NRP1 regulates VEGFA-mediated permeability in an organotypic manner

NRP1 has been studied extensively as a co-receptor of the VEGFA/VEGFR2 pathway, and its ability to modulate VEGFR2 signalling in developmental and pathological angiogenesis has been clearly established. However, results concerning the role of NRP1 in VEGFA-mediated vascular permeability have remained contradictory [24,25,26,27,28,29]. Here, Nrp1fl/fl; Cdh5CreERT2 (Nrp1 iECKO) and Nrp1fl/fl, Cre-negative littermate (Control) mice (Fig. 1A) were used to study the EC-specific role of NRP1 in VEGFA-induced vascular leakage. A 75% reduction in NRP1 protein levels was achieved in lung tissue, chosen for analysis due to the high EC content (Supplementary Fig. 1A). In agreement, immunofluorescent staining and RNA in-situ hybridisation (ISH) analyses showed a significant reduction in NRP1 expression in ECs of the ear and back skin (Fig. 1B, C; Supplementary Fig. 1B–E). Using a highly sensitive assay combining intravital microscopy and atraumatic intradermal microinjection in the ear dermis of Nrp1 iECKO and Nrp1fl/fl control mice, VEGFA-induced vascular leakage was significantly increased in venules following the loss of NRP1 in endothelial cells (Fig. 1D-E; Video 1–2) [30]. Additionally, we measured the rate of dextran extravasation at sites of leakage, which demonstrated that Nrp1 iECKO mice exhibit more profuse leakage at each site (Fig. 1F), indicative of increased disruption of EC-EC junctions. These data were supported by a modified Miles’ assay, with intradermal administration of VEGFA in the ear skin, leading to an increased extravasation of Evans Blue dye in Nrp1 iECKO mice versus their littermate Nrp1fl/fl controls (Fig. 1G). These findings suggest that NRP1 exerts a stabilising role by suppressing VEGFA-induced endothelial junction disruption in the ear dermis. This finding is in contrast to reports showing NRP1 to be a positive regulator of VEGFA-mediated vascular leakage [24, 25, 27].

Fig. 1

NRP1 regulates VEGFA-mediated permeability in an organotypic manner. A Schematic illustration of experimental design to induce recombination in Nrp1fl/fl; Cdh5CreERT2 (Nrp1 iECKO) mice. Nrp1fl/fl were used as control. B and C RNA in-situ hybridisation (ISH) analysis of Nrp1 expression in the ear dermis (B) and back skin (C) of Nrp1 iECKO mice. Images highlight Nrp1 mRNA particles (arrows) specific to CD31-positive vessel area and graphs show quantification of Nrp1 mRNA expression (Nrp1 mRNA particle area/vessel area). Scale bar: 50 μm. D Representative images showing leakage of 2000 kDa FITC-dextran (Pseudo-colour) in response to intradermal VEGFA injection in ear dermis of control and Nrp1 iECKO mice. E Leakage sites per vessel length in response to intradermal VEGFA stimulation in the ear skin of control and Nrp1 iECKO mice. n = 6 mice, two or more acquisitions/mouse. F Quantification of 2000 kDa dextran extravasation over time in the ear skin of control and Nrp1 iECKO mice following intradermal VEGFA stimulation. Black lines represent lines of best fit for the slope between leakage initiation and leakage termination. N ≥ 3 mice, two or more acquisitions/mouse, three or more sites/acquisition. G Evans blue leakage following intradermal administration of VEGFA in the ear skin of control and Nrp1 iECKO mice. Top, representative images. Bottom, quantification of Evans blue extravasation shown as VEGFA-induced leakage fold over PBS control (n ≥ 4 mice). H and I Leakage of fixable 2000 kDa FITC dextran in back skin (H) and trachea (I) after systemic administration of VEGFA in control and Nrp1 iECKO mice Left, representative images. Right, quantification of tracer leakage area/vessel area (n ≥ 8 mice, 2 or more fields of view/mouse). J Evans blue leakage with intradermal administration of VEGFA in the back skin of control and Nrp1 iECKO mice. Top, representative images. Bottom, quantification of Evans blue extravasation shown as VEGFA-induced leakage fold over PBS control (n ≥ 4 mice). Error bars; mean ± SEM. Statistical significance: Two-tailed unpaired Student’s t-test and linear regression with ANCOVA. Scale bar: 100 μm unless stated

One possible explanation for these differences compared to previous reports is that the role of NRP1 may be organ dependent, in line with the growing insights into the distinct properties of ECs in different vessel types and vascular beds [31,32,33]. To investigate a potential organotypic role for NRP1 in EC biology, we studied the consequence of Nrp1 knockout on the barrier properties of ECs in different vascular beds.

Nrp1 iECKO and their littermate controls were assessed, first for their basal permeability properties, revealing that loss of EC NRP1 did not significantly alter basal permeability of a 10 kDa dextran tracer (Supplementary Fig. 2A). To asses VEGFA-induced vascular leakage, selected organs were initially assessed for their susceptibility to VEGFA, chosen based on prior experience of different EC barrier properties [31,32,33,34]. Mice were challenged with systemic administration of fluorescent dextrans with or without VEGFA for 30 min before collecting organs and assessing acute vascular leakage microscopically or following solvent-based extraction of dextran and fluorescence spectroscopy. VEGFA-induced vascular leakage was observed in back skin, trachea, kidney, skeletal muscle and heart (Supplementary Fig. 2B, C). Subsequently, VEGFA-induced vascular leakage was assessed in Nrp1 iECKO tissues, demonstating that loss of NRP1 decreased VEGFA-mediated vascular leakage in the trachea and back skin (Fig. 1H, I), but had no effect on kidney, skeletal muscle and heart irrespective of tracer size (70 kDa and 2000 kDa; Supplementary Fig. 2D-E).

Using a Miles’ assay, endothelial cell NRP1’s positive regulation of VEGFA-VEGFR2 signalling in the back skin was confirmed, with a reduction in VEGFA-induced vascular leakage in Nrp1 iECKO mice (Fig. 1J), in keeping with previous publications [24]. These data collectively suggest that endothelial NRP1 has an organ-specific role in controlling VEGFA-mediated vascular permeability. Importantly, endothelial NRP1 is a positive regulator of VEGFA mediated permeability in the trachea and back skin but a negative regulator in the ear skin.

Global inactivation of NRP1 reduces VEGFA-mediated vascular permeability

Our results here, where NRP1 may play positive (trachea and back skin), negative (ear skin) and passive (kidney, skeletal muscle and heart) roles in VEGFA-induced vascular leakage, were collected using EC-specific knockout mice. In previous studies, mouse models of both EC-specific deletion and a globally expressed NRP1 C-terminal deletion mutant were employed [11, 24]. We thus set out to investigate whether global inactivation of NRP1 might differently modify the VEGFA-induced leakage response.

Nrp1fl/fl mice were crossed with ActbCre mice to generate global Nrp1 knock-out mice (Nrp1 iKO) for which Nrp1fl/fl mice were used as controls. Efficient reduction of NRP1 protein in the Nrp1 iKO was confirmed in lung lysates (Fig. 2A, B). Complete loss of NRP1 could also be seen in the ear and back skin of these mice using immunofluorescent staining (Supplementary Fig. 1B, C). Intravital imaging of the ear dermis of Nrp1 iKO mice showed that global loss of NRP1 suppressed VEGFA-induced vascular leakage (Fig. 2C, D; Video 3–4), in contrast to the increased leakage seen in Nrp1 iECKO (see Fig. 1D–G). Furthermore, global Nrp1 knock-out resulted in a ~ 75% reduction in VEGFA-mediated vascular leakage also in the back skin and tracheal vasculatures, similar to that seen in Nrp1 iECKO mice (Fig. 2E, F). Thus, the global loss of NRP1 results in an alignment of leakage phenotype across the studied tissues.

Fig. 2

Global loss of NRP1 reduces VEGFA-mediated vascular permeability. A Schematic illustration of experimental design to induce recombination in Nrp1fl/fl; ActbCre (Nrp1 iKO) mice. Nrp1fl/fl were used as control. B Western blot and quantification of NRP1 protein levels in lung lysates from tamoxifen-treated control and Nrp1 iKO mice (n ≥ 3 mice). C Representative images showing leakage of 2000 kDa FITC dextran (Pseudo-colour) in response to intradermal VEGFA injection in the ear of control and Nrp1 iKO mice. D Leakage sites per vessel length in response to intradermal VEGFA stimulation in the ear skin of control and Nrp1 iKO mice. N = 3 mice, two or more acquisitions/mouse. E and F Leakage of fixable 2000 kDa FITC dextran in back skin (E) and trachea (F) after systemic administration of VEGFA in control and Nrp1 iKO mice. Left, representative images. Right, quantification of tracer leakage area/vessel area (n ≥ 5 mice, 3 or more fields of view/mouse). G VEGFA-induced leakage in the ear skin of Nrp1 iECKO mice treated intradermally with isotype control or NRP1-VEGFA blocking antibody. Left, representative images. Right, quantification of leakage sites per 100 µm of vessel length (n = 3 mice, ≥ 2 acquisitions/mouse). H and I VEGFA-induced leakage of 2000 kDa dextran in back skin (H) and trachea (I) of Nrp1 iECKO mice treated systemically with isotype control or NRP1-VEGFA blocking antibody. Left, representative images. Right, quantification of tracer area/vessel area (n = 3 mice). Error bars; mean ± SEM. Statistical significance: Two-tailed unpaired Student’s t-test. Scale bar: 100 μm

These conclusions were further explored through the use of an antibody that blocks the binding of VEGFA to NRP1 [35]. Administration of this blocking antibody in Nrp1 iECKO mice, where only endothelial cell NRP1 is removed, resulted in suppressed VEGFA-induced vascular leakage in the ear dermis, while vascular leakage was unchanged in the back skin and trachea (Fig. 2G–I; Video 5–6).

These data illustrate that NRP1 modulates VEGFA signaling in an organotypic and cell-specific manner. Notably, we demonstrate that VEGFA-induced leakage in the back skin and trachea is indistinguishable between global and endothelial-specific NRP1 loss, while there is a contrasting phenotype in the ear skin.

NRP1 is heterogeneously expressed in perivascular cells

NRP1 is known to form a heterocomplex with VEGFR2 upon VEGFA binding. Interestingly, VEGFR2/NRP1/VEGFA can assemble as a juxtacrine trans complex, where NRP1 and VEGFR2 are expressed on the surface of adjacent cells, as well as a cis complex, where NRP1 and VEGFR2 are expressed on the same cell [36]. Importantly, even though the kinetics of trans VEGFR2/NRP1 complex formation is slow, these complexes are stable and produce a distinct signalling output compared to the cis configuration [36]. Thus, NRP1 presented in cis or trans produces differential VEGFR2 signalling output upon VEGFA binding. Given the above findings we reasoned that peri-endothelial distribution of NRP1 could be an important modifier of EC VEGFR2 signalling and possibly explain organotypic differences observed in Nrp1 iECKO mice.

To investigate the pattern of NRP1 expression we employed a Pdgfrβ promoter-driven GFP (Pdgfrβ-EGFP) reporter mouse to visualise PDGFRβ-positive perivascular cells. To visualise ECs, and the localisation of NRP1 in the ear skin and back skin from these mice, tissues were immunostained for CD31 and NRP1. NRP1 expression could be seen in both endothelial and perivascular cells, which was lost in both cell types after global Nrp1 deletion in Nrp1 iKO mice, while it remained expressed in perivascular cells in Nrp1 iECKO mice (Fig. 3A; Supplementary Fig. 1B–E). Both arteriolar (Fig. 3B) and capillary (Fig. 3C) PDGFRβ-positive perivascular cells expressed NRP1. The relative NRP1 expression in perivascular cells compared to EC was consistently higher in the ear skin compared to back skin. Strikingly, the low perivascular NRP1 expression in the back skin resulted in a fourfold difference in the perivascular/EC NRP1 ratio when comparing venules of the back skin with the ear skin (Fig. 3D).

Fig. 3

Perivascular expression of NRP1 is heterogeneous. A Representation of NRP1 expression and method of NRP1 quantification in endothelial and perivascular cells of Pdgfrβ-GFP mice. White dashed lines in right image outlines vascular area, space between white and yellow dashed lines represents perivascular area. Note perivascular, NRP1 expressing cells in between yellow and white lines. B–D Images, left, and quantification, right, showing vascular and perivascular NRP1 expression in arterioles (B), capillaries (C) and venules (D) of ear skin and back skin and its relative expression. Error bars; mean ± SEM. Statistical significance: Two-tailed unpaired Student’s t-test. Scale bar: 50 μm

These data thus show that NRP1 is expressed in perivascular cells of the ear skin and back skin. We find however that the ratio of NRP1 expression between ECs and perivascular cells differs between ear skin and back skin, and between different vessel types. In ear skin the perivascular/EC NRP1 ratio is higher compared to the back skin, which may support a higher ratio of trans NRP1/VEGFR2 relative to cis complexes in the ear skin.

NRP1 distribution modifies VEGFA-mediated signalling and vascular leakage

NRP1 modifies VEGFR2 signalling by controlling its internalisation and intracellular trafficking [37, 38]. The presence of NRP1 in trans however, modifies this dynamic by retaining VEGFR2 on the cell surface for longer time periods, altering its signalling output [36].

We thus wished to investigate whether perivascular NRP1 expression might impact VEGFR2 signalling upstream of vascular leakage, and explain the above described organotypic effects of EC NRP1 loss. For this purpose, Nrp1 iECKO mice were crossed with homozygous Vegfr2Y949F/Y949F mice, to produce Vegfr2Y949F/Y949F;Nrp1 iECKO mice that are deficient in both EC NRP1 expression and VEGFR2 signalling upstream of vascular leakage. In these mice, loss of NRP1 protein was efficiently established in lung tissue (Fig. 4A, B). Analysis of VEGFA-mediated vascular leakage in the ear dermis showed that the increased leakage, induced by the loss of EC NRP1, was abrogated by the concurrent loss of the VEGFR2 phosphosite Y949, known to be required for activation of the cytoplasmic tyrosine kinase Src in response to VEGFA (Fig. 4C, D; Video 7–8). These analyses suggest that, in the ear dermis, EC NRP1 negatively regulates VEGFA-induced vascular leakage by modulation of VEGFR2 activation and phosphorylation.

Fig. 4

NRP1 distribution modifies VEGFA-induced vascular permeability. A Schematic illustration of experimental design to induce recombination in Vegfr2 Y949F/Y949F; Nrp1 iECKO mice. Vegfr2 Y949F/Y949F; Nrp1fl/fl mice were used as control. B Western blot and quantification of NRP1 using lung lysates from tamoxifen-treated control and Vegfr2 Y949F/Y949F; Nrp1 iECKO mice (n = 3 mice). C Representative images showing leakage in response to intradermal VEGFA injection in the ear of control Vegfr2 Y949F/Y949F; Nrp1fl/fl mice and Vegfr2 Y949F/Y949F; Nrp1 iECKO mice. D Leakage sites per vessel length in response to intradermal VEGFA stimulation in the ear skin of control Vegfr2Y949F/Y949F; Nrp1fl/fl and Vegfr2Y949F/Y949F; Nrp1 iECKO mice. Note, data in Nrp1fl/fl and Nrp1 iECKO mice from Fig. 1E are also shown for comparative purposes. Data are normalised to appropriate control (n ≥ 6 mice, two or more acquisitions/mouse). E and F Leakage of 2000 kDa dextran in back skin (E) and trachea (F) of Nrp1fl/fl, Nrp1 iECKO, Vegfr2 Y949F/Y949F and Vegfr2Y949F/Y949F; Nrp1 iECKO mice after systemic administration of VEGFA. Left, representative images. Right, quantification of tracer leakage area/vessel area (n ≥ 3 mice, 2 or more fields of view/mouse). Error bars; mean ± SEM. Statistical significance: Two tailed unpaired Student’s t-test and Two-way ANOVA. Scale bar: 100 μm

We next sought to determine the relationship between NRP1 and VEGFR2 Y949 signalling in the trachea and back skin vasculatures. Leakage in Vegfr2Y949F/Y949F;Nrp1 iECKO mice was induced by systemic VEGFA and fluorescent 2000 kDa dextran administration in both Nrp1 iECKO and Vegfr2Y949F/Y949F;Nrp1 iECKO mice, and their appropriate controls. As we observed previously, loss of EC NRP1 reduced vascular leakage in the back skin and trachea by 75%, an effect that was enhanced further by the VEGFR2 Y949F mutation in tracheal vasculature (Fig. 4E, F). Importantly, leakage in trachea and back skin was similarly inhibited in the Vegfr2Y949F/Y949F model as in the combined Vegfr2Y949F/Y949F;Nrp1 iECKO model, revealing that NRP1’s positive regulation of vascular leakage in these tissues is mediated through VEGFR2 Y949. Altogether, these data show that NRP1 presented in trans by perivascular cells in the ear dermis can signal through VEGFR2 to induce vascular leakage, and that VEGFR2 Y949 is vital for mediating NRP1’s effects on endothelial junctions.

Perivascular NRP1 modifies VEGFA-induced signalling

VEGFA/VEGFR2 signalling mediates Src activation and phosphorylation of VE-Cadherin on Y685, required for vascular permeability [39, 40]. We have also recently shown that PLCγ is an important mediator of VEGFA-induced vascular leakage by allowing the production of nitric oxide through eNOS, which in turn modified Src by tyrosine nitration, required for full Src activity [23]. To clarify the impact of perivascular NRP1 on VEGFA-induced signalling we studied VE-Cadherin and PLCγ phosphorylation in Nrp1 iECKO and Nrp1 iKO mice. Perivascular NRP1 expression present in the ear dermis of Nrp1 iECKO mice was lost in Nrp1 iKO mice (Supplementary Fig. 1B, C). In agreement with the enhanced vascular leakage in the ear dermis of Nrp1 iECKO mice (Fig. 1D–G), VEGFA-induced VE-Cadherin phosphorylation at Y685 was enhanced in Nrp1 iECKO mice versus their littermate control (Fig. 5A). No such induction however was seen in Nrp1 iKO mice (Fig. 5B). In the back skin, VE-Cadherin pY685 levels were induced by VEGFA in the control but not in the Nrp1 iECKO, nor the iKO models (Fig. 5C, D), in accordance with the described permeability phenotype (see Fig. 1, panels H-I). Similarly, PLCγ phosphorylation was potentiated in the ear skin of Nrp1 iECKO but not Nrp1 iKO mice (Supplementary Fig. 3A, B), whereas in the back skin, loss of either endothelial or global NRP1 led to a reduction in PLCγ phosphorylation following intradermal VEGFA stimulation (Supplementary Fig. 3C, D).

Fig. 5

Perivascular NRP1 modifies VEGFA-induced signalling. A and B Phosphorylation of VE-Cadherin (VEC) Y685 in response to intradermal PBS or VEGFA injections in the ear dermis of control Nrp1fl/fl and Nrp1 iECKO (A) or Nrp1 iKO (B) mice. Left, representative images. Right, quantification of phosphorylated VEC Y685 (pVEC685) area per total VEC area after VEGFA stimulation, normalised to PBS in the ear dermis of control Nrp1fl/f and Nrp1 iECKO or Nrp1 iKO mice. N ≥ 3 mice, two or more fields of view/mouse. C and D Phosphorylation of VE-Cadherin Y685 in response to intradermal PBS or VEGFA injections in the back skin of control Nrp1fl/f and Nrp1 iECKO (C) or Nrp1 iKO (D) mice. Left, representative images. Right, quantification of phosphorylated VEC Y685 (pVEC685) area per total VEC area after VEGFA stimulation, normalised to PBS, in the back skin of control Nrp1fl/f and Nrp1 iECKO or Nrp1 iKO mice. N ≥ 3 mice, two or more fields of view/mouse. Error bars; mean ± SEM. Statistical significance: Two-way ANOVA. Scale bar: 50 μm

Taken together these data show that NRP1 is expressed by perivascular cells in a tissue-specific manner and that perivascular NRP1 can be an important modulator of VEGFA/VEGFR2 signalling. In the ear dermis, NRP1 is expressed by both endothelial and perivascular cells, forming cis and trans interactions with VEGFR2, respectively. Whilst the cis EC interaction predominates, loss of EC NRP1 promotes trans NRP1/VEGFR2 interaction and potentiates VEGFA-induced signalling regulating vascular leakage (Fig. 6). Meanwhile, due to a paucity of perivascular NRP1 in the trachea and back skin vasculatures, loss of EC NRP1 leads to a loss of NRP1:VEGFR2 complexes, attenuating VEGFA-regulated vascular leakage.

Fig. 6

Schematic model of NRP1’s spatial effects on VEGFA-induced vascular permeability. With global loss of NRP1, VEGFA/VEGFR2 signalling regulating vascular leakage is suppressed and thus leakage is reduced (left). In the presence of EC NRP1, a VEGFA/VEGFR2/NRP1 complex is formed in cis, which has a rapid and transient effect on VEGFR2 downstream signalling (middle). Loss of EC NRP1 but the presence of perivascular NRP1 leads to formation of a stable VEGFA/VEGFR2/NRP1 complex in trans and enhanced VEGFR2 signalling and increased vascular permeability