RESUMEN
Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.
Asunto(s)
Fibrosis Pulmonar Idiopática/inmunología , Pulmón/patología , Activación de Macrófagos , Macrófagos Alveolares/inmunología , Animales , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Bleomicina/inmunología , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/inmunología , Receptor 1 de Quimiocinas CX3C/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/citología , Pulmón/inmunología , Macrófagos Alveolares/metabolismo , Masculino , Ratones , Análisis de Secuencia de ARN/métodos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Análisis de la Célula Individual/métodos , Regulación hacia ArribaRESUMEN
Endothelial cell (EC) activation underlies many vascular diseases, including pulmonary arterial hypertension (PAH). Several members of the E-twenty six (ETS) family of transcription factors are important regulators of the gene network governing endothelial homeostasis, and their aberrant expression is associated with pathological angiogenesis. The goal of this study was to determine whether deficiencies of the ETS family member, Friend leukemia integration 1 transcription factor (FLI1), and its closest homolog, ETS-related gene (ERG), are associated with PAH. We found that endothelial ERG was significantly reduced in the lung samples from patients with PAH, as well as in chronically hypoxic mice. Functional studies revealed that depletion of ERG or FLI1 in human pulmonary ECs led to increased expression of inflammatory genes, including IFN genes, whereas genes regulating endothelial homeostasis and cell-cell adhesion were down-regulated. Simultaneous knockdown of both ERG and FLI1 had synergistic or additive effects on the expression of these genes, suggesting that ERG and FLI1 coregulate at least a subset of their target genes. Functionally, knockdown of ERG and FLI1 induced cell monolayer permeability with a potency similar to that of vascular endothelial growth factor. Notably, stimulation of ECs with Toll-like receptor 3 ligand poly(I:C) suppressed ERG expression and induced ERG dissociation from the IFNB1 promoter, while promoting signal transducers and activators of transcription 1 (STAT1) recruitment. Consistent with the up-regulation of inflammatory genes seen in vitro, Erg and Fli1 double-heterozygote mice showed increased immune cell infiltration and expression of cytokines in the lung. In conclusion, loss of ERG and FLI1 might contribute to the pathogenesis of vascular lung complications through the induction of inflammation.
Asunto(s)
Endotelio Vascular/metabolismo , Homeostasis , Pulmón/irrigación sanguínea , Proteínas Oncogénicas/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Regulador Transcripcional ERG/metabolismo , Animales , Enfermedad Crónica , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Heterocigoto , Homeostasis/efectos de los fármacos , Homeostasis/genética , Humanos , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipoxia/complicaciones , Hipoxia/genética , Hipoxia/patología , Interferón beta/genética , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Proteínas Oncogénicas/genética , Neumonía/complicaciones , Neumonía/genética , Neumonía/patología , Poli I-C/farmacología , Regiones Promotoras Genéticas/genética , Proteína Proto-Oncogénica c-fli-1/genética , Arteria Pulmonar/patología , Factor de Transcripción STAT1/metabolismo , Regulador Transcripcional ERG/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
To harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Here, model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drug would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native secondary structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that better recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to better maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inmunoconjugados , Neoplasias Pancreáticas , Albúminas , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pulmonary fibrosis is characterized by pathological scaring of the lung. Similar to other fibrotic diseases, scar formation is driven by excessive extracellular matrix deposition by activated, proliferative, and migratory fibroblasts. Currently, the two most widely used chemotaxis and cell migration assays are the scratch assay and the transmembrane invasion assay. Here we present a gap closure assay that employs commercially available cell lines, equipment and reagents and is time efficient as well as straightforward. The protocol uses an Oris pro cell migration assay 96-well plate with a dissolvable plug in the center of each well to create a cell free area at the time of seeding. Cell repopulation of the empty zone is captured via light microscopy at different time points and quantified with free image analysis software. The clear advantages of this assay in comparison to similar protocols are the use of uncomplicated cell culture methods and the ability to image the experiment throughout.
RESUMEN
Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.
Asunto(s)
Asma/metabolismo , Interleucina-17/metabolismo , Contracción Muscular/efectos de los fármacos , Hipersensibilidad Respiratoria/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/efectos de los fármacos , Anciano , Animales , Asma/tratamiento farmacológico , Asma/fisiopatología , Progresión de la Enfermedad , Femenino , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/farmacología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Contracción Muscular/fisiología , Hipersensibilidad Respiratoria/tratamiento farmacológico , Análisis de Secuencia de ARN/métodos , Índice de Severidad de la Enfermedad , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Systemic sclerosis (scleroderma, SSc) is a devastating fibrotic disease with few treatment options. Fumaric acid esters, including dimethyl fumarate (DMF, Tecfidera; Biogen, Cambridge, MA), have shown therapeutic effects in several disease models, prompting us to determine whether DMF is effective as a treatment for SSc dermal fibrosis. We found that DMF blocks the profibrotic effects of transforming growth factor-ß (TGFß) in SSc skin fibroblasts. Mechanistically, we found that DMF treatment reduced nuclear localization of transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP) proteins via inhibition of the phosphatidylinositol 3 kinase/protein kinase B (Akt) pathway. In addition, DMF abrogated TGFß/Akt1 mediated inhibitory phosphorylation of glycogen kinase 3ß (GSK3ß) and a subsequent ß-transducin repeat-containing proteins (ßTRCP) mediated proteasomal degradation of TAZ, as well as a corresponding decrease of TAZ/YAP transcriptional targets. Depletion of TAZ/YAP recapitulated the antifibrotic effects of DMF. We also confirmed the increase of TAZ/YAP in skin biopsies from patients with diffuse SSc. We further showed that DMF significantly diminished nuclear TAZ/YAP localization in fibroblasts cultured on a stiff surface. Importantly, DMF prevented bleomycin-induced skin fibrosis in mice. Together, our work demonstrates a mechanism of the antifibrotic effect of DMF via inhibition of Akt1/GSK3ß/TAZ/YAP signaling and confirms a critical role of TAZ/YAP in mediating the profibrotic responses in dermal fibroblasts. This study supports the use of DMF as a treatment for SSc dermal fibrosis.