RESUMO
Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzyme's catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation.
Assuntos
Compostos de Anilina/farmacologia , Inibidores Enzimáticos/farmacologia , Peroxidase de Eosinófilo/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Peroxidase/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bioensaio , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/química , Combinação de Medicamentos , Endocitose/efeitos dos fármacos , Peroxidase de Eosinófilo/antagonistas & inibidores , Peroxidase de Eosinófilo/genética , Peroxidase de Eosinófilo/metabolismo , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Laminina/química , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peroxidase/antagonistas & inibidores , Peroxidase/genética , Peroxidase/metabolismo , Proteoglicanas/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Eosinophils localize to and release their granule proteins in close association with nerves in patients with asthma and rhinitis. These conditions are associated with increased neural function. In this study the effect of the individual granule proteins on cholinergic neurotransmitter expression was investigated. Eosinophil peroxidase (EPO) upregulated choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. Fluorescently labeled EPO was seen to bind to the IMR-32 cell surface. Both Poly-L-Glutamate (PLG) and Heparinase-1 reversed the up-regulatory effect of EPO on ChAT and VAChT expression and prevented EPO adhesion to the cell surface. Poly-L-arginine (PLA) had no effect on expression of either gene, suggesting that charge is necessary but insufficient to alter gene expression. EPO induced its effects via the activation of NF-κB. MEK inhibition led to reversal of all up-regulatory effects of EPO. These data indicate a preferential role of EPO signaling via a specific surface receptor that leads to neural plasticity.
Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/genética , Peroxidase de Eosinófilo/fisiologia , Neurônios/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colina O-Acetiltransferase/metabolismo , Colinérgicos/metabolismo , Peroxidase de Eosinófilo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Plasticidade Neuronal/genética , Neurônios/efeitos dos fármacos , Ligação Proteica , Células Tumorais Cultivadas , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
We describe the synthesis, properties, and application of two reversible fluorescent probes, mCy-TemOH and Cy-TemOH, for HOBr sensing and imaging in live cells. The two probes contain a hydroxylamine functional group for the monitoring of HOBr oxidation/ascorbic acid reduction events. Confocal fluorescence microscopy has established the HOBr detection in live-cells.