Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture.
OBJECTIVE
Glycosaminoglycans (GAGs) have been implicated in the regulation of outflow resistance of aqueous humor flow through the trabecular meshwork (TM). Their role was further investigated by assessment of the effects of chlorate, an inhibitor of sulfation, and beta-xyloside, which provides a competitive nucleation point for addition of disaccharide units, in anterior segment perfusion culture.


METHODS
Outflow facility was measured in perfused porcine and human anterior organ cultures treated with 20 or 50 mM sodium chlorate, or 1 mM beta-xyloside. Perturbation of extracellular matrix (ECM) components was assessed in paraffin-embedded sections by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells.


RESULTS
Outflow facility increased in porcine eyes with chlorate (3-fold) and beta-xyloside (3.5-fold) treatments. In human eyes, outflow increased approximately 1.5-fold and took longer (>48 hours) to occur. By confocal microscopy, immunostaining for chondroitin and heparan sulfates was observed on edges of human TM beams in nontreated eyes, with intense staining in the juxtacanalicular tissue (JCT) region. In treated eyes, staining of beam edges was severely reduced and was instead found in plaques. Chlorate treatment resulted in a striated pattern of GAG staining in the human JCT region. Fibronectin immunostaining was altered in beta-xyloside-treated eyes, whereas in cell culture, chlorate induced formation of thick fibronectin fibrils, to which tenascin C colocalized.


CONCLUSIONS
Disrupting GAG chain biosynthesis increased outflow facility in perfusion culture and induced atypical ECM molecule interactions in cell culture. This study provides direct evidence of the critical role of GAG chains in regulating outflow resistance in human TM.
Keller
Kate E
KE
Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97239-4197, USA.
Bradley
John M
JM
Kelley
Mary J
MJ
Acott
Ted S
TS
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D002478:Cells, Cultured; D002704:Chlorates / Q000494:pharmacology*; D002809:Chondroitin Sulfates / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006027:Glycosides / Q000494:pharmacology*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D009924:Organ Culture Techniques; D013552:Swine; D019063:Tenascin / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism
D002704:Chlorates; D002809:Chondroitin Sulfates; D005353:Fibronectins; D006025:Glycosaminoglycans; D006027:Glycosides; D006497:Heparan Sulfate; D006820:Hyaluronic Acid; D019063:Tenascin; C010807:xylosides; C032706:sodium chlorate