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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 2, 2003 as doi:10.1096/fj.02-1001fje. |
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Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology,
* Biocenter Oulu and Department of Pathology, University of Oulu, Oulu, Finland;
Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland;
Biocenter Oulu and Department of Pathology, University of Oulu, Department of Ophthalmology, University Hospital of Helsinki, Helsinki, Finland; and
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
2Correspondence: Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland. E-mail: taina.pihlajaniemi{at}oulu.fi
SPECIFIC AIMS
Mice lacking type XVIII collagen have defects in the posterior part of the eye, including delayed regression of the hyaloid vasculature and poor outgrowth of the retinal vessels (EMBO J. 21, 1535, 2002). We report here on further investigations into the role of type XVIII collagen using knockout mice as a model.
PRINCIPAL FINDINGS
1. Rupture of the double layer of iris epithelium in Col18a1-/- mice
Analysis of the Col18a1-/- mouse eyes included the use of preparative biomicroscopy, which revealed abnormal fragmented edges of the irises at the adult stage (Fig. 1
). The corrugated appearance of the pupillary margin (Fig. 1C
), due to anterior extensions of the epithelial layers, was absent in the Col18a1-/- mice (Fig. 1D
). Histological sections showed that the anterior and posterior parts of the iris were separated (Fig. 2
A–D) and that the pigment epithelium of the iris was attached to the lens in the majority of cases and the anterior surface of the iris to the cornea in some (Fig. 2B, D, E
). Dispersed pigment granules or pigment-containing cells could be seen in the anterior part of the mutant eyes, presumably macrophages destroying the dispersed pigment or pigment cells from the disrupted iris; an accumulation of pigment granules was observed in the vitreous body near the inner border of the retina (Fig. 2E, F
).
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The iris is composed of an anterior surface, the iris stroma, and two layers of pigment-containing epithelial cells at the posterior surface. In transmission electron microscopy (EM) the 10-day-old Col18a1-/- mice, with their eyelids still closed, had both iris pigment epithelial cell layers present as in controls. However, as shown for the 14-day-old null mice, the posterior pigment epithelial cell layer begins to detach from the anterior cell layer at the site of apical cell contacts at this stage. In the adult null mutant eyes, the two pigment epithelial cell layers were ruptured apically along the cell contacts. The immunosignals for occludin and cadherin did not differ between the Col18a1-/- and wild-type irises, suggesting that disruption of the iris is not caused by changes in the protein composition of either the tight or adherens junctions in the iris pigment epithelium.
2. Atrophy of the ciliary body epithelial cells in Col18a1-/- mice
Another abnormal eye structure in the adult Col18a1-/- mice was the ciliary body, which lacked its distinctive curved shape (Fig. 2C, D
), even though the ciliary bodies of young Col18a1-/- mice (24 days old) were comparable to those of control mice at the light microscopy level (not shown). All the adult mice studied developed defective ciliary processes with time.
Although light microscopy did not reveal apparent changes in the ciliary processes of the young null mice, EM of ciliary bodies from 12-day-old mice already showed a decreased number of basal infoldings of the ciliary epithelium. The ciliary processes of the adult Col18a1-/- mice were rudimentary, with loss of cavities, and the basal epithelial infoldings were flattened, resulting in a decrease in the surface secreting aqueous humor. Pigmented and nonpigmented epithelial cell layers were both present in the null eyes.
3. Accumulation of extracellular material in basement membrane zones (BM) of iris and ciliary body
Transmission EM studies revealed broadening of the BM zones of iris and ciliary body epithelia and capillaries. In young null mice, the only site where abnormalities could be detected was the BM zone of ciliary body pigmented epithelium, while the BM zones of iris epithelia and capillaries and ciliary body capillaries and nonpigmented epithelium BM zone were comparable to wild-type. In adult mice, extracellular material had strongly accumulated to the BM zone, locating between epithelium and stroma in the iris. Abnormalities could be detected in BM of most of the iris capillaries, ciliary body capillaries, and both ciliary epithelial cell layers. Immunostudies with antibodies against perlecan, type IV collagen, and laminin 
1 showed local increases of immunosignal in the BM zones between iris epithelium and stroma, and in some iris capillaries.
4. Immunofluorescence studies of the iris and ciliary body
To relate the phenotypic changes observed in the Col18-/- mice to the normal occurrence of type XVIII collagen, we examined the location of this collagen in the iris and ciliary body. An antibody recognizing the carboxyl-terminal endostatin domain gave immunosignals in the BM zones of the epithelium and capillaries of the iris and ciliary body whereas an antibody specific to the two longest variants gave signals in the same areas of the iris, but only in the BM zone of the nonpigmented epithelium in the ciliary body.
5. Intraocular pressures
Changes in anterior segment that result in dispersed pigment and attachment of iris to the cornea may endanger the outflow of aqueous humor. Thus, intraocular pressure measurements (IOP) were started at the ages of 3–4 wk, when practically all the null mice had developed disrupted irises. A noninvasive induction/impact tonometer was used, allowing repeated measurements on the same mice. Medians for IOP were no higher in null than in wild-type mice in any age group. The average IOP was lower in the mutants than in the wild-type mice at ages of 5–7 wk and >6 months, probably reflecting poor functioning of the atrophied ciliary bodies.
CONCLUSIONS AND SIGNIFICANCE
The critical role of type XVIII collagen in the development and function of the eye was emphasized by the identification of defects affecting the anterior segment. Having previously described changes in the posterior part of the eye in type XVIII null mice, we report here that the lack of type XVIII collagen results in a defective iris, with separation of the two pigment epithelium layers, atrophy of the ciliary body, and a decrease in IOP with age.
As witnessed by the enhanced adhesion of the posterior iris epithelial cell layer to the lens in null mice and the increased adherence of the hyaloid capillaries to the retina, lack of type XVIII collagen appears to render the BM of the affected structures prone to adherence to neighboring structures. This apparent increased adhesion combined with the increased mechanical force associated with iris function could cause rupturing of the double layer of the iris epithelium in the absence of type XVIII collagen. It is also possible that the lack of this collagen may directly affect epithelial cell–BM interaction, which may cause weakening in the cell–cell contacts via intracellular changes.
Several of the BM zones appeared broader in the null mice than in the controls, and there were signs of the accumulation of extracellular matrix material. This was especially clear in the BM zone between the iris epithelia and stroma and in the BM of the pigmented ciliary epithelium. Our recent studies indicate that a lack of type XVIII collagen causes ultrastructurally visible changes in the BM of other tissues as well, such as the liver and kidney (A. Utriainen et al., unpublished results). The BM between the iris stroma and the pigment epithelia was the only one studied here that is under constant mechanical stress due to mydriasis and miosis. We suggest that mechanical stress has an enhancing role in the accumulation of matrix material in this BM zone. It is known that the carboxyl-terminal NC1 domain of type XVIII collagen and the NC1-derived endostatin domain can interact with several BM components in vitro; the amino-terminal half of the molecule contains several glycosaminoglycan side chains. Thus, it is conceivable that type XVIII collagen may be involved in molecular interactions with some other matrix molecules and that a lack of this collagen disturbs the normal structure of the BM, as observed here.
In contrast to the iris epithelia, where no clear signs of cellular atrophy were detected, the lack of type XVIII collagen results in atrophy of the ciliary nonpigmented epithelial cells. The number of basal infoldings in this epithelium is decreased in young mice and, since the BM zones of the nonpigmented and pigmented ciliary epithelium showed signs of an accumulation of matrix material, we suggest that these abnormal BM are not fully capable of supporting epithelial cell function and may possibly alter the outflow of aqueous humor. Altogether, our findings indicate that type XVIII collagen is needed for the normal development of the ciliary body.
Disorders affecting the anterior eye segment include the Axenfeld-Rieger syndrome and a variety of overlapping phenotypes sharing some of its clinical features, such as the Rieger syndrome, the iridogoniodysgenesis syndrome, iris hypoplasia, Peters’ anomaly, the pigment dispersion syndrome, and familial glaucoma iridogoniodysplasia. Mutations in the transcription factor-encoding genes FKHL7 at 6p25, PITX2/RIEG1 at 4q25, and PAX6 at 11p13 have been demonstrated in several disorders, but other affected genes remain to be identified in various associated loci. In view of the Col18a1-/- mouse phenotype, the type XVIII collagen gene should be considered a new candidate causal factor for anterior segment eye disorders. The human gene for type XVIII collagen is located at 21q22.3 and thus may be implicated in Peters’ anomaly, characterized by corneal abnormalities and a variable degree of iris and lenticular attachment to the cornea. Another intriguing possibility is that this gene may be regulated by some transcription factors known to undergo mutation in various eye anomalies. This may lead to decreases in type XVIII collagen expression; thus, some of the transcription factor defects could be due to insufficient levels of the collagen, shown here to be important for anterior ocular structures.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1001fje 
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