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Purpose. Various proteoglycans are expressed in ocular tissues. We investigated and reviewed the distribution and the potential roles of proteoglycans in cornea, trabecular meshwork, and retinal tissues. Methods. Immunohistochemical studies were performed in rat ocular tissues. The concentration of transforming growth factor (TGF)-P2, which regulates the expression of proteoglycans in aqueous humor from human glaucomatous eyes, was evaluated by enzyme-linked immunosorbent assay (ELISA). In retinal tissues, we examined the localization of 2 soluble nervous tissuespecific chondroitin sulfate proteoglycans, neurocan and phosphacan, by immunohistochemical analysis, then investigated the effect on the neurite outgrowth of cultivated retinal ganglion cells. Results. The expression of chondroitin sulfate in stroma was upregulated at early postnatal stages and reduced during development in rat eyes. In trabecular meshwork tissues, immunohistochemical studies showed the intense expression of decorin. Moreover, elevated levels of TGF-@2in the aqueous humor from glaucomatous patients were observed. In retinal tissues, neurocan and phosphacan were expressed mainly in nerve fiber-rich layers during rat postnatal stages. In vitro, the neurite extension from retinal ganglion cells was inhibited by neurocan and phosphacan. Conclusions. Soluble extracellular proteoglycans in corneal and trabecular meshwork tissues contribute to the stromal transparency in the corneal tissues and the resistance of the aqueous humor outflow in trabecular meshwork tissues. In retinal tissues, chondroitin sulfate and heparan sulfate proteoglycans are not only secreted into the extracellular space of retinal tissues but also expressed in the membrane of the retinal cells, contributing to the neural network formation and the maintenance of the interphotoreceptor matrix. Key Words: Cornea-Decorin-GlycosaminoglycansNeurocan-Neuroglycan C-Phosphacan-ProteoglycansRetinal development-Retinal injury-Trabecular meshwork.
It has been recognized that various proteoglycans are expressed in ocular tissues. It is thought that soluble extracellular proteoglycans in corneal and trabecular meshwork tissues interact with other extracellular matrices and cytokines and contribute to the stromal transparency in the corneal tissues and the resistance of the aqueous humor outflow in trabecular meshwork tissue^.^" On the other hand, chondroitin sulfate and heparan sulfate proteoglycans are not only secreted into the extracellular space of retinal tissues but also expressed in the membrane of the retinal cells, contributing to the neural network formation and the maintenance of the interphoto-
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DOI: 10.1097/01.IC0.0000028325.06702.47
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sion of other SLRPs such as lumican and keratocan. It is suggested that the corneal stroma in biglycan- or decorin-deficient mice may be clear because the binding of dermatan sulfate SLRPs occurs at the d and e bands of collagen, in contrast to the keratan sulfate SLRPs that bind to the a and c bands of collagen.2x
It has been shown that decorin, biglycan, and lumican interact with transforming growth factor (TGF)-P. TGF-P induces the proliferation of corneal stromal fibroblast^,^^ and it is believed that the cytokine is involved in corneal wound healing.3 Also, TGF-P has been hypothesized to play an important role in corneal morphogenesis because, in TGF-P24eficient mice, fewer keratocytes, decreased accumulation of lumican and keratocan, and resultant thinner corneal stroma are observed. Thus, it is likely that these SLRPs regulate the cell response to TGF-P during corneal scamng INVOLVEMENT OF PROTEOGLYCANS IN and development processes. Previous studies reported that, when CORNEAL TRANSPARENCY removed from the stroma, keratocytes rapidly lose the ability to There is much evidence that abnormal accumulation or desecrete keratan ~ulfate.~~ The presence of fibroblast growth faccreased expression of SLRPs induces corneal opacity. For extor-2, however, supports keratan sulfate proteoglycan (lumican, ample, lumican-deficient mice display skin laxity and fragility minecan, and keratocan) synthesis by induction of core-protein resembling certain types of Ehlers-Danlos syndrome.2 In addibiosynthesis.3s Further studies are requikd to elucidate the intertion, the mutant mice develop bilateral corneal opacification where action of the SLRPs with cytokines in corneal wound healing. the regular arrangement of collagen fibrils is d i s t ~ r b e d .The ~~.~~ recessively inherited form (CNA2) of cornea plana, a disorder accompanied by flattened forward convex curvature that causes PROTEOGLYCANS IN THE TRABECULAR decreased refraction, is caused by a homozygous missense mutaMESHWORK AND THEIR tion of keratocan gene (KERA) in 12q.24It has also been reported CLINICAL RELEVANCE that overexpressed keratocan is observed in the corneal stroma of keratoconus patients, which indicates that the overexpression may Trabecular meshwork cells secrete not only collagens or fibroalter the fibrillogenesis in the stroma and lead to the development nectin but also secrete or express chondroitin/dermatansulfate and of keratoconus.2sOn the other hand, decorin- or biglycan-deficient heparan sulfate p r o t e o g l y ~ a n s , 3 which ~ ~ ~ suggests that proteoglymice do not show corneal opacity, although the deficiency induces cans are important contributors to aqueous outflow resistance in abnormal collagen-fiber network, which results in a disorder simithe juxtacanalicular connective tissue.43 Tawara et al. demonlar to Ehlers-Danlos syndrome in decorin-deficient mice and restrated that exfoliation materials in eyes with pseudoexfoliation duced bone mass in biglycan-deficient The disarrangesyndrome contain chondroitin sulfate, dermatan sulfate, and hepament of collagen network induced by the deficiency of decorin or ran sulfate proteoglycans.44 Moreover, Sawaguchi et al. reported that the injection of chondroitinase ABC into the anterior chambiglycan in the corneal stroma may be compensated by the expresComeo, Vol. 21. Suppl. 2. 2002
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bers of monkeys decreased intraocular pressure for 14 days$5 whereas Hubbard et al. found no difference in the intraocular pressures between before and after the injection of chondroitinase ABC.4" Recently, some proteoglycan core proteins in the trabecular meshwork have been reported. Our previous study showed that decorin is distributed in the trabecular meshwork of human eyes4' Furthermore, another SLRP, biglycan, one of the lecticans, versican, and a heparan sulfate proteoglycan, perlecan, are also secreted by trabecular meshwork cells.48 On the other hand, in glaucomatous eyes, elevated levels of TGF-P2 in the aqueous humor have been r e p ~ r t e d . ~ " Moreover, ~'~) trabecular meshwork cells can express this cytokine and its receptors,".'2 It is well known that expression of extracellular matrices is upregulated by the stimulation of TGF-P.53 Since TGF-P promotes the expression of decorin and other proteoglycan~,'~ the interaction of cytokines with proteoglycans may induce the pathologic situation of increased outflow pressure of aqueous humor in glaucomatous eyes.
rnert6' The confocal images were stained with an antineurocan antibody, MAb 1G2. Micrographs show sections stained with hematoxylin eosin (HE). Reproduced with permission of the Association for Research in Vision and Ophthalmology from lnatani M, et al.
Invest Ophthalmol Vis Sci 1999;40:2354.6'
immunoreactivities of neurocan or phosphacan have never been observed in the other ocular tissues, including cornea, trabecular meshwork, or lens. A proteolytic variant of the neurocan core protein is predominantly expressed in postnatal retina, whereas the intact core protein (full-length neurocan) is predominant in embryonal and prenatal retinas."' The phenomenon that the core protein is cleaved as the maturation of neural tissues proceeds is characteristic of neurocan expre~sion.'~."~ Although the proteolytic variant remains highly expressed in adult brain, the expression of either the intact or proteolytic core proteins is barely detected in adult retinal tissues because of the dramatic decrease in neurocan expression after the maturation of the retinal tissues."' Interestingly, the expression of neurocan is intensely upregulated even in adult rat retinal tissues after the stress of transient ischemia."' As in retinal development, the upregulated expression of full-length neurocan is followed by that of a proteolytic variant of neurocan core protein (Fig. 3). Moreover, immunohistochemical analyses revealed that glial Muller cells express neurocan in injured retinal tissue. Several groups have reported that, after mechanical injury to brain tissue, upregulated neurocan expression is also detected in the cortical lesion.""-"' In the lesion, reactive astrocytes produce neurocan. Although it had been believed that neurocan was synthesized by neurons,'Y."9 recent experimental data suggest that, at
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proteoglycan form of phosphacan appears to be an interesting feature in neural retinal tissues. To investigate the significant role of the nonproteoglycan form expressed in the postnatal rat retina, we cultivated retinal ganglion cells on a plate coated with phosphacan.70 The neurite outgrowth was significantly inhibited on the substrate containing phosphacan. Moreover, the inhibitory effect of phosphacan was further promoted by the digestion of chondroitin sulfate linked to the core protein. We performed the same procedure using neurocan, which has an inhibitory effect on neurite outgrowth. This was unaffected by the digestion of chondroitin sulfate. The nonproteoglycan phosphacan, characteristic of retinal tissue during retinal development, may be expressed to inhibit further neurite outgrowth from retinal ganglion cells more effectively than phosphacan bearing chondroitin sulfate.
FIG. 2. lmmunohistochemistry for phosphacan during retinal developrnent.62The confocal images were stained with an antiphosphacan, MAb 684. A: Retinal sections during development (E16-P42) were used for immunohistochemistry. Micrographs show sections stained with hematoxylin eosin (HE). B: lmmunohistochemical image of the optic nerve at P21. The optic nerve was stained as well as the NFL, IPL, and OPL. Note that there is no immunoreactivity in either the choroidaltissue or sclera. ON, optic nerve; SC, sclera; CR, choroidal tissue. Reproduced with permission of the Association for Research in Vision and Ophthalmology from lnatani M, et al. lnvesf Ophthalmol Vis Sci 2000;41:1993?2
least in pathologic situations, neurocan can be expressed by glial cells. Some inve~tigators~~.~' suggest that neurocan may inhibit the sprouting process of the damaged axon and regeneration of the damaged neural network in the mammalian central nervous system. Since neurocan has a strong inhibitory effect to neurite outgrowth in vit1-0;~ it is possible that neurocan may prevent damaged neuronal axons from making abnormal neural network with intact neurons. On the other hand, a significant amount of phosphacan in rat retina does not have any glycosaminoglycan side chains?' A non-
FIG. 3. lmmunoblot analysis of retina subjected to transient ischemia65 The bands of neurocan were barely detected in the control, but then the intensity of immunopositive bands of 220 kDa (fulllength neurocan core protein) and 150 kDa (a proteolytic variant of neurocan core protein) increased slightly at 6 hours after reperfusion. At 24 hours and 72 hours after reperfusion, the intensity of the 220-kDa band, as well as the 150-kDa band, increased markedly. The 220-kDa band was predominant at 24 hours after reperfusion, whereas the intensity of the 150-kDa band became almost the same as that of the 220-kDa band at 72 hours. The closed and open arrow heads show the 220-kDa and 150-kDa bands, respectively. The positions of molecular mass markers are indicated in kDa. Reproduced with permission of the Association for Research in Vision and Ophthalmology from lnatani M, et al. lnvest Ophthalmol Vis Sci2000;41: 2751.65
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After intravitreal injections of p-nitropheny1-beta-Dxylopyranoside (xyloside), a sugar that inhibits chondroitin sulfate proteoglycan synthesis, cone photoreceptor cell outer segment degeneration and shallow retinal detachments have been observed, which suggests that adhesion between the neural retina and retinal pigmented epithelium may be dependent on continuous synthesis of chondroitin sulfate proteoglycans in the lPM.84 Recently, some of the proteoglycans have been identified and characteri~ed.'~-~~ Two major chondroitin sulfate proteoglycans have 150-kDa and 230-kDa core proteins, which are named IMPGI, IPM 150, or SPACR,89-9' and IPM 200 or SPACRCAN.86.88 Moreover, the core proteins of the proteoglycans have the potential hyaluronanbinding motifs of the RHAMM type. It has been reported that hyaluronan is also distributed in the IPM, indicating the interaction of SPACR and SPACRCAN with hyaluronan in the IPM.86.91 In addition, we showed that neuroglycan C is highly expressed in the apical surface of the RPE cells?2 It is suggested that the core protein contains a RHAMM-type hyaluronan-binding motif.93 Neuroglycan C has a transmembrane region in the core protein94 and is bound to the cell membrane of the apical surface of the RPE It is possible that neuroglycan C in the RPE cells may bind hyaluronan in the IPM and may play a role in the adhesion of photoreceptor cells and Miiller cells in the neural retina to RPE cells. Moreover, there are other hyaluronan-binding proteins including CD44, RHAMM, and oxygen-regulated photoreceptor protein-1 in the IPM as well as in the cell membranes of photoreceptor cells, Miiller cells, and RPE c e l l ~ Interestingly, .~~ human SPACWIMPGI gene is mapped to chromosome 6q 13-q 15, whereas the genes of several cone-rod macular dystrophies overlap.95*96Further analyses for 6q-linked retinopathies will be required to identify whether abnormal gene expression of SPACMMPG 1 induces genetic cone-rod macular dystrophies.
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