Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97239, USA.
Invest Ophthalmol Vis Sci. 2011 Jul 7;52(8):5049-57. doi: 10.1167/iovs.10-6948.
Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican's potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated.
Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNA-silencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility.
Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture.
Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes.
Versican 是一种带有大量软骨素硫酸盐 (CS) 糖胺聚糖 (GAG) 侧链的大型蛋白聚糖。为了评估 versican 对房水流出阻力的潜在贡献，评估了其在小梁网 (TM) 中的节段分布以及沉默 versican 基因对流出能力的影响。
荧光量子点 (Qdot) 灌流标记前节流出途径。免疫荧光共聚焦显微镜和定量 RT-PCR 用于确定 versican 蛋白和 mRNA 相对于 Qdot 标记区域的分布。将 shRNA 沉默盒递送至灌流前节培养物中的 TM 细胞的慢病毒传递用于评估 versican 和 CS GAG 链在流出能力中的参与。
TM 细胞摄取 Qdot 显示出人眼和猪眼流出途径的相当大的节段变异性。Qdot 标记的区域水平与 versican 蛋白和 mRNA 水平呈反比；在稀疏 Qdot 标记的区域中 versican 水平相对较高，而在密集标记的区域中则较低。Versican 沉默降低了人眼和猪眼前段的流出能力。然而，CS GAG 生物合成特有的酶 ChGn 的 RNAi 沉默增加了两种物种的流出能力。在灌注培养中沉默 versican 后，TM 近管腔区域中 versican 免疫染色的纤维状模式被破坏。
Versican 似乎是流出阻力的核心组成部分，它可以组织 GAG 和其他 ECM 成分，以促进和控制 TM 中的开放流动通道。然而，这种阻力的确切分子组织在人眼和猪眼中似乎不同。
