopy for aSMA. Immunolabeling (arrowheads) for aSMA is seen on actin microfilaments in the cell containing the vesicle with collagen microfibrils (arrow). This cell is suggested as the myofibroblast. Scale bar: 500 nm.
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cross-linking of tropocollagens. Tropocollagen is formed by removing propeptides from procollagen. Fig. 6 shows that removed propeptides were colocalized with collagen microfibrils in the vesicle. Immunolabeling for aSMA was observed in the cell containing collagen microfibrils (Fig. 7). No immunolabeling was detected when the slides were processed either in the absence of the primary antibody or in the presence of non-immune serum. Discussion It is generally accepted that collagen microfibrils form in the extracellular space (Trelstad and Hayashi, 1979; Davidson and Berg, 1981). Namely, procollagens are secreted out of fibroblasts, and then tropocollagens are formed by removing propeptides. Collagen microfibrils are formed by the regular cross-linking of tropocollagens in the extracellular space (Eyre et al., 1984). In this study, cross-striated constituents were shown in the vesicles of cells. Since the duration and width of the crossstriation were identical to those of collagen microfibrils in the extracellular space, the cross-striated constituents were confirmed as collagen microfibrils. Using the common concept, if collagen microfibrils are found in a cell, they will be absent from the biosynthesis – secretion system and present in the endocytosis – digestion system. Eyden demonstrated cross-striated constituents (collagen microfibrils) identical to those of this study in myofibroblasts of human spindle cell tumors (Eyden, 1989, 1991). He discussed whether vesicles containing collagen microfibrils were lysosomes because they had a distinct submembrane space of uniform width and microvesicles. He also noted that, unless vesicles with collagen microfibrils had these findings, they might be secretory granules in the terminal stage. On the other hand, there are reports that odontoblasts in the incisor teeth of young rats possessed secretory granules containing cross-striated constituents resembling those in this study, but they did not exhibit the complete form of the collagen microfibril (Weinstock and Leblond, 1974; Kajikawa and Kakihara, 1974). In this study, vesicles containing collagen microfibrils connected with the Golgi vesicle and the RER. These findings suggest that the vesicle with collagen microfibrils is present in the synthesis – secretion system. In addition, since these vesicles had no distinct submembrane space of uniform width and microvesicles, they were not considered to be lysosomes. To determine the identity of vesicles containing collagen microfibrils, the vesicle marker was investigated. IEM were performed using the antibody against Rab3A present exclusively on the membrane of secretory granules except for the cytoplasm. Immunolabeling for Rab3A was positive on the membrane of vesicles with collagen microfibrils. It has been demonstrated in various tissues (Castillo et al., 1997; Chaudhuri et al., 2001; Carroll et al., 2001; Yi et al., 2002) that the Rab3A molecule plays an important role in the fusion of secretory granules with the cell membrane in exocytosis.
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Taken together, collagen microfibrils were indicated to be, at least in part, present in secretory granules. The cells were considered to be myofibroblasts since they were revealed to possess abundant actin microfilaments and dense bodies in TEM (Gabbiani, 1977; Eyden, 2001; Ru et al., 2003), and these microfilaments were exhibited to be aSMA-positive in IEM (Hirschel et al., 1971; Komuro, 1990; Gabbiani, 1992; Ina et al., 2002). Myofibroblasts are known as Fthe active fibroblast_ (Gabbiani, 1992) and are well known to overproduce extracellular matrices, including collagen fibers, and to induce fibrosis (Schmitt-Gra¨ff et al., 1993; Essawy et al., 1997). In conclusion, it was indicated that collagen microfibrils were already formed in the cell before collagens were secreted when the cell overproduced collagen. The cells were considered to be myofibroblasts. Acknowledgment We are grateful for the excellent secretarial assistance of Ms. Yukari Goto. References Carroll, K., Ray, K., Helm, B., Carey, E., 2001. Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines. Eur. J. Cell Biol. 80, 295 – 302. Castillo, P.E., Janz, R., Su¨dhof, T.C., Tzounopoulos, T., Malenka, R.C., Nicoll, R.A., 1997. Rab3A is essential for mossy fibre long-term potentiation in the hippocampus. Nature 388, 590 – 593. Chaudhuri, S., Kumar, A., Berger, M., 2001. Association of ARF and Rabs with complement receptor type-1 storage vesicles in human neutrophils. J. Leukocyte Biol. 70, 669 – 676. Davidson, J.M., Berg, R.A., 1981. Posttranslational events in collagen synthesis. Methods Cell Biol. 23, 136 – 199. Engel, J., Prockop, D.J., 1991. The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper. Annu. Rev. Biophys. Chem. 20, 137 – 152. Essawy, M., Soylemezoglu, O., Muchaneta-Kubara, E.C., Shortland, J., Brown, C.B., El Nahas, A.M., 1997. Myofibroblasts and the progression of diabetic nephropathy. Nephrol. Dial. Transplant. 12, 43 – 50. Eyden, B., 1989. Collagen secretion granules in reactive stromal myofibroblasts, with preliminary observations on their occurrence in spindle cell tumours. Virchows Arch., A Pathol. Anat. 415, 437 – 445.
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