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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)
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.
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.
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).
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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