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1.
Cell Tissue Res ; 396(1): 103-117, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38403744

RESUMO

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.


Assuntos
Vitreorretinopatia Proliferativa , Humanos , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Epitélio Pigmentado da Retina , Fator de Crescimento Transformador beta2/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/uso terapêutico , Proteína Morfogenética Óssea 6/farmacologia , Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 6/uso terapêutico , Transição Epitelial-Mesenquimal , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Pigmentos da Retina/uso terapêutico , Movimento Celular
2.
Clinics (Sao Paulo) ; 78: 100241, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37418795

RESUMO

OBJECTIVES: To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR). METHOD: The expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1ß, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1ß, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1ß and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Compared with the surrounding normal tissues, the expression of caspase-1, IL-1ß, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05). CONCLUSIONS: MCP can promote the development of PVR lesions.


Assuntos
Vitreorretinopatia Proliferativa , Humanos , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Interleucina-18/metabolismo , Piroptose , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Citocinas , RNA Mensageiro/metabolismo , Caspases
3.
Biochim Biophys Acta Mol Basis Dis ; 1869(6): 166747, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37207905

RESUMO

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on ß-strands in the protease core 2 domain. Conformational changes induced by Ca2+binding result in these ß-strands forming a ß-sheet and a hydrophobic pocket which docks W286 side chain away from the catalytic cleft, enabling calpain activation based on comparison with the Ca2+-bound CAPN1 protease core. The pathologic variants R243L, L244P, K250N, and R289W are predicted to disrupt the ß-strands, ß-sheet, and hydrophobic pocket, impairing calpain activation. The mechanism by which these variants impair membrane association is unclear. G376S impacts a conserved residue in the CBSW domain and is predicted to disrupt a loop containing acidic residues which may contribute to membrane binding. G267S did not impair membrane association and resulted in a slight but significant increase in autoproteolytic and proteolytic activity. However, G267S is also identified in individuals without NIV. Combined with the autosomal dominant pattern of NIV inheritance and evidence that CAPN5 may dimerize, the results are consistent with a dominant negative mechanism for the five pathogenic variants which resulted in impaired CAPN5 activity and membrane association and a gain-of-function for the G267S variant.


Assuntos
Neuroblastoma , Vitreorretinopatia Proliferativa , Humanos , Calpaína/genética , Calpaína/metabolismo , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/patologia , Mutação
4.
Cell Death Dis ; 14(2): 158, 2023 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-36841807

RESUMO

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of the pathogenesis of proliferative vitreoretinopathy (PVR) that can lead to severe vision loss. Nevertheless, the precise regulatory mechanisms underlying the pathogenesis of PVR remain largely unknown. Here, we show that the expression of death-associated protein-like 1 (DAPL1) is downregulated in PVR membranes and that DAPL1 deficiency promotes EMT in RPE cells in mice. In fact, adeno-associated virus (AAV)-mediated DAPL1 overexpression in RPE cells of Dapl1-deficient mice inhibited EMT in physiological and retinal-detachment states. In a rabbit model of PVR, ARPE-19 cells overexpressing DAPL1 showed reduced ability to induce experimental PVR, and AAV-mediated DAPL1 delivery attenuated the severity of experimental PVR. Furthermore, a mechanistic study revealed that DAPL1 promotes P21 phosphorylation and its stabilization partially through NFκB (RelA) in RPE cells, whereas the knockdown of P21 led to neutralizing effects on DAPL1-dependent EMT inhibition and enhanced the severity of experimental PVR. These results suggest that DAPL1 acts as a novel suppressor of RPE-EMT and has an important role in antagonizing the pathogenesis of experimental PVR. Hence, this finding has implications for understanding the mechanism of and potential therapeutic applications for PVR.


Assuntos
Proteínas de Membrana , Epitélio Pigmentado da Retina , Vitreorretinopatia Proliferativa , Animais , Camundongos , Coelhos , Transição Epitelial-Mesenquimal , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteínas de Membrana/metabolismo
5.
Discov Med ; 34(172): 103-113, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36333112

RESUMO

Proliferative vitreoretinopathy (PVR) is an intractable condition after rhegmatogenous retinal detachment (RD), which is the primary cause of failure in retinal reattachment surgery. This study aimed to investigate the effects of chicken ovalbumin upstream promoter transcriptional factor 1 (COUP-TF1) in the development of proliferative vitreoretinopathy (PVR) both in vitro and in vivo. Adult retinal pigment epithelium cell line was used for in-vitro experiments. Immunocytochemistry assay, real-time quantitative polymerase chain reaction, and Western blot were used to measure the expression of COUP-TF1, alpha-smooth muscle actin (α-SMA), and E-cadherin. Epithelial-mesenchymal transition (EMT) was observed through cell counting kit-8 assay, wound healing tests, and the expression changes of related proteins. PVR rabbit models were established and evaluated by the images of fundus and vitreous cavity, pathological sections, and COUP-TF1 expression. As shown by our results, the proliferation and migration of the COUP-TF1 knockdown cells were reduced compared with the control cells with or without transforming growth factor-ß1 (TGF-ß1) treatment. After TGF-ß1 treatment, α-SMA expression was upregulated in ARPE-19 cells but kept the same in COUP-TF1 knockdown cells. E-cadherin expression was down-regulated in all the groups but the extent of the decrease in COUP-TF1 knockdown cells was smaller. EMT was attenuated in ARPE-19 cells after COUP-TF1 was knocked down. In the in-vivo experiment, PVR severity was attenuated and the retinal detachment rate decreased on the 14th and 28th day in COUP-TF1 knockdown group. In conclusion, COUP-TF1 is related to the development of PVR, and COUP-TF1 knockdown attenuates the progression of PVR. This suggests that COUP-TF1 can be a promising candidate for the treatment of PVR.


Assuntos
Descolamento Retiniano , Vitreorretinopatia Proliferativa , Animais , Coelhos , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Transição Epitelial-Mesenquimal/genética , Fator de Crescimento Transformador beta1/metabolismo , Galinhas/metabolismo , Ovalbumina/metabolismo , Ovalbumina/farmacologia , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologia , Epitélio Pigmentado da Retina/metabolismo , Movimento Celular/genética , Células Cultivadas , Caderinas/genética , Caderinas/metabolismo
6.
Am J Physiol Cell Physiol ; 323(1): C116-C124, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35544697

RESUMO

Retinal pigmented epithelial (RPE) cells play an important role in retinal fibrotic diseases such as proliferative vitreoretinopathy (PVR). The purpose of this study was to elucidate the involvement of dopamine receptor signaling in regulating the fibrotic activation of RPE cells. Dopamine receptor expression, the effect of dopamine on fibrotic activity, and dopamine production were measured in the human RPE cell line ARPE-19. The fibrotic activation of RPE cells was evaluated in response to treatments with selective dopamine receptor agonists and antagonists by measuring gene expression, migration, proliferation, and fibronectin deposition. DRD2 and DRD5 are the dominant dopaminergic receptors expressed in ARPE-19 cells and TGF-ß stimulation enhances the autocrine release of dopamine, which we show further exasperates fibrotic activation. Finally, treatment with D2 dopamine receptor antagonists or D5 dopamine receptor agonists inhibits profibrotic gene expression, migration, proliferation, and fibronectin deposition and thus may serve as effective mechanisms for treating retinal fibrosis including PVR.


Assuntos
Fibronectinas , Vitreorretinopatia Proliferativa , Movimento Celular , Dopamina/metabolismo , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Fibronectinas/metabolismo , Fibrose , Humanos , Receptores Dopaminérgicos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia
7.
Commun Biol ; 5(1): 479, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35589941

RESUMO

Pathological retinal neovascularization (NV) is a clinical manifestation of various proliferative retinopathies, and treatment of NV using anti-VEGF therapies is not selective, as it also impairs normal retinal vascular growth and function. Here, we show that genetic deletion or siRNA-mediated downregulation of IL-33 reduces pathological NV in a murine model of oxygen-induced retinopathy (OIR) with no effect on the normal retinal repair. Furthermore, our fluorescent activated cell sorting (FACS) data reveals that the increase in IL-33 expression is in endothelial cells (ECs) of the hypoxic retina and conditional genetic deletion of IL-33 in retinal ECs reduces pathological NV. In vitro studies using human retinal microvascular endothelial cells (HRMVECs) show that IL-33 induces sprouting angiogenesis and requires NFkappaB-mediated Jagged1 expression and Notch1 activation. Our data also suggest that IL-33 enhances de-ubiquitination and stabilization of Notch1 intracellular domain via its interaction with BRCA1-associated protein 1 (BAP1) and Numb in HRMVECs and a murine model of OIR.


Assuntos
Doenças Retinianas , Vitreorretinopatia Proliferativa , Animais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Humanos , Interleucina-33/genética , Interleucina-33/farmacologia , Camundongos , Neovascularização Patológica/patologia , Oxigênio/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Doenças Retinianas/patologia , Vitreorretinopatia Proliferativa/patologia
8.
Sci Rep ; 11(1): 23989, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34907233

RESUMO

The specific changes linked to de novo development of postoperative PVR have remained elusive and were the object of the underlying study. Vitreous fluid (VF) was obtained at the beginning of vitrectomy from 65 eyes that underwent vitrectomy for primary rhegmatogenous retinal detachment (RRD) without preoperative PVR. Eyes developing postoperative PVR within 6 months after re-attachment surgery were compared to those which did not regarding the preoperative concentrations of 43 cytokines and chemokines in the VF, using multiplex beads analysis. For all comparisons Holm's correction was applied in order to control for multiple comparisons. Twelve out of 65 eyes (18.5%) developed PVR postoperatively. While 12 of the chemokines and cytokines presented concentration differences on a statistical level of p < 0.05 (CXCL5, CCL11, CCL24, CCL26, GM-CSF, IFN-γ, CCL8, CCL7, MIF, MIG/CXCL9, CCL19, and CCL25), CXCL5 was the only cytokine with sufficiently robust difference in its VF concentrations to achieve significance in eyes developing postoperative PVR compared to eyes without PVR. CXCL5 may represent a potent biomarker for the de novo development of postoperative PVR. In line with its pathophysiological role in the development of PVR, it might serve as a basis for the development of urgently needed preventive options.


Assuntos
Quimiocina CXCL5/metabolismo , Complicações Pós-Operatórias , Descolamento Retiniano , Vitreorretinopatia Proliferativa , Corpo Vítreo/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/patologia , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologia , Descolamento Retiniano/cirurgia , Estudos Retrospectivos , Vitreorretinopatia Proliferativa/etiologia , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia
9.
J Cell Mol Med ; 25(21): 10213-10223, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34598306

RESUMO

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes-associated protein (YAP) and the subsequent epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE-19 cells in vitro was studied. After treated with transforming growth factor-ß2(TGF-ß2), the expressions of fibrogenic molecules, YAP activation and the TGF-ß2-Smad signalling pathway in ARPE-19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF-ß2-induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF-ß2-induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF-ß2-induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA-α) and fibronectin. YAP was essential for the TGF-ß2-induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.


Assuntos
Transição Epitelial-Mesenquimal/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/etiologia , Vitreorretinopatia Proliferativa/metabolismo , Proteínas de Sinalização YAP/genética , Animais , Biomarcadores , Linhagem Celular , Movimento Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Fibrose , Humanos , Imuno-Histoquímica , Camundongos , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteínas de Sinalização YAP/metabolismo
10.
Mol Biotechnol ; 63(9): 768-779, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34057656

RESUMO

Retinal diseases are the primary reasons for severe visual defects and irreversible blindness. Retinal diseases are also inherited and acquired. Both of them are caused by mutations in genes or disruptions in specific gene expression, which can be treated by gene-editing therapy. Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) system is a frontier of gene-editing tools with great potential for therapeutic applications in the ophthalmology field to modify abnormal genes and treat the genome or epigenome-related retinal diseases. The CRISPR system is able to edit and trim the gene include deletion, insertion, inhibition, activation, replacing, remodeling, epigenetic alteration, and modify the gene expression. CRISPR-based genome editing techniques have indicated the enormous potential to treat retinal diseases that previous treatment was not available for them. Also, recent CRISPR genome surgery experiments have shown the improvement of patient's vision who suffered from severe visual loss. In this article, we review the applications of the CRISPR-Cas9 system in human or animal models for treating retinal diseases such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), then we survey limitations of CRISPR system for clinical therapy.


Assuntos
Sistemas CRISPR-Cas , Retinopatia Diabética/terapia , Proteínas do Olho/genética , Edição de Genes/métodos , Amaurose Congênita de Leber/terapia , Degeneração Macular/terapia , Retinose Pigmentar/terapia , Vitreorretinopatia Proliferativa/terapia , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Proteínas do Olho/metabolismo , Terapia Genética/métodos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
11.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760200

RESUMO

Proliferative vitreoretinopathy (PVR) is a disease leading to the formation of contractile preretinal membranes (PRMs) and is one of the leading causes of blindness. Connective tissue growth factor (CTGF) has been identified as a possible key determinant of progressive tissue fibrosis and excessive scarring. Therefore, the present study investigated the role and mechanism of action of CTGF in PVR. Immunohistochemical staining was performed to detect the expression of CTGF, fibronectin and collagen type III in PRMs from patients with PVR. The effects and mechanisms of recombinant human CTGF and its upstream regulator, TGF­ß1, on epithelial­mesenchymal transition (EMT) and the synthesis of extracellular matrix (ECM) by retinal pigment epithelium (RPE) cells were investigated using reverse transcription­quantitative PCR, western blotting and a [3H]proline incorporation assay. The data indicated that CTGF, fibronectin and collagen type III were highly expressed in PRMs. In vitro, CTGF significantly decreased the expression of the epithelial markers ZO­1 and E­cadherin and increased that of the mesenchymal markers fibronectin, N­cadherin and α­smooth muscle actin in a concentration­dependent manner. Furthermore, the expression of the ECM protein collagen type III was upregulated by CTGF. However, the trends in expression for the above­mentioned markers were reversed after knocking down CTGF. The incorporation of [3H]proline into RPE cells was also increased by CTGF. In addition, 8­Bromoadenosine cAMP inhibited CTGF­stimulated collagen synthesis and transient transfection of RPE cells with a CTGF antisense oligonucleotide inhibited TGF­ß1­induced collagen synthesis. The phosphorylation of PI3K and AKT in RPE cells was promoted by CTGF and TGF­ß1 and the latter promoted the expression of CTGF. The results of the present study indicated that CTGF may promote EMT and ECM synthesis in PVR via the PI3K/AKT signaling pathway and suggested that targeting CTGF signaling may have a therapeutic or preventative effect on PVR.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Transição Epitelial-Mesenquimal/genética , Pigmentos da Retina/genética , Fator de Crescimento Transformador beta1/genética , Vitreorretinopatia Proliferativa/genética , Western Blotting , Movimento Celular/genética , Matriz Extracelular/genética , Fibronectinas/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/genética , Vitreorretinopatia Proliferativa/patologia
12.
Sci Rep ; 11(1): 966, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441730

RESUMO

Rhegmatogenous retinal detachment (RRD) is an ophthalmic emergency, which usually requires prompt surgery to prevent further detachment and restore sensory function. Although several individual factors have been suggested, a systems level understanding of molecular pathomechanisms underlying this severe eye disorder is lacking. To address this gap in knowledge we performed the molecular level systems pathology analysis of the vitreous from 127 patients with RRD using state-of-the art quantitative mass spectrometry to identify the individual key proteins, as well as the biochemical pathways contributing to the development of the disease. RRD patients have specific vitreous proteome profiles compared to other diseases such as macular hole, pucker, or proliferative diabetic retinopathy eyes. Our data indicate that various mechanisms, including glycolysis, photoreceptor death, and Wnt and MAPK signaling, are activated during or after the RRD to promote retinal cell survival. In addition, platelet-mediated wound healing processes, cell adhesion molecules reorganization and apoptotic processes were detected during RRD progression or proliferative vitreoretinopathy formation. These findings improve the understanding of RRD pathogenesis, identify novel targets for treatment of this ophthalmic disease, and possibly affect the prognosis of eyes treated or operated upon due to RRD.


Assuntos
Retina/patologia , Descolamento Retiniano/patologia , Apoptose/fisiologia , Plaquetas/metabolismo , Plaquetas/patologia , Adesão Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma/metabolismo , Retina/metabolismo , Descolamento Retiniano/metabolismo , Perfurações Retinianas/metabolismo , Perfurações Retinianas/patologia , Transdução de Sinais/fisiologia , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Corpo Vítreo/metabolismo , Corpo Vítreo/patologia , Cicatrização/fisiologia
13.
Acta Ophthalmol ; 99(7): e1070-e1076, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33381918

RESUMO

PURPOSE: To analyse the cytotoxic and antiproliferative effect of methotrexate (MTX) and fluorouracil (5-FU) in vitro on fibroblasts, retinal pigment epithelial (RPE) and photoreceptor cells as an adjunct for reducing the incidence of proliferative vitreoretinopathy (PVR) after rhegmatogenous retinal detachment surgery. METHODS: Methotrexate and 5-FU were dissolved separately in balanced salt solution (BSS) with concentrations ranging from 0-8000 µg/ml and 0-4000 µg/ml, respectively. All solutions were analysed in terms of pH and osmolarity and applied for 1 h to fibroblasts (BJ), RPE (ARPE-19) and photoreceptor (661W) cell lines adherently cultivated in 96-well cell culture plates (10 000 cells/well). 24 h after incubation, the proliferative (BrdU), metabolic (CellTiter-Glo) and apoptotic (Caspase 3/7) activity of the cells were examined in vitro. RESULTS: 5-FU had an antiproliferative effect on BJ and ARPE-19 cells starting from low concentrations (2 µg/ml). However, the viability of 661W cells decreased and apoptosis was induced with increasing 5-FU concentration. In contrast, MTX up to a concentration of 266 µg/ml did neither result in a significant loss of viability nor in increased caspase 3/7 activity of BJ, ARPE-19 and 661W cells and inhibited the proliferation of ARPE-19 already at low concentrations starting from 8 µg/ml. CONCLUSIONS: Methotrexate dissolved in BSS is biocompatible up to a concentration of 266 µg/ml and may act as an intraoperative rinse solution to inhibit RPE proliferation in PVR-diseased eyes. Contrary, the use of 5-FU within the posterior segment of the eye is limited by its cell-damaging effect on photoreceptor cells.


Assuntos
Fluoruracila/efeitos adversos , Metotrexato/efeitos adversos , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/tratamento farmacológico , Apoptose , Células Cultivadas , Fluoruracila/uso terapêutico , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Metotrexato/uso terapêutico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vitreorretinopatia Proliferativa/patologia
14.
FASEB J ; 35(1): e21152, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33151576

RESUMO

Vitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.


Assuntos
Neovascularização Patológica/enzimologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Vasos Retinianos/enzimologia , Corpo Vítreo/enzimologia , Animais , Benzocicloeptenos/farmacologia , Sistemas CRISPR-Cas , Retinopatia Diabética/enzimologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Células HEK293 , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Vasos Retinianos/patologia , Triazóis/farmacologia , Vitreorretinopatia Proliferativa/enzimologia , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/patologia , Corpo Vítreo/patologia , Receptor Tirosina Quinase Axl
15.
PLoS One ; 15(12): e0243626, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347461

RESUMO

Proliferative vitreoretinopathy (PVR) is a progressive disease that develops in a subset of patients who undergo surgery for retinal detachment repair, and results in significant vision loss. PVR is characterized by the migration of retinal pigment epithelial (RPE) cells into the vitreous cavity, where they undergo epithelial-to-mesenchymal transition and form contractile membranes within the vitreous and along the retina, resulting in recurrent retinal detachments. Currently, surgical intervention is the only treatment for PVR and there are no pharmacological agents that effectively inhibit or prevent PVR formation. Here, we show that a single intravitreal injection of the polyether ionophore salinomycin (SNC) effectively inhibits the formation of PVR in a mouse model with no evidence of retinal toxicity. After 4 weeks, fundus photography and optical coherence tomography (OCT) demonstrated development of mean PVR grade of 3.5 (SD: 1.3) in mouse eyes injected with RPE cells/DMSO (vehicle), compared to mean PVR grade of 1.6 (SD: 1.3) in eyes injected with RPE cells/SNC (p = 0.001). Additionally, immunohistochemistry analysis showed RPE cells/SNC treatment reduced both fibrotic (αSMA, FN1, Vim) and inflammatory (GFAP, CD3, CD20) markers compared to control RPE cells/DMSO treatment. Finally, qPCR analysis confirmed that Tgfß, Tnfα, Mcp1 (inflammatory/cytokine markers), and Fn1, Col1a1 and Acta2 (fibrotic markers) were significantly attenuated in the RPE cells/SNC group compared to RPE/DMSO control. These results suggest that SNC is a potential pharmacologic agent for the prevention of PVR in humans and warrants further investigation.


Assuntos
Ionóforos/uso terapêutico , Piranos/uso terapêutico , Vitreorretinopatia Proliferativa/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Injeções Intravítreas , Ionóforos/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Piranos/administração & dosagem , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/patologia
16.
Sci Rep ; 10(1): 20554, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33257736

RESUMO

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Vitreorretinopatia Proliferativa , Adulto , Idoso , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Modelos Animais de Doenças , Emulsões , Feminino , Humanos , Masculino , Coelhos , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia
17.
Sci Rep ; 10(1): 15420, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963317

RESUMO

Uncontrolled retinal pigment epithelial (RPE) cell proliferation/migration contribute to the pathological tractional membrane development in proliferative vitreoretinopathy. Recent studies reported that microRNA (miR)-124 controls various cellular functions via the direct targeting of small Ras homolog family member G (RHOG). Therefore, we investigated the role of the neuron-specific miR-124 and RHOG in RPE cell proliferation/migration. Alterations in miR-124 and RhoG expression, as per cell confluence were evaluated through quantitative real-time PCR and western blotting, respectively. After transfection with miR-124, we quantified RPE cell viability and migration and observed cell polarization and lamellipodia protrusions. We evaluated the expression of RHOG/RAC1 pathway molecules in miR-124-transfected RPE cells. Endogenous miR-124 expression increased proportionally to RPE cell density, but decreased after 100% confluence. Overexpression of miR-124 decreased cell viability and migration, BrdU incorporation, and Ki-67 expression. Inhibition of endogenous miR-124 expression promoted RPE cell migration. Transfection with miR-124 reduced cell polarization, lamellipodia protrusion, and RHOG mRNA 3' untranslated region luciferase activity. Like miR-124 overexpression, RhoG knockdown decreased RPE cell viability, wound healing, and migration, and altered the expression of cell cycle regulators. These results suggest that miR-124 could be a therapeutic target to alleviate fibrovascular proliferation in retinal diseases by regulating RPE proliferation/migration via RHOG.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Células Epiteliais/patologia , MicroRNAs/genética , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/genética , Proteínas rho de Ligação ao GTP/genética , Células Cultivadas , Regulação para Baixo/genética , Humanos , Neurônios/patologia , RNA Mensageiro/genética , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/patologia
18.
Mol Vis ; 26: 517-529, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32818015

RESUMO

Purpose: Interleukin-6 (IL-6) is elevated in intraocular fluid from eyes with proliferative vitreoretinopathy (PVR), but the exact role of the cytokine is still unclear. We investigated the function and mechanism of IL-6 in retinal pigment epithelium (RPE) cell biology in vitro and in a mouse model in vivo. Methods: After treatment with various concentrations of IL-6, RPE cell proliferation was assessed with cell counting kit-8 (CCK-8) assay, and epithelial-mesenchymal transition (EMT) markers were evaluated using western blotting and immunofluorescent staining. The activation of JAK1/STAT3 signaling was determined with western blotting. Moreover, the effects of blockade of IL-6/JAK1/STAT3 signaling were investigated using pharmacological inhibitor S3I-201. For in vivo studies, the PVR model was induced with intravitreal injection of dispase/collagenase in wild-type and IL-6 knockout mice. The severity of PVR was evaluated with histological analysis. The expression of IL-6, gp130, and EMT markers was assessed with quantitative real-time PCR and western blotting. Results: IL-6 statistically significantly induced RPE cell proliferation and EMT in a dose-dependent manner in vitro, which was accompanied by rapid phosphorylation of JAK1 and STAT3. Blockade of the IL-6/JAK1/STAT3 pathway with S3I-201 apparently inhibited RPE proliferation and EMT. Furthermore, IL-6 and gp130 overexpression, and JAK1/STAT3 signaling hyperactivation were detected in the retinas of the wild-type mice at 1, 3, and 7 days after dispase/collagenase injection. Finally, we confirmed that IL-6 deficiency markedly alleviated mouse PVR development via inhibiting EMT. Conclusions: These findings indicate that IL-6 promotes PVR by inducing RPE proliferation and EMT via the JAK1/STAT3 signaling pathway. We provided new evidence that therapeutic strategies to block IL-6 may be beneficial for PVR.


Assuntos
Transição Epitelial-Mesenquimal/genética , Interleucina-6/genética , Janus Quinase 1/genética , Epitélio Pigmentado da Retina/metabolismo , Fator de Transcrição STAT3/genética , Vitreorretinopatia Proliferativa/genética , Ácidos Aminossalicílicos/farmacologia , Animais , Benzenossulfonatos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Interleucina-6/deficiência , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia
19.
Ophthalmic Genet ; 41(4): 358-362, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32429730

RESUMO

BACKGROUND: Axenfeld-Rieger syndrome is characterized by a spectrum of anterior segment dysgenesis involving neural-crest-derived tissues, most commonly secondary to mutations in the transcription factor genes PITX2 and FOXC1. MATERIALS AND METHODS: Single retrospective case report. RESULTS: A full-term infant presented at 5 weeks of age with bilateral Peters anomaly and Axenfeld-Rieger syndrome, with development of atypical features of progressive corneal neovascularization and proliferative vitreoretinopathy. Despite surgical interventions, the patient progressed to bilateral phthisis bulbi by 22 months of age. Genetic testing revealed a novel de novo p.Leu212Valfs*39 mutation in PITX2, leading to loss of a C-terminal OAR domain that functions in transcriptional regulation. CONCLUSIONS: It is important to consider mutations in PITX2 in atypical cases of anterior segment dysgenesis that also present with abnormalities in the angiogenesis of the anterior and posterior segments.


Assuntos
Segmento Anterior do Olho/anormalidades , Neovascularização da Córnea/patologia , Anormalidades do Olho/patologia , Oftalmopatias Hereditárias/patologia , Proteínas de Homeodomínio/genética , Mutação , Fatores de Transcrição/genética , Vitreorretinopatia Proliferativa/patologia , Segmento Anterior do Olho/patologia , Neovascularização da Córnea/complicações , Neovascularização da Córnea/genética , Anormalidades do Olho/complicações , Anormalidades do Olho/genética , Oftalmopatias Hereditárias/complicações , Oftalmopatias Hereditárias/genética , Humanos , Lactente , Masculino , Prognóstico , Estudos Retrospectivos , Vitreorretinopatia Proliferativa/complicações , Vitreorretinopatia Proliferativa/genética , Proteína Homeobox PITX2
20.
Eur Rev Med Pharmacol Sci ; 24(9): 4627-4634, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32432726

RESUMO

OBJECTIVE: Proliferative vitreoretinopathy (PVR) is a complex ocular disease that leads to detached retinas and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells plays a critical role in PVR occurrence. However, the core targets driven by the EMT process that lead to the pathogenesis of PVR remain unclear. In our study, the relationship between embryonic stem cell-expressed Ras (ERas) and EMT in RPE cells was investigated. PATIENTS AND METHODS: The subretinal and epiretinal membrane specimens of human PVR were examined for ERas and hallmarks of autophagy and EMT using Western blotting and immunofluorescence. EMT was induced by transforming growth factor (TGF)-ß1 or epidermal growth factor (EGF) in ARPE-19 cells. Autophagy was inhibited by U0126 or bafilomycin A1 in ARPE-19 cells. RESULTS: ERas was decreased and the classical autophagy biomarker microtubule associated protein 1 light chain 3 alpha (LC3) was upregulated in the subretinal and epiretinal membranes of PVR patients in vivo. Moreover, ERas was downregulated and autophagy was activated in RPE ARPE-19 cells in response to transforming growth factor (TGF)-ß1 and epidermal growth factor (EGF) induction. Finally, overexpression of ERas in RPE cells inhibited autophagy via impaired formation of autophagosomes and lysosomes. CONCLUSIONS: Our study revealed the role of ERas in the pathogenesis of PVR through EMT and provided a novel therapeutic target for PVR prevention and treatment.


Assuntos
Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Proteína Oncogênica p21(ras)/metabolismo , Pigmentos da Retina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Adulto , Idoso , Autofagia/fisiologia , Células Cultivadas , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Vitreorretinopatia Proliferativa/patologia
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