Modulating endothelial function and vascular regeneration through inhibition of microRNA-762 by targeting XAF1 and CMPK2

Aim
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 on vascular endothelial cell function to characterize their potential in promoting a therapeutic avenue for vascular regeneration.

Methods
Expression of miR-762 was analyzed in vivo in different cardiovascular-relevant animal models, as myocardial infarction in C57BL/6J mice by qRT-PCR. Further, miR-762 expression was assessed in human endothelial cells (ECs) and human coronary artery smooth muscle cells (HCASMCs). The role of miR-762 after pre- or anti-miR transfection in cellular functions, such as migration, proliferation, and cell death were assessed. In addition, the angiogenic capacity was quantified by tube formation assays. Potential miR-762 downstream targets were identified by RNA sequencing and validated by qRT-PCR and western blot analysis. Further, immunofluorescence microscopy was performed. 

Results
MiR-762 is significantly upregulated in vivo in ApoE knockout mice (p<0.0001, 2 weeks vs 6 months) feeded by an high fat diet and in a wire-induced restenosis model (p<0.0001, control vs. 21 days after injury) over time. Moreover, miR-762 is significantly upregulated 24 hours after MI in mice in cardiac ECs (p<0.05), while no regulation is observable 72 hours after MI. In vitro, under hypoxic conditions, miR-762 is significantly upregulated in ECs after 24 hours (p<0.05), but not after 72 hours. Transfection with both pre-miR-762 or anti-miR-762 showed a high transfection efficiency. Functional cell analysis revealed that, the level of miR-762 did not affect proliferation. However, downregulation of miR-762 significantly improves migration capacity after 6 hours (p<0.0001) in ECs and significantly reduces apoptosis in ECs (p<0.05). The angiogenic capacity is significantly improved in ECs after transfection with anti-miR (p<0.05). On morphological aspects, the downregulation of miR-762 leads to significantly larger nuclei of ECs (p<0.05). In general, no changes are observed in HCASMCs, and ECs transfected with pre-miR-762.

RNA sequencing revealed that CMPK2 (p<0.0001), XAF1 (p<0.05), and APOL6 (p<0.05) are significantly downregulated by transfection with anti-miR in ECs. These results were confirmed by qRT-PCR. Western blot analyses show that CMPK2 and XAF1 are also significantly downregulated on protein level by downregulation of miR-762 in ECs (p<0.05). Further, the targets are significantly higher expressed in ECs than in HCASMCs (p<0.05).

Conclusion
In summary, we demonstrate that miR-762 is a promising target to improve vascular regeneration after MI that predominantly acts in vascular endothelial cells by indirectly targeting XAF1 and CMPK2. Its therapeutic downregulation improves endothelial cell functions as an enhanced angiogenic, migration capacity and reduced apoptosis. However, to determine the exact way how the downregulation of miR-762 affects cellular pathways, additional experiments will need to be conducted. The next stage of our research will focus on exploring this together with the performance of the first therapeutic in vivo experiments.