Smpd2 m⁶A modification contributes to hypoxic pulmonary hypertension via an alveolar epithelial cell...

Pulmonary vascular remodelling induced by the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is a core pathological feature of hypoxic pulmonary hypertension (HPH), but its exact pathogenesis remains poorly understood. This study aimed to elucidate the functional role and intrinsic regulatory mechanisms of an alveolar epithelial cell (AEC) senescence-associated phenotype in the pathogenesis of HPH.

A 4-week hypoxia-induced HPH rat model and an in vitro hypoxia-induced AEC senescence-associated phenotype model were established. The effects of hypoxia on AECs were analysed by evaluating the senescence-associated phenotype and mitochondrial function. In addition, a Transwell coculture system and AEC-conditioned medium were used to evaluate the effects of hypoxia-exposed AECs with a senescence-associated phenotype on PASMC proliferation. The key signalling pathways associated with the hypoxia-induced AEC senescence-associated phenotype were identified through transcriptome sequencing and subsequent KEGG pathway enrichment analysis. The roles of the Smpd2/ceramide axis in the hypoxia-induced AEC senescence-associated phenotype, PASMC proliferation and HPH progression were explored via the administration of METTL3- and Smpd2-targeting siRNAs, AAV6-shSmpd2 for lung-directed Smpd2 silencing in vivo, the neutral sphingomyelinase (nSMase) inhibitor GW4869, the mitochondria-targeted antioxidant Mito-TEMPO (MT) and the NAD⁺ precursor β-nicotinamide mononucleotide (NMN). Finally, MeRIP-qPCR, RIP-qPCR, mRNA stability assays, and dual-luciferase assays were employed to elucidate the mechanisms of m⁶A RNA methylation in AECs with the senescence-associated phenotype.

Chronic hypoxia induced typical manifestations of HPH in rats, accompanied by a more pronounced AEC senescence-associated phenotype. In cultured human AECs, hypoxia upregulated METTL3, which increased Smpd2 mRNA stability through m⁶A modification recognized by IGF2BP2. These changes were associated with ceramide accumulation and mitochondrial dysfunction and contributed to the AEC senescence-associated phenotype in vitro. AECs with this phenotype released soluble and/or vesicular paracrine signals that were associated with abnormal PASMC proliferation, contributing to vascular remodelling and right ventricular hypertrophy. In cultured human AECs, NMN pretreatment, Smpd2 knockdown, and METTL3 knockdown attenuated the hypoxia-induced AEC senescence-associated phenotype. In hypoxia-exposed rats, NMN supplementation, lung-directed Smpd2 silencing, and pharmacological nSMase inhibition with GW4869 attenuated the AEC senescence-associated phenotype and alleviated HPH progression.

This study provides preclinical evidence that, in hypoxia-exposed rats and cultured human AECs/PASMCs, a hypoxia-induced AEC senescence-associated phenotype may contribute to PASMC proliferation and HPH development. Mechanistically, in cultured human AECs, hypoxia exposure increased METTL3-mediated m⁶A modification of Smpd2 mRNA, which increased its stability in an m⁶A- and IGF2BP2-dependent manner and upregulated Smpd2 expression. The resulting increase in ceramide accumulation was associated with mitochondrial dysfunction and the senescence-associated phenotype in AECs. In this preclinical setting, AECs with this phenotype may further contribute to PASMC proliferation and HPH development. The findings of this study provide preclinical data supporting a candidate mechanism in HPH pathogenesis and identify candidate targets for future validation in human PH and cell-specific in vivo models.