RESUMO
Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.
Assuntos
Angiogênese , Células Endoteliais , Edição de Genes , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Humanos , Angiogênese/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Células Endoteliais/metabolismo , Edição de Genes/métodos , Vetores Genéticos , Neovascularização Patológica/metabolismo , Fosforilação , Retina/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVES: In this article, we provide a database of nonproliferative diabetes retinopathy, which focuses on early diabetes retinopathy with hard exudation, and further explore its clinical application in disease recognition. METHODS: We collect the photos of nonproliferative diabetes retinopathy taken by Optos Panoramic 200 laser scanning ophthalmoscope, filter out the pictures with poor quality, and label the hard exudative lesions in the images under the guidance of professional medical personnel. To validate the effectiveness of the datasets, five deep learning models are used to perform learning predictions on the datasets. Furthermore, we evaluate the performance of the model using evaluation metrics. RESULTS: Nonproliferative diabetes retinopathy is smaller than proliferative retinopathy and more difficult to identify. The existing segmentation models have poor lesion segmentation performance, while the intersection over union (IOU) value for deep lesion segmentation of models targeting small lesions can reach 66.12%, which is higher than ordinary lesion segmentation models, but there is still a lot of room for improvement. CONCLUSION: The segmentation of small hard exudative lesions is more challenging than that of large hard exudative lesions. More targeted datasets are needed for model training. Compared with the previous diabetes retina datasets, the NDRD dataset pays more attention to micro lesions.
Assuntos
Aprendizado Profundo , Retinopatia Diabética , Retinopatia Diabética/diagnóstico , Humanos , Bases de Dados Factuais , Programas de Rastreamento/métodos , Masculino , FemininoRESUMO
BACKGROUND: Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)ß suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRß in RPE cells remained elusive. METHODS: The CRISPR/Cas9 technology was utilized to edit a genomic PDGFRB locus in RPE cells derived from an epiretinal membrane (RPEM) from a patient with PVR, and a retroviral vector was used to express a truncated PDGFRß short of a PDGF-binding domain in the RPEM cells lacking PDGFRß. Western blot was employed to analyze expression of PDGFRß and α-smooth muscle actin, and signaling events (p-PDGFRß and p-Akt). Cellular assays (proliferation, migration and contraction) were also applied in this study. RESULTS: Expression of a truncated PDGFRß lacking a PDGF-binding domain in the RPEM cells whose PDGFRB gene has been silent using the CRISPR/Cas9 technology restores vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. In addition, we show that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunt the vitreous-induced activation of the truncated PDGFRß and Akt as well as the cellular events related to the PVR pathogenesis. These discoveries suggest that in RPE cells PDGFRß can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics. CONCLUSION: The data shown here will improve our understanding of the mechanism by which PDGFRß can be activated by non-PDGFs in the vitreous via an intracellular route of ROS/SFKs and provide a conceptual foundation for preventing PVR by inhibiting PDGFRß transactivation (ligand-independent activation).
Assuntos
Receptor beta de Fator de Crescimento Derivado de Plaquetas , Vitreorretinopatia Proliferativa , Humanos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteínas Proto-Oncogênicas c-akt , Ligantes , Espécies Reativas de Oxigênio/metabolismo , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Movimento CelularRESUMO
BACKGROUND: Orbital blowout fracture is common in ocular trauma. Accurate measurement of orbital volume after fracture is key in improving intraocular correction. OBJECTIVE: This study aims to explore the impact of 3D reconstruction technology in restoring normal exophthalmos in patients with old orbital wall fractures. METHODS: A total of 31 patients were randomly divided into an experimental group (n= 15) and a control group (n= 16). For orbital wall repair and reconstruction, the conventional group used the conventional surgical scheme, and the 3D group used 3D printing technology. RESULTS: There was no statistical difference between the preoperative mean extraocular muscle volume of the healthy eye and the affected eye. However, the mean orbital volume (24.76 vs 27.11, P= 0.005) and mean retrobulbar fat volume (17.53 vs 16.42, P= 0.006) were significantly different between the healthy eye and the affected eye. After an average follow-up of 16 weeks, the differences in pre- and post-surgery exophthalmos in the two groups were 0.42 ± 0.08 mm and 1.63 ± 0.51 mm, respectively. The difference between the two groups was statistically significant (t= 4.42, P= 0.003). The complications were not statistically different. CONCLUSION: Using 3D reconstruction technology preoperatively can significantly improve exophthalmos in patients with old orbital wall fractures.
Assuntos
Enoftalmia , Exoftalmia , Fraturas Orbitárias , Procedimentos de Cirurgia Plástica , Humanos , Enoftalmia/etiologia , Enoftalmia/cirurgia , Imageamento Tridimensional , Exoftalmia/cirurgia , Exoftalmia/complicações , Fraturas Orbitárias/complicações , Fraturas Orbitárias/cirurgia , Estudos RetrospectivosRESUMO
Objective: To investigate the effectiveness of intense pulsed light (M22) in treating patients with meibomian gland dysfunction (MGD) caused by demodex mites. Methods: A total of 100 patients (100 eyes) diagnosed with demodex mites through microscopic examination at Shanxi Bethune Eye Clinic between June 2021 and May 2023 were selected using convenience sampling. The patients were randomly divided into two groups: an experimental group (n=50) and a control group (n=50). The control group received comprehensive treatment consisting of artificial tears, warm compress, anti-inflammatory eye ointment, hypochlorous acid cleansing, okra cotton pad, and meibomian gland massage. In addition to the comprehensive treatment, the experimental group received intense pulsed light (M22) therapy. After 8 weeks of treatment, the mite clearance rate and cure rate of dry eye were measured for both groups. The recurrence rate of dry eye was also observed 4 weeks after discontinuing M22 treatment. Results: The experimental group achieved a mite clearance rate of 88.0%, while the control group had a rate of 58.0%, with a statistically significant difference (χ2 = 5.43, P = 0.017). Regarding the cure rate of dry eye, the experimental group showed a rate of 92.0%, while the control group had a rate of 82.0%, with a statistically significant difference (χ2 = 3.61, P = 0.021). In terms of the recurrence rate of dry eye, the experimental group exhibited a rate of 13.04%, while the control group had a rate of 26.83%, with a statistically significant difference (χ2 = 4.18, P = 0.016). Conclusion: Intense pulsed light (M22) demonstrated superior efficacy in eradicating demodex mites, treating dry eye, and maintaining the treatment's effectiveness compared to comprehensive treatment with medication in patients suffering from meibomian gland dysfunction with demodex mites.
RESUMO
Patients with rhegmatogenous retinal detachment (RRD) require face-down positioning (FDP) for 3-6 months or longer after pars plana vitrectomy (PPV) combined with silicone oil (SO) tamponade. This paper aimed to identify the factors that influenced FDP compliance. This study adopted semi-structured interviews with patients who require FDP after SO tamponade. Constructivist grounded theory was utilized in this study. The qualitative data was analyzed and coded via NVivo 11.0 through open coding, axial coding and selective coding. Twenty-four RRD patients were involved. The interviews yielded five main themes that defined home FDP compliance were identified: posture discomfort, doctor-patient communication, psychological factors, occupational character, and family factors. A theoretical model of the influencing factors of postural compliance of FDP was constructed based on the interview analysis. A variety of factors can affect FDP conformity. We can increase compliance of RRD patients by enhancing comfort, encouraging doctor-patient communication, providing comprehensive care, promoting community-based intervention, and strengthening family education.
Assuntos
Descolamento Retiniano , Humanos , Descolamento Retiniano/cirurgia , Teoria Fundamentada , Acuidade Visual , Vitrectomia , Cooperação do PacienteRESUMO
BACKGROUND: Different splicing of vascular endothelial growth factor (VEGF) gene results in 2 families of VEGF, the proangiogenic isoforms (VEGFxxxa) and the antiangiogenic isoforms (VEGFxxxb). VEGF165b is the major antiangiogenic isoform of VEGF and the most studied member of the VEGFxxxb family so far. OBJECTIVES: To determine the concentration of VEGF165b and VEGF in the aqueous humor (AH) in diabetic eyes with or without diabetic retinopathy (DR) and to address the predictive value of VEGF165b/VEGF ratio for progression of DR. METHODS: AH samples from 20 eyes in healthy controls (CON group), 40 eyes in diabetic patients without DR (nDR group), and 30 eyes in diabetic patients with mild nonproliferative DR (DR group) were collected. All of the patients were followed up for at least 5 years. VEGF165b and VEGF levels of AH samples were measured by enzyme-linked immunosorbent assay (ELISA). The predictive value of the initial VEGF165b/VEGF ratio for progression of DR was studied. RESULTS: The mean concentration of VEGF165b significantly decreased in diabetic eyes vs. controls. The mean concentration of VEGF significantly increased in the DR group vs. the CON group. The VEGF165b/VEGF ratio was significantly lower in diabetic patients compared to the CON group. The VEGF165b/VEGF ratio was significantly lower in diabetic patients compared to the control group. The mean follow-up was 66.1months (range 60-71 months). The risk of DR progression was greater with a lower VEGF165b/VEGF ratio. CONCLUSION: The VEGF165b/VEGF ratio is lower in the AH of DR patients and the decreased ratio of VEGF165b/VEGF predicts DR progression.
Assuntos
Humor Aquoso/metabolismo , Retinopatia Diabética/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Purpose: Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. Methods: The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. Results: AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. Conclusions: AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.
Assuntos
DNA/genética , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Degeneração Macular Exsudativa/genética , Western Blotting , Proliferação de Células , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Reação em Cadeia da Polimerase , RNA Longo não Codificante/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Degeneração Macular Exsudativa/metabolismo , Degeneração Macular Exsudativa/patologiaRESUMO
Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2. We further show that recombinant AAV serotype 1 (rAAV1) transduces ECs of pathologic vessels, and that editing of genomic VEGFR2 locus using rAAV1-mediated CRISPR/Cas9 abrogates angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization. This work establishes a strong foundation for genome editing as a strategy to treat angiogenesis-associated diseases.Abnormal angiogenesis causes many ocular diseases. Here the authors employ CRISPR/Cas9 gene editing technology to silence VEGFR2, a major regulator of angiogenesis, in retinal endothelium and abrogate angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Neovascularização Patológica/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Western Blotting , Células Cultivadas , Neovascularização de Coroide/genética , Neovascularização de Coroide/prevenção & controle , Dependovirus/genética , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neovascularização Patológica/prevenção & controle , Neovascularização Retiniana/genética , Neovascularização Retiniana/prevenção & controleRESUMO
PURPOSE: Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -ß) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -ß and whether suppression of PI5P4Kα and -ß would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). METHODS: PI5P4Kα and -ß encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -ß by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -ß knocked-down ARPE-19 cells. Expression of PI5P4Kα and -ß was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. RESULTS: We found that vitreous enhanced expression of PI5P4Kα and -ß in RPE cells and that knocking down PI5P4Kα and -ß abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -ß in the human PI5P4Kα and -ß knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -ß abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -ß was abundant in epiretinal membranes from PVR grade C patients. CONCLUSIONS: The findings from this study indicate that PI5P4Kα and -ß could be novel therapeutic targets for the treatment of PVR.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Vitreorretinopatia Proliferativa/prevenção & controle , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Coelhos , Descolamento Retiniano/prevenção & controle , Vitreorretinopatia Proliferativa/etiologia , Corpo Vítreo/metabolismoRESUMO
The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.