Exogenous Recombinant Dimeric Neuropilin-1 Is Sufficient to Drive Angiogenesis

Abstract

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A 165 (VEGF-A 165 ), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-␥, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A 165 . This was evidenced by depleting the cell culture of exogenous VEGF-A 165 , and using instead for routine culture VEGF-A 121 , which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A 165 -independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells. Neuropilin-1 (NRP-1)4 is a protein known for playing important functions in neural and vascular systems (reviewed in Refs. 1, 2). Initially, it was described as a regulator of axon collapse and enhancer of angiogenesis. Subsequently, new functions of NRP-1 were characterized and the expanded contemporary view of NRP-1 includes among its functions antigen recognition (3), adhesion via interaction with ␤ integrins (4, 5), activation of latent forms of cytokines (6), control of stem cell differentiation (7-9), and viral infection (10).The majority of studies analyzing NRP-1 function in endothelial cells have focused on the role of the native transmembrane protein. NRP-1 was shown to be abundantly expressed in human, mouse, and chick with highest expression in the vascular endothelium, heart and placenta (11-13). Moreover, it was shown that a homozygous deletion of the Nrp1 gene in mice causes embryonic lethality, because of defects in the vessels and general vascularization (14), while exogenously overexpressed NRP-1 led to formation of excess capillaries and hemorrhages (11).Overexpression of NRP-1 has been observed in the tumor microenvironment, where apart from endothelial cells, the tumor cells themselves were shown to express NRP-1 (15, 16). Current knowledge of NRP-1 places it among the key drivers of angiogenesis (17); however, it must be emphasized that the exact me...

Structures of TraI in solution

“…Our results for TraI generally agree with the TraI molecular envelope calculated from SAXS data by Cheng and colleagues [3]. This envelope is compact and has a volume sufficient to contain most of the TraI molecule.…”

Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells

“…Dose-response experiments identified a specific dose (3.3 nM for Fc-NRP-1 dimer and 500 ng/ml for NRP-1-B) and length of time (4–6 d) for the treatment that gave reproducible results (data not shown and Fig. 3a ) as reported 27,28 . After 4 d of treatment, Fc-NRP-1 dimer significantly increased ( P < 0.01) and NRP-1-B significantly diminished ( P < 0.001) generation of NRP-1 + CD31 + cells ( Fig.…”

“…We modulated NRP-1 expression in these cells with dimeric Fc-NRP-1, a surrogate for membrane NRP-1 (ref. 28), which enhances NRP-1 activity, or the monoclonal antibody, NRP-1-B 27 , which blocks NRP-1 activity. Dose-response experiments identified a specific dose (3.3 nM for Fc-NRP-1 dimer and 500 ng/ml for NRP-1-B) and length of time (4–6 d) for the treatment that gave reproducible results (data not shown and Fig.…”

Neuropilin Functions as an Essential Cell Surface Receptor

“…The Nrp extracellular domain is essential for function. Indeed, the defective angiogenesis observed in the Nrp knock-out mouse can be significantly improved by injecting pregnant mice with a Nrp extracellular domain-Fc fusion (59,60). Both ectodomain shedding and alternative splicing that produces secreted isoforms produce forms of Nrp that can function as endogenous pathway inhibitors (61)(62)(63)(64).…”