RESUMO
Choroidal neovascularization (CNV), a leading cause of blindness in the elderly, is routinely treated with vascular endothelial growth factor (VEGF) inhibitors that have limited efficacy and potentially adverse side effects. An unmet clinical need is to develop novel therapies against other angiogenic factors for alternative or combination treatment to improve efficacy and safety. We recently described secretogranin III (Scg3) as a disease-selective angiogenic factor, causally linked to diabetic retinopathy and acting independently of the VEGF pathway. An important question is whether such a disease-selective Scg3 pathway contributes to other states of pathological angiogenesis beyond diabetic retinopathy. By applying a novel in vivo endothelial ligand binding assay, we found that the binding of Scg3 to CNV vessels in live mice was markedly increased over background binding to healthy choriocapillaris and blocked by an Scg3-neutralizing antibody, whereas VEGF showed no such differential binding. Intravitreal injection of anti-Scg3 humanized antibody Fab (hFab) inhibited Matrigel-induced CNV with similar efficacy to the anti-VEGF drug aflibercept. Importantly, a combination of anti-Scg3 hFab and aflibercept synergistically alleviated CNV. Homozygous deletion of the Scg3 gene markedly reduced CNV severity and abolished the therapeutic activity of anti-Scg3 hFab, but not aflibercept, suggesting a role for Scg3 in VEGF-independent CNV pathogenesis and therapy. Our work demonstrates the stringent disease selectivity of Scg3 binding and positions anti-Scg3 hFab as a next-generation disease-targeted anti-angiogenic therapy for CNV.
Assuntos
Neovascularização de Coroide/metabolismo , Cromograninas/metabolismo , Transdução de Sinais , Animais , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/genética , Cromograninas/genética , Feminino , Fragmentos Fab das Imunoglobulinas/farmacologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP.
Assuntos
Inibidores da Angiogênese/farmacologia , Cromograninas/imunologia , Cromograninas/metabolismo , Neovascularização Patológica/genética , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Animais , Capilares/metabolismo , Cromograninas/antagonistas & inibidores , Cromograninas/genética , Modelos Animais de Doenças , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/efeitos adversos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Neovascularização Retiniana/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Diabetic retinopathy (DR), a leading cause of vision loss in working-age adults, induces mosaic patterns of vasculopathy that may be associated with spatial heterogeneity of intraretinal endothelial cells. We recently reported that secretogranin III (Scg3), a neuron-derived angiogenic and vascular leakage factor, selectively binds retinal vessels of diabetic but not healthy mice. Here, we investigated endothelial heterogeneity of three retinal vascular plexuses in DR pathogenesis and the therapeutic implications. Our unique in vivo ligand binding assay detected a 22.7-fold increase in Scg3 binding to retinal vessels of diabetic mice relative to healthy mice. Functional immunohistochemistry revealed that Scg3 predominantly binds to the DR-stressed CD31- deep retinal vascular plexus but not to the relatively healthy CD31+ superficial and intermediate plexuses within the same diabetic retina. In contrast, VEGF bound to healthy and diabetic retinal vessels indiscriminately with low activity. FITC-dextran assays indicated that selectively increased retinal vascular leakage coincides with Scg3 binding in diabetic mice that was independent of VEGF, whereas VEGF-induced leakage did not distinguish between diabetic and healthy mice. Dose-response curves showed that the anti-Scg3 humanized antibody (hAb) and anti-VEGF aflibercept alleviated DR leakage with equivalent efficacies, and that the combination acted synergistically. These findings suggest: (i) the deep plexus is highly sensitive to DR; (ii) Scg3 binding to the DR deep plexus coincides with the loss of CD31 and compromised endothelial junctions; (iii) anti-Scg3 hAb alleviates vascular leakage by selectively targeting the DR-stressed deep plexus within the same diabetic retina; (iv) combined anti-Scg3 and anti-VEGF treatments synergistically ameliorate DR through distinct mechanisms.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Animais , Camundongos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Células Endoteliais/metabolismo , Diabetes Mellitus Experimental/patologia , Retina/metabolismo , Vasos Retinianos/metabolismoRESUMO
Diabetic retinopathy (DR) is the leading cause of vision loss in working adults in developed countries. The disease traditionally classified as a microvascular complication of diabetes is now widely recognized as a neurovascular disorder resulting from disruption of the retinal neurovascular unit (NVU). The NVU comprising retinal neurons, glia and vascular cells coordinately regulates blood flow, vascular density and permeability to maintain homeostasis. Disturbance of the NVU during DR can lead to vision-threatening clinical manifestations. A limited number of signaling pathways have been identified for intercellular communication within the NVU, including vascular endothelial growth factor (VEGF), the master switch for angiogenesis. VEGF inhibitors are now widely used to treat DR, but their limited efficacy implies that other signaling molecules are involved in the pathogenesis of DR. By applying a novel screening technology called comparative ligandomics, we recently discovered secretogranin III (Scg3) as a unique DR-selective angiogenic and vascular leakage factor with therapeutic potential for DR. This review proposes neuron-derived Scg3 as the first diabetes-selective neurovascular regulator and discusses important features of Scg3 inhibition for next-generation disease-targeted anti-angiogenic therapies of DR.
Assuntos
Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Animais , Cromograninas/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Retina/metabolismo , Retina/patologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. The disease is currently treated with anti-angiogenic biologics, including aflibercept, against vascular endothelial growth factor (VEGF) but with limited efficacy, treatment resistance and requirement for frequent intravitreal injections. Although anti-VEGF gene therapy may provide sustained therapy that obviates multiple injections, the efficacy and side effects related to VEGF pathway targeting remain, and alternative strategies to block angiogenesis independently of VEGF are needed. We recently reported that secretogranin III (Scg3) induces only pathological angiogenesis through VEGF-independent pathways, and Scg3-neutralizing antibodies selectively inhibit pathological but not physiological angiogenesis in mouse proliferative retinopathy models. Anti-Scg3 antibodies synergize dose-dependently with VEGF inhibitors in a CNV model. Here, we report that an adeno-associated virus-8 (AAV8) vector expressing anti-Scg3 Fab ameliorated CNV with an efficacy similar to that of AAV-aflibercept in a mouse model. This study is the first to test an anti-angiogenic gene therapy protocol that selectively targets pathological angiogenesis via a VEGF-independent mechanism. The findings support further safety/efficacy studies of anti-Scg3 gene therapy as monotherapy or combined with anti-VEGF to treat nAMD.
RESUMO
Purpose: The purpose of this study was to investigate the role of lncRNA H19 in epithelial-mesenchymal transition (EMT) and its molecular mechanism in fibrotic cataracts. Methods: TGF-ß2-induced EMT was induced in human lens epithelial cell line (HLECs) and rat lens explants to mimic posterior capsular opacification (PCO) in vitro and in vivo. Anterior subcapsular cataract (ASC) was induced in C57BL/6J mice. The long noncoding RNA (lncRNA) H19 (H19) expression was detected by RT-qPCR. Whole-mount staining of lens anterior capsule was used to detect α-SMA and vimentin. Lentiviruses carrying shRNA or H19 vector were transfected in HLECs to knockdown or overexpress H19. Cell migration and proliferation were characterized by EdU, Transwell, and scratch assay. EMT level was detected by Western blotting and immunofluorescence. The rAAV2 carrying mouse H19 shRNA was injected into ASC model mouse anterior chambers as a gene therapy to determine its therapeutic potential. Results: PCO and ASC models were built successfully. We found H19 upregulation in PCO and ASC models in vivo and in vitro. Overexpression of H19 by lentivirus transfection increased cell migration, proliferation, and EMT. In addition, H19 knockdown by lentivirus suppressed cell migration, proliferation, and EMT levels in HLECs. Moreover, transfection of rAAV2 H19 shRNA alleviated fibrotic area in ASC mouse lens anterior capsules. Conclusions: Excessive H19 participates in lens fibrosis. Overexpression of H19 increases, whereas knockdown of H19 ameliorates HLECs migration, proliferation, and EMT. These results demonstrate H19 might be a potential target for fibrotic cataracts.
Assuntos
Catarata , RNA Longo não Codificante , Animais , Humanos , Camundongos , Ratos , Catarata/genética , Catarata/patologia , Células Epiteliais/metabolismo , Lentivirus , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Fibrose/genética , Fibrose/patologia , Transição Epitelial-Mesenquimal/genéticaRESUMO
Choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. All approved anti-angiogenic drug therapies for CNV target vascular endothelial growth factor (VEGF) but confer limited efficacy. Identification of other CNV-related angiogenic factors will facilitate the development of VEGF-independent alternative therapies. Here, we applied comparative ligandomics to live mice with or without laser-induced CNV for global mapping of CNV-selective endothelial ligands. Secretogranin III (Scg3) previously identified by the same approach as a diabetes-restricted angiogenic factor was mapped with a more than 935-fold increase in binding to CNV vessels compared to healthy choriocapillaris. A novel in vivo ligand binding assay independently confirmed a marked increase in Scg3 binding to CNV vessels, whereas VEGF showed no increase in CNV-selective binding. A new technique of functional immunohistochemistry allowed the visualization and confirmed the increase in in vivo Scg3 binding to CNV vasculatures, including CNV microcapillaries with detailed vascular structures, which was blocked by anti-Scg3 humanized antibody Fab fragment (hFab). The hFab effectively alleviated laser-induced CNV with an efficacy similar to the anti-VEGF drug aflibercept. Homozygous deletion of the Scg3 gene in mice significantly reduced the severity of CNV. Furthermore, the therapeutic activity of anti-Scg3 hFab, but not aflibercept, was abolished in Scg3-/- mice, suggesting the Scg3-dependent nature of the hFab-mediated therapy. These findings suggest that Scg3 plays an important role in CNV pathogenesis and is a promising disease-restricted angiogenic factor for ligand-guided disease-targeted anti-angiogenic therapy of CNV.
Assuntos
Neovascularização de Coroide , Cromograninas , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Cromograninas/metabolismo , Modelos Animais de Doenças , Homozigoto , Lasers , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Deleção de Sequência , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Subsequently to the publication of this paper, the authors have realized that Figs. 2 and 5 have been published containing the same GAPDH control protein bands. After having examined the final proofs of this article, the control blots were indeed different comparing between the figures, and regrettably an error concerning Fig. 2 was made during the final stages of the proof preparation. The corrected version of Fig. 2, including the correct GAPDH protein bands, is shown opposite. Note that the error that occurred with this Figure during production process did not affect the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The Editor of Molecular Medicine Reports apologizes to the authors and to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 19: 927-934, 2019; DOI: 10.3892/mmr.2018.9759].
RESUMO
MATERIALS AND METHODS: LECs were cultured and induced with TGF-ß2 (10 ng/mL). SiRNA against MALAT1 (Si-MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR-204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p. RESULTS: The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-ß2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-ß2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p. CONCLUSIONS: Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by "sponging" miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.
Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica/fisiologia , Cristalino/citologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteína Smad4/genética , Western Blotting , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Vetores Genéticos , Humanos , Hibridização in Situ Fluorescente , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
Autophagy is a major intracellular degradation system that plays an important role in several biological processes. Although some studies indicate that autophagy may play a role in lens degradation and cataracts formation, its underlying mechanism remains to be elucidated. Autophagyrelated gene 4a (ATG4a) cleaves autophagyrelated protein 8 (Atg8) near the C terminus, allowing Atg8 to conjugate with phosphatidylethanolamine via the exposed glycine; although this is pivotal in cancer development, no study has yet linked it to eye diseases. In the present study, the protein expression of ATG4a is significantly upregulated in hydrogen peroxidetreated lens epithelial cells (HLEB3), indicating that ATG4a may play an important role in lens degradation. ATG4a was overexpressed using lentivirus in lens epithelial cells to observe the effect of ATG4a on various phenotypes by transmission electron microscopy, western blotting, EdU incorporation assay, flow cytometry and in situ cell death detection. The results demonstrated that the overexpression of ATG4a could promote autophagy by promoting the adenosine 5'monophosphateactivated protein kinase pathway and inhibiting the Akt pathway. It also upregulated the proliferation and downregulated the apoptosis of lens epithelial cells. Overall, the present study showed that ATG4a plays a vital role in lens degradation and that it could be a potential target in cataract therapies.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proliferação de Células , Cisteína Endopeptidases/metabolismo , Células Epiteliais , Cristalino/citologia , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Catarata/metabolismo , Catarata/patologia , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Purpose: To investigate the functional role of immunoproteasome subunit ß5i in pathologic retinal neovascularization (RNV) and its ability to link the immunoproteasome and autophagy. Methods: Oxygen-induced retinopathy (OIR) was induced in wild-type (WT) and ß5i knockout (KO) mouse pups on a C57BL/6J background. Proteasome catalytic subunit expression and proteasome activity were evaluated by quantitative real-time PCR (qPCR) and proteasome activity. Retinal vascular anatomy and neovascularization were characterized and quantified by retinal vascular flat-mount staining, fluorescence angiography, platelet endothelial cell adhesion molecule (PECAM) immunostaining, and hematoxylin and eosin staining. Correlation factors, including VEGF and ICAM-1, were detected by qPCR. Autophagy was examined by transmission electron microscopy (TEM). Autophagy biomarkers, including LC3, P62, ATG5, and ATG7, were measured by immunostaining and immunoblotting. The protein interaction between ß5i and ATG5 was detected by immunoprecipitation. Results: We observed that ß5i had the greatest effect in WT OIR mice. Fundus fluorescence angiography, retinal flat-mount staining, and PECAM staining revealed that pathologic RNV decreased in ß5i KO OIR mice compared with WT OIR mice. Concurrently, TEM, immunostaining, and immunoblotting showed that autophagy was induced in ß5i KO OIR mice compared to WT OIR mice through increases in autophagosome and LC3 expression and a decrease in P62. Mechanistically, ß5i interacted with ATG5 and promoted its degradation, leading to autophagy inhibition and pathogenic RNV. Conclusions: This study identifies a functional role for ß5i in RNV regulation. ß5i deletion ameliorates RNV and restores autophagy by stabilizing ATG5. These results demonstrate the potential of ß5i to serve as a bridge linking the immunoproteasome and autophagy.
Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Western Blotting , Angiofluoresceinografia , Fundo de Olho , Imunoprecipitação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Neovascularização Retiniana/patologiaRESUMO
Retinal neovascularization (RNV) is a principal cause of visual impairment and blindness worldwide. The present study aimed to investigate how oxidative stress, autophagy and pyroptosis alter in RNV. The oxygeninduced retinopathy (OIR) model was established in C57BL/6J mice by exposing them to a high concentration of oxygen. RNV was clearly visible in the fundus images and was qualitatively analyzed by counting the number of neovascular endothelial cell nuclei at postnatal day 17. Subsequently, the expression of vascular endothelial growth factor (VEGF)A and hypoxiainducible factor1α (HIF1α) at the protein level were measured. Furthermore, oxidative stress was examined using dihydroethidium (DHE) staining, and NADPH oxidase (NOX) 1 and 4 in the retinas were detected using reverse transcriptionquantitative polymerase chain reaction analysis. Additionally, immunostaining of microtubule associated protein 1 light chain 3α (LC3) was performed and the expression levels of the LC3, p62, autophagy protein (Atg)5, Atg7, Atg12, Beclin1, NODlike receptor family pyrin domaincontaining 3 (NLRP3), caspase1, interleukin (IL)1ß, procaspase1 and proIL1ß proteins were determined using western blotting in order to detect pyroptosis and autophagic flux. Autophagosomes were also detected using transmission electron microscopy. The results revealed that VEGFA and HIF1α protein expression levels, the DHEpositive area, and NOX1 and NOX4 mRNA expression levels were significantly increased in the OIR mice. Furthermore, increased levels of NLRP3, caspase1, IL1ß, procaspase1 and proIL1ß proteins demonstrated that pyroptosis was activated. However, an accumulation of p62 and a reduction in the levels of LC3II/I and autophagosomes indicated that autophagic flux was compromised. Therefore, elevated levels of reactive oxygen species and pyroptosis along with attenuated autophagy were demonstrated in the OIR mice. The combination of oxidative stress, pyroptosis and impaired autophagy may serve an important role in the pathophysiology of RNV and may be a potential target to prevent RNV.