Here we describe an evolutionarily conserved miRNA located within an intron of the Slit genes, miR-218, which controls the expression of various Slit-Robo pathway components during development. Dysregulation of Slit-Robo signaling by altered miR-218 levels affects EC function in vitro. We also provide evidence that deficiency in miR-218 dependent Slit-Robo regulation results in aberrant vascular patterning in the mouse retina. Taken together, this study identifies miR-218 as an important regulatory hub, linking transcriptional regulation of Slit genes to post-transcriptional regulation of multiple components of the Slit-Robo pathway, which ultimately influence angiogenesis ().
Proposed mechanism of miR-218 activity during blood vessel patterning
These data are consistent with previous reports of intronic miRNA function, in which the miRNA regulates the same biological process as the protein encoded by the host gene29
. miR-218 may contribute to “fine-tuning” of Slit-Robo pathway genes or generate negative feedback in response to Slit gene activation. It is interesting to speculate that miR-218 may serve to repress the expression of the Robo1/2 receptors in the Slit ligand expressing cells, thereby spatially separating ligand from receptor. Since Robo4 is not a target of miR-218 regulation, it also is possible that miR-218 affects the ratio of Robo1/2 and Robo4 proteins, thereby influencing vascular patterning.
It is currently debated whether the Robo1 and 2 receptors provide a positive or negative influence on EC migration, although Robo4 is generally thought of as a repulsive or stabilizing cue during neural and vascular pathfinding 9, 37
. In our hands, it appears that repression of Robo1/2 and HSPG biosynthetic molecules by miR-218 negatively affects EC migration. In line with these findings, miR-218 was recently reported to also inhibit tumor cell migration and metastasis via repression of Robo138
. The disorganization and reduction of retinal vascular density observed upon miR-218 knockdown appears to be a consequence of migration or cellular adhesion since we did not detect aberrant proliferation (Online Figure VII
The second most enriched biological process targeted by miR-218 includes components of the HS biosynthetic pathway. Here, we show that miR-218 directly targets the C5 epimerase, GLCE, for degradation during vascular development. miR-218 is also predicted to repress 2-O and 6-O sulfotransferases, both of which are critical enzymes, along with GLCE, in regulating the biological activity of HSPGs 13
. HSPGs interact with and regulate the function of many angiogenic growth factors. For example, HSPGs contribute to transport of signaling molecules through the extracellular matrix, formation of growth factor gradients, and stability of ligand-receptor interactions, including Wnt, VEGF, FGF, and Slit-Robo pathways 19, 21, 39–41
. It is conceivable that miR-218 dependent modulation of HSPGs may influence Slit-Robo signaling via multiple mechanisms, including alteration of Slit-Robo affinity or modulating the diffusion radius of Slit from the secreting cell.
Another level of HSPG-dependent regulation involves the localization of HS in the cellular microenvironment. Recent evidence has revealed an important distinction between HSPG presentation to a transmembrane receptor in cis
or in trans
. For example, HS in trans
potentiates VEGFR-mediated angiogenesis; it is hypothesized that HS presented by an adjacent cell delays VEGFR internalization and increases the efficiency and duration of activity of the bound receptor 14
. It is interesting to speculate that miR-218 might influence the HSPG microenvironment, thereby affecting the activity of various growth factor receptors on neighboring cells.
The Slit-Robo signaling pathway was initially identified as a guidance cue controlling the crossing of the midline by axons 42
. Multiple studies report the interdependence of vascular and neural patterning during development 1
. Although our study reveals a direct role of miR-218 in EC migration in vitro, it remains possible that the effect of miR-218 on retinal angiogenesis is at least partially due to an influence on axonal pathfinding. It is also possible that miR-218 contributes to the dynamic regulation of the Robo receptors at the neural midline, directly influencing axonal pathfinding. While miR-218 is robustly expressed in the brain and eye, it is interesting to note that miR-218 is expressed at much lower levels than the Slit1 and 2 genes in bladder, kidney and lungs. This suggests the possibility that miR-218 is inefficiently processed in these tissues, which may result in altered miR-218-dependent Slit-Robo activity.
Slit1 and Robo1 were recently identified as part of the transcriptional network activated during the switch to tumor angiogenesis 43
. This process appears to be especially sensitive to miRNA control. In addition to the present study, miRNA-126, –378, –296, and the 17–92 cluster have been shown to regulate various aspects of vessel formation, remodeling and tumor angiogenesis 44
. Other physiological processes that might respond to alterations in miR-218 levels and Slit-Robo signaling include oxygen-induced retinopathy, light-induced choroidal angiogenesis, VEGF-induced vascular permeability and tumor angiogenesis. Further analysis of miR-218 during vascular pathologies and tumor angiogenesis may define miR-218 as an intriguing candidate for therapeutic manipulation in the treatment of vascular diseases.