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• Research Paper Volume 13, Issue 8 pp 10866-10890

  Pharmacologic activation of autophagy without direct mTOR inhibition as a therapeutic strategy for treating dry macular degeneration

  Relevance score: 5.1766

  Qitao Zhang, Feriel Presswalla, Robin R. Ali, David N. Zacks, Debra A. Thompson, Jason ML. Miller

  Keywords: age-related macular degeneration (AMD), drusen, lipofuscin, retinal pigment epithelium (RPE), autophagy

  Published in Aging on April 19, 2021

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  Dry age-related macular degeneration (AMD) is marked by the accumulation of extracellular and intracellular lipid-rich deposits within and around the retinal pigment epithelium (RPE). Inducing autophagy, a conserved, intracellular degradative pathway, is a potential treatment strategy to prevent disease by clearing these deposits. However, mTOR inhibition, the major mechanism for inducing autophagy, disrupts core RPE functions. Here, we screened autophagy inducers that do not directly inhibit mTOR for their potential as an AMD therapeutic in primary human RPE culture. Only two out of more than thirty autophagy inducers tested reliably increased autophagy flux in RPE, emphasizing that autophagy induction mechanistically differs across distinct tissues. In contrast to mTOR inhibitors, these compounds preserved RPE health, and one inducer, the FDA-approved compound flubendazole (FLBZ), reduced the secretion of apolipoprotein that contributes to extracellular deposits termed drusen. Simultaneously, FLBZ increased production of the lipid-degradation product β-hydroxybutyrate, which is used by photoreceptor cells as an energy source. FLBZ also reduced the accumulation of intracellular deposits, termed lipofuscin, and alleviated lipofuscin-induced cellular senescence and tight-junction disruption. FLBZ triggered compaction of lipofuscin-like granules into a potentially less toxic form. Thus, induction of RPE autophagy without direct mTOR inhibition is a promising therapeutic approach for dry AMD.

• Research Paper Volume 12, Issue 16 pp 16579-16596

  Suppressing endoplasmic reticulum stress-related autophagy attenuates retinal light injury

  Relevance score: 6.438098

  Jing-Yao Song, Bin Fan, Lin Che, Yi-Ran Pan, Si-Ming Zhang, Ying Wang, Victoria Bunik, Guang-Yu Li

  Keywords: oxidative stress, ER stress, autophagy, AMD, PERK

  Published in Aging on August 28, 2020

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  Excessive light exposure is a principal environmental factor, which can cause damage to photoreceptors and retinal pigment epithelium (RPE) cells and may accelerate the progression of age-related macular degeneration (AMD). In this study, oxidative stress, endoplasmic reticulum (ER) stress and autophagy caused by light exposure were evaluated in vitro and in vivo. Light exposure caused severe photo-oxidative stress and ER stress in photoreceptors (661W cells) and RPE cells (ARPE-19 cells). Suppressing either oxidative stress or ER stress was protective against light damage in 661W and ARPE-19 cells and N-acetyl-L-cysteine treatment markedly inhibited the activation of ER stress caused by light exposure. Moreover, suppressing autophagy with 3-methyladenine significantly attenuated light-induced cell death. Additionally, inhibiting ER stress either by knocking down PERK signals or with GSK2606414 treatment remarkably suppressed prolonged autophagy and protected the cells against light injury. In vivo experiments verified neuroprotection via inhibiting ER stress-related autophagy in light-damaged retinas of mice. In conclusion, the above results suggest that light-induced photo-oxidative stress may trigger subsequent activation of ER stress and prolonged autophagy in photoreceptors and RPE cells. Suppressing ER stress may abrogate over-activated autophagy and protect the retina against light injury.

  

  Light exposure increases the level of HO-1 in photoreceptors and RPEs. 661W cells/ARPE-19 cells were cultured in dark conditions or exposed to 1500 Lux light for 1–3 days. The level of HO-1 protein in the whole cell lysate was determined with western blotting, and β-actin was referenced as an internal control. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, **P < 0.01.

  
  
  

  

  NAC treatment suppresses light-induced oxidative stress. 661W cells/ARPE-19 cells were pretreated with NAC (5 mM for 661W cells and 2.5 mM for ARPE-19 cells) or vehicle and cultured under light/dark conditions for 3 days. (A) The intracellular ROS were stained with DCFH-DA fluorescent probe identified by green fluorescence. Scale bar=50 μm. Relative fluorescence intensities were calculated and compared. (B) The GSH/GSSG ratio was measured with a GSH/GSSG Assay Kit. (C) The HO-1 level was determined with western blotting, and β-actin was referred as an internal control. (D) 661W cells pretreated with 5 mM NAC/vehicle were cultured under light/dark conditions for 3 days. ARPE-19 cells pretreated with 2.5 mM NAC/vehicle were cultured under light/dark conditions for 6 days. The cell death percentage was evaluated with PI/Hoechst staining. Scale bar=100μm. The percentage of cell death was calculated as PI-positive cells/total cells%. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, *P < 0.05, **P < 0.01.

  
  
  

  

  Light exposure induces ER stress in photoreceptors and RPEs. 661W cells and ARPE-19 cells were cultured in a dark condition or exposed to 1500 Lux light for 1–3 days after which the levels of ER stress markers were determined by western blotting. β-actin was referenced as an internal control. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, **P < 0.01.

  
  
  

  

  SAL treatment suppresses light-induced ER stress and protects photoreceptors and RPEs. (A) 661W cells/RPE cells were treated with SAL (1 μM, 10 μM, 20 μM and 50 μM) and cultured under 1500 Lux light or dark conditions for the indicated times. The percentage of cell death was evaluated with PI/Hoechst staining. Scale bar=100 μm. (B) The cells were treated with SAL (20 μM for 661W cells; 10 μM for ARPE-19 cells) or vehicle and cultured under light/dark conditions for 3 days, after which the levels of ER stress markers in the whole cell lysate were determined with western blotting, and β-actin was referenced as an internal control. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, NS: no significance, *P < 0.05, **P < 0.01.

  
  
  

  

  NAC treatment suppresses light-induced ER stress in photoreceptors and RPEs. The cells were treated with NAC (5 mM for 661W cells; 2.5 mM for ARPE-19 cells) or vehicle and cultured under light/dark conditions for 3 days, the levels of ER stress markers were determined with western blotting, and β-actin was referenced as an internal control. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, **P < 0.01.

  
  
  

  

  Inhibiting light-induced prolonged autophagy is protective. (A) 661W cells/ARPE-19 cells were cultured in a dark condition or exposed to 1500 Lux light for 1–3 days. The levels of BECN1 and LC3BII in the whole cell lysate was determined with western blotting, and β-actin was referenced as an internal control. (B) After 661W cells and ARPE-19 cells were treated with HCO (20 μM) or vehicle and cultured under light/dark conditions for 3 days, the level of LC3BII and P62 in the whole cell lysate were determined with western blotting, and β-actin was referenced as an internal control. (C) The cells were treated with 3MA (2.5 mM for 661W cells; 1 mM for ARPE-19) or vehicle and cultured under light/dark conditions for 3 days. The level of BECN1 and LC3BII in the whole cell lysate were determined with western blotting, and β-actin was referenced as an internal control. (D) 661W cells pretreated with 2.5 mM 3MA/vehicle were cultured under light/dark conditions for 3 days. ARPE-19 cells pretreated with 1 mM 3MA/vehicle were cultured under light/dark conditions for 6 days. The percentage of cell death was evaluated with PI/Hoechst staining. Scale bar=100 μm. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, *P < 0.05, **P < 0.01.

  
  
  

  

  Inhibiting ER stress suppresses light-related autophagy. 661W cells/ARPE-19 cells were treated with SAL (20 μM for 661W cells; 10 μM for ARPE-19 cells) or vehicle and cultured under light/dark conditions for 3 days. The level of BECN1 and LC3BII in the whole cell lysate were determined with western blotting, and β-actin was referenced as an internal control. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, NS: no significance, *P < 0.05, **P < 0.01.

  
  
  

  

  Inhibiting PERK suppresses light-related autophagy. (A) 661W cells were infected with lentivirus-expressed PERK shRNA (sh-PERK) or negative control shRNA (NC). The level of PERK in the whole cell lysate was determined with western blotting, and β-actin was referenced as an internal control. (B) 661W cells with stable PERK knockdown/ARPE-19 cells treated with GSK (5 μM) or vehicle were cultured under light/dark conditions for 3 days. The target proteins in the whole cell lysate were determined with western blotting, and β-actin was referenced as an internal control. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, NS: no significance, *P < 0.05, **P < 0.01.

  
  
  

  

  Inhibiting PERK blocks autophagic flow and protects the light-damaged cells. (A) 661W cells with stable PERK knockdown and ARPE-19 cells were infected with mCherry-GFP double labeled-LC3B mediated by adenovirus. At 48 h after infection, the ARPE-19 cells were treated with GSK (5 μM) or vehicle. The cells were cultured under 1500 Lux light condition for 3 days and photographed under fluorescence microscopy. Scale bar=20 μm. (B) 661W cells with PERK knockdown were cultured under light/dark conditions for 3 days, but ARPE-19 cells treated with GSK (5 μM) or vehicle were cultured under light/dark conditions for 6 days. The percentage of cell death was evaluated with PI/Hoechst staining. Scale bar=100 μm. Three independent experiments are conducted two weeks apart. The results are presented as the mean± SEM. n (per group) =3, NS: no significance, **P < 0.01.

  
  
  

  

  Suppressing ER stress inhibits prolonged autophagy and protects the retina against light injury. The mice were intraperitoneally injected with a dose of 1 mg/kg once a day for 7 days. On the third day of administration, the mice were exposed to continuous 7000 Lux visible light for 12 h. After light exposure, the mice were fed in the animal room with the normal light/dark cycle. On the fifth day of light exposure, the mice were sacrificed, and the eyeballs were enucleated. (A) The retinas were collected, and target proteins were determined with western blotting. β-actin was referenced as an internal control. Three independent experiments are conducted three weeks apart. The results are presented as the mean± SEM. n (per group) =3, NS: no significance, *P < 0.05, **P < 0.01. (B) The retinas were sectioned and stained with H&E and photographed under a microscope. Scale bar=100 μm; 20 μm. The thickness of the outer nuclear layer (ONL) was measured and quantitatively analyzed. The results are presented as the mean± SEM, n (per group) =6, NS: no significance, **P < 0.01.

  
  
  

• Research Paper Volume 12, Issue 10 pp 9031-9040

  Role of Citicoline in an in vitro AMD model

  Relevance score: 5.7248387

  Sonali Nashine, M. Cristina Kenney

  Keywords: Citicoline, age-related macular degeneration (AMD), neuroprotection, RPE, mitochondria

  Published in Aging on May 29, 2020

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  Citicoline is the exogenous form of the nootropic, Cytidine 5'-diphosphate-choline that exerts its neuroprotective effects in the brain as well as in the eye. The current study characterized the cytoprotective effects of purified Citicoline in transmitochondrial AMD (Age-related Macular Degeneration) RPE cybrid cells which carry diseased mitochondria from clinically characterized AMD patients. The effects of Citicoline were examined via flow cytometry analysis of AnnexinV/ PI-stained cells, IncuCyte live-cell imaging analysis to quantify cells undergoing caspase-3/7-mediated apoptosis, analyses of gene expression profiles of apoptosis, hypoxia, and angiogenesis markers, and measurement of ROS levels and cell viability. Our results demonstrated that Citicoline when added exogenously alleviates apoptotic effects as evidenced by diminished AnnexinV/PI and Caspase-3/7 staining, downregulation of apoptosis genes, enhanced cell viability, and reduced oxidative stress in AMD RPE cybrid cells. In conclusion, our study identified Citicoline as a protector in AMD RPE cybrid cells in vitro. However, further studies are required to establish the merit of Citicoline as a cytoprotective molecule in AMD and to decipher the molecular underpinnings of its mechanism of action in AMD.

  

  (A) AMD Untreated cells’ Representative Annexin V/ PI staining flow cytometry image; (B) AMD Untreated cells’ Representative Annexin V/ PI fluorescence intensity scatter plot; (C) AMD Citicoline-treated cells’ Representative Annexin V/ PI staining flow cytometry image; (D) AMD Citicoline-treated cells’ Representative Annexin V/ PI fluorescence intensity scatter plot; (E) AMD Untreated vs. AMD Citicoline-treated Annexin V/ PI fluorescence intensity quantitation.

  
  
  

  

  (A) BAX gene expression in AMD Untreated and AMD Citicoline-treated cells. (B) Caspase-3 gene expression in AMD Untreated and AMD Citicoline-treated cells. (C) Caspase-9 gene expression in AMD Untreated and AMD Citicoline-treated cells. (D) BCL2L13 gene expression in AMD Untreated and AMD Citicoline-treated cells. (E) Cell viability MTT assay.

  
  
  

  

  (A) Upper and lower panels show Representative Incucyte live-cell images of untreated and Citicoline-treated AMD cells ,respectively, in phase-contrast (first column), stained with NucLight Red (second column), stained with Caspase-3/7 Green (third column), overlap i.e., Caspase-3/7 + NucLight (fourth column), and Merge i.e., Phase-contrast + Caspase-3/7 + NucLight (fifth column). Scale bar = 400 μM.(B) Quantitation of Caspase-3/7 overlap/ Red object count at the 48 h time point. (C) Quantitation of Caspase-3/7 overlap/ Red object count at the 72 h time point.

  
  
  

  

  (A) ROS levels in AMD Untreated and AMD Citicoline-treated cells, (B) HMOX1 gene expression levels in AMD Untreated and AMD Citicoline-treated cells, and (C) HMOX2 gene expression levels in AMD Untreated and AMD Citicoline-treated cells.

  
  
  

  

  (A) HIF-1α gene expression in AMD Untreated and AMD Citicoline-treated cells. (B) VEGF gene expression in AMD Untreated and AMD Citicoline-treated cells.

  
  
  

• Research Paper Volume 12, Issue 7 pp 6151-6171

  Systemic administration of the di-apocarotenoid norbixin (BIO201) is neuroprotective, preserves photoreceptor function and inhibits A2E and lipofuscin accumulation in animal models of age-related macular degeneration and Stargardt disease

  Relevance score: 6.4660587

  Valérie Fontaine, Elodie Monteiro, Mylène Fournié, Elena Brazhnikova, Thinhinane Boumedine, Cécile Vidal, Christine Balducci, Louis Guibout, Mathilde Latil, Pierre J. Dilda, Stanislas Veillet, José-Alain Sahel, René Lafont, Serge Camelo

  Keywords: norbixin, retinal function, A2E, AMD, Stargardt disease

  Published in Aging on April 7, 2020

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  Atrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of Abca4^-/- Rdh8^-/- mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'-cis-norbixin (norbixin), a natural di-apocarotenoid, prepared from Bixa orellana seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in Abca4^-/- Rdh8^-/- mice of different ages, following three experimental designs: “preventive”, “early curative” and “late curative” supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in Abca4^-/- Rdh8^-/- mice following the “early curative” supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment.

  

  Effect of norbixin on ERG and retinal phototoxicity after BLD in BALB/c mice. (A) schematic representation of the protocol design. (B) Scotopic A wave, (C) Scotopic B wave, (D) Photopic B wave, ERG recorded 7 days after BLD. (E) Representative cryosection pictures showing Hoechst 33342 staining of the retinal cell nuclei one week after BLD. (F) Graph showing the number of photoreceptor layers measured along the retina each 200 μm from the optic nerve. (G) Histograms showing the area under the curve (AUC) calculated from the photoreceptor layer quantification and used to perform statistical analyses. IP: intra-peritoneal; no BLD: no blue light damage; no inj.: no injection; OS: outer segment; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Bars represent mean ± s.e.m. with n = 8 per group. ^# or *p<0.05, ^## or **p<0.01, ^### or ***p<0.001, ^#### or ****p<0.0001 compared to non-injected or to vehicle, respectively (One-way ANOVA, Dunnett's post-test).

  
  
  

  

  Kinetics of loss of amplitude of scotopic A and B waves and photopic B wave, of A2E accumulation and of loss of photoreceptor nuclear layers in Abca4^-/- Rdh8^-/- mice aged between 2 and 18 months. (A) Scotopic A wave recorded at a flash intensity of 10 cd.s/m² (n = 30-50). (B) Scotopic B wave recorded at a flash intensity of 10 cd.s/m² (n = 30-50). (C) Photopic B wave recorded at a flash intensity of 30 cd.s/m² (n = 30-50). (D) Kinetics of loss of photoreceptor nuclear layers (n = 7-14). (E) Kinetics of A2E accumulation (n = 15-40). Bars represent mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001 (One-way ANOVA, Dunnett's post-test).

  
  
  

  

  RPE65, cathepsin D and GFAP immunostaining in Abca4^-/- Rdh8^-/- mice of different ages. Pictures showing retinal cryosections of 2-month-old (A, C, E) and 18-month-old (B, D, F) mice captured in bright field (left pictures) or after immunostaining for cathepsin D (A, B), RPE65 (C, D) and GFAP (E, F). OS: outer segment; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.

  
  
  

  

  Effect of norbixin preventive supplementation from 1.5 to 7.5 months in Abca4^-/- Rdh8^-/- mice. (A) schematic representation of the 6-month preventive supplementation protocol design. (B) Scotopic A wave, (C) Scotopic B wave, (D) Photopic B wave, recorded after 6 months of oral supplementation with norbixin in Abca4^-/- Rdh8^-/- mice compared to mice fed with normal chow (vehicle) and to 1.5-month-old mice. (E) Quantification of photoreceptor nuclear layers along the superior and inferior poles of the retina each measured every 200 μm apart from the optic nerve. (F) A2E quantification in eyes from 1.5-month-old Abca4^-/- Rdh8^-/- mice, 7.5-month-old mice fed with normal chow or with norbixin-containing pellets. Bars represent mean ± s.e.m. with n= 8 mice per group (i.e. n=16 eyes per group for ERG). **p<0.01 compared to vehicle (One-way ANOVA, Dunnett's post-test).

  
  
  

  

  Effect of norbixin early curative supplementation from 9 to 15 months in Abca4^-/- Rdh8^-/- mice. (A) Schematic representation of the 6-month early curative supplementation protocol design. (B) Scotopic A wave, (C) Scotopic B wave, (D) Photopic B wave ERG recorded after 6 months of oral supplementation with norbixin in Abca4^-/-Rdh8^-/- mice compared to mice fed with normal chow (vehicle) and to 1.5 and 9-month-old mice. (E) Quantification of photoreceptor nuclear layers along the superior and inferior poles of the retina each measured every 200 μm apart from the optic nerve. (F) A2E quantification in eyes from 9-month-old Abca4^-/- Rdh8^-/- mice, 15-month-old mice fed with normal chow or with norbixin-containing pellets. Bars represent mean ± s.e.m. with n = 6 per group (i.e. n=12 eyes for the norbixin treated group and n=11 eyes for the vehicle treated group for ERG). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to vehicle (One-way ANOVA, Dunnett's post-test).

  
  
  

  

  Effect of norbixin late curative supplementation from 12 to 17 months in Abca4^-/- Rdh8^-/- mice. (A) Schematic representation of the 5-month late curative supplementation protocol design. (B) Scotopic A wave ERG recorded after 5 months of oral supplementation with norbixin in Abca4^-/- Rdh8^-/- mice compared to mice fed with normal chow (vehicle) and to 1.5- and 12-month-old mice. (C) Scotopic B wave. (D) Photopic B wave. (E) Quantification of photoreceptor nuclear layers along the superior and inferior poles of the retina each measured every 200 μm apart from the optic nerve. (F) A2E quantification in eyes from 12-month-old Abca4^-/- Rdh8^-/- mice, 17-month-old mice fed with normal chow or with norbixin-containing pellets. (G) Representative images of lipofuscin content in RPE cells of 17-month-old vehicle and norbixin-treated mice. Large granules of lipofuscin are found in the RPE cytoplasm (white asterisk). (H) Histograms showing the quantified lipofuscin granules expressed by area of 100 μm². (I) Histograms representing the surface of cytoplasm occupied by lipofuscin and expressed in percentage of total cytoplasm surface. Bars represent mean ± s.e.m. with n = 8 per group (i.e. n=16 eyes per group for ERG). *p<0.05, **p<0.01 compared to vehicle (One-way ANOVA, Dunnett's post-test).

  
  
  

• Research Paper Volume 11, Issue 17 pp 6691-6713

  PU-91 drug rescues human age-related macular degeneration RPE cells; implications for AMD therapeutics

  Relevance score: 5.1766

  Sonali Nashine, Sudhakar R. Subramaniam, Marilyn Chwa, Anthony Nesburn, Baruch D. Kuppermann, Howard Federoff, M. Cristina Kenney

  Keywords: age-related macular degeneration (AMD), RPE, PGC-1α, mitochondria, FDA-approved drugs

  Published in Aging on September 2, 2019

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  Since mitochondrial dysfunction is implicated in the pathogenesis of AMD, this study is based on the premise that repurposing of mitochondria-stabilizing FDA-approved drugs such as PU-91, might rescue AMD RPE cells from AMD mitochondria-induced damage. The PU-91 drug upregulates PGC-1α which is a critical regulator of mitochondrial biogenesis. Herein, we tested the therapeutic potential of PU-91 drug and examined the additive effects of treatment with PU-91 and esterase inhibitors i.e., EI-12 and EI-78, using the in vitro transmitochondrial AMD cell model. This model was created by fusing platelets obtained from AMD patients with Rho⁰ i.e., mitochondria-deficient, ARPE-19 cell lines. The resulting AMD RPE cell lines have identical nuclei but differ in their mitochondrial DNA content, which is derived from individual AMD patients. Briefly, we report significant improvement in cell survival, mitochondrial health, and antioxidant potential in PU-91-treated AMD RPE cells compared to their untreated counterparts. In conclusion, this study identifies PU 91 as a therapeutic candidate drug for AMD and repurposing of PU-91 will be a smoother transition from lab bench to clinic since the pharmacological profiles of PU-91 have been examined already.

  

  PU-91 regulates the mitochondrial biogenesis pathway. We used quantitative qRT-PCR to measure the relative mtDNA copy number (A), and the gene expression of markers of the mitochondrial biogenesis pathway such as PGC-1α (B), NRF-1 (C), NRF-2 (D), PPAR-α (E), and PPAR-γ (F). PU-91-treated AMD cybrids (AMD PU-91) had higher mtDNA copy numbers and increased gene expression levels of all the above-mentioned markers (p≤0.05, n=4-5). Data are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05.

  
  
  

  

  PU-91 regulates mitochondrial function. We used the fluorometric JC-1 assay and MitoSOX assay to measure mitochondrial membrane potential and mitochondrial superoxide production, respectively. Treatment with PU-91 led to elevated mitochondrial membrane potential (p≤0.05, n=3) (A) and reduced mitochondrial superoxide production (p≤0.05, n=3) (B) in AMD cybrids (AMD PU-91) compared to the untreated group (AMD UN). Furthermore, PU-91-treated AMD cybrids showed upregulation of the mitochondrial superoxide dismutase, SOD2 gene (p≤0.05, n=5) (C) and reduced expression of HIF1α gene (p≤0.05, n=3-4) (D). (E) PU-91 upregulates MT-RNR2 gene. Using TaqMan probe for the MT-RNR2 gene, qRT-PCR analysis revealed that PU-91 increases MT-RNR2 gene expression by 104% compared to untreated control (p≤0.05, n=5). Data are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05, **p≤0.01.

  
  
  

  

  PU-91 alters mitochondrial GFP fluorescence intensity. Untreated (AMD UN) and PU-91-treated AMD cybrids (AMD PU-91) were stained with CellLight mitochondrial GFP stain followed by confocal imaging of cells. (A) Shows representative bright-field, DAPI, mtGFP, and overlay (DAPI + mtGFP) confocal images. PU-91-treated AMD cybrids had a drastic increase in mtGFP fluorescence intensity compared to the untreated group (p≤0.05, n=3) (B). Data are presented as mean ±SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05.

  
  
  

  

  PU-91 regulates apoptotic cell death. qRT-PCR analysis showed downregulation of apoptotic genes such as Caspase-3 (p≤0.05, n=4) (A) and BAX (p≤0.05, n=4) (B) in AMD cybrids treated with PU-91. Using the MTT assay, it was observed that PU-91-treated AMD cybrids had a higher number of viable cells compared to the untreated group (p≤0.05, n=4) (C). Data are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05, **p≤0.01.

  
  
  

  

  PU-91 regulates apoptotic cell death – Caspase-3/7 staining. (A) Shows representative IncuCyte live-cell images of untreated (AMD UN) and PU-91-treated AMD cybrid cells (AMD PU-91) stained with NucLight Red and Caspase-3/7 Green reagent. (B) Shows quantitation graphs for the 48 h and 72 h time points. Data are presented as mean ±SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05.

  
  
  

  

  PU-91 regulates inflammation and complement. qRT-PCR analysis showed lower gene expression of inflammation markers such as IFNB1 (p≤0.05, n=4) (A), IL-18 (p≤0.05, n=4) (B) in PU-91-treated AMD cybrids (AMD PU-91) compared to untreated AMD cybrids (AMD UN). However, PU-91 upregulated the complement inhibitor CFH gene (p≤0.05, n=3-4) (C). Data are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05.

  
  
  

  

  Effect of PU-91 + EI-12/ EI-78 on cell viability. This figure shows cell viability differences using MTT assay in AMD cells treated with P + EI-12 (A and B) / EI-78 (C and D) at 48 h and 72 h. Data (n=3) are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test were used to measure statistical differences; *p≤0.05. P = PU-91; EI = Esterase Inhibitor.

  
  
  

  

  Effect of PU-91 + EI-12 on gene expression. qRT-PCR analysis showed differential expression of PGC-1α (A), Caspase-3 (B), IL-18 (C), VEGF (D), and SOD2 (E) genes in AMD RPE cells at the 72 h time point. Data (n=3) are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05. P = PU-91; EI = Esterase Inhibitor.

  
  
  

  

  Effect of PU-91 + EI-78 on gene expression. qRT-PCR analysis showed differential expression of PGC-1α (A), Caspase-3 (B), IL-18 (C), VEGF (D), and SOD2 (E) genes in AMD RPE cells at the 72 h time point. Data (n=3) are presented as mean ± SEM and normalized to untreated (UN) AMD cybrids which were assigned a value of 1. Mann-Whitney test was used to measure statistical differences; *p≤0.05. P = PU-91; EI = Esterase Inhibitor.

  
  
  

• Research Paper Volume 11, Issue 13 pp 4323-4337

  HMGB1 and Caveolin-1 related to RPE cell senescence in age-related macular degeneration

  Relevance score: 5.9968004

  Shuo Sun, Bincui Cai, Yao Li, Wenqi Su, Xuzheng Zhao, Boteng Gong, Zhiqing Li, Xiaomin Zhang, Yalin Wu, Chao Chen, Stephen H. Tsang, Jin Yang, Xiaorong Li

  Keywords: A2E, HMGB1, Caveolin-1, RPE cell senescence, AMD

  Published in Aging on July 7, 2019

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  Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is considered a major cause of RPE dysfunction and senescence in age-related macular degeneration (AMD), and N-retinylidene-N-retinylethanolamine (A2E) is the main fluorophore identified in lipofuscin from aged human eyes. Here, human-induced pluripotent stem cell (iPSC)-RPE was generated from healthy individuals to reveal proteomic changes associated with A2E-related RPE cell senescence. A novel RPE cell senescence-related protein, high-mobility group box 1 (HMGB1), was identified based on proteomic mass spectrometry measurements on iPSC-RPE with A2E treatment. Furthermore, HMGB1 upregulated Caveolin-1, which also was related RPE cell senescence. To investigate whether changes in HMGB1 and Caveolin-1 expression under A2E exposure contribute to RPE cell senescence, human ARPE-19 cells were stimulated with A2E; expression of HMGB1, Caveolin-1, tight junction proteins and senescent phenotypes were verified. HMGB1 inhibition alleviated A2E induced cell senescence. Migration of RPE cells was evaluated. Notably, A2E less than or equal to 10μM induced both HMGB1 and Caveolin-1 protein upregulation and HMGB1 translocation, while Caveolin-1 expression was downregulated when there was more than 10μM A2E. Our data indicate that A2E-induced upregulation of HMGB1、Caveolin-1 and HMGB1 release may relate to RPE cell senescence and play a role in the pathogenesis of AMD.

  

  Proteomic mass spectrometry-based measurement of differential expression of HMGB1. (A) The flow chart of shotgun mass spectrometry. (B) Volcano plot illustrating significant differential abundant proteins based on quantitative analysis. The -log₁₀ (P value) was plotted against log₂(fold change A2E treatment/Control). Proteins were significantly upregulated (red dots) or downregulated (green dots) between the A2E treatment and control. The red arrowhead indicates HMGB1.

  
  
  

  

  Experimental validation that blue light exposure of A2E-treated ARPE-19 cells induces HMGB1 upregulation and translocation. (A) An MTT assay was performed on RPE cells treated with different concentrations of A2E with or without blue light photosensitization. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, compared to the control, n=3. (B) FDA/PI staining of RPE cells after in vitro culture for 48 h with 10 μM A2E + blue light (10 min). Most living RPE cells were stained green by fluorescein diacetate (FDA); a few dead cells were stained red bypropidium iodide (PI). (C) Western blot analyses showed that HMGB1 protein expression was higher in 10μM A2E + blue light-treated cells compared to the control and also higher in the blue light treatment, as quantified by densitometry; the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (D) HMGB1 localization in RPE cells was assessed by confocal microscopy after 10μM A2E + blue light treatment. HMGB1 moved from the nucleus (arrow) to the cytoplasm (star) after 10μM A2E + blue light treatment. Nuclei are labelled with DAPI (blue); HMGB1 is stained green.

  
  
  

  

  HMGB1 upregulation and release increase the expression of Caveolin-1. (A) (i) Western blot analyses showed that overexpression of HMGB1 upregulated Caveolin-1; β-actin was used as the loading control; Western blot results were quantified by densitometry, and the results are expressed as a ratio with β-actin. (ii) qPCR analyses showed that overexpression of HMGB1 upregulated Caveolin-1. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (iii) Expression of EGFP and Caveolin-1 was assessed by immunofluorescence in HMGB1-overexpressing RPE cells and negative-control RPE cells. (B) Protein interaction between HMGB1 and Caveolin-1 was revealed by the STRING version 9.1 program. (C) Relative Caveolin-1expression in RPE cell incubated with normal medium, 1μg/ml rHMGB1, 100μM GA, or 1μg/ml rHMGB1+100μM GA, Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (D) Western blot analyses showed that knock-down of HMGB1 downregulated Caveolin-1; Tublin was used as the loading control, western blot results were quantified by densitometry, and the results are expressed as a ratio with Tublin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3.

  
  
  

  

  Overexpression of Caveolin-1 induced ARPE-19 cell senescence and inhibited migration and invasion. (A) Western blot analyses showed that overexpression of Caveolin-1 upregulated Zo-1 and β-catenin; β-actin was used as the loading control. (B) Western blot results were quantified by densitometry, and the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, n=3. (C) qPCR analyses showed that overexpression of Caveolin-1 upregulated Zo-1 and β-catenin. Data are presented as means ± SD; * indicates a p value < 0.05, n=3. (D) Expression of EGFP, Zo-1 and β-catenin was assessed by immunofluorescence in Caveolin-1-overexpressing RPE cells and negative-control RPE cells. (E) Representative microscopic images of β-galactosidase staining in RPE cells showed overexpression of Caveolin-1 in RPE cells compared with that in negative-control RPE cells. Quantification of percentage of cells with positive SA-β-gal staining.Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (F) (i) Wound-healing assays in Caveolin-1-overexpressing RPE cells. (ii). Transwell invasion assays in Caveolin-1-overexpressing RPE cells. (G) (i) The rate of cell migration in different groups was measured at different time points. Note that cell migration was decreased in Caveolin-1-overexpressing RPE cells. (ii) The mean number of invaded cells was assessed in 5 fields. Note that cell invasion was decreased in Caveolin-1-overexpressing RPE cells. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, n=3.

  
  
  

  

  Blue light exposure of A2E-treated ARPE-19 cells increased HMGB1 and Caveolin-1 expression. (A) Western blot assay for HMGB1 and Caveolin-1 in RPE cells treated with a concentration gradient of A2E with or without blue light, quantified by densitometry, and the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (B) Representative microscopic images of β-galactosidase staining in RPE cells with various concentrations of A2E. Quantification of percentage of cells with positive SA-β-gal staining.Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (C) The release of HMGB1 induced by A2E treatment were detected by ELISA assays.

  
  
  

  

  Glycyrrhizic acid alleviated A2E induced cell senescence. (A) An MTT assay was performed on RPE cells treated with different concentrations of GA. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (B)The release of HMGB1 induced by different concentrations of A2E+BL with or without 100μM GA were detected by ELISA assays. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (C) Representative microscopic images of β-galactosidase staining in RPE cells induced by different concentrations of A2E+BL with or without 100μM GA. (D) Quantification of percentage of cells with positive SA-β-gal staining. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (E) Proposed schematic model for strategies for HMGB1 inhibition in response to A2E treatment.

  
  
  

• Research Paper Volume 11, Issue 4 pp 1177-1188

  Nutraceutical effects of Emblica officinalis in age-related macular degeneration

  Relevance score: 4.315921

  Sonali Nashine, Raj Kanodia, Anthony B. Nesburn, Girish Soman, Baruch D. Kuppermann, M. Cristina Kenney

  Keywords: Emblica officinalis, Phyllanthus emblica, Indian gooseberry, Amla, nutraceutical, age-related macular degeneration, AMD

  Published in Aging on February 21, 2019

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  Emblica officinalis Gaetrn (i.e., Phyllanthus emblica/ Indian gooseberry/ Amla) (EO) has been used extensively as a nutraceutical in several diseases since it is known to boost immunity and offers numerous health benefits such as antioxidant, anti-inflammatory, and anti-aging effects. The goal of our study was to test the hypothesis that EO will rescue human AMD RPE transmitochondrial cells from mitochondria-induced cellular damage. AMD RPE transmitochondrial cell lines were created by fusion of mitochondria DNA-deficient APRE-19 (Rho0) cells with platelets isolated from AMD patients, and therefore had identical nuclei but differed in mitochondrial DNA content. These AMD RPE cells were treated with EO extract followed by characterization of effects of EO using cellular and molecular assays. Herein, EO significantly improved live cell number and mitochondrial membrane potential, reduced apoptosis and oxidative stress, down-regulated VEGF, and up-regulated PGC-1α. In conclusion, EO improved cellular and mitochondrial health, thereby playing a key cytoprotective role in AMD in vitro. Further studies are required to examine the mechanisms that mediate the cytoprotective effects of EO.

  

  EO concentration optimization. Bar graph showing the effects of EO on cell death in AMD RPE cybrid cells. No difference was observed between the AMD untreated (bar 1) vs. AMD solvent control (bar 2) groups. Furthermore, no statistically significant difference was observed between untreated (bar 1) and 10 mg/mL EO-treated (bar 3) AMD cybrids. Higher viable cell numbers were observed in EO-treated AMD cybrids at concentrations of 15 mg/mL (bar 4), 20 mg/mL (bar 5), and 25 mg/mL (bar 6). *** indicates p<0.001; ns indicates non-significant p-value. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1. Experiments were performed at the 24 h time-point.

  
  
  

  

  Effect of EO on cell viability. When treated with 25 mg/mL EO, AMD cybrid cells showed consistently increased viable cell numbers at 24 h (A), 48 h (B), and 72 h (C) compared to their untreated counterparts. ** indicates p<0.01. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1.

  
  
  

  

  Effect of EO on Caspase-3/7 and NucLight staining. This figure shows representative IncuCyte live-cell images of untreated and EO-treated AMD cybrid cells stained with NucLight Red and Caspase-3/7 Green reagent (A) and quantitation graphs for Caspase-3/7 Green and NucLight Red staining at the 24 h (B) and 48 h (C) time points. * indicates p<0.05. Data are presented as mean ± SEM, normalized to untreated AMD cybrids, which were assigned a value of 1.

  
  
  

  

  Effect of EO on Caspase-3 and MT-RNR2 gene expression. Treatment of AMD cybrids with EO reduced the gene expression of Caspase-3 (A) and up-regulated MT-RNR2 gene (B). ** indicates p<0.01. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1.

  
  
  

  

  Effect of EO on ROS assay and SOD2 gene expression. Addition of EO lowered ROS levels in AMD cybrids at 24 h (A), 48 h (B), and 72 h (C) time points. (D) shows increased expression of the antioxidant gene, SOD2, as a result of treatment with EO. ** and * indicate p<0.01 and p<0.05 respectively. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1.

  
  
  

  

  Effect of EO on mitochondrial membrane potential and PGC-1α gene expression. This figure shows increased mitochondrial membrane potential in EO-treated AMD RPE cells (A) and increased PGC-1α gene expression in EO-treated AMD cybrids (B). * indicates p<0.05. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1.

  
  
  

  

  Effect of EO on VEGF gene expression and on cell viability and ROS levels in amyloid-β-stressed AMD cells. This figure showed down-regulation of VEGF gene in EO-treated AMD cybrids (A). Pretreatment with EO rescued AMD cybrids from amyloid-β-induced damage as shown by changes in cell viability (B (bar 2 versus bar 5) and ROS levels (C (bar 2 versus bar 5). *, **, and *** indicate p<0.05, p<0.01, and p<0.001 respectively; ns indicates non-significant p-value. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1.

  
  
  

• Research Paper Volume 10, Issue 12 pp 4241-4247

  Circulating insulin-like growth factor-1: a new clue in the pathogenesis of age-related macular degeneration

  Relevance score: 5.202393

  Niccolò Castellino, Antonio Longo, Teresio Avitabile, Andrea Russo, Matteo Fallico, Vincenza Bonfiglio, Mario Damiano Toro, Robert Rejdak, Katarzyna Nowomiejska, Paolo Murabito, Claudio Furino, Michele Reibaldi

  Keywords: age‐related macular degeneration, neovascular AMD, IGF-1, anti‐VEGF, senescence, somatomedin C

  Published in Aging on December 29, 2018

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  In order to investigate Insulin-like growth factor-1 (IGF-1) blood levels in male and female age-matched patients affected by early, intermediate, neovascular age related macular degeneration (AMD) and healthy subjects (no AMD) were enrolled in a prospective, observational study. All patients enrolled were classified according to 4 stages classification of AMD from Age-related eye disease study (AREDS). Each subject underwent a complete ophthalmic examination including best corrected visual acuity (BCVA), applanation tonometry, slit-lamp biomicroscopic examination, color fundus photography, optical coherence tomography (OCT) and, if needed, fluorescein angiography. Overall, 224 anti-VEGF naïve subjects including 56 patients in early AMD group, 56 patients in intermediate AMD group, 56 patients in neovascular AMD group and 56 patients in no AMD group were recruited. For each group 28 male patients and 28 female patients were enrolled. IGF-1 hematic levels were significantly higher (p<0.005) in the neovascular AMD group and in the intermediate AMD group in comparison to no AMD group; no significant difference between early AMD group and no AMD group was found. Our analysis has shown an increment of IGF-1 levels in both neovascular and intermediate stage of AMD supporting the hypothesis that IGF-1 may play a role in the pathogenesis of AMD.

  

  IGF-1 levels in all patients, males and females, in four groups.

  
  
  

  

  Correlations between IGF-1 levels and age in four groups.

  
  
  

• Research Paper Volume 10, Issue 11 pp 3173-3184

  Effects of senescent secretory phenotype acquisition on human retinal pigment epithelial stem cells

  Relevance score: 6.29589

  Raffaella Lazzarini, Michele Nicolai, Vittorio Pirani, Cesare Mariotti, Roberto Di Primio

  Keywords: AMD, RPESCs, age-related diseases, senescence, inflammation

  Published in Aging on November 16, 2018

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  Regenerative medicine approaches based on mesenchymal stem cells (MSCs) are being investigated to treat several aging-associated diseases, including age-related macular degeneration (AMD). Loss of retinal pigment epithelium (RPE) cells occurs early in AMD, and their transplant has the potential to slow disease progression.

  The human RPE contains a subpopulation of cells - adult RPE stem cells (RPESCs) – that are capable of self-renewal and of differentiating into RPE cells in vitro. However, age-related MSC changes involve loss of function and acquisition of a senescence-associated secretory phenotype (SASP), which can contribute to the maintenance of a chronic state of low-grade inflammation in tissues and organs.

  In a previous study we isolated, characterized, and differentiated RPESCs. Here, we induced replicative senescence in RPESCs and tested their acquisition of the senescence phenotype and the SASP as well as the differentiation ability of young and senescent RPESCs.

  Senescent RPESCs showed a significantly reduced proliferation ability, high senescence-associated β-galactosidase activity, and SASP acquisition. RPE-specific genes were downregulated and p21 and p53 protein expression was upregulated.

  These findings document the effects of senescence and SASP acquisition on RPESC differentiation ability and highlight the need for a greater understanding of their role in AMD pathogenesis.

  

  Proliferation rate, β-gal positivity, telomere length, and cell morphology during RPESC replicative senescence. RPESC replicative senescence. (A) Cumulative number of population doublings (CPD) in RPESCs grown to senescence. (B) Percentage of β-gal-positive cells detected during RPESC replicative senescence from P1 to P16. P11, number of culture passages. Data are reported as mean ± SD. *P =0.039. (C) RPESC telomere length during replicative senescence was analyzed from P1 to P18; data are reported as mean ± SD of 3 independent experiments. (D) Morphological analysis of young (P3) and senescent (P16) RPESCs by the TRIC-phalloidin immunofluorescence assay. Senescent RPESCs appear enlarged and flattened. Magnification 20X, scale bar 200 µm. Pictures are representative of 3 independent experiments.

  
  
  

  

  SASP induction in senescent RPESCs. Young (P3) and senescent (P16) RPESCs were maintained in culture for 48 h. The supernatant was analyzed for IL-6 (A), IL-12 (B), IL-17 (C), TNF-α (D), TGFβ1 (E), INF-γ (F), IL-4 (G), IL-10 (H), and IL-13 (I), by ELISA. Data are mean ± SD of 3 independent experiments. *P = from 0.021 to 0.041.

  
  
  

  

  mRNA expression levels of stemness and RPE-specific genes in senescent and young RPESCs. (A) qRT-PCR analysis of the expression levels of stemness genes in senescent (P16) and young (P3) RPESCs. (B) qRT-PCR analysis of the mRNA levels of RPE-specific genes in senescent and young RPESCs. Data are mean ± SD of 3 independent experiments. *P = from 0.026 to 0.036.

  
  
  

  

  Senescence-associated gene expression profile in senescent and young RPESCs. PCR array performed to analyze the mRNA expression levels of senescence-associated genes in 3 senescent (P16) and 3 young (P3) RPESCs. Data are reported as fold change. A 4-fold difference was considered significant and only mRNAs with a ΔΔCt greater than 4 (A) or lower than -4 (B) are reported. *P = from 0.018 to 0.046.

  
  
  

  

  Human p53 and p21 protein expression levels in senescent and young RPESCs. p53 and p21 protein expression levels (A) Western blot analysis (B) and densitometric analysis of blots. Data are mean ± SD of 3 independent experiments. *P = from 0.022 to 0.046. (C) Relative expression levels of mRNA related to p21 and (D) p53 genes in young (P3), pre-senescent (P11) and senescent (P16) RPESCs cells. Data are mean ± SD of 3 independent experiments. *P = from 0.031 to 0.044.

  
  
  

• Research Paper Volume 10, Issue 6 pp 1306-1323

  Loss of NAMPT in aging retinal pigment epithelium reduces NAD⁺ availability and promotes cellular senescence

  Relevance score: 4.666651

  Ravirajsinh N. Jadeja, Folami L. Powell, Malita A. Jones, Jasmine Fuller, Ethan Joseph, Menaka C. Thounaojam, Manuela Bartoli, Pamela M. Martin

  Keywords: retinal pigment epithelium (RPE), aging, age-related macular degeneration (AMD), NAD+, NAMPT, senescence, SIRT1

  Published in Aging on June 12, 2018

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  Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here, we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD⁺)-biosynthetic genes and associated levels of NAD⁺, a principal regulator of energy metabolism and cellular fate, in mouse RPE. NAD⁺ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression, increased expression of senescence markers (p16^INK4a, p21^Waf/Cip1, ApoJ, CTGF and β-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD⁺ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD⁺-enhancing therapies on RPE cell viability. This, we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD⁺ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD⁺ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element.

  

  Changes in RPE NAD+ metabolism with aging in mice. RPE/eye cup was dissected from 2, 12 and 18 months old male C57BL/6J mice to evaluate changes in NAD⁺ metabolism. (A) NAD⁺ content was measured using a commercially available kit. (B) Overview of NAD synthesis pathways in mammals. (C-E) Changes in mRNA expression of enzymes regulating NAD⁺ synthesis were performed by qPCR. (F) Changes in SIRT-1 protein levels were measured by western blotting. Data is presented as mean ± S.E.M for n=5. A representative western blot image from three replicates is shown. mRNA expression of genes were normalized to 18s expression. *p<0.05 compared to 2M (two months old mice). NAMPT; Nicotinamide phosphoribosyltransferase, QPRT; Quinolinate Phosphoribosyltransferase, NMNAT; Nicotinamide mononucleotide adenylyltransferease.

  
  
  

  

  Inhibition of NAMPT activity in human retinal pigment epithelial cells decreases NAD+ levels to induce senescence. Human retinal pigment epithelial cells (ARPE-19) were treated with different doses (0.01-10μM) of a selective NAMPT activity blocker, FK866. Dose-dependent changes in (A) NAD⁺ content (B) cell viability and (C-D) senescence of FK866 treated human RPE cells were evaluated. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated).

  
  
  

  

  FK866 treatments induce changes in the markers of senescence in human retinal pigment epithelial cells. Human retinal pigment epithelial cells (ARPE-19) were treated with different doses (0.01-10μM) of FK866 and changes in the expression of various senescence markers were evaluated by qPCR and western blotting. Dose-dependent changes in (A) p21^Waf/Cip1 mRNA, (B) p16^INK4a and p21^Waf/Cip1 protein and, (C-D) CTGF and ApoJ mRNA levels are shown. A representative western blot image from three replicates is shown. mRNA expression of genes were normalized to 18s expression. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated).

  
  
  

  

  FK866 decreases SIRT-1 expression/activity and increases inflammation in human retinal pigment epithelial cells. Human retinal pigment epithelial cells (ARPE-19) were treated with different doses (0.01-10μM) of FK866 and (A) expression and (B) activity of SIRT1 was evaluated by western blotting and commercially available SIRT1 assay kit respectively. (C-D) Changes in inflammatory markers (IL-6 and IL-8) were evaluated by qPCR. A representative western blot image from three replicates is shown. mRNA expression of genes were normalized to 18s expression. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated).

  
  
  

  

  Time-dependent decline in NAD+ content and induction of senescence in human retinal pigment epithelial cells treated with FK866. Human retinal pigment epithelial cells (ARPE-19) were treated with 10μM FK866 for 24, 48 and 72 hr. Time-dependent changes in (A) NAD⁺ content (B) cell viability and (C-D) senescence of FK866 treated human RPE cells were evaluated. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated).

  
  
  

  

  Time-dependent changes in the markers of senescence in human retinal pigment epithelial cells treated with FK866. Human retinal pigment epithelial cells (ARPE-19) were treated with 10μM FK866 for 24, 48 and 72 hr. and changes in the expression of various senescence markers was evaluated by qPCR and western blotting. Time-dependent changes in (A) p21^Waf/Cip1 mRNA, (B) p16^INK4a and p21^Waf/Cip1 protein and, (C-D) CTGF and ApoJ mRNA levels are shown. A representative western blot image from three replicates is shown. mRNA expression of genes were normalized to 18s expression. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated).

  
  
  

  

  Time-dependent changes in SIRT-1 expression/activity and inflammatory markers in human retinal pigment epithelial cells treated with FK866. Human retinal pigment epithelial cells (ARPE-19) were treated with 10μM FK866 for 24, 48 and 72 hr. and (A) expression and (B) activity of SIRT-1 was evaluated by western blotting, and commercially available SIRT-1 assay kit respectively. (C-D) Changes in inflammatory markers (IL-6 and IL-8) were evaluated by qPCR. (E) Representative images of CellROX and MitoSOX stained 10μM FK866 treated (72 hr.) A representative western blot image from three replicates is shown. mRNA expression of genes were normalized to 18s expression. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated).

  
  
  

  

  Nicotinamide mononucleotide (NMN)treatment preserves NAD⁺ and prevents senescence in human retinal pigment epithelial cells. Human retinal pigment epithelial cells (ARPE-19) were treated with 10μM FK866 alone or in combination with different doses of NMN (0.05-1 mM) for 72 hr. (A) NAD⁺ content was measured using a NAD assay kit. (B) Changes in expression of SIRT1, p16^INK4a and p21^Waf/Cip1 proteins levels were evaluated by western blotting. (C-D) RPE senescence was evaluated by β-galactosidase staining. A representative western blot image from three replicates is shown. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated) and #p<0.05 compared to FK866.

  
  
  

  

  Nicotinamide mononucleotide (NMN)treatment preserves NAD⁺ and prevents senescence in mouse retinal pigment epithelial cells. Primary RPE cells were isolated from 17 days old mouse pups and cultured as described in materials and methods. Mouse primary RPE cells were treated with different doses of FK866 for 5 days to evaluate changes in cell viability, NAD⁺ content, senescence and SIRT1 expression. (A) Cell viability was evaluated by MTT assay. (B and D) NAD⁺ content was measured using a NAD assay kit. (C, E and F) RPE senescence were evaluated by and β-galactosidase staining. (G) Changes in expression of SIRT-1 protein levels were evaluated by western blotting. A representative western blot image from three replicates is shown. Data are presented as mean ± S.E.M for n=3 independent experiments. *p<0.05 compared to CON (Vehicle treated) and #p<0.05 compared to FK866.

  
  
  

  

  In vivo administration of nicotinamide mononucleotide (NMN) prevents FK866-induced NAD⁺ depletion and RPE senescence. Male C57BL/6J mice were injected sub-retinally with 10 μM FK866 (right eye) at day 0 and day 7 and sacrificed on day 15 (7 days after the last dose). RPE/eye cup was then collected for further analysis. Simultaneously, a group of mice were treated with 150 mg/kg NMN (i.p.) for 14 days. Left eye was injected with PBS to use as controls. (A) NAD⁺ content and (B) SIRT-1 expression was evaluated by NAD assay and western blotting respectively. A representative western blot image from three replicates is shown. Data are presented as mean ± S.E.M for n=5. *p<0.05 compared to CON (Vehicle treated) and @p<0.05 compared to FK866.

  
  
  

• Research Paper Volume 9, Issue 1 pp 133-141

  Association of neovascular age-related macular degeneration with month and season of birth in Italy

  Relevance score: 4.5933604

  Antonio Longo, Alessandra Casuccio, Luca Pani, Teresio Avitabile, Salvatore Cillino, Maurizio G. Uva, Vincenza Bonfiglio, Andrea Russo, Guglielmo Parisi, Gilda Cennamo, Claudio Furino, Mariacristina Parravano, Entela Xoxi, Michele Reibaldi

  Keywords: neovascular age-related macular degeneration, neovascular AMD, anti-VEGF, season of birth, month of birth

  Published in Aging on December 19, 2016

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  In order to investigate the influence of season and month of birth on the risk of neovascular age-related macular degeneration (n-AMD) in Italy, we evaluated the month birth and sex of all patients, recorded in the anti-vascular endothelial growth factor (VEGF) monitoring registry of the Italian Medicines Agency, born between 1925–1944, who received intravitreal anti-VEGF injections for n-AMD between January 1, 2013 and July 29, 2015. The numbers of all births in Italy in the same years, extracted from the Italian National Institute of Statistics, were used to calculate the expected number of n-AMD cases. Overall, 45,845 patients (19,207 men, 26,638 women) received intravitreal anti-VEGF for n-AMD; in the same years, 20,140,426 people (10,334,262 male, 9,806,164 female) were born in Italy. Comparing the observed number of n-AMD cases with the expected number of n- AMD cases in each season, we found that the season-specific risk for n-AMD was 2.5% higher for those born in summer (OR=1.03, Bonferroni-corrected P=0.008) and 3% lower for those born in winter (OR=0.96, Bonferroni-corrected P=0.0004). When considering the month of birth, the risk of n-AMD was 5.9% lower for people born in January (OR=0.93, Bonferroni-corrected P=0.0012). The factors causing such differences should be determined.

  

  Pooled analysis of observed/expected births in people with neovascular AMD in Italy between 1925 and 1944 (n = 45845) with 95% confidence intervals.

  
  
  

  

  Odds ratios for people with neovascular AMD being born in different seasons in Italy between 1925 and 1944 (n = 45845) with 95% confidence intervals.

  
  
  

  

  Odds ratios for people with neovascular AMD being born in different months in Italy between 1925 and 1944 (n = 45845) with 95% confidence intervals.

  
  
  

  

  Odds ratios for men and women with neovascular AMD being born in different seasons in Italy between 1925 and 1944 (n = 45845) with 95% confidence intervals.

  
  
  

  

  Odds ratios for men and women with neovascular AMD being born in different months in Italy between 1925 and 1944 (n = 45845) with 95% confidence intervals.

  
  
  

• Research Paper Volume 6, Issue 12 pp 1064-1075

  Pathway activation profiling reveals new insights into Age-related Macular Degeneration and provides avenues for therapeutic interventions

  Relevance score: 4.845792

  Evgeny Makarev, Charles Cantor, Alex Zhavoronkov, Anton Buzdin, Alexander Aliper, Antonei Benjamin Csoka

  Keywords: AMD, Age-related Macular Degeneration, gene expression, Transcriptome profiling, signaling pathway activation strength

  Published in Aging on December 22, 2014

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  Age-related macular degeneration (AMD) is a major cause of blindness in older people and is caused by loss of the central region of the retinal pigment epithelium (RPE). Conventional methods of gene expression analysis have yielded important insights into AMD pathogenesis, but the precise molecular pathway alterations are still poorly understood. Therefore we developed a new software program, “AMD Medicine”, and discovered differential pathway activation profiles in samples of human RPE/choroid from AMD patients and controls. We identified 29 pathways in RPE-choroid AMD phenotypes: 27 pathways were activated in AMD compared to controls, and 2 pathways were activated in controls compared to AMD. In AMD, we identified a graded activation of pathways related to wound response, complement cascade, and cell survival. Also, there was downregulation of two pathways responsible for apoptosis. Furthermore, significant activation of pro-mitotic pathways is consistent with dedifferentiation and cell proliferation events, which occur early in the pathogenesis of AMD. Significantly, we discovered new global pathway activation signatures of AMD involved in the cell-based inflammatory response: IL-2, STAT3, and ERK. The ultimate aim of our research is to achieve a better understanding of signaling pathways involved in AMD pathology, which will eventually lead to better treatments.

  

  PAS values have been calculated according to OncoFinder algorithm. PAS presented on this figure passed the following filters PAS<−1.5 and PAS>1.5 in both datasets. Blue bars represent PAS average for each pathway, and error bars represents standard deviation A. PAS derived from GSE50195 dataset. B. PAS derived from GSE50195 that cell-based inflammatory responses within the RPE-choroid are a core feature of AMD. However, cellular sources and targets of pro-inflammatory secreted factors are still need to be determined along with the regulatory mechanism of the chemokine network.

  
  
  

  

  Complement factor H genetic background (rs1061170 SNP) for PAS values derived from GSE50195 dataset shown for high-risk YH/HH and low-risk YY genotype. Blue shading indicates pathway downregulation; red shading indicates pathway upregulation. Samples with names ending in CTRL indicates control samples; samples with names ending in ARM indicates AMD samples.

  
  
  

  

  Box plots of GSE29801 (right) derived PAS and GSE50195 (left) derived PAS for each pathway. All PAS values for each pathway from two independent data sets are comparable; moreover box plots for GSE50195 derived PAS lay inside of box plots for GSE50195 derived PAS. Box plot whiskers represent min and max values for each pathways.

  
  
  

  

  This figure also serves as a working hypothesis for the pathogenesis of AMD. Proposed steps and interactions are as follows: A. Environmental Stress in the form of aging, obesity, inflammation, or diet causes B. senescence and loss of proliferation of the retinal pigment epithelial cells leading to C. activation of the MAPK, ERK, p38 and AKT pathways in the cytoplasmic components of the cells. This cellular senescence also has several consequences, primary of which are D. upregulation of the SASP, interleukin, and inflammatory cytokine networks, and E. downregulation of the caspase cascade and mitochondrial apoptosis. These pathways also interact; for example the upregulation of the SASP, interleukin, and inflammatory cytokine pathways causes downregulation of the caspase and mitochondrial apoptosis pathways. Green arrows represent upregulated pathways, red arrows represent downregulated pathways, and blue dotted arrows represent connected pathways.

  
  
  

• Research Paper Volume 5, Issue 6 pp 427-444

  Deficiency in the metabolite receptor SUCNR1 (GPR91) leads to outer retinal lesions

  Relevance score: 6.427073

  Sandra Favret, Francois Binet, Eric Lapalme, Dominique Leboeuf, Jose Carbadillo, Tina Rubic, Emilie Picard, Gaelle Mawambo, Nicolas Tetreault, Jean-Sebastien Joyal, Sylvain Chemtob, Florian Sennlaub, John Paul SanGiovanni, Martin Guimond, Przemyslaw Sapieha

  Keywords: AMD, GPR91, geographic atrophy, microglia, succinate, metabolite receptor

  Published in Aging on June 17, 2013

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  Age-related macular degeneration (AMD) is a prominent cause of blindness in the Western world. To date, its molecular pathogenesis as well as the sequence of events leading to retinal degeneration remain largely ill-defined. While the invasion of choroidal neovasculature in the retina is the primary mechanism that precipitates loss of sight, an earlier dry form may accompany it. Here we provide the first evidence for the protective role of the Retinal Pigment Epithelium (RPE)-resident metabolite receptor, succinate receptor 1 (SUCNR1; G-Protein coupled Receptor-91 (GPR91), in preventing dry AMD-like lesions of the outer retina. Genetic analysis of 925 patients with geographic atrophy and 1199 AMD-free peers revealed an increased risk of developing geographic atrophy associated with intronic variants in the SUCNR1 gene. In mice, outer retinal expression of SUCNR1 is observed in the RPE as well as microglial cells and decreases progressively with age. Accordingly, Sucnr1−/− mice show signs of premature sub-retinal dystrophy with accumulation of oxidized-LDL, abnormal thickening of Bruch's membrane and a buildup of subretinal microglia. The accumulation of microglia in Sucnr1-deficient mice is likely triggered by the inefficient clearance of oxidized lipids by the RPE as bone marrow transfer of wild-type microglia into Sucnr1−/− mice did not salvage the patho-phenotype and systemic lipolysis was equivalent between wild-type and control mice. Our findings suggest that deficiency in SUCNR1 is a possible contributing factor to the pathogenesis of dry AMD and thus broaden our understanding of this clinically unmet need.

  

  (A) Immunohistochemistry on sagittal retinal cryosections reveals expression of SUCNR1 in the GCL, INL and RPE. (B-D) Confocal imaging corroborates expression of SUCNR1 (red) in the RPE as confirmed by co-localization with the RPE marker RPE65 (green). Images are representative of 3-4 experiments. (E) Expression profile of Sucnr1 mRNA shows transcripts in RPE and CNS microglia from wild-type mice while an absence of transcripts is noted in samples from Sucnr1−/− mice. (F) Quantitative PCR on RPE isolated from 8, 20 or 43 week old wild-type mice shows a steady decrease of Sucnr1 levels with age (n=4-6). Values are expressed as percentage of controls ± S.E.M., normalized to β-actin standards. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer and RPE: retinal pigment epithelium. Scale bars (A-D): 100μm.

  
  
  

  

  Transmission electron microscopy of RPE/sub-retina the in wild-type (A) and Sucnr1−/− (B) at 20 weeks of age reveals regional disruption of BM and presence nodular debris (asterix). By 43 weeks of age, lipofuscin granules (black arrows) accumulate in the sub-retina of Sucnr1−/− mice (D) while minimal lipofuscin is detected in wild-type mice. Confocal microscopy on retinal cross sections demonstrates accumulation of sub-retinal deposits of oxLDL in Sucnr1−/− mice (F,H,J) while wild-type controls remained ox-LDL free (E,G,I). Images are representative of 3 distinct experiments. (K) Quantitative PCR reveals lower levels of scavenger receptor CD36 in isolated RPE extracts in Sucnr1−/− mice when compared to wild-type controls (n=5-10). Values are expressed as percentage of controls ? S.E.M, normalized to ?-actin standards. *P<0,05. Similar levels of plasma FFA (L) and glycerol (M) were noted in both wild-type and Sucnr1−/− mice suggesting that systemic lipolysis was not affected by SUCNR1. (N-P) Toluidine blue-stained epoxy retinal semi-thin sections show mild degeneration of photoreceptors (20 points of analysis per retina; n=3-4 mice) *P<0,05. Scale bar (A-D): 2μm; (E-J): 100μm; (N,O): 75μm.

  
  
  

  

  Transmission electron microscopy of BM (outlined in yellow) with annotated thickness bar (red). BM is systematically thicker in Sucnr1−/− mice at 8 weeks (A-C), and 20 weeks (D-F). High fat diet exacerbates the noted difference in BM thickness at 20 weeks (G-I). Data is compiled from 15 points of measure per retina in 4 distinct animals. **P<0,01. Scale bar: 1μm.

  
  
  

  

  (A) Migration assays performed in modified Boyden chambers demonstrate that macrophages from Sucnr1−/− mice have compromised chemotaxis towards oxLDL. The migratory potential of macrophages was not enhanced by treatment with succinate (100μM) (n=7). *P<0,05. (B-C) RPE flatmounts extracted at 8 weeks reveal a significant (D) accumulation of microglia in Sucnr1−/− mice when compared to wild-type controls. **P<0,01. Upon receiving high fat diets, the number of sub-retinal microglia did not vary (G) but were bloated (E,F). *P<0,05. Phalloidin stain of RPE flatmounts shows that RPE morphology is intact at 8 weeks in both knockout and controls (H,I), and that microglia (IBA1:green) remain on the retinal side of RPE (J-L). At 20 weeks, microglia (IBA1;green) puncture (M-O) and penetrate the RPE (P,Q). A portion of microglia remain on the retinal side of the RPE at 20 weeks as determined by transmission electron microscopy (R). Scale bar (B,C,E,F): 50μm,(I): 50μm, (J-Q) 20μm, (R): 5μm.

  
  
  

  

  (A) Depiction of bone marrow transplantation scheme. (B) Engraphtment efficiency is confirmed by FACS analysis of host blood and reveals that over 90% of hematopoietic cells in circulation are derived from donor cells as compiled in (C). (D) RPE flatmounts from a CD45.2 positive Sucnr1−/− host shows that donor microglia from CD45.1 mice have traversed and reside in host retinas. (E) Bone marrow transfer from wild-type into Sucnr1−/− hosts results in elevated levels of F480+CD11b+ cells in peripheral blood while transfer of Sucnr1−/− bone marrow into wild-type hosts does not affect numbers of blood-born macrophages. (n=3-4), *P<0,05. (F) Following bone marrow graphting, elevated levels of subretinal microglial were only noted when hosts were deficient in SUCNR1, (n=3) *P<0,05. (G) Graphic depiction of the sequence of events where Sucnr1−/− microglia accumulate in sub-retinal zones subsequent to drusen formation. Lesions of the outer retina then take place.

  
  
  

• Research Paper Volume 5, Issue 1 pp 51-66

  Chronic oxidative stress upregulates Drusen-related protein expression in adult human RPE stem cell-derived RPE cells: A novel culture model for dry AMD

  Relevance score: 7.579614

  David M. Rabin, Richard L. Rabin, Timothy A. Blenkinsop, Sally Temple, Jeffrey H. Stern

  Keywords: RPE, oxidative stress, AMD

  Published in Aging on December 20, 2012

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  Purpose

  The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress.

  Methods

  Primary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny. Confluent cultures of RPESC-derived hRPE and ARPE-19 cells were exposed to a regimen of tert-butylhydroperoxide (TBHP) for 1-5 days. After treatment, gene expression was measured by quantitative PCR (qPCR), protein expression was assessed by immunocytochemistry and transepithelial resistance and cell toxicity were measured.

  Results

  hRPE cells exposed to a regimen of TBHP for 5 days upregulate expression of several molecules identified in drusen, including molecular chaperones and pro-angiogenic factors. 5-day TBHP treatment was significantly more effective than 1-day treatment at eliciting these effects. The extent of hRPE response to 5-day treatment varied significantly between individual donors, nevertheless, 6 transcripts were reliably significantly upregulated. ARPE-19 cells treated with the same 5-day stress regime did not show the same pattern of response and did not upregulate this group of transcripts.

  Conclusions

  RPESC-derived hRPE cells change significantly when exposed to repeated oxidative stress conditions, upregulating expression of several drusen-related proteins and transcripts. This is consistent with the hypothesis that hRPE cells are competent to be a source of proteins found in drusen deposits. Our results suggest that donor-specific genetic and environmental factors influence the RPE stress response. ARPE-19 cells appear to be less representative of AMD-like changes than RPESC-derived hRPE. This adult stem cell-based system using chronic TBHP treatment of hRPE represents a novel in vitro model useful for the study of drusen formation and dry AMD pathophysiology.

  

  (A) Schematic of experimental design used for hRPE and ARPE-19 cells (p, passage; h, hours). hRPE from donors 4 (B-D) and 5 (H,I) as well as ARPE-19 (E-G) cells were collected, plated (B,E), and cultured for 60 days (C,F,H) prior to beginning an experimental course. TBHP treatment begins on day 1. ZO-1 (H,I), tight junction-associated marker (red). White arrows indicate regions suggestive of disrupted tight junctions. Scale bar, 100μm.

  
  
  

  

  (A) hRPE (n=4) release a greater percentage of total intracellular LDH in response to TBHP treatment than ARPE-19 cells (n=4) in the first two days, but both cell types responded similarly over the following days of TBHP treatment (Student's t-test, **, p<0.01). (B) The maximum achievable TER of the hRPE was significantly greater than that of the ARPE-19 under these culture conditions (Student's t-test, **, p<0.01). The TER decreased after multiple TBHP treatments (Student's t-test, *, p<0.05, **, p<0.01; Error bars represent SEM).

  
  
  

  

  hRPE cells were exposed to 500μM TBHP for 1 and 5 days. qPCR for drusen-related transcripts was controlled with S18 ribosomal RNA and GAPDH as housekeeping genes. None of the transcripts assessed were upregulated by hRPE after 1 day (n=4) of TBHP compared to controls. However, hRPE significantly upregulated 11 drusen-related transcripts following 5 days (n=6) of TBHP compared to hRPE treated for 1 day (Welsh's t-test, *, p<0.05; **, p<0.001). Error bars represent standard error of the mean (SEM).

  
  
  

  

  hRPE (n=6) were exposed to 500μM TBHP for 2 hours each day for 5 days and mRNA was quantified using qPCR. (A) Relative quantitation identified 6 transcripts with significantly higher mean expression levels after TBHP treatment compared to controls (Wilcoxon Matched Pairs Test *, p<0.05). hRPE mRNA was quantified relative to housekeeping genes (S18 ribosomal RNA and GAPDH) as well as to the mean of vehicle-treated control hRPE for each transcript across all donor cell lines. (B) 15 of the drusen-related transcripts did not show consistent upregulation after TBHP treatment. hRPE donors are indicated by color. The bar represents the geometric mean.

  
  
  

  

  ARPE-19 cells were exposed to 500μM TBHP for 1 and 5 days. qPCR for drusen-related transcripts was controlled with S18 ribosomal RNA and GAPDH as housekeeping genes. ARPE-19 cells significantly upregulated αA-crystallin and CC9 after 1 day of TBHP treatment (Student's t-test, *, p<0.05). After 5 days of TBHP, ARPE-19 cells down-regulated αA-crystallin and CC9 to below control levels and significantly upregulated βA4-crystallin and Vitronectin (*, p<0.05). Error bars represent standard error of the mean (SEM).

  
  
  

  

  Representative photomicrographs of vehicle-treated hRPE from donor 5(A-E) and ARPE-19 cells (K-O). hRPE (F-J) from donor 5 and ARPE-19 (P-T) cells were exposed to 500 μM TBHP or vehicle in medium for 2 hours/day for 5 days. Changes in cellular morphology can be seen from observing the changes in distribution of cytoplasmic αB-crystallin (G,Q) and the tight-junction associated marker ZO-1 (H,R). Increased expression of Aβ (F), APOJ (I) and APOE (J) was observed in hRPE exposed to TBHP, but not in ARPE-19. Expression is heterogeneous, with some cells expressing notably more than others in the field. Nuclei are labeled with DAPI (blue). White arrowheads indicate pigment deposits. Scale bar, 100μm.

  
  
  

• Research Perspective Volume 3, Issue 9 pp 906-910

  Oxidation as “The Stress of Life”

  Relevance score: 5.146337

  Nikolay L. Malinin, Xiaoxia Z. West, Tatiana V. Byzova

  Keywords: lipid oxidation, oxidative stress, aging, Toll-like receptor 2, AMD, angiogenesis, atherosclerosis

  Published in Aging on September 21, 2011

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  Multiple biological consequences of oxidative stress are known to contribute to aging and aging-related pathologies. It was recently shown that (carboxyalkyl)pyrroles (CAPs), the end products of phospholipid oxidation serve as a novel class of endogenous ligands for Toll-like receptors (TLRs) and promote the process of angiogenesis. In this review, we discuss implications of these findings in the context of age-related pathologies, including tumorigenesis. Accumulation of oxidation products in tissues of aging organisms might create conditions for uncontrolled pathological angiogenesis as seen in patients with age related macular degeneration. CAPs and their receptors, TLRs might also promote the progression of atherosclerotic lesions. Importantly, besides their role in a number of pathologies, oxidative products of phospholipids contribute to tissue repair processes thereby antagonizing the destructive effects of oxidation.

  

  Oxidation of poly-unsaturated fatty acids (exemplified by DHA - docosahexaenoic acid) leads to the generation of intermediate products (HOHA - 4-hydroxy-7-oxo hept-5-enoic acid) and, consequently, accumulation of carboxy-alkyl-pyrrole (CAP) protein adducts. CAPs stimulate TLR1/2 heterodimer on the surface of endothelial cells triggering angiogenesis. In turn, excessive CAP-induced angiogenesis contributes to the age-related macular degeneration and tumor progression. Accumulation of HOHA and CAPs in blood vessel walls may also constitute a substantial component of pathophysiological changes during atherosclerosis.

  
  
  

• Research Paper pp undefined-undefined

  Effect of Humanin G (HNG) on inflammation in age-related macular degeneration (AMD)

  Relevance score: 6.1380835

  Sonali Nashine, Pinchas Cohen, Junxiang Wan, Cristina Kenney

  Keywords: Humanin G, HNG, AMD, inflammation, age-related macular degeneration

  Published in Aging on Invalid Date

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  Inflammation plays a crucial role in the etiology and pathogenesis of AMD (Age-related Macular Degeneration). Humanin G (HNG) is a Mitochondrial Derived Peptide (MDP) that is cytoprotective in AMD and can protect against mitochondrial and cellular stress induced by damaged AMD mitochondria. The goal of this study was to test our hypothesis that inflammation-associated marker protein levels are increased in AMD and treatment with HNG leads to reduction in their protein levels. Humanin protein levels were measured in the plasma of AMD patients and normal subjects using ELISA assay. Humanin G was added to AMD and normal (control) cybrids which had identical nuclei from mitochondria-deficient ARPE-19 cells but differed in mitochondrial DNA (mtDNA) content derived from clinically characterized AMD patients and normal (control) subjects. Cell lysates were extracted from untreated and HNG-treated AMD and normal cybrids, and the Luminex XMAP multiplex assay was used to measure the levels of inflammatory proteins. AMD plasma showed reduced Humanin protein levels, but higher protein levels of inflammation markers compared to control plasma samples. In AMD RPE cybrid cells, Humanin G reduced the CD62E/ E-Selectin, CD62P/ P-Selectin, ICAM-1, TNF-α, MIP-1α, IFN–γ, IL-1β, IL-13, and IL-17A protein levels, thereby suggesting that Humanin G may rescue from mtDNA-mediated inflammation in AMD cybrids. In conclusion, we present novel findings that: A) show reduced Humanin protein levels in AMD plasma vs. normal plasma; B) suggest the role of inflammatory markers in AMD pathogenesis, and C) highlight the positive effects of Humanin G in reducing inflammation in AMD.