MicroRNA-762 inhibition enhances angiogenesis and vascular regeneration by targeting XAF1 and CMPK2

Abstract A well-coordinated angiogenic response becomes crucial to enhance the healing and regeneration following myocardial infarction (MI). Given the absence of effective current therapeutic strategies for promoting post-MI angiogenesis, there is a demand for novel approaches. Recent research has pinpointed microRNAs (miRNAs) as potential regulators of angiogenesis, opening up new avenues for further interventions. This project aims to investigate the effects of microRNA-762 (miR-762) on vascular endothelial function to characterize their potential in promoting a therapeutic avenue for vascular regeneration. miR-762 expression was analyzed in vivo in MI mouse models using qRT-PCR. Its inhibition via anti-miR transfection was evaluated in human endothelial cells (ECs) and fibroblasts. Functional assays assessed migration, proliferation, cell death, and angiogenesis (tube and sprout formation). Potential targets were identified by RNA sequencing and validated via qRT-PCR and western blot. MiR-762 is significantly upregulated in vivo in the ApoE knockout mice (p0.0001, 2 weeks vs 6 months) and in the wire-induced injury model (p0.0001, control vs 10 and 21 days after injury) over time, indicating a first substantial role in vascular pathologies. Consequently, we assessed its regulation after MI in C57BL/6J mice. miR-762 is significantly upregulated 24 hours in cardiac endothelial cells (p0.05), while no regulation is observable after 72 hours. In vitro, comparable results are observable as under hypoxic conditions, miR-762 is significantly upregulated in ECs after 24 hours (p0.05), but not after 72 hours. Functional cell analysis revealed that the level of miR-762 did not affect proliferation. However, downregulation of miR-762 significantly improves migration capacity after after 6 hours (p0.0001) in ECs and fibroblasts and significantly reduces apoptosis in ECs (p0.05). The angiogenic capacity is significantly improved in ECs after inhibition of miR-762 (p0.05). Further, On morphological level, the downregulation of miR-762 leads to significantly larger nuclei of ECs (p0.05). RNA sequencing revealed that CMPK2 (p0.001), XAF1 (p0.05), and APOL6 (p0.01) are significantly downregulated by anti-miR-762 transfection. These results were confirmed by qRT-PCR and western blot analyses (p0.05). All regulated targets are connected to IFNy and the JAK/STAT signaling pathway which throughout shows important regulations as for example STAT1 and IFNyR1. In conclusion, miR-762 inhibition enhances vascular regeneration by improving endothelial migration and angiogenesis, likely through indirect targeting of XAF1 and CMPK2. Further studies will explore the molecular mechanisms and assess the therapeutic potential of miR-762 inhibition in vivo.