Here, we provide evidence that miR-10 regulates endothelial cell behavior and we demonstrate that loss of miR-10 severely affects angiogenesis in vivo. By directly targeting both FLT1 and its soluble splice isoform sFLT1, miR-10 functions to promote KDR-mediated signaling following VEGF exposure, and can titrate pro-angiogenic signaling to fine-tune endothelial cell proliferation, migration, and adhesion.
In zebrafish embryos, we found that loss of miR-10 stalls ISV growth during angiogenesis. ISVs in MO-miR-10-injected embryos had fewer endothelial cells than did controls, indicating reduced proliferation, while increasing miR-10 levels produced ISVs with more endothelial cells but without ISV hyperbranching. Pathfinding of dorsally growing ISVs is controlled somewhat independently of VEGF-A. For example, semaphorin-plexinD1 signaling from the adjacent somites acts repulsively to guide endothelial cells during ISV growth .48
We do not have evidence that increased miR-10 alters these signals, consistent with sprout guidance not being affected in miR-10 overexpressing endothelial cells. However, similar to what was observed previously, augmented angiogenic potential in miR-10 overexpressing larvae expressed in increased proliferative response and thereby in increased endothelial cell number per ISV during angiogenesis, suggesting augmented angiogenic potential.49
Furthermore, miR-10 deficiency in HUVECs disturbed angiogenic behavior in vitro, while increased miR-10 augmented some aspects of angiogenesis. Importantly, the angiogenic potential of miR-10-deficient HUVECs in a matrigel xenograft model was decreased, underscoring the importance of miR-10 in regulating angiogenesis.
We identified FLT1 and its soluble splice variant sFLT1 as direct targets of miR-10, and demonstrated that miR-10 depletion results in elevated FLT1 mRNA levels in zebrafish and increased FLT1 and sFLT1 protein levels in HUVECs. FLT1 is rigidly inserted in the plasma membrane and intrinsically modulates proliferation and migration, while sFLT1 functions extracellularly to spatially modulate VEGF availability and thereby regulate angiogenesis and vessel branching. Mice lacking FLT1 display endothelial cell overgrowth, blood vessel disorganization and hyper-phosphorylated KDR.6
Conversely, FLT1 activation causes defects in angiogenesis and endothelial cell proliferation in response to VEGF coincident with reduced KDR phosphorylation, indicating that FLT1 negatively modulates KDR-mediated pro-angiogenic signaling.8
In HUVECs, we showed that miR-10 depletion and low-dose VEGF stimulation led to decreased phosphorylation of KDR. This confirms that the upregulation of FLT1 and sFLT1 protein by miR-10 knockdown antagonizes KDR stimulation, most likely due to the higher affinity of both FLT1s for VEGF. Additionally, treatment with the KDR-specific inhibitor SU5416 mimicked the miR-10 knockdown phenotype in zebrafish, suggesting that the observed angiogenesis defects in miR-10-deficient zebrafish is caused by diminished KDR function.
Consistent with previous reports, we demonstrated that overexpression of FLT1 in zebrafish causes reduced ISV sprouting with vessels stalled halfway towards the dorsal root.51
This phenotype is identical to that of miR-10-depleted embryos, suggesting that FLT1/sFLT1 upregulation upon miR-10 knockdown critically contributes to the observed angiogenesis defect. In addition to increased cell numbers per ISV, FLT1 deficiency can result in excessive segmental vessel branching, an effect we did not observe in miR-10 duplex injected larvae.51
This discrepancy is likely attributed to the fact that excess miR-10 causes hypomorphic effects by only moderately reducing, rather than completely depleting, FLT1, leaving sufficient protein to influence endothelial cell division without affecting vessel sprouting. Ultimately, the ability to partially rescue the vascular phenotype in miR-10-deficient embryos with concomitant reduction of FLT1 levels suggests that the abnormal angiogenic behavior in endothelial cells lacking miR-10 is, to a great extent, caused by the increase in FLT1 protein.
FLT1 is unlikely to be the only important direct target of miR-10 in endothelial cells, given that FLT1 knockdown incompletely rescues angiogenesis in MO-miR-10-injected zebrafish and incompletely restores proliferation in HUVECs lacking miR-10 at later stages. HOXD10 was described as a very important target of miR-10 in the context of cancer invasion and metastasis.21
In endothelial cells, HOXD10 overexpression can inhibit angiogenesis52
, while Shen et al.24
recently demonstrated that the known angiogenic or anti-angiogenic potential of thrombin or heparin, respectively, is mediated in part through regulation of miR-10b and HOXD10. Our data establish FLT1 as a novel target for miR-10 in endothelial cell biology during development and suggest that miR-10 co-regulates multiple targets to modulate the angiogenic behavior of vascular endothelial cells. Interestingly, recent reports showed that heparin treatment is accompanied by a strong induction of sFLT1 protein.53
We found that high-dose VEGF could not rescue the proliferation defect or the reduced angiogenic behavior of HUVECs lacking miR-10, despite the rescue of KDR phosphorylation. In mice, FLT1-depletion can be partially rescued by expressing an FLT1 variant lacking the tyrosine kinase domain.10
Furthermore, proliferation and branching of vessels derived from FLT1-mutant ESCs can be rescued by soluble sFLT1.8
These findings suggest that FLT1 predominantly functions as a sequestering receptor for VEGF, preventing it from binding to KDR, and that the tyrosine kinase domain may be dispensable for FLT1 function during early development. However, others showed that signaling downstream of FLT1 is particularly important for antagonizing KDR-mediated pro-angiogenic signaling without affecting KDR or MAPK phosphorylation.46
Our data favor a dual role of FLT1/sFLT1 in (1) titrating the dose and spatial availability of VEGF, particularly through sFLT1, and (2) a distinct KDR-antagonizing signaling cascade downstream of FLT1. Further studies are needed to dissect the signaling events downstream of FLT1.
With miR-10, we provide evidence of a miRNA that fine-tunes the tightly balanced process of vascular formation by targeting an important growth factor receptor. Our study establishes miR-10 as an important new target to modulate angiogenesis. As FLT1, sFLT1, and miR-10 are expressed in various tissues and are proposed to be involved in the pathogenesis of various diseases including cancer, studies linking miR-10 levels with FLT1 expression in diseased tissues could provide further mechanistic insights and open new opportunities for novel therapeutic approaches.56–58