m⁶A Modification-mediated GRAP Regulates Vascular Remodeling in Hypoxic Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling induced by human pulmonary arterial smooth muscle cell (HPASMC) proliferation, migration, and apoptosis resistance. m⁶A (N-methyladenosine) is the most prevalent RNA posttranscriptional modification in eukaryotic cells. However, its role in PAH remains elusive. We designed this study to investigate whether m⁶A modification and its effector proteins play a role in pulmonary vascular resistance. Lung samples were used to profile m⁶A concentrations in control subjects and patients with PAH. Bioinformatics analysis, real-time PCR, immunohistochemistry, and Western blotting were used to determine the role of m⁶A effectors in PAH. The biological effects of GRAP modified by m⁶A were investigated using in vitro and in vivo models. Furthermore, RIP-PCR was used to assess the writers and readers of GRAP. In this study, we revealed that m⁶A-modified GRAP mRNA was upregulated in PAH lung samples, cHx/Su-induced mouse models, and hypoxia-stimulated HPASMCs; however, GRAP mRNA and protein were abnormally downregulated. Functionally, overexpression of GRAP drastically alleviated the proliferative and invasive ability of PAH HPASMCs through inhibition of the Ras/ERK signaling pathway in vitro and in vivo. In addition, METTL14 (methyltransferase-like 14) and the m⁶A binding protein YTHDF2 were significantly increased in PAH. Moreover, we found that m⁶A-modified GRAP mRNA was recognized by YTHDF2 to mediate the degradation. GRAP expression was consistently negatively correlated with METTL14 and YTHDF2 in vivo and in vitro. Taken together, for the first time, our findings highlight the function and therapeutic target value of GRAP and extend our understanding of the importance of RNA epigenetics in PAH.