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Author(s): Estrago-Franco, M Fernanda; Moustafa, M Tarek; Riazi-Esfahani, Mohammad; Sapkal, Ashish U; Piche-Lopez, Rhina; Patil, A Jayaprakash; Sharma, Ashish; Falatoonzadeh, Payam; Chwa, Marilyn; Luczy-Bachman, Georgia; Kuppermann, Baruch D; Kenney, M Cristina | Abstract: PurposeTo identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS), complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette smoke, in human retinal pigment epithelial cells (ARPE-19) in vitro.MethodsARPE-19 cells were treated for 24 hours with 200 μM, 100 μM, and 50 μM B(e)p or DMSO (dimethyl sulfoxide)-equivalent concentrations. Some cultures were pre-treated with ROS/RNS inhibitors (NG nitro-L-arginine, inhibits nitric oxide synthase; Apocynin, inhibits NADPH oxidase; Rotenone, inhibits mitochondrial complex I; Antimycin A, inhibits mitochondria complex III) and ROS/RNS levels were measured with a fluorescent H2 DCFDA assay. Multiplex bead arrays were used to measure levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Transforming Growth Factor alpha (TGF-α) and Vascular Endothelial Growth Factor (VEGF). IL-6 levels were also measured by an enzyme-linked immunosorbent assay. Real-time qPCR analyses were performed with primers for C3 (component 3), CFH (inhibits complement activation), CD59 (inhibitor of the complement membrane attack complex (MAC)) and CD55/DAF (accelerates decay of target complement target proteins).ResultsThe ARPE-19 cultures treated with B(e)p showed significantly increased ROS/RNS levels (P l 0.001), which were then partially reversed by 6 μM Antimycin A (19%, P = 0.03), but not affected by the other ROS/RNS inhibitors. The B(e)p treated cultures demonstrated increased levels of IL-6 (33%; P = 0.016) and GM-CSF (29%; P = 0.0001) compared to DMSO-equivalent controls, while the expression levels for components of the complement pathway (C3, CFH, CD59 and CD55/DAF) were not changed.ConclusionThe cytotoxic effects of B(e)p include elevated ROS/RNS levels along with pro-inflammatory IL-6 and GM-CSF proteins. Blocking the Qi site of cytochrome c reductase (complex III) with Antimycin A led to partial reduction in B(e)p induced ROS production. Our findings suggest that inhibitors for multiple pathways would be necessary to protect the retinal cells from B(e)p induced toxicity.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Human Molecular Genetics, 2014, Vol. 23, No. 13 doi:10.1093/hmg/ddu065 Advance Access published on February 28, 2014 Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis pathways: insights into mitochondrial –nuclear interactions M. Cristina Kenney 1,2, ∗ , Marilyn Chwa 1 , Shari R. Atilano 1 , Payam Falatoonzadeh 1 , Claudio Ramirez 1 , Deepika Malik 1 , Mohamed Tarek 1 , Javier Ca´ceres-del-Carpio 1 , Anthony B. Nesburn 1,4 , David S. Boyer 5 , Baruch D. Kuppermann 1 , Marquis Vawter 3 , S. Michal Jazwinski 6 , Michael Miceli 6 , Douglas C. Wallace 7 and Nitin Udar 1 Gavin Herbert Eye Institute, 2 Department of Pathology and Laboratory Medicine, 3 Functional Genomics Laboratory, Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA, 4 Cedars-Sinai Medical Center, Los Angeles, CA, USA, 5 Retina-Vitreous Associates Medical Group, Beverly Hills, CA, USA, 6 Tulane Center for Aging, Tulane University, New Orleans, LA, USA and 7 Children’s Hospital of Philadelphia, Center for Mitochondrial and Epigenomic Medicine, Philadelphia, PA, USA Received December 17, 2013; Revised February 3, 2014; Accepted February 10, 2014 Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other ‘modifiers’ may be involved. Mitochondrial (mt) haplogroups, defined by accu- mulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cyto- plasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways. J cybrids have altered bioenergetic profiles compared with H cybrids. Q-PCR analyses show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA was carried out for all the cybrids to identify haplogroup and non-haplogroup defining SNPs. mtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data sug- gest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mt–nuclear interactions. INTRODUCTION Mitochondria (mt) are unique organelles that have their own DNA. Human mtDNA is maternally inherited and forms a circle of double-stranded DNA with 16 569 nucleotide pairs. The non-coding mt-Dloop (also called the control region) con- tains 1121 nucleotides important for replication and transcrip- tion. The coding region of mtDNA encodes for 37 genes, including 13 protein subunits essential for oxidative phosphorylation (OXPHOS), 2 ribosomal RNAs and 22 transfer RNAs (1 – 3). The mtDNA can be categorized into haplogroups that are defined by a group of single nucleotide polymorphism (SNP) variants that have accumulated over tens of thousands of years and correspond to various geographic populations. While some studies have shown that certain mtDNA hap- logroups can be either protective or increase risk for human dis- eases (4–10), others report no haplogroup association with diseases (11– 13). However, many of these studies identified To whom correspondence should be addressed at: Gavin Herbert Eye Institute, Discovery Center for Eye Research, University of California Irvine, Hewitt Hall, Room 2028, 843 Health Science Road, Irvine, CA 92697, USA. Tel: +1 9498247603; Fax: +1 9498249626; Email: mkenney@uci.edu # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Diabetic retinopathy remains the leading vascular-associated cause of blindness throughout the world. Its treatment requires a multidisciplinary interventional approach at both systemic and local levels. Current management includes laser photocoagulation, intravitreal steroids, and anti-vascular endothelial growth factor (VEGF) treatment along with systemic blood sugar control. Anti-VEGF therapies, which are less destructive and safer than laser treatments, are being explored as primary therapy for the management of vision-threatening complications of diabetic retinopathy such as diabetic macular edema (DME). This review provides comprehensive information related to VEGF and describes its role in the pathogenesis of diabetic retinopathy, and in addition, examines the mechanisms of action for different antiangiogenic agents in relation to the management of this disease. Medline (Pubmed) searches were carried out with keywords “VEGF”, “diabetic retinopathy”, and “diabetes” without any year limitation to review relevant manuscripts used for this article.

Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration M. Cristina Kenney 1 *, Marilyn Chwa 1 , Shari R. Atilano 1 , Janelle M. Pavlis 1 , Payam Falatoonzadeh 1 , Claudio Ramirez 1 , Deepika Malik 1 , Tiffany Hsu 1 , Grace Woo 1 , Kyaw Soe 1 , Anthony B. Nesburn 1,2 , David S. Boyer 3 , Baruch D. Kuppermann 1 , S. Michal Jazwinski 4 , Michael V. Miceli 4 , Douglas C. Wallace 5 , Nitin Udar 1 1 Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, United States of America, 2 Cedars-Sinai Medical Center, Los Angeles, California, United States of America, 3 Retina-Vitreous Associates Medical Group, Beverly Hills, California, United States of America, 4 Tulane Center for Aging and Department of Medicine, Tulane University, New Orleans, Louisiana, United States of America, 5 Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America Abstract Background: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). Methodology/Principal Findings: Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. Conclusion/Significance: Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD. Citation: Kenney MC, Chwa M, Atilano SR, Pavlis JM, Falatoonzadeh P, et al. (2013) Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration. PLoS ONE 8(1): e54339. doi:10.1371/journal.pone.0054339 Editor: Walter Lukiw, Louisiana State University Health Sciences Center, United States of America Received October 1, 2012; Accepted December 10, 2012; Published January 24, 2013 Copyright: s 2013 Kenney et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding provided by The Discovery Eye Foundation www.discoveryeye.org, Lincy Foundation lincyinstitute.unlv.edu/lincy.html, Beckman Macular Research Initiative www.beckmanmacular.org, The Henry Guenther Foundation, Polly and Michael Smith Foundation, Research to Prevent Blindness Foundation www.rpbusa.org, and the National Institute on Aging (AG006168). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mkenney@uci.edu specific SNP variants that have accumulated over tens of thousands of years and correspond to different geographic populations of the world. The H haplogroup is the most common European haplogroup, while the J haplogroup originates from the Northern European region and is defined by SNP variants that are associated with heat production as an adaptation to colder climates [4]. The mtDNA plays an important role in aging and diseases [4– 6]. Specific haplogroups are associated with a variety of eye diseases including age-related macular degeneration (AMD) [7– 10], diabetic retinopathy [11], pseudoexfoliation glaucoma [12,13], primary open-angle glaucoma [14], keratoconus [15], multiple sclerosis-related optic neuritis [16,17], and Leber Introduction Mitochondria provide critical cellular energy using the tricar- boxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and beta-oxidation of fatty acids for metabolism, cell division, production of reactive oxygen species (ROS), and apoptosis. Human mitochondrial (mt) DNA forms a circle of double stranded DNA with 16,569 nucleotide pairs. The non-coding mtDNA Dloop contains 1121 nucleotides and is important for replication and transcription. The coding region of mtDNA encodes for 37 genes including 13 protein subunits essential for OXPHOS, 2 ribosomal RNAs, and 22 transfer RNAs [1–3]. The mtDNA can be categorized into haplogroups that are defined by a set of PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e54339

Herpes simplex virus 1 (HSV-1) and HSV-2 are medically significant pathogens. The development of an effective HSV vaccine remains a global public health priority. HSV-1 and HSV-2 immunodominant “asymptomatic” antigens (ID-A-Ags), which are strongly recognized by B and T cells from seropositive healthy asymptomatic individuals, may be critical to be included in an effective immunotherapeutic HSV vaccine. In contrast, immunodominant “symptomatic” antigens (ID-S-Ags) may exacerbate herpetic disease and therefore must be excluded from any HSV vaccine. In the present study, proteome microarrays of 88 HSV-1 and 84 HSV-2 open reading frames(ORFs) (ORFomes) were constructed and probed with sera from 32 HSV-1-, 6 HSV-2-, and 5 HSV-1/HSV-2-seropositive individuals and 47 seronegative healthy individuals (negative controls). The proteins detected in both HSV-1 and HSV-2 proteome microarrays were further classified according to their recognition by sera from HSV-seropositive clinically defined symptomatic ( n = 10) and asymptomatic ( n = 10) individuals. We found that (i) serum antibodies recognized an average of 6 ORFs per seropositive individual; (ii) the antibody responses to HSV antigens were diverse among HSV-1- and HSV-2-seropositive individuals; (iii) panels of 21 and 30 immunodominant antigens (ID-Ags) were identified from the HSV-1 and HSV-2 ORFomes, respectively, as being highly and frequently recognized by serum antibodies from seropositive individuals; and (iv) interestingly, four HSV-1 and HSV-2 cross-reactive asymptomatic ID-A-Ags, US4, US11, UL30, and UL42, were strongly and frequently recognized by sera from 10 of 10 asymptomatic patients but not by sera from 10 of 10 symptomatic patients ( P < 0.001). In contrast, sera from symptomatic patients preferentially recognized the US10 ID-S-Ag ( P < 0.001). We have identified previously unreported immunodominant HSV antigens, among which were 4 ID-A-Ags and 1 ID-S-Ag. These newly identified ID-A-Ags could lead to the development of an efficient “asymptomatic” vaccine against ocular, orofacial, and genital herpes.