Modulating endothelial function and vascular regeneration through inhibition of microRNA-762

Background and Purpose
In order to enhance the healing and regeneration following myocardial infarction (MI), a well-coordinated angiogenic response becomes crucial. 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 investigation.
This project aims to investigate the effects of microRNA-762 (miR-762) on vascular endothelial function to further characterize their potential to promote a therapeutic avenue for vascular regeneration.

Methods
First, miR-762 was analyzed in vivo in different cardiovascular-relevant animal models, as ApoE knockout mice to mirror atherosclerosis and in the tissue of a wire-induced vascular restenosis model in mice by qRT-PCR. Additionally, miR-762 expression was assessed in human umbilical vein endothelial cells (HUVECs), human coronary artery endothelial cells (HCAECs), and human coronary artery smooth muscle cells (HCASMCs). The role of miR-762 in cellular functions, such as migration and proliferation, was assessed through scratch wound and BrdU assays. Apoptosis was determined by quantifying histone complexes. Potential miR-762 downstream targets were identified from in silico research and their expression levels were evaluated on mRNA level after interfering with miR-762. In consecutive experiments influence of miR-762 on angiogenic capacity was assessed.

Results
MiR-762 is significantly upregulated in vivo in the ApoE knockout mice atherosclerosis model (p<0.0001, 2 weeks vs 6 months) and in the wire-induced injury model (p<0.0001, control vs 10 and 21 days after injury) over time. In vitro, miR-762 is higher expressed in endothelial cells (EC) than in HCASMC. Under hypoxic conditions, miR-762 is significantly upregulated in ECs after 72h (p<0.05). Transfecting with both pre-miR-762 and anti-miR-762 showed high transfection efficiency after 48 hours. The level of miR-762 did not have any effect on proliferation. However, downregulation of miR-762 significantly improved migration capacity after 4 hours (p<0.001) and after 6 hours (p<0.0001) in ECs and reduced apoptosis in ECs. The transfection with pre-miR-762 and anti-miR-762 did not have any influence on the morphological aspects of ECs.
Systematic in silico research identified several targets for miR-762 as FAM3A, NF2, RNF4, PHLDA1, CERS1 and MAP4K2 that may play a role in the context of vascular functions. Based on mRNA expression analysis, significant downregulation of NF2 (p<0.05) was observable after pre-miR transfection as well as significant upregulation of NF2 (p<0.05) after anti-miR transfection.

Conclusion
Based on our findings, it appears that miR-762 could affect endothelial cell function. However, to definitively determine the precise function of miR-762, additional experiments will need to be conducted. The next stage of our research will focus on exploring the role of miR-762 in angiogenesis. Additionally, we will analyze its expression following myocardial infarction in mice.