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Antioxidants (Basel, Switzerland) May 2024This study aims to investigate the role of microRNA let-7f in the dysfunction and degeneration of retinal pigment epithelium (RPE) cells through the induction of...
This study aims to investigate the role of microRNA let-7f in the dysfunction and degeneration of retinal pigment epithelium (RPE) cells through the induction of senescence and oxidative stress. Furthermore, we explore whether let-7f inhibition can protect these cells against sodium iodate (SI)-induced oxidative stress. Oxidative stress and let-7f expression are reciprocally regulated in retinal pigment epithelial cells. Overexpression of let-7f in ARPE-19 cells induced oxidative stress as demonstrated by increased reactive oxygen species (ROS) production as well as senescence. Inhibition of let-7f successfully protected RPE cells from the detrimental effects induced by SI. In addition, let-7f overexpression induced RPE cellular dysfunction by diminishing their migratory capabilities and reducing the phagocytosis of porcine photoreceptor outer segments (POS). Results were further confirmed in vivo by intravitreal injections of SI and let-7f antagomir in C57BL/6 mice. Our results provide strong evidence that let-7f is implicated in the dysfunction of RPE cells through the induction of senescence and oxidative injury. These findings may help to uncover novel and relevant processes in the pathogenesis of dry AMD.
PubMed: 38929085
DOI: 10.3390/antiox13060646 -
International Journal of Molecular... Jun 2024Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an...
Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.
Topics: c-Mer Tyrosine Kinase; Phagocytosis; Animals; Intercellular Signaling Peptides and Proteins; Protein S; Humans; Retina; Mice; Circadian Rhythm; Ligands; Retinal Pigment Epithelium; Receptor Protein-Tyrosine Kinases
PubMed: 38928335
DOI: 10.3390/ijms25126630 -
International Journal of Molecular... Jun 2024Age-related macular degeneration (AMD) is strictly linked to chronic oxidative stress, inflammation, loss of epithelial barrier integrity, and often with abnormal new...
Vitamin D and Sulforaphane Decrease Inflammatory Oxidative Stress and Restore the Markers of Epithelial Integrity in an In Vitro Model of Age-Related Macular Degeneration.
Age-related macular degeneration (AMD) is strictly linked to chronic oxidative stress, inflammation, loss of epithelial barrier integrity, and often with abnormal new blood vessel development. In this study, the retinal epithelial cell line ARPE-19 was treated with pro-inflammatory transforming growth factor-beta (TGF-β) to investigate the activity of vitamin D (VD) and sulforaphane (SF) in abating the consequences of oxidative stress and inflammation. The administration of VD and SF lowered reactive oxygen species (ROS) levels, and abated the related expression of the pro-inflammatory cytokines interleukin-6 and interleukin-8 induced by TGF-β. We evaluated mitochondrial respiration as a source of ROS production, and we discovered that the increased transcription of respiratory elements triggered by TGF-β was prevented by VD and SF. In this model of inflamed epithelium, the treatment with VD and SF also reduced the secretion of VEGF, a key angiogenic factor, and restored the markers of epithelial integrity. Remarkably, all the observed biological effects were potentiated by the co-stimulation with the two compounds and were not mediated by VD receptor expression but rather by the ERK 1/2 pathway. Altogether, the results of this study reveal the powerful synergistic anti-inflammatory activity of SF and VD and lay the foundation for future clinical assessment of their efficacy in AMD.
Topics: Humans; Macular Degeneration; Isothiocyanates; Oxidative Stress; Sulfoxides; Vitamin D; Reactive Oxygen Species; Cell Line; Vascular Endothelial Growth Factor A; Inflammation; Retinal Pigment Epithelium; Epithelial Cells; Transforming Growth Factor beta; Biomarkers; Interleukin-8
PubMed: 38928111
DOI: 10.3390/ijms25126404 -
Bioengineering (Basel, Switzerland) Jun 2024The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative...
The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells.
PubMed: 38927820
DOI: 10.3390/bioengineering11060584 -
Bioengineering (Basel, Switzerland) Jun 2024To characterize transforming growth factor-β (TGF-β) isoform (TGF-β1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and...
To characterize transforming growth factor-β (TGF-β) isoform (TGF-β1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including , , and ; ; ; and , a master regulator for mitochondrial respiration;, tight junction-related molecules, and ; and ; (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-β isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-β1 and TGF-β3 caused a marked increase in TEER values, and TGF-β2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-β isoforms, among which TGF-β1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-β2 and TGF-β3 or by TGF-β1 and TGF-β3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-β isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-β-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-β isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.
PubMed: 38927817
DOI: 10.3390/bioengineering11060581 -
Cells Jun 2024Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs)....
Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of , the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in cells by transduction with gene replacement (ShH10-CMV-) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.
Topics: Choroideremia; Humans; Retinal Pigment Epithelium; Autophagy; Induced Pluripotent Stem Cells; rab GTP-Binding Proteins; Models, Biological; Signal Transduction; Mechanistic Target of Rapamycin Complex 1; Adaptor Proteins, Signal Transducing
PubMed: 38920696
DOI: 10.3390/cells13121068 -
Communicative & Integrative Biology 2024The Dead Sea is unique compared to other extreme halophilic habitats. Its salinity exceeds 34%, and it is getting saltier. The Dead Sea environment is characterized by a... (Review)
Review
The Dead Sea is unique compared to other extreme halophilic habitats. Its salinity exceeds 34%, and it is getting saltier. The Dead Sea environment is characterized by a dominance of divalent cations, with magnesium chloride (MgCl) levels approaching the predicted 2.3 M upper limit for life, an acidic pH of 6.0, and high levels of absorbed ultraviolet radiation. Consequently, only organisms adapted to such a polyextreme environment can survive in the surface, sinkholes, sediments, muds, and underwater springs of the Dead Sea. Metagenomic sequence analysis and amino acid profiling indicated that the Dead Sea is predominantly composed of halophiles that have various adaptation mechanisms and produce metabolites that can be utilized for biotechnological purposes. A variety of products have been obtained from halophilic microorganisms isolated from the Dead Sea, such as antimicrobials, bioplastics, biofuels, extremozymes, retinal proteins, colored pigments, exopolysaccharides, and compatible solutes. These resources find applications in agriculture, food, biofuel production, industry, and bioremediation for the detoxification of wastewater and soil. Utilizing halophiles as a bioprocessing platform offers advantages such as reduced energy consumption, decreased freshwater demand, minimized capital investment, and continuous production.
PubMed: 38919836
DOI: 10.1080/19420889.2024.2369782 -
Cureus May 2024Herpes simplex virus (HSV) infection of the cornea, uvea, and retina is the leading infectious cause of blindness worldwide. This study examined the effects of retinoic...
BACKGROUND
Herpes simplex virus (HSV) infection of the cornea, uvea, and retina is the leading infectious cause of blindness worldwide. This study examined the effects of retinoic acid (RA) on the protein levels of interleukin (IL)-17A and IL-23 cytokines with known proinflammatory effects and toll-like receptor 3 (TLR3) messenger RNA (mRNA) expression in retinal pigment epithelial (ARPE-19) cells treated with HSV-1-infected cell protein 0 (ICP0).
METHODOLOGY
We used 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide assay to calculate the half maximal inhibitory concentration (IC50) doses of RA and ICP0 in ARPE-19 cells. At the end of 24 hours, protein levels of IL-17A and IL-23 were analyzed using enzyme-linked immunosorbent assay. TLR3 mRNA expression levels were also calculated using reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS
RA administration decreased IL-17A levels, which were elevated by ICP0. The IL-23 levels were similar between the ICP0-treated and control groups, but the difference was significant between the ICP0-treated group and RA+ICP0 combination. These results showed that RA can significantly increase IL-23 levels in the presence of ICP0. Although ICP0 dramatically increased TLR3 mRNA expression compared with that in the control group, the RA+ICP0 combination returned TLR3 mRNA expression to a level similar to that in the control group ( = 0.419).
CONCLUSIONS
RA may potentially neutralize HSV-1 ICP0 negative effects in ARPE-19 cells.
PubMed: 38919217
DOI: 10.7759/cureus.61089 -
Proceedings of the National Academy of... Jul 2024To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various species undergo adult reproductive diapause in...
To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various species undergo adult reproductive diapause in response to cool temperatures and/or short day-length. While flies are less active during diapause, it is unclear how adverse environmental conditions affect circadian rhythms and sleep. Here we show that in diapause-inducing cool temperatures, exhibit altered circadian activity profiles, including severely reduced morning activity and an advanced evening activity peak. Consequently, the flies have a single activity peak at a time similar to when nondiapausing flies take a siesta. Temperatures ≤15 °C, rather than photoperiod, primarily drive this behavior. At cool temperatures, flies rapidly enter a deep-sleep state that lacks the sleep cycles of flies at higher temperatures and require high levels of stimulation for arousal. Furthermore, we show that at 25 °C, flies prefer to siesta in the shade, a preference that is virtually eliminated at 10 °C. Resting in the shade is driven by an aversion to blue light that is sensed by Rhodopsin 7 outside of the eyes. Flies at 10 °C show neuronal markers of elevated sleep pressure, including increased expression of Bruchpilot and elevated Ca in the R5 ellipsoid body neurons. Therefore, sleep pressure might overcome blue light aversion. Thus, at the same temperatures that cause reproductive arrest, preserve germline stem cells, and extend lifespan, are prone to deep sleep and exhibit dramatically altered, yet rhythmic, daily activity patterns.
Topics: Animals; Drosophila melanogaster; Sleep; Circadian Rhythm; Rhodopsin; Drosophila Proteins; Photoperiod; Temperature; Light; Diapause, Insect
PubMed: 38917005
DOI: 10.1073/pnas.2400964121 -
Ocular Immunology and Inflammation Jun 2024To characterize the dynamic changes of fundus in Vogt-Koyanagi-Harada (VKH) disease through enhanced spectral-domain optical coherence tomography (EDI-OCT) and explore...
PURPOSE
To characterize the dynamic changes of fundus in Vogt-Koyanagi-Harada (VKH) disease through enhanced spectral-domain optical coherence tomography (EDI-OCT) and explore the predictors of visual prognosis.
METHODS
In this retrospective cohort study, a total of 2152 VKH patients referred to our uveitis center from January 2013 to April 2022 were screened; 151 new-onset VKH patients (299 eyes) and 82 healthy controls (164 eyes) were included. The manifestations of fundus at baseline, 1 month, 3 months, and 12 months after treatment were analysed and their relevance to visual prognosis were evaluated.
RESULTS
After retinal detachment (RD) (97.3%) and optic disc swelling (100%) presented at baseline, retinal reattachment (81.6%) and the granular hyperreflective depositions at the retinal pigment epithelium (RPE) (61.5%) were observed at month 1. The RPE and ellipsoid zone rearrangement accompanying interdigitation zone attenuation (57.9%) was noted finally. Choroidal thickness of patients was higher than that in the controls at baseline and month 1 (both P < 0.001). Best-corrected visual acuity (BCVA) (logarithm of the minimum angle of resolution [logMAR]) (P < 0.001; OR, 4.01), subretinal fibrinoid exudate (P < 0.001; OR, 3.9) and RPE folds ( = 0.001; OR, 2.39) at baseline, and the RD at month 1 (P < 0.001; OR, 3.42) were associated with visual prognosis.
CONCLUSIONS
New-onset VKH patients after treatment exhibited dynamic changes in the fundus especially the outer retina during a 12-month period. The BCVA, subretinal fibrinoid exudate, and RPE folds at baseline, and RD at month May 1, serve as predictors of visual prognosis.
PubMed: 38916535
DOI: 10.1080/09273948.2024.2369940