Effect of amniotic-membrane-derived lumican on cell migration and proliferation of human corneal epithelial cells

Abstract

Purpose : Lumican is a proteoglycan with epithelizing properties that can be extracted from human amniotic membrane (AM). The aim of the present study was to determine the effect of AM-derived (AMD) lumican on cell proliferation and migration of human corneal epithelial (HCE) cells.

Methods : Primary culture of HCE cells was harvested from corneal grafts and expanded in vitro. HCE phenotype (ABCB5+, cytokeratin 19+, and Δp63+) was evaluated using flow cytometry. Lumican was extracted from AM and stored at -20 °C for 12 days until proliferation and cell migration assays were performed. MTT assay was done to determine cell proliferation and wound healing assay for cell migration. In both assays, different doses of AMD lumican (2, 4, and 8 ng/mL) and recombinant human (rh) lumican (1 and 10 ng/mL) were analyzed at 24, 48, and 72 hours. Cell proliferation was calculated as the optical density (OD) at 595 nm, and cell migration as wound closure percentage.

Results : HEC cell proliferation was significantly higher (*p<0.05) at 24 hours with 4 and 8 ng/mL AMD lumican than 2 ng/mL AMD lumican and Rh lumican. However, there was no significant difference between 4 and 8 ng/mL AMD lumican and between 1 and 10 ng/mL Rh lumican, respectively. Contrarily, at 48 hours, 8 ng/mL AMD lumican significantly increased HEC proliferation (*p<0.05) compared to 4 ng/mL AMD lumican. At 72 hours, there were no significant differences between AMD lumican doses. AMD lumican proliferatively affects HEC at 4 and 8 ng/mL AMD lumican from 24 hours.

As regards cell migration, 2 ng/mL AMD lumican significantly increases (*p<0.05) wound closure of HEC at 24, 48, and 72 hours, similarly to complete growth medium. In contrast, 4 and 8 ng/mL AMD lumican at 48 and 24 hours, respectively, significantly decreased wound closure compared to complete growth medium, 2 ng/mL AMD lumican, and Rh lumican. AMD lumican significantly affects HEC migration at 2 ng/mL.

Conclusions : A concentration of AMD lumican between 2 and 4 ng/mL is the optimum dose for cellular proliferation and migration. AMD lumican shows a proliferative and migrative effect on HEC culture, suggesting AMD lumican is a good alternative as an epithelializing treatment.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. AMD lumican proliferatively affects HEC from 24 hours.

 

Figure 2. AMD lumican has a significant effect on HEC migration at 2 ng/mL.