RESUMEN

Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

Asunto(s)

Células Endoteliales/metabolismo , Ferroptosis/efectos de los fármacos , Hipertensión Pulmonar/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Toxinas Bacterianas/metabolismo , Células Cultivadas , Ciclohexilaminas/farmacología , Regulación hacia Abajo/efectos de los fármacos , Ferroptosis/genética , Proteína HMGB1/metabolismo , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/patología , Inflamación/metabolismo , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Monocrotalina/toxicidad , Fenilendiaminas/farmacología , Ratas Sprague-Dawley , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

AIMS: Although interferon regulatory factor 7 (IRF7) has known roles in regulating the inflammatory response, vascular smooth muscle cell proliferation, and apoptosis, its role in the pathogenesis of pulmonary hypertension (PH) is unclear. We hypothesized that IRF7 overexpression could inhibit pulmonary vascular remodeling and slow the progression of PH. MAIN METHODS: IRF7 mRNA and protein levels in the lung samples and pulmonary artery smooth muscle cells (PASMCs) isolated from monocrotaline (MCT)-induced PH rats were assessed. We evaluated the effects of IRF7 on inflammation, proliferation, and apoptosis using an in vivo MCT-induced PH rat model and in vitro methods. KEY FINDINGS: We noted decreased IRF7 mRNA and protein levels in the pulmonary vasculature of MCT-induced PH rats. IRF7 upregulation attenuated pulmonary vascular remodeling, decreased the pulmonary artery systolic pressure, and improved the right ventricular (RV) structure and function. Our findings suggest that nuclear factor kappa-Bp65 (NF-κBp65) deactivation could confer pulmonary vasculature protection, reduce proinflammatory cytokine (tumor necrosis factor-α, interleukin 6) release, and decrease PASMC proliferation and resistance to apoptosis via deactivating transcription factor 3 (ATF3) signaling. ATF3 deactivation induced the downregulation of the proliferation-dependent genes proliferating cell nuclear antigen (PCNA), cyclin D1, and survivin, coupled with increased levels of B cell lymphoma-2-associated X protein (Bax)/B cell lymphoma-2 (Bcl2) ratio, and cleaved caspase-3 in PASMCs. SIGNIFICANCE: Our findings showed that IRF7 downregulation could initiate inflammation via NF-κBp65 signaling, causing PASMC proliferation via ATF3 signaling pathway activation. Therefore, IRF7 could be a potential molecular target for PH therapy.

Asunto(s)

Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/patología , Inflamación/patología , Factor 7 Regulador del Interferón/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Factor de Transcripción Activador 3/metabolismo , Animales , Apoptosis , Caspasa 3/metabolismo , Proliferación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Ciclina D1/metabolismo , Dependovirus/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Hemodinámica , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Inflamación/complicaciones , Pulmón/patología , Pulmón/fisiopatología , Masculino , Monocrotalina , Miocitos del Músculo Liso/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de Señal , Survivin/metabolismo , Regulación hacia Arriba , Remodelación Vascular , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Background: Inflammation and altered immunity contribute to the development of pulmonary arterial hypertension (PH). The alpha 7 nicotinic acetylcholine receptor (α7nAChR) possesses anti-inflammatory activities. The current study was performed to investigate the effects of a selective α7nAChR agonist, PNU-282987, on controlling a monocrotaline (MCT)-induced rat model of PH and explored the underlying mechanisms. Methods: Sprague-Dawley rats were injected with MCT and treated with PNU-282987 at the prevention (starting 1 week before MCT) and treatment (starting 2 weeks after MCT) settings. Four weeks after MCT injection, hemodynamic changes, right ventricular structure, and lung morphological features were assessed. Enzyme-linked immunosorbent assay, Western blot and qRT-PCR were performed to assess levels of inflammatory cytokines and NLRP3 (Nod-like receptor family pyrin domain-containing 3) inflammasome pathway in the rat lung tissues. In addition, the lung macrophage line NR8383 was used to confirm the in vivo data. Results: Monocrotaline injection produced PH in rats and downregulated α7nAChR mRNA and protein expression in rat lung tissues compared to sham controls. Pharmacological activation of α7nAChR by PNU-282987 therapy improved the rat survival rate, attenuated the development of PH as assessed by remodeling of pulmonary arterioles, reduced the right ventricular (RV) systolic pressure, and ameliorated the hypertrophy and fibrosis of the RV in rats with MCT-induced PH. The expression of TNF-α, IL-6, IL-1ß, and IL-18 were downregulated in rat lung tissues, which implied that PNU-282987 therapy may help regulate inflammation. These protective effects involved the inhibition of the NLRP3 inflammasome. In vitro assays of cultured rat lung macrophages confirmed that the anti-inflammation effect of PNU-282987 therapy may contribute to the disturbance of NLRP3 inflammasome activation. Conclusion: Targeting α7nAChR with PNU-282987 could effectively prevent and treat PH with benefits for preventing ongoing inflammation in the lungs of rats with MCT-induced PH by inhibiting NLRP3 inflammasome activation.