RESUMEN
Idiopathic pulmonary fibrosis (IPF) is characterized by abnormal fibroblast accumulation in the lung leading to extracellular matrix deposition and remodeling that compromise lung function. However, the mechanisms of interstitial invasion and remodeling by lung fibroblasts remain poorly understood. The invadosomes, initially described in cancer cells, consist of actin-based adhesive structures that coordinate with numerous other proteins to form a membrane protrusion capable of degrading the extracellular matrix to promote their invasive phenotype. In this regard, we hypothesized that invadosome formation may be increased in lung fibroblasts from patients with IPF. Public RNAseq datasets from control and IPF lung tissues were used to identify differentially expressed genes associated with invadosomes. Lung fibroblasts isolated from bleomycin-exposed mice and IPF patients were seeded with and without the two approved drugs for treating IPF, nintedanib or pirfenidone on fluorescent gelatin-coated coverslips for invadosome assays. Several matrix and invadosome-associated genes were increased in IPF tissues and in IPF fibroblastic foci. Invadosome formation was significantly increased in lung fibroblasts isolated from bleomycin-exposed mice and IPF patients. The degree of lung fibrosis found in IPF tissues correlated strongly with invadosome production by neighboring cells. Nintedanib suppressed IPF and PDGF-activated lung fibroblast invadosome formation, an event associated with inhibition of the PDGFR/PI3K/Akt pathway and TKS5 expression. Fibroblasts derived from IPF lung tissues express a pro-invadosomal phenotype, which correlates with the severity of fibrosis and is responsive to antifibrotic treatment.
Asunto(s)
Fibrosis Pulmonar Idiopática , Podosomas , Ratones , Animales , Podosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Pulmón/patología , Fibrosis Pulmonar Idiopática/metabolismo , Fibroblastos/metabolismo , Fibrosis , Bleomicina/uso terapéuticoRESUMEN
Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA1 (LPAR1). Abundant data indicate that LPA1-dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA1-dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA1. In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.
Asunto(s)
Fibroblastos , Pulmón , Podosomas , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , Fibroblastos/metabolismo , Fibroblastos/patología , Pulmón/patología , Pulmón/metabolismo , Podosomas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Hipoxia de la Célula , Lisofosfolípidos/metabolismo , Indoles/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3â cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Humanos , Inhibidores de Serina Proteinasa/farmacología , Virus de la Influenza A/fisiología , Serina Proteasas/metabolismo , Gripe Humana/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Replicación ViralRESUMEN
INTRODUCTION: Asthma and its associated exacerbation are heterogeneous. Although severe asthma attacks are systematically prescribed corticosteroids and often antibiotics, little is known about the variability of response to these therapies. Blood eosinophils and fractional exhaled nitric oxide (FeNO) are type 2 inflammation biomarkers that have established mechanistic, prognostic and theragnostic values in chronic asthma, but their utility in acute asthma is unclear. We speculate that the clinical and biological response to those treatments varies according to inflammometry and microbiological test results. METHODS AND ANALYSIS: An observational longitudinal pilot study with multimodal clinical and translational assessments will be performed on 50 physician-diagnosed ≥12-year-old asthmatics presenting with an asthma attack and 12 healthy controls, including blood eosinophil count (venous and point-of-care (POC) capillary blood), FeNO and testing for airway infection (sputum cultures and POC nasopharyngeal swabs). People with asthma will be assessed on day 0 and after a 7-day corticosteroid course, with home monitoring performed in between. The primary analysis will be the change in the forced expiratory volume in 1 s according to type 2 inflammatory status (blood eosinophils ≥0.15×109/L and/or FeNO ≥25 ppb) after treatment. Key secondary analyses will compare changes in symptom scores and the proportion of patients achieving a minimal clinically important difference. Exploratory analyses will assess the relationship between clinical, lung function, inflammatory and microbiome parameters; satisfaction plus reliability indices of POC tests; and sex-gender variability in treatment response. Ultimately, this pilot study will serve to plan a larger trial comparing the clinical and biological response to systemic corticosteroids according to inflammatory biomarkers, offering valuable guidance for more personalised therapeutic strategies in asthma attacks. ETHICS AND DISSEMINATION: The protocol has been approved by the Research Ethics Committee of the CIUSSS de l'Estrie-CHUS, Sherbrooke, Quebec, Canada (#2023-4687). Results will be communicated in an international meeting and submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT05870215).
Asunto(s)
Asma , Óxido Nítrico , Humanos , Niño , Proyectos Piloto , Reproducibilidad de los Resultados , Asma/diagnóstico , Asma/tratamiento farmacológico , Biomarcadores , Corticoesteroides/uso terapéutico , Estudios Observacionales como AsuntoRESUMEN
The clk-1 gene of the nematode Caenorhabditis elegans encodes an evolutionarily conserved enzyme that is necessary for ubiquinone biosynthesis. Loss-of-function mutations in clk-1, as well as in its mouse orthologue mclk1, increase lifespan in both organisms. In nematodes, clk-1 extends lifespan by a mechanism that is distinct from the insulin signaling-like pathway but might have similarities to calorie restriction. The evolutionary conservation of the effect of clk-1/mclk1 on lifespan suggests that the gene affects a fundamental mechanism of aging. The clk-1/mclk1 system could allow for the understanding of this mechanism by combining genetic and molecular investigations in worms with studies in mice, where age-dependent disease processes relevant to human health can be modeled.
Asunto(s)
Envejecimiento/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de la Membrana/genética , Animales , Caenorhabditis elegans/genética , Dieta , Humanos , Metabolismo de los Lípidos/genética , Longevidad/genética , Pérdida de Heterocigocidad/genética , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxigenasas de Función Mixta , Mutación , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Temperatura , Ubiquinona/administración & dosificación , Ubiquinona/biosíntesis , Ubiquinona/metabolismoRESUMEN
PURPOSE: To determine the role of eukaryotic translation initiation factor 5A (eIF5A) in TNF-alpha-induced apoptosis of lamina cribrosa (LC) cells. METHODS: LC cells were isolated from optic nerve heads of eyes of two human donors. The cells were treated with TNF-alpha and camptothecin, a TNF synergist, and the incidence of apoptosis was scored by Hoechst staining. Expression of eIF5A protein in response to camptothecin or a combination of camptothecin and TNF-alpha was determined by Western blot analysis. The ability of small inhibitory (si)RNAs directed against eIF5A to protect LC cells from TNF-alpha-induced apoptosis was determined by Hoechst and TUNEL staining of transfected LC cells. RESULTS: TNF-alpha and camptothecin synergized to induce greater than two times more apoptosis in LC cells than when the cells were treated with TNF-alpha or camptothecin separately. Expression of eIF5A protein increased significantly after 8 hours of exposure to TNF-alpha and camptothecin, but not in response to camptothecin alone. siRNAs directed against eIF5A reduced apoptosis of LC cells in response to TNF-alpha and camptothecin by between 35% and 69%, as determined by Hoechst staining. An siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also reduced apoptosis of LC cells by 42%. TUNEL of transfected LC cells treated with TNF-alpha and camptothecin revealed an 80% reduction in apoptosis with siRNA against eIF5A. CONCLUSIONS: TNF-alpha, in synergy with camptothecin, induces apoptosis in human LC cells. eIF5A is upregulated by LC cells in response to TNF-alpha, and siRNAs against eIF5A protect LC cells from apoptosis. Thus, eIF5A appears to be a novel proapoptotic protein in the TNF pathway and a possible target for treatment of glaucoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Disco Óptico/patología , Factores de Iniciación de Péptidos/fisiología , Proteínas de Unión al ARN/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Adolescente , Anciano , Anciano de 80 o más Años , Western Blotting , Camptotecina/farmacología , Células Cultivadas , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Disco Óptico/metabolismo , ARN Interferente Pequeño/metabolismo , Esclerótica/patología , Transfección , Regulación hacia Arriba , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death.