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From the Vascular Medicine and Atherosclerosis Unit,*
Cardiovascular Division, Department of Medicine, Brigham and Women's
Hospital, Harvard Medical School, Boston, Massachusetts, and Geneva
Biomedical Research Institute,
Geneva, Switzerland
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. Moreover, activation of human vascular ECs through
CD40 induced tube formation in a three-dimensional fibrin matrix gel
assay, an effect antagonized by a MMP inhibitor. These results
demonstrated that activation of ECs by interaction with T cells induced
synthesis and release of MMPs and promoted an angiogenic function of
ECs via CD40L-CD40 signaling. As vascular cells at the sites of chronic
inflammation, such as atherosclerotic plaques, express
CD40 and its ligand, our findings suggest that ligation of CD40
on ECs can mediate aspects of vascular remodeling and neovessel
formation during atherogenesis and other chronic immune
reactions.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.17
Activated ECs also express various surface
molecules, including vascular cell adhesion molecule (VCAM)-1,
intercellular adhesion molecule (ICAM)-1, and E-selectin, that promote
recruitment and activation of leukocytes to the sites of
inflammation.18,19
However, it remains unknown whether
contact-dependent interactions of T cells with ECs could promote MMP
expression.
Recently, several reports have established that human vascular ECs
express CD40.20-22
CD40 belongs to the nerve growth factor
receptor family, which includes the TNF receptors and Fas
(CD95).23
These receptors participate in the regulation of
cell proliferation, differentiation, or apoptosis.24,25
CD40 binds CD40 ligand (CD40L, also referred to as gp39 or TRAP), a
cell surface molecule until recently considered restricted to activated
CD4+ T cells.26
CD40L belongs to the TNF
family, which includes TNF-, Fas ligand, and others.23
Initial studies of CD40L-CD40 signaling focused on T-cell-dependent B
cell differentiation and activation.23,24
Several recent
reports have demonstrated that CD40L-CD40 interaction induces EC
activation manifested by increased expression of adhesion
molecules.20-22
Moreover, recent data implicates CD40 and
CD40L in a variety of sites of human chronic immune and inflammatory
conditions, including multiple sclerosis, various neoplasias,
glomerulonephritis, and atherosclerosis.27-32
The present study explored the hypothesis that ligation of CD40 on ECs could induce expression of MMPs and thus contribute to neovessel formation. We demonstrate here that activation of ECs through CD40 ligation, with either membranes from activated CD4+ T cells or human recombinant CD40L (rCD40L), activates MMP-2, induces expression of MMP-9, increases MMP-1 and MMP-3 expression, and stimulates capillary-like tube formation in vitro, a feature of neovascularization. Thus, T cells can stimulate endothelial production or activation of all three classes of MMP and in this manner probably promote neovascularization through a CD40-CD40L contact-dependent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant CD40L (rCD40L) was obtained from Geneva
Biomedical Research Institute (Geneva, Switzerland).33
IL-1ß, TNF-, and basic fibroblast growth factor was obtained from
Endogen (Cambridge, MA). Gelatin was purchased from Bio-Rad (Hercules,
CA) ß-Casein, phorbol 12-myristate 13-acetate (PMA), and polymyxin B
were obtained from Sigma (St. Louis, MO). Rabbit polyclonal anti-human
MMP-1, MMP-3, and MMP-9 antibodies and the MMP inhibitor UK-231,890
were provided by Pfizer Central Research (Kent, UK). Mouse monoclonal
anti-human tissue inhibitor of MMP-1 (TIMP-1) and TIMP-2 were
obtained from Oncogene Science (San Diego, CA). Monoclonal anti-CD40L
antibody and human fibrinogen were purchased from Calbiochem (San
Diego, CA).
Cell Isolation and Culture
Human vascular ECs were isolated from saphenous veins by collagenase treatment and cultured in dishes coated with fibronectin (1.5 µg/cm2; Upstate Biotechnology, Lake Placid, NY) as described elsewhere.34 Cells were maintained in medium 199 (M199; BioWhittaker, Walkersville, MD), supplemented with 1% penicillin/streptomycin (BioWhittaker), 5% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), 100 µg/ml heparin (Sigma), and 50 µg/ml endothelial cell growth factor (Pel-Freez Biological, Rogers, AK) and used throughout passages two to four. Culture media and FBS contained less than 40 pg of endotoxin/ml as determined by chromogenic Limulus amebocyte assay (QLC-1000; BioWhittaker). ECs were characterized by immunostaining with anti-von Willebrand factor monoclonal antibody (Dako, Carpinteria, CA). Vascular ECs were cultured 24 hours before the experiments in M199 supplemented with 0.1% human serum albumin (Immuno-US, Rochester, MI) and in fresh M199 (serum-free) for the experiments.
Freshly isolated human CD4+ T cells were a gift from Dr.
Andrew Lichtman (Brigham and Women's Hospital, Boston, MA). Purity of
CD4+ T cells was 
98%, as determined by FACS analysis
(anti-CD4 monoclonal antibody (MAb) FITC; Calbiochem). Human
CD4+ T cells were activated with PMA (50 ng/ml for 12
hours), and CD40L cell-surface expression was confirmed by FACS
analysis using anti-CD40L MAb FITC (Calbiochem). Activated
CD4+ T cell membranes were prepared as described
previously,35
and membrane fractions were added for
stimulation at a ratio equivalent to 10 T cells to 1 EC. All
experiments presented in this paper were performed in the presence of
Polymyxin B (1 µg/ml).
Zymography
Supernatants of cultured ECs were centrifuged (500 x g for 10 minutes at 4°C), concentrated 10X (Ultrafree centrifugal filter-4; Millipore, Bedford, MA), and separated under nonreducing conditions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg/ml gelatin or casein.36 After electrophoresis, the proteins were renatured by soaking gels (two times for 15 minutes each) in 2.5% Triton X-100 (VWR Scientific, West Chester, PA). Subsequently, gels were incubated (18 hours at 37°C) in 50 mmol/L Tris/HCl (pH 7.4), containing 10 mmol/L CaCl2 and 0.05% Brij 35 (Sigma). To verify the metalloproteinase activity detected by zymography, identical gels were incubated in the above buffer containing either 20 mmol/L EDTA, an inhibitor of MMPs, or 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), an inhibitor of serine proteases. Finally, gels were stained with Coomassie brilliant blue R250 (Sigma; 30 mmol/L in 45% methanol/10% acetic acid) and then destained in 25% methanol/20% acetic acid.
Western Blotting and Immunoprecipitation Analysis
Supernatants of cultured ECs were centrifuged (500 x g for 10 minutes at 4°C), concentrated 10X, separated by standard SDS-PAGE under reducing conditions, and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) using a semi-dry blotting apparatus (3.0 mA/cm2 for 30 minutes; Bio-Rad). Blots were preincubated (2 hours) in 5% defatted dry milk/PBS/0.1% Tween 20 to limit nonspecific binding, and then antibodies were applied in the same buffer. After 1 hour of incubation with the respective primary antibody (1:10,000 anti-MMP-1 or 1:2,000 anti-MMP-3 or anti-MMP-9), blots were washed four times (PBS/0.1% Tween 20), and the secondary peroxidase-conjugated goat anti-rabbit antibody (Jackson Immunoresearch, West Grove, PA) was added (1:20,000) for another hour. Finally, after washing, detection of the antigen was carried out using the enhanced chemiluminescent detection method according to the manufacturer's recommendations (Dupont-NEN, Boston, MA) and subsequent exposure to the membranes to x-ray film.
For immunoprecipitation, cells were washed with methionine/cysteine-free M199 medium (Sigma). Subsequently, the same medium containing 100 µCi/ml [35S]methionine/cysteine was added to the cells for 24 hours. Supernatants of cultured ECs were centrifuged (500 x g for 10 minutes at 4°C), concentrated 10X, and incubated 24 hours (4°C) with nonimmune rabbit serum to reduce nonspecific binding (Vector, Burlingame, CA). After centrifugation (10,000 x g for 10 minutes at 4°C), supernatants were incubated (2 hours at 4°C) with the respective rabbit polyclonal anti-human MMP or TIMP antibody. Then, protein A agarose beads (Gibco, Gaithersburg, MD) were added (2 hours at 4°C). The samples were washed four times in a mixture of 50 mmol/L Tris/HCl, 0.02% SDS, 0.1% Nonidet P-40, 0.5 mol/L NaCl, 5 mmol/L EDTA, 0.1 mmol/L PMSF, 20 µg/ml soybean trypsin inhibitor, 5 µmol/L leupeptin, and 5 µmol/L aprotinin, and thereafter, immunoprecipitated proteins were eluted by heating (10 minutes at 95°C) in reducing SDS-PAGE buffer (65 mmol/L Tris (pH 6.8), 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.0025% bromophenol blue). After centrifugation (500 x g for 10 minutes at 4°C), supernatants of these samples were separated on SDS-PAGE and transferred to PVDF membranes. Membranes were dried and exposed to x-ray film for detection of immunoprecipitated proteins.
To analyze the same supernatants by Western blotting and immunoprecipitation as well as by zymography, we used serum-free conditions for the experiments presented. Serum-free conditions are necessary for zymographic analysis, as serum is known to contain inhibitors of MMP activities. However, we also analyzed MMP expression under serum-containing conditions in Western blot or radioimmunoprecipitation analysis, achieving identical results.
Tubule Formation Assay
Formation of capillary-like tubes by vascular human ECs from
saphenous veins were analyzed using a three-dimensional fibrin matrix
gel assay as described.37
Briefly, endotoxin- and
plasminogen-free human fibrinogen (10 mg/ml; Calbiochem) was dissolved
in serum-free medium (M199; BioWhittaker) and filtered through
0.22-µm filters (Milex-GS, Millipore). Fibrin matrices were prepared
in six-well plates by polymerizing the fibrinogen solution with
-thrombin (2.0 U/ml; Sigma). After polymerization, gels were soaked
in culture medium (M199; BioWhittaker) containing 10% FBS (Atlanta
Biologicals) for 2 hours at 37°C to inactivate the thrombin. Human
vascular saphenous veins ECs were seeded on the surface of the fibrin
gel (50,000 cells/cm2) and overlayered with serum-free
medium M199 (BioWhittaker) for the periods up to 72 hours in the
presence or absence of stimulus described above. Photomicrographs of
the matrix-gel surface as well as of cross sections were taken using a
Nikon VFX-IIA microscope (x40 and x200). For this purpose, fibrin
matrices were washed twice and supernatants removed before
photomicrographs of the gel surface were obtained. Subsequently, the
matrices were carefully removed from the six-well plates, fixed on
clean glass fiber filter strips, and sliced with a fine surgical blade
(Becton Dickinson, Franklin Town, NJ). The slices were washed with PBS
onto microscopic slides, and tubule formation was determined by the
point-hit counting method.
Quantification of Endothelial Capillary-Like Tubule Formation
Photomicrographs of the matrix-gel surface and of the cross sections described above were analyzed using a computer-assisted morphometric analysis system (Optimas 5.2, Optimas Corp., Bothell, WA) by blinded observers. Tube-like structures (>3 µm) were identified on surface photomicrographs, and total tube length was determined for each of four randomly chosen fields. On cross section photomicrographs, the percentage area of capillary-like tubule formation was measured for each of four randomly chosen sections.
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Results |
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We tested the expression of MMPs by human vascular ECs exposed to
membranes isolated from CD40L-bearing CD4+ T cells.
Analysis of serum-free conditioned media of ECs incubated with
membranes of PMA-activated CD4+ T cells revealed induction
and secretion of MMPs by ECs. Gelatin zymography (Figure 1A)
showed constitutive expression of
latent 72-kd gelatinase (MMP-2) by ECs as previously
reported17
not appreciably affected by either membranes of
activated CD4+ T cell or by PMA. Membranes of activated
CD4+ T cells induced activation of MMP-2 by ECs revealed by
the appearance of a 66-kd gelatinolytic band. This treatment also
caused release of the 92-kd gelatinase (MMP-9) by ECs. Western blotting
demonstrated that the small amounts of MMP-1 and MMP-3 secreted by
unstimulated human vascular ECs in culture increased after stimulation
with membranes of activated CD4+ T cells (Figure 1, B and C)
. For MMP-9, Western blot analysis supported the results obtained by
zymography (data not shown). Studies using an anti-CD40L MAb
established the specificity of modulation of MMPs through CD40
ligation. The slight residual MMP-9 expression even after anti-CD40L
pretreatment likely reflects the effect of non-CD40L-dependent T
cell-EC interactions or could result from incomplete blocking by the
antibody used, which was raised against a recombinant CD40L protein.
Preincubation of T cell membranes with IgG control antibody did not
affect MMP expression. Stimulation of ECs with extracts of membranes
from unstimulated CD4+ T lymphocytes as well as activated
CD4+ T cell membranes alone did not induce MMP expression
(data not shown). The expression of MMPs elicited by CD40 ligation on
ECs exceeded that produced by maximal IL-1ß or TNF- stimulation
(data not shown).
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Additional studies used rCD40L to analyze in more detail the
effect of CD40 ligation on ECs and the release of MMPs. Analysis of
serum-free conditioned media showed constitutive expression of latent
72-kd gelatinase (MMP-2) by ECs unaffected by either rCD40L or PMA
(Figure 2A
, left). Supernatants from ECs
stimulated with PMA, an agonist known to induce synthesis and secretion
of MMPs,17
served as a positive control. Recombinant human
CD40L induced activation of MMP-2 (as demonstrated by the appearance of
a 66-kd gelatinolytic band) and caused release of MMP-9 by ECs. Casein
zymography, a method that detects MMP-3 but not MMP-1, showed that
unstimulated human vascular ECs in culture constitutively secrete small
amounts of MMP-3 and that exposure to rCD40L increased the MMP-3
secretion (Figure 2B
, left). The gelatinolytic and caseinolytic
activities were metal dependent as they were completely inhibited by
EDTA (Figure 2, A and B
, center panels) but not by the serine protease
inhibitor PMSF (Figure 2, A and B
, right). Treatment with rCD40L
increased the release of immunoreactive MMP-1 (Figure 2C
, left), MMP-3
(Figure 2C
, center), and MMP-9 (Figure 2C
, right) by human ECs.
Addition of an anti-CD40L MAb blocked the release of MMPs by ECs in
response to rCD40L.
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To characterize further the induction of MMPs by ECs through CD40,
we investigated the release of MMPs as a function of CD40L
concentration and time. Exposure of ECs to rCD40L activated MMP-2,
induced MMP-9 secretion, and increased MMP-3 and MMP-1 expression in a
concentration- and time-dependent manner (Figure 3)
. The induction of MMP-9 through CD40
activation required at least 200 ng/ml rCD40L for 24 hours. For MMP-2
activation and MMP-3 release, EC activation required 600 ng/ml rCD40L
for 24 hours, and 10 µg/ml rCD40L induced maximal MMP release.
Elaboration of immunoreactive MMP-3 and MMP-9 detected by Western blot
analysis had similar concentration and time dependence (data not
shown). Gelatinolytic or caseinolytic activity due to MMP-9, MMP-2, and
MMP-3, respectively, occurred after 6 and 12 hours, with maximal levels
after 24 hours of stimulation with rCD40L. Forty-eight hours of
stimulation with rCD40L produced no further increase (data not shown).
For MMP-1, Western blot analysis confirmed low constitutive expression
of the protein, which increased after 12 hours of exposure to rCD40L
and peaked after 24 hours. Release of MMPs from ECs induced by CD40
ligation and PMA showed similar time dependence (data not shown).
Radioimmunoprecipitation experiments established that rCD40L induced
de novo synthesis of MMP-9 and increased de novo
synthesis of MMP-1 and MMP-3 by ECs (Figure 4)
.
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Western blot analysis showed that serum-free conditioned medium of
unstimulated human vascular ECs contained both TIMP-1 and TIMP-2.
Exposure to rCD40L (24 hours) enhanced the low basal levels of
TIMP-1, as did PMA used as positive control (Figure 5)
. Treatment with either rCD40L or PMA
did not alter TIMP-2 expression. As found for MMPs, TIMP-1 release was
rCD40L time and concentration dependent, rising after 12 hours and
requiring at least 200 ng/ml rCD40L (data not shown).
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To test the potential functional significance of
CD40-induced MMP by vascular ECs, we examined the formation of
capillary-like structures in an in vitro tube formation
assay. Human vascular ECs cultured without stimulation showed neither
morphological change nor tubule formation (Figure 6
, left panels). In contrast, vascular
ECs stimulated with basic fibroblast growth factor (bFGF), a known
angiogenic factor,38
or with rCD40L yielded identical
morphological changes and formation of capillary-like tubules (Figure 6
, center and right panels, respectively). Anti-CD40L antibody
completely blocked this effect. Furthermore, the MMP inhibitor BB-94
markedly reduced capillary-like tubule formation in response to CD40
ligation, consistent with a functional role of the induced MMP in this
angiogenic behavior. In addition, using computer-assisted image
analysis we quantified the vascular endothelial capillary-like tubule
formation (Figure 7)
. Activation of human
vascular ECs through CD40 resulted in morphological changes and
formation of capillary-like structures, similar in response to the
angiogenic factor bFGF. Compared with unstimulated conditions, CD40
ligation induced percentage area and total length of capillary-tubule
formation by 4.5 ± 0.7 and 3.1 ± 0.6 fold, respectively.
These effects were blocked substantially (P <
0.03) by addition of either an anti-CD40L antibody or the MMP inhibitor
BB-94, in accord with the data mentioned above.
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Angiogenesis required ECs to penetrate the ECM, a process that probably involves the secretion of MMPs. In atheroma, the abundant neovessels may provide a portal for leukocyte trafficking as well as a source of intraplaque hemorrhage, which may contribute to the expansion or evolution of these lesions.42,43 The activation of vascular ECs to secrete MMPs may contribute importantly to angiogenesis in atheroma and during tumor growth as well as at other sites of chronic inflammation.
T cells can produce vascular endothelial growth factor,44 which induces MMP expression by ECs.45 In addition to soluble mediators, T cells often signal via contact-dependent pathways. Interactions between T cells and vascular ECs via CD40L-CD40 signals induce expression of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin on ECs,20-22 which may mediate leukocyte recruitment and provide co-stimulation to T cells. We have recently reported that vascular human ECs, smooth muscle cells (SMCs), and human macrophages express functional CD40 and CD40L in vitro and that within atherosclerotic lesions these cells can co-express CD40 and CD40L.32 We and others31,32 also identified CD40L+/CD4+ T cells in human atheroma, suggesting CD40L-CD40 interactions during atherogenesis.46 Moreover, CD40 ligation can augment MMP expression in human vascular SMCs and monocytes/macrophages in vitro.47,48 Therefore, this study investigated the possibility that interactions between T cells and vascular endothelium may induce expression of MMPs by ECs through CD40 ligation and thus favor angiogenesis.
We report here a novel aspect of T cell-endothelial interaction: induction of MMP expression and capillary-like tube formation via CD40L-CD40 signaling. Exposure of human vascular ECs to CD40L, by either membranes of activated CD4+ T cells or rCD40L, augments expression of members of all three families of MMPs. Both sources of CD40L promoted formation of the active form of MMP-2 and induced the expression of MMP-9. Moreover, ligation of CD40 on ECs increased levels of MMP-1 and MMP-3. Besides MMP expression, vascular ECs constitutively express TIMP-1 and TIMP-2 and increase TIMP-1 levels after CD40 ligation. However, within the human atherosclerotic plaque, the balance between MMP and TIMP appears shifted toward predominance of the enzymatic activity.49 Furthermore, we demonstrate here that basal expression of MMPs is not adequate to promote capillary-like tubule formation, whereas CD40 ligation on vascular ECs shifts the balance toward angiogenic functions. The MMPs/TIMP (im)balance at sites of angiogenesis might further be affected by the reversed induction of MMPs during T cell-endothelial interaction. Romanic and Madri demonstrated that T lymphocyte binding to ECs results in increased T cell MMP synthesis,50 thus providing an additional source of matrix-degrading enzymes.
Although cytokines such as IL-1ß or TNF- can induce expression of
MMPs by ECs,17
these cytokines probably do not mediate
expression of MMPs secondarily via CD40L-CD40 signals. Endothelial
cells do not produce TNF- after stimulation with rCD40L (our
unpublished observations). Ligation of CD40 on human ECs does induce
release of functional mature IL-1ß.51
Indeed, the
expression of MMPs by ECs after CD40 ligation exceeded that produced by
maximally effective concentrations of recombinant IL-1ß. These
results suggest that induction of MMPs through CD40 ligation on ECs
occurs independent of soluble cytokine production.
Matrix metalloproteinases degrade the ECM, allowing cells to penetrate and remodel connective tissue. The production of MMPs by ECs contributes to neovascularization, a process crucial in tumor growth, especially in the early phase of metastasis, in formation of granulation tissue, and considered important in atherogenesis. CD40L expressed on CD4+ T cells delivers contact-dependent activating signals to vascular ECs, yielding induction of adhesion molecules and cytokines.20-22 This report provides new evidence that CD40L on T cells can induce expression and activation of MMPs allowing neovascularization via engagement of CD40 on human vascular ECs. The present observations furnish a novel mechanistic link between T cell activation and angiogenesis observed in atherosclerotic lesions as well as at other sites of chronic inflammation. The induction of MMPs in ECs via CD40 ligation may accelerate the digestion of the ECM and facilitate the migration of cells as well as the formation of neovessels, which serve many functions in pathophysiology.
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Acknowledgements |
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Footnotes |
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Supported in part by grants of the NHLBI to P. Libby (HL-56985), the Swiss National Research Found to F. Mach, and the Deutsche Forschungsgemeinschaft to U. Schönbeck (Scho 614/1-1).
F. Mach and V. Schönbeck contributed equally to this work.
Accepted for publication October 11, 1998.
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 T. Suzuki, K. Fukuo, T. Suhara, O. Yasuda, N. Sato, Y. Takemura, M. Tsubakimoto, and T. Ogihara Eicosapentaenoic Acid Protects Endothelial Cells Against Anoikis Through Restoration of cFLIP Hypertension, September 1, 2003; 42(3): 342 - 348. [Abstract] [Full Text] [PDF]
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 C. Contin, V. Pitard, T. Itai, S. Nagata, J.-F. Moreau, and J. Dechanet-Merville Membrane-anchored CD40 Is Processed by the Tumor Necrosis Factor-{alpha}-converting Enzyme: IMPLICATIONS FOR CD40 SIGNALING J. Biol. Chem., August 29, 2003; 278(35): 32801 - 32809. [Abstract] [Full Text] [PDF]
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M. E. J. Reinders, M. Sho, S. W. Robertson, C. S. Geehan, and D. M. Briscoe Proangiogenic Function of CD40 Ligand-CD40 Interactions J. Immunol., August 1, 2003; 171(3): 1534 - 1541. [Abstract] [Full Text] [PDF]
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M. C. Deregibus, S. Buttiglieri, S. Russo, B. Bussolati, and G. Camussi CD40-dependent Activation of Phosphatidylinositol 3-Kinase/Akt Pathway Mediates Endothelial Cell Survival and in Vitro Angiogenesis J. Biol. Chem., May 9, 2003; 278(20): 18008 - 18014. [Abstract] [Full Text] [PDF]
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 S. S. Ahuja, S. Zhao, T. Bellido, L. I. Plotkin, F. Jimenez, and L. F. Bonewald CD40 Ligand Blocks Apoptosis Induced by Tumor Necrosis Factor {alpha}, Glucocorticoids, and Etoposide in Osteoblasts and the Osteocyte-Like Cell Line Murine Long Bone Osteocyte-Y4 Endocrinology, May 1, 2003; 144(5): 1761 - 1769. [Abstract] [Full Text] [PDF]
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L. Nannizzi-Alaimo, V. L. Alves, and D. R. Phillips Inhibitory Effects of Glycoprotein IIb/IIIa Antagonists and Aspirin on the Release of Soluble CD40 Ligand During Platelet Stimulation Circulation, March 4, 2003; 107(8): 1123 - 1128. [Abstract] [Full Text] [PDF]
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P. K. Shah Mechanisms of plaque vulnerability and rupture J. Am. Coll. Cardiol., February 19, 2003; 41(4_Suppl_S): 15S - 22S. [Abstract] [Full Text] [PDF]
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 C.-L. Wang, Y.-T. Wu, C.-A. Liu, M.-W. Lin, C.-J. Lee, L.-T. Huang, and K. D. Yang Expression of CD40 Ligand on CD4+ T-Cells and Platelets Correlated to the Coronary Artery Lesion and Disease Progress in Kawasaki Disease Pediatrics, February 1, 2003; 111(2): e140 - 147. [Abstract] [Full Text] [PDF]
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 T. M. Moore, W. B. Shirah, P. L. Khimenko, P. Paisley, R. N. Lausch, and A. E. Taylor Involvement of CD40-CD40L signaling in postischemic lung injury Am J Physiol Lung Cell Mol Physiol, December 1, 2002; 283(6): L1255 - L1262. [Abstract] [Full Text] [PDF]
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 L. Fuentes, M. Hernandez, F. J. Fernandez-Aviles, M. S. Crespo, and M. L. Nieto Cooperation Between Secretory Phospholipase A2 and TNF-Receptor Superfamily Signaling: Implications for the Inflammatory Response in Atherogenesis Circ. Res., October 18, 2002; 91(8): 681 - 688. [Abstract] [Full Text] [PDF]
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A. E. May, T. Kalsch, S. Massberg, Y. Herouy, R. Schmidt, and M. Gawaz Engagement of Glycoprotein IIb/IIIa ({alpha}IIb{beta}3) on Platelets Upregulates CD40L and Triggers CD40L-Dependent Matrix Degradation by Endothelial Cells Circulation, October 15, 2002; 106(16): 2111 - 2117. [Abstract] [Full Text] [PDF]
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C. Urbich, E. Dernbach, A. Aicher, A. M. Zeiher, and S. Dimmeler CD40 Ligand Inhibits Endothelial Cell Migration by Increasing Production of Endothelial Reactive Oxygen Species Circulation, August 20, 2002; 106(8): 981 - 986. [Abstract] [Full Text] [PDF]
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C. Monaco, E. Andreakos, S. Young, M. Feldmann, and E. Paleolog T cell-mediated signaling to vascular endothelium: induction of cytokines, chemokines, and tissue factor J. Leukoc. Biol., April 1, 2002; 71(4): 659 - 668. [Abstract] [Full Text] [PDF]
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 K. B. Hotary, I. Yana, F. Sabeh, X.-Y. Li, K. Holmbeck, H. Birkedal-Hansen, E. D. Allen, N. Hiraoka, and S. J. Weiss Matrix Metalloproteinases (MMPs) Regulate Fibrin-invasive Activity via MT1-MMP-dependent and -independent Processes J. Exp. Med., January 28, 2002; 195(3): 295 - 308. [Abstract] [Full Text] [PDF]
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 A. Jakubowski, B. Browning, M. Lukashev, I. Sizing, J. S. Thompson, C. D. Benjamin, Y.-M. Hsu, C. Ambrose, T. S. Zheng, and L. C. Burkly Dual role for TWEAK in angiogenic regulation J. Cell Sci., January 15, 2002; 115(2): 267 - 274. [Abstract] [Full Text] [PDF]
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I. Nelissen, I. Ronsse, J. Van Damme, and G. Opdenakker Regulation of gelatinase B in human monocytic and endothelial cells by PECAM-1 ligation and its modulation by interferon-beta J. Leukoc. Biol., January 1, 2002; 71(1): 89 - 98. [Abstract] [Full Text] [PDF]
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U. Schonbeck and P. Libby CD40 Signaling and Plaque Instability Circ. Res., December 7, 2001; 89(12): 1092 - 1103. [Abstract] [Full Text] [PDF]
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A. Bobik and N. Kalinina Tumor Necrosis Factor Receptor and Ligand Superfamily Family Members TNFRSF14 and LIGHT: New Players in Human Atherogenesis Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1873 - 1875. [Full Text] [PDF]
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M. P. Herman, G. K. Sukhova, P. Libby, N. Gerdes, N. Tang, D. B. Horton, M. Kilbride, R. E. Breitbart, M. Chun, and U. Schonbeck Expression of Neutrophil Collagenase (Matrix Metalloproteinase-8) in Human Atheroma: A Novel Collagenolytic Pathway Suggested by Transcriptional Profiling Circulation, October 16, 2001; 104(16): 1899 - 1904. [Abstract] [Full Text] [PDF]
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P. Friedl, S. Borgmann, and E.-B. Brocker Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement J. Leukoc. Biol., October 1, 2001; 70(4): 491 - 509. [Abstract] [Full Text] [PDF]
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P. Libby and U. Schonbeck Drilling for Oxygen: Angiogenesis Involves Proteolysis of the Extracellular Matrix Circ. Res., August 3, 2001; 89(3): 195 - 197. [Full Text] [PDF]
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P. Libby Current Concepts of the Pathogenesis of the Acute Coronary Syndromes Circulation, July 17, 2001; 104(3): 365 - 372. [Full Text] [PDF]
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M. Strasly, F. Cavallo, M. Geuna, S. Mitola, M. P. Colombo, G. Forni, and F. Bussolino IL-12 Inhibition of Endothelial Cell Functions and Angiogenesis Depends on Lymphocyte-Endothelial Cell Cross-Talk J. Immunol., March 15, 2001; 166(6): 3890 - 3899. [Abstract] [Full Text] [PDF]
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 L. Nelimarkka, H. Salminen, T. Kuopio, S. Nikkari, T. Ekfors, J. Laine, L. Pelliniemi, and H. Jarvelainen Decorin Is Produced by Capillary Endothelial Cells in Inflammation-Associated Angiogenesis Am. J. Pathol., February 1, 2001; 158(2): 345 - 353. [Abstract] [Full Text] [PDF]
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F. Tronc, Z. Mallat, S. Lehoux, M. Wassef, B. Esposito, and A. Tedgui Role of Matrix Metalloproteinases in Blood Flow-Induced Arterial Enlargement : Interaction With NO Arterioscler Thromb Vasc Biol, December 1, 2000; 20 (12): e120 - e126. [Abstract] [Full Text] [PDF]
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M. Melter, M. E. J. Reinders, M. Sho, S. Pal, C. Geehan, M. D. Denton, D. Mukhopadhyay, and D. M. Briscoe Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo Blood, December 1, 2000; 96(12): 3801 - 3808. [Abstract] [Full Text] [PDF]
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 U. Schonbeck, G. K. Sukhova, K. Shimizu, F. Mach, and P. Libby Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice PNAS, June 20, 2000; 97(13): 7458 - 7463. [Abstract] [Full Text] [PDF]
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 A. W. Griffioen and G. Molema Angiogenesis: Potentials for Pharmacologic Intervention in the Treatment of Cancer, Cardiovascular Diseases, and Chronic Inflammation Pharmacol. Rev., June 1, 2000; 52(2): 237 - 268. [Abstract] [Full Text] [PDF]
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U. Schonbeck, F. Mach, G. K. Sukhova, M. Herman, P. Graber, M. R. Kehry, and P. Libby CD40 Ligation Induces Tissue Factor Expression in Human Vascular Smooth Muscle Cells Am. J. Pathol., January 1, 2000; 156(1): 7 - 14. [Abstract] [Full Text] [PDF]
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L. Biancone, V. Cantaluppi, M. Boccellino, L. Del Sorbo, S. Russo, A. Albini, I. Stamenkovic, and G. Camussi Activation of CD40 Favors the Growth and Vascularization of Kaposi's Sarcoma J. Immunol., December 1, 1999; 163(11): 6201 - 6208. [Abstract] [Full Text] [PDF]
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C. Phan, A. W. McMahon, R. C. Nelson, J. F. Elliott, and A. G. Murray Activated Lymphocytes Promote Endothelial Cell Detachment from Matrix: A Role for Modulation of Endothelial Cell {beta}1 Integrin Affinity J. Immunol., October 15, 1999; 163(8): 4557 - 4563. [Abstract] [Full Text] [PDF]
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