July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Epigenetic modifications in AMD ARPE-19 cybrids cells regulating CXCL genes
Author Affiliations & Notes
  • Paula Sakemi Fukuhara
    Ophthalmology, University of California Irvine, Irvine, California, Brazil
  • Daniel Hyunjae Lee
    University of California Irvine, Irvine, California, United States
  • Shari Atilano
    University of California Irvine, Irvine, California, United States
  • Marilyn Chwa
    University of California Irvine, Irvine, California, United States
  • Kevin Schneider
    University of California Irvine, Irvine, California, United States
  • Baruch D Kuppermann
    Ophthalmology, University of California Irvine, Irvine, California, Brazil
  • Cristina M Kenney
    Ophthalmology, University of California Irvine, Irvine, California, Brazil
  • Footnotes
    Commercial Relationships   Paula Sakemi Fukuhara, None; Daniel Lee, None; Shari Atilano, None; Marilyn Chwa, None; Kevin Schneider, None; Baruch Kuppermann, None; Cristina Kenney, None
  • Footnotes
    Support  Funding Supported by Discovery Eye Foundation, Polly and Michael Smith, Iris and B. Gerald Cantor Foundation, Roy and Edith Carver Foundation, Max Factor Family Foundation. Supported by an Research to Prevent Blindness Unrestricted Grant.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4005. doi:https://doi.org/
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      Paula Sakemi Fukuhara, Daniel Hyunjae Lee, Shari Atilano, Marilyn Chwa, Kevin Schneider, Baruch D Kuppermann, Cristina M Kenney; Epigenetic modifications in AMD ARPE-19 cybrids cells regulating CXCL genes. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4005. doi: https://doi.org/.

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

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Abstract

Purpose : Angiogenesis and inflammation are features of many retinal diseases. The CXCL families of chemokines are major mediators of the inflammation that are known to recruit neutrophils and promote angiogenesis. Previous studies using transmitochondrial cybrids (cell lines with identical nuclei but mitochondrial (mt) DNA from different individuals) showed that a person’s mtDNA can modulate complement, innate immunity and signaling genes (Kenney et al. BBA, 2013). In addition, the mtDNA variants can mediate methylation profiles and alter transcription for inflammation and angiogenesis genes (Atilano et al Hum Molec Genet. 2015). The present study tests the hypothesis that regulation of the CXCL genes is mediated via epigenetic modifications.

Methods : Cybrids were created by fusing Rho0 ARPE-19 cells (depleted of mitochondria) with platelets isolated from individuals from maternal European mtDNA background (H haplogroup, n=3) or maternal African background (L haplogroup, n=3). Cybrids were plated for 24 hrs, media removed and then replaced with media containing 250µM 5-aza-2’-deoxycytidine (5-aza-dC, Sigma-Alrich, St Louis, MO) for an additional 48 hrs. Media containing 5-aza-dC was replaced each 24 hrs. The RNA was extracted and qRT-PCR performed using primers for CXCL1 (NM_046035), CXCL5 (NM_002994) and CXCL8 (NM_00584, also known as IL8). Statistical differences were measured using Student’s t-test, and significance was determined at P<0.05. The untreated samples were assigned a value of 1.

Results : The cybrids that were demethylated by 5-aza-dC treatment showed significantly increased expression of CXCL1 (386 ± 116, p=0.0084) compared to the untreated cybrids. The CXCL5 transcription increased 4229 ± 1648 fold (p=0.028) while the CXCL8 (IL8) level were also higher (41.7 ± 10.7, p=0.0048).

Conclusions : Untreated cybrid cells that have almost negligible expression levels of CXCL1, -5 or -8 are upregulated significantly when cultures are demethylated with 5-aza-dC. This is the first description of methylation being a major regulator for the CXCL chemokines which are critical for their role in neutrophil recruitment and degranulation, along with angiogenesis, events often found in human retinal diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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