Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Normand, G.
Right arrow Articles by Dreyfus, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Normand, G.
Right arrow Articles by Dreyfus, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Glycobiology Pages 1227-1235  

Neurotrophic growth factors stimulate glycosaminoglycan synthesis in identified retinal cell populations in vitro
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References

Neurotrophic growth factors stimulate glycosaminoglycan synthesis in identified retinal cell populations in vitro

Neurotrophic growth factors stimulate glycosaminoglycan synthesis in identified retinal cell populations in vitro

Guy Normand, David Hicks and Henri Dreyfus1

Laboratoire de Physiopathologie Rétinienne, Clinique Ophtalmologique, CHRU, 1 Place de l'Hôpital, BP 426, 67091 Strasbourg Cedex, France

Received on April 8, 1998; revised on May 4, 1998; accepted on May 4, 1998

Glycosaminoglycans (GAG) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth, but little is known with respect to their regulation through soluble neurotrophic factors. In the present study, we have addressed this issue using cell culture models of three distinct cell populations derived from young rat retinas, namely, purified Müller glia, pigmented epithelium, and neurons respectively. Cultures were maintained in chemically defined media in the presence or absence of either basic fibroblast or epidermal growth factor. In control glial and epithelial cultures, hyaluronic acid dominated the soluble GAG pool, with lesser contributions from dermatan sulfate, chondroitin sulfate, and heparan sulfate (in decreasing order). Retinal neuronal GAG were almost exclusively chondroitin sulfate (~90%). Treatment of glial and epithelial cultures with either factor led to dose-dependent increases in especially hyaluronic acid synthesis (a maximum 6-fold increase relative to control levels), with smaller but consistent changes in chondroitin sulfate. Similar treatment of retinal neurons did not lead to any changes in GAG synthesis. These data indicate that glia and pigment epithelia are the principal sources of GAG components in retina at least in vitro, and that endogenous neurotrophic growth factors can greatly modify GAG synthesis in these two retinal cell populations. Such data suggest that a delicate balance may exist between growth factor availability and glycoconjugate metabolism in vivo, participating in normal or pathological states of the retina.

Key words: retina/basic fibroblast growth factor/epidermal growth factor/glycosaminoglycans/cell culture

Introduction

The current concept of the role of extracellular matrix (ECM) components in the development and maintenance of central nervous system (CNS) tissue has evolved from them playing a passive structural function to a much more dynamic participation in control of numerous physiological functions (reviewed in Reichardt and Tomaselli, 1991). In addition to the glycoproteins present within the ECM, another heterogeneous group of molecules has received increasing attention. These are the proteoglycans (PG) consisting of protein cores to which are attached a variable number of glycosaminoglycan (GAG) side chains. It has become apparent that different forms of PG play important roles in CNS development and repair (Toole, 1976; Herndon and Lander, 1990), exhibiting both stimulatory (Hantaz-Ambroise et al., 1987; Iijima et al., 1991) or inhibitory (Snow et al., 1990; Oohira et al., 1991) influences on neurite outgrowth. A fundamental property of the heparan sulfate (HS) PG is their ability to bind potent neurotrophic growth factors such as basic fibroblast growth factor (bFGF) (Baird, 1994) and transforming growth factor-[beta], where they may act as important regulators of growth factor delivery to target cells (Ruoslahti and Yamaguchi, 1991; Schlessinger et al., 1995).

The vertebrate retina derives from the CNS early in embryogenesis, and following a series of morphogenetic movements eventually forms a folded, laminated tissue bordering the back of the eye. The neural retina proper is closely apposed to the retinal pigmented epithelium (RPE), a monolayer intimately involved in controlling retinal development and turnover (Barnstable, 1990; Raymond and Jackson, 1995). The neural retina itself is composed of relatively few glial and neuronal cell types: the former are formed principally by the Müller glia (RMG) and the latter by first order (photoreceptors), second order (horizontal, bipolar and amacrine cells), and third order (ganglion cells) neurons. Because of its experimental advantages, the retina has been a tissue of choice for studying CNS development and differentiation. Among the aspects studied, the crucial role of neurotrophic factors such as bFGF and epidermal growth factor (EGF) in controlling retinal cell fate, differentiation, phenotype, and survival has been well documented (Anchan et al., 1991; Hicks and Courtois, 1992; Steinberg, 1994; Heidinger et al., 1997). Given the possibility of maintaining distinct retinal cell populations in defined monolayer culture conditions, we were interested in examining GAG metabolism in the absence and presence of exogenous neurotrophic factors. We show here that two non-neuronal elements of the retina, the RPE and RMG, constitute the major sources of retinal GAG, that the majority of this material is formed by nonsulfated hyaluronic acid (HA) but with large amounts of sulfated chondroitin sulfate (CS), and that their metabolism is drastically modified by growth factor treatment.


Figure 1. Phase contrast micrographs of living retinal cell cultures. (a) Second passaged Wistar rat RMG cells. Confluent cells are closely packed and epithelioid-like. (b) First passaged Wistar rat RPE cells. Cells are a mixture of regular and irregular polygons in shape, with flattened peripheries and a prominent nucleus (often binucleate). Cells lack melanin granules in this albino strain. (c) Primary cultures of Wistar rat RN cells. Neurons are present as small islands or dispersed cells interconnected by neurites. Under the conditions used, glial proliferation is limited by the use of chemically defined media and mitotic inhibitors. Scale bar, 30 µm for all panels.

Results

Tissue culture models

Light microscopic and immunocytochemical observations of living and fixed cultures were used to monitor cell morphology and purity of each system. Primary cultures of rat RPE appeared as regular, polygonal cells with a flattened perimeter and prominent nucleus (Figure 1a). These cells were devoid of melanin granules as they originated from albino rats. Based on their morphology and staining with anti-cytokeratin antibodies (unpublished observations), these cultures were >98% RPE. Second passaged cultures of RMG were composed of epithelioid-like or fusiform cells, and again based on immunocytochemical criteria these cultures were 95% pure, with minor contamination from microglia, vascular endothelium, and neurons (<1% each) (Figure 1b). Primary cultures of retinal neurons (RN) were composed of isolated or small islands of rounded neurons interconnected with neuritic processes (Figure 1c). As judged by immunolabeling using glial cell specific antibodies, flattened glial cells accounted for <10% total cell population, even following growth factor treatment (unpublished observations).

Addition of the mitogenic factors bFGF and EGF led to cell number increases in RMG cultures after 48 h: control cultures contained 6.5 × 106 cells, bFGF-treated cultures contained9.3 × 106 cells, and EGF-treated cultures contained 11.2 × 106 cells/100 mm dish, or increases of approximately 40 and 70% following bFGF and EGF addition, respectively.

Glycosaminoglycan synthesis by control cell cultures

Analysis of [3H]-glucosamine uptake into medium (secreted) and cell layer-associated compartments revealed that in both RPE and RMG cultures, the vast majority of total GAG synthetic activity (86-93%) was recovered in the medium (Table I). Of this total secreted GAG content, HA alone represented 48% and 65% of the RMG and RPE pools respectively, while the sulfated GAG were composed mainly of DS (29 and 18%) and CS (19 and 14%) with low amounts of HS (4 and 3%) (Figure 2). Of the small cell associated fraction, HA represented 15 and 45% of RMG and RPE GAG, respectively, while the various sulfated GAG contributed either similar (DS: 31 and 16%, and CS: 27 and 20%) or greater (HS: 27 and 19%) amounts (Figure 2).

Compared to the previously described two cell culture models, primary cultures of RN synthesized relatively small amounts of GAG, about a quarter as much (Table I). However, this synthesis was approximately equally distributed between the medium and cell layer associated fractions. In both compartments, total GAG were present predominantly as CS (>90%) with only equal trace amounts of the other nonsulfated and sulfated species (Fig.2).

Growth factor effects on glycosaminoglycan synthesis

Addition of either bFGF or EGF to the culture medium of RMG or RPE cells led to profound effects on GAG synthesis. Inclusion of increasing concentrations of bFGF or EGF to cultured RMG led to increased synthesis of total GAG, especially within the medium, with increases approaching a plateau of 150% at 10 nM EGF (Table I). Synthetic activity was nearly two fold basal levels at the highest dose of bFGF used. Increases in the cell layer associated fraction were smaller for both growth factors. Single concentrations of either bFGF or EGF were tested on cultured RPE or RN. Again large increases were observed in RPE medium, with 1 nM EGF leading to similar effects as observed for RMG cells, and bFGF treatment producing dramatic 5-fold rises (Table I). On the contrary, bFGF did not elicit significant changes in GAG metabolism within RN cultures, independent of the fraction analyzed. EGF was not tested on RN.

Stimulation of individual GAG classes varied according to the specific class, cell type and growth factor in question. Within RMG cultures, the most dramatic stimulation was observed in the HA content of the medium, increasing a maximum of 3-fold with addition of either factor (Figures 3 and 4, Table II). This stimulation approached saturation at 10 nM with an ED50 ~200 pM, and for bFGF synthesis increased roughly exponentially between 30 and 3000 pM. Higher concentrations of bFGF were not tested. For the sulfated GAG, EGF was more potent than bFGF in stimulating synthesis. EFG-induced CS synthetic activity saturated at <1 nM within the secreted compartment with 2-fold rises. Basic FGF only affected CS synthesis at the highest dose used, 3 nM, the synthetic activity increasing by 100% (Figures 3 and 4, Table II). DS was much less affected by bFGF or EGF treatment, showing maximal 30% increases (Figures 3 and 4, Table II). The trace levels of HS followed profiles similar to those of CS, being stimulated more readily by EGF than bFGF. Growth factor stimulation of radiolabel incorporation into the cell layer associated fraction was very small compared to the soluble fraction, around 1% of maximal incorporation compared to the latter, and changes were not considered as significant. HA increases were small, consistent with its soluble nature. For EGF there were slight effects on all the GAG species within the cell layer associated compartment, with maximal increases of 50-100%, while bFGF did not affect any activity (unpublished observations).

For RPE cells, addition of single doses of 1 nM EGF or 0.6nM bFGF led to three and 5-fold increases respectively in HA levels within the medium (Figure 5, Table III; a maximum of 12-fold relative to control levels for bFGF in one experiment). As for RMG cells, EGF was more effective than bFGF in stimulating synthesis of sulfated GAG, increasing levels by 350%, 300%, and 200% for CS, HS, and DS, respectively (Figure 5a, Table III). Basic FGF stimulated only CS production (150%). In contrast to these pronounced effects on the medium, there were only minimal changes in the majority of GAG located within the cell layer associated fraction. HA seemed to remain more tightly cell associated than for RMG, and was enhanced by EGF (Table III). As observed above, bFGF did not significantly stimulate activities of any GAG in RN cultures (Figure 6, Table III). In all cases, GAG quantification was adjusted for cellular protein levels to compensate for bFGF- and EGF-induced proliferation. Although total radiolabel incorporation varied by 2- to 3-fold between different experiments, relative values of GAG synthesis by the different cell types and with the different growth factors were very reproducible.


Figure 2. Cumulative bar histogram showing percentage distribution of different GAG species in control retinal cell cultures and recovered from the medium or cell layer associated fractions. It can be seen that HA is the major GAG in RMG and RPE media, but a smaller proportion in cell-associated fractions. CS and DS are present in moderate amounts in all fractions, whereas HS is mostly restricted to the cell-associated pool. RN contains almost entirely CS in both compartments. It should be noted that total cell layer associated GAG content was 10-20 times less than in the media.


Figure 3. EGF stimulation of GAG synthesis within the secreted fraction (medium) of RMG cells. HA synthesis shows dose-dependent stimulation, approaching saturation at 10 nM EGF with 4-fold control levels. CS is also stimulated in a dose-dependent manner, as are the trace amounts of HS.


Figure 4. Basic FGF stimulation of GAG synthesis within the secreted fraction of RMG cells. HA synthesis shows dose-dependent stimulation, still rising at 3 nM with 4-fold control levels. CS is upregulated by the highest concentration used, but the other GAG are unaffected.

Comparison of glucosamine and sulfate incorporation

In one series of experiments, incorporation of simultaneously added glucosamine and sulfate into RMG cultures under control and EGF-treated conditions was examined. As before, the great majority of radiolabel was recovered from the medium, for both precursors: 94% of sulfated GAG in both cases. Sulfate was not taken up by nonsulfated HA, but in terms of glucosamine incorporation this GAG was again the dominant species (40% of total GAG). Addition of 0.75 nM EGF led to large increases in glucosamine levels in HA (~200% increase), and as expected no detectable sulfate (Table IV). The uptake of 3H-glucosamine and 35S into total sulfated GAG was stimulated only slightly by EGF in both soluble and insoluble fractions. However, when individual GAG classes were analyzed a reproducible increase in radiolabel incorporation into both medium and cell layer associated CS (28-53% increase depending on the treatment; Table IV) but not into HS or DS was observed.

Table I. Total GAG synthesis in cultured retinal cells under control or growth factor-supplemented conditions
Treatment Retinal Müller glia Retinal pigmented epithelium Retinal neurons
c.p.m.a % Increaseb c.p.m. % increase c.p.m % Increase
Control, Total GAG in medium 54,400
(92.8)		 - 56,700
(85.5)		 - 6200
(42.8)		 -
Control, Total GAG in cells 4200
(7.2)		 - 9600
(14.5)		 - 8300
(57.2)		 -
EGF, Total GAG in medium 136,600
(97.5)		 150 238,000
(92.7)		 320 nd -
EGF, Total GAG in cells 3500
(2.5)		 0 18,800
(7.3)		 100 nd -
bFGF, Total GAG in medium 146,400
(94.6)		 170 253,400
(97.4)		 350 6500
(50.1)		 5
bFGF, Total GAG in cells 8400
(5.4)		 100 6700
(2.6)		 0 6400
(49.9)		 0
Activities are expressed as c.p.m./mg cellular protein, and it can be seen that the large majority of glucosamine is recovered in the medium for RMG and RPE (% total GAG levels shown in parentheses). Both EGF (1 nM) and bFGF (0.6 nM) lead to large increases in secreted GAG levels in RMG (and especially RPE) cells, whereas bFGF does not stimulate such activity in neurons (RN).
acpm/mg protein.
b% increase vs. control.

Table II. Growth factor stimulation of individual GAG classes in the medium of RMG cells
Treatment Hyaluronic acid Heparan sulfate Chondroitin sulfate Dermatan sulfate
c.p.ma % Increaseb c.p.m. % Increase c.p.m. % Increase c.p.m. % Increase
Control EGF 21,600 - 2900 - 9300 - 21,300 -
EGF 0.01 nM 24,900 15 2200 0 6800 0 13,600 0
EGF 0.1 nM 37,300 73 3200 10 13,200 42 25,000 17
EGF 1 nM 67,300 212 5900 103 26,100 181 25,800 21
EGF 10 nM 80,100 271 5600 93 22,800 145 28,100 32
Control bFGF 29,600 - 2400 - 11,100 - 10,100 -
bFGF 0.03 35,900 21 2100 0 9700 0 9100 0
bFGF 0.3 nM 71,200 141 2300 0 11,700 5 8600 0
bFGF 3 nM 107,100 262 2900 21 22,700 105 13,700 36
In two separate experiments, dose-response effects of EGF and bFGF on GAG synthesis were examined. Data are expressed as c.p.m./mg cellular protein, as well as % increase relative to untreated controls. It can be seen that EGF is a generally more effective inducer than bFGF, although both possess similar potency towards HA synthesis.
acpm/mg protein.
b% increase vs. control.

Pertubation of glycosaminoglycan metabolism and RMG migration

Cultured RMG cells under control conditions, or those treated with buffer alone, grew back into tracks denuded by scraping at an approximately equal rate over the 3 day period examined. Pretreatment of control cultures with testicular hyaluronidase increased such movement, leading to more rapid closure of the wound track. Prior boiling of testicular hyaluronidase abolished the effects on glial migration. In contrast, addition of Streptomyces hyaluronidase to RMG did not affect their migration. Treatment with chondroitinase AC slightly increased the rate of migration of RMG, but the difference was not significant. Treatment with HA or CS alone had no effect upon migration (Figure 7).

Discussion

The present study demonstrates that different mammalian retinal cell types in vitro synthesize different types of glycosaminoglycan (GAG), which were predominantly nonsulfated hyaluronic acid (HA) in the soluble fractions of retinal Müller glia and pigmented epithelium (RMG and RPE respectively), and almost entirely chondroitin sulfate (CS) in enriched retinal neuronal (RN) cultures. Addition of either of two polypeptide neurotrophic factors, basic fibroblast and epidermal growth factor (bFGF and EGF, respectively) led to a large stimulation in GAG synthesis by RMG and RPE cells, especially in HA. There was also a reproducible increase in CS but not HS or DS among the sulfated GAG species. In functional assays, only treatment with testicular hyaluronidase modified significantly glial movement. HA was initially thought to simply provide an environment propitious for cellular migration through modifying the hydration state of the extracellular matrix (ECM). Indeed, its levels in the CNS decrease as maturation proceeds, correlating with the decrease in cell movement observed at the end of embryogenesis (Margolis et al., 1975; Toole, 1976; Normand et al., 1985). However, the recent discovery of HA-binding proteins and receptors within distinct regions of the developing and adult central nervous system (CNS) (Jaworski et al., 1994, 1995), and the functional consequences of perturbing HA regulation on neurite outgrowth (Nagy et al., 1995), has led to the belief that they have a more direct role in CNS growth. Within the eye, both the vitreous and the interphotoreceptor matrix (IPM) are reported to contain HA (Meyer and Palmer, 1934; Edwards, 1982; Kaneko, 1987; Hageman and Johnson, 1990), and in the vitreous this GAG is though to be important in maintaining viscoelastic properties (Meyer and Palmer, 1934). RPE have been shown to constitute a major source of CS production in vivo (Tawara et al., 1989), and so represent the main site of subretinal GAG synthesis, whereas RMG may secrete GAG through their endfeet into the vitreous. In the present study, the recovery of most HA activity in the medium of RPE and RMG cell cultures implies it would be secreted principally by these cell types into the appropriate extracellular compartments in vivo, the IPM, choroid and vitreous. RPE show the most HA-dominated profile (~70% total GAG), indicating that this GAG may have important functions in retinal metabolism. The presence of CD44 or Hermes antigen, one of a family of HA-binding proteins (Aruffo et al., 1990), on the apical processes of RMG present within the IPM in vivo (Chaitin et al., 1994) and on RMG cell bodies during development (Chaitin et al., 1996) indicate that these cells may constitute a target for this molecule.

Table III. Growth factor stimulation of individual GAG classes in RPE and RN cells
Treatment Hyaluronic acid Heparan sulfate Chondroitin sulfate Dermatan sulfate
c.p.m.a % Increaseb c.p.m. % Increase c.p.m. % Increase c.p.m. % Increase
RPE, Control, Medium 37,100 - 1800 - 8000 - 9900 -
RPE, Control, Cells 4100 - 1800 - 2000 - 1700 -
RPE, EGF, Medium 163,700 341 6700 272 37,200 365 30,500 208
RPE, EGF, Cells 11,900 190 2500 39 2700 35 1800 6
RPE, bFGF, Medium 223,500 502 2000 11 19,200 140 8700 0
RPE, bFGF, Cells 2100 0 1400 0 2300 15 1000 0
RN, Control, Medium 100 - 150 - 5700 - 250 -
RN, Control, Cells 0 - 200 - 7900 - 250 -
RN, bFGF, Medium 100 0 150 0 6000 5 250 0
RN, bFGF, Cells 0 0 150 0 6000 0 250 0
Averaged data of three separate experiments for RPE, two for RN. As for RMG cells, EGF is a more effective stimulator than bFGF, although the latter is a very potent stimulator of HA production in RPE cells. It can also be seen that changes in the cell layer associated compartment are much less dramatic except for EGF-induced increases in HA.
acpm/mg protein.
b% increase vs. control.

Table IV. Comparison of glucosamine and 35S uptake into RMG cells stimulated with EGF
Treatment 3H-HA (% diff. vs. control) Total 3H-GAG 3H-HS 3H-CS 3H-DS Total 35S-GAG 35S-HS 35S-CS 35S-DS
Control, Medium 651,000 963,600 110,000 349,400 504,300 1,812,300 223,400 636,100 952,800
EGF, Medium 2,006,800 1,100,700 90,300 533,700 476,600 1,776,000 160,400 817,200 798,400
(+208) (+14) (-18) (+53) (-5) (-2) (-28) (+28) (-16)
Control, Cells 21,500 62,300 27,500 17,200 17,500 112,700 54,500 27,400 30,800
EGF, Cells 66,900 72,000 31,300 25,200 15,500 121,700 59,900 36,300 25,400
(+211) (+16) (+14) (+47) (-11) (+8) (+10) (+32) (-18)
In this experiment, [3H]-glucosamine and [35SO4] were added simultaneously to cultured RMG either alone or together with EGF. As seen before, EGF stimulates HA production (detectable only for [3H]-glucosamine incorporation), whereas stimulation of total sulfated GAG is weak. However, analysis of individual sulfated GAG shows that uptake of both glucosamine and sulfate into CS was reproducibly stimulated by 30-50% into both medium and cell associated compartments, while HS and DS were not affected. Data for all treatments are expressed as c.p.m./mg cellular protein. % EGF-induced increases or decreases relative to untreated controls are given in parentheses.


Figure 5. Growth factor stimulation of GAG synthesis within the secreted fraction of RPE cells. The major effect of both EGF and bFGF is to stimulate HA production, by 4- and 6-fold control levels, respectively. EGF also leads to rises in sulfated GAG levels, and bFGF stimulates CS alone.


Figure 6. Basic FGF stimulation of GAG stimulation within the secreted fraction of RN cells. Addition of bFGF does not stimulate any activities.

The different culture models expressed different GAG profiles: RPE were dominated by HA with lesser amounts of CS and DS and trace amounts of HS; RMG showed moderate levels of HA, CS, and DS and again only trace amounts of HS; and RN were very different with virtually nothing but CS. These data contrast with those reported previously for chicken RMG (Threlkeld et al., 1989) and RN (Needham et al., 1988) in vitro, in which HS was the major GAG recovered. However, such differences between mammalian and avian species have been described for other systems. Previous studies on sulfated GAG production by cultured feline RPE also differ from our data in describing HS as the major species (Stramm et al., 1989). Our data resemble more those obtained for human RPE, although in these cultures there was much less HA (Edwards, 1982). Part of the reasons for these differences may be attributed to the different analytical techniques employed in these other studies, such as use of radiolabeled sulfate only, differing identification and recuperation of fractions, sampling at relatively late times, and use of different species and culture methods. Although by our assay techniques HS form only a minor fraction of newly synthesized GAG, they probably play an important role in retinal cellular physiology through their ability to participate in neuronal adhesion (Hantaz-Ambroise et al., 1987; Riopelle and Dow, 1990) and the heparin-binding growth factor signaling pathway (Schlessinger et al., 1995). CS production by mammalian retina and photoreceptor cells (of which our RN cultures contain ~40%, unpublished observations) has also been reported previously (Landers and Hollyfield, 1992), and this GAG is a major structural and functional component of the insoluble cone matrix sheath (Hageman and Johnson, 1987, 1990).


Figure 7. Specific modification of RMG migration by testicular hyaluronidase. RMG movement into a denuded track was measured every 24 h over the space of 72 h, and the histogram represents the distance separating the track borders at 0 (first bar in each series), 24 (second bar in each series), 48 (third bar in each series), and 72 h (fourth bar in each series). Original track width is defined as 100%. Testicular hyaluronidase accelerated cell migration relative to control cultures or those containing buffer alone, and this effect was abolished by prior boiling of the enzyme. However, Streptomyces hyaluronidase had no effect, and chondroitinase AC a slight effect, indicating that it is actually accessory digestion of CS by the testicular enzyme which is responsible for the increased migration. Addition of GAG alone did not modify the response. Abbreviations: H'ase Test, testicular hyaluronidase; H'ase T 100, Pre-boiled testicular hyaluronidase; H'ase Strep, Streptomyces hyaluronidase; C'ase AC, chondroitinase AC. Other abbreviations as in text.

To investigate the possible roles of GAG in retinal cell behavior more directly, we examined the effects of altered GAG metabolism on glial migration. Addition of exogenous HA or CS did not modify RMG migration (which is understandable given the high intrinsic content in the cultures). Digestion of HA by testicular hyaluronidase led to an acceleration in cell migration. However, no effect was observed when experiments were repeated using hyaluronidase purified from Streptomyces. As testicular hyuronidase is less specific than the latter form, being contaminated with other glycolytic enzymes including chondroitinases, this indicates it may be digestion of CS (or other enzyme activities) which perturb cell migration.

A number of reports have demonstrated modulation of GAG metabolism by growth factors, especially transforming growth factor-[beta] (e.g., Bassols and Massagué, 1988; Bachem et al., 1989; D'Angelo and Green, 1991) and also bFGF (Munaim et al., 1991). We chose to examine bFGF and EGF in the present study as much data are available on the biological effects they exert on retinal cells (Anchan et al., 1991; Hicks et al., 1991, for review on FGF; Heidinger et al., 1997). They both stimulate greatly HA synthesis in RMG and RPE cells in vitro. This is not due simply to their mitogenic effects, as values were corrected for increased protein levels, and in addition RPE which proliferate more slowly than RMG cells actually synthesize more HA. The lack of HA in RN cultures further indicates the relative purity of this cell preparation, as significant glial contamination would have been revealed by HA synthesis. Interestingly, there is also consistent stimulation of synthesis of CS but not DS. This is remarkable given the similarity of the two biosynthetic pathways, and further indicates that such growth factor influences are not generalized cell responses but rather modifications of specific metabolic activities. In addition, although their effects are largely similar there are certain differences between EGF and bFGF, such as the broader range of EGF and its pronounced stimulation of DS in RPE cells. As we have shown that these two growth factors also stimulate differentially key aspects of glycolipid synthesis in RMG in vitro (Hicks et al., 1996), one general means by which bFGF and EGF may regulate processes such as cell migration, neuronal process outgrowth, and differentiation (Wagner, 1991; Mazzoni and Kenigsberg, 1992; Baird, 1994) could be through modulating glycoconjugate metabolism.

In conclusion, we have demonstrated that defined in vitro cell populations derived from mammalian retina elaborate distinct GAG, and that these activities can be modulated by growth factors known to be synthesized by in vivo retina. As we have analyzed uniquely the GAG chains, it will be of interest to identify the different PG to which these polysaccharide moieties are attached. Indeed it is possible that growth factor-induced changes in specific PG would have been masked by the general pool. Furthermore, cultured RMG should provide a good model for studying cell signaling events triggered by HA receptor activation in neural cells.

Materials and methods

Reagents

Tissue culture media and supplements were purchased from Gibco Life Sciences, except where indicated. Tissue culture plates were purchased from Costar. Enzymes for GAG digestion were from Sigma.

Tissue culture

RMG cells were isolated and cultured according to previously published methods (Hicks and Courtois, 1990). Briefly, eyeballs from one litter (about 10 individuals) of 7-10 day old Wistar rats were removed immediately following anesthesia and decerebration and soaked overnight in Dulbecco's Modified Eagle's Medium (DMEM). The following morning globes were incubated in DMEM containing trypsin (Difco 1:250, 1 mg/ml) and collagenase (type CS-1, Worthington, 20 U/ml) for 1 h at 37°C, and dissected under an operating microscope in DMEM containing 10% fetal calf serum (FCS) (DMEM/10% FCS). The neural retina and RPE were dissected separately from the posterior eye cup and lens, the former being chopped into small fragments and plated in DMEM/10% FCS in 100 mm petri dishes (contents of ~8 retinas per dish). Over the following 4-5 days in vitro, numerous flat cells migrated from the fragments onto the substrate, and at this stage retinal fragments were removed by vigorous washing with DMEM. Cells were refed with fresh DMEM/10% FCS, and grown to confluence. These flat cells, previously identified by immunocytochemical criteria as RMG (Hicks and Courtois, 1990), were used at the second-passage. Cells were subcultured by rinsing twice in phosphate-buffered calcium-free saline (PBS) followed by addition of 1-2 ml 0.05% trypsin in PBS containing 0.1 mM EDTA. Cells rapidly detached from the surface following swirling at which time excess DMEM/10% FCS was added and the suspension collected and centrifuged at low speed. Viability was determined by trypan blue exclusion, and was routinely >98%. RMG were reseeded in DMEM/10% FCS into either 100mm dishes for GAG analyses, or 6 × 35 mm plates for migration studies, at an initial plating density of 104 cells/cm2.

RPE cultures were prepared as previously described for pigmented rats (Edwards, 1981; Malecaze et al., 1993). Care was taken to cleanly separate the transparent RPE sheets from the surrounding neural retina and posterior eyecup. They were collected and washed twice in PBS, digested briefly in 0.05% trypsin (1 min) and triturated gently to obtain a dispersed cell suspension. An equal volume of DMEM/10% FCS was added, the cells sedimented at low speed and resuspended in fresh DMEM/10% FCS. Viability was determined by trypan blue exclusion, and was routinely >95%. Cells were seeded in the same medium into 60 mm tissue culture dishes (contents of eight eyes per dish). Upon reaching confluence the RPE cells were subcultured as outlined above into 100 mm dishes, and were used at a single passage upon growing to confluence.

For cultures enriched in retinal neurons (RN), retinas were isolated from 4 day old Wistar rats and processed as described previously (Hicks and Courtois, 1992; Heidinger et al., 1997). Following enzymatic digestion (0.05% trypsin in PBS/EDTA for 25 min at 37°C) cells were gently triturated to obtain a fully dissociated suspension and were seeded at a density of 105/cm2 into polylysine precoated 100 mm culture dishes in DMEM/10% FCS. After 15 h in serum-supplemented DMEM to allow cell attachment, cells were gently rinsed twice in serum-free DMEM and the medium replaced by CDM as above supplemented with 10 µM cytosine arabinoside. Cultures were allowed to develop for 4 days in vitro prior to growth factor addition.

Growth factors

Newly confluent RMG and RPE cultures were washed twice in serum-free DMEM and refed with chemically defined medium (CDM) (DMEM supplemented with insulin-transferrin-selenium pre-mix, 40 nM progesterone, 200 µM putrescine, 1 mM pyruvate, 10 µg/ml penicillin/streptomycin) containing either recombinant human bFGF (Pharma Biotechnology, Hannover, Germany) (30-3000 pM) or EGF (tissue culture grade, Upstate Biotechnology Inc., Lake Placid, NY) (5-5000 pM). Simultaneously to growth factor addition, [3H]-glucosamine, 2 µCi/ml (ICN Pharmaceuticals Inc., Costa Mesa, CA), was also included in the culture medium. In a separate series of experiments, [3H]-glucosamine (10 µCi/ml) and Na2[35S]O4 (20 µCi/ml) (ICN Pharmaceuticals Inc., Costa Mesa, CA) were added together at the same time as 0.75 nM EGF. After 48 h, the different cultures were harvested as described below. Cultured RN were treated with growth factors for the final 48 h of the in vitro period. Aliquots of bFGF and EGF were stored at -80°C at 1 µg/ml in DMEM/0.1% bovine serum albumin, and their activity was checked by stimulation of incorporation of [3H]-thymidine into target cells (data not shown).

Glycosaminoglycan analysis

Following growth factor and radiolabeled glucosamine or sulfate incubation, the culture medium was carefully decanted off and pooled from each experimental series (defined as 'medium" for all further analyses). Cells were washed three times in PBS, and following the final wash the adherent cells were scraped with a rubber policeman, suspended in ~2 ml PBS, pooled, and centrifuged at low speed. Aliquots of this fraction (defined as the 'cell layer associated" fraction for all further analyses) were taken for determination of protein content (Lowry et al., 1951). The fractions were then treated separately for determination of radioactive content into the different GAG (3H-glucosamine being incorporated into both sulfated and nonsulfated GAG, and 35SO4 being incorporated only into sulfated GAG). They were initially digested with pronase, 0.25 mg/ml for 48 h at 37°C, and then 2 M NaOH was added to a final concentration of 0.1 M and the preparations left overnight. Residual undigested peptides were precipitated by adding 100% trichloroacetic acid to a final concentration of 6% (w/v). Following centrifugation at 3000 × g for 30 min the supernatants were extensively dialyzed against dd.H2O and concentrated by lyophilization. Labeled total GAG together with added cold GAG as carrier were precipitated by adding three volumes of ethanol containing 1.3% potassium acetate, and samples were stored overnight at -20°C. Precipitates were centrifuged at 3000 × g for 45 min and redissolved in dd.H2O.

Putative GAG-containing material was digested with the following enzymes. Samples were suspended in buffer (0.05 M sodium acetate, 0.15 M NaCl, pH 5.0), hyaluronidase (isolated from Streptomyces hyaluroticus, specific activity 500 U/mg) was added to a concentration of 75 U/ml, and samples were incubated at 37°C for 16 h (Ohya and Kaneko, 1970). Ethanolic potassium acetate (1.3%, as above) was added and the fractions left overnight at -20°C, and then centrifuged as before and the supernatant recovered. Radioactivity within the supernatant was determined by counting in a scintillation counter, and taken as the content in HA. The pellet was resuspended in 800 µl dd.H2O, NaNO2 (100µl) and glacial acetic acid (100 µl) being then added for 90 min at room temperature (Lindahl et al., 1973; Endo and Yosisawa, 1979). Samples were precipitated overnight in cold ethanolic potassium acetate as indicated above and centrifuged, and the supernatant was recovered. The radioactive content of the supernatant was taken as the amount of HS. The pellet was redissolved in dd.H2O and equilibrated in buffer (50 mM Tris-HCl, 50 mM sodium acetate, BSA 0.5 mg/ml) containing chondroitinase AC (0.1 U/ml, buffer pH adjusted to 7.3; Saito et al., 1968). Digestion was performed at 37°C for 4 h, after which the samples were boiled for 6 min and precipitated overnight in cold ethanolic potassium acetate as indicated above, centrifuged and the supernatant recovered. Radioactivity within the supernatant was taken as the amount of CS. The pellet was redissolved in the same buffer, this time containing chondroitinase ABC (0.1 U/ml, buffer pH adjusted to 8.0), and subjected to the same treatments of digestion, boiling, and precipitation. This final radioactive measure was taken as the content of DS (iduronate-rich domains only).

Results presented for RMG are from three independent experiments using 20-30 × 100 mm dishes of confluent cells for each experiment, for RPE from three independent experiments using 5-6 × 100 mm dishes for each experiment, and for RN from two independent experiments using 15 × 100 mm dishes for each experiment.

RMG migration

Confluent cultures of second passaged RMG cells in 6 x 35 mm tissue culture plates were refed with 1.2 ml CDM to which was added 300 µl of PBS containing 0.5 mg/ml bovine serum albumin, either alone or containing the different enzymes to be tested: hyaluronidase type IV-S (from bovine testes, specific activity 750-1500 U/mg), 30 U/ml final concentration; hyaluronidase type IV-S boiled at 100°C for 5 min prior to addition; hyaluronidase lyase (from Streptomyces hyaluroticus, specific activity 500 U/mg), 75 U/ml final concentration; chondroitinase AC (isolated from Flavobacterium heparinum), 0.2 U/ml; or purified GAG (HA from human umbilical cord, and CS A from porcine rib cartilage and CS C from shark cartilage). Initial studies revealed that the usual buffer conditions for optimal hyaluronidase activity (pH 5.0) were toxic for cells, so subsequent experiments were performed in the culture medium at pH ~6.8. The cells were scrape wounded using a trimmed cell scraper, and the average distance between the two wound edges, inversely proportional to the rate of migration, was measured at 0, 24, 48, and 72 h using a calibrated graticule inserted into the inverse microscope eyepiece. The distance at 0 h was standardized to 100% in each experimental series, and all subsequent measures expressed as percentages relative to the initial track width. Mean migration distances were calculated from three independent experiments, measuring three different wells using 10 individual tracings per treatment (Meuillet et al., 1996). In all cases SD values were equal or inferior to 10% of the mean.

Acknowledgments

We thank V. Forster for expert technical assistance. Financial assistance for this work was supplied in part by grants from the Fondation de France, IPSEN, MGEN-INSERM, and ADRET-ALSACE.

Abbreviations

bFGF, basic fibroblast growth factor; CNS, central nervous system; CS, chondroitin sulfate; DMEM, Dulbecco's Modified Eagle's Medium; DS, dermatan sulfate; ECM, extracellular matrix; EGF, epidermal growth factor; FCS, fetal calf serum; GAG, glycosaminoglycan; HA, hyaluronic acid; HS, heparan sulfate; PBS, phosphate-buffered saline; PG, proteoglycan; RMG, retinal Müller glia; RN, retinal neurons; RPE, retinal pigmented epithelium.

References

Anchan ,R.M., Reh,T.A., Angello,J., Balliet,A. and Walker,M. (1991) EGF and TGF-[alpha] stimulate retinal neuroepithelial cell proliferation in vitro. Neuron, 6, 923-936. MEDLINE Abstract

Aruffo ,A., Stamenkovic,I., Melnick,M., Underhill,C.B. and Seed,B. (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell, 61, 1303-1313. MEDLINE Abstract

Bachem ,M.G., Reiss,U., Melchior,U., Se,K.M. and Gressner,A.M. (1989) Transforming growth factors (TGF-[alpha] and TGF-[beta]1) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells. FEBS Lett., 257, 134-137. MEDLINE Abstract

Baird ,A. (1994) Fibroblast growth factors: activities and significance of non-neurotrophin neurotrophic growth factors. Curr. Opinion Neurobiol, 4, 78-86.

Barnstable ,C.J. (1990) Molecular aspects of development of mammalian optic cup and retinal cell types. In Osborne,N.N. and Chader,G.J. (eds.), Progress in Retinal Research, Vol. 10. Pergamon Press, Oxford, pp. 45-67.

Bassols ,A. and Massagué,J. (1988) Transforming growth factor [beta] regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans. J. Biol. Chem., 263, 3039-3045. MEDLINE Abstract

Chaitin ,M.H., Wortham,H.S. and Brun-Zinkernagel,A.M. (1994) Immunohistological localization of CD44 in the mouse retina. Exp. Eye Res., 58, 359-366. MEDLINE Abstract

Chaitin ,M.H., Ankrum,M.T. and Wortham,H.S. (1996) Distribution of CD44 in the retina during development and the rds degeneration. Dev. Brain Res., 94, 92-98.

D'Angelo ,M. and Green,R.M. (1991) Transforming growth factor modulation of glycosaminoglycan production by mesenchymal cells of the developing murine secondary palate. Dev. Biol., 145, 374-378. MEDLINE Abstract

Edwards ,R.B. (1981) The isolation and culturing of retinal pigment epithelium of the rat. Vis. Res., 21, 147-150. MEDLINE Abstract

Edwards ,R.B. (1982) Glycosaminoglycan synthesis by cultured human retinal pigmented epithelium from normal post-mortem donors and a post-mortem donor with retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci., 23, 435-446. MEDLINE Abstract

Endo ,M. and Yosisawa,Z. (1979) A method for microscale isolation of sulfated glycopeptides from tissue. Tohoku J. Exp. Med., 129, 225-231. MEDLINE Abstract

Hageman ,G.S. and Johnson,L.V. (1987) Chondroitin 6-sulfate is a major constituent of primate cone photoreceptor matrix sheaths. Curr. Eye Res., 6, 639-646. MEDLINE Abstract

Hageman ,G.S. and Johnson,L.V. (1990) Structure, composition and function of the retinal interphotoreceptor matrix. In Osborne,N.N. and Chader,G.J. (eds.), Progress in Retinal Research, Vol. 10. Pergamon Press, Oxford, pp. 207-249.

Hantaz-Ambroise ,D., Vigny,M. and Koenig,J. (1987) Heparan sulfate proteoglycan and laminin mediate two different types of neurite outgrowth. J. Neurosci., 7, 2293-2304. MEDLINE Abstract

Heidinger ,V., Hicks,D., Sahel,J. and Dreyfus,H. (1997) Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro. Brain Res., 767, 279-288. MEDLINE Abstract

Herndon ,M.E. and Lauder,A.D. (1990) Diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system. Neuron, 4, 949-961. MEDLINE Abstract

Hicks ,D. and Courtois,Y. (1990) The growth and behaviour of rat retinal Müller cells in vitro. 1. An improved method for isolation and culture. Exp. Eye Res., 51, 119-129. MEDLINE Abstract

Hicks ,D. and Courtois,Y. (1992) Fibroblast growth factor stimulates photoreceptor differentiation in vitro. J. Neurosci., 12, 2022-2033. MEDLINE Abstract

Hicks ,D., Bugra,K., Faucheux,B., Jeanny,J.-C., Laurent,M., Malecaze,F., Mascarelli,F., Raulais,D., Cohen,Y. and Courtois,Y. (1991) Fibroblast growth factors in the retina. In Osborne,N. and Chader,G. (eds.), Progress in Retinal Research, Vol. 11. Pergamon, Oxford, pp. 333-374.

Hicks ,D., Guérold,B. and Dreyfus,H. (1996) Stimulation of endogenous ganglioside metabolism by neurotrophic growth factors in cultured retinal Müller glia. Glia, 16, 316-324. MEDLINE Abstract

Iijima ,N., Oohira,A., Mori,T., Kitabatake,K. and Kohsaka,S. (1991) Core protein of chondroitin sulfate proteoglycan promotes neurite outgrowth in cultures neocortical neurons. J. Neurochem., 56, 706-708. MEDLINE Abstract

Jaworski ,D.M., Kelly,G.M. and Hockfield,S. (1994) BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan binding proteins that is restricted to brain. J. Cell Biol., 125, 495-509. MEDLINE Abstract

Jaworski ,D.M., Kelly,G.M. and Hockfield,S. (1995) The CNS-specific hyaluronan binding protein BEHAB is expressed in ventricular zones coincident with gliogenesis. J. Neurosci., 15, 1352-1362. MEDLINE Abstract

Kaneko ,M. (1987) Interphotoreceptor matrix glycosaminoglycans in bovine eye. Ophthalmic Res., 19, 330-337. MEDLINE Abstract

Landers ,R.A. and Hollyfield,J.G. (1992) Proteoglycans in the mouse interphotoreceptor matrix. VI. Evidence for photoreceptor synthesis of chondroitin sulfate proteoglycan using genetically fractionated retinas. Exp. Eye Res., 55, 345-356. MEDLINE Abstract

Lindhal ,U., Backstrom,G., Jansson,L. and Hallen,A. (1973) Biosynthesis of heparin. II. Formation of sulfamide groups. J. Biol. Chem., 248, 7234-7237.

Lowry ,O.H., Rosebrough,N.J., Farr,A. and Randall,R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275.

Malecaze ,F., Mascarelli,F., Bugra,K., Fuhrmann,G., Courtois,Y. and Hicks,D. (1993) Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium. J. Cell. Physiol., 154, 631-642. MEDLINE Abstract

Margolis ,R.U., Margolis,R.K., Chang,L.B. and Preti,C. (1975) Glycosaminoglycans of brain during development. Biochemistry, 14, 85-88. MEDLINE Abstract

Mazzoni ,E.M. and Kenigsberg,R.L. (1992) Effects of epidermal growth factor in the mammalian central nervous system: its possible implications in brain pathologies and therapeutic applications. Drug Dev. Res., 26, 11-128.

Meuillet ,E., Cremel,G., Dreyfus,H. and Hicks,D. (1996) Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Müller glia. Glia, 17, 206-216. MEDLINE Abstract

Meyer ,K. and Palmer,J. (1934) The polysaccharide of the vitreous humor. J. Biol. Chem., 107, 629-634.

Munaim ,S.I., Klagsbrun,M. and Toole,B.P. (1991) Hyaluronan-dependent pericellular coats of chick embryo limb mesodermal cells: induction by basic fibroblast growth factor. Dev. Biol., 143, 297-302. MEDLINE Abstract

Nagy ,J.I., Hacking,J., Frankenstein,U.N. and Turley,E.A. (1995) Requirement of the hyaluronan receptor RHAMM in neurite extension and motility as demonstrated in primary neurons and neuronal cell lines. J. Neurosci., 15, 241-252. MEDLINE Abstract

Needham ,L.K., Adler,R. and Hewitt,A.T. (1988) Proteoglycan synthesis in flat cell-free cultures of chick embryo retinal neurons and photoreceptors. Dev. Biol., 126, 304-314. MEDLINE Abstract

Normand ,G., Clos,J., Vincendon,G. and Gombos,G. (1985) Postnatal development of rat cerebellum: glycosaminoglycan changes related to variation in water content, cell formation and organ growth. Int. J. Dev. Neurosci., 3, 245-256.

Ohya ,T. and Kaneko,Y. (1970) Novel hyaluronidase from streptomyces. Biochim. Biophys. Acta, 198, 607-609. MEDLINE Abstract

Oohira ,A., Matsui,F. and Katoh Semba,R. (1991) Inhibitory effect of brain chondroitin sulfate proteoglycans on neurite outgrowth from PC12 cells.J. Neurosci., 11, 822-827.

Raymond ,S.M. and Jackson,I.J. (1995) The retinal pigmented epithelium is required for development and maintenance of the mouse retina. Curr. Biol., 5, 1286-1295. MEDLINE Abstract

Riechardt ,L.F. and Tomaselli,K.J. (1991) Extracellular matrix molecules and their receptors: functions in neural development. Annu. Rev. Neurosci., 14, 531-570.

Riopelle ,R.J. and Dow,K.E. (1990) Functional interactions of neuronal heparan sulfate proteoglycans with laminin. Brain Res., 525, 92-100. MEDLINE Abstract

Ruoslahti ,E. and Yamaguchi,Y. (1991) Proteoglycans as modulators of growth factor activities. Cell, 64, 867-869. MEDLINE Abstract

Saito ,H., Yamagata,T. and Suzuki,S. (1968) Enzymatic methods for the determination of small amounts of isomeric chondroitin sulphates. J. Biol. Chem., 243, 1536-1541. MEDLINE Abstract

Schlessinger ,J., Lax,I. and Lemon,M. (1995) Regulation of growth factor action by proteoglycans: what is the role of the low affinity receptors? Cell, 83, 357-360. MEDLINE Abstract

Snow ,D.M., Lemon,V., Carrino,D.A., Caplan,A.I. and Silver,J. (1990) Sulphated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp. Neurol., 109, 111-130. MEDLINE Abstract

Steinberg ,R.H. (1994) Survival factors in retinal degenerations. Curr. Opinion Neurobiol., 4, 515-524.

Stramm ,L.E., Haskins,M.E. and Aguirre,G.D. (1989) Retinal pigment epithelial glycosaminoglycan metabolism: intracellular versus extracellular pathways. In vitro studies in normal and diseased cells. Invest. Ophthalmol. Vis. Sci., 30, 2118-2131. MEDLINE Abstract

Tawara ,A., Varner,H.H. and Hollyfield,J.G. (1989) Proteoglycans in the mouse interphotoreceptor matrix. II. Origin and development of proteoglycans.Exp. Eye Res., 44, 633-642.

Threlkeld ,A., Adler,R. and Hewitt,A.T. (1989) Proteoglycan biosynthesis by chick embryo retina glia-like cells. Dev. Biol., 132, 559-568. MEDLINE Abstract

Toole ,B.P. (1976) Morphogenetic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissues. In Barondes,S.H. (eds.), Neural Recognition Plenum Press, New York, pp. 275-329.

Wagner ,J.A. (1991) The fibroblast growth factors: an emerging family of neural growth factors. Curr. Topics Microbiol. Immunol,. 165, 95-118.

1To whom correspondence should be addressed

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 19 Dec 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. P. Becerra, L. A. Perez-Mediavilla, J. E. Weldon, S. Locatelli-Hoops, P. Senanayake, L. Notari, V. Notario, and J. G. Hollyfield
Pigment Epithelium-derived Factor Binds to Hyaluronan: MAPPING OF A HYALURONAN BINDING SITE
J. Biol. Chem., November 28, 2008; 283(48): 33310 - 33320.
[Abstract] [Full Text] [PDF]

Home page
 Arch OphthalmolHome page
S. Siffroi-Fernandez, A. Cinaroglu, V. Fuhrmann-Panfalone, G. Normand, K. Bugra, J. Sahel, and D. Hicks
Acidic Fibroblast Growth Factor (FGF-1) and FGF Receptor 1 Signaling in Human Y79 Retinoblastoma
Arch Ophthalmol, March 1, 2005; 123(3): 368 - 376.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J. Choi, A. M. Miller, M. J. Nolan, B. Y. J. T. Yue, S. T. Thotz, A. F. Clark, N. Agarwal, and P. A. Knepper
Soluble CD44 Is Cytotoxic to Trabecular Meshwork and Retinal Ganglion Cells In Vitro
Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 214 - 222.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
P deS Senanayake, A Calabro, K Nishiyama, J. Hu, D Bok, and J. Hollyfield
Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface
J. Cell Sci., January 1, 2001; 114(1): 199 - 205.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Normand, G.
Right arrow Articles by Dreyfus, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Normand, G.
Right arrow Articles by Dreyfus, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?