Departments of Surgery and Medical Physiology, Cardiovascular Research Institute, Texas A&M University System Health Science Center, Temple, Texas 76504
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ABSTRACT |
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The hyperpermeability response of
microvessels in inflammation involves complex signaling reactions and
structural modifications in the endothelium. Our goal was to determine
the role of Src-family kinases (Src) in neutrophil-mediated venular
hyperpermeability and possible interactions between Src and endothelial
barrier components. We found that inhibition of Src abolished the
increases in albumin permeability caused by C5a-activated neutrophils
in intact, perfused coronary venules, as well as in cultured
endothelial monolayers. Activated neutrophils increased Src
phosphorylation at Tyr416, which is located in the catalytic domain,
and decreased phosphorylation at Tyr527 near the carboxyl terminus,
events consistent with reports that phosphorylating and transforming
activities of Src are upregulated by Tyr416 phosphorylation and
negatively regulated by Tyr527 phosphorylation. Furthermore, neutrophil
stimulation resulted in association of Src with the endothelial
junction protein -catenin and
-catenin tyrosine phosphorylation.
These phenomena were abolished by blockage of Src activity. Taken
together, our studies link for the first time neutrophil-induced
hyperpermeability to a pathway involving Src kinase activation,
Src/
-catenin association, and
-catenin tyrosine phosphorylation
in the microvascular endothelium.
permeability; transfection; phosphorylation; endothelium; microvessels
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INTRODUCTION |
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VASCULAR ENDOTHELIUM serves as an effective barrier to control the transvascular passage of solutes, fluid, and blood cells. Alterations in the barrier function are involved in thrombogenesis, angiogenesis, inflammation, and ischemia-reperfusion injury. Binding of inflammatory agonists and cells to endothelial cells elicits a multitude of intracellular signaling events, which can result in an increase in endothelial permeability (12, 19, 30, 34). Actomyosin interaction generates contractile forces that pull tightly connected endothelial cells apart, leading to macromolecular efflux. A transmembrane adhesive protein called vascular endothelial (VE)-cadherin forms endothelial adherens junctions (AJ) that inhibit the paracellular leakage of macromolecules. When the equilibrium between these adhesive and contractile forces is altered, barrier dysfunction and leakage occur.
Previous studies have shown that a group of inflammatory cells,
polymorphonuclear leukocytes (PMNs), adhere to and migrate through the
endothelium into surrounding tissues at sites of injury or inflammation
and that this process is associated with permeability increases
(1, 2, 35). We have demonstrated that PMN-induced hyperpermeability occurs concomitantly with an increase in tyrosine phosphorylation of VE-cadherin and -catenin, an important member of
a family of proteins that links the cadherin complex to the actin
cytoskeleton (14, 16, 27). Recently, studies have shown an
apparent link between
-catenin and the Src-family tyrosine kinases
(Src) (17). The Src kinases are known to play a role in
signaling transduction of endothelial barrier dysfunction and angiogenesis (9, 13, 15, 24). Src activity is regulated by
tyrosine phosphorylation at Tyr416, which upregulates the kinase, and
Tyr527, which renders Src less active (24).
Although a copious amount of work has sought to clearly define the
mechanisms responsible for PMN-induced hyperpermeability (1, 3,
27), the molecular targets of activated PMNs and consequential
events in the venular endothelium where leakage occurs are still poorly
understood. This study focuses on possible interactions among PMNs,
tyrosine kinase pathways, and junctional proteins in the regulation of
endothelial cell barrier function. Through transfection of a specific
peptide (SRCi) or administration of PP1, a Src family kinase inhibitor,
we were able to inhibit Src activity; this inhibition significantly
attenuated PMN-induced hyperpermeability responses in both isolated
coronary venules and cultured endothelial cells. A similarly negatively
charged and phosphorylated peptide used as a control had no effect on hyperpermeability. We show that activated PMNs alter the
phosphorylation status of Src with an increase in phosphorylation at
Tyr416 and a decrease at Tyr527. Furthermore, these phosphorylation
changes are blocked with the addition of SRCi. Immunoprecipitation
shows that under PMN-stimulated conditions, the AJ protein -catenin associates with Src. This association occurs concomitantly with
-catenin tyrosine phosphorylation and loss of
-catenin at the cell periphery, and all three of these events are abolished upon Src
inactivation. Taken together, our data demonstrate for the first time a
link among PMNs, the Src tyrosine kinase pathway, and AJ in the
regulation of endothelial cell barrier integrity.
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METHODS |
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Chemicals and drugs.
The chemicals used were human recombinant C5a and phenylarsine oxide
(PAO; Sigma), SRCi and PP1 (Calbiochem), polyclonal anti-Src and
polyclonal anti--catenin (Santa Cruz), polyclonal antiphospho-Src (Tyr416) and (Tyr527) (Cell Signaling), and polyclonal
antiphosphotyrosine (Transduction). SRCi
[Ac-Tyr(PO3H2)-Tyr(PO3H2)-Tyr(PO3H2)-Ile-Glu-OH] competes for binding to SH2 domains (31). Negative control
peptide was
[Ac-Asp-Ser(PO3H2)-Thr(PO3H2)-Val-Ser(PO3H2)-OH].
Isolation and perfusion of microvessels.
Pigs weighing 9-13 kg were sedated with ketamine (2.5 mg/kg im)
and rompun (2.25 mg/kg im), anesthetized with pentobarbital sodium (25 mg/kg iv), and treated with heparin (250 U/kg iv). After a left
thoracotomy, the heart was electrically fibrillated, excised, and
placed in 4°C physiological saline. The left descending artery was
cannulated, and 3 ml of India ink-gelatin-physiological salt solution
were infused to clearly define microvessels. The methods for isolating
and cannulating coronary microvessels have been described previously
(28, 33, 34, 36). Briefly, venules (0.8-1.2 mm,
diameter 20-50 µm) were dissected and transferred to a
cannulating chamber mounted on a Zeiss Axiovert 135 inverted microscope. The vessel was cannulated with a micropipette on each end
and secured with suture; a third, smaller pipette was inserted into the
inflow pipette. The vessel was perfused with either
albumin-physiological salt solution (APSS) through the outer inflow
pipette or APSS containing FITC-albumin through the inner inflow
pipette. The micropipettes were connected to a reservoir that allowed
intraluminal pressure and flow velocity to be adjusted. The vessel
image was displayed on a video monitor, diameter was measured with a
video caliper, and flow velocity was measured with an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University, College Station, TX). Permeability was quantified by measuring FITC-albumin fluorescence in the vessel and in the surrounding area
(33, 34, 36). The apparent solute permeability
coefficient of albumin (Pa) was calculated using the equation Pa = (1/If)(dIf/dt)0(r/2), where
If is the initial step increase in fluorescent
intensity, (dIf/dt)0 is the
initial rate of gradual increase in intensity as solutes diffuse
out of the vessel into the extravascular space, and r
is the venular radius. The venule was perfused at a constant pressure
of 10 cmH2O and a flow velocity of 7 mm/s, and samples were
not used if leakage of FITC-albumin was detected.
Endothelial cell assays.
For permeability studies, human umbilical vein endothelial cells
(HUVEC; Clonetics) were grown on gelatin-coated Costar transwell membranes (VWR) as previously described (27). Cells were
exposed to porcine PMN for 10 min, transfections were carried out for 1 h, and PP1 and DMSO treatments were 10 min. After these
treatments and in the presence of transfection reagent, PMNs, and all
other reagents, FITC-albumin was added to the luminal chamber for 30 min, and samples were collected from both the luminal and abluminal chambers for fluorometry analysis. Readings were converted with a
standard curve to albumin concentrations, which were then used in the
following equation to determine Pa: Pa = [A]V/tA[L]; where [A] is abluminal concentration,
t is time in seconds, A is area of membrane in
cm2, V is volume of abluminal chamber, and [L] is luminal
concentration. For Western analysis, cells were grown on gelatin-coated
60-mm dishes, treated as appropriate, and lysed. Neutrophils were
washed from the dishes before lysis. In each lane of a 6%
polyacrylamide SDS-PAGE, 10 µg of total protein were electrophoresed,
followed by transfer to nitrocellulose membrane. After exposure of
primary antibody and secondary antibody conjugated to horseradish
peroxidase (HRP), bands were detected by using enhanced
chemiluminescence. Bands were quantitated by scanning densitometry. For
immunoprecipitation, 100 µg of total protein were incubated with
either anti-Src, anti--catenin, or anti-phosphotyrosine antibodies
followed by protein A/G conjugated to agarose beads to isolate the
protein(s) of interest before subjection to PAGE. For
-catenin
localization, cells were grown to confluence on coverslips and treated
in the same manner as that for Western analysis. After fixation and
permeabilization, anti-
-catenin antibody was applied, followed
by secondary antibody conjugated to FITC. The coverslips were mounted
on slides for microscopic observation.
Neutrophil isolation.
Porcine neutrophils (PMN) were isolated as previously described
(27). To activate, PMNs were exposed to human recombinant C5a (108 M). In the isolated venule preparations, PMNs
were added to the suffusion bath and, in the case of HUVEC studies,
were added directly on the monolayer, in both instances at a
concentration of 106/ml. Previous studies have shown that
C5a affects endothelial function via neutrophil-dependent pathways yet
does not affect permeability by itself (27).
Protein transfection. To transfect venules with SRCi, vessels were perfused for 1 h with the peptide at 10 µg/ml in the presence of the polyamine transfection reagent TransIT-LT1 (PanVera) at 10 µl/ml. Previous transfection studies using green fluorescent protein and various inhibiting peptides had shown this to be a suitable way to introduce proteins/peptides into intact microvessels (28). Additionally, the TransIT-LT1 alone has no apparent effects on microvascular permeability or vasoreactivity (28). For HUVEC transfection, the SRCi and TransIT-LT1 were added to the cell medium at the same concentrations, and transfection was allowed to proceed for 1 h. Previous studies have shown successful protein transfection of endothelial cells using this technique (26).
Data analysis. In the immunoblot studies, a representative image of Western blots was selected to present. At least three repetitions were performed for each intervention, and optical densities of the protein bands were averaged. Analysis of variance was used to evaluate the significance of intergroup differences in the immunoblot analyses and permeability studies. A value of P < 0.05 was considered significant for the comparisons.
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RESULTS |
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PMN-induced hyperpermeability in venules.
To determine whether Src is involved in neutrophil-mediated
hyperpermeability, coronary venules were transfected with SRCi or
exposed to PP1, a cell-permeable Src inhibitor. As shown in Fig.
1c, when activated with C5a,
PMNs induce a twofold increase in permeability over basal levels. This
increase is not observed with the addition of unstimulated PMNs or C5a
alone (Fig. 1, a and b). Interestingly, when the
SRCi is transfected for 1 h before the addition of activated PMNs,
the hyperpermeability response is attenuated to near basal levels (Fig.
1d). Transfection of a similarly negatively charged and
phosphorylated control peptide (negative control peptide) had no
significant effect on PMN-induced hyperpermeability (Fig.
1e). Furthermore, addition of the cell-permeable Src
inhibitor PP1 blocked PMN-induced hyperpermeability (Fig. 1f), whereas the PP1 vehicle DMSO had no effect (Fig.
1g).
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PMN-induced hyperpermeability in cultured endothelial cells.
To determine that the permeability response of endothelial cell
monolayers is in agreement with that of intact venules, HUVECs were
exposed to activated PMNs after transfection with SRCi or negative
control peptide or exposure to PP1. As shown in Fig. 2b, activated PMNs induced
significant hyperpermeability responses above control levels. In
addition, the supernatant obtained after activated PMNs were
centrifuged had a similar effect on permeability (Fig. 2c).
However, SRCi completely abolished these permeability increases (Fig.
2e). Transfection of the negative control peptide did not
block PMN-induced hyperpermeability (Fig. 2g). In agreement with the effects of SRCi, PP1 also blocked PMN-induced
hyperpermeability (Fig. 2i), whereas the vehicle DMSO had no
effect (Fig. 2k).
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Src tyrosine phosphorylation.
As stated previously, Src is a tyrosine kinase that itself is regulated
by tyrosine phosphorylation. Using HUVECs, we show that activated PMNs
and PAO (a tyrosine phosphatase inhibitor) induce Src phosphorylation
at Tyr416 (Fig. 3A,
lanes 4 and 7), a condition known to activate
Src. This Tyr416 phosphorylation was attenuated in cells that had been
transfected with the SRCi (Fig. 3A, lanes 6 and
8). Figure 3B, lanes 4 and
7, shows that when Src is activated by PMNs or PAO,
phosphorylation at Tyr527 is decreased. Band intensity from three
different experiments was obtained by using scanning densitometry
followed by quantitation using National Institutes of Health image
software. This data showed that the phosphorylation changes of Src
Tyr416 and Tyr527 in response to activated PMNs and PAO are
significantly different than those of control levels (Fig. 3,
D and E). The amount of Src present in
the cells does not appear to vary significantly under any of the test
conditions (Fig. 3C).
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Src activation and -catenin.
Our previous studies (27) had shown a link between
activated PMNs and associated AJ disorganization, whereas others had shown links between AJ proteins and Src (17). Therefore,
we wanted to determine whether interaction among all three of these factors could be detected. First, we found that the AJ protein
-catenin localizes to the cell periphery in confluent monolayers (Fig. 4A). When monolayers are
exposed to activated PMNs, intercellular gaps are formed and
-catenin staining is lost in areas where the cells no longer contact
each other (Fig. 4B). Supernatant from activated PMNs also
induced gap formation (Fig. 4C). Exposure to PP1 or
transfection of SRCi attenuated PMN-induced gap formation (Fig. 4,
D and F). We found that PMN-induced gap formation
is reversible; that is, when the PMNs are washed away from the cells, the monolayer regains its cell-cell contacts (Fig. 4G).
Finally, transfection of a negative control peptide did not prevent
PMN-induced gap formation (Fig. 4H). Next,
immunoprecipitation and immunoblotting studies showed that upon PMN
stimulation of HUVECs,
-catenin interacts with Src, and this
interaction can be abolished by transfection of SRCi (Fig.
5A). Additionally, PMN-induced
-catenin tyrosine phosphorylation is decreased when Src is
inhibited (Fig. 5, C and D), whereas
-catenin
protein levels remain constant (Fig. 5E). These
findings strongly suggest an interaction among activated PMNs, Src, and
-catenin.
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DISCUSSION |
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PMN-induced microvascular leakage has long been linked to the
development of circulatory dysfunction (10). This
paper reports on the effects of PMNs on isolated porcine
coronary venules and cultured HUVECs. Porcine and human PMNs have very
similar oxidative and chemotactic responses, and both show increased
migration upon stimulation by porcine- or human-derived C5a (5,
22, 25). Although there is some debate as to whether or not
human PMNs lead to HUVEC dysfunction, this study and others (4,
11, 32) have shown that activated human PMNs do elicit HUVEC
retraction and transendothelial protein movement. Interestingly, we
found that physical attachment between PMNs and endothelial cells is not required for gap formation and hyperpermeability responses. On the
other hand, loss of the junctional protein -catenin apparently plays
a role in endothelial barrier dysfunction, as seen by our immunofluorescence studies. These phenomena were observed when supernatant from activated PMNs was applied to endothelial monolayers, suggesting that the physical attachment of PMNs to the
endothelial surface or transmigration is not a prerequisite for
endothelial hyperpermeability or AJ disorganization. This result is in
disagreement with another study suggesting that PMN adhesion is
necessary for induction of endothelial hyperpermeability and that
PMN-derived proteases do not modify AJs (7). An
explanation for the discrepancy is the different cellular preparations
and experimental interventions. In particular, PMN activation was
accomplished by using different agents, in our case human recombinant
C5a and PMA in the case of Del Maschio et al. (7). The two
stimulators may cause different endothelial responses. Importantly, our
recent study on isolated venules supports the importance of PMN release
of endothelial activators, rather than physical adherence to the
endothelium, in the induction of microvascular hyperpermeability,
because in the venule study we found that application of C5a-activated
PMNs to the abluminal side of vessels in the absence of adhesion and migration caused a significant increase in permeability
(37). In agreement with this, others have
determined that activated PMNs release factors such as proteases,
cationic proteins, and oxygen radicals that can induce
hyperpermeability responses and degradation of catenins in the
endothelium (6, 8, 20, 29).
Our previous studies had shown definitive effects of PMNs on vascular endothelium using both intact microvessels and cultured cells, in which activated PMNs cause phosphorylation and conformational changes of AJ proteins in association with intercellular gap formation (27, 35). However, signaling events occurring between PMN adhesion and AJ alteration are not well understood. This study, for the first time, links the well-known Src signaling pathway to these processes. We know that regarding Src tyrosine kinases, which have six functional domains, phosphorylation of Tyr527 and interactions between the SH2 and SH3 domains stabilize the inactive form of Src (21). Conversely, phosphorylation of Tyr416 in the activating loop of the kinase domain activates Src (21). Others have shown a Src requirement for vascular permeability in response to vascular endothelial growth factor (9). Our results clearly show that PMN-induced hyperpermeability in both microvessels and endothelial cells can be greatly attenuated through Src inhibition. Activated PMNs increased Src Tyr416 phosphorylation and decreased Tyr527 phosphorylation, two events that are known to upregulate Src activity. These findings suggest that Src is a major component in PMN-mediated endothelial barrier dysfunction.
The precise molecular mechanisms that lead to microvascular leakage
after PMN adhesion are not clearly understood. Previous studies with
endothelial monolayers have shown that activated PMNs induce actin
stress fiber formation, in contrast to unstimulated cells in which most
of the filamentous actin is found at the cell periphery
(27). Apparently, these fibers contact opposite sides of
the cell membrane and induce cellular contraction, which breaks the
contacts between cells and leads to gap formation. Our hypothesis is
that AJ proteins interact with actin stress fibers and this interaction
leads to a disorganization of the AJ and changes in cellular
morphology. Phosphorylation of -catenin, known to link VE-cadherin
to the actin cytoskeleton (14), may be a crucial signaling
event directing such structural changes. The proposed mechanism of
-catenin tyrosine phosphorylation in AJ disorganization is parallel
to the Wnt/
-catenin signaling pathway in which Wnt is found to
stabilize
-catenin by blocking its serine/threonine phosphorylation and subsequent targeting for degradation, leading to
-catenin nuclear localization and transcriptional activation (23). Conversely, absence of Wnt leads to
-catenin
serine/threonine phosphorylation and proteasomal degradation
(18).
One goal of this study was an attempt to correlate activated PMNs with
a signaling pathway involving -catenin that leads to alteration of
AJ components. This study showed that under PMN-stimulated conditions,
-catenin coimmunoprecipitated with Src. This apparent Src/
-catenin association was completely blocked when cells were transfected with SRCi. Additionally, we were able to demonstrate that
PMN-induced
-catenin tyrosine phosphorylation is blocked under
conditions of Src inhibition in cultured endothelial cells. Taken
together, these results suggest that Src and
-catenin interaction and phosphorylation are necessary for PMN-induced hyperpermeability. Perhaps it is Src kinase that directly phosphorylates
-catenin in
response to activated PMNs; this event leads to the disorganization of
the AJ and ultimately endothelial barrier dysfunction. It is apparent
that Src and
-catenin are involved in multiple cellular processes,
and further studies will attempt to more completely understand the
connection between these two proteins and their interactions with other
components central to barrier integrity in the microvascular endothelium.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-61507 and HL-70752 (to S. Y. Yuan) and a VA VISN 17 grant (to J. H. Tinsley). S. Y. Yuan is a recipient of National Institutes of Health Research Career Award K02 HL-03606.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. H. Tinsley, Dept. of Medical Physiology, Texas A&M Univ. System Health Science Center, 702 SW HK Dodgen Loop, Rm. 206F, Temple, TX 76504 (E-mail: jht{at}tamu.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.
July 24, 2002;10.1152/ajpcell.00230.2002
Received 20 May 2002; accepted in final form 17 July 2002.
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