Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/o... more Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and-J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2′-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. 22 tRNAs and 2 rRNAs, that are critical for oxidative phosphorylation (OXPHOS). The mtDNA can be classified into haplogroups, defined by an accumulation of single nucleotide polymorphisms (SNPs), which represent the different geographic origins of populations. Investigations report that the mtDNA haplogroups can be †The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint 'First Authors'.
Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries... more 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 accumulations 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 (cytoplasmic 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 suggest 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.
Background: Mitochondrial dysfunction is associated with the development and progression of age-r... more 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...
To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab... more To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumab-treated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mit...
ABSTRACT PurposeAMD is a leading cause of vision loss in the elderly population. Mitochondrial ge... more ABSTRACT PurposeAMD is a leading cause of vision loss in the elderly population. Mitochondrial genetics is an important area of research that may help us understand the predisposition for AMD between and within racial groups.Previous studies have shown that the mtDNA haplogroup J is associated with AMD, while the H haplogroup is protective. However, the functional consequences of this difference are not understood. We have used cybrids (cytoplasmic hybrids) to study the characteristics and biochemical differences between various haplogroups. Our hypothesis is that cybrids, dissimilar only in their mtDNA haplogroup will behave differently in vitro. MethodsCybrids were created by introducing the mitochondria from human individuals platelets into a host cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). Therefore, all cybrids carry the same nuclear genes but vary only in their mitochondrial content. Cybrid cultures H and J were pelleted, the RNA was isolated and then quantified. RNA samples were reverse
Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ran... more Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Methods Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Results Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumabtreated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mitochondrial membrane potential was seen at 1×, 2× and 10× concentrations of bevacizumab (86.53%, 74.38%, 66.67%; p<0.01) and ziv-aflibercept (73.50%, 64.83% and 49.65% p<0.01) suggestive of early apoptosis at lower doses, including the clinical doses. Conclusions At clinical doses, neither ranibizumab nor aflibercept produced evidence of mitochondrial toxicity or cell death. However, bevacizumab and ziv-aflibercept showed mild mitochondrial toxicity at clinically relevant doses.
ABSTRACT PurposeAge-related Macular Degeneration (AMD) is the leading cause of blindness in peopl... more ABSTRACT PurposeAge-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. MethodsHuman 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
ABSTRACT PurposeMitochondrial DNA haplogroups can be classified according to accumulation of spec... more ABSTRACT PurposeMitochondrial DNA haplogroups can be classified according to accumulation of specific single nucleotide polymorphisms (SNPs). The H haplogroup is of European origin and protective for AMD, the L haplogroup which is of African origin shows only early AMD changes. Our work is to investigate the differences in Reactive Oxygen/Nitrogen Species (ROS/RNS) production and gene expression profiles of cybrids (cytoplasmic hybrids) that have either the H haplogroup or the L haplogroup. MethodsARPE-19 cells lacking mitochondrial DNA (mtDNA) (Rho0) were fused with platelets from individuals with either H or L haplogroup mtDNA as determined by PCR analysis. After culture for 24 hours, ROS/RNS levels were determined with 2`, 7`-dichlorodihydrofluorescein diacetate dye assay. Experiments were repeated twice and performed in 8 wells per sample. RNA was obtained from the H cybrids (n=3) and L cybrids (n=3) and the gene expression patterns were determined using the Affymetrix Human Genome U133 plus 2.0 array. The pat
Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/o... more Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and-J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2′-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. 22 tRNAs and 2 rRNAs, that are critical for oxidative phosphorylation (OXPHOS). The mtDNA can be classified into haplogroups, defined by an accumulation of single nucleotide polymorphisms (SNPs), which represent the different geographic origins of populations. Investigations report that the mtDNA haplogroups can be †The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint 'First Authors'.
Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries... more 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 accumulations 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 (cytoplasmic 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 suggest 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.
Background: Mitochondrial dysfunction is associated with the development and progression of age-r... more 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...
To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab... more To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumab-treated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mit...
ABSTRACT PurposeAMD is a leading cause of vision loss in the elderly population. Mitochondrial ge... more ABSTRACT PurposeAMD is a leading cause of vision loss in the elderly population. Mitochondrial genetics is an important area of research that may help us understand the predisposition for AMD between and within racial groups.Previous studies have shown that the mtDNA haplogroup J is associated with AMD, while the H haplogroup is protective. However, the functional consequences of this difference are not understood. We have used cybrids (cytoplasmic hybrids) to study the characteristics and biochemical differences between various haplogroups. Our hypothesis is that cybrids, dissimilar only in their mtDNA haplogroup will behave differently in vitro. MethodsCybrids were created by introducing the mitochondria from human individuals platelets into a host cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). Therefore, all cybrids carry the same nuclear genes but vary only in their mitochondrial content. Cybrid cultures H and J were pelleted, the RNA was isolated and then quantified. RNA samples were reverse
Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ran... more Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Methods Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Results Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumabtreated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mitochondrial membrane potential was seen at 1×, 2× and 10× concentrations of bevacizumab (86.53%, 74.38%, 66.67%; p<0.01) and ziv-aflibercept (73.50%, 64.83% and 49.65% p<0.01) suggestive of early apoptosis at lower doses, including the clinical doses. Conclusions At clinical doses, neither ranibizumab nor aflibercept produced evidence of mitochondrial toxicity or cell death. However, bevacizumab and ziv-aflibercept showed mild mitochondrial toxicity at clinically relevant doses.
ABSTRACT PurposeAge-related Macular Degeneration (AMD) is the leading cause of blindness in peopl... more ABSTRACT PurposeAge-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. MethodsHuman 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
ABSTRACT PurposeMitochondrial DNA haplogroups can be classified according to accumulation of spec... more ABSTRACT PurposeMitochondrial DNA haplogroups can be classified according to accumulation of specific single nucleotide polymorphisms (SNPs). The H haplogroup is of European origin and protective for AMD, the L haplogroup which is of African origin shows only early AMD changes. Our work is to investigate the differences in Reactive Oxygen/Nitrogen Species (ROS/RNS) production and gene expression profiles of cybrids (cytoplasmic hybrids) that have either the H haplogroup or the L haplogroup. MethodsARPE-19 cells lacking mitochondrial DNA (mtDNA) (Rho0) were fused with platelets from individuals with either H or L haplogroup mtDNA as determined by PCR analysis. After culture for 24 hours, ROS/RNS levels were determined with 2`, 7`-dichlorodihydrofluorescein diacetate dye assay. Experiments were repeated twice and performed in 8 wells per sample. RNA was obtained from the H cybrids (n=3) and L cybrids (n=3) and the gene expression patterns were determined using the Affymetrix Human Genome U133 plus 2.0 array. The pat
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