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1.
Arterioscler Thromb Vasc Biol ; 39(10): 2120-2131, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31434494

RESUMO

OBJECTIVE: Aberrant neovascularization is a leading cause of blindness in several eye diseases, including age-related macular degeneration and proliferative diabetic retinopathy. The identification of key regulators of pathological ocular neovascularization has been a subject of extensive research and great therapeutic interest. Here, we explored the previously unrecognized role of cKIT and its ligand, SCF (stem cell factor), in the pathological ocular neovascularization process. Approach and Results: Compared with normoxia, hypoxia, a crucial driver of neovascularization, caused cKIT to be highly upregulated in endothelial cells, which significantly enhanced the angiogenic response of endothelial cells to SCF. In murine models of pathological ocular neovascularization, such as oxygen-induced retinopathy and laser-induced choroidal neovascularization models, cKIT and SCF expression was significantly increased in ocular tissues, and blockade of cKIT and SCF using cKit mutant mice and anti-SCF neutralizing IgG substantially suppressed pathological ocular neovascularization. Mechanistically, SCF/cKIT signaling induced neovascularization through phosphorylation of glycogen synthase kinase-3ß and enhancement of the nuclear translocation of ß-catenin and the transcription of ß-catenin target genes related to angiogenesis. Inhibition of ß-catenin-mediated transcription using chemical inhibitors blocked SCF-induced in vitro angiogenesis in hypoxia, and injection of a ß-catenin agonist into cKit mutant mice with oxygen-induced retinopathy significantly enhanced pathological neovascularization in the retina. Conclusions; Our data reveal that SCF and cKIT are promising novel therapeutic targets for treating vision-threatening ocular neovascular diseases.


Assuntos
Regulação da Expressão Gênica , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Neovascularização Retiniana/genética , Fator de Células-Tronco/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Inibidores da Angiogênese/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipóxia/complicações , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/genética , Doenças Retinianas/patologia , Doenças Retinianas/fisiopatologia , Transdução de Sinais/genética
2.
Mol Vis ; 23: 823-831, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29225458

RESUMO

Purpose: Vascular endothelial growth factor (VEGF) is a principal mediator of pathological ocular neovascularization, which is the leading cause of blindness in various ocular diseases. As Src, a non-receptor tyrosine kinase, has been implicated as one of the major signaling molecules in VEGF-mediated neovascularization, the present study aimed to investigate whether dasatinib, a potent Src kinase inhibitor, could suppress pathological ocular neovascularization in murine models of oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). Methods: Tube formation, scratch wounding migration, and cell proliferation assays were performed to measure the inhibitory effect of dasatinib on VEGF-induced angiogenesis in human retinal microvascular endothelial cells. Murine models of OIR and laser-induced CNV were used to assess the preventive effect of an intravitreal injection of dasatinib on pathological neovascularization in the retina and choroid. Neovascularization and Src phosphorylation were evaluated with immunofluorescence staining. Results: Dasatinib efficiently inhibited VEGF-induced endothelial proliferation, wounding migration, and tube formation. In mice with OIR and laser injury-induced CNV, eyes treated with a single intravitreal injection of dasatinib exhibited significant decreases in pathological neovascularization compared with that of controls injected with vehicle. The dasatinib-treated OIR mice also showed a decrease in Src phosphorylation in the periretinal tufts. The intravitreal injection of dasatinib did not cause ocular toxicity at the treatment dose administered. Conclusions: These results demonstrated that dasatinib suppressed pathological neovascularization in the mouse retina and choroid. Therefore, dasatinib may be indicated for the treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Dasatinibe/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Feminino , Angiofluoresceinografia , Humanos , Marcação In Situ das Extremidades Cortadas , Injeções Intravítreas , Fotocoagulação a Laser/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Fosforilação , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/metabolismo , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular/efeitos adversos
3.
J Med Chem ; 64(9): 5535-5550, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33902285

RESUMO

Diabetic retinopathy is the leading cause of blindness which is associated with excessive angiogenesis. Using the structure of wondonin marine natural products, we previously created a scaffold to develop a novel type of antiangiogenesis agent that possesses minimized cytotoxicity. To overcome its poor pharmaceutical properties, we further modified the structure. A new scaffold was derived in which the stereogenic carbon was changed to nitrogen and the 1,2,3-triazole ring was replaced by an alkyl chain. By comparing the bioactivity versus cytotoxicity, compound 31 was selected, which has improved aqueous solubility and an enhanced selectivity index. Mechanistically, 31 suppressed angiopoietin-2 (ANGPT2) expression induced by high glucose in retinal cells and exhibited in vivo antiangiogenic activity in choroidal neovascularization and oxygen-induced retinopathy mouse models. These results suggest the potential of 31 as a lead to develop antiangiogenic small-molecule drugs to treat diabetic retinopathy and as a chemical tool to elucidate new mechanisms of angiogenesis.


Assuntos
Inibidores da Angiogênese/farmacologia , Desenho de Fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/uso terapêutico , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Estabilidade de Medicamentos , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêutico
4.
Pharmaceutics ; 13(8)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34452268

RESUMO

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

5.
Exp Mol Med ; 52(10): 1744-1753, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33051573

RESUMO

Dipeptidyl peptidase-4 (DPP-4) inhibitors are used for the treatment of type 2 diabetes mellitus (DM). Recent studies have shown that beyond their effect in lowing glucose, DPP-4 inhibitors mitigate DM-related microvascular complications, such as diabetic retinopathy. However, the mechanism by which pathological retinal neovascularization, a major clinical manifestation of diabetic retinopathy, is inhibited is unclear. This study sought to examine the effects of evogliptin, a potent DPP-4 inhibitor, on pathological retinal neovascularization in mice and elucidate the mechanism by which evogliptin inhibits angiogenesis mediated by vascular endothelial growth factor (VEGF), a key factor in the vascular pathogenesis of proliferative diabetic retinopathy (PDR). In a murine model of PDR, an intravitreal injection of evogliptin significantly suppressed aberrant retinal neovascularization. In human endothelial cells, evogliptin reduced VEGF-induced angiogenesis. Western blot analysis showed that evogliptin inhibited the phosphorylation of signaling molecules associated with VEGF-induced cell adhesion and migration. Moreover, evogliptin substantially inhibited the VEGF-induced activation of adenosine 5'-diphosphate ribosylation factor 6 (Arf6), a small guanosine 5'-triphosphatase (GTPase) that regulates VEGF receptor 2 signal transduction. Direct activation of Arf6 using a chemical inhibitor of Arf-directed GTPase-activating protein completely abrogated the inhibitory effect of evogliptin on VEGF-induced activation of the angiogenic signaling pathway, which suggests that evogliptin suppresses VEGF-induced angiogenesis by blocking Arf6 activation. Our results provide insights into the molecular mechanism of the direct inhibitory effect of the DPP-4 inhibitor evogliptin on pathological retinal neovascularization. In addition to its glucose-lowering effect, the antiangiogenic effect of evogliptin could also render it beneficial for individuals with PDR.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Piperazinas/farmacologia , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Inibidores da Angiogênese/farmacologia , Animais , Modelos Animais de Doenças , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Fosforilação , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Transdução de Sinais/efeitos dos fármacos
6.
Hypertension ; 76(6): 1778-1786, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33100045

RESUMO

Loss of BMP (bone morphogenic protein) signaling induces a phenotype switch of pulmonary arterial smooth muscle cells (PASMCs), which is the pathological basis of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Here, we identified FGF12 (fibroblast growth factor 12) as a novel regulator of the BMP-induced phenotype change in PASMCs and elucidated its role in pulmonary vascular remodeling during PAH development. Using murine models of PAH and lung specimens of patients with PAH, we observed that FGF12 expression was significantly reduced in PASMCs. In human PASMCs, FGF12 expression was increased by canonical BMP signaling. FGF12 knockdown blocked the antiproliferative and prodifferentiation effect of BMP on human PASMCs, suggesting that FGF12 is required for the BMP-mediated acquisition of the quiescent and differentiated PASMC phenotype. Mechanistically, FGF12 regulated the BMP-induced phenotype change by inducing MEF2a (myocyte enhancer factor 2a) phosphorylation via p38MAPK signaling, thereby modulating the expression of MEF2a target genes involved in cell proliferation and differentiation. Furthermore, we observed that TG (transgenic) mice with smooth muscle cell-specific FGF12 overexpression were protected from chronic hypoxia-induced PAH development, pulmonary vascular remodeling, and right ventricular hypertrophy. Consistent with the in vitro data using human PASMCs, FGF12 TG mice showed increased MEF2a phosphorylation and a substantial change in MEF2a target gene expression, compared with the WT (wild type) controls. Overall, our findings demonstrate a novel BMP/FGF12/MEF2a pathway regulating the PASMC phenotype switch and suggest FGF12 as a potential target for the development of therapeutics for ameliorating pulmonary vascular remodeling in PAH.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Remodelação Vascular/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células/genética , Células Cultivadas , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley
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