Recombinant Myocilin Purified From Human Trabecular Meshwork Cells Increases Outflow Resistance in Human Anterior Segments but Only in the Presence of Aqueous Humor

Abstract

Abstract: : Purpose:We have previously shown that recombinant myocilin purified from a prokaryotic expression system increased outflow resistance in human anterior segments. This study was performed to determine if full–length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance following infusion into human anterior segments. Methods: A Feline Immunodeficiency Virus vector that contained an internal cassette expressing both full–length myocilin (amino acids 1–504 fused to a C–terminal tag containing histidine and V5 epitopes) and puromycin N–acetyl–transferase was used to transduce a transformed trabecular meshwork cell line (TM5; gift from Dr. Abe Clark, Alcon Labs). Positive expressing cells were selected with puromycin. Purification of recombinant myocilin was performed on the media from TM5–myocilin expressing cells using nickel ion affinity chromatography. Control purifications were performed on media from TM5 cells not overexpressing myocilin. Anterior segments of human eyes were placed in organ culture and perfused with either DMEM (Dulbecco’s Modified Eagle’s Media) or DMEM containing 50% porcine aqueous humor. One eye received an anterior exchange with recombinant myocilin while the fellow eye received an equal volume of control. Results: Recombinant myocilin increased outflow resistance by 81% ± 57% (mean ± SD) in human anterior segments that had been incubated in porcine aqueous humor while the fellow control eye increased outflow resistance by 20% ± 30% (n=9, p=0.005). As little as 2µg of myocilin was able to increase outflow resistance (101% ± 70%, n=5). Maximal outflow resistance was obtained 9–16 hours following infusion and remained above baseline for 4–5 days. Recombinant myocilin did not change outflow resistance in human anterior segments incubated in DMEM (10% ± 13%, n=6). However, recombinant myocilin did increase outflow resistance in DMEM incubated eyes, but only after recombinant myocilin was incubated with porcine aqueous humor prior to infusion (112% ± 71%, n=6, p=0.013). Conclusions: Myocilin purified from human trabecular meshwork cells increased outflow resistance in human anterior segments, but only after incubation with aqueous humor. Recombinant myocilin appears to form a complex in aqueous that enables it to bind specifically within the trabecular meshwork.

Keywords: trabecular meshwork • outflow: trabecular meshwork • intraocular pressure