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J Biol Chem, Vol. 273, Issue 52, 35371-35380, December 25, 1998
From the Division of Infectious Diseases, Departments of Pathology
and Medicine, Department of Veterans Affairs Medical Center, University
of Maryland School of Medicine, Baltimore, Maryland 21201
Bacterial lipopolysaccharide or endotoxin induces
actin reorganization, increased paracellular permeability, and
endothelial cell detachment from the underlying extracellular matrix
in vitro. We studied the effect of endotoxin on
transendothelial albumin flux and detachment of endothelial cells
cultured on gelatin-impregnated filters. The endotoxin-induced changes
in endothelial barrier function and detachment occurred at doses and
times that were compatible with endotoxin-induced apoptosis. Since the
actin cytoskeleton and cell-cell and cell-matrix adhesion all
participate in the regulation of the paracellular pathway and
cell-matrix interactions, we studied whether protein components of the
actin-linked adherens junctions were modified in response to endotoxin.
Components of cell-cell ( Gram-negative bacteremia is often complicated by systemic vascular
collapse, disseminated intravascular coagulation, and vascular leak
syndromes (1-4). One common element to these complications is
endothelial cell (EC)1 injury
and/or dysfunction. Evidence exists that a constituent of the bacterial
cell envelope, endotoxin or lipopolysaccharide (LPS), is responsible
for much of the EC injury associated with Gram-negative sepsis (1, 2,
4, 5). Although much of LPS-induced vascular pathophysiology can be
explained through host responses to LPS (1), evidence exists for a
direct role for LPS (6-9). ECs, which line the vasculature, are the
first host tissue barrier seen by circulating LPS, and LPS has been localized to the endothelium (10). Furthermore, in the absence of
nonendothelial-derived host mediator systems, LPS directly induces
numerous EC responses in vitro (11, 12).
The EC response to LPS includes actin reorganization (6, 8), monolayer
barrier dysfunction (8, 9, 13, 14), and cell detachment from the
underlying extracellular matrix (ECM) (15, 16). In EC, the actin
cytoskeleton is tethered to two distinct adherens junctions, the zonula
adherens (ZA) and focal adhesions (FA), which mediate cell-cell and
cell-matrix interactions, respectively (17-20). These junctions each
contain a transmembrane protein that is linked to the actin
cytoskeleton via a multiprotein complex of cytoplasmic structural and
signaling proteins. The ZA transmembrane protein, cadherin, which
homotypically binds to cadherin molecules on neighboring cells, is
linked to the actin cytoskeleton through at least three cytoplasmic
proteins, LPS has been shown to induce EC apoptosis both in vivo and
in vitro (26-28). In LPS-challenged mice, widespread EC
apoptosis, an event which could only be partially blocked by
recombinant tumor necrosis factor Materials--
The caspase inhibitor peptide,
Z-VAD-fluoromethylketone (Z-VAD) and the negative control peptide,
Z-FA-fluoromethylketone (Z-FA), were purchased from Calbiochem. LPS
phenol-extracted from Escherichia coli serotype 0111:B4,
polymyxin B (PMB) sulfate, staphylococcal enterotoxin B (SEB),
herbimycin A, and dimethyl sulfoxide (Me2SO) were obtained
from Sigma. Recombinant human TNF- EC Culture--
Bovine pulmonary artery ECs (American Type
Culture Collection, Rockville, MD) were cultured in Dulbecco's
modified Eagle's medium (Sigma) enriched with 20% fetal bovine serum
(FBS) (HyClone Laboratories), L-glutamine (5 mM), nonessential amino acids, and vitamins in the presence
of penicillin (50 units/ml) and streptomycin (50 µg/ml) (Sigma) as
described (8, 9). Only cells from passages two through seven were studied.
Assay of Transendothelial Albumin Flux--
Transendothelial
[14C]bovine serum albumin (BSA) flux was assayed as
described (6-9). ECs (2 × 105 cells/chamber) were
cultured for 72 h on gelatin-impregnated polycarbonate filters
(13-mm diameter, 0.4-µm pore size) (Nuclepore Inc., Pleasanton, CA)
mounted in polystyrene chemotactic chambers (ADAPS, Inc., Dedham, MA)
inserted into the wells of 24-well plates. The base-line barrier
function of each monolayer was determined by applying an equivalent and
reproducible amount of tracer molecule, [14C]BSA (1.1 pmol/0.5 ml; Sigma), to each upper compartment for 1 h at
37 °C, after which the lower compartment was counted for 14C activity. Only EC monolayers retaining Filter Detachment Assay--
Gelatin-impregnated polycarbonate
filters (25-mm diameter, 0.4-µm pore size) (Nuclepore) were mounted
in chemotactic chambers (ADAPS) that were inserted into the wells of
6-well plates. The chambers were seeded with 3.5 × 105 EC and cultured for 72 h. Following treatment, the
supernatant and one wash from each well were pooled and the cells
counted in triplicate. The remaining monolayer was trypsin-detached
(0.5 mg/ml, 15 min) and the cells counted. Filters were stained with Coomassie Brilliant Blue to document complete cell detachment. Percent
detachment was expressed as (total cells in the supernatant and
wash)/(total cells in supernatant, wash, and detached from filter) × 100%.
Immunoblotting of Adherens Junction Proteins--
Confluent EC
monolayers were washed with ice-cold phosphate-buffered saline
containing 1 mM sodium orthovanadate, lysed with ice-cold
modified radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, protease
inhibitor mixture tablet (Boehringer Mannheim), 1 µg/ml pepstatin, 1 µg/ml type 1 DNase, 1 mM vanadate, 50 mM
NaF), scraped, transferred to microcentrifuge tubes, and centrifuged
(16,000 × g, 10 min, 4 °C). The supernatants (20 µg of protein/lane) were resolved by SDS-PAGE on an 8-16% Tris
glycine gradient gel (Novex Inc., San Diego, CA) and transferred to
polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA). Blots
were blocked with 3% dry milk and then incubated with anti- Immunoprecipitation (IP) of Adherens Junction Proteins--
EC
were lysed with modified RIPA buffer and the lysates incubated
overnight at 4 °C with anti-pan cadherin, anti- Detection of Apoptosis--
LPS-induced EC apoptosis was assayed
by both DNA laddering and TUNEL (terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling) assays
(35). To assess DNA laddering, EC harvested with a cell scraper from
treated monolayers were centrifuged (200 × g, 5 min)
and resuspended in 200 µl of phosphate-buffered saline. DNA was
isolated using the Apoptotic DNA Ladder kit (Boehringer Mannheim).
Isolated DNA was incubated with RNase, DNase-free (2 µg/ml, 20 min,
room temperature; Boehringer Mannheim), and 2 µg of DNA was resolved
on a 2% agarose gel containing 0.5 µg/ml ethidium bromide. DNA in
the gel was visualized by ultraviolet (UV) light and photographed with
Polaroid positive/negative film (Polaroid Corp., Cambridge, MA).
For the TUNEL assay, ECs cultured in the wells of chamber slides (Nalge
Nunc International Corp., Naperville, IL) were treated, fixed in 4%
paraformaldehyde, and permeabilized (0.1% Triton X-100, 0.1% sodium
citrate; 3 min at 4 °C). Each well was exposed to 100 µl of TUNEL
reaction mixture (Boehringer Mannheim) in a humidified chamber for
1 h at 37 °C in the dark. The slides were rinsed and mounted
with anti-fade mounting medium (Vector Laboratories Inc., Burlingame
CA) under glass coverslips. The monolayers were photographed through a
Zeiss Axioscop 20 microscope (× 100 oil, 1.3 N.A., Plan Neofluar
objective). For phase contrast microscopy, ECs were visualized through
a Zeiss inverted microscope (× 10, 0.25 N.A., F-Achromat PH1 objective).
Statistical Methods--
For time-dependent
measurements (transendothelial albumin flux, filter detachment assays,
and relative state of paxillin tyrosine phosphorylation), the mean
response for each experimental group was compared with its respective
control utilizing the Student's t test. For other
measurements, analyses of variance was used to compare the mean
responses among experimental and control groups. The Tukey post
hoc comparison test was used to determine between which groups
significant differences existed. For all statistical methods employed,
a p value of <0.05 was considered significant.
Dose- and Time-dependent Effect of LPS on Transendothelial
[14C]BSA Flux and EC Detachment--
LPS exposure for
6 h increased transendothelial [14C]BSA flux in a
dose-dependent manner (Fig.
1A). The mean (±S.E.)
pretreatment transendothelial [14C]BSA flux was
0.017 ± 0.001 pmol/h (n = 94), and the mean
(±S.E.) [14C]BSA transfer across naked filters without
endothelial monolayers was 0.215 ± 0.015 pmol/h
(n = 16). LPS at concentrations Dose- and Time-dependent Effect of LPS-induced EC
Apoptosis--
LPS exposure for 4 h induced apoptosis in a
dose-dependent manner as assayed by DNA fragmentation into
multiples of approximately 180 base pairs (bp) (Fig.
2A). DNA fragmentation was
observed at LPS concentrations Dose- and Time-dependent Effect of LPS-induced Cleavage
of ZA and FA Component Proteins--
After establishing that LPS
induces EC apoptosis and that the dose and time requirements for this
event were compatible with those for EC barrier function and
detachment, adherens junction proteins that mediate cell-cell and
cell-substrate adhesion were studied. Western analysis of LPS-exposed
EC revealed a dose- and time-dependent cleavage of the ZA
proteins, Caspase Inhibition Blocks LPS-induced Protein Cleavage and
Apoptosis--
The caspase inhibitor peptide, Z-VAD (36, 37), was
tested for its ability to block LPS-induced cleavage of adherens
junction proteins. The peptide inhibited LPS-induced cleavage of
Effect of Caspase Inhibition on LPS-induced Disruption of the
Endothelial Monolayer--
Since caspase inhibition protected against
LPS-induced cleavage of ZA proteins, the ability of Z-VAD to block
LPS-induced increments in [14C]BSA was studied (Fig.
6). At concentrations that could block both LPS-induced apoptosis and cleavage of FA and ZA proteins, the
caspase inhibitor, Z-VAD (100 µM), did not abrogate
LPS-induced loss of barrier function. Since LPS-induced opening of the
endothelial paracellular pathway can be blocked by protein tyrosine
kinase (PTK) inhibition (6), we studied whether PTK inhibition with herbimycin A could also block LPS-induced cleavage events. Herbimycin A
(1 µM) protected against LPS-induced increments in
[14C]BSA flux (Fig. 6) but did not block cleavage of
adherens junction proteins (Fig. 6, inset).
Influence of Effect of Caspase Inhibition on LPS-induced EC
Detachment--
Since caspase inhibition blocked cleavage of FA
proteins, the ability of the caspase inhibitor peptide to block
LPS-induced EC detachment was studied (Fig.
8). Introduction of Z-VAD (100 µM) protected against LPS-induced (100 ng/ml; 4 h)
EC detachment (Fig. 8A) as well as morphological changes
associated with apoptosis (Fig. 8B). The PTK inhibitor,
herbimycin A (1 µM), conferred only partial protection
against LPS-induced detachment (Fig. 8A). EC exposed to LPS
(100 ng/ml, 4 h) displayed widespread cell rounding and detachment
from the underlying substrate (Fig. 8B, v). These changes were diminished by the Z-VAD peptide (100 µM)
(Fig. 8B, vi) and to a lesser extent by
herbimycin A (1 µM) (Fig. 8B, vii) but not by the negative control peptide, Z-FA (100 µM)
(Fig. 8B, viii).
Influence of FAK Cleavage on Its Protein-Protein
Interactions--
FAK, a PTK localized to specialized areas of
cell-substrate adhesion, binds to and tyrosine phosphorylates another
FA protein, paxillin (39, 40). IP of paxillin co-immunoprecipitated
full-length FAK but not the cleavage product (Fig.
9A). The anti-FAK antibody that recognizes both the full-length FAK and its cleavage product binds
to the region of FAK (amino acids 354-533) that contains its catalytic
domain. Paxillin binds to the COOH terminus of FAK (amino acids
919-1042). These findings indicate that LPS-induced cleavage of FAK
leads to the disassociation of paxillin from the kinase domain of FAK.
We then wanted to determine whether this disassociation affected the
tyrosine phosphorylation state of paxillin. In EC incubated with LPS
(100 ng/ml) for increasing exposure times, tyrosine phosphorylation of
paxillin decreased over time, whereas in medium controls it remained
constant (Fig. 9B). This LPS-induced decrease in tyrosine
phosphorylation of paxillin was prevented by co-administration of the
caspase inhibitor, Z-VAD (100 µM) (Fig. 9C).
LPS exposures of The Lipid A Portion of LPS Induces Cleavage of the ZA and FA
Proteins--
LPS, as the name implies, is composed of both lipid and
polysaccharide components (41). We have previously demonstrated that
the lipid A component of LPS is the bioactive portion responsible for
inducing EC barrier dysfunction (7). To determine whether lipid A also
induces cleavage of adherens junction proteins, we co-administered LPS
with either PMB or ENP, two structurally distinct proteins that bind to
and neutralize lipid A (7) (Fig.
10A). At concentrations that
completely block LPS-induced increments in [14C]BSA flux,
ENP (1 µg/ml) and PMB (10 µg/ml) were able to protect against
LPS-induced cleavage of p130Cas, FAK, SEB and TNF- In this report, EC monolayers cultured on ECM-coated filters were
used in two parallel experimental systems to demonstrate comparable
dose and time requirements for LPS-induced increments in
transendothelial [14C]BSA flux and EC detachment. Our
present and previous studies as well as studies by other investigators
have demonstrated that LPS concentrations of LPS-induced loss of barrier function and EC detachment occurred at LPS
concentrations and exposure times that failed to induce any detectable
cytotoxicity as measured by either trypan blue exclusion or lactate
dehydrogenase release (8, 9, 15). In the present studies, EC apoptosis
as measured by DNA laddering, TUNEL staining, and caspase-mediated
proteolysis could be detected after LPS exposures as brief as 2 h.
Using the more sensitive 51Cr-release assay, small but
consistent increases in 51Cr release could be demonstrated
only after exposures of LPS-induced proteolysis was selective for certain ZA and FA proteins.
The ZA components, The cleavage of all four proteins was inhibited in a
dose-dependent manner by the cell-permeable, irreversible
caspase inhibitor, Z-VAD. Asp in the P1 position of the
peptide confers specificity for caspases; its lack of an amino acid in
the P4 position permits inhibitory activity for several
caspase family members (36). Z-VAD has been previously reported to
block Fas-induced apoptosis, caspase 1 activity, and activation of
caspase 3 (36, 37). A peptide reported to inhibit the cysteine
protease, cathepsin B (38), failed to completely block LPS-induced
cleavage events. Other protease inhibitors, including those directed
against trypsin, serine, thiol, and aspartic proteases, have previously
been reported to be ineffective in blocking LPS-induced EC injury and
detachment (15). The Z-VAD peptide was also able to abrogate
LPS-induced EC DNA laddering and TUNEL staining at the same
concentration that blocked caspase-mediated cleavage events. Recently,
a DNase that degrades DNA during apoptosis has been shown to be
activated following caspase cleavage of its inhibitor (49). Therefore, it is not surprising that caspase inhibition also blocks LPS-induced DNA fragmentation and morphological changes within the nucleus.
Since caspase inhibition could block LPS-induced cleavage of ZA and FA
proteins, we investigated whether it also blocked two LPS-induced EC
responses that reflect EC-EC and EC-ECM integrity, barrier dysfunction,
and EC detachment, respectively. The Z-VAD peptide did not block
LPS-induced increments in transendothelial [14C]BSA flux.
We have previously reported that PTK inhibition protects against
LPS-induced actin depolymerization, opening of the paracellular pathway, and loss of barrier function (6). The mechanism(s) of
protection and the phosphoprotein(s) that participate in LPS-induced barrier dysfunction remain unknown. Herbimycin A blocked
transendothelial [14C]BSA flux but not cleavage of
adherens junction proteins. In addition, LPS treatment of human
pulmonary artery EC, which are more resistant to LPS-induced injury
(50), resulted in barrier dysfunction in the absence of detectable
protein cleavage.2 In
contrast, EC detachment was completely blocked by caspase inhibition,
whereas PTK inhibition only provided partial protection. That PTK
inhibition protects against LPS-induced barrier dysfunction but not
protein cleavage, whereas caspase inhibition blocks proteolysis and EC
detachment but not barrier dysfunction, suggests that LPS-induced EC
detachment and barrier dysfunction are mediated through distinct pathways. Whether the ability of PTK inhibition to partially protect against EC detachment is mediated through the preservation of intercellular adherens junctions remains unknown.
The functional impact of LPS-induced cleavage events on protein-protein
interactions was studied (Fig. 11).
Antibodies raised against the NH2 terminus of In contrast to Increasing evidence suggests that FA integrity and cell adhesion are
necessary for cell survival (61, 62). Integrins not only activate many
signaling pathways associated with cell proliferation, their activation
has also been reported to be anti-apoptotic (61-63). Ligation of the
The LPS molecule, as the name implies, is composed of a lipid
conjugated to a polysaccharide chain. Lipid A has previously been shown
to be the active portion of the LPS molecule capable of stimulating
CD14-bearing cells (68) as well as the non-CD14-bearing EC (7). PMB and
ENP, derived from the bacterium Bacillus polymyxa and the
horseshoe crab Limulus polyphemus, respectively, both bind
to and neutralize the lipid A portion of LPS and block several LPS-induced EC responses (7). In the present report, we found that
preadsorption with either agent protected against LPS-induced cleavage
of all four adherens junction proteins. Thus, lipid A, a lipid that is
novel in nature, induces EC caspase activation. Although these findings
might have suggested that such proteolytic events during septic
processes were limited to LPS exposure, the same cleavage events were
clearly evoked by other agents as well. SEB, which is derived from the
Gram-positive organism, Staphylococcus aureus, and TNF- In summary, LPS induces caspase activation, cleavage of ZA and FA
proteins, barrier dysfunction, and EC detachment, all at comparable
doses and exposure times. There was no evidence of disruption of either
ZA protein-protein interactions or loss of barrier function as a result
of caspase-mediated proteolysis. Cleavage of FAK, however, resulted in
the dissociation of its catalytic kinase domain from the FA protein,
paxillin, and a resultant decrease in the level of paxillin tyrosine
phosphorylation. These events were blocked by caspase inhibition which
also blocked EC detachment. These findings suggest a bifurcation in the
pathways through which LPS influences cell-cell adhesion and the
endothelial paracellular pathway versus EC adhesion to the
underlying ECM. Finally, these findings have ramifications for more
than Gram-negative septic processes as other exogenous and endogenous
mediators can induce similar protein cleavage events.
*
This work was supported in part by the Office of Research
and Development, Department of Veterans Affairs, and U. S. Army Medical Research and Development Command Grant DAMD 17-91-Z-1004.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.
§
To whom correspondence should be addressed: Medical Service (111),
Rm. 5D-139, Dept. of Veterans Affairs Medical Center, 10 N. Greene St.,
Baltimore, MD 21201. Tel.: 410-605-7182; Fax: 410-605-7914.
The abbreviations used are:
EC, endothelial cell(s); LPS, lipopolysaccharide; ECM, extracellular matrix; ZA, zonula
adherens; FA, focal adhesion; FAK, focal adhesion kinase; TNF- 2
D. D. Bannerman, unpublished data.
Bacterial Lipopolysaccharide Disrupts Endothelial Monolayer
Integrity and Survival Signaling Events through Caspase Cleavage of
Adherens Junction Proteins*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
- and 
-catenin) and cell-matrix (focal
adhesion kinase and p130Cas) adherens junctions were
cleaved by caspases activated during apoptosis with dose and time
requirements that paralleled those seen for barrier dysfunction and
detachment. Cleavage of focal adhesion kinase led to its dissociation
from the focal adhesion-associated signaling protein, paxillin,
resulting in reduced paxillin tyrosine phosphorylation. Inhibition of
caspase-mediated cleavage of these proteins protected against
detachment but not opening of the paracellular pathway. Therefore,
endotoxin-induced disruption of endothelial monolayer integrity and
survival signaling events is mediated, in part, through caspase
cleavage of adherens junction proteins.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-, -, and -catenin (21-23). The integrin family of
transmembrane proteins localized to FA binds to the RGD-containing
sequence found in certain ECM proteins (24). Integrins are linked to the actin cytoskeleton via a cytoplasmic complex of proteins comprised of vinculin, tensin, talin, -actinin, paxillin, focal adhesion kinase (FAK), and p130Cas (19, 20, 25).
(TNF-) binding protein, has
been demonstrated (28). LPS also directly induces EC activation of
caspases in vitro, in the absence of endogenous mediators
derived from non-EC sources, including TNF- (29). Caspases, which
are cysteine proteases activated during apoptosis, cleave a limited
number of cell proteins including the adherens junction components,
-catenin, -catenin, and FAK (30-34). In the present report, we
have studied the ability of LPS to induce directly EC apoptosis, and we
have identified key adherens junction proteins that are cleaved as a
result of LPS-induced caspase activation. Since EC monolayer integrity
is dependent on intact cell-cell and cell-matrix junctions, we have
considered how cleavage of adherens junction proteins contributes to
the loss of barrier function and EC detachment. Finally, the impact of
FAK cleavage on protein-protein interactions, tyrosine phosphorylation
events, and anti-apoptotic signaling was studied.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(specific activity 
2 × 107 units/mg) was purchased from Endogen, Inc. (Woburn,
MA). Endotoxin-neutralizing protein (ENP) was a gift from the
Associates of Cape Cod (Woodshole, MA).
97% of the
tracer were then treated and again assayed for transendothelial
[14C]BSA flux.
-catenin
(1.0 µg/ml), anti--catenin (0.5 µg/ml), anti--catenin (0.13 µg/ml), anti-focal adhesion kinase (FAK) (0.25 µg/ml),
anti-p120Cas (0.3 µg/ml), anti-p130Cas (0.25 µg/ml) (all purchased from Transduction Laboratories Inc., Lexington,
KY), anti--catenin NH2 terminus (1:5000 dilution; generous gift of Dr. Barry M. Gumbiner of the Memorial Sloan-Kettering Cancer Center), or anti-pan cadherin (6.6 µg/ml; Sigma) antibodies for 1 h. The blots were incubated with horseradish
peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG)
(0.13 µg/ml; Transduction Labs), developed with enhanced
chemiluminescence (Amersham Pharmacia Biotech), and exposed to Kodak
X-Omat Blue film (NEN Life Science Products). To ensure equal protein
loading, the blots were stained with Fast Green (Sigma) and then
stripped with 100 mM 2-mercaptoethanol, 2% sodium dodecyl
sulfate, 62.5 mM Tris-HCl (pH 6.7) at 70 °C for 30 min,
washed, blocked, and reprobed with anti--tubulin murine monoclonal
antibody (0.5 µg/ml; Boehringer Mannheim) followed by horseradish
peroxidase-conjugated anti-mouse IgG (0.13 µg/ml) (Transduction Labs).
-catenin, anti-APC
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or anti-paxillin
antibodies (2.5 µg of antibody/500 µg of total protein). The
resultant immune complexes were immobilized by incubation with either
anti-mouse or anti-rabbit IgG cross-linked to agarose (Sigma) for
2 h at 4 °C and centrifuged. The pellet was washed three times
with modified RIPA buffer, boiled in 2× sample buffer (5× sample
buffer: 62.5 mM Tris-HCl (pH 6.8), 2.5% SDS, 25%
glycerol, 1% -mercaptoethanol, and 0.1% bromphenol blue), and
immunoblotted as described above. In other experiments, blots of EC
paxillin immunoprecipitates were incubated for 1 h with a
biotinylated 4G10 anti-phosphotyrosine antibody (0.7 µg/ml; Upstate
Biotechnology Inc., Lake Placid, NY) followed by horseradish
peroxidase-conjugated streptavidin (0.5 µg/ml; Upstate Biotechnology
Inc.). To monitor efficiency of IP and protein loading, blots
containing paxillin immunoprecipitates were stripped and reprobed with
anti-paxillin antibody (0.025 µg/ml; Transduction Labs). The blots
were analyzed by laser densitometry (Molecular Dynamics Corp.,
Sunnyvale, CA), and the phosphotyrosine signal was normalized to
paxillin. The relative amount of paxillin tyrosine phosphorylation
following LPS exposure was expressed relative to the simultaneous
medium controls. For those monolayers exposed to LPS (100 ng/ml; 6 h) in the presence of the Z-VAD caspase inhibitor, the relative amount of paxillin tyrosine phosphorylation was expressed relative to that
detected in EC exposed to Z-VAD alone.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 ng/ml increased
[14C]BSA flux compared with the simultaneous medium
control. The LPS-induced effect appeared to plateau at concentrations
of 100 ng/ml. The LPS effect on endothelial barrier function was also time-dependent (Fig. 1B). LPS exposures (10 ng/ml) of 2 h increased [14C]BSA flux compared with the
simultaneous medium control. For the EC detachment studies, the mean
(±S.E.) total cell counts (supernatant + wash + filter) from
LPS-exposed monolayers and media controls were not significantly
different at all time points tested (data not shown). The mean (±S.E.)
percent detachment for cells exposed for 6 h to LPS 10 ng/ml was
increased compared with the simultaneous medium controls (Fig.
1C); at 1 ng/ml the LPS effect approached but did not reach
statistical significance (p = 0.0748). The mean
(±S.E.) percent detachment of cells exposed to LPS 10 ng/ml was
increased at 2 h compared to the simultaneous medium controls (Fig.
1D). Therefore, endothelial barrier dysfunction and EC
detachment could be each induced by comparable LPS concentrations and
exposure times.
View larger version (51K):
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Fig. 1.
Dose- and time-dependent effect
of LPS on transendothelial [14C]BSA flux and EC
detachment. Vertical bars represent mean (±S.E.)
transendothelial [14C]BSA flux in pmol/h (A
and B) or percent EC detachment from gelatin-impregnated
filters (C and D) immediately after a 6-h
exposure to increasing concentrations of LPS (A and
C), or after increasing exposure times to a fixed
concentration of LPS (10 ng/ml) or medium alone (B and
D). n for each experimental condition is notated
within each bar. * = significantly increased compared with
the simultaneous medium control.
1 ng/ml. The LPS-induced effect
appeared to plateau at concentrations of 30 ng/ml. LPS-induced
apoptosis was also time-dependent (Fig. 2B). DNA
laddering was observed at LPS exposures (100 ng/ml) of 2 h.
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Fig. 2.
LPS-induced EC DNA fragmentation. EC
were incubated with either increasing concentrations of LPS for 6 h (A) or a fixed concentration of LPS (100 ng/ml) (+) or
medium alone ( 
) for increasing exposure times (B). EC DNA
was isolated and subjected to gel electrophoresis in a 2% agarose gel
stained with ethidium bromide. Bands were visualized with UV light. DNA
molecular weight markers, expressed as bp, are indicated to the
left of each gel. Each gel is representative of three
separate experiments.
-catenin and -catenin (Fig.
3). The other components of the ZA,
cadherin, -catenin, and p120Cas, remained intact (Fig.
3). At concentrations 3 ng/ml, 6-h LPS exposures induced proteolysis
of both -catenin and -catenin (Fig. 3A). Furthermore,
LPS exposure (100 ng/ml) times of 2 h were required for the cleavage
events (Fig. 3B). -Catenin cleavage generated a 70-kDa
fragment; -catenin was cleaved into two distinct fragments of 74 and
64 kDa. The 74 kDA -catenin cleavage product appeared at lower LPS
concentrations than the 64-kDa product. The FA proteins, FAK and
p130Cas, were similarly cleaved in a dose- (Fig.
4A) and
time-dependent (Fig. 4B) manner. Proteolysis of
FAK and p130Cas generated 96- and 33-kDa cleavage products,
respectively. Two other FA proteins, paxillin and talin, remained
intact, and their relative abundance did not change (data not shown).
The relative abundance of the cytoskeletal protein, -tubulin,
remained constant throughout the LPS exposure (Fig. 4, A and
B).
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Fig. 3.
Dose- and time-dependent effect
of LPS on cleavage of ZA component proteins. EC were incubated
with either increasing concentrations of LPS for 6 h
(A) or a fixed concentration of LPS (100 ng/ml) (+) or
medium alone ( ) for increasing exposure times (B). EC
lysates were immunoblotted with antibodies raised against cadherin,
p120Cas, -catenin, -catenin, or -catenin.
Molecular mass (in thousands) is indicated by arrows to the
right. Each blot is representative of three separate
experiments.
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[in a new window]
Fig. 4.
Dose- and time-dependent effect
of LPS on cleavage of FA component proteins. EC were incubated
with either increasing concentrations of LPS for 6 h
(A) or a fixed concentration of LPS (100 ng/ml) (+) or
medium alone ( ) for increasing exposure times (B). EC
lysates were immunoblotted with anti-p130Cas or anti-FAK
antibodies and then stripped and reprobed for -tubulin. Molecular
mass (in thousands) is indicated by arrows to the
right. Each blot is representative of three separate
experiments.
-catenin, -catenin, FAK, and p130Cas in a
dose-dependent manner at concentrations of 12.5
µM; almost complete inhibition was seen with 100 µM Z-VAD (Fig.
5A and data not shown). The
generation of the 64-kDa -catenin fragment was blocked at lower
concentrations of the caspase inhibitor than the 74-kDa fragment. A
negative control peptide (Z-FA) which inhibits the cysteine protease,
cathepsin B (38), failed to completely block LPS-induced cleavage of
-catenin and FAK. Although it appeared to block the generation of
the 64-kDa fragment of -catenin, it had no inhibitory effect on the
generation of the 74-kDa fragment. Since Z-VAD could block LPS-induced
protein cleavage, the ability of this peptide to block LPS-evoked
apoptosis was assessed by two independent assays, DNA laddering (Fig.
5B) and TUNEL (Fig. 5C). LPS-induced (100 ng/ml,
4 h) EC DNA fragmentation was completely blocked by the Z-VAD (100 µM) peptide (Fig. 5B). The ability of this
peptide to inhibit LPS-induced apoptosis was confirmed by the TUNEL
assay (Fig. 5C). Visualization with fluorescence microscopy of EC exposed to LPS (100 ng/ml, 4 h) revealed cells that were preferentially labeled with the fluorescent nucleotide (Fig. 5C, iv-vi). The labeled chromatin in these cells was condensed, and apoptotic bodies were evident. EC exposed to LPS in the presence of
Z-VAD (Fig. 5C, iii), Z-VAD alone (Fig. 5C, ii),
or medium (Fig. 5C, i) displayed no fluorescent signal.
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Fig. 5.
Caspase inhibition protects against
LPS-induced protein cleavage and apoptosis. EC were incubated for
4 h with medium, caspase inhibitor (Z-VAD; 100 µM),
LPS (100 ng/ml), LPS in the presence of increasing concentrations of
Z-VAD, or LPS in the presence of Z-FA (100 µM), a
negative control (NC) peptide (A). The caspase
inhibitor or negative control peptide was introduced 2 h prior to
and throughout the 4-h LPS exposure. EC lysates were immunoblotted with
antibodies raised against -catenin, -catenin, or FAK. Each blot
is representative of three separate experiments. Apoptosis was detected
by DNA laddering (B) and TUNEL staining (C). EC
were incubated for 4 h with medium, caspase inhibitor (Z-VAD; 100 µM), LPS (100 ng/ml), or LPS with Z-VAD. EC DNA was
electrophoresed through a 2% agarose gel stained with ethidium bromide
(B). Bands were visualized with ultraviolet light. DNA
molecular weight markers, expressed as bp, are indicated to the
left. In other experiments, EC monolayers were fixed,
permeabilized, and assessed for apoptosis by the TUNEL assay
(C) (i, medium control; ii, Z-VAD;
iii, LPS with Z-VAD; and iv-vi, LPS alone).
Arrows and arrowheads indicate apoptotic bodies
and condensed nuclear chromatin, respectively. Magnification, × 450.
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Fig. 6.
Effect of caspase inhibition on LPS-induced
increments in transendothelial albumin flux. Vertical
bars represent mean (±S.E.) transendothelial
[14C]BSA flux in pmol/h immediately after a 4-h exposure
to medium, Z-VAD (100 µM), herbimycin A (Herb
A; 1 µM), LPS (100 ng/ml), LPS + Z-VAD, and LPS + herbimycin A. For all studies, Z-VAD and herbimycin A were introduced 2 and 16 h, respectively, prior to and throughout the LPS challenge.
*, significantly increased compared with media alone; **, significantly
decreased compared with LPS alone but not significantly increased
compared with media alone. n for each experimental group is
indicated in each bar. In other experiments, EC seeded on
cell culture dishes were treated identically as above and immunoblotted
with anti- -catenin antibodies (inset). Each lane number
in brackets corresponds with the same number and treatment
under each vertical bar. The blot is representative of three
separate experiments.
-Catenin Cleavage on Its Protein-Protein
Interactions--
As a first test as to whether -catenin cleavage
affected sequences that are established binding sites for
-catenin-binding proteins, antibodies raised against either the COOH
terminus (amino acids 571-781) or the NH2 terminus (amino
acids 6-138) were used to probe blots of lysates of EC exposed to LPS
(100 ng/ml; 6 h) or medium alone (Fig.
7A). The antibody directed
against the NH2 terminus failed to recognize the 70-kDa
fragment, localizing the cleavage site(s) to the NH2
terminus. IP of -catenin, which binds to the NH2
terminus of -catenin, or IP of cadherin or APC, both of which bind
to the COOH terminus of -catenin, co-immunoprecipitated both the
full-length and truncated forms of -catenin (Fig. 7B). -Catenin and cadherin each also bound the two -catenin cleavage products (data not shown).
View larger version (43K):
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Fig. 7.
Effect of LPS-induced -catenin cleavage on
-catenin-protein interactions. Lysates of EC exposed for 6 h to LPS (100 ng/ml; +) or medium alone () were immunoblotted with
anti--catenin antibodies raised against either the COOH- or
NH2 terminus (A). In other experiments, lysates
of EC similarly exposed to LPS or medium alone were incubated with
anti-cadherin, anti--catenin, or anti-APC antibodies followed by
anti-mouse or anti-rabbit IgG cross-linked to agarose (B).
The immunoprecipitated proteins were resolved by SDS-PAGE, transferred
to polyvinylidene fluoride membrane, and immunoblotted with an antibody
raised against the COOH terminus of -catenin. The left
panel represents whole cell lysates probed with anti--catenin
antibody. Each blot is representative of three separate
experiments.
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Fig. 8.
Caspase inhibition protects against
LPS-induced EC detachment. A, vertical bars
represent mean (±S.E.) percent EC detachment from gelatin-impregnated
filters immediately after a 4-h exposure to medium, Z-VAD (100 µM), herbimycin A (Herb A; 1 µM), LPS (100 ng/ml), LPS + Z-VAD, or LPS + herbimycin A. For all studies, Z-VAD and Herb A were introduced 2 and 16 h,
respectively, prior to and throughout the LPS challenge. *,
significantly increased compared with medium alone; **, significantly
decreased compared with LPS alone but not significantly increased
compared with medium alone; ***, significantly decreased compared with
LPS alone and significantly increased compared with medium alone.
n for each experimental group is indicated in each
bar. B, in other studies, ECs cultured in plastic
dishes were incubated for 4 h with (i) medium, (ii) Z-VAD (100 µM), (iii) herbimycin A (1 µM), (iv) a
control peptide, Z-FA (100 µM), (v) LPS alone (100 ng/ml), (vi) LPS + Z-VAD, (vii) LPS + herbimycin A, or (viii) LPS + Z-FA and then visualized by phase contrast microscopy. Magnification, × 35.
4 h decreased tyrosine phosphorylation of paxillin
compared with the simultaneous medium controls, and at 6 h, the
Z-VAD peptide conferred ~90% protection against this decrease (Fig.
9D).
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Fig. 9.
Effect of FAK cleavage on its association
with and tyrosine phosphorylation of paxillin. Lysates of EC
exposed for 6 h to LPS (100 ng/ml; +) or medium alone ( ) were
immunoprecipitated with anti-paxillin antibodies and immunoblotted with
a murine monoclonal antibody raised against FAK (A). The
left two lanes represent whole cell lysates probed with
anti-FAK antibody. In other experiments, paxillin was
immunoprecipitated from lysates of EC after increasing exposure times
to LPS (100 ng/ml; +) or media alone () (B) or to EC
treated for 6 h with LPS (100 ng/ml) or medium, in the presence or
absence of Z-VAD (100 µM) (C). Blots of these
paxillin immunoprecipitates were probed with biotinylated
anti-phosphotyrosine antibody (4G10). All blots (A, B, and
C) were stripped and reprobed for paxillin (*IB).
Laser densitometry to quantitate B and C is shown
(D). Each blot is representative of three separate
experiments.
-catenin, and
-catenin (Fig. 10A and data not shown).
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Fig. 10.
LPS-induced EC protein cleavage is blocked
by lipid A neutralizing proteins and mimicked by EC exposure to either
SEB or TNF- . EC were incubated for 4 h with medium, ENP (1 µg/ml), PMB (10 µg/ml), LPS (100 ng/ml), or LPS in the presence of
ENP or PMB (A). In other experiments, EC were incubated for
4 h with either media, LPS (100 ng/ml), SEB (100 µg/ml), or
TNF- (500 units/ml) (B). EC lysates were immunoblotted
with either anti--catenin or anti-FAK antibodies. Each blot is
representative of three separate experiments.
Also Induce Cleavage of ZA and FA Component
Proteins--
To determine whether cleavage of EC adherens junction
proteins was specific to LPS, we incubated EC with concentrations of either SEB (100 µg/ml) or TNF- (500 units/ml) which induce
comparable barrier dysfunction to that seen with 100 ng/ml LPS (Fig.
10B). Both agonists were able to induce identical cleavage
of p130Cas, FAK, -catenin, and -catenin (Fig.
10B and data not shown).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
10 ng/ml and LPS
exposure times of 2 h are required to induce a wide range of EC
responses including actin reorganization, intercellular gap formation,
loss of barrier function, and EC detachment (8, 9, 13-15). Although
this 2-h stimulus-to-response lag time is sufficient for new protein synthesis to occur, neither LPS-induced barrier dysfunction (8) nor EC
detachment (15) was blocked by prior protein synthesis inhibition.
Furthermore, the time requirements for the LPS-induced loss of barrier
function are not unlike those described after recombinant TNF- (42,
43), interleukin-1 (44), or SEB (45) exposures.
4 h (9, 15). Interestingly, LPS-induced
opening of the paracellular pathway and EC detachment each precede
51Cr release (9, 15). The detection of 51Cr
release following prolonged LPS exposures may reflect the later stages
of apoptosis when plasma membrane integrity is often compromised (46).
- and -catenin, were cleaved, whereas -catenin, p120Cas, and cadherin were not. The two
substrates for activated caspases are both members of the Armadillo
family of proteins and share 65% sequence homology (47, 48). Although
a cleavage product could not be detected for p120Cas, a
more distantly related member of the Armadillo family, its total
relative abundance inconsistently decreased with increasing LPS
exposure times. Of the FA proteins studied, only FAK and
p130Cas were cleaved. To our knowledge, this is the first
time that p130Cas has been identified as a substrate for caspases.
-catenin
failed to recognize the lower molecular mass band demonstrating that
the truncated form lacked the NH2 terminus. The
-catenin-binding site on -catenin has been localized to amino
acids 120-151 in the NH2 terminus (51), and the cadherin
and APC-binding sites are located in the COOH-terminal Armadillo
repeats (52). It was anticipated that APC and cadherin could bind to
the lower molecular mass cleavage product of -catenin. Unexpectedly,
-catenin also bound to the cleavage product suggesting that the
caspase cleavage site is proximal to the -catenin-binding site.
NH2-terminal deletion mutants of -catenin lacking amino
acids 1-89 retain normal binding to both cadherin and -catenin
(53). Recently, growth factor deprivation has been shown to induce
caspase-mediated cleavage of EC -catenin into successively smaller
fragments over time (33). Those fragments that appeared at 4 h
still bound to -catenin, whereas the smallest fragment which only
appeared after 8 h did not. In serum-starved fibroblasts,
Brancolini et al. (34) described a -catenin cleavage
product that displayed decreased binding to -catenin. Although
-catenin cleavage occurred as early as 2 h, the decreased
association of - and -catenins was reported only after 12 h.
LPS-induced -catenin cleavage, barrier dysfunction, and EC
detachment were each detectable at 
4h when ZA multiprotein complex integrity was maintained. It is conceivable that prolonged LPS
exposure generates smaller -catenin fragments unable to bind -catenin or to couple the ZA to the actin cytoskeleton. Two previous studies have presented evidence for the existence of multiple caspase
cleavage sites within -catenin, some of which differ in their
specificity for various caspase family members (33, 34). Furthermore,
one recent report has shown that distinct inducers of apoptosis differ
in their ability to promote caspase-mediated cleavage of a given
substrate (54). That apoptosis induced by LPS exposure and growth
factor withdrawal each activates caspases which differentially cleave
-catenin is not surprising. Although disruption of
-catenin-protein interactions was not detected, this does not
necessarily mean cell-cell adhesion remained competent. A human gastric
cancer cell line expressing mutant -catenin with an
NH2-terminal deletion of amino acids 28-134 retained its
ability to bind -catenin and cadherin (55). However, these cells
displayed reduced intercellular adhesion that was restored upon
expression of full-length -catenin. As EC approach confluence and
form stable ZA junctions, -catenin replaces -catenin as the
protein linking cadherin to -catenin (56). Our findings that
-catenin is a substrate for caspase-mediated proteolysis and that
its cleavage products retain the ability to bind -catenin are in
agreement with a previous study (33).
View larger version (68K):
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Fig. 11.
Hypothetical schema for LPS-induced
disruption of endothelial monolayer integrity. LPS-induced loss of
endothelial barrier function and EC detachment may be mechanistically
linked to the activation of PTKs and/or caspases. LPS induces the
activation of EC PTKs and a transient state of increased protein
tyrosine phosphorylation. PTK inhibition protects against LPS-induced
intercellular gap formation, actin depolymerization, and loss of
barrier function. LPS also activates caspases that cleave the zonula
adherens proteins, - and -catenin, as well as the focal adhesion
proteins, focal adhesion kinase (FAK) and p130Cas. The
cleavage of - and -catenin does not appear to alter their binding
to -catenin. Cleavage of FAK leads to the dissociation of its kinase
domain from another focal adhesion constituent protein and FAK
substrate, paxillin, resulting in decreased tyrosine phosphorylation of
the latter. This may, in turn, disrupt the ability of focal adhesions
to participate in cell adherence to the underlying extracellular matrix
and/or disrupt integrin-mediated, cell-survival signaling.
- and -catenin proteolysis, FAK cleavage resulted
in its inability to associate with at least one other protein, paxillin. The FAK cleavage product, which contains the catalytic kinase
domain, was unable to associate with paxillin which binds to the COOH
terminus of FAK. Compatible with these findings, two distinct caspase
cleavage sites have been identified between the kinase domain and the
FA targeting domain, the latter of which overlaps with the
paxillin-binding site (30, 31). Since FAK phosphorylates paxillin on
Tyr-118, in vitro (40), we studied whether the state of
paxillin tyrosine phosphorylation was affected by FAK cleavage. We have
previously shown increased tyrosine phosphorylation of paxillin in EC
following a brief LPS exposure (1 h) (6, 7) temporally proximal to
the caspase-mediated FAK cleavage described here. LPS exposure times
(2 h) that resulted in FAK cleavage and EC detachment were associated
with a time-dependent decrease in the state of paxillin
tyrosine phosphorylation. Moreover, this decrease was blocked by
caspase inhibition. Therefore, caspase-mediated cleavage of FAK leads
to dissociation of its catalytic domain from paxillin resulting in a
decreased state of paxillin tyrosine phosphorylation. Evidence exists
that tyrosine phosphorylation of FA proteins, including paxillin, is
important for FA formation and cell attachment (25, 57-60). First,
adherent cells have increased phosphotyrosine-containing proteins
localized to the FA (58), and more specifically, tyrosine
phosphorylation of FAK and paxillin increases as cells adhere to the
underlying ECM (57). Second, PTK inhibition blocks FA formation (57).
Finally, overexpression of a protein tyrosine phosphatase prevents cell
spreading and FA formation (59), whereas protein tyrosine phosphatase
inhibition promotes FAK and paxillin tyrosine phosphorylation, stress
fiber formation, and FA assembly (60).
v3 integrin, the primary integrin found
in mature EC FA, promotes cell survival through suppression of the
bax-induced apoptosis pathway (64), and in proliferating EC,
v3 antagonism induces apoptosis (65).
Since integrin engagement and/or activation are vital for cell
survival, disruption of the structural and/or signaling elements that
couple integrins to the cell interior may arrest cell proliferation and
promote apoptosis. In fact, displacement of endogenous FAK from FA by
microinjection of a FAK mutant that contains the FA-targeting COOH
terminus but lacks the catalytic kinase domain diminishes serum-induced
DNA synthesis (66). Interestingly, caspase-mediated cleavage of FAK
generates a COOH-terminal fragment not unlike the above mentioned
COOH-terminal construct which competes with FAK for binding to the FA
complex (31, 66). Finally, reduction of cellular levels of FAK with antisense oligonucleotides induces cell detachment and apoptosis (67).
,
an established mediator of the acute phase response to injury that is
elevated during both Gram-negative and Gram-positive bacterial
infections, each were able to induce cleavage of the same four adherens
junction proteins cleaved following exposure to LPS. The cleavage
products generated by proteolysis of all four proteins were of the same
molecular mass as those detected following LPS exposure. These combined
data suggest that cleavage of adherens junction proteins and disruption
of tyrosine phosphorylation signaling events occur in response to
diverse apoptotic stimuli. Furthermore, the elevated levels of TNF-
observed during sepsis may synergistically enhance the sensitivity of
EC to LPS- and/or SEB-induced apoptosis.
FOOTNOTES
Recipient of the Department of Defense Augmentation Award for
Science and Engineering Research Training.
, tumor necrosis factor ; PMB, polymyxin B; SEB, staphylococcal
enterotoxin B; Me2SO, dimethyl sulfoxide; ENP, endotoxin
neutralizing protein; FBS, fetal bovine serum; DMEM, Dulbecco's
modified Eagle's medium; BSA, bovine serum albumin; RIPA, radioimmunoprecipitation assay; IgG, immunoglobulin G; IP, immunoprecipitation; TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling; PTK, protein tyrosine kinase; bp, base pairs; APC, familial adenomatous
polyposis coli gene product; Z-, benzyloxycarbonyl-.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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I. Petrache, A. D. Verin, M. T. Crow, A. Birukova, F. Liu, and J. G. N. Garcia Differential effect of MLC kinase in TNF-{alpha}-induced endothelial cell apoptosis and barrier dysfunction Am J Physiol Lung Cell Mol Physiol, June 1, 2001; 280(6): L1168 - L1178. [Abstract] [Full Text] [PDF]
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 Y.-H. Ling, Y. Zhong, and R. Perez-Soler Disruption of Cell Adhesion and Caspase-Mediated Proteolysis of {beta}- and {gamma}-Catenins and APC Protein in Paclitaxel-Induced Apoptosis Mol. Pharmacol., March 1, 2001; 59(3): 593 - 603. [Abstract] [Full Text]
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G. Zysk, B. K. Schneider-Wald, J. H. Hwang, L. Bejo, K. S. Kim, T. J. Mitchell, R. Hakenbeck, and H.-P. Heinz Pneumolysin Is the Main Inducer of Cytotoxicity to Brain Microvascular Endothelial Cells Caused by Streptococcus pneumoniae Infect. Immun., February 1, 2001; 69(2): 845 - 852. [Abstract] [Full Text] [PDF]
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E. O. Harrington, A. Smeglin, J. Newton, G. Ballard, and S. Rounds Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis Am J Physiol Lung Cell Mol Physiol, February 1, 2001; 280(2): L342 - L353. [Abstract] [Full Text] [PDF]
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L. Xing, C. Ge, R. Zeltser, G. Maskevitch, B. J. Mayer, and K. Alexandropoulos c-Src Signaling Induced by the Adapters Sin and Cas Is Mediated by Rap1 GTPase Mol. Cell. Biol., October 1, 2000; 20(19): 7363 - 7377. [Abstract] [Full Text]
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 J.-F. Martini, C. Piot, L. M. Humeau, I. Struman, J. A. Martial, and R. I. Weiner The Antiangiogenic Factor 16K PRL Induces Programmed Cell Death in Endothelial Cells by Caspase Activation Mol. Endocrinol., October 1, 2000; 14(10): 1536 - 1549. [Abstract] [Full Text]
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 J. N. Lavoie, C. Champagne, M.-C. Gingras, and A. Robert Adenovirus E4 Open Reading Frame 4-induced Apoptosis Involves Dysregulation of Src Family Kinases J. Cell Biol., September 5, 2000; 150(5): 1037 - 1056. [Abstract] [Full Text] [PDF]
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 J. Albon, A. B. Tullo, S. Aktar, and M. E. Boulton Apoptosis in the Endothelium of Human Corneas for Transplantation Invest. Ophthalmol. Vis. Sci., September 1, 2000; 41(10): 2887 - 2893. [Abstract] [Full Text]
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 L. Chandrasekaran, C.-Z. He, H. Al-Barazi, H. C. Krutzsch, M. L. Iruela-Arispe, and D. D. Roberts Cell Contact-dependent Activation of alpha 3beta 1 Integrin Modulates Endothelial Cell Responses to Thrombospondin-1 Mol. Biol. Cell, September 1, 2000; 11(9): 2885 - 2900. [Abstract] [Full Text]
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S. F. Law, G. M. O'Neill, S. J. Fashena, M. B. Einarson, and E. A. Golemis The Docking Protein HEF1 Is an Apoptotic Mediator at Focal Adhesion Sites Mol. Cell. Biol., July 15, 2000; 20(14): 5184 - 5195. [Abstract] [Full Text]
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 T. Stefanec Endothelial Apoptosis: Could It Have a Role in the Pathogenesis and Treatment of Disease? Chest, March 1, 2000; 117(3): 841 - 854. [Abstract] [Full Text] [PDF]
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S. Kook, S. R. Shim, S. J. Choi, J. Ahnn, J. I. Kim, S. H. Eom, Y. K. Jung, S. G. Paik, and W. K. Song Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells Mol. Biol. Cell, March 1, 2000; 11(3): 929 - 939. [Abstract] [Full Text]
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J. C. Mills, N. L. Stone, and R. N. Pittman Extranuclear Apoptosis: The Role of the Cytoplasm in the Execution Phase J. Cell Biol., August 23, 1999; 146(4): 703 - 708. [Abstract] [Full Text] [PDF]
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D. D. Bannerman, J. C. Tupper, W. A. Ricketts, C. F. Bennett, R. K. Winn, and J. M. Harlan A Constitutive Cytoprotective Pathway Protects Endothelial Cells from Lipopolysaccharide-induced Apoptosis J. Biol. Chem., April 27, 2001; 276(18): 14924 - 14932. [Abstract] [Full Text] [PDF]
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