Exp.Eye Res. (1991) 53, 753-758

Hyaluronan TERUO Department



of Ophthalmology,




(Received 24 September

School of Medicine, Japan





1990 and accepted in revised form 3 December

City, Osaka 589,


Hyaluronan (hyaluronic acid), well-known for its viscoelastic properties, is also recognized as a biological signal to cells. Using organ cultures of the rabbit cornea, we investigated the effectsof hyaluronan on the migration of cornea1epithelium. The addition of hyaluronan to the culture medium increased the length of the path of the cornea1 epithelial layer in a dose-dependent fashion. Other glycosaminoglycans (chondroitin, chondroitin sulphate. keratan sulphate and heparan sulphate) were also tried, but only hyaluronan exhibited a stimulatory effect on cornea1epithelial migration. The effectsof hyaluronan and fibronectin or epidermal growth factor (EGF)were additive: the addition of antisera against fibronectin or against EGF did not alter the stimulatory effect of hyaluronan. These results demonstrate that hyaluronan stimulates cornea1epithelial migration by mechanism(s) different from those of fibronectin and EGF. Key words: cornea ; epithelial migration ; hyaluronan : organ culture.

1. Introduction

The first event in epithelial wound closure is the migration of cornea1 epithelial cells to the defects. Fibronectm and epidermal growth factor (EGF)stimulate cornea1 epithelial migration, and clearly play important roles in the restoration of the epithelium (Ho et al., 1974; Nishida et al., 1983a, 1984; Watanabe, Nakagawa and Nishida, 1987 ; Nishida, 1988 ; Schultz, Davis and Eiferman, 1988 ; Mathers et al., 1989; Tripathi, Kwait and Tripathi, 1990). The immediate resurfacing of epithelial defectsmay require the orchestrated responsesof cornea1epithelial cells to extracellular matrix proteins and growth factors. The intraocular use of hyaluronan represented a revolutionary advance in ophthalmic surgery (Miller and Stegmann, 1981; Balazs and Denlinger, 1989). Its viscoelastic properties permit ophthalmic surgeons to manoeuvre surgical instruments and the intraocular lens atraumatically during the cataract surgery. Extraocular use of hyaluronan has also been reported to protect cornea1epithelial cells, especially in dry eye patients (Polack and McNiece, 1982 ; DeLuise and Peterson, 19 84 ; Limberg et al., 19 8 7 ; Laflamme and Swieca, 1988 ; Nelson and Farris, 1988 ; Wysenbeek et al., 1988 : Chung, Fagerholm and Linstrom, 1989). Hyaluronan is an important constituent of the extracellular matrix and is thought to play an important role in various physiological phenomena, such as wound healing, inflammation, and devel* For correspondenceand reprint requestsat: Departmentof Ophthalmology, Kiii University School of Medicine, 377-2 OhnoHigashi, Osaka-Sayama City, Osaka 589, Japan.



opment (Forrester and Balazs, 1980; Hakansson, Hallgren and Venge, 1980 ; Weigel, Fuller and LeBoeuf, 1986; Weigel et al., 1988). Since hyaluronan is now recognized as a biological signal for cells, we were interested in its role in maintaining the integrity of the cornea1epithelium. In this study, we investigated the effects of hyaluronan on the migration of cornea1 epithelial cells in tissue culture. 2. Materials and Methods

Albino rabbits weighing 2-3 kg were obtained from Hokusetsu Sangyo (Settsu, Osaka, Japan) ; TC-199 culture medium was from the Research Foundation for Microbial Diseases of Osaka University (Suita, Osaka, Japan) ; plastic multi-well culture dishes (24 wells, no. 3424) were from Costar (Cambridge, MA). Chick hyaluronan from cockscomb (sodium hyaluronate, average molecular weight : 6 70 kDa) was from Santen Pharmaceutical Co. (Osaka, Japan). This hyaluronan preparation was free from growth factor(s) activity by measuring the incorporation of [3H]thymidine into the cultured epithelial cells of rabbit cornea. Chondroitin sulphate and chondroitin from whale cartilage, Keratan sulphate sodium salt from bovine cornea, and heparan sulphate sodium salt from bovine kidney were obtained from Seikagaku Kogyo, Tokyo, Japan. Human plasma fibronectin was from the New York Blood Center (New York, NY). Recombinant human EGF was from Earth Pharmaceutical Co. (Akoh, Hyogo, Japan). The purity of both fibronectin and EGF were assessed by SDS polyacrylamide gel electrophoresis and high performance liquid chromatography. 0 1991 Academic Press Limited


The rabbits were killed by an intravenous overdoseof pentobarbital. Eyes were enucleated, a sclerocorneal section was excised, washed several times with sterile phosphate-buffered saline (PBS)and cut into 2 x 4 mm blocks with a razor blade. The blocks were cultured as describedpreviously [with a slight modification (Nishida et al., 1983a)] in multi-well culture dishes for 24 hr, except where noted below, in a humidified incubator under 5% CO, at 37°C. The effect of hyaluronan (1 mg ml-l culture medium, except where noted otherwise) on the migration of rabbit cornea1epithelium was studied in a series of experiments. In the first, the cornea1 block was incubated with TC-199 culture medium (1 ml in all experiments), alone or with hyaluronan, for 6, 12, 18 or 24 hr. Next we investigated the effectof hyaluronan in different concentrations (0.1, 0.3 or 1.0 mg ml-‘). To investigate whether or not the stimulatory effect of hyaluronan is unique among various other glycosaminoglycans, the stimulatory effect of hyaluronan (0.1, 0.3 and 1.0 mg ml-*) was compared with those of chondroitin sulphate (0.1. 0.3 and 1.0 mg ml-‘), chondroitin (0.1, 0.3 and 1.0 mg ml-‘), keratan sulphate (0.1, 0.3 and 1.0 mg mill) and heparan sulphate (0.1, 0.3 and 1.0 mg ml-‘). Since both fibronectin and EGF stimulate cornea1 epithelial migration (Nishida et al., 1983a), we intended to determine whether the stimulatory action of hyaluronan was synergic with, or additive to, fibronectin or EGF. Accordingly, cornea1 blocks were cultured with unsupplemented medium or media containing fibronectin (10 or 100 yg ml-‘, fibronectin plus hyaluronan, EGF (0.3 or 3 ng ml-‘), or EGF plus hyaluronan. Then we examined the effectsof guineapig antiserum against fibronectin and rabbit antiserum against EGF on hyaluronan-stimulated epithelial migration. Migration paths were compared after culture with : (1) unsupplemented medium ; (2) normal serum ; (5 mg ml-‘) or anti-EGF (3) anti-fibronectin (0.1 mg ml-l) antiserum ; (4) hyaluronan ; (5) hyaluronan plus antiserum ; or (6) hyaluronan plus normal serum. Epithelial migration has two phases; a lag phase and a migratory phase. To determine whether hyaluronan exerts its effect at either or both of these phases, we changed culture media during the 24-hr incubation period using a medium that contained hyaluronan during only the first or the last 12 hr. Controls were cultured for 24 hr without hyaluronan or for 24 hr with hyaluronan. Similar experiments exposed the cornea1blocks to hyaluronan for the first 3 or 6 hr of the 24-hr incubation period. The length of the path of epithelium was determined as reported previously (Nishida et al., 1983a). In brief, the cultivated cornea1blocks from rabbits were washed with PBSand fixed overnight with a mixture of 100% ethanol and glacial acetic acid (95 : 5) at 4°C. The blocks were dehydrated through graded ethanol, immersed in xylene, and embedded in paraffin: three



4 pm sections were cut, 300 pm apart, from each block so that epithelial migration down the cut side of the block, towards the endothelial side, could be determined. After deparaffinization, the specimens were stained with haematoxylin-eosin and observed under a light microscope (Model BH-2, Olympus, Tokyo, Japan). Photographs were taken with black and white film (Neopan F, Fuji Films, Tokyo, Japan) with the aid of an automatic exposure metre. and the length of the path of epithelial migration was measured on each photograph. Results were expressed as a mean ( ~s.E.M.) for six determinations and statistical analysis was calculated using the Student’s t-test. 3. Results

Figure 1 shows the effects of the addition of hyaluronan on the length of the path of the cornea1 epithelial layer at various periods of cultivation. No appreciable epithelial migration was observed 6 hr after the initiation of the cultivation. The length of the path of epithelial layer started to increase after a 12hr cultivation, and thereafter it increased proportionally to the incubation period. Addition of hyaluronan to the culture medium resulted in significantly greater increases than were seen in the cultures without hyaluronan, after 12 (P < O.Ol), 18 (P < 0.001) and 24 (P < 0.01) hr in culture. These results demonstrated that hyaluronan stimulated the migration of the cornea1 epithelial layer. Incubation for 24 hr with hyaluronan at various concentrations demonstrated that the length of the path of the cornea1 epithelial layer increased proportionally to the concentration of hyaluronan (Fig. 2). Of the different glycosaminoglycans tested, only hyaluronan increased the length of the path of the cornea1epithelial layer (Fig. 3). Chondroitin sulphate, chondroitin, and keratan sulphate, tested on concen-

Hours in culture

FIG. 1. Chronologicalchangesin the length of the path of cornea1epithelium cultured in unsupplementedTC-199 (O), and TC-199containing hyaluronan [l mg ml-‘: (@)I. Barsshow S.E.M. of six measurements.NS.Not significant; (*) P < O-01: (**)P < 0.001 overthoseculturedin unsupplementedTC-199.






Effects offibronectin (FN) on the stimulutory action of hyaluronan (HA) in corned epithelial migration



Migration (pm)


Unsupplemented TC- 19 9 10 pg ml-’ FN 100 ,ug ml-’ FN 1 mg ml-l HA 1 mg ml-l HA + 10 pg ml-’ FN 1 mgml-’ HA+lOO~gml~l FN

570+ 54 613+46 751+34 769+35 852k17 944f26

lOOi9~5 108k8.1 132+6.0* 135+6.1* 149$3*Ot 166f4.6tS

of hyoluronan (mg mi’)

FIG. 2. The dose effects of hyaluronan on the stimulation of cornea1 epithelial migration after 24 hr cultivation. NS. Not significant: (*) P < 001; (**) P < O-005 over those cultured without hyaluronan.

Results were expressedas a mean k S.E.M. for six determinations. (*) P < 0.02 ; (t) P < DO05 compared with unsupplemented TC199: ($J P < PO5 compared with 1 mg ml-’ HA.


Effects of EGF on the stimulatory action of hyaluronan (HA) in cornea1epithelial migration


of glycosaminoglycans

(mg mi’l

FIG. 3. The stimulatory effect of various concentrations of hyaluronan or other glycosaminoglycans on the length of the path of the cornea1 epithelial layer: (0-O) hyaluronan; (n----n) chondroitin sulphate; (A---A) chondroitin; (U---U) keratan sulphate; (0 -0 ) heparan sulphate. Data are expressed as percentage of the length of the path of the cornea1 blocks cultured in unsupplemented TC-199 for 24 hr. Bars show S.E.M.of six measurements. (*) P < 0.01; (**) P < 0.005: (***) P < 0.001 over those cultured in unsupplemented TC- 19 9.

up to 1 mg ml-l, did not affect the length of the path: heparan sulphate actually decreased the length of the path of the cornea1 epithelial layer in a dose-dependent fashion. As expected, both fibronectin and EGFincreased the length of the path of cornea1epithelium in the absence of hyaluronan. The addition of hyaluronan to the culture medium further increased epithelial migration in an additive manner (Tables I and II). As shown in Table III, the addition of antiserum against fibronectin decreasedthe epithelial migration significantly (412 pm, as compared with 608 pm in control cultures). No significant effect was observed in the presenceof normal serum. When hyaluronan was added to the culture medium, the length of the path of cornea1 epithelium increased by 125 pm. Even in the trations


Migration km) .-~-

Unsupplemented TC-19 9 0.3 ng ml-’ EGF 3 ng ml-l EGF 1 mg ml-l HA 1 mg ml-’ HA+0.3 ng ml-’ EGF 1 mg ml-l HA+ 3 ng ml-’ EGF

558k 14 533f40 677*18 671+27 680+41 802+27

% .~~ 100_+2.5 96 If: 7.2 121+3.2$ 120+4.8t 122k7.3” 144+4.8t§

Results were expressedas a mean + S.E.M. for six determinations. (*) P i 0.05; (f) P < 0.005: ($) P < 0001 compared with unsupplemented TC-199: (4) P < 0.01 compared with 1 mg ml-’ HA.


Eflects of guinea-pig antiserum against FN (5 mg ml-l) or normal guinea-pig serum (5 mg ml-‘) on the stimulator-y action ofhyaluronan (HA, 1 mg ml-l) in cornea1epithelial migration Medium

Migration (pm)

Unsupplemented TC- 199 Anti-FN Normal serum HA HA + anti-FN HA + normal serum

608f16 4121t24 642 f 39 733+31 558fll 776+21

% lOOf2.6 68 Jr 3.9$ 106f 6.4 121+5.lt 92 f l+S$§ 128k3.5’

Results were expressedas a mean + S.E.M. for six determinations. (*) P < 0.02; (t) P < 0.01: ($) P < 0.001 compared with unsupplemented TC-I 99: (4) P < 0.001 compared with 1 mg ml-’ HA.

presenceof antiserum against fibronectin or of normal serum, the addition of hyaluronan increased the epithelial migration by 146 and 134 pm, respectively. Addition of antiserum against EGF did not affect the stimulatory action of hyaluronan (Table IV).




Effects of rabbit antiserum against EGF (01 mg ml-l) or normal rabbit serum (0.1 mg ml-‘) on the stimulatory action of hyaluronan (HA, 1 mg ml-‘) in cornea1epithelial migration

Migration Medium

UnsupplementedTC-199 Anti-EGF Normal serum HA HA + anti-EGF HA + normal serum



644+25 633+_46 647+20 773f42 784k 32 790+27

loo+ 3-9 98k7.1 lOO+ 3.1 120$6.5* 122 f 5.0* 123+4.2*

Results were expressed as a mean + S.E.M. for six determinations. (*) P < 0.05 compared with unsupplemented TC-199.



Ejffcts on cornea1 epithelial migration of the timing of exposure to hyaluronan with a 24-h incubation

Exposureperiod (W

Migration OLm)



568+26 724+28 729+_21 714+42

lOOk4.6 127+4,9t 128+_3.7$ 126 + 7.4*

O-24 o-12 12-24

Results were expressed as a mean + S.E.M. for six determinations. (*) P < @05; (f) P < DOl: ($) P < 0.001 compared with those cultured in unsupplemented TC-199.

Hyaluronan exhibited full stimulatory activity when it was present during either the first or the last 12 hr of a 24-hr cultivation (Table V). The measured increase in epithelial migration in cultures exposed to hyaluronan for 12 hr at either phase did not differ from the increase in cultures with hyaluronan for 24 hr continuously. Exposure of the cornea to hyaluronan for 3 hr did not result in any stimulatory effect observable at the end of the 24-hr cultivation ; 536 f 40 pm (no exposure to hyaluronan) and 538 + 3 1 pm (3 hr exposure). However, when the duration of exposure exceeded 6 hr, the length of the path of cornea1 epithelium increased significantly (698 + 32, 713 + 20 and 719f24pm for 6, 9 and 12 hr exposure, respectively) and was almost the same as in cultures exposedto hyaluronan for 24 hr continuously (7 15 f 25 ,um). 4. Discussion

Becausethe cornea is avascular, maintenance of its structure depends on complex, orchestrated interactions of the extracellular matrix and growth factors. The present paper demonstrated that hyaluronan


stimulates the migration of cornea1 epithelium in organ culture and that the mechanism of action of hyaluronan differs from that of fibronectin or EGF. Cornea1epithelial migration is the first step among various wound-healing processes. Fibronectin and EGF,shown previously to stimulate cornea1epithelial migration in the same organ culture system used in the present report (Nishida et al.. 1983a : Watanabe et al., 1987). also facilitate the closure of cornea1 epithelial defects in vivo (Frati et al., 19 72 ; Savage and Cohen, 19 73 ; Daniele et al., 19 79 ; Nishida et al., 1983a, b; Arturson, 1984; Nishida et al., 1984; Petroutsos et al., 1984 ; Spigelman, Vernot and Deutsch, 1985). Similarly, we have found that instillation of hyaluronan (1 mg ml-l) in vivo stimulated cornea1wound closure, after n-heptanol debridement, in a dose-dependentfashion (unpubl. res.). The stimulatory effect of hyaluronan was dosedependent and unique among the various glycosaminoglycans tested. Keratan sulphate is a major constituent of the normal cornea1 stroma (about 65 %). and chondroitin sulphate is also present (about 30%) (Hassell et al., 1983; Friend, 1987). When the cornea is wounded, the amount of keratan sulphate in the scar is reduced and the amount of chondroitin sulphate increases (Kitano and Goldman, 1966). However, as reported here, neither chondroitin sulphate, chondroitin. nor keratan sulphate affected the migration of the cornea1 epithelium. Furthermore. heparan sulphate inhibited epithelial migration in a dose-dependent manner. We cannot explain how heparan sulphate retards epithelial migration. Perhaps heparan sulphate, which binds to the fibronectin molecule, interferes with the biological activity of fibronectin. No relationship was observed between the stimulatory action of glycosaminoglycans on the cornea1 epithelium and the presence of sulphate radicals, since neither chondroitin sulphate nor chondroitin affected epithelial migration. The stimulatory effect of hyaluronan was not inhibited by the antisera against fibronectin and EGF,suggesting that the mode of action of hyaluronan might be different from that of fibronectin or that of EGF. In our previous experience with this cornea1culture model, the rate of epithelial migration was extremely low for the first 12 hr (lag phase) but was active during the last 12 hr (migrating phase) of a 24-hr culture period. Our present findings with hyaluronan were similar to our previous results with fibronectin. which could also stimulate epithelial migration when it was added during the lag or the migratory phase. On the other hand, hyaluronan differed from fibronectin in demonstrating no proportional linear relationship between the duration of exposure to hyaluronan and the length of the epithelial path at the end of the 24hr culture. Unlike hyaluronan, EGF is effective only in the lag phase and produced no stimulatory effect on cornea1 epithelial migration when added to the culture




medium during the migrating phase (Nishida et al., 1990). EGFdid not stimulate epithelial migration if it was present in the culture medium for 6 hr; however, exposure of the cornea to EGF for more than 9 hr produced its full stimulatory effect (Nishida et al., 1990). The present results show that hyaluronan also requires a minimum period of exposure (6 hr) to produce its stimulatory effect. If, hyaluronan stimulates epithelial motility, it suggests that there may be binding sites or receptors for this material on the epithelial cells, as has been proposed for fibronectin and EGF(Frati et al., 19 72 ; Trinkaus-Randall, Newton and Franzblau, 1990). Further attempts to identify a hyaluronan binding site or receptors in the cornea1 epithelial cells should be made. Although further investigation is needed before we understand the precise mechanism(s) of the action of hyaluronan, our findings demonstrate that hyaIuronan, like fibronectin and EGF, is involved in the repair of cornea1 epithelial defects. Acknowledgements We would like to thank Dr B. Horowitz of the New York Blood Center, New York, NY for the kind gift of human plasma fibronectin. We also thank Earth Pharmaceutical Co. for the supply of recombinant human EGF and antibody against recombinant human EGF and Santen Pharmaceutical Co. for the supply of hyaluronan (sodium hyaluronate). This research was supported in part by a grant from the Ministry of Education, Culture and Science of Japan and by a grant from Osaka Eye Bank, Osaka, Japan.

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Hyaluronan stimulates corneal epithelial migration.

Hyaluronan (hyaluronic acid), well-known for its viscoelastic properties, is also recognized as a biological signal to cells. Using organ cultures of ...
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