Proteoglycans in the Eye

Corneo 2l(Suppl. 2): S62469, 2002. 0 2002 Lippincolt Williams & Wilkins. Inc., Philadelphia

Review Article

Proteoglycans in the Eye

Hidenobu Tanihara, M.D., Masaru Inatani, M.D., Takahisa Koga, M.D., Tsuyoshi Yano, M.D., and Akira Kimura, M.D.

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 tissue- specific chondroitin sulfate proteoglycans, neurocan and phosphacan, by immunohistochemical analysis, then investigated the effect on the neurite outgrowth of cultivated retinal ganglion cells. Re- sults. 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-@2 in 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. Conclu- sions. 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-Glycosaminoglycans- Neurocan-Neuroglycan C-Phosphacan-Proteoglycans- Retinal development-Retinal injury-Trabecular meshwork.

Submitted March 26, 2002. Accepted April 26, 2002. From the Department of Ophthalmology (H.T., T.K., T.Y., A.K.), Ku-

mamoto University School of Medicine, Kumamoto, and the Department of Ophthalmology and Visual Sciences (M.I.), Kyoto University Graduate School of Medicine, Kyoto, Japan.

This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan.

Address correspondence and reprint requests to Dr. H. Tanihara, De- partment of Ophthalmology, Kumamoto University School of Medicine, 1 - 1 - 1 Honjo, Kumamoto 860-8556, Japan. E-mail: tanihara@ pearl.ocn.ne.jp

MOLECULAR STRUCTURE OF PROTEOGLY CANS

Proteoglycans are composed of a core protein molecule to which glycosaminoglycans are covalently linked as side chains.'.* The glycosaminoglycans are large carbohydrates that are composed of repeating disaccharide units and occur in four main forms. Based on their glycosaminoglycan side chains, proteoglycans are classi- fied into chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate proteoglycans. The core proteins consist of multiple structurally different domains so that proteoglycans can interact with various biologically active molecules through the glycosaminoglycans and the particular domains of core proteins, which suggests that proteoglycans are multifunctional molecules.3

Thirty or more structurally different core proteins have been isolated to date and characterized from vertebrate tissues. Al- though the distribution and function of proteoglycans had been studied previously, based on the characterization of their glycosaminoglycans, the molecular cloning of proteogl ycan core proteins after the early 1990s has enabled us to classify proteoglycans into subfamilies, such as the lectican and syndecan families, on the basis of the structure of the core proteins and to evaluate the molecular function of the core proteins. Many proteoglycans are secreted from cells and then oriented in the extracellular space. Others exist in the cell membrane because of the transmembrane domain or the glycosylphosphatidylinositol (GPI) domain of the core proteins. It is considered that membrane-bound proteoglycans serve as receptors for growth factors and transmit signals to intra- cellular moIecu1es.4~

INTRAOCULAR PROTEOGLYCANS AND THEIR FEATURES

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-

S62 DOI: 10.1097/01 .IC0.0000028325.06702.47

PROTEOGLYCANS IN THE EYE S63

receptor matrix (IPM). Thus, interestingly, the variation and characterization of the proteoglycan core proteins as well as glycosaminoglycans in retinal tissues appear to be different from those in non-neuronal ocular tissues, including the cornea. In this article, we review the roles of proteoglycans in the eye.

IDENTIFICATION OF CORNEAL PROTEOGLYCANS

Corneal stroma is a rich source of keratan sulfate and chondroitiddermatan sulfate proteoglycans.8 It is believed that they are essential for the maintenance of corneal transparency. Cuprolinic blue staining and subsequent electron microscopic studies have shown that the distribution of proteoglycans is associated with collagen fibers in the corneal stroma? Moreover, the changed expression and the disarranged distribution of these proteoglycans and collagen fibers observed during corneal wound healing indicates the possibility of an interaction between the proteoglycans and the collagens.lns” Since the early 1990s, the core proteins of the proteoglycans in the corneal tissues have been success ive ly iden t i f i ed . Lumican ,12 kera tocan ,13 mimecad~steoglycin,~~ and fibromodulin” are the major keratan sulfate proteoglycans of the corneal stroma, and decorin and biglycan are the major chondroitiddermatan sulfate proteoglycans.16 During corneal development, secretion of keratan sulfate proteoglycans occurs soon after migrating neural crest cells enter the primary corneal stroma.I7 In our immunohistochemical studies, the expression of chondroitin sulfate was found in stroma and was upregulated at early postnatal stages and reduced during development in rat eyes (unpublished data). All the major proteoglycans in the corneal stroma belong to the family of the small leucine-rich proteoglycans (SLRPs).’* The SLRPs bind to fibrillar collagens including I, 11, 111, V, VI, and XIV.I9 It is thought that corneal transparency is dependent on the arrangement of collagen fibrils via binding to SLRPs within the corneal stroma.20

INVOLVEMENT OF PROTEOGLYCANS IN CORNEAL TRANSPARENCY

There is much evidence that abnormal accumulation or decreased expression of SLRPs induces corneal opacity. For ex- ample, lumican-deficient mice display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome.2’ In addition, the mutant mice develop bilateral corneal opacification where 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 decreased refraction, is caused by a homozygous missense mutation of keratocan gene (KERA) in 12q.24 It has also been reported that overexpressed keratocan is observed in the corneal stroma of keratoconus patients, which indicates that the overexpression may alter the fibrillogenesis in the stroma and lead to the development of keratoconus.2s On the other hand, decorin- or biglycan-deficient mice do not show corneal opacity, although the deficiency induces abnormal collagen-fiber network, which results in a disorder similar to Ehlers-Danlos syndrome in decorin-deficient mice and reduced bone mass in biglycan-deficient The disarrange- ment of collagen network induced by the deficiency of decorin or biglycan in the corneal stroma may be compensated by the expres-

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

CORNEAL PROTEOGLYCANS AND WOUND HEALING

In general, proteoglycans play important roles in regulation of cellular adhesion, proliferation, and migration. Because these basic cellular behaviors play a part in wound healing, it has been rehy- pothesized that corneal proteoglycans are involved in corneal wound healing. In corneal wound healing, altered expression of keratan sulfate and chondroitin sulfate proteoglycans,” as well as so-called large proteoglycans, have been shown by a series of animal experiments.3o.” From the viewpoint of proteoglycan core proteins, increased production of perlecan and reduced expression of lumican are shown in corneal scar tissues.32 This phenomenon may be explained by the changed character of the keratocytes recruited to the wounded regions. Also, it is likely that the alter- ation in proteoglycan expression results in corneal epithelial adhesion and migration to the wounded region, because delayed corneal epithelial wound healing is shown in ‘lumican-deficient mice.’3

CYTOKINES AND CORNEAL PROTEOGLYCANS

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 and development processes. Previous studies reported that, when removed from the stroma, keratocytes rapidly lose the ability to secrete keratan ~ulfate.”~’~ The presence of fibroblast growth factor-2, however, supports keratan sulfate proteoglycan (lumican, minecan, and keratocan) synthesis by induction of core-protein biosynthesis.3s Further studies are requikd to elucidate the interaction of the SLRPs with cytokines in corneal wound healing.

PROTEOGLYCANS IN THE TRABECULAR MESHWORK AND THEIR CLINICAL RELEVANCE

Trabecular meshwork cells secrete not only collagens or fibro- nectin but also secrete or express chondroitin/dermatan sulfate and heparan sulfate p ro teog ly~ans ,3~~~ which suggests that proteoglycans are important contributors to aqueous outflow resistance in the juxtacanalicular connective tissue.43 Tawara et al. demonstrated that exfoliation materials in eyes with pseudoexfoliation syndrome contain chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans.44 Moreover, Sawaguchi et al. reported that the injection of chondroitinase ABC into the anterior cham-

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S64 H. TANIHARA ET AL.

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 rep~r ted .~"~ '~) 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.

IDENTIFICATION OF RETINAL PROTEOGLYCANS IN 1980s AND EARLY 1990s

In the 1980s, researchers extracted proteoglycans from retinal tissues by rinsing retinal tissues with physiologically balanced saline and guanidine HCI. Chondroitin sulfate proteoglycans are extracted mainly from the extracellular matrices of the retinal tissues, whereas heparan sulfate proteoglycans exist either in the basal lamina or the cell membrane.''-'x The results indicate that chondroitin sulfate proteoglycans participate in the construction of the organized extracellular matrix in the neural retina, and heparan sulfate proteoglycans constitute a part of basal membranes as extracellular matrix and bind the cell membrane through the transmembrane domain or GPI domain. In the early 1990s, the core protein genes of proteoglycans expressed in the central nervous tissues were isolated and ~haracterized.~-' It has been demonstrated that proteoglycans in the central nervous system tissues have unique structures of core proteins that are different from those expressed in non-neural tissues.

TWO MAJOR NERVOUS TISSUE-SPECIFIC PROTEOGLYCANS: NEUROCAN

AND PHOSPHACAN

We have studied 2 soluble chondroitin sulfate proteoglycans, neurocan and phosphacan, because they are exclusively expressed in developing rat brain as the major components of chondroitin sulfate proteoglycan~.~~ It is believed that neurocan and phosphacan regulate neurite outgrowth during the development of the central nervous system via binding adhesion molecules in the membrane of neuronal cells such as Ng-CAML1 and axonin-I/TAG- I ."O

Our immunohistochemical analyses showed that neurocan and phosphacan are highly expressed in nerve fiber-rich layers, such as the nerve fiber layer (NFL), inner plexiform layer (IPL), and outer plexiform layer (OPL) in postnatal rat retina, whereas the expression levels in the embryonal rat retinas are faint but distributed throughout the retina (Figs. 1 and 2).61,h2 As retinal development is completed, the immunoreactivity is decreased. Moreover, the

FIG. 1. lmmunohistochemistry for neurocan during retinal develop- rnert6' The confocal images were stained with an antineurocan antibody, MAb 1 G2. 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 1 999;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."' Interest- ingly, 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

Coriteo. V d . 21, Siippl. 2, 2002


proteoglycan form of phosphacan appears to be an interesting fea- ture 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 effec- tively than phosphacan bearing chondroitin sulfate.

FIG. 2. lmmunohistochemistry for phosphacan during retinal devel- oprnent.62 The confocal images were stained with an antiphospha- can, MAb 684. A: Retinal sections during development (E16-P42) were used for immunohistochemistry. Micrographs show sections stained with hematoxylin eosin (HE). B: lmmunohistochemical im- age 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 choroidal tissue 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 isch- emia65 The bands of neurocan were barely detected in the control, but then the intensity of immunopositive bands of 220 kDa (full- length 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 po- sitions of molecular mass markers are indicated in kDa. Reproduced with permission of the Association for Research in Vision and Oph- thalmology from lnatani M, et al. lnvest Ophthalmol Vis Sci2000;41: 2751 .65

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INVOLVEMENT OF CHONDROITIN SULFATE PROTEOGLY CANS IN RETINAL DISEASES

Until now, there have been no reports about the direct relation- ship between abnormal expression of soluble chondroitin sulfate proteoglycans such as neurocan and phosphacan and retinal diseases. Versican, a member of the lectican family including neurocan, is also highly expressed in the NFLs during retinal development.7' Perveen et al. reported that Wagner syndrome, an autoso- ma1 dominant vitreoretinopathy characterized by chorioretinal atrophy, cataract, and retinal detachment, is linked to 5q14.3, where the gene encoding versican lies?2 It is possible that the versican gene may be a candidate for Wagner syndrome.

Fukuchi et al. demonstrated that small chondroitiddermatan sulfate proteoglycans, which are cuprolinic blue-positive, are associated with collagen fibers within the lamina cribrosa, and that accumulation and enlargement of collagen-associated proteoglycan filaments are seen in the lamina cribrosa of monkey glaucomatous model eyes, accompanied by the destruction of collagenous beams.73 The SLRPs such as decorin are bound to collagens in the extracellular matrix of tissues.74 Their data may reflect upregula- tion of SLRPs after retinal injury. It also has been reported that the expression of decorin is induced by mechanical injury to brain tissues.75 Thus, we examined the expression of decorin after transient retinal when the inner retinal layers, such as the NFL, ganglion cell layer, and IPL, are damaged. Our immunohistochemical results using anti-glial fibrillary acidic protein and anti- s-loop antibodies, which are glial cell markers, indicated that glial cells proliferate in the damaged inner retinal layers. At 7 days after retinal reperfusion, decorin immunoreactivities are upregulated in the inner retinal layers. Furthermore, in similar transient retinal ischemia models, the upregulated expression of numerous cytokines, such as interleukin-1 and TGF-P, was n~ted.'~.~' These cytokines have been shown to upregulate numerous constituents of extracellular matrices, including d e ~ o r i n . ~ ~ . ' ~ Decorin may thus be upregulated by cellular response to secreted cytokines in retinas undergoing reperfusion and may be involved in the repair process in injured neural tissues.

Recently, it has been reported that nyctalopin, a GPI-anchored membrane-bound proteoglycan that is a member of the SLRP family, is expressed in tissues including retina, brain, testis, muscle, and kidney.80*8' In situ hybridization analyses indicate expression in photoreceptor cells, retinal ganglion cells, and the cells in the inner nuclear layer." Interestingly, the gene mutation in patients with X-linked congenital stationary night-blindness was found in a candidate gene, NYX, which encodes a nyctalopin.80*8' The point mutation present in patients from 2 large pedigrees is predicted to eliminate the portion of nyctalopin required for establishing the GPI anchor and potentially results in the protein being soluble rather than membrane-tethered."

THE ROLE OF CHONDROITIN SULFATE PROTEOGLYCANS IN THE IPM

It is known that the IPM and the apical surface of retinal pigment epithelium (RPE) cells are stained by cuprolinic blue." Be- cause the staining is eliminated by treatment with chondroitinase ABC, it is thought that chondroitin sulfate proteoglycans are abun- dantly present in the IPM and on the apical surface of RPE celks3

After intravi t real inject ions of p-nitropheny1-beta-D- xylopyranoside (xyloside), a sugar that inhibits chondroitin sulfate proteoglycan synthesis, cone photoreceptor cell outer segment de- generation 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 character i~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 hyaluronan- binding 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 over- lap.95*96 Further analyses for 6q-linked retinopathies will be required to identify whether abnormal gene expression of SPACMMPG 1 induces genetic cone-rod macular dystrophies.

THE INTERACTION OF PROTEOGLYCANS WITH HYALURONAN DURING

RETINAL DEVELOPMENT

There have been many reports suggesting that the interaction of hyaluronan-binding proteins with hyaluronan may be essential to the formation and maintenance of the photoreceptor cells. Now, we are focusing on the distribution of neuroglycan C during retinal development. In postnatal rat retinas, the distribution is in the NFL, IPL, and OPL. not in the IPM.92 Our immunoeleclron microscopic analysis showed that the immunoreactivity is localized on the cell membrane of the neurites of retinal neuronal cells. Additionally, immunohistochemical analysis on cultivated retinal ganglion cells derived from postnatal rat eyes showed that neuroglycan C is highly localized in spiny neurites budding from long extended neurites of the retinal ganglion cells. Bremer and Rasquin demonstrated that hyaluronan is transiently detectable in the inner layers of retinas during retinal development using a hyaluronan-binding protein as a probe for hya l~ronan?~ In general, hyaluronan is highly expressed during the development of mammalian tissues including the central nervous tissues.98 It is possible that hyaluronan in the developing neural retina may regulate the outgrowth of neurites via binding to neuroglycan C in the cell membrane of neurites. Interestingly, it has been reported that hyaluronan is con- centrated in the perineuronal nets and the node of Ranvier in the brain t i ~ s u e s ? ~ Penneural nets are reticular networks present on

Cornea. Vol. 21. Suppt. 2, 2002


the surface of cell bodies and proximal dendrites of certain popu- lation of neurons. loo The reticular network represents extracellular matrices surrounding the synapse regions. It is thought that perineural nets act as a cell-surface barrier that blocks the formation of new synaptic contacts by incoming axons.'oI It is known that neurocan is also distributed in the perineural nets."' Since neurocan, which contains the hyaluronan-binding domain in the core p r~ te in , "~ is transiently expressed in the NFLs during retinal development,6' it is likely that the complex of neurocan and hyaluronan may be involved in the network formation through the binding of neurocan to cell adhesion molecules on the neurites from the retinal neuronal cells. We will continue to perform further studies on the interaction of hyaluronan with proteoglycans and their roles in retinal neural network formation.

SIGNIFICANCE OF INVESTIGATING PROTEOGLYCANS IN THE EYE

As described in this review, ocular proteoglycans participate in important regulatory mechanisms to maintain normal function and structure of the eye from the cornea to the retina. Recent devel- opments in molecular and cell biologic studies have enabled us to identify molecular structures and functions of proteoglycans. Based on the knowledge of molecular mechanisms of ocular proteoglycans, further studies will show us the detailed information of pathologic phenomena in the eye and shed new light on our un- derstanding of numerous ocular diseases.

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Proteoglycans in the Eye

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