RESUMEN
Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.
Asunto(s)
Sistemas CRISPR-Cas , Neovascularización de la Córnea , Modelos Animales de Enfermedad , Edición Génica , Células Endoteliales de la Vena Umbilical Humana , Factor A de Crecimiento Endotelial Vascular , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/terapia , Neovascularización de la Córnea/metabolismo , Animales , Edición Génica/métodos , Ratones , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Sistemas CRISPR-Cas/genética , Ratones Endogámicos C57BL , Proliferación Celular/genéticaRESUMEN
OBJECTIVE: To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization. METHODS: The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 µM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at ï¼80 â until histological study or protein extraction. RESULTS: Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC. CONCLUSION: This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.
Asunto(s)
Quemaduras Químicas , Neovascularización de la Córnea , Emodina , Humanos , Ratones , Animales , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Quemaduras Químicas/tratamiento farmacológico , Quemaduras Químicas/metabolismo , Quemaduras Químicas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Simulación del Acoplamiento Molecular , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Transducción de Señal , Células Endoteliales de la Vena Umbilical Humana , Inflamación/tratamiento farmacológico , Modelos Animales de EnfermedadRESUMEN
PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.
Asunto(s)
Neovascularización de la Córnea , Fibrosis , Terapia Genética , Factores de Crecimiento Nervioso , Serpinas , Animales , Conejos , Córnea/patología , Córnea/metabolismo , Neovascularización de la Córnea/terapia , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/patología , Neovascularización de la Córnea/metabolismo , Decorina/genética , Decorina/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Fibrosis/terapia , Terapia Genética/métodos , Vectores Genéticos , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Serpinas/genética , Serpinas/metabolismoRESUMEN
Purpose: Corneal neovascularization (CNV) impairs corneal transparency and visual acuity. The study aims to deepen our understanding of the molecules involved in CNV induced by alkali burns, facilitate a better grasp of CNV mechanisms, and uncover potential therapeutic targets. Methods: Eighty-four mice were selected for establishing CNV models via alkali burns. On days 3, 7, and 14 after the burns, corneal observations and histological investigations were conducted. An integrated analysis of RNA sequencing (RNA-seq)-based transcriptomics and label-free quantitative proteomics was performed in both normal and burned corneas. Bioinformatics approaches, encompassing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, were applied to discern differentially expressed genes (DEGs) and crucial signaling pathways. Four potentially CNV-related genes were validated using quantitative real-time PCR (qRT-PCR) and Western blot. Results: Significant CNV was observed on the seventh day. Forty-one genes were differentially expressed in neovascularized corneas, with 15 upregulated and 26 downregulated at both mRNA and protein levels. Bioinformatics analysis revealed that these DEGs participated in diverse biological processes, encompassing retinol and retinoic acid metabolism, neutrophil chemotaxis, and actin filament assembly, along with significant enrichment pathways like cytochrome P450, tyrosine, and phenylalanine metabolism. The upregulation of lymphocyte cytosolic protein 1 (LCP1) and cysteine and glycine-rich protein 2 (CSRP2) genes and the downregulation of transglutaminase 2 (TGM2) and transforming growth factor-beta-induced (TGFBI) genes were confirmed. Conclusions: We analyzed gene expression differences in mouse corneas 7 days after alkali burns, finding 41 genes with altered expression. The exact role of these genes in CNV is not fully understood, but exploring angiogenesis-related molecules offers potential for CNV treatment or prevention.
Asunto(s)
Quemaduras Químicas , Neovascularización de la Córnea , Animales , Ratones , Neovascularización de la Córnea/genética , Quemaduras Químicas/genética , Proteómica , Neovascularización Patológica , Perfilación de la Expresión Génica , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Corneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo. METHODS: A PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo. RESULTS: METTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo. CONCLUSION: Our findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.
Asunto(s)
Neovascularización de la Córnea , Herpesvirus Humano 1 , Queratitis Herpética , Animales , Ratones , Humanos , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/patología , Herpesvirus Humano 1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vía de Señalización Wnt , Queratitis Herpética/patología , Neovascularización Patológica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , ARN Mensajero/genética , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
BACKGROUND: Corneal neovascularization (CNV) can be caused by chemical burns. Macrophages are involved in angiogenesis and lymphangiogenesis during CNV. The aim of this study was to investigate whether Wilms' tumor 1-associated protein (WTAP) is involved in macrophage recruitment and VEGF secretion via N6-methyladenosine (m6A) modification. METHODS: A CNV mouse model was established by corneal alkali burn. Tumor necrosis factor alpha (TNF-α) was used to stimulate vascular endothelial cells. m6A immunoprecipitation qPCR was used to determine the enrichment of m6A levels in mRNAs. The H3K9me3 enrichment in the promoter region of CC motif chemokine ligand 2 (CCL2) was detected by chromatin immunoprecipitation assay. The WTAP inhibition in vivo was performed using the adeno-associated virus. RESULTS: In the alkali burn corneal tissues, angiogenesis and lymphangiogenesis were promoted as CD31 and LYVE-1 expressions were elevated, and the number of macrophages as well as WTAP expression were increased. Under the TNF-α stimulation, WTAP promoted the recruitment of endothelial cells to macrophages by promoting CCL2 secretion. Mechanistically, WTAP affected the enrichment of H3K9me3 at the CCL2 promoter by regulating the m6A level of SUV39H1 mRNA. The in vivo experiment showed that VEGFA/C/D secretion of macrophages was reduced after WTAP interference. Mechanistically, WTAP regulated the translational efficiency of HIF-1α via m6A modification. CONCLUSION: WTAP affected macrophage recruitment to endothelial cells via regulation of H3K9me3-mediated CCL2 transcription. WTAP also affected macrophage secretion of VEGFA/C/D via m6A-mediated translation regulation of HIF-1α. Both pathways were involved in the WTAP regulation of angiogenesis and lymphangiogenesis during CNV.
Asunto(s)
Quemaduras Químicas , Neovascularización de la Córnea , Ratones , Animales , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células Endoteliales/metabolismo , Quemaduras Químicas/metabolismo , Quemaduras Químicas/patología , Macrófagos/metabolismoRESUMEN
This study was to explore the inhibitory effect of bromfenac sodium (BF) / chitosan (CS) nanoparticles (NPs) on corneal neovascularization (CNV). 45 New Zealand white rabbits provided by The First Affiliated Hospital of Jinan University were randomly divided into a control group (group A, n = 15), 0.1% BF aqueous solution treatment group (group B, n = 15), and 0.1% BF/CS-NPs suspension treatment group (group C, n = 15). A rabbit corneal alkali burn model was established. The average particle size of BF/CS-NPs with different BF concentrations was mainly 341.6 ± 12.9 nm - 548.7 ± 15.4 nm; and the Zeta potential distribution was 24.3 ± 2.5 mV - 35.7 ± 4.3 mV. When the initial concentration of BF was 1.5 mg/mL, the maximum drug loading was 57.35 ± 5.26%. The area of CNV in group C was significantly lower than that in groups B and A, and the differences were statistically significant (P < 0.05). At 6, 12, 18, and 24 days after surgery, the mRNA expression levels in cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) gene were compared after standardized by ß-actin; group A had the highest expression level, followed by group B, and group C had the lowest expression level, showing statistically significant differences (P < 0.05). The BF/CS-NPs granules prepared in this study had stable physical and chemical properties and had a good sustained-release effect, and the release duration can be as long as 48 hours. BF/CS-NPs can inhibit the formation of CNV at different time points after alkali burn, and reduce the expression of VEGF and COX-2 in corneal tissue after alkali burn.
Asunto(s)
Quemaduras Químicas , Neovascularización de la Córnea , Quemaduras Oculares , Animales , Benzofenonas , Bromobencenos , Quemaduras Químicas/tratamiento farmacológico , Quemaduras Químicas/metabolismo , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Ciclooxigenasa 2/genética , Modelos Animales de Enfermedad , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/tratamiento farmacológico , Quemaduras Oculares/metabolismo , Conejos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Corneal neovascularization (CRNV) is a severe threat to the vision of people. MicroRNA-335 (miR-335) has the function of facilitating angiogenesis. However, whether miR-335 regulates the progression of CRNV remains unclear. METHODS: The miR-335 expressions in CRNV rats induced by corneal suture and HUVECs induced by b-FGF were detected by quantitative real-time PCR. For the miR-335 function, wound healing and tube formation assays were performed. For the miR-335 mechanism, a dual-luciferase reporter gene assay was conducted. Besides, for the epidermal growth factor receptor (EGFR) function, Cell Counting Kit-8 and wound healing assays were performed. Meanwhile, the rescue assay was used to assess the miR-335/EGFR function in the migration and angiogenesis of b-FGF-treated HUVECs. RESULTS: Functionally, the miR-335 knockdown weakened the migration and angiogenesis of b-FGF-treated HUVECs, while the miR-335 overexpression showed an opposite trend. Mechanistically, miR-335 interacted with EGFR and negatively regulated the expression of EGFR. The rescue assay illustrated that miR-335 regulated the migration and angiogenesis of b-FGF-treated HUVECs through EGFR. CONCLUSIONS: In general, our data confirmed that miR-335 facilitated the process of CRNV by targeting EGFR.
Asunto(s)
Neovascularización de la Córnea , Receptores ErbB , MicroARNs , Animales , Movimiento Celular , Proliferación Celular , Neovascularización de la Córnea/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , RatasRESUMEN
Given the implications of the problem of neovascularization on ocular health, as well as the growth in the number of cases, the purpose of the present study has been testing the efficacy of siRNAs (small interfering RNA) designed to silence Hypoxia Inducible Factor -1α (HIF-1α) and to demonstrate that their use stops neovascularization in a model of corneal burn. Corneal wounds in the limbic zone were made in the eyes of New Zealand white rabbits. Topical applications of siRNAs were done the next day to the wound for four consecutive days and eyes were examined with a slit lamp. Evaluation of neovascularization progress was done by analyzing images by ImageJTM and to determine the neovascular area in Matlab ® was used. At the same time, a rabbit corneal cell line was used for in vitro study of hypoxia exposure and Western blot analysis of the cell's extracts were done. Under normal cell culture oxygenation, the expression of HIF-1α was lower than that observed under hypoxic conditions. After 2 h of hypoxia, there was a significant increase in the HIF-1α expression, effect that was maintained up to 6 h. The increased in HIF-1α was mimicked by a cell permeable prolyl-4-hydroxylase inhibitor. Cobalt chloride showed no capacity to increase HIF-1α in vitro. The effect of three different siRNA on HIF-1α was tested after 4 h of hypoxia. siRNA#1 was able to silence 80% of HIF-1α expression, siRNA#2 and siRNA#3 reduce the expression in 45% and 40% respectively. In addition, the three siRNA were tested in a corneal model of neovascularization. scrambledsiRNA#2 was the most effective inhibitor of blood vessel production, followed by siRNA#3 and siRNA#1. Compared to the scrambled siRNA (100% of blood vessel generation), siRNA#2 blocked the presence of blood vessels by 83 ± 2%, siRNA#3 inhibited 45 ± 7% and siRNA#1 only inhibited 18 ± 5%. The necessary time to observe the 50% of effect showed values of NV50 of 10.2 ± 2.4 days for the scrambled siRNA, 9.1 ± 1.4 for siRNA#1, 6.5 ± 1.85 for siRNA#2 and 4.8 ± 1.8 days for siRNA#3. In conclusion, the topical application of siRNA towards HIF-1α seems to be an effective and reliable method to stop neovascularization.
Asunto(s)
Neovascularización de la Córnea , Administración Tópica , Animales , Hipoxia de la Célula , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/terapia , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Patológica , ARN Interferente Pequeño/genética , Conejos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Corneal neovascularization (CNV) is a common disease that affects the vision ability of more than 1 million people annually. Small interfering RNA (siRNA) delivery nanoparticle platforms are a promising therapeutic modality for CNV treatment. However, the efficient delivery of siRNA into cells and the effective release of siRNA from delivery vehicles in a particular cell type challenge effective RNAi clinical application for CNV suppression. This study reports the design of a novel reactive oxygen species (ROS)-responsive lipid nanoparticle for siRNA delivery into corneal lesions for enhanced RNAi as a potential CNV treatment. We demonstrated that lipid nanoparticles could efficiently deliver siRNA into human umbilical vein endothelial cells and release siRNA for enhanced gene silencing by using the upregulated ROS of CNV to promote lipid nanoparticle degradation. Moreover, the subconjunctival injection of siRNA nanocomplexes into corneal lesions effectively knocked down vascular endothelial growth factor expression and suppressed CNV formation in an alkali burn model. Thus, we believe that the strategy of using ROS-responsive lipid nanoparticles for enhanced RNAi in CNV could be further extended to a promising clinical therapeutic approach to attenuate CNV formation.
Asunto(s)
Neovascularización de la Córnea , Nanopartículas , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Liposomas , Oxígeno/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The aim of this study was to analyze the single and combined effects of N-acetyl cysteine (NAC) and doxycycline (Dox) on the inflammatory and angiogenic factors in the rat model of alkali-burned cornea. Rats were treated with a single and combined 0.5% NAC and 12.5 µg/mL Dox eye drops and evaluated on days 3, 7, and 28. In the corneas of various groups, the activity of Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes was assessed. The expression of inflammatory factors (TNF-α, Rel-a, and CXCL-1) and angiogenic factors (VEGF-a, MMP2, and MMP9) was measured using real-time polymerase chain reaction. The antioxidant enzyme activities decreased substantially 3 days after injury with sodium hydroxide (NaOH). NAC and combined NAC+ Dox topical treatments increased the SOD enzyme activity on day 28 (P < 0.05). The expression of TNF-α and Rel-a genes following single and combined treatment of NAC and Dox decreased significantly on days 7 and 28 (P < 0.05). The mRNA level of angiogenic factors and corneal neovascularization (CNV) level declined in NaOH-injured rats treated with Dox (P < 0.05). The topical treatment of Dox could attenuate inflammation and CNV complications. However, NAC treatment may not reduce the expression of angiogenic genes.
Asunto(s)
Quemaduras Químicas , Neovascularización de la Córnea , Quemaduras Oculares , Acetilcisteína/metabolismo , Acetilcisteína/farmacología , Álcalis/metabolismo , Álcalis/farmacología , Inductores de la Angiogénesis/metabolismo , Inductores de la Angiogénesis/farmacología , Animales , Quemaduras Químicas/complicaciones , Quemaduras Químicas/tratamiento farmacológico , Quemaduras Químicas/metabolismo , Córnea/metabolismo , Neovascularización de la Córnea/etiología , Neovascularización de la Córnea/genética , Modelos Animales de Enfermedad , Doxiciclina/metabolismo , Doxiciclina/farmacología , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/complicaciones , Quemaduras Oculares/tratamiento farmacológico , Ratas , Hidróxido de Sodio/metabolismo , Hidróxido de Sodio/farmacología , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Our previous study revealed that mesenchymal stem cells (MSCs) inhibited angiogenesis via miRNA-mediated repression of prospero homeobox 1 (PROX1). This study aimed to verify whether miR-340-5p participates in the therapeutic effect of MSCs on corneal neovascularization (CNV) via repressing PROX1 and epithelial membrane protein 2 (EMP2). MATERIALS AND METHODS: The rat CNV model was established by corneal alkali burn. The binding relationship between miR-340-5p and 3'-untranslational regions (3'UTRs) of EMP2 and PROX1 was confirmed using dual-luciferase reporter assay. After culturing corneal epithelial cells (CECs) using MSC supernatants, the vascular endothelial growth factor (VEGF) level in CEC supernatants and the CEC viability were detected. The role of miR-340-5p in the therapeutic effect of MSC on CNV was determined via lentivirus-mediated miR-340-5p intervention in vivo. RESULTS: The expression of miR-340-5p was reduced and EMP2 and PROX1 were increased in CNV corneal tissues. The lentivirus-mediated overexpression of miR-340-5p inhibited the expressions of EMP2 and PROX1. The dual-luciferase reporter assay confirmed that miR-340-5p could bind with the 3'UTRs of EMP2 and PROX1. miR-340-5p was enriched in MSC supernatants and the culture of CECs using MSC supernatants increased the miR-340-5p expression in CECs. After being cultured in miR-340-5p-knocking down MSC supernatants, the expressions of EMP2 and PROX1 were increased, and the VEGF level and CEC viability were restored. The in vivo experiments also indicated that the therapeutic effect of MSCs was mediated by miR-340-5p. CONCLUSIONS: miR-340-5p mediates the therapeutic effect of MSCs on CNV via binding and repressing the expressions of EMP2 and PROX1.
Asunto(s)
Neovascularización de la Córnea , Quemaduras Oculares , Células Madre Mesenquimatosas , MicroARNs , Animales , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/terapia , Quemaduras Oculares/inducido químicamente , Proteínas de Homeodominio/genética , Glicoproteínas de Membrana/genética , MicroARNs/genética , Ratas , Proteínas Supresoras de Tumor/genética , Factor A de Crecimiento Endotelial VascularRESUMEN
PURPOSE: Corneal injury may cause neovascularization and lymphangiogenesis in cornea which have a detrimental effect to vision and even lead to blindness. Bone morphogenetic protein 4 (BMP4) regulates a variety of biological processes, which is closely relevant to the regulation of corneal epithelium and angiogenesis. Herein, we aimed to evaluate the effect of BMP4 on corneal neovascularization (CNV), corneal lymphangiogenesis (CL), corneal epithelial repair, and the role of BMP4/Smad pathway in these processes. METHODS: We used MTT assay to determine the optimal concentration of BMP4. The suture method was performed to induce rat CNV and CL. We used ink perfusion and HE staining to visualize the morphological change of CNV, and utilized RT-qPCR and ELISA to investigate the expression of angiogenic factors and lymphangiogenic factors. The effects of BMP4 and anti-VEGF antibody on migration, proliferation and adhesion of corneal epithelium were determined by scratch test, MTT assay and cell adhesion test. RESULTS: BMP4 significantly inhibited CNV and possibly CL. Topical BMP4 resulted in increased expression of endogenous BMP4, and decreased expression of angiogenic factors and lymphangiogenic factors. Compared with anti-VEGF antibody, BMP4 enhanced corneal epithelium migration, proliferation and adhesion, which facilitated corneal epithelial injury repair. Simultaneously, these processes could be regulated by BMP4/Smad pathway. CONCLUSIONS: Our results demonstrated unreported effects of BMP4 on CNV, CL, and corneal epithelial repair, suggesting that BMP4 may represent a potential therapeutic target in corneal injury repair.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Lesiones de la Cornea/genética , Neovascularización de la Córnea/etiología , Sustancia Propia/patología , Regulación de la Expresión Génica , ARN/genética , Cicatrización de Heridas , Animales , Proteína Morfogenética Ósea 4/biosíntesis , Movimiento Celular , Proliferación Celular , Células Cultivadas , Lesiones de la Cornea/complicaciones , Lesiones de la Cornea/patología , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/patología , Sustancia Propia/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , ARN/metabolismo , Ratas , Ratas WistarRESUMEN
Purpose: Paxillin (PXN) is a key component of focal adhesions and plays an important role in angiogenesis. The aim of the present study was to investigate the effect of PXN in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were transfected with PXN overexpression and PXN interference vectors. Biochemical detection was used to detect adenosine triphosphate and lactic acid production. The morphology of mitochondria was observed under an electron microscope, and flow cytometry was conducted to measure mitochondrial membrane potential. Transwell experiments were used to detect the migration and tube formation ability of each group of cells. The expression of hexokinase (HK)1, HK2, glucose transporter 1 (GLUT1), phosphorylated phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, and phosphorylated mechanistic target of rapamycin (mTOR) was evaluated by western blot. Results: PXN silencing reduced the levels of lactic acid and adenosine triphosphate, downregulated HK1, HK2, and GLUT1, suppressed PI3K/AKT/mTOR signaling activation, and inhibited VEGF-A-induced mitochondria injury in VEGF-A-induced HUVECs. We also determined that miR-145-5p decreased the VEGF-A-induced expression of PXN and inhibited the invasion and angiogenesis of HUVECs. Also, miR-145-5p inhibition blocked the protective effect of PXN interference on VEGF-A-induced HUVEC injury. Furthermore, PXN interference significantly decreased lactic acid and adenosine triphosphate levels, inhibited PI3K/AKT/mTOR activation, and decreased the levels of HK1, HK2, and GLUT1 in VEGF-A-treated mouse corneal. Conclusions: The results indicate that PXN silencing inhibited the VEGF-A-induced invasion and angiogenesis of HUVECs via regulation of cell metabolism and mitochondrial damage, suggesting that PXN may be a potential target for antiangiogenic therapies.
Asunto(s)
Neovascularización de la Córnea/genética , Regulación de la Expresión Génica , MicroARNs/genética , Factor A de Crecimiento Endotelial Vascular/efectos adversos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neovascularización de la Córnea/inducido químicamente , Neovascularización de la Córnea/metabolismo , Humanos , MicroARNs/biosíntesis , Paxillin/biosíntesis , Paxillin/genética , ARN/genética , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVES: Vascular endothelial growth factor A (VEGFA) is one of the major factors initiating and regulating angiogenesis. LncRNA taurine up-regulated gene 1 (TUG1) has been implicated in the pathological neovascularization. The aim of this study is to explore the function of TUG1 in regulating VEGFA-mediated angiogenesis in endothelial cells. METHODS: A total of 12 corneal neovascularization (CRNV) samples were collected form patient undergoing corneal transplantation at Tongji Hospital, Wuhan, China. qRT-PCR and Western blotting were performed to examine gene expression and protein levels. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro angiogenesis model. CCK-8 proliferation assay was used to determine cell proliferation capacity and wound healing was performed to analyze cell migration ability. Dual luciferase reporter assay was used for functional interaction validation between miR-505-3p and its targets. The in vitro angiogenic potential was evaluated by tube formation assay. RESULTS: TUG1 and VEGFA were upregulated in CRNV tissues and VEGFA-treated HUVECs. TUG1 knockdown inhibited proliferation, migration and tube formation capacity of HUVECs. TUG1 regulated the angiogenesis of HUVECs by modulating VEGFA expression through targeting miR-505-3p. CONCLUSIONS: Our results suggest that lncRNA TUG1 promotes the angiogenesis of HUVECs through modulating miR-505-3p/VEGFA axis.
Asunto(s)
Córnea/irrigación sanguínea , Neovascularización de la Córnea/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , MicroARNs/metabolismo , Neovascularización Fisiológica , ARN Largo no Codificante/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Células Cultivadas , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/patología , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , MicroARNs/genética , ARN Largo no Codificante/genética , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Corneal neovascularization (CoNV) can cause abnormal blood vessels to grow in the transparent cornea, leading to various sight-threatening eye diseases. MicroRNAs are known to play essential roles in the regulation of numerous biological functions. We try to clarify the role of a specific microRNA, miR497, which has been shown to regulate the growth of tumor cells and angiogenesis on the basis of available data. However, the association between miR-497 and vascularized cornea remains unclear. Therefore, it is urgently needed to understand the molecular mechanism of miR497 in the progress of corneal neovascularization. Animal model of CoNV was established in wildtype (WT) C57BL/6 mice, CRISPR/Cas9 mediated miR-497 knockout (KO) and overexpressed (TG) C57BL/6 mice. MiR-497, expressed in corneas, was actively involved in alkali burn-induced corneal neovascularization via targeting STAT3 and negatively regulating its expression, attenuating macrophage infiltration and M2 polarization. Knockdown of miR-497 enhanced the formation of corneal angiogenesis through targeting STAT3 and facilitating its expression, promoting recruitment of macrophages, while overexpression of miR-497 restrained blood vessel sprouting via regulating downstream STAT3 and VEGFA expression, reducing macrophage activation and inhibiting M2 polarization. Moreover, miR-497 knockout-mediated damage effect can be rescued through the inhibition of STAT3 signaling. Mechanically, miR-497 might serve as a potential strategy for pathological corneal neovascularization via macrophage through the IL-6/STAT3/VEGFA signaling pathway.
Asunto(s)
Neovascularización de la Córnea/prevención & control , Interleucina-6/metabolismo , Activación de Macrófagos/inmunología , MicroARNs/administración & dosificación , Factor de Transcripción STAT3/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , MicroARNs/genética , Transducción de SeñalRESUMEN
Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn-/-) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn-/- and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn-/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn-/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn-/- mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn-/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn-/- mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn-/- mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn-/- mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.
Asunto(s)
Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/fisiopatología , Decorina/fisiología , Actinas/metabolismo , Animales , Neovascularización de la Córnea/genética , Endoglina/genética , Endoglina/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.
Asunto(s)
Córnea/patología , Neovascularización de la Córnea/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Animales , Córnea/metabolismo , Neovascularización de la Córnea/patología , Eliminación de Gen , Humanos , Linfangiogénesis , Ratones Endogámicos C57BL , ARN Mensajero/genética , Receptores Tipo I de Factores de Necrosis Tumoral/análisis , Receptores Tipo II del Factor de Necrosis Tumoral/análisisRESUMEN
We have previously used the genetic diversity available in common inbred mouse strains to identify quantitative trait loci (QTLs) responsible for the differences in angiogenic response using the corneal micropocket neovascularization (CoNV) assay. Employing a mouse genome-wide association study (GWAS) approach, the region on chromosome 15 containing Basp1 was identified as being significantly associated with angiogenesis in inbred strains. Here, we developed a unique strategy to determine and verify the role of BASP1 in angiogenic pathways. Basp1 expression in cornea had a strong correlation with a haplotype shared by mouse strains with varied angiogenic phenotypes. In addition, inhibition of BASP1 demonstrated a dosage-dependent effect in both primary mouse brain endothelial and human microvascular endothelial cell (HMVEC) migration. To investigate its role in vivo, we knocked out basp1 in transgenic kdrl:zsGreen zebrafish embryos using a widely adopted CRISPR-Cas9 system. These embryos had severely disrupted vessel formation compared to control siblings. We further show that basp1 promotes angiogenesis by upregulating ß-catenin gene and the Dll4/Notch1 signaling pathway. These results, to the best of our knowledge, provide the first in vivo evidence to indicate the role of Basp1 as an angiogenesis-regulating gene and opens the potential therapeutic avenues for a wide variety of systemic angiogenesis-dependent diseases.
Asunto(s)
Neovascularización de la Córnea/patología , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Neovascularización Patológica/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Animales , Movimiento Celular , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/genética , Vía de Señalización Wnt , Pez CebraRESUMEN
AIMS: The therapeutic potential of Hedgehog (Hh) signalling agonists for vascular diseases is of growing interest. However, molecular and cellular mechanisms underlying the role of the Hh signalling in vascular biology remain poorly understood. The purpose of the present article is to clarify some conflicting literature data. METHODS AND RESULTS: With this goal, we have demonstrated that, unexpectedly, ectopically administered N-terminal Sonic Hh (N-Shh) and endogenous endothelial-derived Desert Hh (Dhh) induce opposite effects in endothelial cells (ECs). Notably, endothelial Dhh acts under its full-length soluble form (FL-Dhh) and activates Smoothened in ECs, while N-Shh inhibits it. At molecular level, N-Shh prevents FL-Dhh binding to Patched-1 (Ptch1) demonstrating that N-Shh acts as competitive antagonist to FL-Dhh. Besides, we found that even though FL-Hh ligands and N-Hh ligands all bind Ptch1, they induce distinct Ptch1 localization. Finally, we confirmed that in a pathophysiological setting, i.e. brain inflammation, astrocyte-derived N-Shh acts as a FL-Dhh antagonist. CONCLUSION: The present study highlights for the first time that FL-Dhh and N-Hh ligands have antagonistic properties especially in ECs.