RESUMO
Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.
Assuntos
Hipertensão Arterial Pulmonar , Transdução de Sinais , Remodelação Vascular , Animais , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/fisiologia , Ratos , Humanos , Hipertensão Arterial Pulmonar/fisiopatologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Masculino , Modelos Animais de Doenças , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Camundongos , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A , Proliferação de Células/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/metabolismo , Lisina/análogos & derivadosRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterised by exuberant tissue remodelling and associated with high unmet medical needs. Outcomes are even worse when IPF results in secondary pulmonary hypertension (PH). Importantly, exaggerated resistance to cell death, excessive proliferation and enhanced synthetic capacity are key endophenotypes of both fibroblasts and pulmonary artery smooth muscle cells, suggesting shared molecular pathways. Under persistent injury, sustained activation of the DNA damage response (DDR) is integral to the preservation of cells survival and their capacity to proliferate. Checkpoint kinases 1 and 2 (CHK1/2) are key components of the DDR. The objective of this study was to assess the role of CHK1/2 in the development and progression of IPF and IPF+PH. METHODS AND RESULTS: Increased expression of DNA damage markers and CHK1/2 were observed in lungs, remodelled pulmonary arteries and isolated fibroblasts from IPF patients and animal models. Blockade of CHK1/2 expression or activity-induced DNA damage overload and reverted the apoptosis-resistant and fibroproliferative phenotype of disease cells. Moreover, inhibition of CHK1/2 was sufficient to interfere with transforming growth factor beta 1-mediated fibroblast activation. Importantly, pharmacological inhibition of CHK1/2 using LY2606368 attenuated fibrosis and pulmonary vascular remodelling leading to improvement in respiratory mechanics and haemodynamic parameters in two animal models mimicking IPF and IPF+PH. CONCLUSION: This study identifies CHK1/2 as key regulators of lung fibrosis and provides a proof of principle for CHK1/2 inhibition as a potential novel therapeutic option for IPF and IPF+PH.
Assuntos
Hipertensão Pulmonar , Fibrose Pulmonar Idiopática , Animais , Fibroblastos/metabolismo , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Miócitos de Músculo Liso/metabolismoRESUMO
Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.
Assuntos
Fármacos Cardiovasculares/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/prevenção & controle , Miócitos de Músculo Liso/efeitos dos fármacos , Trifluoperazina/farmacologia , Animais , Antipsicóticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Hemodinâmica/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Hipóxia/induzido quimicamente , Hipóxia/genética , Hipóxia/fisiopatologia , Indóis/administração & dosagem , Monocrotalina/administração & dosagem , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Pirróis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Survivina/genética , Survivina/metabolismoRESUMO
Syncytin genes are envelope genes of retroviral origin that have been exapted for a role in placentation. They are involved in the formation of a syncytial structure (the syncytiotrophoblast) at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one, as observed in humans and all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely, syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryo survival. Their cognate cellular receptors, which are necessary to mediate cell-cell fusion and syncytiotrophoblast formation, are still unknown. By devising a sensitive method that combines a cell-cell fusion assay with the screening of a mouse cDNA library, we succeeded in identifying the glycosylphosphatidylinositol (GPI)-anchored membrane protein lymphocyte antigen 6E (Ly6e) as a candidate receptor for SynA. Transfection of cells with the cloned receptor led to their fusion to cells expressing SynA, with no cross-reactive fusion activity with SynB. Knocking down Ly6e greatly reduced SynA-induced cell fusion, thus suggesting that Ly6e is the sole receptor for SynA in vivo Interaction of SynA with Ly6e was further demonstrated by a competition assay using the soluble ectodomain of Ly6e. Finally, reverse transcription-quantitative PCR (RT-qPCR) analysis of Ly6e expression on a representative panel of mouse tissues shows that it is significantly expressed in the mouse placenta together with SynA.IMPORTANCE Syncytin genes are envelope genes of endogenous retroviruses, co-opted for a physiological function in placentation. Syncytins are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising a sensitive in vitro fusion assay that enables the high-throughput screening of normalized cDNA libraries, we identified the long-sought receptor for syncytin-A (SynA), a mouse syncytin responsible for syncytiotrophoblast formation at the maternofetal interface of the mouse placenta. This protein, Ly6e (lymphocyte antigen 6E), is a GPI-anchored membrane protein, and small interfering RNA (siRNA) experiments targeting its deletion as well as a decoy assay using a recombinant soluble receptor show that Ly6e is the necessary and sufficient partner of SynA. Its profile of expression is consistent with a role in both ancestral endogenization of a SynA founder retrovirus and present-day placenta formation. This study provides a powerful general method to identify genes involved in cell-cell fusion processes.
Assuntos
Antígenos Ly/metabolismo , Fusão Celular , Proteínas da Gravidez/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos Ly/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Testes Genéticos/métodos , Camundongos , Receptores de Superfície Celular/genéticaRESUMO
Single-domain antibodies, referred to as VHH (variable heavy chains of heavy chain-only antibodies) or in their commercial name as nanobodies, are potent tools for the detection of target proteins in biological samples. They have the advantage of being highly stable, specific, and sensitive, with affinities reaching the nanomolar range. We utilized this tool to develop a rapid detection method that discriminates cells infected with Rift Valley fever virus (RVFV), based on the intracellular detection of the viral nonstructural NSm protein localized on the outer membrane of mitochondria. Here we describe how NSm-specific VHHs have been produced, cloned, and characterized, highlighting their value in RVFV research and diagnosis. This work may also raise interest in other potential applications such as antiviral therapy.
Assuntos
Febre do Vale de Rift , Vírus da Febre do Vale do Rift , Anticorpos de Domínio Único , Proteínas não Estruturais Virais , Vírus da Febre do Vale do Rift/imunologia , Anticorpos de Domínio Único/imunologia , Humanos , Febre do Vale de Rift/imunologia , Febre do Vale de Rift/diagnóstico , Febre do Vale de Rift/virologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Antivirais/imunologiaRESUMO
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
RESUMO
Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.
Assuntos
Hipertensão Pulmonar , Animais , Cromatina/metabolismo , Feto/metabolismo , Humanos , Pulmão/patologia , Camundongos , Artéria Pulmonar/patologia , Interferência de RNA , Fatores de Transcrição/metabolismo , Remodelação Vascular/genéticaRESUMO
HYPOTHESIS: SENS-401 (R-azasetron besylate) is effective against severe acoustic trauma-induced hearing loss. BACKGROUND: SENS-401 has calcineurin inhibiting properties and attenuates cisplatin-induced hearing loss in a rat model. Cisplatin-induced and acoustic trauma-induced hearing loss share common apoptotic pathways. METHODS: The dose-response relationship of SENS-401 (6.6âmg/kg BID, 13.2âmg/kg BID, 26.4âmg/kg QD) and treatment time-window (13.2âmg/kg BID starting 24, 72, and 96âh posttrauma) versus placebo for 28 days were evaluated in a male rat model of severe acoustic trauma-induced hearing loss (120âdB SPL, 2âh) using auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) measures followed by cochlear outer hair cell (OHC) counting with myosin-VIIa immunolabeling. RESULTS: All SENS-401 doses improved ABR threshold shift and recovery, reaching statistical significance (pâ<â0.05) for ABR threshold recoveries after 28-days treatment. DPOAE amplitude loss and recovery improved markedly for 13.2âmg/kg BID SENS-401, reaching significance after 14 days (pâ<â0.05). Significant improvements in ABR threshold shifts/recovery and DPOAE amplitude loss occurred with up to 96-hours delay in initiating SENS-401 (pâ<â0.05), and in DPOAE amplitude recovery with up to 72-hours delay (pâ<â0.05). Significantly more surviving OHCs were present after SENS-401 treatment compared with placebo after 24 to 96-hours delay posttrauma, with up to 5.3-fold more cells in the basal cochlea turn. CONCLUSIONS: In vivo data support the otoprotective potential of twice daily oral SENS-401. Improvements in hearing loss recovery make SENS-401 a promising clinical candidate for acoustic trauma-induced hearing loss, including when treatment is not initiated immediately.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Perda Auditiva Neurossensorial/tratamento farmacológico , Oxazinas/farmacologia , Estimulação Acústica/efeitos adversos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Cisplatino/toxicidade , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Perda Auditiva Neurossensorial/induzido quimicamente , Masculino , Emissões Otoacústicas Espontâneas/efeitos dos fármacos , Oxazinas/administração & dosagem , Oxazinas/uso terapêutico , Ratos , Ratos WistarRESUMO
Biomarkers in easy-to-access body fluid compartments, such as blood, are commonly used to assess health of various organ systems in clinical medicine. At present, no such biomarkers are available to inform on the health of the inner ear. Previously, we proposed the outer-hair-cell-specific protein prestin, as a possible biomarker and provided proof of concept in noise- and cisplatin-induced hearing loss. Our ototoxicity data suggest that circulatory prestin changes after inner ear injury are not static and that there is a temporal pattern of change that needs to be further characterized before practical information can be extracted. To achieve this goal, we set out to 1) describe the time course of change in prestin after intense noise exposure, and 2) determine if the temporal patterns and prestin levels are sensitive to severity of injury. After assessing auditory brainstem thresholds and distortion product otoacoustic emission levels, rats were exposed to intense octave band noise for 2â¯h at either 110 or 120â¯dB SPL. Auditory function was re-assessed 1 and 14 days later. Blood samples were collected at baseline, 4, 24, 48, 72â¯h and 7 and 14 days post exposure and prestin concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Functional measures showed temporary hearing loss 1 day after exposure in the 110â¯dB SPL group, but permanent loss through Day 14 in the 120â¯dB SPL group. Prestin levels temporarily increased 5% at 4â¯h after 120â¯dB SPL exposure, but not in the 110â¯dB SPL group. There was a gradual decline in prestin levels in both groups thereafter, with prestin being below baseline on Day 14 by 5% in the 110â¯dB group (NS) and more than 10% in the 120â¯dB SPL group (pâ¯=â¯0.043). These results suggest that there is a temporal pattern of change in serum prestin level after noise-induced hearing loss that is related to severity of hearing loss. Circulatory levels of prestin may be able to act as surrogate biomarker for hearing loss involving OHC loss.
Assuntos
Perda Auditiva Provocada por Ruído/sangue , Transportadores de Sulfato/sangue , Animais , Limiar Auditivo/fisiologia , Biomarcadores/sangue , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/fisiologia , Perda Auditiva Provocada por Ruído/patologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Humanos , Masculino , Emissões Otoacústicas Espontâneas/fisiologia , Ratos Wistar , Fatores de TempoRESUMO
HYPOTHESIS: SENS-401, an oral clinical-stage drug, may reduce cisplatin-induced hearing loss and cochlear damage in an in vivo model. BACKGROUND: Cisplatin is commonly associated with hearing loss, causing significant learning and behavioral difficulties in the pediatric cancer population, and for which there are currently no clinical solutions. SENS-401 has previously been shown to improve acoustic trauma-induced hearing loss in vivo. METHODS: The effect of SENS-401 (R-azasetron besylate) on cisplatin IC50 values was evaluated in a panel of cisplatin-sensitive cell lines (NIH:OVCAR-3, SK-N-AS, NCI-H460, FaDu). Auditory brainstem response and distortion product otoacoustic emission tests were performed in a rat model of cisplatin-induced hearing-loss (8âmg/kg, day 1) at baseline, and after 14 days of SENS-401 (6.6, 13.2, 26.4âmg/kg/d). Cochlear outer hair cells were counted after immunolabeling for myosin-VIIa. RESULTS: Cisplatin cytotoxicity was not impacted by the addition of SENS-401 (up to 10âµM) in any of the cell types evaluated. In vivo, all SENS-401 doses significantly improved auditory brainstem response threshold shift (up to 30âdB) and distortion product otoacoustic emission amplitude loss (up to 19âdB) over placebo. Body weight and survival were not significantly different between rats receiving placebo and those receiving 26.4âmg/kg SENS-401. Significantly more surviving outer hair cells were present after SENS-401 treatment compared with placebo (pâ<â0.001), with up to 11-fold more in the basal turn of the cochlea. CONCLUSION: In vivo and in vitro data support the otoprotective potential and tolerability of SENS-401 without impacting chemotherapeutic potential. Oral SENS-401 is a promising candidate for treating cisplatin-induced ototoxicity.