Macular Corneal Dystrophy

Macular Corneal Dystrophy Studies of Sulfated Glycosaminoglycans in Corneal Explant and Confluent Stromal Cell Cultures Gordon K. Klintworth, MD, PhD, and Clayton F. Smith, BS

The inherited disorder macular corneal dystrophy (.MCD), a localized corneal mucopolysaccharidosis, is currently thought to result from an inability to catabolize corneal keratan sulfate (keratan sulfate 1). As studies on isolated cells have provided insight into metabolic abnormalities in other inherited disorders, we investigated cultured corneal fibroblasts from 4 patients with MCD from several standpoints. Lines of corneal fibroblasts with MCD could not be distinguished from controls with cytochemical methods known to stain the naturally occurring accumulations. In contrast to cultured fibroblasts from patients with mucopolysaccharidoses Type I-H (Hurler syndrome) and Type II (Hunter syndrome), corneal fibroblasts from patients with MCD did not accumulate abnormal quantities of "S-sulfate-labeled glycosaminoglycans, but like normal corneal and cutaneous fibroblasts reached a state of equilibrium within 2 days. Also, the rate at which sulfated glycosaminoglycans were removed from cultured corneal fibroblasts in MCD by secretion and degradation more closely resembled that of normal cells than those with the systemic mucopolysaccharidoses. The secretion of sulfated glycosaminoglycans into the nutrient medium by corneal fibroblasts from patients with MCD occurred at a linear rate comparable to that of other cells studied. The aforementioned data, nonetheless, remain consistent with the hypothesis that MCD is an inherited disorder of keratan sulfate I (corneal keratan sulfate) catabolism, as isolated corneal fibroblasts in contrast to corneal explants synthesize little or no keratan sulfate in culture. In view of the latter, we also compared the profile of 'S-labeled glycosaminoglycans produced by a corneal explant from a patient with MCD with that normally synthesized by human corneal explants. The latter synthesized and secreted a population of '5S-sulfate-labeled glycosaminoglycans with properties of keratan sulfate. Considerably less material with these attributes was identified with the same analytic techniques in the cornea with MCD or in its surrounding medium after the abnormal cornea had been incubated under identical conditions. In addition to manifesting an impaired synthesis of corneal keratan sulfate-like material, the cornea with MCD produced a greater percentage of chondroitin5-6sulfate than normal. These findings suggest that the synthesis of corneal keratan sulfate and other glycosaminoglycans may be altered in MCD. (Am J Pathol 89:167-182, 1977)

NIACULAR CORNEAL DYSTROPHY (MCD) is a rare inherited disease in which opacities usually first become evident in both corneas at about the age of puberty. Initiallv irregular, ill-defined areas of diffuse From the Department of Pathology. Duke University Medical Center. Durham. North Carolina Presented in part at the Sixty-first Annual M1eeting of the Federation of American Societies for Experimental Biology, Chicago. Ill. April 197. Supported in part by Grant 1 ROI-EY00146 from the US Public Health Sersvice Accepted for publication June 28. 1977 Address reprint requests to Dr. Gordon K Klintworth. Department of Pathology. Duke University Mledical Center. Durham. NC 27710X 167

168

KLINTWORTH AND SMITH

American Journal of Pathology

clouding appear in the central cornea. With time, they progressively become confluent and eventually involve the entire corneal stroma. The disease ctulminates in severe visual impairment, usuallv before the fifth decade. Vision can be restored by, corneal grafting, and the excised corneal tisstue is tv pified morphologicallv by distinctive abnormalities. Intracvtoplasmic accumtulations that have tentatively been identified cvtochemicallv as glvcosaminoglvcans occur within the corneal fibroblasts (also known as keratocvtes) and usually also in the endothelium of the cornea.' -' Deposits of material with similar cvtochemical characteristics aggregate extracellularlv betw een the collagen fibers and commonly within portions of Descemet s membrane, which very often lhas excrescences on its inner sturface (cornea guttata). As MICD appears to be a localized mucopolv saccharidosis,8 9 w e sttudied ctulttured corneal fibroblasts from patients wvith this disease with techni(lutes that have vielded valuable data about several inherited disordlers of glvcosaminoglvcan metabolism such as mucopolvsaccharicdosis Type I-H (Hurler syndrome) and mucopolvsaccharidosis Type II (Hunter svndrome).'6 In addition, wve investigated the sulfated glvcosaminoglvcans produced by a corneal explant of a patient with \MCD. Materials and Methods Studies on Cultured Fibroblasts Cell Lines

Corneal btuttons from 3 patients with MICD were placed in Ringer's balanced salt solultion within sterile containers immediately following penetrating keratoplasty (NILE, a 43-year-old woman. Family 13; HLS, a 45-vear-old man, Family 3; MISN, a 32-year-old woman, Family 3-the family numbnbers correspond to those in the registry (if patients wvith mactilar corneal dystrophy in the United States that is maintained by the senior auithor). The diagnosis was histologically confirmed by light and electron microscopy on portions of each corneal button. The corneal epithelium and endothelium were removed from a (lutadrant of the excised corneal button by gentle scraping with a scalpel blade. Cuilttures of corneal fibroblasts were established from the corneal stroma in tissue cuiltture flasks (Falcon, Oxnard, Calif.) in Eagle's minimtum essential medium (NMENI) with 10% fetal calf seruim, 100 uinits ml of penicillin, 100 gg ml of streptomvcin (all obtained fronm Gibco, Grand Island, N.Y. ); 10 m\I N-2-h\cdroxveth\vIpiperazitne-.\'-2-etlhaiesulfonic acid (H EPES) buffer (Calbiochem, Sian Diego, Calif.) was adlded to the medliuim to increase pH stalilitv of the CO2-bicarbonate b)uffering system. An addlitional cell line (G\1 1123) derived from the cornea of JXV a 41-year-old woman vvith MNICD (Family 21) was obtained from the Human Genetic Mutant Cell Repository, Institute for Nledical Research (Camden, N.J.). All cultures wvere incubated at pH 7.2 to 7.4 in a huimidified tissue culttire incuibator at 37 C in an atmosphere of 3Y C02-95% air. They were usually fed twvice a week wvith Eagle's minimuim essential medium with 10%7 fetal calf seruimn (but without antibiotics). The inorganic stulfate in the medium (not counting the calf sertum contributtion) was 0.81 mM "liter. For control studies. fibroblast cultures were established fromn inormal cortneal tissue that

Vol. 89, No. 1

MACULAR CORNEAL DYSTROPHY

169

October 1977

l1a(I been excisedi from eves that were entu1leated witlh a retinoblastoma (8-inotihi-oleld girl ). Coats' disease (I I-montih-old boy), andc a melanoma (76-year-old nman ) andl fronm the skiin of individuals of variable age. Abnormal ctitaineotis fibroblast lines were also obtained frorim the Htiman Genetic Mutant Celi Repository. Instittite for Miedical Research from p)atientis with mucopolvsacchariclosis Type I-H (Htirler syndrome) (liiies (;\1-2 ant (;\I-413) and n-mucopolvsaccharidcosis Type II (Huinter svndrome) (lin1es (Gl -615 and(lGI-690). Cytochem istry

For liglht microscopy, culttured cells in a confluent state were tryplsinize(l an(d pipette( into petri dishes containing sterile precleaned glass micro slides 3 inches X 1 inchl (1 .0 mn thick). These were incubated, like the parent culttures, in minimtum essential medltiun at pH 7.2 to 7.4 in a hujmidlifiedl tissuie ctultuire incuibator at 37 C in an atmosphere of .5%- ('IO29.5% air as described above. Twenty-four houirs, later the attaclhedc cells were fix cd 1 immersion in formalin and staine(l with hematoxvlin andt e osin, periodic acid-Schliff toluidine blue, alcian blue (pH 2.5), Or Hale's coll(oidal irotl nimethiod 177atdCI mnlo111ted( for microscopic examniniation. In some instances the staining was performed after prior incuibation of the unfixedl cells with testicular hvaluironidase or clhotndlroititn AI3C lvase in concentrations equivalent to those uisedI in chemical analyses (see analytical procedures below ). Incorporation of 35SQ4 Into Glycosaminoglycans

The incorporation of intrdcellular 35S sulfate by culttured fibroblasts from patients with NMCD, mucopolysaccharidosis Ty pe I-H, mucopolvsaccharidosis Ty-pe II as vvell as by normal cutaneous and c(ornepl fibroblasts wvas investigated. As glycosaminoglvcans are the only macromolecules that become significantly labeled with inorganic sulfate,'8'20 the simple assay system of Fratantoni et al.'6 was employed. Fibroblasts were added to petri dishes and incubated in 6 ml Eagle's minimum essential medium supplemented with I0%4 fetal calf serum, to which had been added 3 to 10 IuCi carrier-free H2S3504,ml. (New England Nuclear Corp., Boston, Mass.). ICD fibroblasts wvere studied in medium to which w as added 100 units /ml penicillin and 100 ,ggml streptomycin (twvo cell lines) and in medium that did not contain antibiotics (twvo cell lines). The labeled medium wvas removed at different intervals (I to 7 days) and dialxzed for 4 hours against 0.1 NI (NH4)2S04, followed by running tap 'water for 20 hours. The cells svere washed with isotonic saline until the washing contained no label. Following this the cells wvere cletachled from the petri dishes by treathnent with 0.4%c trypsin (Worthington Biochemical Corp., Freehold, N.J.) in saline at 37 C for 13 minutes. centrifuged, and extracted four times with boiling 8W0%e ethanol (1 mintute boiling in 2 ml ethanol each time). Unincorporated 35S04 and low-molecular-wveight compounds were removed by the dialysis ancd ethanol extraction. An ali(quot of the dialyzed medium was counted directly. The extractec cell residue was dissolv ecl by gentle heating in 10%W soditum hydroxide for (letermination of protein by the method of Lowry Ut (.j21 or by a modified biuiret methodI that was less sensitive to interference from nonprotein stubstances.22 The radioacti\ity of the specimen s-as also established by liquid scintillation counting. Removal of Prelabeled Intracellular Sulfated Glycosaminoglycans

To determine the rate at 'which the intracellutlar stulfatedl glveosaminogly cans w ere removed from cells by secretion and degradation, fibroblasts were inctubatedl for 2 dlays in 35S04-labeled mediim. The radioactive medium was removed, and the cells were washed, trvpsinized, and replated and incubated in unlabeled mediutm for I to 3 days. Radioactive measuirements were determined in a Beckman LS-250 li(quidl scintillationi spectrometer uitilizing a 30%f a(quieouis solultion of Insta-gel (Packar(d Instrunmenit Co., Downer's Grove. 111.) as scintillator. The couinting efficiency %vas 88%.

170



Studies on Explants

Seeing that isolated corneal fibroblasts, in contrast to freshly excised corneal explants, secrete little or no keratan sulfate in culture,28 we compared the profile of 3 S-labeled glycosaminoglycans produced by a corneal explant from a patient with MCD with that normally synthesized by human corneal explants. A portion of a corneal button excised at the time of keratoplasty from 1 patient with MCD (MSN, a 52-year-old woman, Family 3) was incubated at 37 C in a humidified tissue culture incubator in an atmosphere of 5 % C02-95% air in phosphate-buffered saline (pH 7.3) containing 750 ,uCi IIS/ml for 1 hour. As the total incorporation of radioactive isotope in corneal glycosaminoglycans is enhanced by the presence of the corneal epithelium and endothelium,23 both of these layers were maintained on the explant. After 1 hour of pulse labeling, the corneal button was immediately placed in Eagle's minimum essential medium with 10% fetal calf serum containing 10 mM HEPES buffer and no antibiotics. After 4 days' incubation at 37 C in this medium, the sulfated glycosaminoglyeans in the button and medium were analyzed. For control purposes, normal corneal tissue, excised from eyes that were enucleated with retinoblastomas (8-month-old girl, TH; 14-month-old girl, AD), Coats' disease (11-monthold boy, JE), and melanomas (54-year-old woman, LB; 76-year-old man, HS) were similarly studied. Analytical Procedures

Explants and their nutrient media were heat denatured for 10 minutes at 90 to 100 C, digested with 0.04 mg/ml of pronase (Grade B) (Calbiochem) at 37 C in 0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.0) with 0.004 M CaCl2 and 4% ethanol for 48 hours. Cold trichloracetic acid was then added to a concentration of 5%, and the precipitates were removed by centrifugation. The supernatants were extracted five times with an equal volume of ether and once with four volumes of chloroform/methanol (2: 1), then dialyzed against deionized water. The characteristics of the labeled glycosaminoglycans in the supernatants were determined by several techniques. 1. Part of these dialyzed preparations were chromatographed on Dowex 1-X2 (Cl-) without further treatment. Other portions of the material were incubated at 37 C for 18 hours with 0.1 to 0.2 units/ml of chondroitin. ABC lyase (EC 4.2.2.4) (Miles Laboratories, Elkhart, Ind.) in 0.05 M Tris buffer (pH 8.0), or with 0.01 mg/ml of bovine testicular hyaluronidase (EC 3.2.1.35) (Sigma Chemical Company, St. Louis, Mo.) in 0.15 M NaCl with 0.1 M NaH2PO4, (pH 5.3). Enzymatic digestions were carried out in the presence of 0.02% sodium azide to prevent bacterial growth. These digests were applied to 0.5 x 5 cm Dowex 1-X2 (Cl-) (AG 1-X2, 200 to 400 mesh, Bio-Rad Laboratories, Richmond, Calif.) columns and eluted stepwise with increasing concentrations of NaCl (0 to 5 M).24 Fractions of 20 to 50 ml were collected. In each case the salt concentration was increased to the next step when the eluted radioactivity dropped to 20 counts/min/ml or less above background. It was usually necessary to equilibrate the columns with each salt concentration for several hours or overnight to collect most of the "tail" of radioactive material at that concentration. This was particularly significant above 2 M NaCl. Radioactivity measurements on portions of column eluents were conducted in a Beckman LS-250 liquid scintillation spectrometer utilizing a 50% aqueous solution of Insta-gel (Packard Instrument Company, Inc., Downers Grove, Ill.) as scintillator. The counting efficiency was 88%. Chondroitin ABC lyase-resistant material eluted by 0.5 to 2 M salt solutions was pooled, dialyzed against distilled water, and lyophilized. The 3 M eluent was similarly treated. The lyophilized samples were dissolved in 1.0 ml. of 0.05 M Tris/HCl (pH 7.2). The susceptibility of these fractions to keratan sulfate-endogalactosidase 25 (kindly provided by Dr. Kiyoshi Nakazawa, Meijo University, Japan) was tested by adding this glycosidase (0.01 ml of 150 units/ml) in this same Tris buffer to aliquots and incubating them for 48 hours at 37 C (fresh enzyme was added after 24 hours). Equivalent portions that served as controls received boiled enzyme or only buffer. The reaction was terminated by heating

Vol. 89, No. 1 October 1977


171

the specimens in a boiling w-ater bath for 5 minutes. The degradatix e effect of the enzyme w-as determined bv chromatographing control and enzy me-treated specimens on Sephadex G-50, (Pharmacia Fine Chemicals Inc., Piscatawav, N.J.). 2. Aliquots of solutions containing labeled glycosaminoglycans mvere concentrated hb lyophilization and were analyzed by descending chromatography on Wrhatman :3\11\ paper in a solvent system of l-butanol-acetic acid-I N ammonia (2: 3: 1, *v) according to the method of Saito et al.' Specimens were chromatographed with and without prior trc Iment with chondroitin ABC lyase (EC 4.2.2.4), chondroitin AC lvase (EC 4 2 2 3) (!'ies Laboratories), or bovine testicular hy-aluronidase (EC 3.2.1.35) (Sigma Chemical Company). Thirty- to fifty-microliter samples were incubated in 0.1 unit of chondroitin ABC lvase. 0.3 units of chondroitin AC lvase, or 13 units testicular hyaluronidase for 18 hours at .37 C. The sulfated and nonsulfated disaccharides were readily separated in this system. Standard -1-di-4S [2-acetamido-2-deoxy-.3-O-(D-gluco-4-enepy ranos! luronic acid)4-O-sulfo-D-galactose], A-di-6S [2-acetamido-2-deoxv-3-0-(D-gluco-4-enepx ranosy-luronic acid )-6-0-sulfo-D-galactose], and A-di-OS [2-acetamido-2-deoxv--3-0(D-gluco-4-enepx ranosyluronic acid-D-galactose] (Miles Laboratories), were chromatographed along with the labeled samples. The locations of the standards wvere observed under ultraviolet light and after spraying the chromatograms with aniline hydrogen phthalate reagent and heating at 110 C.27 The positions of the labeled disaccharides derived from the corneal explants and the nutrient media were established by cutting the strips into segments and measuring the radioactivity of each strip using liquid scintillation counting. :3. Additional characterization of the glycosaminoglycans wsas carried out by treating lyophilized samples with nitrous acid to selectively degrade N-sulfated glycosaminoglycans, like heparan sulfate and heparin.2--" Portions of labeled glycosaminoglycans in 2 ml of distilled water were mixed with 1 ml of 1 N h\drochloric acid and 1 ml of freshl%prepared 20%7e n-butyl nitrite (v v-) (Kodak, Rochester. N.Y.) in absolute ethanol and allowed to react in an open vessel with gentle shaking for 2 hours at room temperature. The reaction was stopped by neutralizing with 1 ml of I N sodium hydroxide. This method completely degrades heparin and heparan sulfates by a single treatment.31 Follow-ing this, the specimens mvere dried under nitrogen, dissolved in distilled %vater, and passed through a 1.6 by 35 cm Sephadex G-50 column in 0.5 NM Tris (h\-droxymethil)aminomethane (Tris) buffer (pH 7.3). One-milliliter fractions were collected. As controls. samples of M"S-sulfatelabeled material that had not been treated with buts-l nitrite mvere similarlv chromatographed. The material not affected by nitrous acid w-as pooled. lyophilized. and redissolved in :3 ml of 0.0l \1 Tris buffer (pH 8.0), and part of it was digested w-ith chondroitin ABC lIase (EC 4.2.2.4, 0.1.33 unit ml; Miles Laboratories) for 18 hours at 37 C. Portions that mwere and were not treated with this enz%-me were chromatographed on Sephadex G.S0. In control samples, second treatment with this enzyme caused no additional degradation of material resistant to the initial treatment. Material excluded from Sephadex C-50 after treatment w-ith pronase, nitrous acid, and chondroitin ABC lvase was pooled and lyophilized. Specimens were dissolved in 1.0 ml of 0.05 Tris HCl (pH 7.2) and the susceptibility of this material to degradation by keratan sulfate-endogalactosidase -v as determined as described above. 4. The glycosaminoglycans were further analy zed by electrophoresis performed at 250 V and 3 to 4 mA for 32 hours at room temperature on cellulose acetate strips using a Beckman Microzone Electrophoresis system (Beckman Instruments, Inc.. Fullerton. Calif.) b%- a modification of the method of Seno et al." The buffer was 0.2 MI calcium acetate (pH 7.25) and ethylene glycol in a ratio of 3:2. Follosing electrophoresis the strips w-ere fixed and stained for 20 minutes with 0.2%c alcian blue in 6%c acetic acid and or cut into 1-mm segments and analyzed for radioactivity. Appropriate standards of hyaluronic acid, dermatan sulfate, chondroitin sulfates, heparin, heparan sulfate. and keratan sulfate w-ere analyzed under the same conditions. Autoradiographv wsas performed on some strips bv applying them to Osray RPI x-ray film (Agfa-Gevaert, Belgium) and developing them in the dark for variable periods of time.


172


Results Studies on Cultured Fibroblasts

The cultured corneal fibroblasts from the patients with MCD and the systemic mucopolysaccharidoses Type I-H (Hurler syndrome) and Type II (Hunter syndrome) stained inconsistently with cytochemical methods for glycosaminoglycans. Cytoplasmic inclusions in some cells yielded an affinity for alcian blue, colloidal iron, and periodic acid-Schiff, and manifested metachromasia with toluidine blue. In contrast to the corneal tissue obtained at the time of keratoplasty, the intracytoplasmic accumulations within cultured corneal fibroblasts from patients with MCD were not resistant to testicular hyaluronidase and chondroitin ABC lyase digestion. Moreover, many normal cultured corneal fibroblasts also contained accumulations within their cytoplasm with the same cytochemical characteristics as the corneal cells cultured from subjects with MCD. Metachromatic granules were identified within some cultured corneal fibroblasts with MCD, but these were qualitatively and quantitatively indistinguishable from those found in normal corneal and cutaneous fibroblasts. Incorporation of Intracellular 355 Sulfate

In conformity with the work of Fratantoni et al.,'6 cultured fibroblasts from patients with mucopolysaccharidosis Type I-H and mucopolysaccharidosis Type II accumulated intracellular 35S sulfate at a linear rate over a period of 4 days (Text-figure 1). Cutaneous and corneal fibroblasts 0 C6000-

5000 E E

TEXT-FIGURE 1-Incorporation of intra-

4000

celluilar 35S sulfate bv cultured fibroblasts from patients with systemic mucopolysacchari+doses, (Hurler) (open triangles),

O

3000 ° 3000-

.°

/

2000

CL

r- 1000-

6'n 13

2

3

Time (days)

/

Type I-H Type 11 (Hunter) (solid triangles), macular

corneal dystrophy (solid circles), and normal individuials (open circles).



173

from normal individuals reached a steady state within 2 days. In contrast to patients with systemic mucopolysaccharidoses, in 4 patients with MCD, a comparable abnormal accumulation of 35S sulfate labeled glycosaminoglycans did not occur in the corneal fibroblasts. Removal of Prelabeled Intracellular Sulfated Glycosaminoglycans

The radioactive glycosaminoglycan content of cultured human corneal and cutaneous fibroblasts declined with time, and normal cells only contained about 16.5% of the labeled material by 3 days. In mucopolvsaccharidosis Type I-H and Type II, 80% or more of the labeled material was still present within cutaneous fibroblasts after 3 days. The corneal fibroblasts from patients with NICD more closely resembled normal cells than those from patients w-ith systemic mucopolvsaccharidoses (Text-figure 2). Secretion of Glycosaminoglycans

Sulfated glycosaminoglycans were secreted into the medium by all cells studied at a linear rate over the time period investigated. The rate of secretion bv fibroblasts from patients with NICD, mucopolysaccharidosis

90

270-

TEXT-FIGIRE 2-Remosal of prelabeled intracellular sulfated glycosaminoglycans by cultured fibroblasts from individuals w-ith macular corneal dystrophv (solid circles). s%-stemic mucopolysaccharidoses T-pe 1-H (Hurler s\-ndrome) (open triangles) and Type II (Huinter s-ndrome) -solid triangles. and normal controls (open circles).

o - 60-

\ ' * 50

\ \

\

\ 30

Time (days)



174

Type I-H, and mucopolysaccharidosis Type II did not differ significantly from the controls (Text-figure 3). Studies With Corneal Explants

All normal corneal extracts synthesized a prominent fraction of 35S labeled glycosaminoglycans, which eluted from Dowex 1-X2 (Cl-) with 3 M sodium chloride after digestion with chondroitin ABC lyase (Textfigure 4). In individual cases the amount varied, being 19.9%, 8.7%, and 6.8% in the corneas obtained from subjects that were 8 months, 11 months, and 76 years old, respectively. Approximately 85% of this fraction was susceptible to keratan sulfate-endogalactosidase. Almost 98% of the glycosaminoglycans produced by normal corneal explants which eluted with lower concentrations of sodium chloride were degraded by chondroitin ABC lyase or keratan sulfate-endogalactosidase. Althought corneal keratan sulfate has traditionally been identified by ion exchange chromatography on Dowex 1 (Cl-) by its elution in 3 M NaCl fractions, we, like Hart,30 identified a considerable amount of keratan sulfate-endogalactosidase-sensitive material that eluted at lower salt concentrations. Ninety-three percent of the 0.5 to 2 M portion of the chondroitin ABC 1000 A

900

0

A /

800

2/

E

700-

E6

600__ 500 c

A

o

oU / ^ E

XA / ^

o02~~~~~~~~~

,8 400

o_

TEXT-FIGUCRE 3-Secretion of intno the me(liim bh fibroblasts from patienits with macolar corneal

soilfated glycosaminoglycans

X*

dvstrophy (solid circles), systemic Imtocopolysaccharidoses, Type H (open triangles), rype II (solid triangles), and normal controls (open circles).

0

Time (days)



175

lyase-resistant fraction was degraded by keratan sulfate-endogalactosidase. In striking contrast to normal human corneal explants, the explant from the patient with MCD produced different sulfated glycosaminoglyeans. Noteworthy was the observation that virtually all of the MS-labeled glycosaminoglycans could be eluted from Dowex 1-X2 (Cl-) by sodium chloride with a molarity of less than 2. Unlike the explants of normal human corneas, only 0.9%-a quantity within the experimental error of the analytic technique -luted with 3 M sodium chloride where keratan sulfate normally appears. Almost all of the labeled glycosaminoglycans synthesized by the cornea with MCD could be degraded with chondroitin ABC lyase or keratan sulfate-endogalactosidase. A small fraction susceptible to nitrous acid was identified in the MCD and control explants. A comparison of the sulfated glycosaminoglycans produced by the corneal explants with MCD and normal human corneas is summarized in Table

v

-5S C

TEXr-FIGuRES 4 and 5-Elu-

tion profile from Dowex 1-X2 (Cl-) of TMS-sulfate-labeled gl cosaminogly cans synthesized bv normal human corneal explants (based on average observations from three different corneas, TH, JE, HS) (4), and corneal explant from individual with macular corneal dystrophy (5) after incubation in S"labeled medium. The solid line indicates the elution pattern without chondroitin ABC Ivase (CHase ABC) digestion, while dotted line indicates the effect of this enzvme. Most of the 0.5 to 2 NI portion of the chondroitin ABC hIase-resistant fractions was degraded by keratan sulfate-endogalactosidase.

u

M NoCI

I 7V soSO

0D

I'

.60 I I

I'I I1 I

0

.1 50 I I 2 II I I I 40 I1 I

I'

0.;23 20

A% 1% It UUL

CL5 1% c

LWO I P%

SU L 2.0

I I

.1

- -

3.0

M NoCI

4--0

5.0

176



Table 1-Sulfated Glycosaminoglycans Produced by Normal and Macular Corneal Dystrophy Explants*

Normal Macular corneal dystrophy

LB

AD

JE

Chondroitin-4-sulfate Dermatan sulfate

33.0 ± 0.3% 4.4 ± 0.1% 48.3 ± 0.5%

8.8 ± 0.1 1.6 ± 0.1 27.6 ± 0.3

12.4 ± 0.1 3.2 ± 0.1 38.4 ± 0.4

6.3 ± 0.1 9.5 ± 0.1 36.6 ± 0.4

Keratan sulfate-like materialt Fraction a Fractionb Fractions a + b Heparan sulfate-like materialt Unidentified fractions

7.0 ± 1.0% 1.0±1.0% 8.0 ± 2.0% 2.3 ± 1.0% 4.0 ± 1.0%

34.5 ± 0.3

15.9 ± 0.2 15.1 ±0.2 31.0 ± 0.3 9.8 ± 0.1 5.2 ± 0.1

32.2 ± 0.3

Glycosaminoglycan

Chondroitin-6-sulfate

17.2±0.2 51.7 i 0.5 5.9 i 0.1 4.4 ± 0.1

9.3±0.1 41.5 i 0.4 2.8 ± 0.1 3.3 i 0.1

* Expressed as percentage S68 incorporation as caiculated from results obtained with several analytic techniques. t Chondroitin ABC lyase and nitrous acid resistant but susceptible to keratan sulfateendogalactosidase. Fractions a and b include material that elutes from Dowex-1 X2 (Cl-) with 0 to 2 M and 3 to 5 M salt concentrations, respectively. t Degraded by nitrous acid.

1. Aside from manifesting impaired corneal keratan sulfate synthesis, the cornea with MCD produced a greater percentage of chondroitin-6sulfate than normal. Analyses of the nutrient media revealed similar glycosaminoglycans to the explants, but the quantities of labeled material were less than in the corneal tissues. Discussion

The basic abnormality in MCD clearly resides in the nuclear DNA. In 1964, Klintworth and Vogel 8 proposed that this disease is a genetically determined metabolic storage disorder manifestly restricted to the cornea and characterized by an intracellular and extracellular accumulation of excessive quantities of glycosaminoglycans (mucopolysaccharides). This view was based on a consideration of the genetic, light and electron microscopic morphologic attributes, and the histochemical characteristics of the disease. The accumulations stain positively with periodic acid-Schiff,2 4 8,11,12,14,33 35 alcian blue,12"1636 metachromatic dyes,3'4'8"11,12 and possess an affinity for colloidal iron.2-5,8,11,1,534 Aside from these light microscopic cytochemical properties, the accumulations can be stained for electron microscopic examination with the periodic acidSchiff-thiocarbohydrazide-silver proteinate 36 and periodic acid-silver metheneamine techniques.3839 Subsequent investigations have provided additional support for the concept that MCD is a localized mucopolysaccharidosis. 1-5,9,11-15,33,34 Also, the fact that corneal stromal and endothelial cell synthesize glycosaminoglycans 23,40 makes it reasonable to accept the



177

premise that the corneal deposits in MCD are products of its own cellular elements.9 By drawing from the analogy between NMCD and the systemic mucopolvsaccharidoses, one is led to suspect an enzymatic defect in the degradation of one or more comeal glycosaminoglycans. The cornea contains several of these polysaccharides which warrant consideration, namely keratan sulfate, chondroitin-4-sulfate (chondroitin sulfate A), chondroitin-6-sulfate (chondroitin sulfate C), and low sulfated glycosaminoglycans.23 The persistence of the staining qualities of the accumulations after testicular hyaluronidase digestion 4,8 precludes hyaluronic acid, chondroitin-4-sulfate, and chondroitin-6-sulfate as significant components, while their failure to become digested by chondroitin ABC lyase rules out dermatan sulfate, too.4' B3y exclusion, the number one suspect is keratan sulfate, which accounts for the greatest quantity of the sulfated glycosaminoglycans in the normnal comea. This contention is supported by the affinity of the deposits for alcian blue at low pH with magnesium chloride concentrations of up to 0.8 M-an attribute of keratan sulfate.44' Also, of the aformentioned histochemical quartet that demonstrate the deposits, the periodic acid-Schiff may react with keratan sulfate, while chondroitin sulfates, hyaluronic acid, and other glvcosaminoglvcans are not usually visualized by this reaction.-",-3 A storage disease involving comeal keratan sulfate wvould account for the apparent limitation of the disease to the comea. From a clinical standpoint the disease is restricted to corneal opacification. If lesions of comparable size exist in other nontransparent structures they could easily be asvmptomatic and overlooked in tissue sections stained with hematoxvlin and eosin. To date a detailed examination of all tissues in patients with MCD has not been performed. Particularly important would be an examination of cartilage-a tissue with manv chemical similarities to the cornea. Be this as it mav, histologic observations on several tissues (skin, breast, fallopian tube, ovary, uterus, oral mucosa, thyroid) excised from patients for miscellaneous diseases and a single autopsy, albeit be incomplete, have not disclosed lesions in tissues other than the cornea.S 34 Keratan sulfate exists in cartilage and bone as well as in the cornea, but it is now recognized that corneal keratan sulfate (keratan sulfate I) is unique to the cornea and differs in severpil respects from cartilaginous keratan sulfate (keratan sulfate II).3 42- The predominant structural feature of both types of keratan sulfate is a core portion consisting of sulfated galactose and N-acetyl-glucosamine residue>. With corneal keratan sulfate, the carbohydrate polynr¢r is linked by alkali-stable N-glvcoside bonds to asparagine residues to core protein.42'45 The carbohydrate-pep-

178



tide bond of cartilaginous keratan sulfate, on the other hand, appears to involve N-acetylgalactosamine O-glycosidically linked to serine and threonine residues by an alkali-labile bond." The average length of the polymer chains, the degree of branching, and the susceptibility to keratan-endogalactosidase degradation are dissimilar in corneal and cartilaginous keratan sulfate.31'42 Significant differences also exist in the predominant amino acids and in the relative amounts of glucosamine, galactosamine, and sialic acid. Graf et al.35 have argued that the propensity of the deposits to stain with periodic acid-Schiff and alcian blue favors the conclusion that the stored material is a glycoprotein rather than an acid mucopolysaccharide (glycosaminoglycan). But negative cytochemical reactions for proteins 4,8 attest against a significant proteinaceous component in the stored material. To some extent the question of whether the disease involves a glycoprotein or a glycosaminoglycan is academic. The polymers of keratan sulfate, like those of other glycosaminoglycans, are linked covalently to core protein. Also, despite the traditional consideration of keratan sulfates as glycosaminoglycans, there is an increasing amount of evidence to indicate that it would be more realistic to regard them as sulfated glycoproteins. They lack uronic acid, a constituent of other glycosaminoglycans, and contain sugars characteristic of glycoproteins including galactose, glucosamine, galactosamine, mannose, sialic acid, and fucose." The literature contains very few reports on the application of tissue culture techniques to MCD.33'47"8 Danes 47 studied cultured fibroblasts from the cornea, conjunctiva, and skin of 6 patients with MCD and reported that the uronic content was similar to controls. Francois et al.33 maintained that they could detect differences between normal corneal fibroblasts and those from individuals with MCD by culturing cells in medium containing the vital dye acridine orange. We failed to confirm their observations in a study involving cultured corneal fibroblasts from 2 patients with MCD and appropriate controls.49 In many inherited diseases, attention has been given in the past to the presence of metachromatic granules in cultured cells.48 As pointed out under Results, we observed metachromatic granules in some cultured fibroblasts from patients with MCD and the systemic mucopolysaccharidoses at.a pH that does not diminish the catabolism of sulfated glycosaminoglyeans,50 but we also identified indistinguishable granules in cultured fibroblasts from normal corneas and skin. In an unpublished study, Cotlier et al.51 explored the activity of several glycosidases (lysosomal enzymes degradative for certain glycosaminoglycans and glycoproteins) in cultured limbal conjunctival fibroblasts from a patient with MCD. They detected a relative



179

deficiency of a-galactosidase, wvhich they also found to be defective in a fragment of corneal tissue. However, this observation could not be confirmed in other specimens from MCD-affected patients. As the synthesis of keratan sulfate by the cornea of man and other species either ceases or markedly decreases in culture,L 4o 525 our failure to obtain 3S kinetic data comparable to the systemic mucopolysaccharidoses Type I-H (Hurler syndrome) and Type II (Hunter svndrome) in cultured corneal fibroblasts with NMCD remains consistent with the contention that this disease is a disorder of keratan sulfate I catabolism. If cultured corneal fibroblasts do not synthesize significant quantities of keratan sulfate, its intracvtoplasmic accumulation obviously will not occur, even if keratan sulfate cannot be degraded because of an inherited deficiency of a critical enzy me. Why the DNA in comeal fibroblasts should curtail its ability to express the gene for keratan sulfate synthesis in culture remains to be determined. Relevant to this point is the observation that the synthesis of some specific marcomolecules by other differentiated cell types does not take place in vitro unless adequate conditions are obtained.55-59 Our knowledge of the mechanisms controlling the synthesis and degradation of corneal glycosaminoglvcans unfortunately remains rudimentan-. The minute amount of material studied precluded a precise identification of all glycosaminoglvcans synthesized by the corneal explants. The chondroitin ABC Ivase-resistant fraction of 35S labeled glvcosaminoglvcans synthesized by corneal explants which was eluted from Dowex 1-x2 (ClI-) by 3 NM sodium chloride is believed to be keratan sulfate for several reasons: a) with the analytic procedure employed, keratan sulfate elutes from Dowex I-X2 (Cl-) primarily at 3 M NaCl and chondroitin sulfate at 1 3M NaCl,24 " b) as shown in the present investigation and by others," most of the 3 N1 fraction that is resistant to nitrous acid and also chondroitin ABC lyase digestion is sensitive to keratan sulfate-endogalactosidase; and c) the same fraction is not produced by sclera-a tissue Which lacks keratan sulfate23 Our findings on the corneal explant w-ith MCD, taken w-ith the aforementioned, suggest that the synthesis of keratan sulfate and other sulfated glycosaminoglvcans are altered in MICD. References 1. Bliimcke S. Thiel HJ. Niedorf HR: Licht- und elektronenmikroskopische Untersuchungen iuber die fleckformige Hornhautdystrophie. Ophthalmologica 164:35-49. 1972 2. Francois J. Hanssens NM, Teuchy H, Sebruyns N: Ultrastructural findings in corneal macular dystrophy (Groenouw II Type). Ophthalmol Res 1:80-98, 19735 :3. Francois J. 'ictoria-Troncoso V: Histopathogenic study of the macular dystrophy

180



of the cornea: Fehr's dystrophy or Groenouw's Type II. Ophthalmol Res 7:261-269, 1975

4. Garner A: Histochemistry of corneal macular dystrophy. Invest Ophthalmol 8:475-483, 1969 5. Herrmann J, Meythaler H: Licht- und electronenmikroskopische Untersuchungen bei Dystrophia corneae maculosa. Albrecht von Graefes Arch Klin Ophthalmol 181:165-178, 1971

6. Jones ST, Zimmerman LE: Macular dystrophy of the cornea (Groenouw type II): Clinicopathologic report of two cases with comments concerning its differential diagnosis from lattice dystrophy (Biber-Haab-Dimmer). Am J Ophthalmol 47:1-16, 1959

7. Jones ST, Zimmerman LE: Histopathologic differentiation of granular, macular and lattice dystrophies of the cornea. Am J Ophthalmol 51:394-410, 1961 8. Klintworth GK, Vogel FS: Macular corneal dystrophy: An inherited acid mucopolysaccharide storage disease of the corneal fibroblast. Am J Pathol 45:565-586, 1964 9. Klintworth GK: Current concepts on the ultrastructural pathogenesis of macular and lattice corneal dystrophies. Birth Defects 7:27-31, 1971 10. Lorenzetti DWC, Kaufman HE: Macular and lattice dystrophies and their recurrences after keratoplasty. Trans Am Acad Ophthalmol Otolaryngol 71:112-118, 1967 11. Morgan G: Macular dystrophy of the cornea. Br J Ophthalmol 50:57-67, 1966. 12. Seitz R, Goslar HG: Beitrag zur Klinik, Morphologie und Histochemie der verschiedenen Formen von Hornhautdystrophie. Klin Monatsbl Augenheilkd 147:673-691, 1965 13. Snip RC, Kenyon KR, Green WR: Macular corneal dystrophy: Ultrastructural pathology of corneal endothelium and Descemet's membrane. Invest Ophthalmol 12:88-97, 1973 14. Teng CC: Macular dystrophy of the cornea: A histochemical and electron microscopic study. Am J Ophthalmol 62:436-454, 1966 15. Tremblay M, Dube I: Macular dystrophy of the cornea: Ultrastructure of two cases. Can J Ophthalmol 8:47-53, 1973 16. Fratantoni JC, Hall CW, Neufeld EF: The defect in Hurler's and Hunter's syndromes: Faulty degradation of mucopolysaccharides. Proc Natl Acad Sci USA 60:699-706, 1968 17. McManus JFA, Mowry RW: Staining Methods: Histologic and Histochemical, New York, Paul B, Hoeber, Inc., 1960 18. Bostrom H, Aqvist S: Utilization of S35-labelled sodium sulfate in the synthesis of chondroitin sulfuric acid, taurine, methionine, and cystine. Acta Chem Scand 6:1557-1559, 1952 19. Jennings MA, Florey HW: Autoradiographic observations on the mucous cells of the stomach and intestine. Q J Exp Physiol 41:131-152, 1956 20. Pasternak CA, Kent PW: Biosynthesis of intestinal mucins. II. Incorporation of [35S] sulphate by guinea-pig colon in vitro. Biochem J 68:452-457, 1958 21. Lowry OH, Rosebrough NJ, Farr Al, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 22. Doetsch K, Gadsden RH: Determination of urinary total protein by use of gel filtration and a modified biuret method. Clin Chem 21:778-781, 1975 23. Klintworth GK, Smith CF: A comparative study of extracellular sulfated glycosaminoglycans synthesized by rabbit corneal fibroblasts in organ and confluent cultures. Lab Invest 35:258-263, 1976 24. Schiller S, Slover GA, Dorfman A: A method for the separation of acid mucopolysaccharides: Its application to the isolation of heparin from the skin of rats. J Biol Chem 236:983-987, 1961



181

23. Nakazaw-a K, Suzuki S: Purification of keratan sulfate-endogalactosidase and its action on keratan sulfates of different origin. J Biol Chem 250:912-917, 1975 26. Saito H, Yamagata T, Suzuki S: Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem 243:1536-1542, 1968 27. Partridge SM: Aniline hydrogen phthalate as a spraying reagent for chromatography of sugars. Nature 164:443, 1949 28. Cifonelli JA, King J: The distribution of 2-acetamido-2-deoxv-D-glucose residues in mammalian heparins. Carbohydr Res 21:173-186, 1972 29. Cifonelli JA, King J: Structural studies on heparins with unusually high N-acetylglucosamine contents. Biochim Biophys Acta 320:331-340, 1973 :30. Hart GW: Biosynthesis of glycosaminoglycans during corneal development. J Biol Chem 251:6513-6521, 1976 31. Conrad G%W, Hart GW: Heparan sulfate biosynthesis by embnronic tissues and primary fibroblast populations. Dev Biol 44:253-269, 1975. 32. Seno N, Anno K, Kondo K, Nagase S, Saito S: Improved method for electrophoretic separation and rapid quantitation of isomeric chondroitin sulfates on cellulose acetate strips. Anal Biochem 37:197-20'2, 1970 .3:3. Francois J, Victoria-Troncosco V, Maudgal PC. Victoria-Ihler A: Study of the lysosomes by vital stains in normal keratocytes and in keratocytes from macular dystrophy of the cornea. Invest Ophthalmol 15:599-605, 1976 :34. Ghosh N,MMcCulloch C: Niacular corneal dystrophy. Can J Ophthalmol 8:515-526, 1973 :35. Graf B, Pouliquen Y, Frouin NI-A, Faure J-P, Offret G: Cytochemical study of macular dystrophy of the cornea (Groenouw- II): An ultrastructural study. Exp Eye Res 18:163-169, 1974 ,36. Quintarelli G: Mlethods for the histochemical identification of acid mucopolysaccharides: A critical evaluation. Chemical Physiology of Nlucopolvssaccharides. International Symposium on The Chemical Physiology of NMucopolysaccharides, NMilan, 1965. Edited by G Quintarelli. Boston, Little, Brown & Co.. 1968 37. Zugibe FT: N\ucopolv-saccharides of the arterial wall. J Histochem Cvtochem 1 1:35-39, 1963 :38. Klintworth GK, NMcCracken JS: Ultrastructure of human comeal diseases. Electron NMicroscopy in Human NMedicine. Edited by JV' Johannessen. New York, NlcGrawHill Book Co.. 1977 (In press) 39. Tripathi RC, Ashton N: Application of electron microscopy to the study of ocular inbom errors of metabolism. Birth Defects 12:69-104, 1976 40. Yue BYJT, Baum JL, Silbert JE: The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells. Biochem J 158:567-573, 1976 41. Klintworth GK: Unpublished observations, 1977 42. Bhavanandan V'P, NMeyer K: Studies on keratosulfates: NMethylation and partial acid hydrolysis of bovine comeal keratosulfate. J Biol Chem 242:4352-4359, 1967 43. Hirano S, NMever K: Enzymatic degradation of corneal and cartilaginous keratosulfates. Biochem Biophys Res Commun 44: 1371-1375, 1971 44. Mathews NIB, Cifonelli JA: Comparative biochemistry of keratosulfates. J Biol Chem 240:4140-4145, 1965 45. Seno N, NMever K, Anderson B, Hoffman P: Variations in keratosulfates. J Biol Chem 240:1005-1010, 1965 46. Spiro RG: Glycoproteins: Their biochemistry, biology and role in human disease. N Engl J MIed 281:991-1001, 1969 47. Danes BS: Corneal clouding in the genetic mucopolysaccharidoses: A cell culture study. Clin Genet 4:1-7. 1973 48. Klintw-orth GK: Tissue culture in the inherited corneal dystrophies: Possible applications and problems. Birth Defects 12:115-132, 1976

182



49. Klintworth GK, Hawkins HK, Smith CF: Acridine orange particles in cultured fi)roblasts: A comparative study of macular corneal dystrophy, systemic mucopolvsaccharidoses and normal controls. (Unpublished data) 50. Lie SO, McKusick VA. Neufeld EF: Simulation of genetic mucopolysaccharidoses in normal humani fibroblasts by alteration of pH of the medium. Proc Natl Acad Sci USA 69:2361-2363, 1972 51. C,otlier E, Yamashiroya H, Hughes WF: Unpublished observations 52. (Conrad GW, Dorfman A: Synthesis of sulfated mucopolvsaccharides by chick corneal fibroblasts in vitro. Exp Eye Res 18:421-433, 1974 53. Gradinger MC, Schwater-Huibner ME: Biosynthesis of glycosaminoglycans bL mammaliarn corneal epithelium and fibroblasts in vitro. I. Isolation and fractionation differences of GA(; from the two cell types. Albrecht von Graefes Arch Klin Oplhthalinol 196:9-19, 1975 54. (Gra(dinger MIC, Sclhwater-Hflbner ME: Biosynthesis of glycosaminoglycans bL marnmalian corneal epithelium and fibroblasts in vitro. II. Approach to specify the GAG from the two cell tvpes. Albrecht von Graefes Arch Klin Ophthalmol 196:21-30, 1975 55. Okada TS, Eguchi G, Takeichi M: The expression of differentiation bv chicken lens epithelium in in vitro cell culture. Dev Growth Diff 13:323-336, 1971 56. Mlichalopoulos G, Pitot HC: Primary culture of parenchymal liver cells on collagen membranes: NIorphological and biochemical observations. Exp Cell Res 94:70-78, 1975 57. Rodesch F: Differentiation, contact inhibition and intercellular communication in retinial pigment cells. Exp Cell Res 76:55-62, 1973 58. Bissell DM, Hammaker LE, Meyer UA: Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J Cell Biol 59:722-734, 1973 59. Phillips MJ, Oda M, Edwards VD, Greenberg GR, Jeejeebhoy KN: Ultrastructural and functional studies of cultured hepatocytes. Lab Invest 31:533-542, 1974

Acknowledgments We are grateful to Dr. K. Nakazawa for a gift of keratan sulfate-endogalactosidase and to Drs. \M. B. 'Mathews, J. A. Cifonelli, and L. Roden for providing reference standards of several glvcosaminoglycans ob)tained from animal connective tissues.