Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function
2003; Springer Nature; Volume: 23; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7600014
ISSN1460-2075
AutoresAlexander G. Marneros, Douglas R. Keene, Uwe Hansen, Naomi Fukai, Karen S. Moulton, Patrice L Goletz, Gennadiy Moiseyev, Basil S. Pawlyk, Willi Halfter, Sucai Dong, Masao Shibata, Tiansen Li, Rosalie K. Crouch, Peter Brückner, Bjørn R. Olsen,
Tópico(s)Retinal and Macular Surgery
ResumoArticle11 December 2003free access Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function Alexander G Marneros Corresponding Author Alexander G Marneros Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Douglas R Keene Douglas R Keene Portland Research Center, Shriners Hospitals for Children, Portland, OR, USA Search for more papers by this author Uwe Hansen Uwe Hansen Department of Physiological Chemistry and Pathophysiology, University of Münster, Münster, Germany Search for more papers by this author Naomi Fukai Naomi Fukai Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Karen Moulton Karen Moulton Department of Surgery, Children's Hospital, Boston, MA, USA Search for more papers by this author Patrice L Goletz Patrice L Goletz Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Gennadiy Moiseyev Gennadiy Moiseyev Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Basil S Pawlyk Basil S Pawlyk Massachusetts Eye and Ear Infirmary, Boston, MA, USA Search for more papers by this author Willi Halfter Willi Halfter Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Sucai Dong Sucai Dong Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Masao Shibata Masao Shibata Medical & Biological Laboratories Co., Ina-City, Japan Search for more papers by this author Tiansen Li Tiansen Li Massachusetts Eye and Ear Infirmary, Boston, MA, USA Search for more papers by this author Rosalie K Crouch Rosalie K Crouch Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Peter Bruckner Peter Bruckner Department of Physiological Chemistry and Pathophysiology, University of Münster, Münster, Germany Search for more papers by this author Bjorn R Olsen Bjorn R Olsen Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alexander G Marneros Corresponding Author Alexander G Marneros Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Douglas R Keene Douglas R Keene Portland Research Center, Shriners Hospitals for Children, Portland, OR, USA Search for more papers by this author Uwe Hansen Uwe Hansen Department of Physiological Chemistry and Pathophysiology, University of Münster, Münster, Germany Search for more papers by this author Naomi Fukai Naomi Fukai Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Karen Moulton Karen Moulton Department of Surgery, Children's Hospital, Boston, MA, USA Search for more papers by this author Patrice L Goletz Patrice L Goletz Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Gennadiy Moiseyev Gennadiy Moiseyev Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Basil S Pawlyk Basil S Pawlyk Massachusetts Eye and Ear Infirmary, Boston, MA, USA Search for more papers by this author Willi Halfter Willi Halfter Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Sucai Dong Sucai Dong Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Masao Shibata Masao Shibata Medical & Biological Laboratories Co., Ina-City, Japan Search for more papers by this author Tiansen Li Tiansen Li Massachusetts Eye and Ear Infirmary, Boston, MA, USA Search for more papers by this author Rosalie K Crouch Rosalie K Crouch Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Peter Bruckner Peter Bruckner Department of Physiological Chemistry and Pathophysiology, University of Münster, Münster, Germany Search for more papers by this author Bjorn R Olsen Bjorn R Olsen Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Alexander G Marneros 1, Douglas R Keene2, Uwe Hansen3, Naomi Fukai1, Karen Moulton4, Patrice L Goletz5, Gennadiy Moiseyev5, Basil S Pawlyk6, Willi Halfter7, Sucai Dong7, Masao Shibata8, Tiansen Li6, Rosalie K Crouch5, Peter Bruckner3 and Bjorn R Olsen1 1Department of Cell Biology, Harvard Medical School, Boston, MA, USA 2Portland Research Center, Shriners Hospitals for Children, Portland, OR, USA 3Department of Physiological Chemistry and Pathophysiology, University of Münster, Münster, Germany 4Department of Surgery, Children's Hospital, Boston, MA, USA 5Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA 6Massachusetts Eye and Ear Infirmary, Boston, MA, USA 7Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA 8Medical & Biological Laboratories Co., Ina-City, Japan *Corresponding author. Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115, USA. Fax: +1 617 432 0638; E-mail: [email protected] or [email protected] The EMBO Journal (2004)23:89-99https://doi.org/10.1038/sj.emboj.7600014 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks. Introduction The retinal pigment epithelium (RPE) is essential for vision, supplying 11-cis retinal to photoreceptors, and performing the daily phagocytosis of the shed distal tips of the photoreceptor outer segments. Abnormalities of the RPE are found in human age-related macular degeneration (ARMD), the major cause of blindness in the Western world, and are associated with morphological changes at the Bruch's membrane/RPE interface with pathological sub-RPE deposit formation, termed basal laminar deposits (Curcio and Millican, 1999). They precede the atrophic as well as the exudative type of ARMD (Sarks, 1976). Early-type basal laminar deposits contain a predominantly amorphous electron-dense material, composed of excess basement membrane (BM) material produced by the RPE (van der Schaft et al, 1994). The pathogenetic mechanisms involved in basal laminar deposits formation in early ARMD remain unknown, in part because of the lack of a convincing mouse model. To study the role of Bruch's membrane abnormalities for RPE function and deposit formation, we investigated the eyes of mice lacking collagen XVIII/endostatin. This collagen is a component of almost all vascular and epithelial BMs (Muragaki et al, 1995), and is also a component of Bruch's membrane (Fukai et al, 2002). Collagen XVIII molecules contain 10 triple-helical (COL) domains that are separated by non-triple-helical (NC) regions (Oh et al, 1994a). A potent inhibitor of angiogenesis, endostatin (O'Reilly et al, 1997) is a proteolytic fragment of the C-terminal NC1 domain of collagen XVIII. Little is known about the physiological role of collagen XVIII and endostatin. Col18a1−/− mice are viable and fertile, but show developmental defects in hyaloid vessel regression (Fukai et al, 2002). Inactivating mutations in the human gene for collagen XVIII, COL18A1, have been identified in patients with Knobloch syndrome (Sertie et al, 2000), who have progressive retinal degeneration, high myopia, and occipital encephalocele. The underlying histopathological changes in the eyes of Knobloch syndrome patients are unknown, and the disease mechanism is not understood. Eye abnormalities in Knobloch syndrome patients suggest an important role of collagen XVIII and/or endostatin in ocular structures, and imply that there are functional alterations in ocular BMs of these patients and of Col18a1−/− mice. In this study, we demonstrate that collagen XVIII/endostatin is essential for the function of the RPE. Aged Col18a1−/− mice have reduced visual function with pathological electroretinograms (ERGs). Histologically, we find massive age-dependent accumulation of electron-dense deposits between the RPE and Bruch's membrane. The deposits contain excess BM material and are similar to basal laminar deposits. These changes are associated with an abnormal vitamin A metabolism in the RPE. Rhodopsin content in the retina is reduced, explaining the progressive loss of vision. Glial fibrillary acidic protein (GFAP) expression is increased in the neural retina in association with RPE and photoreceptor abnormalities, as seen in ARMD (Guidry et al, 2002). The RPE abnormalities suggest an important role of this collagen for BM function. To gain insights into the structural basis for this role, we used immuno-electron microscopy (immuno-EM) to determine the ultrastructural organization of collagen XVIII/endostatin in Bruch's membrane and in other BMs, and show that this collagen is anchored in a polarized fashion in perlecan-containing BM scaffolds. Results Lack of collagen XVIII/endostatin results in an age-dependent attenuation of visual function We performed electroretinography experiments with dark-adapted Col18a1−/− mice and wild-type littermates at 2 and 16 months of age. All Col18a1−/− mice had abnormal visual function, with ERGs showing significantly reduced a- and b-wave amplitudes, and prolonged implicit times (Figures 1A and B). Attenuation of visual function increased with age, 16-month-old Col18a1−/− mice having only about 48% of normal b-wave amplitudes (Figure 1B). Figure 1.ERGs of Col18a1−/− and wild-type littermates show reduced a- and b-wave amplitudes in mutant mice. (A) Representative ERGs from 2-month-old wild-type and mutant mice. The b-wave amplitude average and standard deviation are indicated. (B) Representative ERGs from 16-month-old wild-type and mutant mice. Download figure Download PowerPoint Morphological RPE abnormalities in Col18a1−/− mice with age-dependent formation of sub-RPE deposits Examination of the retina in living Col18a1−/− animals by fluorescence angiography revealed that the retinal vessels were perfused (not shown) and that there was no atrophy of the retina. The eyes of these animals were examined by light and electron microscopy (EM). Bruch's membrane showed no disruption and stained positively for BM components, including laminin and type IV collagen. In wild-type mice, extensive interdigitations were seen between the apical villi of RPE cells and the photoreceptor outer segments (Figure 2A). In contrast, in aged Col18a1−/− mice, morphological abnormalities of the RPE and the outer segments of the photoreceptors could be seen (Figures 2B and C). The outer segments of the photoreceptors appeared disorganized and abnormally bent in mutant mice, and a reduced interdigitation of the apical villi of the RPE with the photoreceptors was apparent when compared to wild-type littermates. Despite the reduced interdigitation, cell polarity of the RPE was maintained in the mutant mice, with apical villi and basal cell membrane infoldings present. In addition, immunohistochemical staining for the polarization marker ezrin showed no difference between mutant and wild-type mice (not shown). Figure 2.Col18a1−/− mice show age-dependent morphological abnormalities of the RPE and sub-RPE deposits. (A) Electron micrograph of the RPE region (white double-headed arrow) of a wild-type littermate (16-month-old) with no sub-RPE deposits at the basement membrane (BM) and normal interdigitation (arrow) of the apical villi of the RPE with the photoreceptor outer segments. Scale bar, 1.5 μm. (B) Reduced interdigitation of the apical villi of the RPE with the photoreceptor outer segments (arrow) in a 16-month-old Col18a1−/− mouse. Abnormal deposits are visible at the basal RPE (black double-headed arrow). Scale bar, 1.5 μm. (C) Increased sub-RPE deposits (black double-headed arrow) in a 22-month-old Col18a1 null mouse. The diameter of the remaining RPE cell (white double-headed arrow) is reduced in relation to the sub-RPE deposits. Scale bar, 1.5 μm. (D) The RPE of a 16-month-old wild-type littermate shows no deposits between the basal infoldings (arrow) or membranous debris. Scale bar, 0.5 μm. (E) Higher magnification of electron-dense deposits (arrow) between the basal infoldings of the RPE in mutant mice (16-month-old). Scale bar, 1 μm. (F) Membranous debris (arrowhead) of RPE basal infoldings in aged mutant mice (16-month-old). Amorphous material is indicated by an arrow. Scale bar, 0.3 μm. (G) Electron-dense amorphous sub-RPE deposits (arrow) with membranous debris (arrowhead) in a 16-month-old Col18a1−/−/Hspg2Δ3/Δ3 mutant mouse. Scale bar, 0.5 μm. Download figure Download PowerPoint In a number of mouse strains, including C57Bl/6 mice examined here, subtle age-dependent accumulation of amorphous electron-dense material between the basal infoldings of the RPE can be seen by EM. However, in aged Col18a1−/− mice the accumulation of such material was dramatically increased when compared to wild-type littermates (Figure 2D), with the material occupying the entire sub-RPE space (Figures 2E and F). This amorphous electron-dense material contained vesicles and membranous debris, and was continuous with the lamina densa of the RPE BM, which had the same electron density when examined by EM. The RPE basal infoldings were wider in mutant mice than in wild-type mice that had no deposits. In 22-month-old mutant mice, the sub-RPE deposits occupied a larger area than the entire RPE diameter (Figure 2C). The deposits were found throughout the entire sub-RPE space of the eye, including the peripheral retinal region. These deposits observed in aged Col18a1−/− mice show striking morphological similarities to basal laminar deposits found in aged human eyes with early ARMD (van der Schaft et al, 1994). Widened RPE basal infoldings and irregular apical villi, as observed in these mutant mice, are also found in human eyes with basal laminar deposits (van der Schaft et al, 1994). In 2-month-old Col18a1−/− mice, we did not observe sub-RPE deposit formation. Thus, the deposit formation in Col18a1−/− mice is an age-dependent process, which is associated with the progressive attenuation of visual function. Since collagen XVIII is a heparan sulfate proteoglycan (HSPG) (Halfter et al, 1998), we tested if the deposits are due to the lack of collagen XVIII protein or due to a reduced heparan sulfate (HS) content of Bruch's membrane, by examining sub-RPE deposit formation in mice with further depleted HS content of Bruch's membrane. These mice lack Col18a1 and also exon 3 of the perlecan gene, resulting in a loss of attachment sites for three HS side chains of perlecan (Hspg2Δ3/Δ3), but have essentially normal levels of perlecan core protein (Rossi et al, 2003). We found that sub-RPE deposits in the Col18a1−/−/Hspg2Δ3/Δ3 mice were similar in size and morphology as in Col18a1−/− mice (Figure 2G). These findings suggest that the formation of sub-RPE deposits is a consequence of the lack of collagen XVIII protein and not due to a reduced HS content of Bruch's membrane. Although the possibility of a compensatory upregulation of other HSPGs at the BM site in double-mutant mice—and therefore an HS content like in Col18a1−/− mice—cannot be entirely excluded, this seems unlikely based on the previously reported observation of more severe lens degeneration in double-mutant mice, when compared to Hspg2Δ3/Δ3 mice (Rossi et al, 2003). While the lens in Col18a1−/− mice shows no degeneration, in double-mutant mice lens degeneration appears earlier than in Hspg2Δ3/Δ3 mice, suggesting that the additional lack of HS in the lens capsule is not compensated by upregulation of other HSPGs in the BM. An increased accumulation of electron-lucent debris and lipids within Bruch's membrane has been observed in ApoE−/− mice (Dithmar et al, 2000). To test if the extent of sub-RPE deposit formation in Col18a1−/− mice is influenced by the accumulation of debris and lipids within Bruch's membrane, we crossed Col18a1−/− mice with ApoE−/− mice, and examined the formation of sub-RPE deposits in the double-mutant mice. We found that the extent and onset of deposit formation in Col18a1−/−/ApoE−/− mice was not significantly different from Col18a1−/− mice (Figure 3). This suggests that sub-RPE deposit formation in Col18a1−/− mice is a process that is independent of the accumulation of abnormal lipid material into Bruch's membrane. Figure 3.Sub-RPE deposit formation occurs independently of accumulation of lipids in Bruch's membrane. (A) Early sub-RPE deposits (arrow) in a 7-month-old Col18a1−/− mouse. Scale bar, 0.5 μm. (B) Electron-lucent material (arrowheads) in Bruch's membrane (BM) in a 7-month-old ApoE−/−/Col18a1−/− mouse that shows similar sub-RPE deposits (arrow) as seen in Col18a1−/− mice. Scale bar, 0.5 μm. Download figure Download PowerPoint Sub-RPE deposits in Col18a1−/− mice contain excess BM material To test if the sub-RPE deposit formation in aged Col18a1−/− mice may serve as a model for basal laminar deposits formation in early ARMD, we aimed to characterize the composition of these deposits and compare them to basal laminar deposits. It has been demonstrated by immuno-EM that basal laminar deposits in early ARMD contain excess BM material, including type IV collagen (van der Schaft et al, 1994). Based on the morphological similarities of the observed sub-RPE deposits in aged Col18a1−/− mice to basal laminar deposits, we speculated that the deposits result from abnormal accumulation of BM material produced by the RPE. We performed immuno-EM experiments with polyclonal antibodies against BM components and other proteins produced by the RPE, such as TIMP-3 or ApoE. We found that the sub-RPE deposits labeled strongly for the BM component type IV collagen (Figures 4A and B). In addition, high-magnification EM images suggested the presence of the BM component type VIII collagen in the deposits (Figure 4C), based on the observation of typical hexagonal arrays as formed by collagen VIII molecules within Descemet's membrane or in vitro (Sawada et al, 1990). In situ hybridization for Col8a1 and Col8a2 demonstrated that murine RPE cells express type VIII collagen (Figure 4D). Figure 4.Sub-RPE deposits in Col18a1−/− mice contain excess BM material. (A) Immuno-EM labeling of an 18-month-old Col18a1−/− mouse eye with polyclonal anti-type IV collagen antibodies. The BM (ECBM) of the endothelial cells (EC) of the choroid layer shows heavy labeling, as well as the electron-dense sub-RPE deposits (BLD). The RPE BM is indicated (arrow). Collagen fibers (CF) can be seen within Bruch's membrane. Scale bar, 250 nm. (B) Higher magnification of sub-RPE deposits that were labeled for type IV collagen. Scale bar, 250 nm. (C) Arrows indicate area of hexagonal ultrastructure observed within areas of sub-RPE deposits in Col18a1−/− mice that resemble type VIII collagen. Scale bar, 0.5 μm. (D) In situ hybridization for Col8a2 shows a positive purple stain of corneal endothelium (arrow), and also for RPE cells (line). Retina (R) and lens (L) are indicated. Magnification × 10. Download figure Download PowerPoint Thus, sub-RPE deposits in Col18a1−/− mice resemble basal laminar deposits not only in their morphology but also in their molecular composition. Based on these findings, we suggest that Col18a1−/− mice may serve as a model for studying mechanisms of basal laminar deposits formation. Aged Col18a1−/− mice have reduced retinyl esters in the RPE and decreased rhodopsin content in the retina Sub-RPE deposit formation in early ARMD is believed to interfere with transport processes and metabolism of the RPE, such as with the uptake or processing of vitamin A. Vitamin A is modified in the RPE in order to provide 11-cis retinal to the photoreceptors, which is required for vision. We speculated that the abnormal deposit formation in aged Col18a1−/− mice might interfere with vitamin A uptake or processing. In order to test this hypothesis, we measured retinyl ester content in the RPE of mutant and control mice, these esters being RPE storage forms of vitamin A, and found significantly reduced retinyl ester content in the RPE of aged Col18a1−/− mice with values of less than 20% compared to those of wild-type littermates (Figure 5A). In 2-month-old mutant mice, no deposits were detected, and consistent with this observation, the retinyl ester content was not significantly reduced in the RPE of these mice. Serum retinoid levels showed no difference between mutant and wild-type mice (data not shown), excluding a systemic retinoid abnormality in Col18a1−/− mice as the reason for the reduced RPE retinyl esters. Figure 5.Aged Col18a1−/− mice have reduced retinyl esters in the RPE, decreased rhodopsin contents in the retina, and reduced RPE65 protein. (A) Total endogenous retinyl ester content is reduced in 16-month-old Col18a1−/− mice when compared to wild-type littermates, but not in young mutant mice (2-month-old). The contents of retinyl esters are indicated as pmol/eye. Values are mean±s.e.m. of 2–3 separate experiments. We confirmed in a series of independent HPLC analyses with C57Bl/6 mice that the observed age-dependent increase of retinyl ester contents in the RPE of C57Bl/6 control mice is normal. (B) Measurements of rhodopsin contents (pmol/eye) in 2- and 16-month-old wild-type and mutant mice. Aged mutant mice show a significant reduction of rhodopsin contents in the retina. Values are mean±s.e.m. of 3–4 separate experiments. (C) Western blot of whole-eye homogenates of 18-month-old Col18a1−/− mice (KO) and matched wild-type control animals (WT). Numbers indicate densitometric measurements for β-actin (loading control) and RPE65 labeling, demonstrating a decrease of RPE65 protein in KO mice. Recombinantly produced RPE65 protein (RPE65) served as control sample. Download figure Download PowerPoint Based on these data, we hypothesized that the reduction of retinyl esters in the RPE in aged Col18a1−/− mice might be associated with an insufficient supply of 11-cis retinal to photoreceptors, and thus result in reduced rhodopsin concentrations in the retina. We measured rhodopsin content in the eyes of dark-adapted Col18a1−/− mice and wild-type littermates, and observed a significant reduction of rhodopsin in the retinas of aged Col18a1−/− mice (Figure 5B). Reduced levels of RPE65 protein in the RPE of aged Col18a1−/− mice We performed high-dose vitamin A administration experiments with aged Col18a1−/− and control mice, and measured the effect of vitamin A on visual sensitivity by ERGs. If the sub-RPE deposits are the rate-limiting factor for vitamin A uptake into the RPE, one would expect that high systemic concentrations of vitamin A would increase vitamin A uptake into the RPE and consequently lead to increased ERG amplitudes in the mutant mice. Systemic administration of five times higher doses of vitamin A than were sufficient to increase ERG amplitudes in vitamin A-deprived wild-type animals (Katz et al, 1993) did not lead to a significant increase in ERG amplitudes in aged Col18a1−/− mice. B-wave amplitudes of baseline ERGs in a group of 16-month-old mutant mice were 397±112 μV, and 48 h after intramuscular administration of 40 μg vitamin A, b-wave amplitudes had not significantly changed (450±192 μV). Administration of higher doses of vitamin A and ERG measurements up to 96 h after injections failed to detect a significant increase of b-wave amplitudes in aged Col18a1−/− mice. These findings suggest that the reduced retinyl esters in the RPE and rhodopsin levels in the neural retina are, at least in part, a consequence of an RPE/neural retina dysfunction. To provide further evidence for this hypothesis, we measured RPE65 protein levels in the eyes of aged mutant and control mice. RPE65 is essential for the formation of 11-cis retinal (Redmond et al, 1998), and has recently been demonstrated to be a major membrane-associated retinoid binding protein of the RPE (Jahng et al, 2003). In western blot experiments, we found in the eyes of aged Col18a1−/− mice a reduction of RPE65 protein levels to about a third when compared with age-matched controls (Figure 5C). Thus, lack of collagen XVIII results in RPE dysfunction with an abnormal vitamin A metabolism, associated with decreased levels of RPE65 and retinyl esters, and in reduced retinal rhodopsin levels with attenuation of visual function. Abnormalities in the neural retina of Col18a1−/− mice Photoreceptors depend on a proper function of the RPE. Retinal abnormalities have been described in ARMD eyes, with an increased expression of GFAP in Müller cells (Guidry et al, 2002). Based on the observed morphological changes of the distal photoreceptors and the RPE abnormalities in Col18a1−/− mice, we examined if the retina showed further pathological changes. We found an increased expression of GFAP in the retina of Col18a1−/− mice (Figure 6). GFAP expression was highest at local areas in the retina where photoreceptors appeared disorganized. Figure 6.Increased expression of GFAP in the neural retina of Col18a1−/− mice. (A) Immunofluorescence labeling of GFAP (using an FITC-conjugated secondary antibody) in a section of an 18-month-old wild-type mouse. Some labeling for GFAP can be seen at the inner limiting membrane region of the retina (arrows), but no GFAP was detected in the photoreceptor layer (arrowhead). Magnification × 40. (B) Increased GFAP labeling is found in the photoreceptor layer (arrowhead) and the inner limiting membrane region (arrow) in an 18-month-old Col18a1−/− mouse eye. Local disorganization of photoreceptors can be observed as well (region between arrow and arrowhead). Magnification × 40. (C) Overlay image of the same region as in (B). Pigmented cells (arrow) accumulate at regions of photoreceptor disorganization (arrowhead) at the retinal–vitreal interface. Magnification × 20. (D) Western blot showing increased GFAP protein in an 18-month-old Col18a1−/− mouse eye (densitometric value: 1436) in comparison to the wild-type control sample (densitometric value: 471). β-Actin was used as a loading control (KO densitometric value: 605; WT densitometric value: 504). M: marker lane. Download figure Download PowerPoint We previously described F4/80-positive macrophage-like pigmented cells that migrate out of the iris in aged Col18a1−/− mice (Marneros and Olsen, 2003), but not in young mutant mice. What leads to the migration of these cells in aged mutant mice is unclear. Here we find that these macrophage-like cells accumulate at the retinal/vitreous border at areas of heavily increased GFAP expression and local photoreceptor disorganization (Figure 6). This observation suggests that the age-dependent RPE dysfunction and retinal changes attract these macrophage-like cells. In conclusion, lack of collagen XVIII leads to functional RPE abnormalities with the formation of excess BM-like material as basal laminar-like deposits under the RPE, an altered vitamin A metabolism of the RPE, and neural retina changes with reduced rhodopsin levels that result in an attenuation of visual function with pathological ERGs (Figure 7). The observed RPE abnormalities and loss of visual function in aged mutant mice, most likely due to the effect of the lack of collagen XVIII/endostatin in the underlying Bruch's membrane, suggest that the absence of this collagen might cause altered properties of Bruch's membrane and induce functional changes in the RPE. To better understand how collagen XVIII/endostatin may function within Bruch's membrane and other BMs, we determined the ultrastructural organization of collagen XVIII molecules in BMs. Figure 7.Schematic model of RPE and retinal abnormalities in Col18a1−/− mouse eyes in comparison to normal eyes. Sub-RPE deposits in mutant mice are associated with reduced RPE65 protein and reduced retinyl esters in the RPE, reduced retinal rhodopsin content, photoreceptor abnormalities, and increased retinal GFAP expression. Download figure Download PowerPoint Ultrastructural localization of collagen XVIII/endostatin in Bruch's membrane and other BMs We performed immuno-EM of Bruch's membrane with a
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