Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture
Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture
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DOI：
10.1167/iovs.07-0903
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发表时间：
2008-06-01
影响因子：
4.4
通讯作者：
Acott, Ted S.
中科院分区：
文献类型：
--
作者：
Keller, Kate E.;Bradley, John M.;Acott, Ted S.
目的.糖胺聚糖（GAG）参与调节通过小梁网（TM）的房水流出阻力。通过评估氯酸盐（硫酸化抑制剂）和β-木糖苷（为添加二糖单位提供竞争性成核点）在眼前节灌注培养中的作用，进一步研究了它们的作用。在用20或50 mM氯酸钠或1 mM β-木糖苷处理的灌注猪和人前部器官培养物中测量流出设施。通过免疫荧光和共聚焦显微镜在石蜡包埋切片中评估细胞外基质（ECM）组分的扰动。对培养的TM细胞进行平行实验。氯酸盐（3倍）和β-木糖苷（3.5倍）治疗的猪眼流出设施增加。在人眼中，流出增加约1.5倍，发生时间更长（> 48小时）。通过共聚焦显微镜，软骨素和硫酸乙酰肝素的免疫染色观察到的边缘上的人TM梁在未处理的眼睛，与强烈的染色在小管组织（JCT）区域。在治疗的眼睛中，光束边缘的染色严重减少，而是在斑块中发现。氯酸盐处理导致人JCT区域中GAG染色的条纹图案。纤维连接蛋白的免疫染色在β-木糖苷处理的眼睛中改变，而在细胞培养中，氯酸盐诱导形成厚的纤维连接蛋白原纤维，腱生蛋白C共定位。破坏GAG链生物合成增加了灌注培养中的流出便利性，并诱导了细胞培养中的非典型ECM分子相互作用。本研究提供了GAG链在调节人TM流出阻力中的关键作用的直接证据。
PURPOSE. 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.