187 research outputs found
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Bioenergetics Consequences of Mitochondrial Transplantation in Cardiomyocytes.
Background Mitochondrial transplantation has been recently explored for treatment of very ill cardiac patients. However, little is known about the intracellular consequences of mitochondrial transplantation. This study aims to assess the bioenergetics consequences of mitochondrial transplantation into normal cardiomyocytes in the short and long term. Methods and Results We first established the feasibility of autologous, non-autologous, and interspecies mitochondrial transplantation. Then we quantitated the bioenergetics consequences of non-autologous mitochondrial transplantation into cardiomyocytes up to 28 days using a Seahorse Extracellular Flux Analyzer. Compared with the control, we observed a statistically significant improvement in basal respiration and ATP production 2-day post-transplantation, accompanied by an increase in maximal respiration and spare respiratory capacity, although not statistically significantly. However, these initial improvements were short-lived and the bioenergetics advantages return to the baseline level in subsequent time points. Conclusions This study, for the first time, shows that transplantation of non-autologous mitochondria from healthy skeletal muscle cells into normal cardiomyocytes leads to short-term improvement of bioenergetics indicating "supercharged" state. However, over time these improved effects disappear, which suggests transplantation of mitochondria may have a potential application in settings where there is an acute stress
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A two-step method for identifying photopigment opsin and rhodopsin gene sequences underlying human color vision phenotypes.
PurposeTo present a detailed, reliable long range-PCR and sequencing (LR-PCR-Seq) procedure to identify human opsin gene sequences for variations in the long wavelength-sensitive (OPN1LW), medium wavelength-sensitive (OPN1MW), short wavelength-sensitive (OPN1SW), and rhodopsin (RHO) genes.MethodsColor vision was assessed for nine subjects using the Farnsworth-Munsell 100 hue test, Ishihara pseudoisochromatic plates, and the Rabin cone-contrast threshold procedure (ColorDX, Konan Medical). The color vision phenotypes were normal trichromacy (n = 3), potential tetrachromacy (n = 3), dichromacy (n = 2), and unexplained low color vision (n = 1). DNA was isolated from blood or saliva and LR-PCR amplified into individual products: OPN1LW (4,045 bp), OPN1MW (4,045 bp), OPN1SW (3,326 bp), and RHO (6,715 bp). Each product was sequenced using specific internal primer sets. Analysis was performed with Mutation Surveyor software.ResultsThe LR-PCR-Seq technique identified known single nucleotide polymorphisms (SNPs) in OPN1LW and OPN1MW gene codons (180, 230, 233, 277, and 285), as well as those for lesser studied codons (174, 178, 236, 274, 279, 298 and 309) in the OPN1LW and OPN1MW genes. Additionally, six SNP variants in the OPN1MW and OPN1LW genes not previously reported in the NCBI dbSNP database were identified. An unreported poly-T region within intron 5(c.36+126) of the rhodopsin gene was also found, and analysis showed it to be highly conserved in mammalian species.ConclusionsThis LR-PCR-Seq procedure (single PCR reaction per gene followed by sequencing) can identify exonic and intronic SNP variants in OPN1LW, OPN1MW, OPN1SW, and rhodopsin genes. There is no need for restriction enzyme digestion or multiple PCR steps that can introduce errors. Future studies will combine the LR-PCR-Seq with perceptual behavior measures, allowing for accurate correlations between opsin genotypes, retinal photopigment phenotypes, and color perception behaviors
Use of Intravitreal Triamcinolone in the Treatment of Macular Edema Related to Retinal Vein Occlusion
ObjectiveTo analyze the increasing trend of intravitreal triamcinolone (IVTA) use in the treatment of retinal vein occlusion-related macular edema.MethodsWe performed MEDLINE/PUBMED searches (September 1984 - December 2007) to identify articles containing the keywords macular edema and triamcinolone. Case reports, reviews and abstracts were identified from references in the reviewed literature. This review focuses on literature published during the past 7 years with more than two-thirds of the articles that we reviewed being printed during the past 5 years. These reports analyzed the success of IVTA in the treatment of macular edema over a 12 month course of time.ResultsThe majority of studies suggested promising results for short time periods (4-6 months) after IVTA treatments. However, long term results were not encouraging.ConclusionsThe success of IVTA therapy for short durations has been the impetus for development of sustained release devices to be used in the treatment of macular edema associated with various retinal diseases including edema related to retinal vein occlusion
Humanin G (HNG) protects age-related macular degeneration (AMD) transmitochondrial ARPE-19 cybrids from mitochondrial and cellular damage.
Age-related macular degeneration (AMD) ranks third among the leading causes of visual impairment with a blindness prevalence rate of 8.7%. Despite several treatment regimens, such as anti-angiogenic drugs, laser therapy, and vitamin supplementation, being available for wet AMD, to date there are no FDA-approved therapies for dry AMD. Substantial evidence implicates mitochondrial damage and retinal pigment epithelium (RPE) cell death in the pathogenesis of AMD. However, the effects of AMD mitochondria and Humanin G (HNG), a more potent variant of the mitochondrial-derived peptide (MDP) Humanin, on retinal cell survival have not been elucidated. In this study, we characterized mitochondrial and cellular damage in transmitochondrial cybrid cell lines that contain identical nuclei but possess mitochondria from either AMD or age-matched normal (Older-normal (NL)) subjects. AMD cybrids showed (1) reduced levels of cell viability, lower mtDNA copy numbers, and downregulation of mitochondrial replication/transcription genes and antioxidant enzyme genes; and (2) elevated levels of genes related to apoptosis, autophagy and ER-stress along with increased mtDNA fragmentation and higher susceptibility to amyloid-β-induced toxicity compared to NL cybrids. In AMD cybrids, HNG protected the AMD mitochondria, reduced pro-apoptosis gene and protein levels, upregulated gp130 (a component of the HN receptor complex), and increased the protection against amyloid-β-induced damage. In summary, in cybrids, damaged AMD mitochondria mediate cell death that can be reversed by HNG treatment. Our results also provide evidence of Humanin playing a pivotal role in protecting cells with AMD mitochondria. In the future, it may be possible that AMD patient's blood samples containing damaged mitochondria may be useful as biomarkers for this condition. In conclusion, HNG may be a potential therapeutic target for treatment of dry AMD, a debilitating eye disease that currently has no available treatment. Further studies are needed to establish HNG as a viable mitochondria-targeting therapy for dry AMD
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European mtDNA Variants Are Associated With Differential Responses to Cisplatin, an Anticancer Drug: Implications for Drug Resistance and Side Effects.
Background: Cisplatin, a powerful antitumor agent, causes formation of DNA adducts, and activation of apoptotic pathways. Presently, cisplatin resistance develops in up to 70% of patients but the underlying molecular mechanism(s) are unclear and there are no markers to determine which patients will become resistant. Mitochondria play a significant role not only in energy metabolism but also retrograde signaling (mitochondria to nucleus) that modulates inflammation, complement, and apoptosis pathways. Maternally inherited mitochondrial (mt) DNA can be classified into haplogroups representing different ethnic populations that have diverse susceptibilities to diseases and medications. Methods: Transmitochondrial cybrids, where all cell lines possess identical nuclear genomes but either the H (Southern European) or J (Northern European) mtDNA haplogroups, were treated with cisplatin and analyzed for differential responses related to viability, oxidative stress, and expression levels of genes associated with cancer, cisplatin-induced nephrotoxicity and resistance, apoptosis and signaling pathways. Results: The cisplatin-treated-J cybrids showed greater loss of cell viability along with lower levels of reactive oxygen species and mitochondrial membrane potential compared to cisplatin-treated-H cybrids. After cisplatin treatment, J cybrids showed increased gene expression of BAX, CASP3, and CYP51A, but lower levels of SFRP1 compared to untreated-J cybrids. The cisplatin-treated-H cybrids had elevated expression of CDKN1A/P21, which has a role in cisplatin toxicity, compared to untreated-H cybrids. The cisplatin-treated H had higher transcription levels of ABCC1, DHRS2/HEP27, and EFEMP1 compared to cisplatin-treated-J cybrids. Conclusions: Cybrid cell lines that contain identical nuclei but either H mtDNA mitochondria or J mtDNA mitochondria respond differently to cisplatin treatments suggesting involvement of the retrograde signaling (from mitochondria to nucleus) in the drug-induced cell death. Varying toxicities and transcription levels of the H vs. J cybrids after cisplatin treatment support the hypothesis that mtDNA variants play a role in the expression of genes affecting resistance and side effects of cisplatin
Mechanism of Action of Risuteganib for Retinal Diseases through Protection of Retinal Pigment Epithelium (RPE) and Enhancement of Mitochondrial Functions
Purpose : Risuteganib is a novel synthetic peptide that has advanced through Phase II clinical trials, showing promising efficacy in retinal diseases, including dry age-related macular degeneration (AMD) and diabetic macular edema (DME). This study is to explore the mechanism of action (MOA) of risuteganib by uncovering its functional target(s) and the associated cell layer.
Methods : Fluorescent staining of mouse and rat retinal cryo-sections was performed with risuteganib-dye conjugates and compared with control peptide. Protective effects against oxidative stress was studied in ARPE-19 cell line challenged with tert-Butyl Hydroperoxide (tBHP) using WST-1 assay and Caspase 3/7 apoptosis assay. Mitochondrial bioenergetics were measured using Seahorse XF cell mito stress test.
Results : Peptide-dye staining of animal retinal tissue indicated preferential localization of risuteganib in the RPE layer. 24hr risuteganib pretreatment significantly rescued ARPE-19 cells from tBHP-induced oxidative stress in WST-1 assay (p<0.05) and Caspase 3/7 apoptosis assay (p<0.01). Seahorse bioenergetics measurement of ARPE-19 cells showed that risuteganib dose-dependently enhanced mitochondrial basal, maximal and ATP-related respirations of RPE cells.
Conclusions : Oxidative stress is one of the hallmarks of retinal diseases AMD and DME, and is associated with impaired RPE function. The observations of preferential binding to RPE layers in retina and the protection of mitochondrial function in RPE cells against oxidative stress in vitro, suggest that the clinically observed therapeutic effect of risuteganib in dry AMD and DME may be associated with supporting RPE cells and maintaining mitochondrial stability and function. Such a novel MOA of risuteganib could lead to new strategies for treatment of retinal diseases
Mechanism of Action of Risuteganib for Retinal Diseases through Protection of Retinal Pigment Epithelium (RPE) and Enhancement of Mitochondrial Functions
Purpose : Risuteganib is a novel synthetic peptide that has advanced through Phase II clinical trials, showing promising efficacy in retinal diseases, including dry age-related macular degeneration (AMD) and diabetic macular edema (DME). This study is to explore the mechanism of action (MOA) of risuteganib by uncovering its functional target(s) and the associated cell layer.
Methods : Fluorescent staining of mouse and rat retinal cryo-sections was performed with risuteganib-dye conjugates and compared with control peptide. Protective effects against oxidative stress was studied in ARPE-19 cell line challenged with tert-Butyl Hydroperoxide (tBHP) using WST-1 assay and Caspase 3/7 apoptosis assay. Mitochondrial bioenergetics were measured using Seahorse XF cell mito stress test.
Results : Peptide-dye staining of animal retinal tissue indicated preferential localization of risuteganib in the RPE layer. 24hr risuteganib pretreatment significantly rescued ARPE-19 cells from tBHP-induced oxidative stress in WST-1 assay (p<0.05) and Caspase 3/7 apoptosis assay (p<0.01). Seahorse bioenergetics measurement of ARPE-19 cells showed that risuteganib dose-dependently enhanced mitochondrial basal, maximal and ATP-related respirations of RPE cells.
Conclusions : Oxidative stress is one of the hallmarks of retinal diseases AMD and DME, and is associated with impaired RPE function. The observations of preferential binding to RPE layers in retina and the protection of mitochondrial function in RPE cells against oxidative stress in vitro, suggest that the clinically observed therapeutic effect of risuteganib in dry AMD and DME may be associated with supporting RPE cells and maintaining mitochondrial stability and function. Such a novel MOA of risuteganib could lead to new strategies for treatment of retinal diseases
Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro
Purpose: 7-ketocholesterol (7kCh), a natural byproduct of oxidation in lipoprotein deposits, is implicated in the pathogenesis of diabetic retinopathy and age-related macular degeneration (AMD). This study was performed to investigate whether several clinical drugs can inhibit 7kCh-induced caspase activation and mitigate its apoptotic effects on retinal cells in vitro.
Method: Two populations of retinal cells, human retinal pigment epithelial cells (ARPE-19) and rat neuroretinal cells (R28), were exposed to 7kCh in the presence of the following inhibitors: Z-VAD-FMK (pan-caspase inhibitor), simvastatin, memantine, epicatechin, and Z-IETD-FMK (caspase-8 inhibitor) or Z-ATAD-FMK (caspase-12 inhibitor). Caspase-3/7, -8, and -12 activity levels were measured by fluorochrome caspase assays to quantify cell death. IncuCyte live-cell microscopic images were obtained to quantify cell counts.
Results: Exposure to 7kCh for 24 hours significantly increased caspase activities for both ARPE-19 and R28 cells (P < 0.05). In ARPE cells, pretreatment with various drugs had significantly lower caspase-3/7, -8, and -12 activities, reported in % change in mean signal intensity (msi): Z-VAD-FMK (48% decrease, P < 0.01), memantine (decreased 47.8% at 1 μM, P = 0.0039 and 81.9% at 1 mM, P < 0.001), simvastatin (decreased 85.3% at 0.01 μM, P < 0.001 and 84.8% at 0.05 μM , P < 0.001) or epicatechin (83.6% decrease, P < 0.05), Z-IETD-FMK (68.1% decrease, P < 0.01), and Z-ATAD-FMK (47.7% decrease, P = 0.0017). In contrast, R28 cells exposed to 7kCh continued to have elevated caspase- 3/7, -8, and -12 activities (between 25.7% decrease and 17.5% increase in msi, P > 0.05) regardless of the pretreatment.
Conclusion: Several current drugs protect ARPE-19 cells but not R28 cells from 7kChinduced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases
cGAS Drives Noncanonical-Inflammasome Activation in Age-Related Macular Degeneration
Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits DICER1 deficiency, resultant accumulation of endogenous Alu-retroelement RNA, and NLRP3-inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human-cell-culture and mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β production and gasdermin D-dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-β, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of cGAS in responding to mobile-element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial-damage-induced inflammasome activation, expand the immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness
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