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Trabecular Meshwork Glycosaminoglycans in Human and Cynomolgus Monkey Eye Ted S

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Trabecular Meshwork Glycosaminoglycans in Human and Cynomolgus Monkey Eye Ted S Trabecular Meshwork Glycosaminoglycans in Human and Cynomolgus Monkey Eye Ted S. Acorr,*t Mary Wesrcorr,* Michael 5. Posso,* and E. Michael Van Duskirk* The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non- human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate elec- trophoresis. For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed. The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical. The human TM contains hyaluronic acid (HA), chondroitin- 4-sulfate and/or 6- sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used. Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 29.0% HA, 14.1% CS, 21.5% DS, 20.3% KS, and 15.0% HS. The cynomolgus monkey trabecular GAGs include: 12.8% HA, 14.3% CS, 15.2% DS, 42.1% KS, and 15.6% HS. Invest Ophthalmol Vis Sci 26:1320-1329, 1985 The preponderance of evidence suggests that the We have examined the extracellular matrix GAGs primary site of aqueous outflow resistance resides of the human and nonhuman primate trabecular within the trabecular meshwork and possibly within meshwork as the initial step in studies intended to clar- the deep portion of the corneoscleral meshwork and/ ify the role that GAGs play in the regulation of aqueous or the amorphous juxtacanalicular basement mem- outflow. In essentially all of the tissues studied to date, brane near the canal of Schlemm.1"3 Extracellular ma- the GAGs are found as components of larger proteo- trix appears to comprise a significant portion of this glycans and proteoglycan aggregates; the GAGs appear tissue and probably of the aqueous outflow barrier in to function only as a part of these larger molecules.14 primates.3"13 Quantitative aqueous perfusion5-6 and The GAG distribution profile of the trabecular mesh- histochemical7"13 studies with degradative enzymes work of enucleated primate eyes was determined as a have lead to the suggestion that glycosaminoglycans prelude to studying these more complex proteoglycans (GAGs) may be a key physiologic component of the and to serve as reference points for studies of the reg- resistance to the outflow of aqueous humor. The GAGs ulation of the biosynthetic and degradative activities may reduce the functional diameter of the flow chan- of the human trabecular meshwork in explant organ nels through the deep corneoscleral intertrabecular culture. spaces and/or regulate flow through the juxtacanali- cular basement membrane. Recently, significant biochemical advances in the Materials and Methods techniques for characterizing GAGs, and the proteo- Materials glycans of which they are usually a component, have 14 been reported. Micro techniques have also been de- Human eye bank eyes were obtained from 6 to 72 veloped, which facilitate the analysis of GAGs from 15 20 hr after enucleation and used immediately or stored small tissues. " These advances make the analysis of frozen at -20°C. Cynomolgus monkey (Macacafas- the unlabeled GAGs from the primate trabecular 420 cicularis) and a few Rhesus monkey (Macaca mulatto) meshwork feasible. eyes were enucleated at autopsy and used immediately or stored frozen at -20°C. Hyaluronidase (Hyaluro- nate lyase, EC 4.2.2.1, from Streptomyces hyaluroly- From the Departments of Ophthalmology* and Biochemistry,! School of Medicine, Oregon Health Sciences University, Portland, ticus), Chondroitinase AC (Chondroitin sulfate AC Oregon. lyase, EC 4.2.2.5, from Arthrobacler aurescens), Chon- Supported in part by grants from the USPHS (EY-03279), the droitinase ABC (Chondroitin sulfate ABC lyase, EC American Health Assistance Foundation: National Glaucoma Re- 4.2.2.4, from Proteus vulgaris), Keratanase (Keratan search Program, and Research to Prevent Blindness. sulfate endo-B-galactopyranosyl-glycanohydrolase, Submitted for publication: March 30, 1984. from Pseudomonas species), Heparitinase (heparitin Reprint requests: Ted S. Acott, PhD, Department of Ophthal- mology, LI05, 3181 SW Sam Jackson Park Road, Portland, OR sulfate lyase, EC 4.2.2.8, from Flavobacterium hepar- 97201. inum), keratan sulfate, chondroitin-4-sulfate, chon- 1320 Downloaded from iovs.arvojournals.org on 09/25/2021 No. 10 PRIMATE TRADEOJLAR MESH WORK GLYCOSAMINOGLYCANS / Acorr er ol. 1321 droitin-6-sulfate, dermatan sulfate, heparan sulfate, and 10% ethanol16; and scanned without clearing on an hyaluronic acid were purchased from Miles Labora- Helena Laboratories Rapid-Scan (Beaumont, TX) or tories, Inc; Elkhart, IN (prepared by Seikagaku Kogyo; a Beckman model CDS-200 densitometer. Areas under Tokyo, Japan); papain, cysteine hydrochloride, hyal- the curves were determined on a Zeiss Digiplan MOP- uronidase (from bovine testes), heparin, deoxyribo- 3 image analyzer (Carl Zeiss, Inc., Oberkochen, West nuclease I (EC 3.1.21.1) and ribonuclease A (EC Germany). Uniform quantities of standards, usually 3.1.27.5) were purchased from Sigma Chemical Co (St. hyaluronic acid and chondroitin-4-sulfate, were in- Louis, MO); bovine testicular hyaluronidase was also cluded on all runs for quantitative comparisons be- purchased from Worthington (Freehold, NJ); Bio-Gel tween runs. In separate experiments, a "relative dye P-6DG (desalting gel) was purchased from Bio-Rad binding factor" for Alcian Blue staining and interaction Laboratories, Richmond, CA; Alcian Blue 8GX was was determined for each type of commercially available purchased from Aldrich Chemicals (Milwaukee, WI) GAG applied to CAE strips. Several concentrations of and n-butyl nitrite was purchased from American Sci- each GAG were scanned densitometrically and the entific (McGraw Park, IL). All other chemicals were areas were used to evaluate linearity within the ranges the best Reagent Grade available. of interest and to obtain estimates of the dye binding factors of each type of GAG relative to HA for com- parison with unknowns. Methods Identification of GAGs by sequential enzymatic digestions: Measurement of the GAG profiles of the Extraction of GAGs from tissues: The trabecular respective eye structures was accomplished by a mod- meshwork, cornea, iris, ciliary body and a 5-mm ring ified version of methods previously described.151719 of the sclera were removed from enucleated eye bank Enough sample or standard to produce good visible or cynomolgus monkey eyes and separated under a bands when stained after CAE using 0.25-/zl applica- Wild M5D (Heerbrugg, Switzerland) dissecting scope. tions, was dissolved in 30 /x\ of water and a 5-n\ aliquot The tissues were blotted and weighed. Lipids were ex- was removed for electrophoresis on CAE. The remain- tracted for 24 hr with one change, 10 volumes each, ing 25 /xl were diluted to 100 /A final volume with the of chloroform:methanol (2:1). The tissues were rinsed first enzyme buffer to give the final concentrations given in acetone, dried, weighed, and digested with papain below, the enzyme digestion was completed and the for 24 to 48 hr at 65 °C. The papain incubation buffer reaction was terminated by the addition of 3 volumes was 5 mM ethylenediaminetetraacetic acid (EDTA), 2 of cold 5 mM potassium acetate in ethanol. The GAGs mM cysteine hydrochloride, 200 mM sodium acetate were precipitated over night at -20°C, centrifuged at at pH 5.5, and contained 50 units of papain per mil- 12,000 X g for 90 min at 4°C, the supernatant was liliter and 1 ml of incubation mixture per 10 eyes. The removed and the pellet was dried and resuspended in papain and other protein remaining were precipitated 25 n\ of water. A 5-/il aliquot was removed for CAE by incubation with 10% (final volume) trichloroacetic and the remainder was diluted to 100 /nl with the next acid (TCA) for 2 hr at 4°C and were centrifuged for enzyme solution; the process was repeated removing 15 min at 4°C and 12,000 X g; the pellet was rinsed a 5-/A aliquot and reducing the sample volume until once and the pooled supernatants were applied to Bio- all the steps were completed. We eliminated the TCA gel P-6DG (desalting-gel) columns to separate TCA and precipitation step after each treatment161719 because other small compounds from the GAGs. The high mo- we found that our dermatan sulfate standards were in- lecular weight fractions were pooled, the GAGs were completely precipitated from 10% TCA solutions by precipitated overnight at -20°C with three volumes of ethanol (data not shown). absolute ethanol containing 5% potassium acetate and The sequence of treatment buffers (final concentra- centrifuged for 90 min at 4°C and 12,000 X g. This tions), enzymes, incubation conditions (including our partially purified fraction was then redissolved in water modifications of reported methods), and components for cellulose acetate electrophoresis (CAE) or sequential degraded were: enzymatic digestion. Cellulose acetate electrophoresis: The various GAGs 1. Streptomyces hyaluronidase (5 TR units) in 20 and standards were applied to cellulose acetate strips mM sodium acetate, 2 mM EDTA, 150 mM sodium (Microzone Plus, Beckman Instruments, Fullerton, chloride, pH 5.0, was incubated with the sample for 5 CA) with a 0.25-/xl applicator and electrophoresed in hr at 60°C to degrade hyaluronic acid.2122 a Beckman Microzone model R-101 electrophoresis 2. Chondroitinase AC (1 unit) in 20 mM Tris (tris cell at room temperature for 2 hr at 4 mA using 0.3 (hydroxymethyl) aminomethane), 50 mM sodium ac- M cadmium acetate buffer (pH 4.1).1618 Strips were etate, 100 mM sodium chloride, pH 8.0, was incubated stained for 15 min in 1% Alcian Blue, 5 mM sodium with the sample for 5 hr at 37°C to degrade chon- acetate in 50% ethanol; destained in 5% acetic acid in droitin-4 and 6-sulfates.2324 Downloaded from iovs.arvojournals.org on 09/25/2021 1022 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1985 Vol.
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