MSCs, which offer immense potential for cell-based tissue regeneration, have the capability to differentiate along vascular cell lineages [1
]. Previously, we have shown that multipotent human MSCs express NRP-1 and PDGFRs, but not VEGFRs, and that PDGFs regulate MSC proliferation and migration, and the smooth muscle-specific cytoskeleton [2
]. PDGFRα is an essential regulator of mesenchymal tissue formation in early embryonic development [38
], and both PDGFRs contribute to vessel-wall development and remodelling following injury [42
]. The essential contribution of NRP-1 to vascular development and neovascularization is also well documented [11
] and, although its mechanisms of action remain incompletely understood, it is thought to regulate cell-surface-receptor clustering and signalling in a ligand-dependent manner. Our discovery that NRP-1 regulates the phosphorylation and signalling responses of PDGFRs, especially PDGFRα, sheds important light on fundamental cellular mechanisms of tissue development and neovascularization.
NRP-1 co-immunoprecipitated and co-localized with p-PDGFRs, and this association was significantly increased in the presence of growth-factor ligands, indicating that the PDGFR cross-talk with NRP-1 that we have identified may occur through a receptor-bridging mechanism. Indeed, in-vitro
-binding studies indicate that PDGFRα and NRP-1 do not interact directly, but PDGF ligands, PDGF-AA, PDGF-BB and VEGF-A165
, all bind NRP-1 (Supplementary Figure S4 at http://www.BiochemJ.org/bj/427/bj4270029add.htm
). PDGF-AA-mediated PDGFRα responses were particularly dependent upon NRP-1, implying that NRP-1 may be indispensable for PDGFRα function in tissue development and remodelling. PDGFRβ dependence on NRP-1 was also significant, so NRP-1 must regulate PDGFRβ-dependent smooth muscle cell migration, proliferation and differentiation during vessel-wall maturation and repair.
While NRP-1 is a transmembrane protein, immunofluorescence analysis demonstrated that the majority of NRP-1 in permeabilized MSCs and HUVECs was localized intracellularly. Exposure to VEGF-A165
has been shown to promote NRP-1 on the surface of HUVECs to internalize, with immunofluorescence analysis of the permeablized HUVECs demonstrating NRP-1 predominantly localized around perinuclear regions [49
]. Thus a similar mechanism resulting in rapid ligand-induced NRP-1 internalization may occur in MSCs.
MSCs readily formed extensive networks in Matrigel™ and CAM assays, highlighting their potential to contribute to blood-vessel formation. Co-localization of NRP-1 with phosphorylated PDGFRs occurred prominently in these networks, and the essential role for NRP-1/PDGFR cross-talk in network formation was confirmed by knockdown of NRP-1 which caused dramatically reduced PDGFR phosphorylation and grossly disrupted network formation. Prominent pericellular co-localization of NRP-1 with PDGFRα during early network formation suggests that this relationship is particularly important in initiating cellular changes leading to network formation. In MSCs, VEGF-A also induced NRP-1/PDGFR co-localization, similar to VEGF-A-induced NRP-1 co-localization with VEGFR2 in HUVECs. Since in HUVECs, NRP-1 co-localized with both VEGFR2 and PDGFRs in response to VEGF-A, NRP-1/PDGFR cross-talk is likely to contribute to endothelial functions mediated by VEGF-A. Our MSCs do not express VEGFRs, so their response to PDGFs and VEGF-A ligands is channelled through PDGFRs, but in endothelial and other cells expressing both VEGFRs and PDGFRs, the relative abundance of each receptor, local ligand concentrations and receptor affinities may combine to modify NRP-1-dependent receptor signals.
The essential contribution of PDGFRα to the formation of embryonic mesoderm and mesenchymal tissues is well documented [34
], and we have demonstrated a high PDGFRα/PDGFRβ ratio in our MSCs [4
]. PDGFRα-null mice die at around E10 (where E is embryonic day) due to vascular and other defects [40
], whereas conditional null mice highlight that both PDGFRs are essential for early yolk sac vascular development [41
]. The NRP-1-knockout mouse is also embryonic lethal, with major yolk sac and embryonic vascular defects, dying between E10 and E12.5 [50
]. Thus the functional cross-talk between NRP-1 and both PDGFRs, especially PDGFRα, that we have identified, suggests a fundamental developmental relationship between these receptors.
In summary, in the present study we have shown ligand-dependent cross-talk between NRP-1 and phosphorylated PDGFRs that controls receptor signalling, migration, network formation and proliferation of MSCs. We have thus identified NRP-1 as an essential co-receptor for PDGFR signalling, which may critically contribute to the formation of blood vessels and other mesenchymal tissues. This mechanism may be exploited in the application of MSCs in tissue regeneration.