MicroRNAs (miRs) are small noncoding RNAs which negatively regulate the expression of targeted mRNA transcripts. Some miRs are already known to modulate angiogenesis, including miR-503 which we showed contributes towards defective post-ischemic angiogenesis in diabetes. miR-503 is under the control of the p75 neurotrophin receptor (p75NTR), which is upregulated in endothelial cells (ECs) of diabetic models and contributes to impaired post-ischaemic reparative angiogenesis in diabetes.Purpose
The aims of this study were: 1) to elucidate the miR signature of p75NTR in ECs; and 2) to characterize the impact of p75NTR—regulated miRs on EC function.Methods
Human umbilical vein ECs (HUVECs) were infected with Ad.p75NTR or Ad.Null and used in a miR array. Array results were confirmed by qPCR using validated miR primers. To mimic advanced diabetes, where hyperglycaemia is accompanied by tissue starvation, HUVECs were cultured in high D-glucose (HG, 25 mM) and low growth factors (LGF). To modulate miR expression, HUVECs were transfected with pre-miR precursor, anti-miR inhibitor or negative control. Transfected cells were prompted in proliferation (BrdU incorporation) and in vitro angiogenesis on Matrigel assays. Predicted miR target genes were searched using the databases Microcosm and TargetScan. Finally, relative miR-expression was quantified in limb muscles of type 2 diabetic db/db mice and non-diabetic controls.Results
Relative expression of miR-30c-2* was upregulated three-fold in p75NTR—transduced HUVECs (p < 0.01 vs. Null-HUVECs) and five-fold in HG/LGF compared to culture in normal conditions (p < 0.01). miR-503 (already studied by us) and miR-30c2* were the only miRs upregulated in p75NTR—HUVECs. Overexpression of miR-30c-2* in HUVECs impaired both proliferation and angiogenesis (40% decrease in proliferation and 25% decrease in tube length of Matrigel network, p < 0.01 for both). In addition, miR-30c-2* was upregulated three-fold in the adductor muscles of db/db mice (p < 0.05 vs. non-diabetic controls). Bioinformatics analysis identified the cell cycle regulator MCM7 as a putative miR-30c-2* target. MCM7 mRNA levels were decreased in p75NTR—HUVECs (by 57% vs. Null-HUVECs, p < 0.05) and in HUVECs cultured in HG/LGF. Overexpression of miR-30c-2* also reduced MCM7 mRNA (by 64%, p < 0.01).Conclusion
Our data suggest that the p75NTR-induced miRNA miR-30c-2* may act as an anti-angiogenic mediator in the context of diabetes by inhibiting the cell cycle regulator MCM7. miR-30c-2* may prove to be a novel therapeutic target for diabetes-induced impairment of post-ischaemic reparative neovascularisation.