An In-Vitro Study of the Effect of Ultraviolet Radiation on Growth Patterns and Gene Expression of Human ARPE-19 Cybrid H and J, and their Implications with Age-related Macular Degeneration (AMD)

Abstract

 

Purpose

 

Studies have shown that mitochondrial DNA (mtDNA) plays an important role in the aging process. Various haplogroups of mtDNA have been classified based on variable combinations of single nucleotide polymorphisms (SNPs). Cybrids (cytoplasmic hybrids) have identical nuclei but mitochondria of different haplogroups. Although both haplogroups H and J represent European-Caucasian populations, individuals with haplogroup J have a higher susceptibility to develop AMD and H is protective. The aim of this study was to evaluate the effect of UV radiation (a known environmental risk factor for AMD) on cell growth patterns and gene expression in various cellular pathways in H and J cybrids.

 

Methods

 

ARPE-19 cells were depleted of mitochondria (Rho0) and fused with platelets isolated from haplogroup H or J subjects to create individual cybrids. Cybrids were treated with 10mJ of UV radiation. Growth patterns of cybrids H and J were studied over 14 days in both UV-treated and untreated groups. RNA extraction and cDNA synthesis were done at 0, 72 and 120 hours, after exposure. Various pro-apoptotic genes RARA (Retinoic Acid Receptor Alpha), BBC3 (BLC2 Binding Protein) and BLC2L13 (BCL2 like 13) (both BBC3 and BCL2L13 induce mitochondrial outer membrane permeabilization and caspase activation); pro-inflammatory gene IL33 (Interleukin-33); and angiogenic gene TGF-A (Transforming Growth Factor-Alpha) were studied using Q-PCR.

 

Results

 

A stepladder growth pattern was seen in UV-treated groups, which was significantly different than control groups. J cybrids showed more growth than H cybrids at all studied time points. At 120 hours, both UV-treated cybrids H and J showed increase in gene expression levels for pro-apoptotic genes RARA, BBC3 and BCL2L13, but cybrid H showed a greater increase. UV-treated cybrid J showed higher expression of pro-inflammatory gene IL-33 and angiogenic gene TGF-A as compared to UV-treated cybrid H. Table 1: Fold differences relative to untreated cybrid H (0 hours)

 

Conclusions

 

Our study shows that SNP variations in mtDNA may alter nuclear gene expression and cellular responses to stress, and contribute to differential genetic risk factors for AMD.

 

 

Fold differences relative to Untreated Cybrid H (0 Hours)

 

Keywords: 412 age-related macular degeneration • 600 mitochondria • 533 gene/expression