RESUMEN
Pulmonary hypertension (PH) is characterized by vascular remodeling predominantly driven by a phenotypic switching in pulmonary artery smooth muscle cells (PASMCs). However, the underlying mechanisms for this phenotypic alteration remain incompletely understood. Here, we identified that RNA methyltransferase METTL3 is significantly elevated in the lungs of hypoxic PH (HPH) mice and rats, as well as in the pulmonary arteries (PAs) of HPH rats. Targeted deletion of Mettl3 in smooth muscle cells exacerbated hemodynamic consequences of hypoxia-induced PH and accelerated pulmonary vascular remodeling in vivo. Additionally, the absence of METTL3 markedly induced phenotypic switching in PASMCs in vitro. Mechanistically, METTL3 depletion attenuated m6A modification and hindered the processing of pri-miR-143/145, leading to a downregulation of miR-143-3p and miR-145-5p. Inhibition of hnRNPA2B1, an m6A mediator involved in miRNA maturation, similarly resulted in a significant reduction of miR-143-3p and miR-145-5p. We demonstrated that miR-145-5p targets Krüppel-like factor 4 (KLF4) and miR-143-3p targets fascin actin-bundling protein 1 (FSCN1) in PASMCs. The decrease of miR-145-5p subsequently induced an upregulation of KLF4, which in turn suppressed miR-143/145 transcription, establishing a positive feedback circuit between KLF4 and miR-143/145. This regulatory circuit facilitates the persistent suppression of contractile marker genes, thereby sustaining PASMC phenotypic switch. Collectively, hypoxia-induced upregulation of METTL3, along with m6A mediated regulation of miR-143/145, might serve as a protective mechanism against phenotypic switch of PASMCs. Our results highlight a potential therapeutic strategy targeting m6A modified miR-143/145-KLF4 loop in the treatment of PH.
Asunto(s)
Adenosina , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Metiltransferasas , MicroARNs , Miocitos del Músculo Liso , Arteria Pulmonar , Factor 4 Similar a Kruppel/metabolismo , Animales , MicroARNs/genética , MicroARNs/metabolismo , Arteria Pulmonar/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Miocitos del Músculo Liso/metabolismo , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratas , Fenotipo , Masculino , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Músculo Liso Vascular/metabolismo , Ratones Endogámicos C57BL , Remodelación Vascular/genética , Ratas Sprague-Dawley , HumanosRESUMEN
BACKGROUND: Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. METHODS: Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. RESULTS: The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-ß1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. CONCLUSIONS: Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.
Asunto(s)
Adenosina , Células Endoteliales , Transición Epitelial-Mesenquimal , Hipertensión Pulmonar , Factores de Transcripción de Tipo Kruppel , Metiltransferasas , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Humanos , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Metilación , Ratones Endogámicos C57BL , Cadherinas/metabolismo , Cadherinas/genética , Masculino , Remodelación Vascular/genética , Células CultivadasRESUMEN
Hypoxia-induced pulmonary hypertension (HPH) is a fatal cardiovascular disease characterized by an elevation in pulmonary artery pressure, resulting in right ventricular dysfunction and eventual heart failure. Exploring the pathogenesis of HPH is crucial, and small noncoding RNAs (sncRNAs) are gaining recognition as potential regulators of cellular responses to hypoxia. In this study, we conducted a comprehensive analysis of sncRNA profiles in eight tissues of male HPH rats using high-throughput sequencing. Our study unveiled several sncRNAs, with the brain, kidney, and spleen exhibiting the highest abundance of microRNA (miRNA), tRNA-derived small RNA (tDR), and small nucleolar RNA (snoRNA), respectively. Moreover, we identified numerous tissue-specific and hypoxia-responsive sncRNAs, particularly miRNAs and tDRs. Interestingly, we observed arm switching in miRNAs under hypoxic conditions and a significant increase in the abundance of 5' tRNA-halves among the total tDRs during hypoxia. Overall, our study provides a comprehensive characterization of the sncRNA profiles in HPH rats.