RESUMEN
Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.
Asunto(s)
Artritis Reumatoide/genética , Lesión Pulmonar/genética , Arginina Deiminasa Proteína-Tipo 2/genética , Fibrosis Pulmonar/genética , Sindecano-2/genética , Animales , Anticuerpos Antiproteína Citrulinada/genética , Artritis Reumatoide/complicaciones , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Bleomicina/toxicidad , Citrulinación/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/complicaciones , Lesión Pulmonar/patología , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/patología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Factor de Transcripción Sp1/genéticaRESUMEN
BACKGROUND: Acute lung injury (ALI) is a common lung disorder that affects millions of people every year. The infiltration of inflammatory cells into the lungs and death of the alveolar epithelial cells are key factors to trigger a pathological cascade. Trophoblast stem cells (TSCs) are immune privileged, and demonstrate the capability of self-renewal and multipotency with differentiation into three germ layers. We hypothesized that intratracheal transplantation of TSCs may alleviate ALI. METHODS: ALI was induced by intratracheal delivery of bleomycin (BLM) in mice. After exposure to BLM, pre-labeled TSCs or fibroblasts (FBs) were intratracheally administered into the lungs. Analyses of the lungs were performed for inflammatory infiltrates, cell apoptosis, and engraftment of TSCs. Pro-inflammatory cytokines/chemokines of lung tissue and in bronchoalveolar lavage fluid (BALF) were also assessed. RESULTS: The lungs displayed a reduction in cellularity, with decreased CD45+ cells, and less thickening of the alveolar walls in ALI mice that received TSCs compared with ALI mice receiving PBS or FBs. TSCs decreased infiltration of neutrophils and macrophages, and the expression of interleukin (IL) 6, monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) in the injured lungs. The levels of inflammatory cytokines in BALF, particularly IL-6, were decreased in ALI mice receiving TSCs, compared to ALI mice that received PBS or FBs. TSCs also significantly reduced BLM-induced apoptosis of alveolar epithelial cells in vitro and in vivo. Transplanted TSCs integrated into the alveolar walls and expressed aquaporin 5 and prosurfactant protein C, markers for alveolar epithelial type I and II cells, respectively. CONCLUSION: Intratracheal transplantation of TSCs into the lungs of mice after acute exposure to BLM reduced pulmonary inflammation and cell death. Furthermore, TSCs engrafted into the alveolar walls to form alveolar epithelial type I and II cells. These data support the use of TSCs for the treatment of ALI.
Asunto(s)
Lesión Pulmonar Aguda , Trofoblastos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/terapia , Células Epiteliales Alveolares , Animales , Líquido del Lavado Bronquioalveolar , Lipopolisacáridos , Pulmón , Ratones , Ratones Endogámicos C57BL , Células MadreRESUMEN
Rationale: CD148/PTRJ (receptor-like protein tyrosine phosphatase η) exerts antifibrotic effects in experimental pulmonary fibrosis via interactions with its ligand syndecan-2; however, the role of CD148 in human pulmonary fibrosis remains incompletely characterized.Objectives: We investigated the role of CD148 in the profibrotic phenotype of fibroblasts in idiopathic pulmonary fibrosis (IPF).Methods: Conditional CD148 fibroblast-specific knockout mice were generated and exposed to bleomycin and then assessed for pulmonary fibrosis. Lung fibroblasts (mouse lung and human IPF lung), and precision-cut lung slices from human patients with IPF were isolated and subjected to experimental treatments. A CD148-activating 18-aa mimetic peptide (SDC2-pep) derived from syndecan-2 was evaluated for its therapeutic potential.Measurements and Main Results: CD148 expression was downregulated in IPF lungs and fibroblasts. In human IPF lung fibroblasts, silencing of CD148 increased extracellular matrix production and resistance to apoptosis, whereas overexpression of CD148 reversed the profibrotic phenotype. CD148 fibroblast-specific knockout mice displayed increased pulmonary fibrosis after bleomycin challenge compared with control mice. CD148-deficient fibroblasts exhibited hyperactivated PI3K/Akt/mTOR signaling, reduced autophagy, and increased p62 accumulation, which induced NF-κB activation and profibrotic gene expression. SDC2-pep reduced pulmonary fibrosis in vivo and inhibited IPF-derived fibroblast activation. In precision-cut lung slices from patients with IPF and control patients, SDC2-pep attenuated profibrotic gene expression in IPF and normal lungs stimulated with profibrotic stimuli.Conclusions: Lung fibroblast CD148 activation reduces p62 accumulation, which exerts antifibrotic effects by inhibiting NF-κB-mediated profibrotic gene expression. Targeting the CD148 phosphatase with activating ligands such as SDC2-pep may represent a potential therapeutic strategy in IPF.
Asunto(s)
Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/genética , Pulmón/metabolismo , Animales , Antibióticos Antineoplásicos/toxicidad , Autofagia/efectos de los fármacos , Autofagia/genética , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Técnicas In Vitro , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Noqueados , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Fragmentos de Péptidos/farmacología , Fenotipo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal , Sindecano-2/farmacología , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The impact of lipotoxicity on the development of lung fibrosis is unclear. Saturated fatty acids, such as palmitic acid (PA), activate endoplasmic reticulum (ER) stress, a cellular stress response associated with the development of idiopathic pulmonary fibrosis (IPF). We tested the hypothesis that PA increases susceptibility to lung epithelial cell death and experimental fibrosis by modulating ER stress. Total liquid chromatography and mass spectrometry were used to measure fatty acid content in IPF lungs. Wild-type mice were fed a high-fat diet (HFD) rich in PA or a standard diet and subjected to bleomycin-induced lung injury. Lung fibrosis was determined by hydroxyproline content. Mouse lung epithelial cells were treated with PA. ER stress and cell death were assessed by Western blotting, TUNEL staining, and cell viability assays. IPF lungs had a higher level of PA compared with controls. Bleomycin-exposed mice fed an HFD had significantly increased pulmonary fibrosis associated with increased cell death and ER stress compared with those fed a standard diet. PA increased apoptosis and activation of the unfolded protein response in lung epithelial cells. This was attenuated by genetic deletion and chemical inhibition of CD36, a fatty acid transporter. In conclusion, consumption of an HFD rich in saturated fat increases susceptibility to lung fibrosis and ER stress, and PA mediates lung epithelial cell death and ER stress via CD36. These findings demonstrate that lipotoxicity may have a significant impact on the development of lung injury and fibrosis by enhancing pro-death ER stress pathways.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácido Palmítico/toxicidad , Fibrosis Pulmonar/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Antígenos CD36/deficiencia , Antígenos CD36/fisiología , Células Epiteliales/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ácido Palmítico/administración & dosificación , Ácido Palmítico/farmacocinética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patologíaRESUMEN
OBJECTIVE: Venous valves are essential but are prone to injury, thrombosis, and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs nonvalve sites to elucidate biologic differences associated with vein valves. METHODS: Tissue explants of fresh human saphenous veins were prepared, and the migration of SMCs from explants of valve sinus vs nonvalve sinus areas was measured. Proliferation and death of SMCs were determined by staining for Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling. Proliferation and migration of passaged valve vs nonvalve SMCs were determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs nonvalve intima-media was determined by RNA sequencing. RESULTS: Valve SMCs demonstrated greater proliferation in tissue explants compared with nonvalve SMCs (19.3% ± 5.4% vs 6.8% ± 2.0% Ki67-positive nuclei at 4 days, respectively; mean ± standard error of the mean, five veins; P < .05). This was also true for migration (18.2 ± 2.7 vs 7.5 ± 3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P < .0001). Cell death was not different (39.6% ± 16.1% vs 41.5% ± 16.0% terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, respectively, at 4 days, five veins). Cultured valve SMCs also proliferated faster than nonvalve SMCs in response to platelet-derived growth factor subunit BB (2.9 ± 0.2-fold vs 2.1 ± 0.2-fold of control, respectively; P < .001; n = 5 pairs of cells). This was also true for migration (6.5 ± 1.2-fold vs 4.4 ± 0.8-fold of control, respectively; P < .001; n = 7 pairs of cells). Blockade of fibroblast growth factor 2 (FGF2) inhibited the increased responses of valve SMCs but had no effect on nonvalve SMCs. Exogenous FGF2 increased migration of valve but not of nonvalve SMCs. Unlike in the isolated, cultured cells, blockade of FGF2 in the tissue explants did not block migration of valve or nonvalve SMCs from the explants. Thirty-seven genes were differentially expressed by valve compared with nonvalve intimal-medial tissue (11 veins). Peptide-mediated inhibition of SEMA3A, one of the differentially expressed genes, increased the number of migrated SMCs of valve but not of nonvalve explants. CONCLUSIONS: Valve compared with nonvalve SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathologic lesion formation in valves. Whereas FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in the valve wall tissue remain unclear but may be among the differentially expressed genes discovered in this study. One of these genes, SEMA3A, mediates a valve-specific inhibitory effect on the injury response of valve SMCs.