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1.
Life Sci ; 241: 117146, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31816325

RESUMO

AIMS: Diabetic retinopathy (DR) is the main cause of blindness in adults and investigating new therapeutic targets for DR is necessary. This study aimed to investigate the effect of high-mobility group box 1 (HMGB1) protein and its mechanism in diabetic retinopathy (DR) were investigated. MAIN METHODS: Human retinal endothelial cells (HREC) were uesd for chip-seq. Sprague Dawley (SD) rats were randomly divided into control group, HMGB1 group, diabetes mellitus (DM) combined with HMGB1 siRNA group, and DM group. Next, eyeballs were removed and retinas were detached for western blot. The DM model of cell was built by increasing the glucose concentration in cell culture medium. The regulation of HMGB1 was achieved by short hairpin (sh)-HMGB1 transfection, then, the transfected cells were harvested for luciferase assay, western blot and qRT-PCR analyses as well as proliferation and apoptosis detection. KEY FINDINGS: Chip-seq and luciferase assay showed the possible transcription factor functions of HMGB1 and IKB-α was one of the HMGB1 binding sites. In vivo and in vitro results indicated high expression of HMGB1 and NF-kB and low expression of IKB-α in DR and the expression of IKB-α and NF-kB was regulated by HMGB1. Moreover, cell assays showed that HMGB1 inhibited cell proliferation and promoted apoptosis. SIGNIFICANCE: The results from the present study showed that HMGB1 may be involved in the pathogenesis of DR as a transcription factor through NF-kB pathway. Therefore, blockade of HMGB1 may be a new method for the treatment of DR.


Assuntos
Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteína HMGB1/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteína HMGB1/genética , Humanos , Masculino , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , Ratos , Ratos Sprague-Dawley , Retina/citologia , Retina/metabolismo
2.
Medicine (Baltimore) ; 98(51): e18333, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31860985

RESUMO

To determine characteristics of diabetic macular edema patients with serous retinal detachment (SRD).We classified naïve diabetic macular edema (DME) patients with or without SRD, and compared their baseline characteristics; intravitreal bevacizumab (IVB) responsiveness; aqueous concentrations of IL (interleukin)-1ß, -2, -8, -10, -17, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF). In addition, factors associated with the existence of SRD were identified.Of the 64 DME patients, 14 had SRD. The average levels of aqueous VEGF and PlGF were significantly higher in the SRD group than in the control group (P = .022 and P = .041, respectively). The best-corrected visual acuity (BCVA) and central subfield thickness (CST) did not differ significantly between the 2 groups at baseline or after 3 consecutive monthly IVBs. In multivariate logistic regression analysis, the level of aqueous VEGF was the only factor associated with the existence of SRD (odds ratio: 1.03; P = .038).Rather than aqueous inflammatory cytokines, levels of aqueous VEGFs were associated with the occurrence of SRD in DME patients. In terms of prognosis, the existence of SRD was not related with BCVA or CST changes.


Assuntos
Humor Aquoso/metabolismo , Bevacizumab/uso terapêutico , Retinopatia Diabética/tratamento farmacológico , Edema Macular/tratamento farmacológico , Descolamento Retiniano/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Estudos de Casos e Controles , Retinopatia Diabética/metabolismo , Feminino , Humanos , Interleucina-1beta/metabolismo , Injeções Intravítreas , Edema Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Placentário/metabolismo , Prognóstico , Retina/patologia , Descolamento Retiniano/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acuidade Visual/efeitos dos fármacos
3.
Exp Eye Res ; 189: 107851, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655041

RESUMO

Diabetic retinopathy (DR), a common microvascular complication of diabetes, is reported to be the leading cause of blindness worldwide. In our previous study, we found that the Raf kinase inhibitor protein (RKIP) is significantly decreased in vitreous humor of proliferative diabetic retinopathy (PDR) patients, which indicated that RKIP might play a role in the development of PDR. To investigate the role of RKIP in PDR, stable overexpression and knockdown of RKIP in Human retinal capillary endothelial cells (HRCECs) were generated by using lentivirus constructs. Then, the glucose-induced cell viability, migration, angiogenesis, and (endothelial to mesenchymal transition) EndMT were determined in the RKIP-wide type (WT), -knocking down (KD) and -overexpression (OE) HRCECs. The results showed that, compared with the RKIP-WT groups, the glucose-induced cell viabilities, migration and angiogenesis were significantly increased in the RKIP-KD groups, while significantly decreased in the RKIP-OE groups. Besides, compared with the control groups, CD31 and vWF were upregulated, while α-SMA was downregulated in the RKIP-KD groups, while CD31 and vWF were downregulated, while α-SMA was upregulated in the RKIP-OE groups induced by glucose. In conclusion, our results showed that RKIP negatively regulates glucose-induced cell viability, migration, angiogenesis, and EndMT in HRCECs, suggesting that the downregulation of RKIP in the vitreous humor of PDR patients might contribute to the development of DR.


Assuntos
Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Glucose/farmacologia , Proteína de Ligação a Fosfatidiletanolamina/genética , Vasos Retinianos/metabolismo , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/patologia , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Humanos , Proteínas do Tecido Nervoso , Proteína de Ligação a Fosfatidiletanolamina/biossíntese , RNA/genética , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia
4.
Exp Eye Res ; 189: 107830, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31593688

RESUMO

Reactive oxygen species (ROS) act through multiple pathways to induce apoptosis of retinal capillary pericytes, which is an early marker and the primary cause of the progression of diabetic retinopathy. However, the specific molecular mechanisms behind ROS-induced retinal capillary pericyte loss in diabetic retinopathy remains elusive. In this study, we investigated the molecular regulation and effects of DJ-1/PARK7 on oxidative stress and injury of rat retinal pericytes (RRPs). To perform the research, RRPs were isolated from rat retina and cultured in medium with for 2 days: control group (5.6 mM glucose), high glucose group (30 mM glucose), hypertonic group (5.6 mM glucose + 24.4 mM mannitol). We found decreased expression of DJ-1 and increased apoptosis of RRPs in high glucose group. To further study the role of DJ-1, four groups were divided as follows: normal control group (5.6 mM glucose), high glucose (30 mM glucose), empty vector control group (pcDNA3.1,30 mM glucose), DJ-1 overexpression group (pcDNA3.1-myc-DJ-1,30 mM glucose). DJ-1, P53, p-P53, cleaved caspase-3, manganese superoxide dismutase (MnSOD), catalase (CAT) and PI3K/Akt/mTOR signaling pathway in each group was detected by Western Blot. RRPs apoptosis was detected by Terminal-deoxynucleoitidyl Transferase mediated Nick End Labeling (TUNEL) and 4'6- diamidino-2-phenylindole (DAPI). Mitochondrial function was detected by jc-1 and fluorescent probes DCFH-DA was used to determine reactive oxygen species (ROS). We found that high glucose (30 mM) lasting two days can induce significant apoptosis of RRPs, increase ROS production and expressions of p-p53 and active caspase-3, impair mitochondrial function, decrease the activities of MnSOD and CAT, and decrease expression of DJ-1, p-AKT and p-mTOR. In contrast, DJ-1/PARK7 overexpression significantly increases expression of DJ-1, p-AKT and p-mTOR, increases expression and activities of MnSOD and CAT, improves mitochondrial function, decreases expression of apoptotic gene protein p-p53 and active caspase-3, reduces ROS production and reduces the apoptotic rate of RRPs induced by high glucose. These results suggest that DJ-1 may play a role in protecting RRPs from high glucose induced-oxidative injury. DJ-1 might improve mitochondrial function, inhibit ROS production and enhance antioxidant capacity to reduce apoptosis of retinal pericytes through the PI3K/AKT/mTOR signaling pathway which may be related to early pathogenesis of diabetic retinopathy.


Assuntos
Proteínas de Transporte/genética , Retinopatia Diabética/genética , Regulação da Expressão Gênica , Pericitos/metabolismo , Proteína Desglicase DJ-1/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genética , Animais , Apoptose , Western Blotting , Proteínas de Transporte/biossíntese , Células Cultivadas , Diabetes Mellitus Experimental , Retinopatia Diabética/metabolismo , Retinopatia Diabética/prevenção & controle , Glucose/toxicidade , Masculino , Estresse Oxidativo , Pericitos/patologia , Proteína Desglicase DJ-1/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/biossíntese
5.
Zhonghua Yan Ke Za Zhi ; 55(10): 769-776, 2019 Oct 11.
Artigo em Chinês | MEDLINE | ID: mdl-31607066

RESUMO

Objective: To identify differentially expressed proteins between the patients with proliferative diabetic retinopathy (PDR) and vitreous floaters, and explore treatment target for PDR based on isobaric tags for relative and absolute quantification (iTRAQ) LC-MS/MS Proteomics. Method: Vitreous samples were collected from 28 eyes of patients with PDR and 4 eyes with vitreous floaters, which served as controls. For quantitative proteomics, vitreous samples were combined and proteins extracted and labeled with iTRAQ peptide-tagging reagents. Samples were fractionated by liquid chromatography (LC), analyzed by tandem mass spectrometry (MS/MS) and Gene Ontology (GO) analyses performed on differentially expressed proteins identified in the PDR samples. Results: In the PDR vitreous, 26 proteins were identified that were differentially expressed when compared to the controls. Of these, 7 showed a significant increase (P<0.05) and 19 a significant decrease (P<0.05)in expression in PDR patients. These included some high abundance proteins including Retinoic acid receptor reactive protein 2 (PDR 1=85.0, PDR 2=83.0, Control 1=119.6, Control 2=120.2, FC=0.710, P=0.001), Semaphorin-4B(PDR 1=64.4, PDR 2=68.8, Control 1=135.4, Control 2=146.0, FC=0.473, P=0.023), Apolipoprotein B (PDR 1=104.4, PDR 2=106.6, Control 1=89.0, Control 2=85.3, FC=1.211, P=0.024), and Heat shock protein 70 (PDR 1=69.3, PDR 2=75.0, Control 1=137.7, Control 2=138.3, FC=0.523, P=0.026), which are closely related to the pathological mechanism of PDR. GO analysis clustered the differentially expressed genes into three major functional domains: Biological Processes, Molecular Function and Cellular Component. Differential gene expression was found in the categories of cellular metabolism, organonitrogen compound and carbohydrate derivative metabolic processes, transferase activity and transmembrane signaling receptor activity. KEGG Pathway analysis indicate that Chemerin signaling through Akt, Sema4B signaling via PI3K, and HIF-1α signal pathways were all altered in the PDR samples. Conclusions: In this study we identified variations in expression of genes extensively involved in key biological processes in the retina including neovascularization, cellular metabolism and transmembrane signaling, which provide new insights into the pathophysiology of PDR. Extracellular matrix was degraded and endothelial cell migration was induced by Chemerin, in addition, the destruction of blood-retinal barrier and neuronal apoptosis were induced by ApoB. Chemerin and ApoB accelerated the development of PDR. Sema 4B participated in vascular protection, HSP70 conducted anti-apoptosis. These two cytokines protected the retinal neurovascular in PDR patients. Therefore, Chemerin, Sema 4B, ApoB and HSP70 may be the treatment target for PDR. (Chin J Ophthalmol, 2019, 55:769-776).


Assuntos
Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Corpo Vítreo/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Quimiocinas/metabolismo , Cromatografia Líquida/métodos , Diabetes Mellitus Tipo 2/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Corpo Vítreo/química
6.
Invest Ophthalmol Vis Sci ; 60(13): 4215-4223, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618425

RESUMO

Purpose: To elucidate the mechanism whereby miR-590-3p regulates pyroptosis in diabetic retinopathy (DR). Methods: Human retinal microvascular endothelial cells (HRMECs) incubated with high glucose (HG) were used to establish cell models, and the expression levels of miR-590-3p, caspase-1, IL-1ß, NLRP1, NOX4, TXNIP, NLRP3, and ROS were determined. Additionally, miR-590-3p was altered using a mimic or an inhibitor, and siRNAs targeting NLRP1 and NOX4 were applied to explore the regulatory mechanism of miR-590-3p in DR. The relationships between miR-590-3p and NLRP1/NOX4 also were investigated using a luciferase reporter assay. Furthermore, vitreous tissue samples were collected to confirm pyroptosis in clinical DR. Results: Downregulated miR-590-3p and upregulated NLRP1/NOX4 levels were observed in a cell culture model of DR. Inhibiting miR-590-3p upregulated NLRP1, the NOX4/ROS/TXNIP/NLRP3 pathway, and caspase-1. NLRP1 and NOX4 were confirmed as direct target genes of miR-590-3p. The overexpression of miR-590-3p or knockdown of NLRP1 and NOX4 increased cell activity and suppressed pyroptosis. Intriguingly, the upregulation of IL-1ß induced the downregulation of miR-590-3p by lowering the DNA promoter activity of pri-miR-590. Conclusions: HG induced pyroptosis in a cell culture model of DR, and the downregulation of miR-590-3p promoted pyroptotic death by targeting NLRP1 and activating the NOX4/ROS/TXNIP/NLRP3 pathway via an IL-1ß-mediated positive feedback loop.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , MicroRNAs/fisiologia , NADPH Oxidase 4/metabolismo , Piroptose/fisiologia , Células Cultivadas , Humanos , MicroRNAs/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
7.
Invest Ophthalmol Vis Sci ; 60(13): 4084-4096, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31574534

RESUMO

Purpose: To investigate whole transcriptional differences between proliferative diabetic retinopathy (PDR) neovascular membranes (NVMs) and retinas, and the regulatory genes participating in retinal neovascularization in PDR. Methods: We used high-throughput sequencing technology to capture the whole-genome gene expression levels of all participants, including 23 patients with PDR or branch retinal vein occlusion (BRVO), 3 normal retinal samples, and 2 retinal samples from type II diabetic (T2D) eyes by donation, followed by analyses of expression patterns using bioinformatics methods, then validation of the data by in situ hybridization and Western blotting. Results: We showed that transcriptional profiles of the NVMs were distinct from those of the retinas. Angiogenesis growth factors VEGFC, ANGPT1, ANGPT2, and EFNB2, and their receptors FLT4, TIE1, TIE2, and EPHB4, respectively, were overexpressed. Expression of VEGFA was highly upregulated in T2D retina, but low in the NVMs, while angiogenesis transcription factors, including ETS1 and ERG, were coordinately upregulated in NVMs. Conclusions: This study described a PDR neovascularization model in which pathological retina-secreted vascular endothelial growth factor A (VEGFA) enhanced the expression of a set of angiogenesis transcription factors and growth factors, to cooperatively induce the retinal neovascularization. Based on these results, novel potential therapeutic targets and biomarkers for PDR treatment and diagnosis are suggested.


Assuntos
Angiopoietina-1/metabolismo , Retinopatia Diabética/metabolismo , Efrina-B2/metabolismo , Neovascularização Retiniana/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Humanos , Receptor EphB4/metabolismo , Receptor de TIE-1/metabolismo , Receptor TIE-2/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Oclusão da Veia Retiniana/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
8.
Invest Ophthalmol Vis Sci ; 60(12): 3842-3853, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31529081

RESUMO

Purpose: Outer blood retinal barrier breakdown is a neglected feature of diabetic retinopathy (DR). We demonstrated that the agonism of the δ opioid receptor (DOR) by epicatechin preserves the tight junction proteins in ARPE-19 cells under diabetic conditions. Presently, we aimed to evaluate the possible role of the DOR on the maintenance of tight junction of RPE layer and on the early markers of experimental DR. Methods: DR markers and external retinal tight junction proteins were evaluated in CL57B diabetic mice submitted to intravitreous injection of short hairpin RNA (shRNA)-DOR (108 transducing units [TU]/mL) treated or not with DOR agonist (0.05 g/animal/d of epicatechin in drinking water) for 16 weeks. The presence of DOR in human retina from postmortem eyes from diabetic and nondiabetic donors were also performed. Results: DOR is present in RPE layer and in neuro retina. The treatment with DOR agonist prevented the upregulation of the early markers of retinopathy (glial fibrillary acidic protein, VEGF) and the downregulation of pigment epithelium-derived factor, occludin, claudin-1, and zonula occludens-1 tight junction expressions. The silencing of DOR in retina of diabetic mice partially abolished the protective effects of epicatechin. In human retina specimens, DOR is present throughout the retina, similarly in nondiabetic and diabetic donors. Conclusions: This set of experiments strongly indicates that the DOR agonism preserves RPE tight junctions and reduces the early markers of retinopathy in model of diabetes. These novel findings designate DOR as a potential therapeutic tool to treat DR with preservation of the RPE tight junction proteins.


Assuntos
Catequina/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Retinopatia Diabética/prevenção & controle , Receptores Opioides delta/agonistas , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/metabolismo , Idoso , Animais , Glicemia/metabolismo , Western Blotting , Claudina-1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Impedância Elétrica , Proteínas do Olho/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fatores de Crescimento Neural/metabolismo , Ocludina/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Receptores Opioides delta/metabolismo , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
9.
Graefes Arch Clin Exp Ophthalmol ; 257(11): 2429-2436, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31512044

RESUMO

PURPOSE: Diabetic retinopathy (DR) is a complex eye disease associated with diabetes mellitus. It is characterized by three pathophysiological components, namely microangiopathy, neurodegeneration, and inflammation. We recently reported that intraperitoneal administration of BNN27, a novel neurosteroidal microneurotrophin, reversed the diabetes-induced neurodegeneration and inflammation in rats treated with streptozotocin (STZ), by activating the NGF TrkA and p75 receptors. The aim of the present study was to investigate the efficacy of BNN27 to protect retinal neurons when applied topically as eye drops in the same model. METHODS: The STZ rat model of DR was employed. BNN27 was administered as eye drops to diabetic Sprague-Dawley rats for 7 days, 4 weeks post-STZ (70 mg/kg) injection. Immunohistochemistry and western blot analyses were employed to examine the viability of retinal neurons in control, diabetic, and diabetic-treated animals and the involvement of the TrkA receptor and its downstream signaling ERK1/2 kinases, respectively. RESULTS: BNN27 reversed the STZ-induced attenuation of the immunoreactive brain nitric oxide synthetase (bNOS)- and tyrosine hydroxylase (TH)-expressing amacrine cells and neurofilament (NFL)-expressing ganglion cell axons in a dose-dependent manner. In addition, BNN27 activated/phosphorylated the TrkA receptor and its downstream prosurvival signaling pathway, ERK1/2 kinases. CONCLUSIONS: The results of this study provide solid evidence regarding the efficacy of BNN27 as a neuroprotectant to the diabetic retina when administered topically, and suggest that its pharmacodynamic and pharmacokinetic profiles render it a putative therapeutic for diabetic retinopathy.


Assuntos
Desidroepiandrosterona/administração & dosagem , Diabetes Mellitus Experimental , Retinopatia Diabética/tratamento farmacológico , Retina/patologia , Administração Tópica , Animais , Western Blotting , Desidroepiandrosterona/farmacocinética , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/metabolismo , Relação Dose-Resposta a Droga , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/metabolismo , Resultado do Tratamento
10.
Int J Mol Sci ; 20(19)2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557880

RESUMO

Diabetic retinopathy (DR), a sight-threatening neurovasculopathy, is the leading cause of irreversible blindness in the developed world. DR arises as the result of prolonged hyperglycemia and is characterized by leaky retinal vasculature, retinal ischemia, retinal inflammation, angiogenesis, and neovascularization. The number of DR patients is growing with an increase in the elderly population, and therapeutic approaches are limited, therefore, new therapies to prevent retinal injury and enhance repair are a critical unmet need. Besides vascular endothelial growth factor (VEGF)-induced vascular proliferation, several other mechanisms are important in the pathogenesis of diabetic retinopathy, including vascular inflammation. Thus, combining anti-VEGF therapy with other new therapies targeting these pathophysiological pathways of DR may further optimize treatment outcomes. Technological advancements have allowed for high-throughput proteomic studies examining biofluids such as aqueous humor, vitreous humor, tear, and serum. Many DR biomarkers have been identified, especially proteins involved in retinal inflammatory processes. This review attempts to summarize the proteomic biomarkers of DR-associated retinal inflammation identified over the last several years.


Assuntos
Retinopatia Diabética/complicações , Retinopatia Diabética/metabolismo , Proteoma , Proteômica , Retinite/etiologia , Retinite/metabolismo , Biomarcadores , Líquidos Corporais/metabolismo , Retinopatia Diabética/diagnóstico , Humanos , Processamento de Proteína Pós-Traducional , Proteômica/métodos
11.
Mater Sci Eng C Mater Biol Appl ; 105: 110092, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546391

RESUMO

Naltrexone (NTX) is a potent opioid growth factor receptor (OGFR) antagonist proved to be useful for treatment of ocular surface complications. The aim of this work was to explore the feasibility of designing NTX-imprinted 2-hydroxyethyl methacrylate-based hydrogels for sustained drug release on the ocular surface. Acrylic acid (AAc) and benzyl methacrylate (BzMA) were chosen as functional monomers able to form binding cavities mimicking OGFR binding sites for NTX. Imprinted hydrogels containing functional monomers loaded higher amounts of NTX compared to non-imprinted ones by simple soaking in drug aqueous solution. In addition, possibility of carrying out the loading and sterilization processes in one step was investigated. NTX release was evaluated both under agitated sink conditions and in a microfluidic flow chamber mimicking the hydrodynamic conditions of the eye, namely the small volume of lachrymal fluid and its renovation rate. Sustained release profiles together with adequate swelling degree (46 to 57% w/w), light transparency (over 85%) and oxygen permeability may make these hydrogels suitable candidates to NTX-eluting contact lenses. NTX-loaded and non-loaded discs successfully passed the chorioallantoic membrane test for potential ocular irritation and were cytocompatible with human mesenchymal stem cells. Finally, NTX-imprinted hydrogels tested in the bovine corneal permeability assay provided therapeutically relevant amounts of NTX inside the cornea, reaching drug levels similar to those attained with a concentrated aqueous solution in spite the discs showed sustained release.


Assuntos
Córnea/metabolismo , Retinopatia Diabética/tratamento farmacológico , Hidrogéis , Naltrexona , Animais , Bovinos , Embrião de Galinha , Córnea/patologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Humanos , Hidrogéis/química , Hidrogéis/farmacocinética , Hidrogéis/farmacologia , Naltrexona/química , Naltrexona/farmacocinética , Naltrexona/farmacologia , Permeabilidade
12.
Exp Eye Res ; 188: 107789, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31491425

RESUMO

Although there have been no previous reports on the pH of the human vitreous body, it has been highly theorized that it changes in patients with diabetes mellitus (DM). In humans, it is necessary to measure the vitreous pH in vitro, which is an important point that presents a major problem, as vitreous pH immediately changes when exposed to air. The purpose of this present study was to report our recent development of an in vitro method for measuring vitreous pH via the combination of 27-gauge (G) vitreous surgery and a blood gas analyzer, as well as our investigative findings on whether or not there is a difference of pH depending on the presence of diabetes mellitus (DM). This cross-sectional study involved 30 subjects [18 subjects without DM (DM-) and 12 subjects with DM (DM+)] with no previous history of ophthalmologic surgery. The DM+ group included 6 cases of proliferative diabetic retinopathy (PDR) and 6 cases of non-PDR (NPDR). The DM- Group was comprised of patients with a macular hole or idiopathic epiretinal membrane. The DM+ Group included patients not only with macular hole or idiopathic epiretinal membrane but also diabetic macular edema, however, patients with obvious vitreous hemorrhage were excluded. In all patients, a vitreous specimen was anaerobically obtained at the start of 27G pars plana vitrectomy, with a venous blood sample being collected immediately prior to surgery. Between the DM- and DM+ subjects, pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), K+, Na+, Ca2+, Cl-, lactate, and glucose were compared. In the items in which a significant difference was found between DM- and DM+, the values between the PDR and NPDR cases were also compared. Our findings showed no significant difference in vitreous and venous-blood pH between the DM- and DM+ subjects. The vitreous biochemical data revealed that Ca2+ significantly reduced and lactate and glucose significantly increased in DM+ compared to DM-. Thus, we compared Ca2+, lactate, and glucose between the PDR and NPDR cases. Although glucose did not significantly change, Ca2+ significantly decreased and lactate significantly increased in the PDR cases. The venous biochemical data revealed that only glucose significantly increased in DM+. The data in all investigated items was found to be significantly different between the vitreous and venous samples. Our findings revealed that lactate increases and Ca2+ decreases in the vitreous body of DM patients, especially those with PDR, probably due to the increased production of lactic acid. However, although the production of lactic acid increased, the pH remained at a nearly constant value, thus suggesting that the human vitreous body has a high buffering capacity.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Corpo Vítreo/metabolismo , Idoso , Gasometria , Cálcio/metabolismo , Dióxido de Carbono/metabolismo , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismo , Pressão Parcial
13.
Invest Ophthalmol Vis Sci ; 60(12): 4063-4073, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31560762

RESUMO

Purpose: The balance of neuronal excitation and inhibition is important for proper retinal signaling. A previous report showed that diabetes selectively reduces light-evoked inhibition to the retinal dim light rod pathway, changing this balance. Here, changes in mechanisms of retinal inhibitory synaptic transmission after 6 weeks of diabetes are investigated. Methods: Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ, 75 mg/kg), and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell voltage-clamp recordings of electrically evoked inhibitory postsynaptic currents from rod bipolar cells and light-evoked excitatory postsynaptic currents from A17-amacrine cells were made in dark-adapted retinal slices. Results: Diabetes shortened the timecourse of directly activated lateral GABAergic inhibitory amacrine cell inputs to rod bipolar cells. The timing of GABA release onto rod bipolar cells depends on a prolonged amacrine cell calcium signal that is reduced by slow calcium buffering. Therefore, the effects of calcium buffering with EGTA-acetoxymethyl ester (AM) on diabetic GABAergic signaling were tested. EGTA-AM reduced GABAergic signaling in diabetic retinas more strongly, suggesting that diabetic amacrine cells have reduced calcium signals. Additionally, the timing of release from reciprocal inhibitory inputs to diabetic rod bipolar cells was reduced, but the activation of the A17 amacrine cells responsible for this inhibition was not changed. Conclusions: These results suggest that reduced light-evoked inhibitory input to rod bipolar cells is due to reduced and shortened calcium signals in presynaptic GABAergic amacrine cells. A reduction in calcium signaling may be a common mechanism limiting inhibition in the retina.


Assuntos
Sinalização do Cálcio/fisiologia , Retinopatia Diabética/metabolismo , Células Bipolares da Retina/metabolismo , Células Amácrinas/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Estimulação Luminosa , Estreptozocina , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo
14.
Exp Eye Res ; 189: 107813, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31560926

RESUMO

In this study, we aimed to investigate whether exosomes derived from platelet-rich plasma (PRP-Exos) can regulate hyperglycemia-induced retinal injury via targeting the TLR4 signaling pathway. We studied the effects of PRP-Exos on retinal endothelial injury in diabetic rats and human retinal endothelial cells (HRECs) in vitro. Isolated PRP-Exos were observed by transmission electron microscopy and flow cytometry. Samples were obtained from the retinas of rats and cultured HRECs after treatment to analyze reactive oxygen species levels. Immunofluorescence and Western blotting were conducted to assess the levels of adhesion molecules and the TLR4 signaling pathway. The content of CXCL10 in PRP-Exos was analyzed by Western blot. The plasma level of PRP-Exos was greatly increased in diabetic rats. In cultured HRECs, PRP-Exos induced the production of malonyldialdehyde(MDA) and reactive oxygen species(ROS) and inhibited the activity of superoxide dismutase(SOD). Further analysis showed that the activation of the TLR4 pathway by PRP-Exos played a pivotal role in regulating inflammation. The inhibition of the TLR4 pathway by TAK-242 had a robust protective effect on PRP-Exo-induced retinal endothelial injury in vitro and vivo. In addition, PRP-Exo-derived CXCL10 led to retinal endothelial injury, and antagonizing CXCL10 with a CXCL10-neutralizing antibody dramatically attenuated such injury. In summary, PRP-Exos mediate hyperglycemia-induced retinal endothelial injury by upregulating the TLR4 signaling pathway.


Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética/genética , Endotélio Vascular/metabolismo , Exossomos/metabolismo , Regulação da Expressão Gênica , Hiperglicemia/metabolismo , Receptor 4 Toll-Like/genética , Animais , Apoptose , Western Blotting , Células Cultivadas , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Endotélio Vascular/ultraestrutura , Humanos , Hiperglicemia/patologia , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Plasma Rico em Plaquetas/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/ultraestrutura , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
15.
Korean J Ophthalmol ; 33(4): 326-332, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31389208

RESUMO

PURPOSE: To evaluate tear film function in patients with diabetes mellitus (DM) using tear film osmolarity (TFO) measurements compared to other tear film function tests. METHODS: DM patients without any history of ocular surface disorder but with potential effects on the tear film were enrolled in this cross-sectional study. Data including dry eye symptoms, duration of DM, stage of diabetic retinopathy and blood hemoglobin A1c levels were recorded. Tear film break-up time (TBUT) and basic tear secretion (Schirmer test) were assessed. TFO was determined using the Tearlab Osmolarity System. The outcome measures were the difference between the mean values of TBUT, basic tear secretion and TFO in both the study and control groups. RESULTS: We recruited 51 DM patients and 20 control subjects with a mean age of 51.2 (range, 21 to 70) and 48.5 (range, 24 to 70) years, respectively. A total of 27 patients (53%) and 11 controls (55%) reported dry eye symptoms (p = 0.668). The mean TBUT was 10.2 ± 4.8 seconds in the study group versus 10.5 ± 2.8 seconds in controls, which was not significantly different (p = 0.747). The mean Schirmer test score was 8.1 ± 4.3 mm in the patients versus 10.1 ± 3.0 mm in the controls (p = 0.069). The mean TFO was 294.1 ± 12.9 mosmol/L in the patients versus 291.4 ± 14.5 mosmol/L in the controls (p = 0.456). It was significantly higher in patients with poor glycemic control determined by hemoglobin A1c > 8% (p = 0.003). TFO had a positive correlation with the duration of DM (p = 0.030) but not with the stage of diabetic retinopathy (p = 0.944). However, TFO showed a significant relationship with dry eye symptoms (p = 0.001). CONCLUSIONS: TFO is impaired in patients with uncontrolled DM and is better correlated with glycemic control and dry eye symptoms than the TBUT and Schirmer tests.


Assuntos
Diabetes Mellitus/metabolismo , Retinopatia Diabética/metabolismo , Síndromes do Olho Seco/metabolismo , Lágrimas/química , Adulto , Idoso , Estudos Transversais , Retinopatia Diabética/complicações , Retinopatia Diabética/diagnóstico , Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/etiologia , Feminino , Hemoglobina A Glicada/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Concentração Osmolar , Adulto Jovem
16.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31375001

RESUMO

In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the first time, the induced pluripotent stem-cell-derived retinal pigment epithelium (hiPSC-RPE) cell lines derived from type 2 diabetics and healthy control patients were utilized to assess the effects of glucose concentration on the cellular functionality. We show that both type 2 diabetic and healthy control hiPSC-RPE lines differentiate and mature well, both in high and normal glucose concentrations, express RPE specific genes, secrete pigment epithelium derived factor, and form a polarized cell layer. Here, type 2 diabetic hiPSC-RPE cells had a decreased barrier function compared to controls. Added insulin increased the epithelial cell layer tightness in normal glucose concentrations, and the effect was more evident in type 2 diabetics than in healthy control hiPSC-RPE cells. In addition, the preliminary functionality assessments showed that type 2 diabetic hiPSC-RPE cells had attenuated autophagy detected via ubiquitin-binding protein p62/Sequestosome-1 (p62/SQSTM1) accumulation, and lowered pro- matrix metalloproteinase 2 (proMMP2) as well as increased pro-MMP9 secretion. These results suggest that the cellular ability to tolerate stress is possibly decreased in type 2 diabetic RPE cells.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Retinopatia Diabética/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Epitélio Pigmentado da Retina/patologia , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Glucose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Permeabilidade , Epitélio Pigmentado da Retina/metabolismo
17.
Mol Med Rep ; 20(4): 3719-3727, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432194

RESUMO

The major ophthalmic complication in patients with diabetes is diabetic retinopathy (DR), which is one of the major eye diseases that causes blindness. It is well established that the occurrence and duration of DR is positively correlated with duration of diabetes. Advanced glycation end product (AGE) accumulation in patients with diabetes is one factor that leads to the development of DR. However, the underlying mechanisms remain unclear. In the present study, the role of phosphoinositide 3­kinase/protein kinase B (Akt) signaling in AGE­induced DR development was investigated. An in vitro experimental system was used to study the effects of AGEs on human retinal capillary endothelial cells (HRCECs) and Müller cells. Flow cytometry, MTT, western blotting and BrdU incorporation assays were performed. Reverse transcription­quantitative polymerase chain reaction was used to measure the expression of angiogenesis­associated genes. Functional assays of angiogenesis, including HRCEC invasion and tube formation assays. It was demonstrated that the expression of receptor for AGEs was upregulated in HRCECs and Müller cells following treatment with AGEs. AGE treatment did not affect Müller cell viability, but enhanced HRCEC viability. Akt inhibition increased cell apoptosis and death in HRCECs. AGE treatment upregulated the expression of pro­angiogenic genes, which was suppressed by Akt inhibitor treatment. In addition, Akt inhibitor treatment suppressed HRCEC invasion and tube formation ability. The present study suggested that Akt­mediated signaling may serve critical roles in the development of DR due to the accumulation of AGEs. Akt may be a potential therapeutic target in DR.


Assuntos
Retinopatia Diabética/patologia , Células Endoteliais/patologia , Produtos Finais de Glicação Avançada/metabolismo , Vasos Retinianos/patologia , Apoptose , Linhagem Celular , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Produtos Finais de Glicação Avançada/análise , Humanos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Retina/metabolismo , Retina/patologia , Vasos Retinianos/metabolismo
18.
Invest Ophthalmol Vis Sci ; 60(8): 2942-2949, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284311

RESUMO

Purpose: Diabetic macular edema (DME) is characterized by an accumulation of fluid in the macula due to diabetic retinopathy. Currently, anti-VEGF drugs are the standard treatment worldwide for DME. This study aimed to assess whether the existence of epiretinal membrane (ERM) affects anti-VEGF efficacy, due to reduced permeability of the antibody through the ERM. Methods: We retrospectively examined clinical data of DME patients who underwent anti-VEGF treatment and evaluated whether clinical differences existed between DME eyes with ERM and those without ERM. We then created an in vitro ERM model using MIO-M1, ARPE-19, and NTI-4 cells on Transwell membranes and evaluated antibody permeability through this in vitro ERM model using fluorescently labeled antibodies. Results: Central retinal thickness (CRT) change between before and 1 month after first anti-VEGF treatment, as well as final CRT and final visual acuity 12 months after first anti-VEGF treatment, significantly differed between DME eyes with ERM and those without ERM. The in vitro ERM model led to production of collagen I in a manner similar to that of human ERM specimens. Fluorescence intensity of the lower chamber of the in vitro ERM model was significantly reduced in a dose-dependent manner. Conclusions: Clinical data analysis indicated that the existence of ERM in DME eyes lowered the efficacy of anti-VEGF treatment. Reduced antibody permeability through the in vitro ERM model suggested ERM presence was associated with resistance to anti-VEGF treatment in DME eyes with ERM.


Assuntos
Inibidores da Angiogênese/farmacocinética , Retinopatia Diabética/tratamento farmacológico , Membrana Epirretiniana/metabolismo , Edema Macular/tratamento farmacológico , Modelos Biológicos , Ranibizumab/farmacocinética , Idoso , Biomarcadores/metabolismo , Células Cultivadas , Retinopatia Diabética/metabolismo , Resistência a Medicamentos , Feminino , Humanos , Injeções Intravítreas , Edema Macular/metabolismo , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Estudos Retrospectivos , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Vitrectomia
19.
Biosci Biotechnol Biochem ; 83(9): 1655-1662, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31272285

RESUMO

Diabetic retinopathy (DR) is a leading cause of new-onset blindness. Recent studies showed that protecting retinal ganglion cells (RGCs) from high glucose-induced injury is a promising strategy for delaying DR. This study is to investigate the role of miR-145-5p in high glucose-induced RGC injury. Here, RGCs were randomly divided into low glucose and high glucose groups. PCR assay showed miR-145-5p was significantly upregulated in high glucose group. Transfection of miR-145-5p inhibitor decreased pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) levels, elevated cell viability and proliferation, as well as suppressed cell apoptosis by ELISA, MTT, EdU proliferation, colony formation and flow cytometry assays, respectively. Moreover, dual-luciferase reporter assay confirmed FGF5 as a target gene of miR-145-5p. FGF5 knockdown could partially reverse the protective effects of miR-145-5p on RGC-5 cells. In conclusion, our results demonstrated that inhibition of miR-145-5p might be a neuroprotective target for diabetes mellitus-related DR. Abbreviations: DR: diabetic retinopathy; RGCs: retinal ganglion cells; miR-145-5p: microRNA-145-5p; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; FGF: fibroblast growth factor; ATCC: American Type Culture Collection; WT: wild type; MUT: mutant type.


Assuntos
Sobrevivência Celular , Retinopatia Diabética/patologia , Regulação para Baixo , Fator 5 de Crescimento de Fibroblastos/metabolismo , MicroRNAs/metabolismo , Células Ganglionares da Retina/metabolismo , Apoptose , Linhagem Celular , Citocinas/metabolismo , Retinopatia Diabética/metabolismo , Fator 5 de Crescimento de Fibroblastos/genética , Glucose/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Células Ganglionares da Retina/citologia
20.
Int J Mol Sci ; 20(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319465

RESUMO

A large number of preclinical studies suggest the involvement of resveratrol in the prevention and treatment of eye diseases induced by oxidative stress and inflammation. We tested the hypothesis that resveratrol influences many pathways of in vitro and in vivo models of diabetic retinopathy through a systematic literature review of original articles. The review was conducted in accordance with the PRISMA guidelines. A literature search of all original articles published until April 2019 was performed. The terms "resveratrol" in combination with "retina", "retinal pathology", "diabetic retinopathy" and "eye" were searched. Possible biases were identified with the adopted SYRCLE's tool. Eighteen articles met inclusion/exclusion criteria for full-text review. Eleven of them included in vitro experiments, 11 studies reported in vivo data and 3 studies described both in vitro and in vivo experiments. Most of the in vivo studies did not include data that would allow exclusion of bias risks, according to SYRCLE's risk of bias tool. Both in vitro and in vivo data suggest anti-apoptotic, anti-inflammatory and anti-oxidative actions of resveratrol in models of diabetic retinopathy. However, results on its anti-angiogenic effects are contradictory and need more rigorous studies.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Resveratrol/uso terapêutico , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Humanos , Camundongos
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