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1.
J Neurochem ; 153(3): 390-412, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31550048

RESUMO

Retinal hypoxia triggers abnormal vessel growth and microvascular hyper-permeability in ischemic retinopathies. Whereas vascular endothelial growth factor A (VEGF-A) inhibitors significantly hinder disease progression, their benefits to retinal neurons remain poorly understood. Similar to humans, oxygen-induced retinopathy (OIR) mice exhibit severe retinal microvascular malformations and profound neuronal dysfunction. OIR mice are thus a phenocopy of human retinopathy of prematurity, and a proxy for investigating advanced stages of proliferative diabetic retinopathy. Hence, the OIR model offers an excellent platform for assessing morpho-functional responses of the ischemic retina to anti-angiogenic therapies. Using this model, we investigated the retinal responses to VEGF-Trap (Aflibercept), an anti-angiogenic agent recognizing ligands of VEGF receptors 1 and 2 that possesses regulatory approval for the treatment of neovascular age-related macular degeneration, macular edema secondary to retinal vein occlusion and diabetic macular edema. Our results indicate that Aflibercept not only reduces the severity of retinal microvascular aberrations but also significantly improves neuroretinal function. Aflibercept administration significantly enhanced light-responsiveness, as revealed by electroretinographic examinations, and led to increased numbers of dopaminergic amacrine cells. Additionally, retinal transcriptional profiling revealed the concerted regulation of both angiogenic and neuronal targets, including transcripts encoding subunits of transmitter receptors relevant to amacrine cell function. Thus, Aflibercept represents a promising therapeutic alternative for the treatment of further progressive ischemic retinal neurovasculopathies beyond the set of disease conditions for which it has regulatory approval. Cover Image for this issue: doi: 10.1111/jnc.14743.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Degeneração Retiniana/tratamento farmacológico , Vasos Retinianos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Neurônios Dopaminérgicos/patologia , Feminino , Isquemia/tratamento farmacológico , Isquemia/patologia , Masculino , Camundongos , Microvasos/patologia , Rede Nervosa/patologia , Proteínas Recombinantes de Fusão/farmacologia , Degeneração Retiniana/patologia , Vasos Retinianos/patologia , Sistema Vasomotor/efeitos dos fármacos , Sistema Vasomotor/patologia
2.
J Cell Mol Med ; 23(4): 2362-2371, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30680928

RESUMO

The mechanism underlying vasoproliferative retinopathies like retinopathy of prematurity (ROP) is hypoxia-triggered neovascularisation. Nerve growth factor (NGF), a neurotrophin supporting survival and differentiation of neuronal cells may also regulate endothelial cell functions. Here we studied the role of NGF in pathological retinal angiogenesis in the course of the ROP mouse model. Topical application of NGF enhanced while intraocular injections of anti-NGF neutralizing antibody reduced pathological retinal vascularization in mice subjected to the ROP model. The pro-angiogenic effect of NGF in the retina was mediated by inhibition of retinal endothelial cell apoptosis. In vitro, NGF decreased the intrinsic (mitochondria-dependent) apoptosis in hypoxia-treated human retinal microvascular endothelial cells and preserved the mitochondrial membrane potential. The anti-apoptotic effect of NGF was associated with increased BCL2 and reduced BAX, as well as with enhanced ERK and AKT phosphorylation, and was abolished by inhibition of the AKT pathway. Our findings reveal an anti-apoptotic role of NGF in the hypoxic retinal endothelium, which is involved in promoting pathological retinal vascularization, thereby pointing to NGF as a potential target for proliferative retinopathies.


Assuntos
Anticorpos Neutralizantes/farmacologia , Neovascularização Patológica/terapia , Fator de Crescimento Neural/antagonistas & inibidores , Retinopatia da Prematuridade/terapia , Apoptose/efeitos dos fármacos , Células Endoteliais , Humanos , Injeções Intraoculares , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fator de Crescimento Neural/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Retina/efeitos dos fármacos , Retina/patologia , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/patologia , Proteína X Associada a bcl-2/genética
3.
Thromb Haemost ; 119(3): 439-448, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30620991

RESUMO

The replication stress inflicted on retinal endothelial cells (ECs) in the context of hypoxia-induced pathological neovascularization during proliferative retinopathy is linked with activation of the deoxyribonucleic acid (DNA) repair response. Here, we studied the effect of deficiency of the DNA damage response adaptor 53BP1, which is an antagonist of homologous recombination (HR), in the context of proliferative retinopathy. In the model of retinopathy of prematurity (ROP), 53BP1-deficient mice displayed increased hypoxia-driven pathological neovascularization and tuft formation, accompanied by increased EC proliferation and reduced EC apoptosis, as compared with 53BP1-sufficient mice. In contrast, physiological retina angiogenesis was not affected by 53BP1 deficiency. Knockdown of 53BP1 in ECs in vitro also resulted in enhanced proliferation and reduced apoptosis of the cells under hypoxic conditions. Additionally, upon 53BP1 knockdown, ECs displayed increased HR rate in hypoxia. Consistently, treatment with an HR inhibitor reversed the hyper-proliferative angiogenic phenotype associated with 53BP1 deficiency in ROP. Thus, by unleashing HR, 53BP1 deletion increases pathological EC proliferation and neovascularization in the context of ROP. Our data shed light to a previously unknown interaction between the DNA repair response and pathological neovascularization in the retina.


Assuntos
Proliferação de Células , Células Endoteliais/metabolismo , Recombinação Homóloga , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Retinopatia da Prematuridade/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Predisposição Genética para Doença , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Knockout , Morfolinas/farmacologia , Fenótipo , Pirróis/farmacologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/prevenção & controle , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
5.
J Clin Invest ; 127(10): 3624-3639, 2017 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-28846069

RESUMO

Hematopoietic stem cells (HSCs) remain mostly quiescent under steady-state conditions but switch to a proliferative state following hematopoietic stress, e.g., bone marrow (BM) injury, transplantation, or systemic infection and inflammation. The homeostatic balance between quiescence, self-renewal, and differentiation of HSCs is strongly dependent on their interactions with cells that constitute a specialized microanatomical environment in the BM known as the HSC niche. Here, we identified the secreted extracellular matrix protein Del-1 as a component and regulator of the HSC niche. Specifically, we found that Del-1 was expressed by several cellular components of the HSC niche, including arteriolar endothelial cells, CXCL12-abundant reticular (CAR) cells, and cells of the osteoblastic lineage. Del-1 promoted critical functions of the HSC niche, as it regulated long-term HSC (LT-HSC) proliferation and differentiation toward the myeloid lineage. Del-1 deficiency in mice resulted in reduced LT-HSC proliferation and infringed preferentially upon myelopoiesis under both steady-state and stressful conditions, such as hematopoietic cell transplantation and G-CSF- or inflammation-induced stress myelopoiesis. Del-1-induced HSC proliferation and myeloid lineage commitment were mediated by ß3 integrin on hematopoietic progenitors. This hitherto unknown Del-1 function in the HSC niche represents a juxtacrine homeostatic adaptation of the hematopoietic system in stress myelopoiesis.


Assuntos
Proteínas de Transporte/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mielopoese , Nicho de Células-Tronco , Estresse Fisiológico , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Moléculas de Adesão Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliais/metabolismo , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout
6.
Nat Immunol ; 18(6): 654-664, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28414311

RESUMO

In obesity, inflammation of white adipose tissue (AT) is associated with diminished generation of beige adipocytes ('beige adipogenesis'), a thermogenic and energy-dissipating function mediated by beige adipocytes that express the uncoupling protein UCP1. Here we delineated an inflammation-driven inhibitory mechanism of beige adipogenesis in obesity that required direct adhesive interactions between macrophages and adipocytes mediated by the integrin α4 and its counter-receptor VCAM-1, respectively; expression of the latter was upregulated in obesity. This adhesive interaction reciprocally and concomitantly modulated inflammatory activation of macrophages and downregulation of UCP1 expression dependent on the kinase Erk in adipocytes. Genetic or pharmacological inactivation of the integrin α4 in mice resulted in elevated expression of UCP1 and beige adipogenesis of subcutaneous AT in obesity. Our findings, established in both mouse systems and human systems, reveal a self-sustained cycle of inflammation-driven impairment of beige adipogenesis in obesity.


Assuntos
Adipócitos Bege , Adipogenia/imunologia , Tecido Adiposo Branco/imunologia , Diferenciação Celular/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Células 3T3-L1 , Adipócitos/imunologia , Adipócitos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Adesão Celular/imunologia , Dieta Hiperlipídica , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação , Feminino , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Integrina alfa4/genética , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Monócitos/imunologia , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Gordura Subcutânea , Linfócitos T/imunologia , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adulto Jovem
7.
Thromb Haemost ; 117(6): 1150-1163, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28447099

RESUMO

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of ß2-integrin-dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1-deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1-proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted ß2-integrin-dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the ß2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1-dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.


Assuntos
Proteínas de Transporte/metabolismo , Endotélio Vascular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Inflamação/metabolismo , Isquemia/metabolismo , Leucócitos/fisiologia , Retinopatia da Prematuridade/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Adesão Celular , Moléculas de Adesão Celular , Movimento Celular , Modelos Animais de Doenças , Extremidades/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Isquemia/imunologia , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , RNA Interferente Pequeno/genética , Retinopatia da Prematuridade/imunologia
9.
Mol Cell Biol ; 36(3): 376-93, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26572826

RESUMO

Angiogenesis is a central regulator for white (WAT) and brown (BAT) adipose tissue adaptation in the course of obesity. Here we show that deletion of hypoxia-inducible factor 2α (HIF2α) in adipocytes (by using Fabp4-Cre transgenic mice) but not in myeloid or endothelial cells negatively impacted WAT angiogenesis and promoted WAT inflammation, WAT dysfunction, hepatosteatosis, and systemic insulin resistance in obesity. Importantly, adipocyte HIF2α regulated vascular endothelial growth factor (VEGF) expression and angiogenesis of obese BAT as well as its thermogenic function. Consistently, obese adipocyte-specific HIF2α-deficient mice displayed BAT dysregulation, associated with reduced levels of uncoupling protein 1 (UCP1) and a dysfunctional thermogenic response to cold exposure. VEGF administration reversed WAT and BAT inflammation and BAT dysfunction in adipocyte HIF2α-deficient mice. Together, our findings show that adipocyte HIF2α is protective against maladaptation to obesity and metabolic dysregulation by promoting angiogenesis in both WAT and BAT and by counteracting obesity-mediated BAT dysfunction.


Assuntos
Adipócitos/patologia , Tecido Adiposo Marrom/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Deleção de Genes , Obesidade/genética , Obesidade/fisiopatologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/irrigação sanguínea , Tecido Adiposo Marrom/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Inflamação/complicações , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Neovascularização Fisiológica , Obesidade/complicações , Obesidade/metabolismo , Termogênese , Proteína Desacopladora 1 , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Thromb Haemost ; 114(6): 1241-9, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26311310

RESUMO

In proliferative retinopathies, like proliferative diabetic retinopathy and retinopathy of prematurity (ROP), the hypoxia response is sustained by the failure of the retina to revascularise its ischaemic areas. Non-resolving retina ischaemia/hypoxia results in upregulation of pro-angiogenic factors and pathologic neovascularisation with ectopic, fragile neovessels. Promoting revascularisation of the retinal avascular area could interfere with this vicious cycle and lead to vessel normalisation. Here, we examined the function of endothelial junctional adhesion molecule-C (JAM-C) in the context of ROP. Endothelial-specific JAM-C-deficient (EC-JAM-C KO) mice and littermate JAM-C-proficient (EC-JAM-C WT) mice were subjected to the ROP model. An increase in total retinal vascularisation was found at p17 owing to endothelial JAM-C deficiency, which was the result of enhanced revascularisation and vessel normalisation, thereby leading to significantly reduced avascular area in EC-JAM-C KO mice. In contrast, pathologic neovessel formation was not affected by endothelial JAM-C deficiency. Consistent with improved vessel normalisation, tip cell formation at the interface between vascular and avascular area was higher in EC-JAM-C KO mice, as compared to their littermate controls. Consistently, JAM-C inactivation in endothelial cells resulted in increased spreading on fibronectin and enhanced sprouting in vitro in a manner dependent on ß1-integrin and on the activation of the small GTPase RAP1. Together, endothelial deletion of JAM-C promoted endothelial cell sprouting, and consequently vessel normalisation and revascularisation of the hypoxic retina without altering pathologic neovascularisation. Thus, targeting endothelial JAM-C may provide a novel therapeutic strategy for promoting revascularisation and vessel normalisation in the treatment of proliferative retinopathies.


Assuntos
Endotélio Vascular/fisiopatologia , Molécula C de Adesão Juncional/deficiência , Neovascularização Patológica/fisiopatologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/fisiopatologia , Vitreorretinopatia Proliferativa/fisiopatologia , Animais , Adesão Celular , Hipóxia Celular , Linhagem Celular , Tamanho Celular , Extensões da Superfície Celular , Modelos Animais de Doenças , Células Endoteliais , Endotélio Vascular/patologia , Fibronectinas , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta1/fisiologia , Isquemia/fisiopatologia , Molécula C de Adesão Juncional/fisiologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/etiologia , Especificidade de Órgãos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Interferência de RNA , RNA Interferente Pequeno/genética , Vasos Retinianos/ultraestrutura , Proteínas rap1 de Ligação ao GTP/fisiologia
11.
Arthritis Rheumatol ; 67(12): 3279-85, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26245636

RESUMO

OBJECTIVE: Endothelial cell activation by tumor necrosis factor (TNF) and associated leukocyte infiltration are hallmarks of vasculitis. The aim of this study was to investigate the potential role of the cellular stress-associated endothelial X-box binding protein 1 (XBP-1) transcription factor in TNF-induced endothelial cell inflammation and vasculitis. METHODS: Mice with an endothelial cell-specific XBP-1 deficiency were used in a modified local Shwartzman reaction (LSR) model of TNF-induced small vessel vasculitis. To address the contribution of XBP-1 to the TNF-mediated inflammatory response in endothelial cells, we examined the activation of XBP-1 expression by TNF as well as the effect of XBP-1 knockdown in endothelial cells on TNF-induced signaling, proinflammatory gene expression, and leukocyte-endothelial cell adhesion. RESULTS: The active spliced form of XBP-1 in endothelial cells was triggered by TNF. In addition, endothelial XBP-1 contributed to the sustained TNF-triggered NF-κB-dependent transcriptional activation of proinflammatory molecules, which was associated with leukocyte-endothelial cell adhesion. In the LSR model, endothelial cell-specific XBP-1-deficient mice displayed significantly less vascular damage, accompanied by reduced perivascular neutrophil infiltration, as compared with wild-type mice. CONCLUSION: Endothelial XBP-1 is activated by TNF and regulates leukocyte-endothelial cell adhesion in vitro as well as neutrophil infiltration and vascular damage in murine vasculitis.


Assuntos
Adesão Celular/genética , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , NF-kappa B/metabolismo , Infiltração de Neutrófilos/genética , Fenômeno de Shwartzman/genética , Fatores de Transcrição/genética , Vasculite/genética , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Fatores de Transcrição de Fator Regulador X , Fenômeno de Shwartzman/imunologia , Fatores de Transcrição/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/imunologia , Proteína 1 de Ligação a X-Box
12.
Mol Psychiatry ; 20(7): 880-888, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25385367

RESUMO

Inflammation in the central nervous system (CNS) and disruption of its immune privilege are major contributors to the pathogenesis of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor, which inhibits integrin-dependent leukocyte adhesion. Here we show that Del-1 contributes to the immune privilege status of the CNS. Intriguingly, Del-1 expression decreased in chronic-active MS lesions and in the inflamed CNS in the course of EAE. Del-1-deficiency was associated with increased EAE severity, accompanied by increased demyelination and axonal loss. As compared with control mice, Del-1(-/-) mice displayed enhanced disruption of the blood-brain barrier and increased infiltration of neutrophil granulocytes in the spinal cord in the course of EAE, accompanied by elevated levels of inflammatory cytokines, including interleukin-17 (IL-17). The augmented levels of IL-17 in Del-1-deficiency derived predominantly from infiltrated CD8(+) T cells. Increased EAE severity and neutrophil infiltration because of Del-1-deficiency was reversed in mice lacking both Del-1 and IL-17 receptor, indicating a crucial role for the IL-17/neutrophil inflammatory axis in EAE pathogenesis in Del-1(-/-) mice. Strikingly, systemic administration of Del-1-Fc ameliorated clinical relapse in relapsing-remitting EAE. Therefore, Del-1 is an endogenous homeostatic factor in the CNS protecting from neuroinflammation and demyelination. Our findings provide mechanistic underpinnings for the previous implication of Del-1 as a candidate MS susceptibility gene and suggest that Del-1-centered therapeutic approaches may be beneficial in neuroinflammatory and demyelinating disorders.


Assuntos
Axônios/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte/metabolismo , Bainha de Mielina/metabolismo , Neuroimunomodulação/fisiologia , Medula Espinal/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proteínas de Ligação ao Cálcio , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Proteínas de Transporte/genética , Moléculas de Adesão Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Granulócitos/patologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Neuroimunomodulação/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Índice de Gravidade de Doença , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia
13.
Int J Cancer ; 135(9): 2054-64, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24676840

RESUMO

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine-producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC-associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX-mediated mechanism and opposing influences of hypoxia-inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene-manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max-mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re-expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX-related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.


Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Células Cromafins/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Paraganglioma/patologia , Feocromocitoma/patologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/genética , Western Blotting , Ciclo Celular , Diferenciação Celular , Células Cromafins/metabolismo , Perfilação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mutação/genética , Paraganglioma/genética , Paraganglioma/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
14.
Int J Radiat Biol ; 90(8): 700-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24512568

RESUMO

PURPOSE: In this work we examined the presence of the neural stem cell biomarker Hairy and Enhancer of Split 3 (Hes3) in the anterior eye segment and in the aberrant growth condition of the conjunctiva pterygium. Further, we studied the response of Hes3 to irradiation. MATERIALS AND METHODS: Adult mouse and human corneoscleral junction and conjunctiva, as well as human pterygium were prepared for immunohistochemical detection of Hes3 and other markers. Total body irradiation was used to study the changes in the pattern of Hes3 expression. RESULTS: The adult rodent and human eye as well as pterygium, contain a population of cells expressing Hes3. In the human eye, Hes3-expressing (Hes3+) cells are found predominantly in the subconjunctival space spanning over the limbus where they physically associate with blood vessels. The cytoarchitecture of Hes3 + cells is similar to those previously observed in the adult central nervous system. Furthermore, irradiation reduces the number of Hes3 + cells in the subconjunctival space. In contrast, irradiation strongly promotes the nuclear localization of Hes3 in the ciliary body epithelium. CONCLUSIONS: Our results suggest that a recently identified signal transduction pathway that regulates neural stem cells and glioblastoma cancer stem cells also operates in the ocular surface, ciliary body, and in pterygium.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Olho/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Pterígio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos da radiação , Olho/irrigação sanguínea , Olho/efeitos dos fármacos , Olho/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Terapia de Alvo Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/efeitos da radiação , Pterígio/tratamento farmacológico , Pterígio/fisiopatologia , Proteínas Repressoras
15.
J Immunol ; 191(8): 4367-74, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24043887

RESUMO

Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. The complement anaphylatoxin C5a receptor (C5aR) has been implicated in inflammatory processes and as regulator of macrophage activation and polarization. However, the role of C5aR in obesity and AT inflammation has not been addressed. We engaged the model of diet-induced obesity and found that expression of C5aR was significantly upregulated in the obese AT, compared with lean AT. In addition, C5a was present in obese AT in the proximity of macrophage-rich crownlike structures. C5aR-sufficient and -deficient mice were fed a high-fat diet (HFD) or a normal diet (ND). C5aR deficiency was associated with increased AT weight upon ND feeding in males, but not in females, and with increased adipocyte size upon ND and HFD conditions in males. However, obese C5aR(-/-) mice displayed improved systemic and AT insulin sensitivity. Improved AT insulin sensitivity in C5aR(-/-) mice was associated with reduced accumulation of total and proinflammatory M1 macrophages in the obese AT, increased expression of IL-10, and decreased AT fibrosis. In contrast, no difference in ß cell mass was observed owing to C5aR deficiency under an HFD. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.


Assuntos
Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Resistência à Insulina/imunologia , Macrófagos/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Adipócitos/imunologia , Adipócitos/metabolismo , Animais , Complemento C5a/metabolismo , Gorduras na Dieta/imunologia , Gorduras na Dieta/metabolismo , Feminino , Fibrose/imunologia , Inflamação/imunologia , Células Secretoras de Insulina/metabolismo , Interleucina-10/biossíntese , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/imunologia , Obesidade/metabolismo , Receptor da Anafilatoxina C5a/biossíntese , Receptor da Anafilatoxina C5a/imunologia , Regulação para Cima
16.
Blood ; 116(22): 4395-403, 2010 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-20625009

RESUMO

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.


Assuntos
Complemento C3/imunologia , Complemento C5/imunologia , Imunidade Inata , Neovascularização Patológica/imunologia , Retina/patologia , Retinopatia da Prematuridade/imunologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Complemento C3/genética , Complemento C5a/imunologia , Deleção de Genes , Humanos , Recém-Nascido , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/imunologia , Retina/imunologia , Retinopatia da Prematuridade/patologia , Fatores de Crescimento do Endotélio Vascular/imunologia
17.
Blood ; 114(8): 1707-16, 2009 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-19411631

RESUMO

EphrinB transmembrane ligands and their cognate EphB receptor tyrosine kinases regulate vascular development through bidirectional cell-to-cell signaling, but little is known about the role of EphrinB during postnatal vascular remodeling. We report that EphrinB is a critical mediator of postnatal pericyte-to-endothelial cell assembly into vascular structures. This function is dependent upon extracellular matrix-supported cell-to-cell contact, engagement of EphrinB by EphB receptors expressed on another cell, and Src-dependent phosphorylation of the intracytoplasmic domain of EphrinB. Phosphorylated EphrinB marks angiogenic blood vessels in the developing and hypoxic retina, the wounded skin, and tumor tissue, and is detected at contact points between endothelial cells and pericytes. Furthermore, inhibition ofEphrinB activity prevents proper assembly of pericytes and endothelial cells into vascular structures. These results reveal a role for EphrinB signaling in orchestrating pericyte/endothelial cell assembly, and suggest that therapeutic targeting of EphrinB may prove useful for disrupting angiogenesis when it contributes to disease.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Células Endoteliais/fisiologia , Efrinas/fisiologia , Neovascularização Fisiológica/fisiologia , Pericitos/fisiologia , Animais , Animais Recém-Nascidos , Vasos Sanguíneos/metabolismo , Células da Medula Óssea/fisiologia , Adesão Celular/genética , Células Cultivadas , Células Endoteliais/metabolismo , Efrina-B2/antagonistas & inibidores , Efrina-B2/genética , Efrina-B2/fisiologia , Efrinas/genética , Efrinas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Fisiológica/genética , Pericitos/metabolismo , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Receptores da Família Eph/fisiologia , Transdução de Sinais/fisiologia
18.
Nat Med ; 15(5): 553-8, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19377486

RESUMO

H2A histone family member X (H2AX, encoded by H2AFX) and its C-terminal phosphorylation (gamma-H2AX) participates in the DNA damage response and mediates DNA repair. Hypoxia is a physiological stress that induces a replication-associated DNA damage response. Moreover, hypoxia is the major driving force for neovascularization, as the hypoxia-mediated induction of vascular growth factors triggers endothelial cell proliferation. Here we studied the role of the hypoxia-induced DNA damage response in endothelial cell function and in hypoxia-driven neovascularization in vivo. Hypoxia induced replication-associated generation of gamma-H2AX in endothelial cells in vitro and in mice. Both in cultured cells and in mice, endothelial cell proliferation under hypoxic conditions was reduced by H2AX deficiency. Whereas developmental angiogenesis was not affected in H2afx(-/-) mice, hypoxia-induced neovascularization during pathologic proliferative retinopathy, in response to hind limb ischemia or during tumor angiogenesis was substantially lower in H2afx(-/-) mice. Moreover, endothelial-specific H2afx deletion resulted in reduced hypoxia-driven retina neovascularization and tumor neovascularization. Our findings establish that H2AX, and hence activation of the DNA repair response, is needed for endothelial cells to maintain their proliferation under hypoxic conditions and is crucial for hypoxia-driven neovascularization.


Assuntos
Endotélio Vascular/fisiopatologia , Histonas/deficiência , Histonas/genética , Neovascularização Patológica/genética , Vasos Retinianos/fisiopatologia , Animais , Dano ao DNA , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Deleção de Genes , Membro Posterior , Humanos , Hidroxiureia/farmacologia , Hipóxia/genética , Hipóxia/fisiopatologia , Isquemia/genética , Isquemia/fisiopatologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/prevenção & controle , Fosforilação , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/fisiologia , Veias Umbilicais/fisiopatologia
19.
Invest Ophthalmol Vis Sci ; 50(3): 1454-63, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19060272

RESUMO

PURPOSE: To determine the localization of JAM-C in human RPE and characterize its functions. METHODS: Immunofluorescence, Western blot, and PCR was used to identify the localization and expression of JAM-C, ZO-1, N-cadherin, and ezrin in cultures of human fetal RPE (hfRPE) with or without si-RNA mediated JAM-C knockdown and in adult native RPE wholemounts. A transepithelial migration assay was used to study the migration of leukocytes through the hfRPE monolayer. RESULTS: JAM-C localized at the tight junctions of cultured hfRPE cells and adult native RPE. During initial junction formation JAM-C was recruited to the primordial cell-cell contacts and after JAM-C knockdown, the organization of N-cadherin and ZO-1 at those contacts was disrupted. JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by reduced apical staining of ezrin. JAM-C inhibition significantly decreased the chemokine-induced transmigration of granulocytes but not monocytes through the hfRPE monolayer. CONCLUSIONS: JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. It is important for tight junction formation in hfRPE, possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell-cell contacts, and has a role in the polarization of hfRPE cells. Finally, JAM-C promotes the basal-to-apical transmigration of granulocytes but not monocytes through the hfRPE monolayer.


Assuntos
Moléculas de Adesão Celular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Antígenos CD/metabolismo , Western Blotting , Caderinas/metabolismo , Ensaios de Migração de Leucócitos , Polaridade Celular , Células Cultivadas , Citocinas/farmacologia , Proteínas do Citoesqueleto/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Granulócitos/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Monócitos/fisiologia , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/farmacologia , Epitélio Pigmentado da Retina/embriologia , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1
20.
Science ; 322(5904): 1101-4, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-19008446

RESUMO

Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus-1) as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1 deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/- mice displayed significantly higher neutrophil accumulation in lipopolysaccharide-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1 double-deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.


Assuntos
Proteínas de Transporte/fisiologia , Adesão Celular , Células Endoteliais/fisiologia , Monócitos/fisiologia , Infiltração de Neutrófilos , Neutrófilos/fisiologia , Pneumonia/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular , Molécula 1 de Adesão Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Migração e Rolagem de Leucócitos , Ligantes , Lipopolissacarídeos/imunologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Peritonite/imunologia , Proteínas Recombinantes de Fusão/metabolismo
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