Perturbation of hyaluronan synthesis in the trabecular meshwork and the effects on outflow facility.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Casey Eye Institute, Oregon Health & Science University, Portland, 97239, USA. gregorka@ohsu.edu
Kate E
KE
Keller
Ying Ying
YY
Sun
Yong-Feng
YF
Yang
John M
JM
Bradley
Ted S
TS
Acott
D001082:Aqueous Humor / Q000502:physiology*; D015153:Blotting, Western; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*
D012333:RNA, Messenger; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases