Post-transcriptional modifications play pivotal roles in various pathological processes and ischemic disorders. However, the role of N7-methylguanosine (m7G), particularly m7G in mRNA, on post-ischemic angiogenesis remains largely unknown. Here, we identified that methyltransferase like 1 (METTL1) was a critical candidate responsible for a global decrease of m7G within mRNA from the ischemic tissues. The in vivo gene transfer of METTL1 improved blood flow recovery and increased angiogenesis with enhanced mRNA m7G upon post-ischemic injury. Increased METTL1 expression using plasmid transfection in vitro promoted HUVECs proliferation, migration, and tube formation with a global increase of m7G in mRNA. Mechanistically, METTL1 promoted VEGFA mRNA translation in an m7G methylation-dependent manner. Our findings emphasize a critical link between mRNA m7G and ischemia and provide a novel insight of targeting METTL1 in the therapeutic angiogenesis for ischemic disorders, including peripheral arterial disease.

Background: Post-ischemic angiogenesis is critical for blood flow recovery and ischemic tissue repair. N6-methyladenosine (m6A) plays essential roles in numerous biological processes. However, the impact and connected mechanism of m6A on post-ischemic angiogenesis are not fully understood. Methods: AlkB homolog 5 (ALKBH5) was screened out among several methyltransferases and demethylases involved in dynamic m6A regulation. Cardiac microvascular endothelial cells (CMECs) angiogenesis and WNT family member 5A (WNT5A) stability were analyzed upon ALKBH5 overexpression with adenovirus or knockdown with small interfering RNAs in vitro. The blood flow recovery, capillary, and small artery densities were evaluated in adeno-associated virus (AAV)-ALKBH5 overexpression or ALKBH5 knockout (KO) mice in a hind-limb ischemia model. The same experiments were conducted to explore the translational value of transient silencing of ALKBH5 with adenovirus. Results: ALKBH5 was significantly upregulated in hypoxic CMECs and led to a global decrease of m6A level. ALKBH5 overexpression further reduced m6A level in normoxic and hypoxic CMECs, impaired proliferation, migration, and tube formation only in hypoxic CMECs. Conversely, ALKBH5 knockdown preserved m6A levels and promoted angiogenic phenotypes in hypoxic but not in normoxic CMECs. Mechanistically, ALKBH5 regulated WNT5A expression through post-transcriptional mRNA modulation in an m6A-dependent manner, which decreased its stability and subsequently impeded angiogenesis in hypoxic CMECs. Furthermore, ALKBH5 overexpression hindered blood flow