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1.
J Biol Chem ; 286(52): 44965-75, 2011 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-22072717

RESUMO

Pathological neovascularization occurs when a balance of pro- and anti-angiogenic factors is disrupted, accompanied by an amplifying inflammatory cascade. However, the interdependence of these responses and the mechanism triggering the initial angiogenic switch have remained unclear. We present data from an epithelial debridement model of corneal neovascularization describing an initial 3-day period when a substantial component of neovascular growth occurs. Administration of selective inhibitors shows that this initial growth requires signaling through VEGFR-2 (vascular endothelial growth factor receptor-2), independent of the accompanying inflammatory response. Instead, increased VEGF production is found prominently in repair epithelial cells and is increased prior to recruitment of neutrophil/granulocytes and macrophage/monocytes. Consequently, early granulocyte and monocyte depletion has little effect on corneal neovascularization outgrowth. These data indicate that it is possible to pharmacologically uncouple these mechanisms during early injury-driven neovascularization in the cornea and suggest that initial tissue responses are coordinated by repair epithelial cells.


Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/metabolismo , Epitélio/metabolismo , Animais , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/patologia , Epitélio/patologia , Feminino , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Monócitos/metabolismo , Monócitos/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
2.
J Med Chem ; 61(4): 1622-1635, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29400470

RESUMO

A noninvasive topical ocular therapy for the treatment of neovascular or "wet" age-related macular degeneration would provide a patient administered alternative to the current standard of care, which requires physician administered intravitreal injections. This manuscript describes a novel strategy for the use of in vivo models of choroidal neovascularization (CNV) as the primary means of developing SAR related to efficacy from topical administration. Ultimately, this effort led to the discovery of acrizanib (LHA510), a small-molecule VEGFR-2 inhibitor with potency and efficacy in rodent CNV models, limited systemic exposure after topical ocular administration, multiple formulation options, and an acceptable rabbit ocular PK profile.


Assuntos
Administração Tópica , Indóis/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Degeneração Macular Exsudativa/tratamento farmacológico , Animais , Neovascularização de Coroide , Descoberta de Drogas , Indóis/farmacocinética , Indóis/uso terapêutico , Soluções Oftálmicas , Inibidores de Proteínas Quinases , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Coelhos , Roedores , Relação Estrutura-Atividade
3.
J Med Chem ; 58(23): 9273-86, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26568411

RESUMO

The benefit of intravitreal anti-VEGF therapy in treating wet age-related macular degeneration (AMD) is well established. Identification of VEGFR-2 inhibitors with optimal ADME properties for an ocular indication provides opportunities for dosing routes beyond intravitreal injection. We employed a high-throughput in vivo screening strategy with rodent models of choroidal neovascularization and iterative compound design to identify VEGFR-2 inhibitors with potential to benefit wet AMD patients. These compounds demonstrate preferential ocular tissue distribution and efficacy after oral administration while minimizing systemic exposure.


Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Degeneração Macular Exsudativa/tratamento farmacológico , Administração Oral , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacocinética , Animais , Corioide/efeitos dos fármacos , Corioide/patologia , Neovascularização de Coroide/patologia , Feminino , Humanos , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Ratos , Degeneração Macular Exsudativa/patologia
4.
Invest Ophthalmol Vis Sci ; 55(10): 6525-34, 2014 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-25205860

RESUMO

PURPOSE: We attempted to reproduce published studies that evaluated whether the following factors influence choroidal neovascularization (CNV) induced by laser photocoagulation in murine retinas: small interfering RNA (siRNA), cobra venom factor, complement factors C3 and C5, and complement receptor C5aR. In addition, we explored whether laser-induced CNV in mice was influenced by the vendor of origin of the animals. METHODS: Reagents or genotypes reported by others to influence CNV in this model were assessed using our standard procedures. Retrospective analyses of control or placebo mice in many experiments were done to evaluate whether the CNV area induced by laser photocoagulation varied according to vendor. RESULTS: Administration of the following agents did not have a substantial impact on the CNV induced by laser burns in mice: siRNA, low-molecular-weight inhibitor of the C5a receptor (PMX53), or cobra venom factor. Jackson Laboratory (JAX) mice lacking either C3 or C5 had increased neovascularization compared to non-littermate JAX wild-type controls. Taconic mice lacking C3 had reduced CNV compared to non-littermate Taconic wild-type control mice. A retrospective analysis of vehicle-treated wild-type C57BL/6 mice used as controls across 132 experiments conducted from 2007 to 2010 revealed that mice purchased from JAX or from Charles River produced less neovascularization than mice from Taconic. CONCLUSIONS: We present our recommended methods for conducting experiments with the mouse laser-induced CNV model to enhance reproducibility and minimize investigator bias.


Assuntos
Neovascularização de Coroide/patologia , Fotocoagulação a Laser/efeitos adversos , Epitélio Pigmentado da Retina/patologia , Animais , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/genética , Modelos Animais de Doenças , Feminino , Angiofluoresceinografia , Fundo de Olho , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Epitélio Pigmentado da Retina/metabolismo , Estudos Retrospectivos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
PLoS One ; 9(10): e111472, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25343517

RESUMO

Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others.


Assuntos
Neovascularização Patológica/metabolismo , Pirróis/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Lasers , Leucócitos/metabolismo , Camundongos Endogâmicos C57BL , Retina/metabolismo , Retina/patologia , Receptor 2 Toll-Like/agonistas
6.
Cell Stem Cell ; 7(1): 127-33, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20621056

RESUMO

Successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor. Therapeutic advances therefore necessitate the characterization of the cells that are able to propagate a tumor in vivo. We show an important link between tumor genotype and isolation of tumor-propagating cells (TPCs). Three mouse models of the most common form of human lung cancer each had TPCs with a unique cell-surface phenotype. The cell-surface marker Sca1 did not enrich for TPCs in tumors initiated with oncogenic Kras, and only Sca1-negative cells propagated EGFR mutant tumors. In contrast, Sca1-positive cells were enriched for tumor-propagating activity in Kras tumors with p53 deficiency. Primary tumors that differ in genotype at just one locus can therefore have tumor-propagating cell populations with distinct markers. Our studies show that the genotype of tumor samples must be considered in studies to identify, characterize, and target tumor-propagating cells.


Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Animais , Citometria de Fluxo , Genótipo , Técnicas In Vitro , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Modelos Biológicos , Células Tumorais Cultivadas
7.
Cell ; 121(6): 823-35, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15960971

RESUMO

Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). Identified at the bronchioalveolar duct junction, BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties. Furthermore, BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo. These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma. Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma, this work points to BASCs as the putative cells of origin for this subtype of lung cancer.


Assuntos
Adenocarcinoma Bronquioloalveolar/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Pulmonares/patologia , Alvéolos Pulmonares/patologia , Células-Tronco/patologia , Adenocarcinoma Bronquioloalveolar/metabolismo , Animais , Carcinógenos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Genes ras/fisiologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos , Alvéolos Pulmonares/efeitos dos fármacos , Proteína C Associada a Surfactante Pulmonar/metabolismo , Células-Tronco/metabolismo , Uteroglobina/metabolismo
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