Purchase this article with an account.
Javier Cáceres del Carpio, Mohamed Tarek, Claudio Ramirez, Payam Falatoonzadeh, Marilyn Chwa, Deepika Malik, S Michal Jazwinski, Miceli Michael, Baruch Kuppermann, Cristina Kenney; Differences in Reactive Oxygen Species (ROS) Production and Expression of Genes from the Complement and Inflammatory Pathways Between H and K Haplogroup Cybrids. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4579.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Age-related Macular Degeneration (AMD) is the leading cause of blindness in people older than 50 in developed countries. Mitochondrial DNA (mtDNA) polymorphisms, that are maternally-inherited and are classified into haplogroups, can be associated with AMD. Individuals with haplogroup H, have less risk of developing AMD. Haplogroup K is related to AMD in some families. In this study we hypothesize that different haplogroups can affect nuclear gene expression and influence the production of ROS. We established a cybrid (cytoplasmic hybrid) model that has human RPE cells with identical nuclei but different mitochondria (H vs K haplogroups) and compared the differences in ROS production and expression of genes involved in the complement and inflammation pathways.
Human ARPE-19 cells without mitochondria (Rho0) were fused with platelets from individuals that had either H or K haplogroup genetic background. The haplogroup profiles were determined with PCR and restriction enzyme digestion. The ROS assay was performed with 2'7' dichlorodihydrofluorescein diacetate (H2DCFDA) dye. Gene expression profiles were determined using Quantitative-PCR (Q-PCR) for selected genes of the complement and inflammatory pathways: Complement Component 3 (C3), Complement Factor H (CFH), CD59, CD55, Transforming Growth Factor Alpha (TGFA), Transforming Growth Factor Beta 2 (TGFB2), Interleukin 6 (IL-6) and Retinoic Acid Receptor Alpha 1 (RARA1).
ROS production for haplogroup K cybrids was 68.78±7.07% compared to H, which had been normalized to 100% (p=0.02). Expression levels for complement and inflammation genes were significantly lower in K haplogroup cybrids compared to the H cybrids: C3, 0.07 fold, p<0.001; CFH, 0.3 fold, p=0.02; CD59, 0.69 fold, p=0.012; EFEMP1, 0.17 fold, p=0.003. Results for CD55, TGFA, TGFB2, IL-6 and RARA1 were not statistically significant.
Cybrids may provide a useful model to study the effects of mtDNA variants on cell behavior and gene expression. Our findings suggest that H and K cybrids have significantly different expression profiles for C3, a major component of the alternative complement system, and two inhibitors of the complement pathway, CFH and CD59, which potentially may influence the development of AMD.
This PDF is available to Subscribers Only