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Kate E. Keller, John M. Bradley, Ying Ying Sun, Ted S. Acott; Perturbation Of Hyaluronan Synthesis By HAS Gene Silencing In The Trabecular Meshwork And The Effects On Outflow Facility. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4633.
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© ARVO (1962-2015); The Authors (2016-present)
Hyaluronan (HA) plays an important role in the regulation of outflow resistance through the trabecular meshwork (TM) in many species. However, the contribution of HA to human outflow resistance remains controversial. Three HA synthase (HAS) genes have been identified, HAS 1-3. Here we evaluated the contribution of HAS’s to outflow facility in human and porcine TMs.
Two methods were used to reduce HA synthesis: shRNA silencing lentivirus were generated to knockdown expression of HAS1 and HAS2 and 1 mM 4-methylumbelliferone (4MU) was used to inhibit HAS2 and HAS3 synthases. The effects of HAS gene silencing and 4MU treatment on outflow facility was assessed in anterior segment perfusion culture. Flow patterns before and after 4MU treatment were labeled using red (655 nm) and green (585 nm) fluorescent quantum dots (Qdots), respectively, and confocal microscopy. HA levels in human (HTM) and porcine (PTM) cell cultures were quantitated by ELISA assay in response to TNFα and TGFß2 treatments and mechanical stretch, while HA staining in PTM cells was investigated using biotinylated HA binding protein (HAbp).
Lentiviral delivery of HAS1 and HAS2 silencing vectors to human anterior segments decreased outflow facility, while infection of porcine eyes increased outflow facility. A similar response was observed for 4MU treatment. Qdot analysis of flow patterns before and after 4MU treatment showed an expanded area of green staining suggesting that inhibition of HA synthesis hindered penetration of Qdots deep into the TM. By ELISA assay, mechanical stretch reduced cell-associated HA in TM cells, while TNFα and TGFß2 increased cell-associated HA in PTM but not HTM cells. By immunofluorescence, HAbp was found to extensively label TNFα- and TGFß2-treated PTM cells, with the apparent formation of HA cables.
Perturbation of HA synthesis by inhibition or HAS gene knockdown has opposite effects on human and porcine outflow facility, implying species differences in the composition and/or structural organization of the outflow resistance. This study provides the first conclusive evidence for a role of HA in the human outflow pathway.
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