The synthesis of glycosaminoglycans by corneal stroma cells in culture

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. AN rights o/ reproduction in my form reserved

Experimental Cell Research 88 (1974) 193-197

THE SYNTHESIS OF GLYCOSAMINOGLYCANS STROMA CELLS IN CULTURE

BY CORNEAL

I. M. DAHL, W. JOHNSEN, A. ANSETH and H. PRYDZ Department of Ophthalmology and Institute of Medical Biology, University of Tromse, Tromsa, Norway

SUMMARY Primary cultures of stroma cells from rabbit cornea have been established. In medium supplemented with serum the cells divide and produce glycosaminoglycans which are excreted into the medium. The glycosaminoglycans produced seemed to consist of about 10 % keratan sulphate I, about 20 % chondroitin sulphate and 60-70 % hyaluronic acid. No significant variations in the composition were observed during the growth cycle. The degree of sulphation increased with the age of the culture from about one sulphate group per 10 hexosamine residues to about one per 3 residues.

Cornea1 opacities frequently complicate lesions and disorders affecting the cornea1 stroma. Earlier observations have suggested [l, 2, 31that whereas stromal cells from normal cornea synthesize keratan sulphate I and chondroitin 4-sulphate, stromal cells from cornea afflicted by opacities also synthesize dermatan sulphate. The main structural difference between chondroitin 4-sulphate and dermatan sulphate is the occurrence of D-glucuronic acid in the former and L-iduronit acid in the latter. In order to study the regulation of the synthesis of these macromolecules we have established primary cultures of fibroblasts from rabbit cornea1 stroma. Some characteristics of these cells and the carbohydrate part of the glycosaminoglycans formed in vitro are described here.

30 ml Falcon flasks (Falcon Plastics, Osnard, Calif.) containing 8 ml Eagle Minimum Essential Medium (GIBCo, Grand Island, N.Y.) with 10% calf serum (GIBCO), glycine (final conc.‘O.l mM), serine (final cont. 0.1 mM), streptomycin (final cont. 50 pug/ml) and penicillin G (final cont. 30 ,cg/ml) added. Outgrowth of fibroblasts appeared after 2 days. After 8 days the medium was poured off and the cell sheets dispersed with 0.25 % trypsin CDifco. Detroit. Mich.) in phosphate-buffered -EDT&saline (NaCl 8 g/l, KC1 0.2 g/l, KH,PO,, 0.2 g/l, NasHPOd 1.15 g/l, EDTA-diNa 0.2 g/l) at 37°C for 15 min. The cells were centrifuged, washed with fresh culture medium and resuspended to let the remaining pieces of tissue sediment. The singlecell supernatant was pipetted off and seededinto Falcon flasks (about 1OScells/flask) or Roux bottles (about lw cells/flask). After 3-4 transfers some cells were frozen and stored in liquid nitrogen in 1 ml ampoules.

Cell counts Cells were counted on duplicate flasks in a Coulter counter and in a Biirker haemocytometer with trypan blue to estimate viability. DNA was extracted by a slight modification of the method of Munro & Fleck [16] and measured by diphenylamine according to Burton [7].

Isolation of glycosaminoglycans

MATERIALS

AND METHODS

Preparation of primary cultures Cornea1 tissue was removed aseptically from anaesthesized rabbits, cut to small pieces and incubated in 13” -

741818

Used medium was collected after various intervals of growth and centrifuged (10000 g/2”C/15 min) to remove dead cells and debris. One volume of medium was mixed with 0.3 vol of 2 % cetylpyridinium chloride (CPC) (Sigma, St Louis, MO.) and left for 2 h at room temperature. The sediment after centrifugation (27000 Exptl well Res 88 (1974)

194 Dahl et al.

Fig. 1. Unstained stromal cells from rabbit cornea grown on coverslips in Leighton tubes for 2 days. Primary culture established 22 days earlier. x 500.

g/25”C/30 min) was dissolved in 0.1 vol of 60% isopropanol, reprecipitated by adding 0.4 vol of 1 % sodium acetate in ethanol and left at 4°C overnight. The sediment after centrifugation (15 000 g/2”C/l5 min) was washed with ethanol and dissolved in 0.1 M sodium acetate buffer pH 5.5 containing 5 mM cysteine and 5 mM EDTA. Activated papain in the same EDTA-cysteine-sodium acetate buffer was added (final cont. 0.5 mg/ml) to digest the glycosaminoglycans for 18-24 h at 60°C. If necessary the digestion was repeated. The supernatant after centrifugation (27 000 g/2”C/l5 min) was made 3 mM in Na,SO,. CPC (2% in 3 mM Na,SO,) was added drop-wise under stirring until no more precipitate appeared. After centrifugation (27 000 g/2”C/15 min) the sediment was rapidly washed twice with distilled water. dissolved by dropwise addition of 2 N KCI, repreciuitated bv overnight incubation with 8 vol of ethanol/ H,O/acefic acid-124 : 7 : 1 (v/v/v)] and centrifuged. The final sediment was washed with 75 % ethanol.

Analysis of glycosaminoglycans The carbohydrates were analysed as described [lo]. Galactose and galactosamine gave almost the same colour yield in the galactose oxidase reaction. Hexuranic acid was determined by the method of Bitter & Muir [5] or Blumenkrantz & Asboe-Hansen [6]. Exptl Cell Res 88 (1974)

Hexosamines were determined by the modified ElsonMorgan method [15] and also in the Jeol JLC-6AH amino acid analyser. Total glycosaminoglycans in the medium were estimated by Alcian Blue [18] with chondroitin sulphate (Sigma, type A) as standard. Electroohoresis was carried out in 4 % formic acid on cellulose acetate strips at 2 mA/strip (l&l 5 V/cm) for 3 h. We also tried the svstems described bv Haruki & Kirk [II] and Seno et al. [17]. Sulphate was determined by the method of Antonopoulos [4]. Unused medium was treated identically with used medium and served as blank in all determinations.

Hyaluronidase

treatment

The final precipitate after glycosaminoglvcan isolation was dissolved in 0.1 M-sodium a&:ate buffer pH 5.0 containing 0.1 M NaCl. Hyaluronidase (Sigma, type VI) (final cont. 50 pg/ml) was added and the samples incubated with shaking for 3 h at 37°C in a water bath. Glycosaminoglycans were then precipitated with CPC (final cont. 0.5 %) for 1.5 h at room temperature and the precipitate centrifuged at 27 300 g/30 min/25”C. The sediment was washed twice with ethanol. The supematant and sediment were analysed as described above. The total loss of uranic acid in the two washing fluids amounted to 5-15 % of that present in the material incubated with hyaluronidase.

195

Synthesis of glycosaminoglycans by cornea1 stroma cells

RESULTS AND DISCUSSIGN Cell cultures

The stromal cells resembled fibroblasts (fig. 1). They had a generation time of 25-30 h in medium with 10 % serum and contained about 6.6 ,ug DNA/cell. The cells required serum for growth and reached a density of about lo8 cells/flask (area 25 cmz). After 26-30 transfers (5-6 months) a ‘crisis’ appeared. Most cells loosened or died and a new strain with a generation time of about 16 h emerged. Untransformed cells which had undergone only a few transfers showed good viability after storage in liquid nitrogen for one year. The present studies were carried out with cells which had undergone fewer than 23 transfers. Glycosaminoglycans in growth medium

Electrophoretic analysis in 4% formic acid of the glycosaminoglycans isolated from used growth medium showed two bands which had no exact correspondence with any available standard. Using the other two systems, in most cases one and never more than two bands were seen. The glycosaminoglycans in the medium appeared in the void volume when 10 ml were filtered through BioGel P-200 (2 x 30 cm) and the eluted fractions analysed for uranic acid. The molecular radii of these compounds were therefore fairly large in aqueous solution, probably at least 50-60 A. Fresh growth medium con-

-1

600 400 200

000 00 00 00 00

Fig. 2. Abscissa: incubation time (days); ordinate: (left) cell number per Roux flask (x 1O-p) (o-e): (right) Alcian Blue-binding material (rig/ml -of used medium). Columns: Amount of Alcian Blue-binding material accumulated in medium during 2 days of

groW!i* Alctan Blue-binding material excreted by stromal cells from rabbit cornea after various intervals of growth.

tained very little Alcian Blue-binding material. The amount of such material found in medium harvested at various intervals after the start of new cultures (fig. 2) was greater in the late log phase and beginning of the stationary phase (after day 6) than in early log phase. The amount of glycosaminoglycans excreted to the medium per day was twice as high in

Table 1. Composition of isolated glycosaminoglycan material Moles/100 moles of carbohydrate Interval of cell growth (days)

Number of expts

O-3 22

3 58

8-10 11-13

2 3

Hexosamine

Uranic acid

Galactose

31 (19-46) 34 (17-45) (24-43) 40 (3347) 38 (2746)

48 (23-68) 43 54 (36-74) (19-59) 52 (45-58) 48 (42-59)

21 (13-31) 31 13 (5-26) (23-38) 9 (8-10) 14 (12-15) Exptl Cell Res 88 (1974

196 Dahl et al. Table 2. Percentage of glycosaminoglycan components released by hyaluronidase treatment Growth period (days)

Hexuronic acid % released

Hexosamine Galactosea

resistant material is markedly increased. On the assumption that keratan sulphate 1 is completely resistant to hyaluronidase and that all the hexosamine in the resistant material is glucosamine, the amount of keratan sul-

phate I present may be calculated to about 8-10% of the total. The amount of galactose l-3 82 92 45 presentin the isolated undigested glycosamino5-8 17 95 42 11-13 79 91 38 glycan material would, however, suggestthat keratan sulphate made up twice as much. 0 The galactosevalues are calculated by assuming that galactosamineis 93 % releasedby hyaluronidase. Alternatively, the hyaluronidase sensitive galactose was releasedfrom other substances. stationary phase as in log phase, even when From the composition of the total glycocalculated per lo5 cells. The estimated saminoglycan material the rest most likely amounts of the various glycosaminoglycan co,nsistsof about 20 % chondroitin sulphate components added up to 90-116 % of the and 60-70% hyaluronic acid and these were total determined as Alcian Blue-binding probably the two bands seen in electromaterial. However, due to the presence of phoresis. carbohydrate material probably of nonThe average molar ratio of sulphate/hexoglycosaminoglycan nature in the supernatant, samine was 0.08 in glycosaminoglycans isoit is difficult to evaluate how completely lated from medium used from the 4th to the CPC and Alcian Blue will precipitate the 6th day of culture, and increased to 0.14 for various glycosaminoglycans. The composition material from the 6th to the 8th day, 0.24 for of the material isolated from medium in material from the 8th to the 10th day and which cells were grown for various periods 0.36 for material from the 11th to the 13th of their growth cycle (table 1) may therefore day. Thus, the glycosaminoglycans are apparnot be quantitatively correct. The glucos- ently secretedin a more completely sulphated amine/galactosamine ratio was found to be form when the cell cultures have reached the stationary phase. about 3.5 in the amino acid analyser. Cultures have been established of rabbit After hyaluronidase treatment most of the (Volkert, referred to in [12]), calf [14], chick glycosaminoglycan-components appeared in the supernatant (table 2). However, there was embryo [8] and human [8] cornea1 cells, always some undigested material. Testicular mainly for virus cultivation. Conrad [8] studied the incorporation of hyaluronidase converts hyaluronic acid, chondroitin 4-sulphate and chondroitin 6- various radioactive precursors into glycosulphate to oligosaccharides while keratan saminoglycans by chick cornea1 cells. He sulphate is not broken down. Accordingly, found evidence for the synthesis of chondrowe find that the resistant glycosaminoglycan(s) itin sulphate and keratan sulphate by the contain more galactosethan the hyaluronidase same clones of cells and observed that the degree of sulphation increased with the age sensitive material. It is evident that the cells produce some of the culture. Our findings agree closely keratan sulphate I, since the isolated glyco- with those of Conrad [8] except that we did saminoglycans contain galactose and the not observe a decline in glycosaminoglycan fraction of galactose in the hyaluronidase- synthesis during the log phase. Exptl Cell Res 88 (1974)

Synthesis of glycosaminoglycans by cornea1 stroma cells

Danes [9] used cornea1 cell cultures from patients with various mucopolysaccharidoses to study the production of glycosaminoglycans. She found their genetic metabolic defects to be demonstrable in vitro as is the case with fibroblasts from other organs. The regulatory mechanisms involved in bringing about the epimerization of D-glucuronic to L-iduronic acid [13] may also be available for study in an in vitro system. This work was supported by the Norwegian Council for Science and the Humanities.

REFERENCES 1. Anseth, A & Fransson, L-A, Exptl eye res 8 (1969) 302. 2. Anseth, A, Exptl eye res 8 (1969) 310. 3. - Ibid 8 (1969) 438.

197

4. Antonopoulos, C A, Acta them Stand 16 (1962)

1521. 5. Bitter. T & Muir. H M. Anal biochem 4 (1962) .

330. 6. Blumenkrantz, N & Asboe-Hansen, G, Anal biochem 54 (1973) 484. Burton, K, Biochem j 62 (1956) 315. ifi Conrad, G W, Dev biol 21 (1970) 611. 9: Danes, B S, Clin gen 4 (1973) 1. 10. Gladhaug Berre, A, Idsterud, B, Christensen, T B, Holm, T & Prydz, H, Biochem j 135 (1973) 791. 11. Haruki, F & Kirk, J E, Biochim biophys acta 136 (1967) 391. 12. Lee&y, J, Science 149 (1965) 633. 13. Lindahl, U & Backstrom, G, Biochem biophys res commun 46 (1972) 985. 14. Ludwig, H, Paulsen, J & Kaminjolo, J S, Z med mikrobiol immunol 155 (1969) 133. 15. Maver. M M. in Kabat. E A & Maver. M M. Exit1 immunochemistry p. 505, 2nd edh. Charles C. Thomas, Springfield, Ill. (1961). 16. Munroe, H N & Fleck, A, Meth biochem anal 14 (1966) 13. 17. Seno, N; Ariizumi, K, Nagase, S & Anno, K, J biochem 72 (19721 479. 18. Whiteman, P, Biochem j 131 (1973) 343. Received March 4, 1974

Exptl Cell Res 88 (1974)