June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Differences in Reactive Oxygen Species (ROS) Production and Expression of Genes from the Complement and Inflammatory Pathways Between H and K Haplogroup Cybrids
Author Affiliations & Notes
  • Javier Cáceres del Carpio
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Mohamed Tarek
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Claudio Ramirez
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Payam Falatoonzadeh
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Marilyn Chwa
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Deepika Malik
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • S Michal Jazwinski
    Tulane Center for Aging, Tulane University, New Orleans, LA
  • Miceli Michael
    Tulane Center for Aging, Tulane University, New Orleans, LA
  • Baruch Kuppermann
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Cristina Kenney
    Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA
  • Footnotes
    Commercial Relationships Javier Cáceres del Carpio, None; Mohamed Tarek, None; Claudio Ramirez, None; Payam Falatoonzadeh, None; Marilyn Chwa, None; Deepika Malik, None; S Michal Jazwinski, None; Miceli Michael, None; Baruch Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech (C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech (C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva (C), ThromboGenics (C); Cristina Kenney, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4579. doi:
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      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

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