Abstract
Purpose :
Pseudoexfoliation glaucoma (PEXG) is a form of open angle glaucoma that is characterized by outflow resistance dysregulation and elevated intraocular pressure (IOP). The LOXL1-AS1 gene codes for a long non-coding RNA, whereby polymorphisms decrease its expression and are associated with risk of PEXG. Since LOXL1-AS1 influences the transcription of many genes, we hypothesize that LOXL1-AS1 in outflow cells regulates the expression of gene-products known to participate in outflow resistance regulation, including extracellular matrix (ECM), cell adhesion and mechanotransduction signaling proteins.
Methods :
Knockdown of LOXL1-AS1 expression was achieved in primary cultures of human trabecular meshwork (TM) and Schlemm’s canal (SC) cells using a targeted Ad-shRNA, or by introducing targeted siRNA into HLE-B3 cells. Knockdown efficiency of LOXL1-AS1 and ECM gene expression was assessed using qPCR. Western blots were used to monitor ECM and mechanotransduction protein expression after LOXL1-AS1 knockdown, comparing to scrambled controls. Lastly, morphological changes due to LOXL1-AS1 knockdown in HLE-B3, SC, and TM cells were evaluated by calculating semi-axis ratios using FIJI.
Results :
Knockdown of LOXL1-AS1 in HLE-B3 cells significantly altered the expression of seven ECM proteins (p<0.05, n=3), but did not change phosphorylation status of candidate mechanotransduction proteins AKT, MAPK, FAK (p>0.05, n=3). Experiments in one TM cell strain show that knockdown of LOXL1-AS1 results in dysregulation of ECM target genes including, but not limited to, MMP2, MMP14, MMP28, and MYC. In SC cells, knockdown of LOXL1-AS1 led to significant expression changes in ECM target genes including neuronal adhesion protein, vascular adhesion protein, integrin alpha 2, and laminin gamma 1 (p<0.05, n=4). With LOXL1-AS1 knockdown, SC cells significantly increased the proportion of phosphorylated AKT (p=0.008, n=4), but no changes were observed with phosphorylation status of FAK or MAPK. Knockdown of LOXL1-AS1 resulted in a shortening of the major axis of SC cells (p=0.006, n=3).
Conclusions :
LOXL1-AS1 regulates genes involved in ECM remodeling and mechanotransduction signaling in human ocular cells. These data indicate that LOXL1-AS1 has a regulatory role in outflow resistance homeostasis, and represents a potential target for PEXG therapies.
This is a 2021 ARVO Annual Meeting abstract.