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Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin and plakoglobin were observed.
Adhesion, not transmigration, was reported as sufficient to trigger
this disruption. In parallel, when such monolayers were lysed and
subjected to protein gel electrophoresis, VE-cadherin and associated
components were shown to be partly degraded. Other experiments showed
that mAb against VE-cadherin resulted in greater PMN transmigration in
vivo and in vitro (6, 7, 10, 11).
These results gave rise to a model where VE-cadherin acts as a
gatekeeper for passage of macromolecules and of inflammatory
leukocytes. It was proposed that leukocyte adhesion triggered a signal
that led to VE-cadherin complex disassociation, allowing leukocytes to
migrate through the gap so formed (8, 9). This hypothesis
was brought into question by subsequent reports. The degradation of
-catenin and plakoglobin during PMN
adhesion/transmigration as assessed by protein gel
electrophoresis or immunofluorescence was shown to be a
postpreparation artifact. However, the role of VE-cadherin was not
addressed (12). PMN contain substantial amounts and
varieties of proteases (13) and activated PMN or purified
PMN elastase have been reported to cleave VE-cadherin
(14). Thus, the protocols to stop transmigration and then
observe the monolayer were incapable of completely inactivating PMN
proteases. Accordingly, our group examined transendothelial migration
of monocytes and a monocytic leukocyte tumor cell line, U937, both of
which contain fewer proteases than PMN and lack the serine protease PMN
elastase (15). In this study, it was reported that focal
and transient loss of VE-cadherin was associated with transmigration,
mAb that inhibited transmigration (but not leukocyte adhesion) blocked
this loss, and that gaps in VE-cadherin may reseal after transmigration
was complete. However, like earlier studies, postfixation degradation
of junctional proteins remained an issue, and it was not possible to
determine the exact sequence of events or kinetics during
transmigration in this end point-type assay.
Alternative hypotheses of transmigration were put forward by others. It was proposed that endothelial cells have preexisting gaps in both their adherens and tight junctions at corners where three endothelial cells meet, and that PMN preferentially transmigrate in this region (16, 17). In this model, PMN transmigration did not require disruption or loss of junctional components. However, these studies also relied on postfixation analysis, although the authors used an improved fixation protocol. In another model it was reported that chemoattractant-induced PMN transmigration occurred predominantly through the endothelial cell body, rather than at junctions between adjacent endothelial cells, as assessed by fixation and electron microscopy (18). In this model also, no disruption of junctional components was expected.
To resolve these conflicting data, we devised a system using VE-cadherin linked to an enhanced GFP (EGFP) tag. This cDNA construct was placed in a recombinant adenoviral (AdV) expression vector, such that endothelial cells were transduced at high efficiency (19). From similar experiments with E-cadherin in epithelial cells (20), we reasoned that this method would allow direct visualization of VE-cadherin during leukocyte transmigration and thus avoid postpreparation artifacts because the entire process is observed in real time in physiological fluid shear stress conditions. Using this approach, we show that VE-cadherin fused to GFP mimics the native molecule, and we document transient and reversible changes in VE-cadherin localization during leukocyte transmigration. In this in vitro flow system, PMN and monocyte transmigration occurs at small preexisting gaps in VEcadGFP or requires de novo gap formation in this component, but is always paracellular. De novo gaps are only formed during the act of transmigration, and are not triggered by leukocyte-endothelial adhesion. Lastly, we present evidence that gap formation may be due to physical displacement of VEcadGFP in the lateral plane of the adherens junction, suggesting that proteolysis is not necessary for leukocyte transmigration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVECs were isolated and cultured as previously described
(15). Human PMN (>95% pure) were isolated from whole
blood obtained from healthy volunteers by venous puncture as previously
described (21). PBMC were isolated by
centrifugation on Ficoll-Hypaque (Lymphocyte separation medium;
Organon Teknika, Durham, NC). Contaminating RBC were eliminated by
hypotonic lysis. Instead of purified monocytes, the PBMC
fraction was used in transmigration assays for ease of preparation.
Typically, this procedure yielded 
30% monocytes. The other major
population in PBMC preparations is lymphocytes, which do not
transmigrate in this flow system (15).
Antibodies
TEA 1/31 (murine anti-human VE-cadherin, IgG1; Immunotech,
Westbrook, ME) was conjugated to Alexa 538 (Molecular Probes, Eugene,
OR) for immunofluorescence studies. Anti -catenin mAb (RDI-BCATENIN,
murine, IgG1) was purchased from Research Diagnostics (Flanders, NJ);
anti-plakoglobin (PG5.1, murine, IgG2b) was purchased from
BioDesign (Carmel, NY). An anti-junction adhesion molecule (JAM)
polyclonal rabbit antiserum was commercially produced to order, to a
peptide corresponding to the carboxyl-terminal 19 aa of human JAM
(Primm Laboratories, Cambridge, MA).
Construction of EGFP-tagged VE-cadherin (VEcadGFP)
Human VE-cadherin cDNA in a Bluescript II vector was obtained as
a gift from Dr. Shintaro Suzuki (Aichi Human Service Center, Aichi,
Japan). Site-directed mutagenesis (Transformer kit; Clontech
Laboratories, Palo Alto, CA) was used to repair a frame shift mutation
in the cytoplasmic tail, and then to replace the terminal stop codon
with a single base deletion. An EagI site immediately
downstream was used to fuse this construct with EGFP (pEGFP-N2 plasmid;
Clontech). The resulting construct encoded full-length VE-cadherin (737
aa in the mature protein), a linker of 6 aa, and a carboxyl-terminal
tag of EGFP (239 aa) attached to the cytoplasmic tail (Fig. 1
A). Constructs were confirmed by DNA sequencing. Native
VE-cadherin migrates at 140 kDa in SDS-PAGE. The fusion protein was
anticipated to migrate at 170 kDa with the added mass of the EGFP
tag, as confirmed by gel electrophoresis (Fig. 1C).
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Our group has previously used AdV vectors to transduce adhesion molecules such as VCAM-1 and E-selectin into human endothelium (19, 22). Accordingly, the VEcadGFP cDNA was transferred to an AdV expression vector, and high titer virus stocks were produced (23). These stocks were titrated for expression in HUVEC as follows: HUVEC were plated at subculture 2 at a ratio of 1:4 on gelatin-coated plastic 12-well plates. Twenty-four hours later, cells were infected with varying doses of AdV vector, and cultured for another 5 days. Single cell suspensions of HUVEC were then prepared from the 12-well plates using nonenzymatic cell dissociation buffer (Life Technologies, Grand Island, NY) and analyzed by flow cytometry for the EGFP tag. A dose of virus was chosen that routinely caused 60–80% of endothelial cells to express the VEcadGFP at 5 days of infection. Flow cytometric analysis showed that VEcadGFP AdV infection resulted in an increase of expression of the VE-cadherin epitope by 50%. Monolayers were morphologically identical with sham-infected endothelial cells by phase contrast microscopy and by modified Wright-Giemsa stain (Hema 3 Stain; Fisher Scientific, Pittsburgh, PA), and AdV infection did not result in endothelial activation as evidenced by lack of induction of E-selectin, VCAM-1, or up-regulation of ICAM-1 (data not shown and Ref. 19).
Immunoprecipitation and Western blot
HUVEC were transduced with VEcadGFP as described above, and
cultured for 5 days. The monolayers were cell surface biotinylated
(Biotinylation kit; Amersham, Arlington Heights, IL) and lysed as
previously described (9). Equal aliquots of lysate were
immunoprecipitated with mAb against VE-cadherin, -catenin,
plakoglobin, or an anti-JAM polyclonal antiserum. Samples were
resolved on 8% SDS-PAGE, transferred to nitrocellulose, and probed
with streptavidin-horseradish peroxidase. Bands were detected
using ECL (Amersham, Piscataway, NJ) and autofluorography.
Endothelial paracellular permeability assays
Assays were conducted using a modification of previously published techniques (24). Briefly, transwell inserts (0.4-µm pore size, 6.5-mm diameter; Costar, Cambridge, MA) were coated with 0.1% gelatin, and HUVEC were plated at a concentration of 2.5 x 104 cells/well and infected the next day with a dose of virus shown to cause 60–80% expression by flow cytometry. Four days later, fluorescein-conjugated dextran (70 kDa molecular mass, anionic; Molecular Probes) was added at 500 µg/ml in HBSS without phenol red, and the bottom chamber was replaced with HBSS. After 1 h at 37°C, the insert was removed, and the amount of fluorescence in the bottom chamber was measured using a fluorescence plate reader (CytoFluor II; PerSeptive Biosystems, Cambridge, MA). A positive control of thrombin at 2 U/ml was used to confirm normal response of HUVEC to this agonist (data not shown).
Endothelial cell transwell static transmigration assays
Costar transwells (3-µm pore size, 6.5-mm diameter) were
plated and infected as for permeability assays above. After culture for
3–4 days, medium in upper and lower chambers was replaced with TNF-
(Genzyme, Cambridge, MA) at 25 ng/ml for 4 h. PMN prepared from
peripheral blood of normal volunteers were labeled with Cell Tracker
Green (Molecular Probes) at 0.1 µM, and added to the upper chamber at
2.5 x 105 per well in M199 medium. PMN were
allowed to transmigrate at 37°C for varying periods of time, and then
the upper chamber was removed to stop the assay. Transmigrated cells in
the bottom chamber were counted using a fluorescence plate reader.
Endothelial cell flow transmigration assays
HUVEC (passage 2) were plated at 25% confluence on
fibronectin-coated 25-mm glass coverslips and infected 24 h later
with VEcadGFP AdV vector. After 5 days in culture, confluent HUVEC
monolayers were activated with human rTNF- at 25 ng/ml for 4–6 h.
Freshly isolated PMN were resuspended at 0.5 x
106 cells/ml and drawn across HUVEC monolayers in
a parallel plate flow chamber as previously described
(15). Leukocytes were allowed to interact with the
monolayer, and fields were counted using a x40 phase lens at 3, 6, 9,
and 12 min. Transmigrated PMN were distinguished from those interacting
with the apical surface by their phase-dark morphology. Percent
transmigrated = transmigrated cells/total interacting cells
x 100.
Real-time flow fluorescence microscopy
HUVEC (passage 2) were plated at 25% confluence on
fibronectin-coated 25-mm glass coverslips and infected 24 h later
with VEcadGFP AdV vector. After 5 days in culture, confluent HUVEC
monolayers were activated with human rTNF- at 25 ng/ml for 4–6 h.
Freshly isolated PMN or PBMC were labeled for 10 min at 37°C in M199
medium, using Cell Tracker Orange at a 0.1 µM concentration
(Molecular Probes). Labeled leukocytes were resuspended at 0.5 x
106 cells/ml and were drawn across HUVEC
monolayers in a parallel plate flow chamber as previously described
(15). Leukocytes were allowed to roll and adhere to the
monolayer at 20–25°C, which is permissive for leukocyte rolling and
arrest but not transmigration. After sufficient leukocytes had
accumulated on the apical surface, the apparatus was warmed to 37°C
to permit transmigration. Images were collected using a Bio-Rad
(Hercules, CA) confocal apparatus (MRC 1024, Kr/Ar), because its laser
illumination and wide aperture objectives provided good sensitivity
along with computer-aided image acquisition. To gather information from
a broad focal plane, the laser iris was opened to maximum. Using this
technique, red fluorescent leukocytes were visible both above the
monolayer as well as below, indicating that the focal plane was broad
enough to visualize VEcadGFP in the entire thickness of the HUVEC
monolayer. Every 15 s, sequential red channel, green channel, and
differential interference contrast (DIC) images were recorded
digitally, typically for 20 min. Images were analyzed and processed
using Confocal Assistant 4.02 shareware, Todd Clark Brelje, Adobe
Photoshop 5.5, and Adobe ImageReady 2.0 (Adobe Systems, San Jose, CA).
Images are representative of multiple experiments on different days for
both PMN and monocytes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two-color fluorescence microscopy showed that VEcadGFP localized
to endothelial intercellular junctions (Fig. 1
B, A–C), similar to
endogenous VE-cadherin in control monolayers (D). Note that VEcadGFP
was also detected intracellularly (A), whereas Ab staining of these
nonpermeabilized monolayers detected only surface Ag (B). Distribution
of other junctional proteins including platelet endothelial cell
adhesion molecule, JAM, -catenin, and F-actin was similar in
VEcadGFP AdV vector-transduced and control monolayers (data not
shown).
VEcadGFP associated with -catenin and plakoglobin
The cytoplasmic domain of wild-type VE-cadherin forms a complex
with cytosolic proteins - and -catenin, and plakoglobin in
confluent vascular endothelial cell monolayers (25). To
determine whether attachment of a cytoplasmic EGFP tag alters this
complex, confluent HUVEC monolayers were cell surface biotinylated,
lysed, and then subjected to immunoprecipitation and SDS-PAGE. A mAb
against the extracellular domain of VE-cadherin (TEA 1/31)
immunoprecipitated a biotinylated species of 140 kDa in uninfected
HUVEC (Fig. 1C, white arrowhead). VEcadGFP transduction
resulted in a new species of 170 kDa, due to the 27-kDa EGFP tag
(Fig. 1C, black arrowhead). Immunoprecipitation with
-catenin or plakoglobin caused coprecipitation of both VE-cadherin
and VEcadGFP as detected by surface biotinylation, indicating that both
proteins associated with these catenins. A negative control shows that
a polyclonal antiserum to another junctional protein, JAM, did not
associate with -catenin, plakoglobin, VE-cadherin, or VEcadGFP.
Overexpression of VEcadGFP caused enhanced barrier function in HUVEC
To determine whether VEcadGFP contributed to paracellular
permeability similar to wild-type VE-cadherin (2), HUVEC
monolayers were transduced with different viral constructs. Fig. 1D shows that overexpression of either VEcadGFP or wild-type
VE-cadherin in AdV vector constructs resulted in a drop in monolayer
permeability to fluorescein-dextran, as compared with control
uninfected HUVEC or HUVEC transduced with control EGFP AdV vector.
Thus, infection with VEcadGFP AdV vector significantly enhanced HUVEC
barrier function.
Overexpression of VEcadGFP did not significantly alter PMN transmigration kinetics under static or flow conditions
HUVEC were grown on transwell inserts (for the static assay) or
glass coverslips (for the flow assay) and infected with VEcadGFP. They
were then induced with TNF-, and PMN transmigration was measured as
described in Materials and Methods. Fig. 1, E and
F, shows that VEcadGFP infection did not significantly alter
transmigration over a range of time points as compared with uninfected
HUVEC.
Real-time visualization of leukocyte transmigration through HUVEC transduced with VEcadGFP
Using the automated microscopy system described in Materials
and Methods, we followed PMN as they transmigrated through a 4- to
6-h TNF--activated HUVEC monolayer, collecting images every 15
s, both in two-color fluorescence and in DIC. Analysis of these paired
images from multiple experiments showed that in many cases, adherent
PMN approached a continuous junction of VEcadGFP, a gap was formed in
the wall, and then the PMN migrated through the resulting gap (Fig. 2).4 In
other cases, PMN migrated through small preexisting gaps in VEcadGFP
(Fig. 4B). Monocytes
were observed to behave similarly (Fig. 3). From these experiments it was not
possible to determine whether these gaps in VEcadGFP were populated
with endogenous wild-type VE-cadherin. However, even for migration
through preexisting gaps, leukocyte passage typically coincided with a
significant widening of the gap. Typical gap dimensions during
transmigration events were 4–6 µm in size. In a few cases, two
different leukocytes consecutively transmigrated, one behind the other,
through the same gap, where the second leukocyte migrated before the
pore created by the first had completely closed. Of a total of 52
transmigration events viewed in entirety, PMN transmigration was
associated with de novo gap formation in 19 instances; in the remaining
33 events, PMN migrated through a preexisting gap in VEcadGFP. Of 32
monocyte transmigration events, 18 were associated with de novo gap
formation; the rest migrated through preexisting VEcadGFP gaps. It may
be that overexpression of VEcadGFP alters this ratio; however, it is
reasonable to conclude that at least part of the time leukocyte
transmigration is associated with new gap formation.
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Others have reported that PMN transmigration occurs primarily at
corners of three or more endothelial cells, where they observed small
gaps in cadherin staining (16). In the current
experimental system, tricellular gaps were not consistently observed in
either endogenous wild-type VE-cadherin or in VEcadGFP. Instead, there
were occasional small gaps in junctional staining, which occurred both
at multicellular corners, and at bicellular junctions. Furthermore,
both PMN and monocytes migrated through multicellular corners (53 and
26%, respectively; Fig. 4A) and also through bicellular
junctions (47 and 74%; Figs. 2, 3, and 5). When migration occurred at
multicellular corners, it occurred at preexisting gaps as well as
through fresh gap formation. In the current in vitro system, no
evidence of transcellular transmigration was observed. At the
resolution provided by the experimental apparatus, all transmigration
events occurred at lateral junctions, through gaps in VEcadGFP.
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After leukocyte transmigration through a gap, VEcadGFP was found
to fill in and thus reseal the gap formed. This resealing process took
5 min on the average for both PMN and monocytes, and ranged from 1:45
to 11 min. Interestingly, even gaps that were present before approach
of a leukocyte were capable of sealing after transmigration (Fig. 4B), suggesting that endothelial junctional proteins are
dynamic and not fixed.
In several cases of transmigration, it was possible to monitor
VE-cadherin gap formation and closing to a higher degree of clarity
(Fig. 5). Here it was observed that as a
gap was formed in VEcadGFP, there appeared a simultaneous clustering of
green fluorescent label in the junction proximal to the gap. After
transmigration, the bunched up VEcadGFP gradually diffused laterally
back into the gap, analogous to the opening and closing of a curtain.
Gap formation was not due to vertical displacement of VEcadGFP, because
the focal plane was broad enough to visualize the entire monolayer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin and plakoglobin. Overexpression of
VEcadGFP, like overexpression of VE-cadherin, caused a decrease in
endothelial permeability and, therefore, enhanced barrier function.
VEcadGFP did not alter the rate or extent of PMN transmigration
assessed under flow or static conditions. Thus by biochemical and
functional analyses, VEcadGFP mimicked the behavior of native
endothelial VE-cadherin. This construct was used in real-time
fluorescent imaging to extend our previous studies and to analyze
whether leukocyte transmigration led to alterations in VE-cadherin
distribution under flow conditions. Direct real-time observation of VEcadGFP during leukocyte transmigration under flow yielded several interesting findings. We report exclusively paracellular leukocyte transmigration, migration through tricellular corners and bicellular junctions, and migration through both preexisting gaps and through de novo gap formation. Because the current experimental system uses real-time observation of leukocytes and endothelial cells under flow, it avoids the limitations of previous studies such as static assays and postfixation artifacts, and yet is sensitive to small localized and temporary changes in protein localization that may not be appreciated by bulk biochemical analysis.
Although widespread proteolysis of VE-cadherin as a result of PMN adhesion has been reported (8, 9), we have no evidence that this occurred with real-time imaging. Instead, the current system clearly documents formation of gaps (or widening of preexisting gaps) at a distal step, immediately before transmigration. Gap formation or widening is frequently accompanied by a clustering effect of VEcadGFP at adjacent regions of lateral junctions. After transmigration, this clustered material gradually diffuses back to refill the junction. These observations lead us to hypothesize that gap formation or widening allows the endothelium to accommodate a transmigrating leukocyte. Leukocyte-endothelial interaction and cross-linking of endothelial surface proteins have been shown to induce intracellular signals in endothelium (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38). Gap formation or widening may be a result of signaling from such an interaction, or in response to a soluble factor released by leukocytes. Alternatively, it is possible that gap formation may occur as a passive process. From studies of a related protein, E-cadherin, lateral diffusion of cadherin molecules is thought to occur in a time scale of minutes (20). It is possible that as a leukocyte approaches a lateral junction it may extend a pseudopod, which simply forces aside dimers of VE-cadherin that bridge the junction. If there is already a small gap in VE-cadherin, the leukocyte may use this means to widen it sufficiently to permit its passage. The leukocyte then transmigrates through the resulting gap, maintaining it by physically interrupting the junction. Throughout leukocyte passage, we observed that VEcadGFP was in close apposition to the leukocyte perimeter. After transmigration is completed, once more there is no obstruction to the lateral movement of VE-cadherin, and the displaced material returns to close the gap. Like other members of the cadherin family, VE-cadherin is likely to derive adhesive strength by cooperative binding and tethering to the actin cytoskeleton as recently suggested by atomic force microscopy experiments (39). To allow free lateral movement to accommodate leukocyte transmigration, VE-cadherin may need to undergo decoupling from the cytoskeleton. This may occur as an endothelial response to leukocyte signaling, or alternatively, leukocytes may be able to recognize portions of the endothelial junction that are already undergoing remodeling in response to migration, shear stress, or other stimuli. Alternatively, it is possible that VE-cadherin movement is directed by rearrangement of the cytoskeleton. These hypotheses will be tested in future studies.
In the current study, only the EGFP tag at the carboxyl-terminus of
VEcadGFP is detected, thus it remains possible that partial proteolysis
of other portions of the construct may have occurred while the
fluorescent marker is pushed aside. However, physical displacement is
at least partly responsible for gap formation. Gap formation and
closing were also observed where a transduced endothelial cell lay next
to another expressing no detectable VEcadGFP, and where two endothelial
cells both expressed much lower levels (data not shown). Although these
examples display less well, they suggest along with the results shown
in Fig. 1, E and F, that the effects reported
here are not dependent on overexpression of VEcadGFP.
Our group previously reported gaps in VE-cadherin staining at sites where leukocytes were adherent, presumably about to transmigrate using fixation and subsequent immunofluorescent microscopy techniques (15). We concluded that a correlation existed between the position and frequency of gaps and leukocyte transmigration. Thus, leukocytes capable of robust transmigration caused large numbers of gaps in VE-cadherin staining, whereas leukocytes incapable of transmigration did not. However, it was not possible to definitively conclude that a leukocyte was actually going to transmigrate through a particular gap, or even that the gap occurred before fixation and staining. In the current study we confirm and extend our previous findings. First, we document changes in VEcadGFP distribution as a leukocyte is actively transmigrating under physiologic flow conditions. Second, these gaps are in close apposition to the migrating leukocyte and imply a tight fit of the leukocyte within the endothelial junction. Third, the current study indicates a specific order of events as the leukocyte prepares to transmigrate, and kinetics for gap formation and subsequent closure. Finally, leukocyte movement to the lateral junctions is not in itself sufficient to trigger gap formation. The corollary is that without exception, leukocyte migration required either de novo gap formation or widening of a preexisting gap in VE-cadherin.
PMN transmigration of in vitro cultured HUVEC has been reported to
occur predominantly at tricellular corners, where gaps were reported in
staining of both VE-cadherin and occludin (16, 17, 38). We
did not observe consistent gaps in junctional VEcadGFP (or in
VE-cadherin staining) at tricellular corners in the current system.
However, we did find that PMN migrated 50% of the time through
tricellular corners through both de novo and preexisting gaps.
Interestingly, even preexisting gaps were enlarged by the leukocyte
during the actual process of transmigration. In contrast to PMN,
monocytes preferentially migrated through bicellular junctions. We
infer that different leukocyte types may exhibit site preferences for
transmigration, and speculate that different vascular beds may behave
likewise. The somewhat lower level of PMN transmigration at tricellular
corners in the present system may reflect differences in junctional
structure, as Burns et al. (16) use medium that enhance
occludin expression and the number of tight junctional strands formed.
Our current model system did not address tight junctional components;
however, previous reports have found that tight junction "strands"
can be rapidly increased in small vessels in vivo in response to
certain stimuli (40), suggesting that these structures are
dynamic, like adherens junctions. Venous endothelium has been reported
to express less occludin than arterial endothelium (41);
however, we are not aware of studies addressing the expression of tight
junctional components in postcapillary venules. We speculate that tight
junctional components may also dissociate or diffuse out of the way to
accommodate transmigrating leukocytes, and that these gaps may need to
occur in concert with VE-cadherin to coordinate formation of a passage
through the entire junctional complex. These questions will require
development of reagents allowing real-time visualization of multiple
junctional components, rather than relying on postfixation end
point-type assays.
In the present study, monocytes preferred bicellular junctions, whereas PMN migrated through both bi- and tricellular junctions. Less strikingly, monocytes showed a moderate preference for migration through de novo gaps, whereas PMN showed a moderate preference for preexisting gaps. These results suggest potential differences in mechanisms of transmigration between the two cell types, possibly due to differential use of adhesion molecules or chemokines.
VE-cadherin has been proposed to function as a gatekeeper at endothelial junctions. Thus, VE-cadherin mAbs have been well documented to enhance leukocyte transmigration while increasing monolayer permeability to macromolecules (decreased barrier function) (6, 7, 10, 11). We were surprised to find that overexpression of VEcadGFP (or VE-cadherin) in HUVEC did not alter PMN transmigration in static or flow conditions, although barrier function was increased. From these results it appears that improved barrier function to macromolecules does not necessarily equate with reduced transmigration, suggesting a more complicated mechanism beyond simply a generalized "gatekeeper" function for VE-cadherin.
In conclusion, we present data showing real-time changes in VE-cadherin as a result of paracellular transmigration of PMN and monocytes across cytokine-treated endothelial monolayers. The current system avoids postfixation artifacts and difficulties in visualization of small localized effects in VE-cadherin that may not be observed by protein chemistry techniques used in previous studies. Although it is possible that the movement of VEcadGFP does not accurately reflect that of endogenous wild-type VE-cadherin, this is unlikely because no difference in localization or function was observed. The technique described here may serve as a useful model for leukocyte transmigration studies and may be used in the future to track transmigration in different endothelial beds, and to follow trafficking of VE-cadherin in response to other stimuli, such as thrombin or shear stress. Lastly, it may become possible to track multiple components of the lateral junction simultaneously, to gain an understanding of the dynamic response of the entire complex and its role in endothelial behavior and responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sunil Shaw, LMRC 414, 221 Longwood Avenue, Boston, MA 02115. E-mail address: sshaw{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: VE-cadherin, vascular endothelial-cadherin; VEcadGFP, VE-cadherin/green fluorescent protein fusion construct; EGFP, enhanced GFP; PMN, polymorphonuclear cell; AdV, adenoviral; JAM, junction adhesion molecule; DIC, differential interference contrast.
4 The on-line version of this article contains supplemental material.
Received for publication February 20, 2001. Accepted for publication June 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin, and -catenin with vascular endothelial cadherin (VE-cadherin). J. Cell Biol. 129:203.This article has been cited by other articles:
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  A. Rabodzey, P. Alcaide, F. W. Luscinskas, and B. Ladoux Mechanical Forces Induced by the Transendothelial Migration of Human Neutrophils Biophys. J., August 1, 2008; 95(3): 1428 - 1438. [Abstract] [Full Text] [PDF]
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K. A. Kelly, J. R. Allport, A. M. Yu, S. Sinh, E. H. Sage, R. E. Gerszten, and R. Weissleder SPARC is a VCAM-1 counter-ligand that mediates leukocyte transmigration J. Leukoc. Biol., March 1, 2007; 81(3): 748 - 756. [Abstract] [Full Text] [PDF]
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