Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224-6801
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ABSTRACT |
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The mechanisms responsible for the divergent physiological responses of endothelial cells to vascular endothelial growth factor (VEGF) are incompletely understood. We hypothesized that VEGF elicits increased endothelial permeability and cell migration via differential activation of intracellular signal transduction pathways. To test this hypothesis, we established a model of VEGF-induced endothelial barrier dysfunction and chemotaxis with bovine pulmonary endothelial cells. We compared the effects of VEGF on transendothelial electrical resistance (TER), actin cytoskeletal remodeling, and chemotaxis of lung endothelial cells and then evaluated the role of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK)1/2 in VEGF-mediated endothelial responses. The dose response of pulmonary arterial and lung microvascular endothelial cells to VEGF differed when barrier regulation and chemotaxis were evaluated. Inhibition of tyrosine kinase, phosphoinositol 3-kinase, or p38 MAPK significantly attenuated VEGF-mediated TER, F-actin remodeling, and chemotaxis. VEGF-mediated decreased TER was also significantly attenuated by inhibition of ERK1/2 MAPK but not by inhibition of fetal liver kinase-1 (flk-1) or Src kinase. In contrast, VEGF-mediated endothelial migration was not attenuated by ERK1/2 inhibition but was abolished by inhibition of either flk-1 or Src kinase. These data suggest potential mechanisms by which VEGF may differentially mediate physiological responses in vivo.
vascular endothelial growth factor; endothelial cell chemotaxis; endothelial cell permeability; endothelial barrier dysfunction; mitogen-activated protein kinase
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INTRODUCTION |
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VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), also termed vascular permeability factor, is a 34- to 46-kDa disulfide-linked dimeric protein (39, 45) first isolated and purified from tumor cells because of its ability to markedly increase vascular permeability to plasma proteins (63). VEGF is also a potent endothelial cell mitogen (39, 45), is chemotactic for both endothelial cells (51, 60) and monocytes (3), and plays a critical role in both angiogenesis and vasculogenesis (8, 20, 48, 49, 76).
Numerous investigators have confirmed the importance of VEGF during normal vascular development (8, 20, 49, 76), and it has also received attention in pathological processes such as ischemia, primarily because of its angiogenic activity. Increased expression of VEGF (31, 46) and its endothelial cell receptors fms-like receptor tyrosine kinase and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1) (46) has been demonstrated acutely after cardiac (31, 46) and cerebral (42) ischemia in animal models, and it was recently shown that serum levels of VEGF increased in patients with active myocardial ischemia then decreased within 30 min of intervention to recanalize occluded vessels (62). The stimulus for increased VEGF expression during ischemia of organs of the systemic circulation is thought to be hypoxia (42, 68).
One of the hallmarks of acute pulmonary ischemic injury is increased vascular permeability (5, 6, 30, 41), and neovascularization occurs after longer periods of unilateral pulmonary arterial ischemia in vivo (10, 47). Despite similarities that exist with ischemia-reperfusion injury in other solid organs, the mechanisms of ischemic lung injury are likely to be distinct. One important difference between systemic and pulmonary ischemia is that pulmonary ischemia is not necessarily associated with tissue hypoxia if the lung is inflated with oxygen while blood flow is impaired, as is the case, for example, with lung preservation for human lung transplantation. Our laboratory (5, 6) and others (30) previously demonstrated that the vascular injury that occurs in the ischemic pulmonary vasculature is independent of hypoxia.
Our laboratory (7, 38) also recently demonstrated oxygen-independent upregulation of VEGF in association with increased pulmonary vascular permeability during both global ischemia of isolated lungs and unilateral pulmonary arterial occlusion in vivo. To further evaluate the potential role of VEGF during lung ischemia, we developed a model to study the mechanisms of VEGF-mediated endothelial barrier dysfunction in vitro and compared the effects of VEGF on pulmonary endothelial permeability and migration, two possible physiological consequences of increased VEGF expression during ischemia. The pathways by which VEGF mediates cell proliferation (29, 34, 43, 54, 56, 65) and migration (28, 36, 51, 58) have been characterized in some detail, whereas the signaling pathways leading to enhanced endothelial cell permeability have been less extensively evaluated (9, 13, 40, 73). We explored the role of mitogen-activated protein kinases (MAPKs) in differentially mediating VEGF-induced pulmonary vascular endothelial barrier dysfunction and chemotaxis because both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 are activated by VEGF and are involved in VEGF-mediated endothelial cell proliferation (43, 54) and migration (28, 60), respectively.
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METHODS |
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Reagents
Recombinant human VEGF165 (rhVEGF) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The p38 inhibitors SB-202190 and SB-203180, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-290042, the Src kinase inhibitor PP2, the tyrosine kinase inhibitor genistein, and the flk-1 inhibitor SU-1498 were purchased from Calbiochem (San Diego, CA). UO-126 was obtained from Promega (Madison, WI). Rabbit anti-phospho-p44/42 MAPK, rabbit anti-phospho-p38 MAPK, and p38 MAPK antibodies were purchased from New England Biolabs (Beverly, MA). Monoclonal anti-pan ERK antibody was obtained from Transduction Laboratories (Lexington, KY). MAPK-activated protein kinase (MAPKAPK)-2 and 27-kDa heat shock protein antibodies were purchased from StressGen (Victoria, BC, Canada). In vitro kinase assay kits for ERK and p38 were purchased from Cell Signaling Technologies (Beverly, MA). Texas Red-phalloidin and SlowFade mounting media were purchased from Molecular Probes (Eugene, OR). Tris-glycine (12%) gels were purchased from Invitrogen (Carlsbad, CA). Transwell chemotaxis chambers (6.5-mm diameter, 8-µm pore size) were purchased from Costar (Cambridge, MA). Chemotaxis filters were soaked in 20 µg/ml of rat tail collagen (Boehringer Mannheim, Indianapolis, IN) in 0.1% acetic acid for 1 h at room temperature and dried overnight. Triton X was purchased from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.Bovine pulmonary arterial endothelial cells (BPAECs) frozen at passage 16 were obtained from the American Type Culture Collection (CCL 209; Manassas, VA), cultured in complete medium, and used at passages 19-24, as previously described by our laboratory (22). Bovine lung microvascular endothelial cells (BLMVECs) at passage 3 were purchased from VEC TECHNOLOGIES (Troy, NY) and were used at passages 5-9. All endothelial cell cultures were maintained in DMEM (Life Technologies, Rockville, MD) supplemented with 20% (vol/vol) colostrum-free bovine serum (Irvine Scientific, Santa Ana, CA), 15 µg/ml of endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), 1% antibiotic and antimycotic solution (10,000 U/ml of penicillin, 10 µg/ml of streptomycin, 25 µg/ml of amphotericin B; KC Biologicals, Lenexa, KS), 0.1 mM nonessential amino acids (Life Technologies), and heparin (1,000 U/ml; Pharmacia and Upjohn, Kalamazoo, MI). The endothelial cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2-95% air and grew to contact-inhibited monolayers with typical cobblestone morphology. Cells from each primary flask were detached with 0.05% trypsin, resuspended in fresh culture medium, and passaged into 35-mm dishes for MAPK phosphorylation and activity determinations, eight-well gold microelectrode chambers for measurement of transendothelial electrical resistance (TER), or Transwell chemotaxis chambers for migration measurements. Cells were washed three times and then transferred to medium (as described above) that did not contain endothelial cell growth supplement and contained only 2% FBS for 22-24 h before initiation of the experimental protocols.
Measurement of TER
The dose-response of pulmonary conduit and microvascular endothelial cells to rhVEGF and the effects of MAPK, PI3K, Src kinase, flk-1, and tyrosine kinase inhibition on VEGF-mediated permeability were assessed by measurement of TER with the use of electric cell-substrate impedance sensing (Applied BioPhysics, Rochester, NY) as previously described by our laboratory (23). Briefly, cells were seeded onto gold microelectrodes (Applied Biophysics) and grown to confluence. The size of the small gold electrode (For some experiments, TER was resolved into components reflecting
resistance to current flow beneath the cell layer () and resistance
to current flow between adjacent cells (Rb)
(24) with the methods of Giaever and Keese
(27), which model the endothelial monolayer
mathematically. This analysis allowed evaluation of the relative
contribution of alterations in the net state of cell-matrix adhesion
(
) and cell-cell integrity (Rb) to the
overall decrease in TER induced by VEGF.
Measurement of Endothelial Cell Chemotaxis
Assessment of endothelial cell migration was performed as recently described (16) with minor modifications. Endothelial cells (bovine macrovascular and microvascular) were dislodged after brief trypsinization and dispersed into homogeneous single-cell suspensions that were washed extensively with medium 199-0.1% acid-free BSA (migration medium) and resuspended in the same medium at 106 cells/ml. To assess migration from established monolayers, cells (105) were dispersed onto collagen-coated chemotaxis filters that partitioned the Transwell inserts into upper and lower chambers. Migration medium (300 µl) was placed in the lower chambers, and the cells were allowed to adhere for 1 h at 37°C. In some experiments, the medium on both sides of the filters was replaced with specific antagonists for 30 min after cell attachment. The medium in the lower chambers was then removed, and cells were challenged by the addition of 300 µl of fresh migration medium containing chemoattractants to the lower chamber. Unless otherwise indicated, migration was allowed to proceed for 2 h at 37°C. The cells that remained attached to the upper surface of the filters were carefully removed with cotton swabs, and the cells that migrated to the lower surface of the filters were fixed and stained with a Diff-Quik staining set (Dade Behring, Newark, DE) according to the manufacturer's recommendations. The filters were allowed to dry under a vented hood, carefully removed from the Transwell chambers, mounted on glass slides with Cytoseal mounting medium (VWR, Bridgeport, NJ), and finally examined by light microscopy (×200, Nikon Eclipse TE 300). Images of at least six consecutive fields lying horizontally across each filter were captured by a Sony digital photo camera (DKC 5000). The number of migrating cells per field was enumerated, and the average was calculated. Data are expressed as the number of migrating cells per field and represent the mean ± SE values from three separate experiments performed in duplicate within each experiment unless specified otherwise.Evaluation of Actin Cytoskeletal Rearrangement
Actin cytoskeletal remodeling occurs during cell migration and is intimately linked with endothelial cell barrier regulation (22). We therefore performed imaging of endothelial gap formation and F-actin organization, as previously described (69), after fixing the endothelial cell monolayers with 5% paraformaldehyde in phosphate-buffered saline containing 25 mM Tris, pH 7, on ice for 10 min. Cells were rinsed thoroughly in Tris-buffered saline (pH 7.6) then permeabilized by treatment with Triton (0.2%) in rinse buffer. After the cells were rinsed three times, they were incubated with 1% BSA in rinse buffer at room temperature for 1 h followed by incubation with 1 U/ml of Texas Red-phalloidin (Molecular Probes) to identify F-actin by fluorescence microscopy. After three washes with PBS, the coverslips were mounted and analyzed with a Nikon video imaging system consisting of a phase-contrast inverted microscope, equipped with a set of objectives and filters for immunofluorescence, connected to a digital camera and processor. The images were recorded and saved on a Pentium II PC for further management with Adobe Photoshop software.Evaluation of MAPK Activation
Western blot analysis. MAPK activation was determined as previously described (70) by the immunoblotting of endothelial cell lysates with specific phospho-ERK and phospho-p38 antibodies that indicate the enhanced catalytic activity of the enzymes. Briefly, after VEGF challenge, cells were lysed in 2× Laemmli SDS sample buffer and boiled for 5 min. The lysates were separated by 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes for Western immunoblotting as our laboratory previously described (70). ERK1/2 phosphorylation was detected with 1 µg/ml of rabbit anti-phospho-p44/42 MAPK; p38 MAPK phosphorylation was determined by rabbit anti-phospho-p38 MAPK (1 µg/ml); total ERK protein was detected by monoclonal anti-pan ERK antibody (50 ng/ml); and total p38 MAPK protein was detected by rabbit anti-p38 MAPK antibody (1 µg/ml). After incubation with peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, 1:10,000 dilution; Sigma or goat anti-mouse IgG, 1:10,000 dilution; Bio-Rad), immunoreactive proteins were visualized with an enhanced chemiluminescence detection system according to the manufacturer's directions (Amersham).
In vitro kinase assays.
VEGF-mediated activation of p38 and ERK was confirmed with in vitro
kinase assays. After exposure to rhVEGF (200 ng/ml), BLMVECs were
harvested under nondenaturing conditions, washed with PBS, then lysed
in cell lysis buffer (Cell Signaling Technologies) containing
phenylmethylsulfonyl fluoride. Phosphorylated p38 was immunoprecipitated with immobilized phospho-p38 MAPK antibody, and then
an in vitro kinase assay was performed according to the manufacturer's
protocol (Cell Signaling Technologies). Samples were analyzed for p38
kinase activity by Western blot analysis, probing with
phospho-activating transcription factor-2 antibody (Cell Signaling
Technologies). To evaluate ERK activation, a p44/42 MAPK assay kit from
Cell Signaling Technologies was employed. Briefly, immobilized
phospho-p44/42 MAPK antibody was used to selectively immunoprecipitate
active ERK1/2 from cell lysates. The resulting immunoprecipitate was
then incubated in vitro with an Ets domain protein (Elk)-1 fusion
protein in the presence of ATP and kinase buffer per the
manufacturer's instructions. Phosphorylation of Elk-1 was assessed by
Western blot analysis with a phospho-Elk antibody. The effects of
UO-126 on inhibition of ERK1/2 were also confirmed with this assay.
Because the pyridinylimidazole compounds inhibit p38 MAPK by complexing
with p38 MAPK in the ATP-binding region and do not prevent
phosphorylation of p38 (74), the effects of SB-202190 on
the activation of p38 in response to VEGF were evaluated by
immunoprecipitating the cell lysates with anti-MAPKAPK2 antibody
(MAPKAPK2 is activated by p38), immobilizing the immunoprecipitated protein with protein A/G PLUS-agarose (Santa Cruz Biotechnology), then
incubating the immunoprecipitates in vitro with 27-kDa heat shock
protein (HSP-27), a known MAPKAPK2 substrate, in the presence of kinase
buffer (Cell Signaling Technologies) and 5 µCi of
-32P. Activation or inhibition of HSP-27 was
then determined by Western blot analysis and autoradiography.
Statistical Analysis
Differences in TER of endothelial cells in response to increasing concentrations of rhVEGF were determined with split-plot analysis of variance. The effects of genistein, SU-1498, MAPK, PI3K, and Src kinase inhibitors on TER were compared with one-way analysis of variance. Differences in cell migration in response to rhVEGF in the presence and absence of pharmacological inhibitors were evaluated with two-way analysis of variance. When significant variance ratios were obtained, least significant differences were calculated to allow comparison of individual group means. Differences were considered significant for P values ![]() |
RESULTS |
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Effects of VEGF on In Vitro Endothelial Barrier Function and Actin Cytoskeletal Rearrangement
As shown in Fig. 1, VEGF caused a dose- and time-dependent decrease in TER of both BPAECs (A) and BLMVECs (B). Endothelial barrier dysfunction was maximal 30-40 min after VEGF administration, with complete recovery of barrier function in both cell types by 60 min at concentrations of
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Effects of VEGF on Endothelial Cell Chemotaxis
In contrast to transendothelial permeability, the dose response of BPAECs and BLMVECs to rhVEGF differed when endothelial cell chemotaxis was evaluated. Migration of both conduit and microvascular lung endothelial cells was maximal at a VEGF concentration of 10 ng/ml and decreased at higher VEGF concentrations (Fig. 4). However, the magnitude of endothelial cell chemotaxis in response to rhVEGF was more pronounced in BPAECs compared with that in BLMVECs.
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Role of MAPK Activation in VEGF-Induced Barrier Dysfunction, Actin Rearrangement, and Chemotaxis
Administration of permeability-enhancing concentrations of rhVEGF to BLMVECs was also accompanied by activation of both p38 and ERK1/2 MAPK, as demonstrated by both Western blot (Fig. 5) and in vitro kinase (Fig. 5) assays. As shown in Fig. 6, VEGF-mediated decreased TER of BLMVECs was attenuated by pretreatment with the tyrosine kinase inhibitor genistein (10 µM; A), by inhibition of p38 MAPK with the pyridinylimidazole inhibitor SB-202190 (20 µM; C), and by inhibition of ERK1/2 with the MAPK kinase (MEK) inhibitor UO-126 (10 µM; D), suggesting that this response was mediated by tyrosine kinases and involved both p38 and ERK MAPK activation. Inhibition of Src kinase with PP2 (10 µM; F) or of flk-1 with SU-1498 (10 µM; B) did not significantly attenuate VEGF-mediated alterations in TER.
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Inhibition of both ERK1/2 and p38 MAPK also attenuated actin stress
fiber formation in response to VEGF (200 ng/ml) as shown in Fig.
7. The effects of MAPK inhibition were
confirmed with in vitro kinase assays that demonstrated attenuation of
VEGF-mediated ERK activation by UO-126 (Fig.
8A) and p38 activation by
SB-202190 (Fig. 8B). Transendothelial permeability was also
attenuated by inhibition of PI3K with LY-290042 (50 µM; Fig.
6E) in this model. Pretreatment with LY-290042 (Fig.
9) only minimally decreased activation of
ERK but markedly attenuated p38 MAPK activity, suggesting that the
effects of PI3K inhibition are primarily via attenuation of downstream
p38 MAPK activation in response to VEGF.
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As shown in Fig. 10, the chemotaxis of
BPAECs in response to rhVEGF (10 ng/ml) was attenuated by pretreatment
with genistein (10 µM; A), SU-1498 (10 mM; B),
SB-203180 (20 µM; C), LY-290042 (50 µM; E),
and PP2 (10 mM; F), consistent with previous reports that
flk-1-mediated p38 activation is an important component of VEGF-induced
endothelial migration (60). Interestingly, despite the
finding that UO-126 inhibited actin stress fiber and interendothelial gap formation in confluent pulmonary microvascular endothelial monolayers exposed to 200 ng/ml of rhVEGF, MEK inhibition did not alter
VEGF-mediated endothelial chemotaxis (Fig. 10D).
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DISCUSSION |
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VEGF, a potent endothelial cell mitogen (39, 45), has been found to have multiple physiological and pathophysiological effects, including mediation of endothelial barrier dysfunction (13, 18, 19, 33, 40, 59, 71, 73) and its role as a chemotactic factor for both endothelial cells (28, 37, 58) and monocytes (3). Recent studies have also suggested that VEGF may act as a survival factor for endothelial (21, 26, 72), hematopoietic (44), and epithelial (36) cells. VEGF has been shown to play a critical role in normal vascular development (8, 20, 49, 76) and vascular remodeling under a number of pathophysiological circumstances, including ischemia (1, 48). VEGF is highly expressed in the lung under basal conditions (50), and studies (14, 25, 55, 67) have suggested a significant role for VEGF in pulmonary vascular remodeling in response to hypoxia, monocrotaline, and congenital heart disease. Our laboratory (7, 38) previously demonstrated oxygen-independent upregulation of VEGF expression in response to ischemia in the ventilated lung and a potential role for VEGF in increased pulmonary vascular permeability under these conditions.
Although the signal transduction pathways leading to VEGF-induced endothelial proliferation and migration have been studied extensively (28, 29, 34, 37, 51, 53, 54, 56, 58, 60), the cellular mechanisms mediating endothelial barrier dysfunction in response to VEGF have been less thoroughly evaluated (9, 13, 73). Previous studies have suggested that ERK1/2 and p38 MAPK are key mediators of signal transduction in response to VEGF (53). Interestingly, both ERK1/2 and p38 MAPK activation have been demonstrated after cardiac (12, 57, 66) and cerebral (64) ischemia or ischemia-reperfusion, and other studies (2, 75) have suggested that these MAPKs may be critical mediators of the injury that occurs in response to ischemia in these organs. The mechanisms by which MAPK inhibition attenuates ischemia-reperfusion injury of systemic organs is incompletely understood but may be linked with the inhibition of inflammatory responses (4).
As a first step to understanding the potential role of VEGF in
ischemic lung injury, we sought to develop an in vitro model to
determine how VEGF induces specific pulmonary vascular endothelial responses and to understand whether differential MAPK signaling might
be involved in determining these disparate responses. Our studies
demonstrate a differential concentration dependence of both conduit and
microvascular pulmonary endothelial cells to VEGF when barrier
regulatory and chemotactic responses were evaluated. Both large- and
small-vessel endothelial cell barrier function, assessed by continuous
measurement of TER, declined with increasing concentrations of VEGF.
Barrier dysfunction was transient at low concentrations of VEGF (10
ng/ml) but only partially reversible at higher concentrations of VEGF.
These data are consistent with previous reports of VEGF-mediated
increased endothelial cell permeability in vitro as assessed by
histological (18, 19, 59) and physiological (9, 13,
33, 40, 71) parameters. Our data extend these previous studies
by implicating alterations of cell-cell contact (Rb) rather than endothelial cell detachment
from the extracellular matrix (
) as the cause of endothelial barrier
dysfunction induced by VEGF, findings that are consistent with previous
reports (13, 18, 40) demonstrating a potential role for
endothelial cell junctional protein disorganization in VEGF-mediated
enhanced permeability.
When permeability responses to VEGF were compared with the concentration-response curves for endothelial cell chemotaxis to VEGF, there were clear differences not explained by differences in cell type because BPAECs and BLMVECs were used for evaluation of both responses. Chemotaxis was maximal at a VEGF concentration that caused a trivial, reversible decrease in endothelial barrier function (10 ng/ml) and was attenuated at higher VEGF concentrations, strongly suggesting that enhanced endothelial permeability in response to VEGF occurs independently of endothelial cell migration. This finding is consistent with a recent report (49) that high VEGF expression levels during development were linked with vasculogenesis and permeability, whereas lower levels were associated with angiogenesis and cell migration. The dose response of pulmonary vascular endothelial cells to VEGF when evaluating endothelial chemotaxis in our studies is similar to that recently demonstrated by Kanno et al. (37) in human umbilical vein endothelial cells. Those investigators suggested that 10-fold higher concentrations of VEGF were required to maximally induce endothelial cell proliferation, compared with chemotaxis, but did not evaluate the effects of VEGF on permeability. Only one recent study (28) has directly compared the ability of VEGF to mediate endothelial migration and permeability. Although these investigators evaluated chemotaxis both in vitro and in vivo, endothelial barrier function was assessed only with an in vivo assay (Miles assay). This study (28) suggested that stimulation of KDR/flk-1 (VEGF receptor 2) mediated both endothelial chemotaxis and permeability; however, the specific signaling pathways activated by KDR/flk-1 that might explain differential physiological responses were not evaluated. Interestingly, our data suggest that pharmacological inhibition of flk-1 had no significant effect on endothelial barrier dysfunction in vitro in response to VEGF, although it did abolish VEGF-mediated endothelial cell chemotaxis. A recent study by Clauss et al. (11) suggested a permissive role for transmembrane tumor necrosis factor in mediating VEGF-induced hyperpermeability in human umbilical vein endothelial cells and in skin vasculature, raising the possibility that VEGF may initiate cellular signaling via membrane proteins other than its well-characterized endothelial cell receptors, although this study reported expression of transmembrane tumor necrosis factor in normal dermal endothelium but not in lung vasculature.
VEGF has been demonstrated to activate a number of mediators of signal
transduction in endothelial cells both in vitro and in vivo (26, 28, 32, 43, 52; for summary see Ref. 53). Our studies demonstrate that VEGF-mediated endothelial barrier dysfunction was
attenuated by inhibition of PI3K, p38 MAPK, or MEK/ERK. The effects of
PI3K inhibition appear to be mediated primarily by inhibition of
downstream p38 MAPK activation because VEGF-induced ERK activation was
not dramatically altered by PI3K inhibition. Simultaneous
administration of both p38 and MEK/ERK inhibitors did not abolish this
protective effect (data not shown), suggesting that activation of p38
is not directly upstream of ERK in a signaling cascade or vice versa.
ERK1/2 has been implicated in VEGF-mediated endothelial barrier
dysfunction by several investigators (35, 40), and our
laboratory (70) recently demonstrated that the ERK1/2
pathway is a key regulator in the barrier regulatory response of bovine
endothelial cells to phorbol esters. The involvement of phospholipase
C-/protein kinase C in VEGF-induced alterations in permeability has
been controversial (9, 13, 40, 73), and we could not
identify previous studies that have addressed a potential role for p38
MAPK in enhanced endothelial permeability in response to VEGF. Our
studies suggest that inhibition of either VEGF-mediated ERK1/2 or p38
activation has similar consequences on increased endothelial
permeability, a finding not described in other models of endothelial
barrier regulation.
As previously reported by others (28, 37, 51, 58, 60), in contrast to VEGF-mediated barrier dysfunction, endothelial cell chemotaxis in response to VEGF was attenuated by inhibition of tyrosine kinase, flk-1, PI3K, and p38 but not by inhibition of MEK/ERK. Interestingly, inhibition of MEK/ERK failed to prevent VEGF-mediated cell migration despite the finding that actin cytoskeletal remodeling induced by VEGF was attenuated by MEK inhibition. These data provide additional evidence for differential MAPK signaling in VEGF-induced permeability and chemotaxis. Whereas both responses required p38 activation, ERK1/2 activity was only essential in VEGF-mediated endothelial barrier regulation. Our results are consistent with previous reports (37, 60) suggesting that the effects of VEGF on actin stress fiber formation and endothelial cell migration are mediated by independent pathways involving both p38 activation and enhanced actin polymerization as well as phosphorylation of focal adhesion kinase and assembly of focal adhesions. Our studies also confirmed that inhibition of Src kinase, which inhibits focal adhesion assembly, attenuated VEGF-mediated endothelial chemotaxis, yet Src kinase inhibition did not alter the permeability response to VEGF. It has been shown that VEGF-mediated endothelial chemotaxis (51, 58) and actin stress fiber formation (51) may also depend on activation of a pathway involving the serine threonine kinase Akt and its downstream effector nitric oxide. Thus cell migration in response to VEGF is likely to result from activation of multiple overlapping pathways, and inhibition of actin stress fiber formation alone is not sufficient to prevent this response.
One potential limitation of these studies may be the use of pharmacological inhibitors to investigate pathways of signal transduction. The pharmacological agents used in the present study were administered at concentrations with well-established in vitro specificity (15, 17). In addition, the specificity of these drugs for inhibition of p38 and ERK1/2 MAPK in our studies was confirmed with two independent assays (data not shown). Although a pharmacological approach to protein kinase inhibition has been criticized because of possible nonspecific effects of such agents, others have suggested that an adequate picture of signal transduction is best performed in living cells subjected to a minimal amount of manipulation (61). Furthermore, the use of genetic approaches for modulating signal transduction pathways (e.g., overexpression of dominant negative gene constructs) is limited when evaluating physiological responses such as endothelial permeability because low transfection efficiency and membrane-altering properties of currently available transfection reagents make assessment of barrier properties of transfected cells difficult.
In summary, our studies compare the cellular mechanisms regulating barrier dysfunction and chemotaxis of both conduit and microvascular pulmonary endothelial cells in response to VEGF. The dose-response curves for enhanced permeability and cell migration differed, and these physiological processes involved activation of overlapping but distinct signaling pathways. These data suggest a potential explanation of how VEGF differentially mediates physiological responses in vivo, because concentration-dependent activation of specific signal transduction pathways may determine the balance of effects of VEGF on the vascular endothelium under a number of pathophysiological circumstances, including ischemia. Understanding the cellular mechanisms by which VEGF mediates pulmonary endothelial cell barrier dysfunction and chemotaxis will allow more critical evaluation of the role of this pluripotent growth factor in both normal lung development and ischemic vascular injury and remodeling.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-60628 (to P. M. Becker) and HL-68064 (to A. Verin and J. G. N. Garcia).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. M. Becker, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Rm. 4B72, Baltimore, MD 21224-6801 (E-mail: pbecker{at}mail.jhmi.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 May 2001; accepted in final form 6 August 2001.
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