Effects of two SERPINS on ARPE-19 Cells after Acute Induced-NaIO₃ Damage

Abstract

Purpose : Oxidative stress is one of the risk factors associated in neurodegenerative diseases such as age-related macular degeneration (AMD). One hallmark of AMD is degeneration of the retinal pigment epithelium (RPE) that eventually leads to blindness. Sodium iodate (NaIO₃) induces a selective retinal degeneration targeting the RPE by triggering oxidative injury. Two serpins, pigment epithelium-derived factor (PEDF) and protease nexin-1 (PN-1), have neurotrophic properties and are present extracellularly in the retina. While PEDF lacks protease inhibitory activity, PN-1 is a serine protease inhibitor. The aim of the study is to investigate the effects of PEDF and PN-1 on RPE treated with a selective oxidative agent

Methods : Human ARPE-19 cells were seeded in 96-well plates. After 16h of serum starvation, the cells were pretreated with full-length recombinant human PEDF, PN-1 and PN-1-[R346A], an altered version at its serpin reactive center, as well as synthetic peptides derived from PEDF termed 34mer, 44mer and 17mer, and from PN-1 termed 17mer and incubated at 37C for 2 h. Freshly prepared NaIO₃ solutions were added to the cultures and incubated at 37C for 2 h. After changing the media and incubating for 16 h, cytotoxicity was determined by measuring the product of the cytoplasmic Lactate DeHydrogenase (LDH) enzyme in the media. Media with serum was replaced and incubated for 48 h before cell viability was determined by measuring the intracellular ATP levels

Results : PEDF and PN-1 decreased the activity of LDH in the cultures to 30 and 50% respectively, relative to 100% of untreated cultures. They increased cell viability to 142% and 132%, respectively, relative to untreated cultures. The PEDF peptides 17mer, 44mer and PN-1 peptide 17mer decreased the LDH levels to 60% - 74%, whereas they increased cell viability, respect to untreated cultures, by 1.5–fold. The inactive serpin PN-1-[R346A] decreased cytotoxicity and increased cell viability similar to its wild type counterpart PN-1. However, the PEDF 34mer had no significant effect on cytotoxicity or viability of ARPE-19 damaged with NaIO3

Conclusions : The two serpins PEDF and PN-1 share protective properties against NaIO₃-mediated injury. A small fragment from both proteins with 17 residues contains a biological active region. The inhibition of serine proteases is dispensable for the protection by these two serpins

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.