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Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.
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Axônios , Traumatismos do Nervo Óptico , Animais , Camundongos , Axônios/metabolismo , Gânglios Espinais/metabolismo , Mamíferos , Regeneração Nervosa/genética , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.
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Hepatite Alcoólica , Humanos , Escherichia coli , Imunoglobulina A , Autoanticorpos , Imunoglobulina G , Imunoglobulina MRESUMO
Vanadium (V) is an essential mineral element in animals, but excessive V can lead to many diseases, affecting the health of humans and animals. However, the molecular crosstalk between mitochondria-associated endoplasmic reticulum membranes (MAMs) and inflammation under V exposure is still at the exploratory stage. This study was conducted to determine the molecular crosstalk between MAMs and inflammation under V exposure in ducks. In this study, duck hepatocytes were treated with NaVO3 (0 µM, 100 µM, and 200 µM) and 2-aminoethyl diphenyl borate (2-APB) (IP3R inhibitor) alone or in combination for 24 h. The data showed that V exposure-induced cell vacuolization, enlarged intercellular space, and decreased density and viability. Meanwhile, hydrogen peroxide (H2O2), malonaldehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reactive oxygen species (ROS) levels were upregulated under V treatment. In addition, excessive V could lead to a marked reduction in the MAMs structure, destruction of the membrane structure and overload of intracellular Ca2+ and mitochondrial Ca2+. Moreover, V treatment resulted in notable upregulation of the levels of MAMs-relevant factors (IP3R, Mfn2, Grp75, MCU, VDAC1) but downregulated the levels of IL-18, IL-1ß, and lactate dehydrogenase (LDH) in the cell supernatant. Additionally, it also significantly elevated the levels of inflammation-relevant factors (NLRP3, ASC, caspase-1, MAVS, IL-18, IL-1ß, and TXNIP). However, the inhibition of IP3R expression attenuated the V-induced variations in the above indicators. Collectively, our results revealed that the maintenance of calcium homeostasis could protect duck hepatocytes from V-induced inflammation injury via MAMs.
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Cálcio , Patos , Humanos , Animais , Cálcio/metabolismo , Patos/metabolismo , Vanádio , Interleucina-18/metabolismo , Peróxido de Hidrogênio , Galinhas/metabolismo , Retículo Endoplasmático/metabolismo , Hepatócitos/metabolismo , Cálcio da Dieta/metabolismo , Inflamação/induzido quimicamente , Inflamação/veterinária , Inflamação/metabolismoRESUMO
Human retinal organoid transplantation could potentially be a treatment for degenerative retinal diseases. How the recipient retina regulates the survival, maturation, and proliferation of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice and conducted histology and single-cell RNA sequencing alongside time-matched cultured retinal organoids. Unexpectedly, we observed human cells that migrated into all recipient retinal layers and traveled long distances. Using an unbiased approach, we identified these cells as astrocytes and brain/spinal cord-like neural precursors that were absent or rare in stage-matched cultured organoids. In contrast, retinal progenitor-derived rods and cones remained in the subretinal space, maturing more rapidly than those in the cultured controls. These data suggest that recipient microenvironment promotes the maturation of transplanted photoreceptors while inducing or facilitating the survival of migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for potential cell-based treatments of retinal diseases.
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Degeneração Retiniana , Análise da Expressão Gênica de Célula Única , Humanos , Camundongos , Animais , Diferenciação Celular/fisiologia , Retina , Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana/terapia , Organoides/transplanteRESUMO
Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Neovascularização Retiniana , Humanos , Camundongos , Animais , Acriflavina/metabolismo , Acriflavina/farmacologia , Acriflavina/uso terapêutico , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-ß) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKß) that selectively targets NF-κB signaling, can modulate TGF-ß/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKß inhibition on RPE-EMT-associated factors using a second IKKß inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.
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Epitélio Pigmentado da Retina , Vitreorretinopatia Proliferativa , Humanos , Epitélio Pigmentado da Retina/metabolismo , Transição Epitelial-Mesenquimal , Quinase I-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismoRESUMO
The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We sought to determine if there was antibody deposition in SAH livers and whether antibodies extracted from SAH livers were cross-reactive against both bacterial antigens and human proteins. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissue from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Employing human proteome arrays, we profiled the antibodies extracted from explanted SAH, alcoholic cirrhosis (AC), nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), autoimmune hepatitis (AIH), hepatitis B virus (HBV), hepatitis C virus (HCV) and HD livers and found that antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins as autoantigens. The use of an E. coli K12 proteome array revealed the presence of unique anti- E. coli antibodies in SAH, AC or PBC livers. Further, both Ig and E. coli captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion and focal adhesion (IgG). Except IgM from PBC livers, no common autoantigen was recognized by Ig and E. coli captured Ig from AC, HBV, HCV, NASH or AIH suggesting no cross-reacting anti- E. coli autoantibodies. The presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in the liver may participate in the pathogenesis of SAH.
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Cadmium (Cd), a persistent and harmful heavy metal in the environment, can accumulate in the kidneys and cause nephrotoxicity. Selenium (Se) is a beneficial natural element that alleviates the toxicity of Cd. To ascertain the relationship between the protective mechanism of Se against Cd nephrotoxicity and ferroptosis and pyroptosis, we randomly divided 48 sheep into four groups and treated them with Cd chloride and/or sodium selenite for 50 days. The data confirmed that Cd apparently resulted in impaired kidney histology and function, depletion of GSH and nicotinamide adenine dinucleotide phosphate contents and CAT and SOD activities, elevation of MDA level, as well as the reduction in selenoprotein mRNA (GPX1, GPX4, TXNRD1, SELP) levels and GPX4 protein level and immunofluorescence intensity. Meanwhile, Cd induced ferroptosis by causing iron overload, up-regulating PTGS2, NCOA4, TFR1, and LC3B mRNA levels and PTGS2 and LC3B-II/LC3B-I protein levels, reducing SLC7A11 and FTH1 mRNA and protein levels, and enhancing the immunofluorescence co-localization of FTH1/LC3B. Moreover, it was also found that Cd triggered pyroptosis, which was evidenced by the increase of NLRP3 immunohistochemical positive signal, GSDMD-N immunofluorescence intensity, IL-1ß and IL-18 release and the levels of pyroptosis-related mRNA (NLRP3, ASC, Caspase-1, GSDMD, IL-1ß and IL-18) and proteins (NLRP3, Caspase-1p20, GSDMD-N, IL-1ß and IL-18). Notably, Se increased the expression level of GPX4 and the transcription factors TFAP2c and SP1, and ameliorated Cd-induced changes in aforementioned factors. In conclusion, GPX4 utilization by Se might be required to alleviate Cd-induced ferroptosis and pyroptosis in sheep kidney.
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Ferroptose , Selênio , Animais , Ovinos , Cádmio/metabolismo , Selênio/farmacologia , Interleucina-18/metabolismo , Piroptose , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ciclo-Oxigenase 2/metabolismo , Rim/patologia , Caspase 1/metabolismo , RNA Mensageiro/metabolismoRESUMO
Cadmium (Cd) is a poisonous metal element that causes mitochondrial dysfunction. Selenium (Se) can reduce the damage of Cd to various organs of animals, but the protective mechanism of Se in Cd-induced lung injury has not been fully elucidated. For purpose of further illustrating the specific mechanism of Se alleviated Cd-triggered pulmonary toxicity, 48 sheep were divided into 4 groups, of which the sheep in the treatment group were taken 1 mg/kg body weight (BW) of Cd, 0.34 mg/kg BW of Se, and 0.34 mg Se + 1 mg/kg BW of Cd by intragastric administration for 50 d, respectively. The results indicated that Cd caused inflammatory cell infiltration and alveolar wall thickening, which facilitated mitochondrial vacuolation and formation of mitophagosomes in lung tissues. Simultaneously, Cd treatment impaired the antioxidant capacity of sheep lung tissue. Additionally, Cd treatment down-regulated the expression levels of mitochondrial biogenesis and mitochondrial fusion, but up-regulated the levels of mitochondrial fission and mitophagy mediated by FUNDC1. Moreover, the immunofluorescence co-localization puncta of LC3B/COX IV, LC3B/FUNDC1 were increased after Cd treatment. Nevertheless, co-treatment with Se improved effectively the above variation caused by Cd exposure. In summary, Se could mitigate Cd-generated mitophagy through FUNDC1-mediated mitochondrial quality control pathway in the lungs of sheep.
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Cádmio , Selênio , Animais , Ovinos , Cádmio/metabolismo , Selênio/farmacologia , Selênio/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologia , Mitofagia , Mitocôndrias , Pulmão/metabolismoRESUMO
The accumulation of copper (Cu) in the organisms could lead to kidney damage by causing mitochondrial dysfunction. Given that mitochondria are one of the targets of Cu poisoning, this study aimed to investigate the role of mitophagy in Cu-induced mitochondrial dysfunction in renal tubular epithelial cells to understand the mechanism of Cu nephrotoxicity. Hence, the cells were treated with different concentrations of Cu sulfate (CuSO4 ) (0, 100, and 200 µM), and mitophagy inhibitor (Cyclosporine A, 0.5 µM) and/or 200 µM CuSO4 in the combination for 12 h. Results showed that Cu caused mitochondrial swelling, vacuoles, and cristae fracture; increased the number of mitochondrial and lysosome fluorescent aggregation points; upregulated the mRNA levels of mitophagy-associated genes (LC3A, LC3B, P62, BNIP3, NIX, OPTN, NDP52, Cyp D LAMP1, and LAMP2) and protein levels of LC3II/LC3I, BNIP3, and NIX, downregulated the mRNA and protein levels of P62; reduced the mitochondrial membrane potential (MMP), ATP content, mitochondrial respiratory control rate (RCR), mitochondrial respiratory control rate (OPR), and the mRNA and protein levels of PGC-1α, TOMM20, and Mfn2, but increased the mRNA and protein levels of Drp1. Besides, cotreatment with Cu and CsA dramatically decreased the level of mitophagy, but increased mitochondrial division, further reduced MMP, ATP content, RCR, and OPR, mitochondrial fusion and thereby reduced mitochondrial biogenesis. Taken together, these data indicated that Cu exposure induced BNIP3/NIX-dependent mitophagy in duck renal tubular epithelial cells, and inhibition of mitophagy aggravated Cu-induced mitochondrial dysfunction.
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Patos , Mitofagia , Animais , Mitofagia/genética , Patos/genética , Patos/metabolismo , Cobre/toxicidade , Cobre/metabolismo , Mitocôndrias/metabolismo , Células Epiteliais/metabolismo , RNA Mensageiro/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using PirtEGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.
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Neuralgia , RNA Citoplasmático Pequeno , Ratos , Camundongos , Masculino , Feminino , Animais , Gânglios Espinais/metabolismo , Transcriptoma , Análise de Célula Única , Cálcio/metabolismo , Ratos Sprague-Dawley , Neuralgia/metabolismo , Neurônios/metabolismo , Hiperalgesia/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Weaning transition generally impairs the immune system, inducing immune disturbance, which may be associated with post-weaning diarrhea and high mortality in piglets. The spleen is a pivotal lymphatic organ that plays a key role in the establishment of the immune system. Traditional Chinese medicine (TCM) prescriptions, XiaoJianZhong (XJZ) and Jiansananli-sepsis (JSS), are widely used prescriptions for treating spleen damage and diarrhea. Here, we hypothesized that XJZ and JSS maintain the spleen physiological function by ameliorating antioxidant capacity and inflammatory response in weaned piglets. In this study, 18 weaned piglets were assigned to the Control, XJZ and JSS groups. By hematoxylin and eosin staining, hematological analysis, flow cytometric analysis, qRT-PCR and western blot, the effects of both TCM prescriptions on the spleen antioxidant defense system and inflammatory pathway were explored. Results showed that both TCM treatment significantly ameliorated the weaning-induced morphological damage in piglets, as evidenced by clearer and more perfect spleen histology, as well as higher relative area of white pulp. Meanwhile, both XJZ and JSS exerted better blood parameters, as supported by the changes of monocyte level and lymphocyte subpopulations CD4+/CD8+ ratio. Furthermore, the levels of inflammatory markers, IL1ß, IL6, IL8, and TNF-α in the spleen were markedly decreased after supplemented with both TCM prescriptions. Importantly, the inhibition of nuclear factor-kappaB (NF-κB) and its downstream effector genes (IL6, IL8, and TNF-α) in both XJZ and JSS treatment groups further confirmed alleviation of inflammatory responses in the spleen. In addition, both XJZ and JSS enhanced the antioxidant capacity of the spleen by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-activated antioxidant defense system. Notably, the results of PCA and network correlation analysis indicated that XJZ and JSS treatment altered the expression profiles of inflammatory and antioxidant-related factors in the spleen of weaned-piglets, which may involve the synergy of NF-κB and Nrf2 signaling pathways. In summary, our study showed that TCM prescriptions, XJZ and JSS could ameliorate inflammatory response and antioxidant capacity in the spleen by synergistically regulating NF-κB and Nrf2 signaling pathways in piglets.
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In order to study the synergistic effects of organic and inorganic thickening agents on the rheological properties of cement paste, the rheological parameters, thixotropy cement-paste containing limestone powder (LP), re-dispersible polymer powder (RPP), and hydroxypropyl methylcellulose ether (HPMC) were investigated using the Anton Paar MCR 102 rheometer at different resting times. The early-age hydration process, hydration products, and microstructure were also analyzed with scanning electron microscopy (SEM) and thermogravimetry analyses (TGA). The results showed that the addition of LP, RPP, and HPMC affected the rheological properties of cement paste, but the thickening mechanism between organic and inorganic thickening agents was different. The small amount of LP increased the plastic viscosity but decreased the yield stress of cement paste due to its dense filling effect. Adding 1% of RPP improved the thixotropic property of cement paste by 50%; prolonging the standing time could improve the thixotropic performance by as much as two times. Only 0.035% HPMC added to the cement paste increased the plastic viscosity by 20%, while the yield stress increased nearly twice. The more HPMC added, the more significant effect it showed. Cement paste compounds with LP, RPP, and HPMC balanced the yield stress and plastic viscosity and improved the thixotropy. The C-L6-R1.0-H0.035 paste presented as a pseudoplastic, its rheological indexes were close to one, and it was hardly affected by the resting time. The composite superposition effect of organic and inorganic thickening agents reduced the impact of resting time for all pastes. As the organic thickening component inhibited the hydration more than the LP promoted the hydration of the cement paste, indicating that the C-L6-R1.0-H0.035 paste remained in the particle fusion stage after curing for three days, as shown by the SEM images.
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BackgroundTo reduce the treatment burden for patients with neovascular age-related macular degeneration (nvAMD), emerging therapies targeting vascular endothelial growth factor (VEGF) are being designed to extend the interval between treatments, thereby minimizing the number of intraocular injections. However, which patients will benefit from longer-acting agents is not clear.MethodsEyes with nvAMD (n = 122) underwent 3 consecutive monthly injections with currently available anti-VEGF therapies, followed by a treat-and-extend protocol. Patients who remained quiescent 12 weeks from their prior treatment entered a treatment pause and were switched to pro re nata (PRN) treatment (based on vision, clinical exam, and/or imaging studies). Proteomic analysis was performed on aqueous fluid to identify proteins that correlate with patients' response to treatment.ResultsAt the end of 1 year, 38 of 122 eyes (31%) entered a treatment pause (≥30 weeks). Conversely, 21 of 122 eyes (17%) failed extension and required monthly treatment at the end of year 1. Proteomic analysis of aqueous fluid identified proteins that correlated with patients' response to treatment, including proteins previously implicated in AMD pathogenesis. Interestingly, apolipoprotein-B100 (ApoB100), a principal component of drusen implicated in the progression of nonneovascular AMD, was increased in treated patients who required less frequent injections. ApoB100 expression was higher in AMD eyes compared with controls but was lower in eyes that develop choroidal neovascularization (CNV), consistent with a protective role. Accordingly, mice overexpressing ApoB100 were partially protected from laser-induced CNV.FundingThis work was supported by the National Eye Institute, National Institutes of Health grants R01EY029750, R01EY025705, and R01 EY27961; the Research to Prevent Blindness, Inc.; the Alcon Research Institute; and Johns Hopkins University through the Robert Bond Welch and Branna and Irving Sisenwein professorships in ophthalmology.ConclusionAqueous biomarkers could help identify patients with nvAMD who may not require or benefit from long-term treatment with anti-VEGF therapy.
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Inibidores da Angiogênese/administração & dosagem , Apolipoproteína B-100/metabolismo , Neovascularização de Coroide , Proteínas do Olho/metabolismo , Degeneração Macular , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/metabolismo , Feminino , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Masculino , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background: Leisure provides opportunities for urban and rural residents to relax, recover their vitality, and improve their personal growth, development, and well-being. However, the impact of the leisure participation process, types, obstacles, participation motivation, and satisfaction on health is not very clear, especially the impact of leisure behavior on health, and is worthy of in-depth discussion. Objective: The objective of this study was to explore the impact of social capital and leisure participation on the self-rated health of urban residents in China so as to provide an important reference for national health promotion activities. Methods: the questionnaire on the relationship between social capital, leisure behavior, and self-rated health was compiled by ourselves. The residents participating in leisure and fitness in 25 residential fitness centers in Chengdu were investigated in the morning and evening, and the obtained data were processed by exploratory and confirmatory factor analysis. Results: (1) Social capital had no direct influence on leisure hindrance; leisure motivation and leisure participation had no direct influence on self-rated health. (2) Leisure satisfaction has a direct positive impact on self-rated health, while leisure hindrance has a significant negative impact on self-rated health. (3) Social capital has a direct positive impact on leisure satisfaction, and social capital has a direct positive impact on self-rated health. Leisure satisfaction plays an intermediary role in the path of social capital affecting self-rated health, and the intermediary force exceeds the direct impact of social capital on self-rated health. Conclusion: The effect of leisure satisfaction on self-rated health is higher than that of social capital, and it plays an intermediary role in the impact path of social capital on self-rated health. Therefore, how to make urban community residents with different backgrounds obtain leisure satisfaction through leisure activities is an important topic of national health promotion.
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Capital Social , China , Nível de Saúde , Humanos , Atividades de Lazer , População UrbanaRESUMO
Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell-based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag-based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration-associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease.
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Transição Epitelial-Mesenquimal , Epitélio Pigmentado da Retina/metabolismo , Carcinogênese , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Embrionárias , Humanos , Células-Tronco Pluripotentes Induzidas , ProteomaRESUMO
Purpose: RPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems. Methods: EMT of human stem cell-derived RPE monolayers was induced by either enzymatic dissociation or modulation of TGF-ß signaling. Transcriptomic analysis of cells at different stages of EMT was performed by RNA-sequencing, and select findings were confirmed by reverse transcription quantitative PCR and immunostaining. An ingenuity pathway analysis (IPA) was performed to identify signaling pathways and regulatory networks associated with EMT. Results: Proteocollagenolytic enzymatic dissociation and cotreatment with TGF-ß and TNF-α both induce EMT in human stem cell-derived RPE monolayers, leading to an increased expression of mesenchymal factors and a decreased expression of RPE differentiation-associated factors. Ingenuity pathway analysis identified the upstream regulators of the RPE-EMT regulatory networks and identified master switches and nodes during RPE-EMT. Of particular interest was the identification of widespread dysregulation of axon guidance molecules during RPE-EMT progression. Conclusions: The temporal transcriptome profiles described here provide a comprehensive resource of the dynamic signaling events and the associated biological pathways that underlie RPE-EMT onset. The pathways defined by these studies may help to identify targets for the development of novel therapeutic targets for the treatment of retinal disease.
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Transição Epitelial-Mesenquimal/fisiologia , Células-Tronco Pluripotentes/citologia , Epitélio Pigmentado da Retina/metabolismo , Transcriptoma/fisiologia , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais , Fatores de TranscriçãoRESUMO
ßA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as ßA3 and ßA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that ßA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of ßA3/A1-crystallin in metabolism of retinal astrocytes. We found that ßA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but ßA3-crystallin does not. Loss of ßA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited ßA1-knockdown (KD) mice, but not in ßA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified ßA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced ßA1-crystallin and higher levels of PTP1B in the vitreous humor.
Assuntos
Astrócitos/enzimologia , Retinopatia Diabética/enzimologia , Metabolismo Energético , Glucose/metabolismo , Mitocôndrias/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Retina/enzimologia , Cadeia A de beta-Cristalina/metabolismo , Animais , Astrócitos/patologia , Estudos de Casos e Controles , Células Cultivadas , Cristalinas/genética , Cristalinas/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Ratos Sprague-Dawley , Retina/patologia , Cadeia A de beta-Cristalina/genéticaRESUMO
In addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here, we show that knocking out myosin IIA and IIB (myosin IIA/B) in retinal ganglion cells alone, either before or after optic nerve crush, induces significant optic nerve regeneration. Combined Lin28a overexpression and myosin IIA/B knockout lead to an additive promoting effect and long-distance axon regeneration. Immunostaining, RNA sequencing, and western blot analyses reveal that myosin II deletion does not affect known axon regeneration signaling pathways or the expression of regeneration-associated genes. Instead, it abolishes the retraction bulb formation and significantly enhances the axon extension efficiency. The study provides clear evidence that directly targeting neuronal cytoskeleton is sufficient to induce significant CNS axon regeneration and that combining altered gene expression in the soma and modified cytoskeletal dynamics in the axon is a promising approach for long-distance CNS axon regeneration.
Assuntos
Nervo Óptico/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Miosinas , Regeneração Nervosa , Células Ganglionares da Retina/metabolismoRESUMO
Single-cell RNA-sequencing (scRNA-seq) provides new opportunities to gain a mechanistic understanding of many biological processes. Current approaches for single cell clustering are often sensitive to the input parameters and have difficulty dealing with cell types with different densities. Here, we present Panoramic View (PanoView), an iterative method integrated with a novel density-based clustering, Ordering Local Maximum by Convex hull (OLMC), that uses a heuristic approach to estimate the required parameters based on the input data structures. In each iteration, PanoView will identify the most confident cell clusters and repeat the clustering with the remaining cells in a new PCA space. Without adjusting any parameter in PanoView, we demonstrated that PanoView was able to detect major and rare cell types simultaneously and outperformed other existing methods in both simulated datasets and published single-cell RNA-sequencing datasets. Finally, we conducted scRNA-Seq analysis of embryonic mouse hypothalamus, and PanoView was able to reveal known cell types and several rare cell subpopulations.