Our work establishes two major findings: 1) NRP1 expression occurs early in the differentiation of both murine and human stem cells to endothelial cells, and 2) Both the Semaphorin and VEGF binding functions of NRP1 are required for the differentiation of stem cells to ECs in the vasculogenesis model system. The findings illustrate the utility of the stem cell model for testing effects on vasculogenesis, and indicate a surprising role for both the Semaphorin and VEGF binding activities of NRP1 in vasculogenesis.
Our results show that NRP1 is expressed early in the differentiation of stem cells, in cells that have begun the process of forming vascular, hematopoietic and cardiac precursors (mesodermal cell types). Prior findings in the literature suggested that NRP1 expression occurs early in development, as the NRP1 and 2 double knockout mouse is lethal after 8.5 days of development due to failure of blood vessel formation (9
). Since mesoderm formation occurs during 6–7 days of development, and prior work has shown that VEGFR2 expression begins during this period in a population of cells that form both blood vessels and the earliest hematopoietic cells (4
), it seemed plausible that NRP1 expression would begin due to its function as a co-receptor for VEGFR2. Studies in Zebrafish have also shown that NRP1 was expressed at the end of gastrulation and during somitogenesis, stages of development where vasculogenesis occurs, and is a comparable time point to our ES cell model (18
). Additionally, our experiments show that NRP1 expression occurs early in both murine and human stem cells, illustrating that this event is conserved across species.
A secondary finding of our results was that the growth factors BMP4 and basic fibroblast growth factor are sufficient for expression of NRP1, while the BMP antagonist Noggin inhibits NRP1 expression. These findings are not surprising in that the same conditions are sufficient to drive expression of VEGFR2 from murine and human stem cells. However, the stem cell model of vasculogenesis may facilitate further investigation of the mechanisms controlling NRP1 expression. This is of clinical relevance as NRP1 expression has been documented in several types of cancer, is associated with worse outcomes with cancer, and the regulation of NRP1 expression in cancer may represent a therapeutic target for cancer therapy (19
NRP1 function blocking antibodies significantly decreased the number of endothelial cells derived from differentiating stem cells () and antagonism of both the Semaphorin and VEGF binding activities resulted in decreased endothelial cell formation, a novel finding. Prior work in several developmental and tumor angiogenesis models indicated that antagonism of the VEGF binding function of NRP1 primarily blocked vessel formation in these contexts (21
). Our results are novel in that they indicate that both the VEGF and Semaphorin binding domains of NRP1 appear to be required for differentiation of endothelial cells from stem cells. Our findings are somewhat limited by the fact that the NRP1 function blocking antibodies did not completely inhibit differentiation of endothelial cells. The reason for this is not known, however it is possible that NRP2 expression is also present in a portion of the cells and that NRP2 may have an overlapping function with NRP1 in the differentiation of stem cells to endothelial cells. The NRP1 function blocking antibodies used in these experiments have no effect on NRP2 function (21
Prior results have indicated that antagonism of the VEGF binding activity of NRP1 only modestly impaired VEGFR2 activation. Work by Pan and coworkers indicate that the Semaphorin and VEGF domain binding antibodies disrupt formation of the VEGF/NRP1/VEGFR2 signaling complex (21
). Therefore, the effect of both VEGF and Semaphorin binding function blocking NRP1 antibodies are likely acting to inhibit signaling events that are coupled to NRP1 and VEGFR2 complex formation, or events downstream of this complex, but not simply by impairing VEGFR2 activity alone. These findings suggest an important function of the VEGFR2/NRP1 molecular complex in the initial differentiation of stem cells to endothelial cells, and is a novel mechanism of this pathway, as known components of VEGF signaling downstream of VEGFR2 (ERK, p38 and AKT) are unaffected by either of the blocking antibodies in proliferating endothelial cell cultures. One likely candidate for the downstream effects of the VEGF/VEGFR2/NRP1 signaling complex is Neuropilin Interacting Protein (NIP), also known as GIPC, a PDZ domain containing protein that interacts directly with NRP1 (22
). However, the interaction of GIPC with the VEGF/VEGFR2/NRP1 complex have not been extensively explored to identify a precise mechanism, and the role of this signaling complex in vasculogenesis remains open to further investigation. Greater understanding of the VEGF/VEGFR2/NRP1 signaling complex and events outside of canonical VEGF signaling represents the next advance in our understanding of VEGF signaling and further development of therapeutics in this field. Additionally, extension of our finding that NRP1 expression and function as a critical event in de novo endothelial cell formation to adult stem cell models including tissue specific cancer stem cells and cardiac stem cells may represent a therapeutic target in ischemic and malignant vasculogenesis.
In conclusion, our results indicate that NRP1 is expressed early on in the differentiation of stem cells to endothelial cells, and that NRP1 function is required for the differentiation of ECs from stem cells. Surprisingly, both the Semaphorin and VEGF binding functions of NRP1 are required for EC formation suggesting that events downstream of VEGF/VEGFR2/NRP1 complex formation and not canonical VEGF signaling alone are required for this process.