In this study we sought to identify the balance of angiogenic receptors, their regulation by ligand, and to apply the cell-by-cell analysis to identify any variation in receptor density across endothelial cells. When ensemble averaged surface densities were analyzed, the order of magnitude of each receptor was similar across the blood macrovascular, blood microvascular and lymphatic microvascular cells studied, with levels of VEGFR1, VEGFR2, and VEGFR3 being one order of magnitude less than the levels of NRP1. Furthermore, our studies revealed that each of the endothelial cells had a significantly higher surface density of VEGFR2 compared to VEGFR1 (p<0.001); only one study (Scatchard analysis) reports VEGFR1 and VEGFR2 densities supporting this finding, with 4200 VEGFR1/HUVEC and 12,400 VEGFR2/HUVEC [57
]; however, these densities are approximately 2-fold higher than our observations. On average, LECs have a higher surface density of VEGFR3 than VEGFR1 and VEGFR2. This was consistent with the functions of these receptors, VEGFR3 being the major signaling receptor for lymphangiogenesis [53
]. The NRP1 densities were found to be an order of magnitude higher than any of the VEGFRs. These high levels of NRP1 are consistent with the previous studies of NRP1 surface density and total NRP1 protein [21
]. Furthermore, the blood endothelial cells presented twice the number of NRP1 compared to lymphatic endothelial cells, suggesting a stronger role for NRP1 in blood endothelium.
All endothelial cells tested responded similarly to the sustained VEGF165
treatment: increasing VEGFR1 surface expression and decreasing VEGFR2 surface density. The long-term VEGF-C treatment caused the downregulation of VEGFR2 and VEGFR3 in HUVECs and LECs. The downregulation of VEGFR2 by VEGF165
and the downregulation of VEGFR2 and VEGFR3 by VEGF-C is consistent with the ligand-induced internalization and degradation exhibited by tyrosine kinase receptors [18
]. However, the upregulation of VEGFR1 that we observe goes against this principle, and further supports the premise that VEGFR1 generally serves as an anti-angiogenic receptor.
Despite their fundamental role in lining blood and lymphatic vessels, endothelial cells display heterogeneity in morphology, protein expression, and function [29
]. Furthermore, stochasticity in gene expression can lead to heterogeneity in signaling within homogenous cell populations [31
], thereby affecting angiogenic signaling. Using cell-by-cell analysis, we characterize such variability in receptor densities across the endothelial cells and find that in addition to regulating receptor density, elevated VEGF165
levels increase the heterogeneity of VEGFR1 surface density while decreasing the heterogeneity of VEGFR2 surface density, while VEGF-C decreases the heterogeneity of VEGFR2 and VEGFR3 surface densities (). These VEGF-induced shifts in density and heterogeneity have significant implications to the proportion of signaling that occurs through heterodimerized versus homodimerized receptors. Recently, VEGF mediated dimerization of VEGFR2/3, VEGFR2/2, and VEGFR3/3 was reported in human saphenous ECs, with the short-term VEGF-A treatment (8 min) inducing VEGFR2/2 homodimerization, and short-term VEGF-C inducing VEGFR2/3 and VEGFR3/3 dimerization [59
]. The VEGF-induced shift in receptor balance following long-term treatment may similarly shift the percentage of heterodimeric and homodimeric complexes, increasing VEGFR1/2, due to the increased availability of VEGFR1, and decreasing VEGFR2/3, VEGFR2/2, and VEGFR3/3 complexes due to their collective downregulation. Since the homodimers of VEGFR2/2 display pro-angiogenic signaling, while heterodimers between VEGFR1/2 are not only functional but may affect pro-angiogenic signaling through VEGFR2 [60
], further research should identify the role of long-term VEGF treatment in regulating the balance of these angiogenic receptors.
Fig. 5 Schematic of VEGF165 regulation of receptor density and distribution. As VEGFA165 levels increase, VEGFR1 surface density increases heterogeneity of VEGFR1 surface density, decreases VEGFR2 heterogeneity and surface density and decreases NRP1 surface (more ...)
The cell-by-cell analysis also uncovered the VEGF-induced downregulation of NRP1 in all endothelial cells tested and an upregulation of VEGFR3 in MECs, all of which were masked through averaging. The downregulation of NRP1 is consistent with NRP1 being a pro-angiogenic receptor. As such, recent anti-angiogenic cancer therapies target NRP1 [63
]. Furthermore, the cell-by-cell analysis revealed slight ligand-induced changes in receptor densities, which may affect angiogenic signaling that requires further examination (VEGF165
on VEGFR3 in MECs, VEGF-C on NRP1 in HUVECs and VEGFR1 in HUVECs and LECs).
We hypothesized that quantifying the surface density of angiogenic receptors (VEGFR1, VEGFR2, and NRP1) would inform us on the balance between pro-angiogenic (VEGFR2) and anti-angiogenic or modulatory (VEGFR1) signaling. The 30–80% decrease in pro-angiogenic surface receptors (VEGFR2 and NRP1) following the VEGF165
treatment, ~65% decrease in pro-lymphangiogenic surface receptors (VEGFR2 and VEGFR3) following the VEGF-C treatment, and 40–70% increase in anti-angiogenic surface receptor (VEGFR1) following the VEGF165
treatment, suggests that in the long-term, VEGFRs are either upregulated or downregulated in response to VEGF to achieve an angiogenic balance or angiostasis. A recent study reported similar VEGF regulation of total VEGFR1 and VEGFR2 in endothelial cells and determined pathways critical for these regulatory effects [67
]. Future work should determine whether in vivo application of VEGF165
also pushes the system to angiostasis. If so, then the application of VEGF for pro-angiogenic therapy could be combined with a therapy that concomitantly increases the VEGFR2 and NRP1 surface densities while decreasing the VEGFR1 surface density. Possible approaches may include targeting the proteins regulating VEGFR2 degradation such as the JNK/c-Jun pathway [67
], c-Cbl [18
], or Grb10 [68
]. Alternatively, the machinery mediating VEGFR2 internalization may be targeted, such as vascular endothelial cadherin or dynamin [69
]. Altogether, these data provide a fundamental understanding of how VEGFRs affect the angiogenic balance and can serve as the basis of angiogenesis modeling studies.
To our knowledge, this is the first application of quantitative flow cytometry to characterize cell surface density of angiogenic receptors. In this study, we used several cell types, including endothelial macrovascular, microvascular and lymphatic cells. In the future this technique could be applied to characterize receptor density in various tissues under physiological and pathological conditions.