Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
2.
Eur J Immunol ; 54(1): e2350626, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37837385

RESUMO

To better understand the stoichiometry of CD95L required to trigger apoptotic and nonapoptotic signals, we generated several CD95L concatemers from dimer to hexamer conjugated via a flexible link (GGGGS)2 . These ligands reveal that although the hexameric structure is the best stoichiometry to trigger cell death, a dimer is sufficient to induce the apoptotic response in CD95-sensitive Jurkat cells. Interestingly, only trimeric and hexameric forms can implement a potent Ca2+ response, suggesting that while CD95 aggregation controls the implementation of the apoptotic signal, both aggregation and conformation are required to implement the Ca2+ pathway.


Assuntos
Apoptose , Receptor fas , Humanos , Apoptose/fisiologia , Proteína Ligante Fas , Células Jurkat
3.
Immunity ; 45(1): 209-23, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27438772

RESUMO

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.


Assuntos
Sinalização do Cálcio , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fosfolipase C gama/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Receptor fas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fosfolipase C gama/genética , Domínios e Motivos de Interação entre Proteínas/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transcriptoma , Migração Transendotelial e Transepitelial , Receptor fas/genética
4.
J Anat ; 244(4): 620-627, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38214341

RESUMO

Imaging techniques in anatomy have developed rapidly over the last decades through the emergence of various 3D scanning systems. Depending on the dissection level, non-contact or tactile contact methods can be applied on the targeted structure. The aim of this study was to assess the inter and intra-observer reproducibility of an ArUco-based localisation stylus, that is, a manual technique on a hand-held stylus. Ten fresh-frozen, unembalmed adult arms were used to digitalise the glenoid cartilage related to the glenohumeral joint and the contour of the clavicle cartilage related to the acromioclavicular joint. Three operators performed consecutive digitalisations of each cartilage contour using an ArUco-based localisation stylus recorded by a single monocular camera. The shape of each cartilage was defined by nine shape parameters. Intra-observer repeatability and inter-observer reproducibility were computed using an intra-class correlation (ICC) for each of these parameters. Overall, 35.2 ± 2.4 s and 26.6 ± 10.2 s were required by each examiner to digitalise the contour of a glenoid and acromioclavicular cartilage, respectively. For most parameters, good-to-excellent agreements were observed concerning intra-observer (ICC ranging between 0.81 and 1.00) and inter-observer (ICC ranging between 0.75 and 0.99) reproducibility. To conclude, through a fast and versatile process, the use of an ArUco-based localisation stylus can be a reliable low-cost alternative to conventional imaging methods to digitalise shoulder cartilage contours.


Assuntos
Articulação do Ombro , Ombro , Adulto , Humanos , Reprodutibilidade dos Testes , Variações Dependentes do Observador , Cartilagem
6.
Semin Cancer Biol ; 60: 121-131, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31176682

RESUMO

CD95 (also known as Fas) is the prototype of death receptors; however, evidence suggests that this receptor mainly implements non-apoptotic signaling pathways such as NF-κB, MAPK, and PI3K that are involved in cell migration, differentiation, survival, and cytokine secretion. At least two different forms of CD95 L exist. The multi-aggregated transmembrane ligand (m-CD95 L) is cleaved by metalloproteases to release a homotrimeric soluble ligand (s-CD95 L). Unlike m-CD95 L, the interaction between s-CD95 L and its receptor CD95 fails to trigger apoptosis, but instead promotes calcium-dependent cell migration, which contributes to the accumulation of inflammatory Th17 cells in damaged organs of lupus patients and favors cancer cell invasiveness. Novel inhibitors targeting the pro-inflammatory roles of CD95/CD95 L may provide attractive therapeutic options for patients with chronic inflammatory disorders or cancer. This review discusses the roles of the CD95/CD95 L pair in cell migration and metastasis.


Assuntos
Proteína Ligante Fas/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Receptor fas/metabolismo , Apoptose , Cálcio/metabolismo , Citoesqueleto/metabolismo , Citotoxicidade Imunológica , Proteína Ligante Fas/genética , Homeostase , Humanos , Imunomodulação , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/patologia , Neoplasias/terapia , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Ligação Proteica , Transdução de Sinais , Receptor fas/genética
7.
Nat Chem Biol ; 14(12): 1079-1089, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30429604

RESUMO

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.


Assuntos
Inflamação/prevenção & controle , Peptidomiméticos/farmacologia , Fosfolipase C gama/metabolismo , Células Th17/efeitos dos fármacos , Receptor fas/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/etiologia , Masculino , Camundongos Mutantes , Simulação de Acoplamento Molecular , Peptidomiméticos/química , Fosfolipase C gama/genética , Domínios Proteicos , Ritonavir/química , Ritonavir/farmacologia , Relação Estrutura-Atividade , Células Th17/metabolismo , Células Th17/patologia , Tiazóis/química , Tiazóis/farmacologia , Receptor fas/genética
8.
Bioorg Med Chem Lett ; 29(16): 2094-2099, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31301931

RESUMO

The death receptor CD95 (also known as Fas) induces apoptosis through protein/protein association and the formation of the death-inducing signaling complex. On the other hand, in certain biological conditions, this receptor recruits different proteins and triggers the formation of another complex designated motility-inducing signaling complex, which promotes cell migration and inflammation. This pathway relies on a short sequence of CD95, called calcium-inducing domain (CID), which interacts with the phospholipase PLCγ1. To better understand how CID/PLCγ1 interaction occurs, we synthesized different α-AA peptides mimicking CID. Some of these peptidomimetics are as potent as the natural peptide to disrupt the CID/PLCγ1 interaction and cell migration, and showed improved pharmacokinetic properties. We also generated biotinyl- and palmitoyl-labelled peptidomimetics, useful chemico-biological tools to further explore the pro-inflammatory signal of CD95, which plays an important role in the pathogenesis of lupus and other autoimmune diseases.


Assuntos
Peptidomiméticos/farmacologia , Fosfolipase C gama/metabolismo , Multimerização Proteica/efeitos dos fármacos , Receptor fas/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Biotina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ligação Proteica
9.
Biophys J ; 112(1): 87-98, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076819

RESUMO

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca2+-bound CaM but not Ca2+-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.


Assuntos
Transferência de Energia , Medições Luminescentes , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Técnicas Biossensoriais , Calmodulina/metabolismo , Células HEK293 , Temperatura Alta , Humanos
10.
J Immunol ; 195(5): 2207-15, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26202984

RESUMO

The anti-CD20 mAb, rituximab, is routinely used to treat B cell malignancies. However, a majority of patients relapse. An improvement in the complete response was obtained by combining rituximab with chemotherapy, at the cost of increased toxicity. We reported that rituximab induced the colocalization of both the Orai1 Ca(2+) release-activated Ca(2+) channel (CRAC) and the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 with CD20 and CD95 into a cluster, eliciting a polarized store-operated Ca(2+) entry (SOCE). We observed that blocking this Ca(2+) entry with downregulation of Orai1, pharmacological inhibitors, or reducing calcemia with hypocalcemic drugs sensitized human B lymphoma cell lines and primary human lymphoma cells to rituximab-induced apoptosis in vitro, and improved the antitumoral effect of rituximab in xenografted mice. This revealed that Ca(2+) entry exerted a negative feedback loop on rituximab-induced apoptosis, suggesting that associating CRAC channel inhibitors or hypocalcemic agents with rituximab may improve the treatment of patients with B cell malignancies. The calcium-dependent proteins involved in this process appear to vary according to the B lymphoma cell type, suggesting that CRAC-channel targeting is likely to be more efficient than calcium-dependent protein targeting.


Assuntos
Apoptose/efeitos dos fármacos , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Linfoma não Hodgkin/tratamento farmacológico , Rituximab/farmacologia , Receptor fas/metabolismo , Animais , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Western Blotting , Canais de Cálcio/genética , Linhagem Celular Tumoral , Difosfonatos/farmacologia , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Humanos , Imidazóis/farmacologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Proteínas de Membrana/metabolismo , Camundongos Knockout , Microscopia Confocal , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Interferência de RNA , Rituximab/administração & dosagem , Molécula 1 de Interação Estromal , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido Zoledrônico
11.
Diabetologia ; 58(4): 749-57, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25403481

RESUMO

AIMS/HYPOTHESIS: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an important role in glucose-induced signalling and glucose homeostasis. METHODS: We used pharmacological and genetic approaches in beta cells to determine secretion and calcium metabolism. Furthermore, Adcy8 knockout mice were characterised. RESULTS: In clonal beta cells, inhibitors of adenylyl cyclases or their downstream targets reduced the glucose-induced increase in cytosolic calcium and insulin secretion. This was reproduced by knock-down of ADCY8, but not of ADCY1. These agents also inhibited glucose-induced increase in cytosolic calcium and electrical activity in primary beta cells and similar effects were observed after ADCY8 knock-down. Moreover, insulin secretion was diminished in islets from Adcy8 knockout mice. These mice were glucose intolerant after oral or intraperitoneal administration of glucose whereas their levels of glucagon-like peptide-1 remained unaltered. Finally, we knocked down ADCY8 in the ventromedial hypothalamus to evaluate the need for ADCY8 in the central regulation of glucose homeostasis. Whereas mice fed a standard diet had normal glucose levels, high-fat diet exacerbated glucose intolerance and knock-down mice were incapable of raising their plasma insulin levels. Finally we confirmed that ADCY8 is expressed in human islets. CONCLUSIONS/INTERPRETATIONS: Collectively, our findings demonstrate that ADCY8 is required for the physiological activation of glucose-induced signalling pathways in beta cells, for glucose tolerance and for hypothalamic adaptation to a high-fat diet via regulation of islet insulin secretion.


Assuntos
Adenilil Ciclases/metabolismo , Glicemia/metabolismo , Células Secretoras de Insulina/enzimologia , Adenilil Ciclases/deficiência , Adenilil Ciclases/genética , Animais , Cálcio/metabolismo , Linhagem Celular , Dieta Hiperlipídica , Modelos Animais de Doenças , Genótipo , Intolerância à Glucose/sangue , Intolerância à Glucose/enzimologia , Homeostase , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Núcleo Hipotalâmico Ventromedial/enzimologia
12.
PLoS Biol ; 9(6): e1001090, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21713032

RESUMO

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²âº/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.


Assuntos
Movimento Celular/imunologia , Proteína Ligante Fas/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células HEK293 , Humanos , Lúpus Eritematoso Sistêmico/sangue , Pseudópodes/fisiologia , Transdução de Sinais , Migração Transendotelial e Transepitelial/fisiologia , Receptor fas/imunologia , Receptor fas/metabolismo , Quinases da Família src/fisiologia
13.
Proc Natl Acad Sci U S A ; 108(47): 19072-7, 2011 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-22065776

RESUMO

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca(2+) release-activated Ca(2+) channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca(2+) influx is fundamental to recruiting the Ca(2+)-dependent protein kinase C (PKC) ß2 to the DISC. PKCß2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca(2+) and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.


Assuntos
Apoptose/fisiologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Complexos Multiproteicos/metabolismo , Proteína Quinase C/metabolismo , Receptor fas/metabolismo , Western Blotting , Caspase 10/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Citometria de Fluxo , Humanos , Imunoprecipitação , Microscopia Confocal , Proteína ORAI1 , Proteína Quinase C beta , Estatísticas não Paramétricas
14.
Biomed Pharmacother ; 174: 116552, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38599061

RESUMO

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.


Assuntos
Conexina 43 , Miócitos de Músculo Liso , Fator de Crescimento Neural , Artéria Pulmonar , Animais , Humanos , Masculino , Ratos , Células Cultivadas , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Junções Comunicantes/efeitos dos fármacos , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fator de Crescimento Neural/metabolismo , Fosforilação , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Ratos Wistar , Receptor trkA/metabolismo
15.
Br J Pharmacol ; 180(21): 2802-2821, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37351910

RESUMO

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a cardiovascular disease characterised by an increase in pulmonary arterial (PA) resistance leading to right ventricular (RV) failure. Reactive oxygen species (ROS) play a major role in PH. OP2113 is a drug with beneficial effects on cardiac injuries that targets mitochondrial ROS. The aim of the study was to address the in vivo therapeutic effect of OP2113 in PH. EXPERIMENTAL APPROACH: PH was induced by 3 weeks of chronic hypoxia (CH-PH) in rats treated with OP2113 or its vehicle via subcutaneous osmotic mini-pumps. Haemodynamic parameters and both PA and heart remodelling were assessed. Reactivity was quantified in PA rings and in RV or left ventricular (LV) cardiomyocytes. Oxidative stress was detected by electron paramagnetic resonance and western blotting. Mitochondrial mass and respiration were measured by western blotting and oxygraphy, respectively. KEY RESULTS: In CH-PH rats, OP2113 reduced the mean PA pressure, PA remodelling, PA hyperreactivity in response to 5-HT, the contraction slowdown in RV and LV and increased the mitochondrial mass in RV. Interestingly, OP2113 had no effect on haemodynamic parameters, both PA and RV wall thickness and PA reactivity, in control rats. Whereas oxidative stress was evidenced by an increase in protein carbonylation in CH-PH, this was not affected by OP2113. CONCLUSION AND IMPLICATIONS: Our study provides evidence for a selective protective effect of OP2113 in vivo on alterations in both PA and RV from CH-PH rats without side effects in control rats.


Assuntos
Insuficiência Cardíaca , Hipertensão Pulmonar , Disfunção Ventricular Direita , Ratos , Animais , Hipertensão Pulmonar/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ventrículos do Coração/metabolismo , Artéria Pulmonar , Insuficiência Cardíaca/metabolismo , Hipóxia/complicações , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Disfunção Ventricular Direita/metabolismo , Função Ventricular Direita , Modelos Animais de Doenças
16.
Cells ; 11(9)2022 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-35563744

RESUMO

Study of the initial steps of the CD95-mediated signaling pathways is a field of intense research and a long list of actors has been described in the literature. Nonetheless, the dynamism of protein-protein interactions (PPIs) occurring in the presence or absence of its natural ligand, CD95L, and the cellular distribution where these PPIs take place render it difficult to predict what will be the cellular outcome associated with the receptor engagement. Accordingly, CD95 stimulation can trigger apoptosis, necroptosis, pyroptosis, or pro-inflammatory signaling pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphatidylinositol-3-kinase (PI3K). Recent data suggest that CD95 can also activate pattern recognition receptors (PRRs) known to sense damage-associated molecular patterns (DAMPs) such as DNA debris and dead cells. This activation might contribute to the pro-inflammatory role of CD95 and favor cancer development or severity of chronic inflammatory and auto-immune disorders. Herein, we discuss some of the molecular links that might connect the CD95 signaling to DAMP sensors.


Assuntos
Transdução de Sinais , Receptor fas , Alarminas , Apoptose/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Receptor fas/metabolismo
17.
Cells ; 11(15)2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35954193

RESUMO

In intrapulmonary arteries (IPAs), mechanical forces due to blood flow control vessel tone, and these forces change during pulmonary hypertension (PH). Piezo1, a stretch-activated calcium channel, is a sensor of mechanical stress present in both endothelial cells (ECs) and smooth muscle cells (SMCs). The present study investigated the role of Piezo1 on IPA in the chronic hypoxia model of PH. Rats were raised in chronically hypoxic conditions for 1 (1W-CH, early stage) or 3 weeks (3W-CH, late-stage) of PH or in normoxic conditions (Nx). Immunofluorescence labeling and patch-clamping revealed the presence of Piezo1 in both ECs and SMCs. The Piezo1 agonist, Yoda1, induced an IPA contraction in Nx and 3W-CH. Conversely, Yoda1 induced an endothelial nitric oxide (eNOS) dependent relaxation in 1W-CH. In ECs, the Yoda1-mediated intracellular calcium concentration ([Ca2+]i) increase was greater in 1W-CH as compared to Nx. Yoda1 induced an EC hyperpolarization in 1W-CH. The eNOS levels were increased in 1W-CH IPA compared to Nx or 3W-CH PH and Yoda1 activated phosphorylation of Akt (Ser473) and eNOS (Ser1177). Thus, we demonstrated that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i, endothelial-dependent hyperpolarization, and Akt-eNOS pathway activation in the early stage of PH.


Assuntos
Hipertensão Pulmonar , Animais , Células Endoteliais/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/metabolismo , Ratos , Vasoconstrição/fisiologia
18.
Cells ; 11(18)2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-36139373

RESUMO

Expression of the nerve growth factor NGF is increased in pulmonary hypertension (PH). We have here studied whether oxidative stress and inflammation, two pathological conditions associated with transforming growth factor-ß1 (TGF-ß1) in PH, may trigger NGF secretion by pulmonary arterial (PA) cells. Effects of hydrogen peroxide (H2O2) and interleukin-1ß (IL-1ß) were investigated ex vivo on rat pulmonary arteries, as well as in vitro on human PA smooth muscle (hPASMC) or endothelial cells (hPAEC). TßRI expression was assessed by Western blotting. NGF PA secretion was assessed by ELISA after TGF-ß1 blockade (anti-TGF-ß1 siRNA, TGF-ß1 blocking antibodies, TßRI kinase, p38 or Smad3 inhibitors). TßRI PA expression was evidenced by Western blotting both ex vivo and in vitro. H2O2 or IL-1ß significantly increased NGF secretion by hPASMC and hPAEC, and this effect was significantly reduced when blocking TGF-ß1 expression, binding to TßRI, TßRI activity, or signaling pathways. In conclusion, oxidative stress and inflammation may trigger TGF-ß1 secretion by hPASMC and hPAEC. TGF-ß1 may then act as an autocrine factor on these cells, increasing NGF secretion via TßRI activation. Since NGF and TGF-ß1 are relevant growth factors involved in PA remodeling, such mechanisms may therefore be relevant to PH pathophysiology.


Assuntos
Hipertensão Pulmonar , Fator de Crescimento Transformador beta1 , Animais , Anticorpos Bloqueadores , Células Endoteliais/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Hipertensão Pulmonar/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Fator de Crescimento Neural/metabolismo , Estresse Oxidativo , Artéria Pulmonar/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Crescimento Transformador beta1/metabolismo
19.
Biomolecules ; 12(7)2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35883510

RESUMO

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.


Assuntos
Canais de Cátion TRPV , Canais de Potencial de Receptor Transitório , Cálcio/metabolismo , Células HEK293 , Humanos , Hipóxia/metabolismo , Técnicas de Patch-Clamp , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
20.
Antioxidants (Basel) ; 11(5)2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35624710

RESUMO

The development and use of nanomaterials, especially of nickel oxide nanoparticles (NiONPs), is expected to provide many benefits but also has raised concerns about the potential human health risks. Inhaled NPs are known to exert deleterious cardiovascular side effects, including pulmonary hypertension. Consequently, patients with pulmonary hypertension (PH) could be at increased risk for morbidity. The objective of this study was to compare the toxic effects of NiONPs on human pulmonary artery endothelial cells (HPAEC) under physiological and pathological conditions. The study was conducted with an in vitro model mimicking the endothelial dysfunction observed in PH. HPAEC were cultured under physiological (static and normoxic) or pathological (20% cycle stretch and hypoxia) conditions and exposed to NiONPs (0.5-5 µg/cm2) for 4 or 24 h. The following endpoints were studied: (i) ROS production using CM-H2DCF-DA and MitoSOX probes, (ii) nitrite production by the Griess reaction, (iii) IL-6 secretion by ELISA, (iv) calcium signaling with a Fluo-4 AM probe, and (v) mitochondrial dysfunction with TMRM and MitoTracker probes. Our results evidenced that under pathological conditions, ROS and nitrite production, IL-6 secretions, calcium signaling, and mitochondria alterations increased compared to physiological conditions. Human exposure to NiONPs may be associated with adverse effects in vulnerable populations with cardiovascular risks.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA