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Circulation Research. 2005;96:1257-1265
Published online before print May 26, 2005, doi: 10.1161/01.RES.0000171756.13554.49
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(Circulation Research. 2005;96:1257.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Neuropilin-1 Regulates Vascular Endothelial Growth Factor–Mediated Endothelial Permeability

Patrice M. Becker, Johannes Waltenberger, Robin Yachechko, Tamara Mirzapoiazova, James S.K. Sham, Chun Geun Lee, Jack A. Elias, Alexander D. Verin

From the Division of Pulmonary and Critical Care Medicine (P.M.B., R.Y., T.M., J.S.K.S., A.D.V.), Johns Hopkins University School of Medicine, Baltimore, Md; University Hospital Maastricht and Cardiovascular Research Institute Maastricht (CARIM) (J.W.), Maastricht, the Netherlands; and the Division of Pulmonary and Critical Care Medicine (C.G.L., J.A.E.), Yale University School of Medicine, New Haven, Conn.

Correspondence to Dr Patrice M. Becker, Johns Hopkins Asthma and Allergy Center, Room 4B74, 5501 Hopkins Bayview Cir, Baltimore, MD 21224. E-mail pbecker1{at}jhmi.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.


Key Words: vascular endothelial growth factor receptor-2 • transendothelial electrical resistance • chemotaxis • mitogen-activated protein kinase • acute lung injury


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular endothelial growth factor (VEGF) was first isolated and purified from tumor cells because of its ability to markedly increase vascular permeability to plasma proteins.1 VEGF is expressed normally in most tissues, including lung,2,3 with abundant expression of VEGF in alveolar cells of normal rats.3,4 Published data support a significant role for VEGF in the development of acute lung injury and acute respiratory distress syndrome (ARDS) after various insults.5–10 In the adult lung, VEGF may promote vascular injury6–9,11 via effects on increased endothelial permeability. On the other hand, VEGF stimulates endothelial cell proliferation12 and chemotaxis,13 potential reparative responses to pulmonary vascular injury. Because cellular responses to VEGF may be either injurious or reparative, understanding how the endothelium transduces specific VEGF-mediated signals is critical.

Vascular endothelial cells express 2 tyrosine kinase receptors for VEGF, termed VEGF receptor 1 (VEGFR1)14 and VEGF receptor 2 (VEGFR2).15 Intermolecular cross-talk between VEGFR1 and VEGFR2 has been reported,16–18 and represents one possible pathway for differential regulation of VEGF signals. Endothelial cells also express neuropilin-1 (Npn-1), a class 3 semaphorin receptor, which binds VEGF19 in an isoform-specific manner.20–23 The importance of Npn-1 in vasculogenesis was confirmed by recent murine studies demonstrating that constitutive or endothelial-specific Npn-1 deletion led to disrupted cardiac and vascular development, with embryonic lethality.24,25 Additionally, the phenotype of Npn-1 transgenic mice was similar to that reported for mice constitutively overexpressing VEGF,26 with excess capillary formation, dilated blood vessels, extensive hemorrhage, and embryonic lethality,27 suggesting a possible role for Npn-1 in enhanced vascular permeability as well as vasculogenesis and angiogenesis.

Npn-1 has a short intracellular domain with no predicted signaling functions.28 In vitro, Npn-1 can complex with VEGFR1 and VEGFR223,29,30 and may potentiate VEGFR2 activation.23,30 Prior evidence supports a clear role for VEGFR2 in VEGF-mediated chemotaxis and proliferation,13,31,32 but the cell surface receptors mediating VEGF-induced endothelial permeability have been less extensively evaluated.33,34 Although several studies support a role for VEGFR2 in VEGF-induced vascular barrier dysfunction,33,34 previous studies from our laboratory35 and others36 suggest that VEGFR2 may not be sufficient to elicit endothelial permeability responses to VEGF. Because upstream events that differentially activate VEGF receptors can modulate the physiologic response of endothelium to VEGF,17,18 we hypothesized that Npn-1 might be a required component of the pathway regulating VEGF-induced vascular permeability. To test this hypothesis, we determined the effects of Npn-1 overexpression and inhibition on VEGF-mediated endothelial cell permeability, and VEGFR2 activation, in vitro. To understand whether the effects of Npn-1 were specific to VEGF-induced vascular barrier dysfunction, we evaluated whether Npn-1 overexpression and inhibition comparably altered VEGF-mediated endothelial-cell chemotaxis. Finally, the effects of Npn-1 inhibition on VEGF-induced pulmonary vascular permeability were measured in vivo, by studying mice that overexpress VEGF in inducible, lung-specific fashion.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Reagents and Endothelial Cell Culture
See the online data supplement available at http://circres.ahajournals.org.

Endothelial Cell Transfection
Npn-1 was subcloned from pMT21 into pcDNAHygro, then porcine aortic endothelial cells (PAE) stably expressing VEGFR2 (PAE/KDR)32 were transfected with 0.2 to 0.4 µg DNA using Lipofectamine (Invitrogen, Carlsbad, Calif), according to the manufacturer’s instructions. Growth media containing 0.2 mg/mL Hygromycin (Calbiochem, San Diego, Calif), or 0.5 mg/mL G418 and Hygromycin, was used to select for clones expressing Npn-1 or both VEGFR2 and Npn-1 (PAE/KDR/Npn-1), respectively. Stably transfected cells were identified by Western blot analysis of cell lysates. Shown in Figure 1, PAE did not express Npn-1 or VEGFR2 before transfection. PAE/KDR expressed VEGFR2, but not Npn-1, whereas PAE/Npn-1 expressed Npn-1, but not VEGFR2. PAE/KDR/Npn-1 expressed both VEGFR2 and Npn-1. Similarly, human pulmonary artery endothelial cells (HPAE) expressed both VEGFR2 and Npn-1. No PAE-derived cell line expressed either VEGFR1 or Npn-2, whereas HPAE expressed VEGFR1, but not Npn-2 (data not shown).



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  		Figure 1. Expression of VEGFR2 and Npn-1, measured by Western blot analysis, in PAE and PAE stably transfected with Npn-1 (PAE/Npn), VEGFR2 (PAE/KDR), or both Npn-1 and VEGFR2 (PAE/KDR/Npn-1). Shown for comparison is expression of Npn-1 and VEGFR2 in HPAE.

Measurement of Transendothelial Electrical Resistance
Effects of rhVEGF on barrier function of stably transfected PAE, or HPAE, were assessed by measurement of transendothelial electrical resistance (TER) using electric cell-substrate impedance sensing (Applied Biophysics), as previously described35 (see online data supplement).

Western Blot Analysis
VEGFR2, and p38 and ERK1/2 MAP kinase activation, were determined as previously described9,35 (see online data supplement).

Measurement of Endothelial Cell Chemotaxis
Assessment of endothelial cell chemotaxis was performed as previously described,35 with minor modifications. Data represent the mean±SE value from 3 separate experiments performed in duplicate within each experiment (see online data supplement).

Confocal Microscopy
After VEGF treatment, endothelial cell monolayers were processed for immunofluoresence microscopy as previously described.35 Cover slips were mounted and analyzed using a Zeiss LSM510 confocal microscope with a Plan-Neofluor 40x oil objective (NA=1.3). Z-stacks of 2-dimensional immunofluorescent images of VEGFR2 and actin were recorded simultaneously using the multitrack mode to prevent bleed-through. Images were processed, and 3-D images were reconstructed using LSM 510 software (version 3.2 SP2) and then stored for further management using Adobe Photoshop software.

Animal Protocols
The CC10-rtTA-VEGF mice used in this study were generated as previously described37,38 (see online data supplement). Npn-1 neutralizing antibodies or nonimmune rabbit IgG (50 µg IP) were administered every 24 hours, then mice were euthanized under general anesthesia after 48 hours of exposure to doxycycline. The left lung was rapidly excised and weighed then placed in a desiccating oven (65°C) and weighed daily for 7 days until lung dry weight stabilized for calculation of wet/dry lung weight ratios. The right lung was snap-frozen in liquid nitrogen, then stored at –80°C until the tissue was homogenized for protein extraction and Western blot analysis. CC10-rtTA-VEGF mice receiving no doxycycline and wild-type littermates receiving 48 hours doxycycline served as additional controls.

Statistical Analysis
Statistical comparisons were made using analysis of variance (see online data supplement). When significant variance ratios were obtained, the least significant differences were calculated to allow comparison of individual group means. Differences were considered significant for probability values ≤0.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effects of Npn-1 Overexpression on Endothelial Cell Permeability
To determine the effects of Npn-1 on VEGF-mediated endothelial barrier dysfunction, TER of PAE/Npn, PAE/KDR, and PAE/KDR/Npn-1 was measured after administration of VEGF165. As shown in Figure 2A, TER of PAE/KDR/Npn-1 decreased in a dose-dependent manner after concentrations of VEGF165 between 10 to 200 ng/mL. In contrast, VEGF165 did not alter permeability of PAE/KDR (Figure 2B) or PAE/Npn-1 (Figure 2C). Shown in Figure 2B and 2D, TER of PAE/KDR/Npn-1 decreased 14±6% within 36 minutes of 200 ng/mL VEGF165 administration, as compared with a 4±3% increase in TER of PAE/KDR (P<0.0001). The decreased TER in PAE/KDR/Npn-1 was time-dependent and sustained, reaching a nadir of –49±8% 6 hours after administration of VEGF165. In contrast, the maximal decrease in TER in PAE/KDR 6 hours after the same concentration of VEGF165 was –8±8% (P<0.0001). Shown in Figure 2C and 2E, the response of PAE/Npn-1 was comparable to that of PAE/KDR, with a {Delta}TER of 3.3±2.8% and –6.5±4.2% after 36 and 360 minutes exposure to 200 ng/mL VEGF165.



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  		Figure 2. A, TER of PAE/KDR/Npn-1 decreased in dose- and time-dependent fashion after VEGF165 treatment (P<0.0001) (n=4 to 6 per group; arrow represents time of VEGF administration). B, Change in TER of PAE/KDR (open circles) and PAE/KDR/Npn-1 (closed squares) after 200 ng/mL VEGF165. TER of PAE/KDR was not altered by VEGF, whereas TER of PAE/KDR/Npn-1 decreased significantly after VEGF administration (n=5 to 6 per group). C, Change in TER of PAE/Npn-1 (open triangles) and PAE/KDR/Npn-1 (closed squares) after 200 ng/mL VEGF165. TER of PAE/Npn-1 was not altered by VEGF, whereas TER of PAE/KDR/Npn-1 decreased significantly after VEGF administration (n=5 to 8 per group). D, Mean change in TER 36 and 360 minutes after administration of 200 ng/mL VEGF165 to PAE/KDR (open bars) and PAE/KDR/Npn-1 (closed bars) cells (n=5 to 6 per group). E, Mean changes in TER 36 and 360 minutes after administration of 200 ng/mL VEGF165 to PAE/Npn-1 (open bars) and PAE/KDR/Npn-1 (closed bars) cells (n=5 to 8 per group).

The effects of permeability-enhancing concentrations of VEGF165 on activation of VEGFR2 also differed. Shown in Figure 3, treatment of both PAE/KDR and PAE/KDR/Npn-1 with 200 ng/mL VEGF165 resulted in rapid phosphorylation of VEGFR2, but the amount of phosphorylated VEGFR2 (p-VEGFR2) was greater, and activation was more rapid, in PAE/KDR/Npn-1 as compared with PAE/KDR. This difference was paralleled by increased and sustained activation of the downstream signaling molecules, ERK1/2 and p38 mitogen activated protein kinase (MAPK), in PAE/KDR/Npn-1, as compared with PAE/KDR (middle and bottom panels of Figure 3). Shown in Figure 3, differences in VEGFR2 and MAPK activation were not attributable to increased amounts of VEGFR2 or MAPK protein as a consequence of Npn-1 expression.



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  		Figure 3. Time course of VEGFR2, p38, and ERK1/2 MAPK activation after 200 ng/mL VEGF165. Activation of VEGFR2 and p38 and ERK1/2 MAPK was increased, and activation was more sustained, in PAE/KDR/Npn-1 than PAEKDR. Results are representative of 3 separate experiments.

To determine whether Npn-1 altered the cellular distribution of activated VEGFR2, confocal microscopy of confluent endothelial cell monolayers was performed after administration of permeability-enhancing concentrations of VEGF165. Cells were double immunostained for p-VEGFR2 and cellular actin. Shown in Figure 4A and 4B are representative results from 3 separate experiments. In both figures, sagittal sections reconstructed from Z-stack images are shown in the top panels. The remaining panels represent 2-D scans at varying levels within the monolayer (apical, medial, and basal). The panels on the left side of each figure represent control cells not exposed to rhVEGF, whereas the panels on the right side of each figure represent cells fixed 5 minutes after administration of VEGF165 (200 ng/mL). In control untreated PAE/KDR (Figure 4A), little p-VEGFR2 expression was seen. VEGF treatment resulted in increased actin stress fiber formation, with p-VEGFR2 appearing at the tips of actin stress fibers. In contrast, as shown in Figure 4B, p-VEGFR2 expression was increased under control conditions in PAE/KDR/Npn-1, and there appeared to be more depolymerized cellular actin present. After VEGF165 administration, there was a pronounced increase in the expression of p-VEGFR2 in PAE/KDR/Npn-1. In addition to increased expression of p-VEGFR2 at tips of actin stress fibers, polarity-dependent differential expression of p-VEGFR2 was seen, such that activated VEGFR2 was prominently localized to the apical cell membrane (Figure 4B, top).



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  		Figure 4. A, Confocal microscopy images of PAE/KDR under control conditions (left) and 5 minutes after administration of 200 ng/mL VEGF165 (right). Cells were immunostained for phospho-VEGFR2 (green) and actin (red). p-VEGFR2 colocalizes with tips of actin stress fibers and increases after administration of rhVEGF (arrows in bottom right panel) but demonstrates no polarity-dependence of expression. B, Confocal microscopy images of PAE/KDR/Npn-1, under control conditions (left) and 5 minutes after administration of 200 ng/mL VEGF165 (right). Administration of rhVEGF resulted in increased p-VEGFR2 when compared with PAE/KDR. In addition to increased p-VEGFR2 at tips of actin stress fibers (arrows in bottom right panel), images demonstrate polarity-dependent differential localization of p-VEGFR2 to the apical membrane after VEGF (arrowheads in top right panel). Note is also made of increased p-VEGFR2, and increased depolymerized actin, under control conditions. Results are representative of 3 separate experiments.

To compare the effect of Npn-1 expression on VEGF-induced endothelial cell permeability with previous reports in the literature regarding how Npn-1 alters VEGF-mediated endothelial cell migration, the effects of VEGF165 on chemotaxis of PAE/KDR and PAE/KDR/Npn-1 was compared. As shown in Figure 5, basal chemotaxis did not differ between these 2 cell types. Although Npn-1 expression enhanced chemotaxis in response to VEGF, as has been previously reported,19 it was not required, as both PAE/KDR and PAE/KDR/Npn-1 migrated efficiently in response to VEGF.



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  		Figure 5. VEGF165-mediated chemotaxis of PAE/KDR/Npn-1 was enhanced relative to PAE/KDR, but Npn-1 expression was not required for this response (n=6 per group).

Effects of Npn-1 Inhibition on VEGF-Mediated Lung Endothelial Cell Permeability In Vitro
To determine whether Npn-1 inhibition attenuated VEGF-mediated pulmonary endothelial cell permeability, 200 ng/mL VEGF165 was administered to HPAE after pretreatment with Npn-1 or VEGFR2 neutralizing antibodies, alone or in combination, or nonimmune IgG. Pretreatment with Npn-1 neutralizing antibodies resulted in attenuation of endothelial barrier dysfunction at all time points after VEGF165 treatment (Figure 6A, top). In contrast, VEGFR2-neutralizing antibodies did not prevent decreased TER after VEGF165, although VEGFR2-neutralization may have resulted in a less sustained effect of VEGF165 on endothelial barrier function (P=0.025; Figure 6A, middle). The combination of both Npn-1 and VEGFR2 neutralizing antibodies appeared the most effective strategy for attenuating VEGF-induced HPAE permeability (Figure 6A, bottom). In contrast with permeability, VEGFR2-neutralization completely attenuated VEGF-mediated chemotaxis of HPAE (P<0.0001), whereas Npn-1 inhibition only partially blocked this response (Figure 6B).



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  		Figure 6. A, Neutralizing antibodies for Npn-1 significantly attenuated increased permeability of HPAE in response to 200 ng/mL VEGF165 (top; n=3 to 5 per group). VEGFR2-neutralizing antibodies did not attenuate VEGF165-mediated barrier dysfunction of HPAE, although VEGFR2 inhibition resulted in less sustained decrease in TER (middle; n=3 to 5 per group). The combination of both Npn-1 and VEGFR2 neutralizing antibodies completely attenuated rhVEGF-induced permeability of HPAE (bottom; n=4 per group). Arrows represent the addition of VEGF. B, In contrast to permeability, VEGF165-mediated chemotaxis was completely blocked by pretreatment of HPAE with neutralizing antibodies against VEGFR2, whereas Npn-1 neutralization was only partially effective at attenuating this response (n=6 per group).

To confirm a role for Npn-1 in mediated vascular barrier dysfunction in human lung endothelial cells, we compared the effects of 2 VEGF isoforms, VEGF121 and VEGF165, on TER of HPAE. VEGF165, used for all of the other experiments in this study, ligates all VEGF receptors expressed on these cells, including VEGFR1, VEGFR2, and Npn-1. In contrast, VEGF121 binds VEGFR1 and VEGFR2, but not Npn-1. Consistent with the effects of Npn-1 neutralization on VEGF-induced barrier dysfunction, VEGF121 had no effect on TER of HPAE, when compared with comparable concentrations of VEGF165 (Figure 7).



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  		Figure 7. In contrast to VEGF165, administration of VEGF121 (200 ng/mL), a VEGF isoform that does not bind Npn-1, did not alter TER of HPAE (n=4 per group).

Effects of Npn-1 Inhibition on VEGF-Mediated Vascular Permeability In Vivo
To determine whether Npn-1 modulated VEGF response in vivo, the effects of Npn-1 neutralizing antibodies on pulmonary vascular permeability and VEGFR2 activation were determined in mice inducibly overexpressing human VEGF165 in lung-specific fashion.37,38 Pulmonary VEGF expression was induced by administration of doxycycline in drinking water for 48 hours, immediately after injection of rabbit anti-rat Npn-1 neutralizing antibodies (50 µg IP Q24 hoursx2 doses), or comparable amounts of nonimmune rabbit IgG. Pulmonary vascular permeability, estimated by measurement of lung wet/dry weight ratio, increased significantly after induction of VEGF in IgG-treated animals (Figure 8A). In contrast, Npn-1 neutralization significantly attenuated VEGF-mediated vascular leak (P=0.04). Shown in Figure 8B, Western blot analysis of whole-lung homogenates demonstrated that Npn-1 neutralizing antibodies significantly attenuated VEGFR2 phosphorylation, and p38 and ERK1/2 MAPK activation, after the induction of VEGF.



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  		Figure 8. A, Lung wet/dry weight ratio increased in CC10-rtTA-VEGF mice after induction of pulmonary VEGF165 expression with doxycycline. This increase in lung water was attenuated by pretreatment with {alpha}-Npn-1 (50 µg IP; n=4 to 6 per group). B, Representative Western blot analysis of whole-lung homogenates from the same mice demonstrated that {alpha}-Npn-1 decreased activation of VEGFR2, and p38, and ERK1/2 MAPK after pulmonary VEGF induction. Expression of total lung Npn-1 protein did not differ between groups (n=4 per group).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Mounting experimental evidence supports a significant role for VEGF in the development of acute lung injury. Kaner et al demonstrated that overexpression of VEGF in murine lung, using adenoviral transfection, was associated with profound pulmonary edema.7 These data are supported by recent studies in transgenic mice, where lung-specific overexpression of VEGF165 caused hemorrhage11 and edema38 formation. Additionally, increased pulmonary VEGF expression was shown in response to inhaled lipopolysaccharide (LPS), in association with increased BAL protein concentration and neutrophil influx.8 These reports are consistent with our published studies, which demonstrated that increased pulmonary vascular permeability induced by ventilated pulmonary ischemia in both ferret and mouse lungs was linked with significant upregulation of VEGF.6,9 Emerging clinical studies have confirmed these experimental data. Recent reports demonstrated increased expression of VEGF in preserved human-lung allografts, in association with increased wet/dry lung weight ratios,5,39 and plasma VEGF was shown to increase during the first 48 hours after presentation in 40 ARDS patients.10

Npn-1 binds VEGF, and in vitro studies suggest that this receptor can enhance VEGF signaling through VEGFR2,30 as well as perhaps through other pathways.40 Our data suggest that Npn-1 is required for VEGF-mediated vascular barrier dysfunction, as PAE expressing only VEGFR2 exhibited no permeability change in response to VEGF165. In contrast, coexpression of Npn-1 with VEGFR2 completely restored VEGF-induced endothelial leak in a dose-dependent manner. Stable transfection of PAE with Npn-1 alone was insufficient to cause VEGF-mediated endothelial barrier dysfunction. Consistent with these findings, inhibition of Npn-1 attenuated VEGF165-mediated barrier dysfunction of HPAE, which express both VEGFR2 and Npn-1, as well as VEGFR1. Interestingly, although a few prior studies in other model systems suggested that VEGF121 could increase vascular permeability,51–53 our data demonstrate that VEGF121 (which binds VEGFR1 and VEGFR2, but not Npn-1) did not increase permeability of human lung endothelial cells in vitro. A requirement for Npn-1 in VEGF-mediated vascular leak is supported by recent studies suggesting that VEGF-E variants that retain VEGFR2 binding, but lack Npn-1 binding, do not cause increased vascular permeability.41,42

To determine whether the effects of Npn-1 were specific for VEGF-induced endothelial permeability, we evaluated the consequences of Npn-1 expression and inhibition on VEGF-mediated endothelial cell chemotaxis. In contrast to barrier dysfunction, stable expression of VEGFR2 alone was sufficient to allow chemotaxis in response to VEGF165. Consistent with this, inhibition of VEGFR2 completely prevented the chemotaxis of HPAE to VEGF. These data support several prior studies demonstrating a critical role for VEGFR2 in VEGF-induced endothelial chemotaxis and proliferation.13,18,31 As has been previously reported, stable coexpression of Npn-1 and VEGFR2 enhanced VEGF-mediated endothelial chemotaxis,19,43 and Npn-1 inhibition partially attenuated human pulmonary artery VEGF-mediated endothelial cell migration. These data suggest that although Npn-1 may modulate endothelial chemotaxis to VEGF, it is not required to elicit this response. In contrast, Npn-1 appears to be critical for VEGF-mediated endothelial permeability.

Our data also demonstrate that Npn-1 coexpression resulted in increased activation of ERK1/2 and p38 MAPK in response to permeability-enhancing concentrations of VEGF. We, and others, previously reported that inhibition of either ERK1/235,44 or p3835 MAPK attenuated VEGF-induced endothelial cell barrier dysfunction in vitro. The finding that Npn-1 expression enhanced activation of p38 and ERK 1/2 MAPK therefore supports the hypothesis that Npn-1 is an important component of the pathway mediating endothelial permeability in response to VEGF. However, the current study was designed to evaluate whether Npn-1/VEGFR2 complexes determine specificity of the cellular response to VEGF, and did not investigate the specific intracellular signaling events mediating endothelial barrier dysfunction.

Although the VEGF receptors and signaling pathways implicated in VEGF-induced endothelial cell mitogenicity and migration have been extensively studied,13,31,32 the mechanisms of VEGF-mediated endothelial permeability enhancement have been less comprehensively evaluated.33,34 The literature is inconsistent regarding the specific VEGF receptors transducing VEGF-mediated vascular leak,42,45,46 and the majority of in vitro studies of endothelial permeability have used varying concentrations of the 165 isoform of VEGF.33,44,47–49 In addition, permeability assays vary between studies, and may therefore reflect differing aspects of vascular barrier dysfunction.50 No currently available in vitro permeability assay can adequately reproduce the in vivo situation. On the other hand, in vivo permeability assays may be influenced by factors other than permeability. For example, the Miles assay commonly used to assess dermal vascular permeability measures the rate of tracer movement into tissue, and may depend on blood flow, capillary pressure, and vascular surface area, as well as permeability. Similarly, lung wet/dry weight, the assay reported in this study, may increase either because of increased lung water, vascular surface area, or both. Because of these limitations, results of different assays of permeability may yield discrepant results.

Although methods for estimating permeability in vitro and in vivo are not directly comparable, in the current study results of in vivo experiments were consistent with in vitro experiments. Administration of Npn-1 neutralizing antibodies prevented the development of pulmonary edema after the induction of VEGF expression in transgenic mice expressing human VEGF165 in externally regulable, lung-specific fashion. This protection from VEGF-induced increased lung water was linked with attenuation of VEGFR2, ERK1/2, and p38 MAPK activation, measured by Western blot analysis in whole lung homogenates. Although the increased wet/dry lung weight ratio elicited by VEGF induction was small, it was statistically significant and consistent with prior reports of VEGF-induced vascular leak in these animals.38 It should be noted that lung wet/dry weight ratio may increase secondary to increased vascular permeability, increased vascular surface area, or both. To minimize any contribution of the latter to this measurement, we chose to evaluate the effects of Npn-1 inhibition at an early time point (48 hours) of VEGF induction. This choice was based on published data that pulmonary VEGF expression in CC10-rtTA-VEGF transgenic mice was not maximal until 48 hours of doxycycline induction, whereas longer periods (3 to 5 days) of VEGF induction stimulated angiogenesis, which might increase vascular surface area and complicate estimation of permeability.37

To evaluate the involvement of VEGFR2 in Npn-1–mediated endothelial barrier dysfunction, we compared VEGFR2 activation after permeability-enhancing concentrations of VEGF in cells expressing VEGFR2 alone and cells expressing both VEGFR2 and Npn-1. Coexpression of Npn-1 and VEGFR2 potentiated phosphorylation of VEGFR2, measured both by Western blot and immunocytochemistry. Furthermore, confocal microscopy demonstrated that Npn-1 altered the cellular distribution of activated VEGFR2, such that VEGF induced polarity-dependent localization of p-VEGFR2 to the apical cell membrane. Additionally, in vivo experiments suggested that Npn-1 inhibition attenuated VEGFR2 activation. These data extend published studies demonstrating that Npn-1 can complex with VEGFR2 in vitro23,29,30 and may potentiate VEGFR2 activation.23,30 On the other hand, VEGFR2 expression alone was insufficient to increase permeability of PAE in response to VEGF, and VEGFR2 inhibition did not attenuate VEGF-mediated barrier dysfunction of HPAE. These data suggest that enhanced endothelial permeability in response to VEGF may involve signaling through pathways40–42 in addition to those activated by VEGFR2. Because human lung endothelial cells express VEGFR1 in addition to VEGFR2 and Npn-1, future studies will evaluate whether VEGFR1 modulates the effects of Npn-1 on VEGF-induced vascular leak. A possible role for VEGFR1 in vascular permeability was suggested by a recent report that snake venom vascular endothelial growth factor (TfsvVEGF) preferentially phosphorylates VEGFR1 over VEGFR2 and enhances permeability assessed by the Miles assay.46 This study did not report whether TfsvVEGF bound Npn-1.

Taken together, these data demonstrate an important role for Npn-1 in VEGF-induced vascular permeability and support the hypothesis that increased Npn-1 expression or activation represents one pathway by which diverse physiologic responses of vascular endothelium to VEGF may be differentially mediated. Increased lung VEGF expression can cause pulmonary edema, the primary clinical manifestation of ARDS. Our results suggest that strategies targeting Npn-1 may lead to the development of novel therapeutic approaches to attenuate the injurious consequences of VEGF in this complex clinical disorder.


*    Acknowledgments
 
This work was funded by NIH HL60628 (P.M.B.), PO1-HL56389, HL74744, and HL64242 (J.A.E.), and by a grant from the European Commission to J.W. (QLK3-CT-2002-01955).


*    Footnotes
 
This manuscript was sent to Peter Libby, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received December 21, 2004; resubmission received April 13, 2005; revised resubmission received May 12, 2005; accepted May 17, 2005.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983; 219: 983–985.[Abstract/Free Full Text]
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