Keller Kate E, Sun Ying Ying, Yang Yong-Feng, Bradley John M, Acott Ted S
Casey Eye Institute, Oregon Health & Science University, Portland, 97239, USA.
Invest Ophthalmol Vis Sci. 2012 Jul 9;53(8):4616-25. doi: 10.1167/iovs.12-9500.
Hyaluronan (HA) is a major component of the aqueous outflow pathway. However, the contribution of HA to human outflow resistance remains unclear. Three HA synthase genes (HAS1-3) have been identified. Here, we evaluate the contribution of each of the HAS proteins to outflow facility in anterior segment perfusion culture.
Two methods were used to reduce HA synthesis: 1 mM 4-methylumbelliferone (4MU) was used to inhibit all HAS synthases and shRNA silencing lentivirus was generated to knock down expression of each HAS individually. Quantitative RT-PCR, Western immunoblotting and an HA ELISA assay were used to assess HAS mRNA and protein levels and HA concentration, respectively. The effects of 4MU treatment and HAS gene silencing on outflow facility were assessed in human and porcine perfusion culture.
Quantitative RT-PCR and Western immunoblotting showed a reduction of each HAS in response to their respective silencing and 4MU treatment. HA concentration was concomitantly reduced. Treatment with 4MU decreased outflow facility in human anterior segments but increased outflow facility in porcine eyes. Lentiviral delivery of HAS1 and HAS2 silencing vectors caused similar opposite effects on outflow facility. Silencing of HAS3 did not significantly affect outflow resistance in either species.
This is the first conclusive evidence for a significant role of HA in the human outflow pathway. HA chains synthesized by HAS1 and HAS2 contribute to outflow resistance, while hyaluronan produced by HAS3 does not appear to play a significant role.
透明质酸（HA）是房水流出途径的主要成分。然而，HA对人体流出阻力的作用仍不清楚。已鉴定出三种透明质酸合酶基因（HAS1 - 3）。在此，我们评估每种HAS蛋白对眼前段灌注培养中流出易度的作用。
采用两种方法降低HA合成：使用1 mM 4 - 甲基伞形酮（4MU）抑制所有HAS合酶，并制备shRNA沉默慢病毒分别敲低每种HAS的表达。分别使用定量RT - PCR、Western免疫印迹和HA ELISA测定法评估HAS mRNA和蛋白水平以及HA浓度。在人和猪的灌注培养中评估4MU处理和HAS基因沉默对流出易度的影响。
定量RT - PCR和Western免疫印迹显示，每种HAS在各自沉默和4MU处理后表达降低。HA浓度随之降低。4MU处理降低了人眼前段的流出易度，但增加了猪眼的流出易度。HAS1和HAS2沉默载体的慢病毒递送对流出易度产生了类似的相反影响。HAS3的沉默对两种物种的流出阻力均无显著影响。
这是HA在人体流出途径中起重要作用的首个确凿证据。由HAS1和HAS2合成的HA链有助于流出阻力，而由HAS3产生的透明质酸似乎未发挥重要作用。
