Methylation regulates Lysyl oxidase like 1 (LOXL1) expression in Pseudoexfoliation Glaucoma

Abstract

Purpose : Lysyl oxidase like 1 (LOXL1) catalyzes collagen and elastin crosslinking and its dysregulation is associated with pseudoexfoliation syndrome (PXF) and pseudoexfoliation glaucoma (PXFG). LOXL1 expression is downregulated in PXFG. We hypothesise that this may be via DNA methylation, previously reported by our group as a method of epigenetic regulation in glaucoma. We wished to investigate LOXL1 levels in Human Tenons Fibroblasts (HTFs) and aqueous (AqH) from PXF, PXFG and cataract controls (CAT) and to establish if DNA methylation was responsible for altering LOXL1 expression when driven by modifying conditions like oxidative stress and hypoxia.

Methods : Patients with PXF, PXFG and CAT were prospectively recruited and cells were propagated from subconjunctival Tenons capsules isolated during surgery (n=73). LOXL1 expression was measured by quantitative Polymerase Chain Reaction (qPCR) and Western Blot and LOXL1 activity by ELISA. Global DNA methylation was measured by ELISA and DNA Methyltransferase 1/3a (DNMT1/3a) by qPCR. DNMT3a binding to the LOXL1 promoter was investigated by Chromatin Immunoprecipitation. DNA methylation was inhibited using 5-azacitidine (5-aza/0.3μM). To investigate environmental influencers of DNA methylation, CAT HTFs were cultured under hypoxic (1%O₂) and oxidative stress conditions (200μM H₂O₂) and LOXL1 expression measured as above. Clinical information was recorded in a RedCap™ database.

Results : LOXL1 expression was decreased in PXFG vs. CAT (n=3, p<0.01) as it was in CAT HTFs cultured under hypoxic (n=3, p<0.05) and oxidative stress conditions (n=3, P<0.01). LOXL1 activity was increased in the AqH of PXFG vs. CAT (n=20, P<0.01). Lipid peroxidation was increased in PXFG compared with CAT and PXF (n=3, P<0.01). Global DNA methylation and DNMT expression were increased in PXFG vs. CAT (n=3, p<0.05) and DNMT inhibition with 5-aza restored LOXL1 expression (n=3, P<0.05).

Conclusions : LOXL1 expression is decreased in PXFG. This may be due to gene silencing via DNA methylation and cellular stress conditions may initiate this process. Methylation inhibition could represent a future therapeutic target for PXFG.

This is a 2020 ARVO Annual Meeting abstract.