RESUMO
BACKGROUND: Recently, the IMPRES study revealed that systemic imatinib improves exercise capacity in patients with advanced pulmonary arterial hypertension. Imatinib blocks the tyrosine kinase activity of the platelet-derived growth factor (PDGF)-receptor (PDGFR), acts antiproliferative and relaxes pulmonary arteries. However so far, the relaxant effects of imatinib on pulmonary veins (PVs) and on the postcapillary resistance are unknown, although pulmonary hypertension (PH) due to left heart disease (LHD) is most common and primarily affects PVs. Next, it is unknown whether activation of PDGFR alters the pulmonary venous tone. Due to the reported adverse effects of systemic imatinib, we evaluated the effects of nebulized imatinib on the postcapillary resistance. METHODS: Precision-cut lung slices (PCLS) were prepared from guinea pigs. PVs were pre-constricted with Endothelin-1 (ET-1) and the imatinib-induced relaxation was studied by videomicroscopy; PDGF-BB-related vascular properties were evaluated as well. The effects of perfused/nebulized imatinib on the postcapillary resistance were studied in cavine isolated perfused lungs (IPL). Intracellular cAMP/cGMP was measured by ELISA in PVs. RESULTS: In PCLS, imatinib (100 µM) relaxed pre-constricted PVs (126%). In PVs, imatinib increased cAMP, but not cGMP and inhibition of adenyl cyclase or protein kinase A reduced the imatinib-induced relaxation. Further, inhibition of KATP-channels, [Formula: see text]-channels or Kv-channels diminished the imatinib-induced relaxation, whereas inhibition of NO-signaling was without effect. In the IPL, perfusion or nebulization of imatinib reduced the ET-1-induced increase of the postcapillary resistance. In PCLS, PDGF-BB contracted PVs, which was blocked by imatinib and by the PDGFR-ß kinase inhibitor SU6668, whereas inhibition of PDGFR-α (ponatinib) had no significant effect. Conversely, PDGFR-ß kinase inhibitors (SU6668/DMPQ) relaxed PVs pre-constricted with ET-1 comparable to imatinib, whereas the PDGFR-α kinase inhibitor ponatinib did not. CONCLUSIONS: Imatinib-induced relaxation depends on cAMP and on the activation of K+-channels. Perfused or nebulized imatinib significantly reduces the postcapillary resistance in the pre-constricted (ET-1) pulmonary venous bed. Hence, nebulization of imatinib is feasible and might reduce systemic side effects. Conversely, PDGF-BB contracts PVs by activation of PDGFR-ß suggesting that imatinib-induced relaxation depends on PDGFR-ß-antagonism. Imatinib combines short-term relaxant and long-term antiproliferative effects. Thus, imatinib might be a promising therapy for PH due to LHD.
Assuntos
Mesilato de Imatinib/administração & dosagem , Proteínas Proto-Oncogênicas c-sis/metabolismo , Veias Pulmonares/efeitos dos fármacos , Veias Pulmonares/fisiologia , Resistência Vascular/fisiologia , Vasodilatação/fisiologia , Animais , Becaplermina , Relação Dose-Resposta a Droga , Feminino , Cobaias , Inibidores de Proteínas Quinases/administração & dosagem , Resistência Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagemRESUMO
A disintegrin and metalloproteinase 8 (ADAM8) has been identified as a signature gene associated with moderate and severe asthma. Studies in mice have demonstrated that the severity of asthma can be reduced by either transgenic knock-out or by antibodies blocking ADAM8 function, highlighting ADAM8 as potential drug target for asthma therapy. Here, we examined the therapeutic effect of an ADAM8 inhibitor peptide (BK-1361) that specifically blocks cellular ADAM8 activity in ovalbumin-sensitized and challenged Balb/c mice. We found that BK-1361 (25 µg/g body weight) attenuated airway responsiveness to methacholine stimulation by up to 42%, concomitantly reduced tissue remodeling by 50%, and decreased inflammatory cells (e.g. eosinophils down by 54%)/inflammatory factors (e.g. sCD23 down by 50%)/TH2 cytokines (e.g. IL-5 down by 70%)/ADAM8-positive eosinophils (down by 60%) in the lung. We further verified that BK-1361 specifically targets ADAM8 in vivo as the peptide caused significantly reduced levels of soluble CD23 in wild-type but not in ADAM8-deficient mice. These findings suggest that BK-1361 blocks ADAM8-dependent asthma effects in vivo by inhibiting infiltration of eosinophils and TH2 lymphocytes, thus leading to reduction of TH2-mediated inflammation, tissue remodeling and bronchial hyperresponsiveness. Taken together, pharmacological ADAM8 inhibition appears as promising novel therapeutic strategy for the treatment of asthma.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Asma/tratamento farmacológico , Asma/imunologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Citocinas/metabolismo , Inflamação/patologia , Proteínas de Membrana/antagonistas & inibidores , Peptídeos Cíclicos/uso terapêutico , Células Th2/imunologia , Proteínas ADAM/deficiência , Proteínas ADAM/metabolismo , Animais , Antígenos CD/metabolismo , Asma/patologia , Asma/fisiopatologia , Brônquios/efeitos dos fármacos , Brônquios/patologia , Brônquios/fisiopatologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Líquido da Lavagem Broncoalveolar , Contagem de Células , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/complicações , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Cloreto de Metacolina , Camundongos Endogâmicos BALB C , Camundongos Knockout , Peptídeos Cíclicos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de IgE/metabolismo , Solubilidade , Células Th2/efeitos dos fármacosRESUMO
Brain-intrinsic degenerative cascades have been proposed to be an initial factor driving lesion formation in multiple sclerosis (MS). Here, we identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the mouse forebrain. Female C57BL/6 mice were fed cuprizone for 3 weeks, followed by a period of 2 weeks on normal chow to induce the formation of lesion foci in the forebrain. Subsequent immunization with myelin oligodendrocyte glycoprotein 35-55 peptide, which induces myelin autoreactive T cells in the periphery, resulted in massive immune cell recruitment into the affected forebrain. Additional adoptive transfer experiments together with flow cytometry analysis underline the importance of brain-derived signals for immune cell recruitment. This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and MS lesion formation. Additional studies have to address the signaling cascades and mechanistic processes that form the top-down communication between the affected brain area, neurovascular unit, and peripheral immune cells. SIGNIFICANCE STATEMENT: We identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the forebrain. Thus, immune cell recruitment might be a second step during the formation of new inflammatory lesions in multiple sclerosis. A better understanding of factors regulating neurodegeneration-induced immune cell recruitment will pave the way for the development of novel therapeutic treatment strategies.
Assuntos
Linfócitos/fisiologia , Monócitos/fisiologia , Doenças Neurodegenerativas/patologia , Prosencéfalo/patologia , Transferência Adotiva , Animais , Complexo CD3/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Quelantes/toxicidade , Cuprizona/toxicidade , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Adjuvante de Freund/toxicidade , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Glicoproteína Mielina-Oligodendrócito/imunologia , Doenças Neurodegenerativas/induzido quimicamente , Fragmentos de Peptídeos/imunologia , Toxina Pertussis/toxicidadeRESUMO
ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics.
Assuntos
Proteína ADAM10/antagonistas & inibidores , Proteína ADAM17/antagonistas & inibidores , Proteína ADAM10/química , Proteína ADAM17/química , Linhagem Celular Tumoral , Bases de Dados de Compostos Químicos , Glicosilação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
ADAM17 is implicated in several debilitating diseases. However, drug discovery efforts targeting ADAM17 have failed due to the utilization of zinc-binding inhibitors. We previously reported discovery of highly selective nonzinc-binding exosite-targeting inhibitors of ADAM17 that exhibited not only enzyme isoform selectivity but synthetic substrate selectivity as well ( J. Biol. Chem. 2013, 288, 22871). As a result of SAR studies presented herein, we obtained several highly selective ADAM17 inhibitors, six of which were further characterized in biochemical and cell-based assays. Lead compounds exhibited low cellular toxicity and high potency and selectivity for ADAM17. In addition, several of the leads inhibited ADAM17 in a substrate-selective manner, which has not been previously documented for inhibitors of the ADAM family. These findings suggest that targeting exosites of ADAM17 can be used to obtain highly desirable substrate-selective inhibitors. Additionally, current inhibitors can be used as probes of biological activity of ADAM17 in various in vitro and, potentially, in vivo systems.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Sondas Moleculares , Proteína ADAM17 , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Expression of the C4-specific phosphoenolpyruvate carboxylase (C4-PEPC) gene in maize (Zea mays) is regulated in a tissue-specific manner, but affected by light and nutrient availability. We manipulated these stimuli in a combinatorial manner and analyzed concomitant changes in histone acetylation of the nucleosomes associated with the C4-PEPC gene in relation to transcriptional activity and steady-state mRNA levels. Whereas the transition from the lowest activity to an intermediate activity was observed in the absence of histone acetylation, the light-induced boost to full activity was associated with strong enhancement of the acetylation of both histones H3 and H4 limited to the gene region. Once activated by light, prolonged darkness was necessary to reduce both transcription and, in parallel, histone acetylation. Unexpectedly, histone acetylation was also induced in bundle sheath cells, although the transcriptional activity did not respond to illumination in this tissue. Furthermore, we were able to down-regulate the promoter by nitrogen depletion in the light without any decrease in the hyperacetylation of histone H4. When plants kept in prolonged darkness were nitrogen depleted and then exposed to light, transcription was not induced, but the promoter chromatin became hyperacetylated. We suggest a model where inhibition of a histone deacetylase in the light triggers H4 hyperacetylation at the C4-PEPC gene promoter regardless of the transcriptional activity of the gene. Our data indicate that an understanding of the interplay between histone modification and transcription requires analysis of signal integration on promoters in vivo.