The development of tools to control the cell activity after transplantation is of utmost importance in Regenerative Medicine. For clinical efficacy, it is imperative that cells survive and engraft into the host tissue. However, a significant number of cells (typically >70% in few days) die or are lost within hours after transplantation. Therefore, the implementation of new strategies to promote cell survival after transplantation is crucial.
Both cord blood-derived endothelial progenitor cells (CB-EPCs) and VEGF-A have been proposed as therapeutic tools to induce angiogenesis for the treatment of ischemic diseases. VEGF-A promotes the formation of microvessels that differ structurally and functionally depending on its presentation. Free VEGF forms big, leaky vessels while immobilized VEGF facilitates the formation of highly branched networks. Immobilization of VEGF alters VEGFR-2 signaling and subsequent downstream pathways. Improved understanding of the molecular targets underlying the action mechanism of immobilized VEGF might help to develop new platforms to increase post-transplantation cell survival and regenerative capacity.
We showed that conjugated VEGF modulates cord blood-derived endothelial cell (CB-EC) activity by decreasing the expression miR-17 both in vitro and in vivo. To mimic the down-regulation of miR-17 by immobilized VEGF and understand the effect of miR-17 down-regulation on cell survival, CB-ECs were transfected with antagomiR-17. AntagomiR-17 increased CB-EC survival at least 1.5 times (n = 6) compared to scrambled antagomiR or pro-angiogenic miRNAs reported in the literature (e.g. miR-424 and miR-132) and sprout formation on Matrigel at least 2 times (n = 5) under hypoxia conditions (0.1% O2). In vivo, antagomiR-17 accelerated hemodynamic recovery of the whole limb (n = 12) in unilateral limb ischemia obtained by occlusion of the left femoral artery (Figure 1).
Blood flow recovery evaluated by Laser Doppler analysis was significantly higher 21 days after surgery in antagomiR-17 group compared to scrambled antagomiR and EBM-2 groups. Immunohistochemical analyses showed around 1.4 times increase in the capillary density of skeletal muscle in antagomiR-17 condition. In order to determine the gene target and potential pathway involved in the biological effect of antagomiR-17, next generation mRNA sequencing and data mining were used. Gene expression results and siRNA experiments on CB-ECs revealed ZNF652 and CDKN1A as the targets of antagomiR-17-mediated EC survival. Interestingly, CDKN1A was reported in the literature as an anti-apoptotic molecule.
Overall, our results show that the immobilized VEGF increases the EC survival both in vitro and in vivo by downregulating miR-17, of which main gene targets are CDKN1A and ZNF652 to exert its function in the survival of the cells.
- vascular endothelial growth factor
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