© 1999 American Heart Association, Inc.
Original Contribution |
Vascular Endothelial Growth Factor Induces Activation and Subcellular Translocation of Focal Adhesion Kinase (p125FAK) in Cultured Rat Cardiac Myocytes
From the Departments of Cardiovascular Medicine (N.T., Y.S., K.T., T.K., Y.Y.) and Nephrology and Endocrinology (E.N.), Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo; Institute for Adult Diseases (N.T., Y.S.), Asahi Life Foundation, Shinjuku-ku, Tokyo; Department of Immunology (Y.S.), School of Medicine, Juntendo University, Bunkyo-ku, Tokyo; and Department of Molecular Biology (H.S.), Osaka BioScience Institute, Suita, Osaka, Japan.
Correspondence to Naoyuki Takahashi, Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail takan-tky{at}umin.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Vascular endothelial growth factor (VEGF) has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation. Recently, we found that VEGF activated the mitogen-activated protein kinase cascade in cultured rat cardiac myocytes. To elucidate how VEGF affects adhesive interaction of cardiac myocytes with the extracellular matrix (ECM), one of the important cell functions, we investigated the molecular mechanism of activation of focal adhesion-related proteins, especially focal adhesion kinase (p125FAK), in cultured rat cardiac myocytes. We found that the 2 VEGF receptors, KDR/Flk-1 and Flt-1, were expressed in cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation. VEGF induced tyrosine phosphorylation and activation of p125FAK as well as tyrosine phosphorylation of paxillin; this was accompanied by subcellular translocation of p125FAK from perinuclear sites to the focal adhesions. This VEGF-induced activation of p125FAK was inhibited partially by the tyrosine kinase inhibitors genistein and tyrphostin. Activation of p125FAK was accompanied by its increased association with adapter proteins GRB2, Shc, and nonreceptor type tyrosine kinase p60c-src. Furthermore, we confirmed that VEGF induced a significant increase in adhesive interaction between cardiac myocytes and ECM using an electric cell-substrate impedance sensor. These results strongly suggest that p125FAK is one of the most important components in VEGF-induced signaling in cardiac myocytes, playing a critical role in adhesive interaction between cardiac myocytes and ECM.
Key Words: signal transduction • growth substance • cardiac myocyte • cell adhesion
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is an important regulator of endothelial cell proliferation, migration, and permeability and is secreted from tumor cells and cells exposed to hypoxia.1 2 3 4 VEGF has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation, as well as in tumor neovascularization.5 VEGF has been reported to be induced by hypoxia in several cell types in vitro,4 6 including cardiac myocytes.7 8 Levy et al7 demonstrated that cardiac myocytes synthesize and secrete VEGF in response to hypoxia in vitro. We previously reported that serum level of VEGF was markedly elevated in patients with acute myocardial infarction and it rapidly returned to basal level after reperfusion therapy.9 This indicates that most of the tissue cells, including cardiac myocytes, are exposed to high levels of VEGF in conditions such as acute myocardial infarction, as well as that VEGF is one of the most sensitive indicators of hypoxia. Recently, we found that VEGF activated mitogen-activated protein kinases (MAPKs), S6 kinase (p90rsk), and Raf-1 in cultured rat cardiac myocytes.10 This indicates that cardiac myocytes themselves are also target cells for VEGF.
Adhesive interactions between cells and the extracellular matrix (ECM) are known to be mediated by the integrin family.11 The cell-ECM interaction plays a fundamental role in regulating cellular behaviors such as migration, proliferation, and differentiation, especially for cardiac myocytes to perform continuously repetitive contractions and to adapt to external stresses such as hypoxia.12 Recent studies suggest that integrins transduce extracellular signals across the plasma membrane.11 13 The identification of focal adhesion kinase (p125FAK) provided the first evidence for the activation of an intracellular signaling molecule by integrins.14 15 16 p125FAK is a nonreceptor protein tyrosine kinase that is widely expressed in different cell types and phosphorylated on tyrosine residues accompanied with formation of focal adhesions. p125FAK has also been proved to be involved in signal transduction from cell surface receptors for neuropeptides and growth factors.17 18 19 20 21 Recently, Abedi and Zachary22 have reported that VEGF induces tyrosine phosphorylation of p125FAK and paxillin, another focal adhesion protein, in endothelial cells, as well as increases immunofluorescent staining of them in focal adhesions, which suggests that adhesion between endothelial cells and ECM may be modulated by VEGF stimulation. In this study, we investigated whether VEGF modulates adhesion between cardiac myocytes and ECM, as is the case with endothelial cells, analyzing the activation of focal adhesion-related proteins, especially p125FAK, in cultured rat cardiac myocytes. To confirm that VEGF modulates cell adhesion, we evaluated the state of cell adhesion to ECM using an electric impedance sensor. Here we show that VEGF induces activation of p125FAK, accompanied by subcellular translocation of p125FAK from perinuclear sites to focal adhesions, as well as its increased association with adapter proteins GRB2-, Shc-, and nonreceptor-type tyrosine kinase p60c-src. VEGF also induced tyrosine phosphorylation of paxillin, a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers. Furthermore, VEGF definitely accelerated the adhesion of cardiac myocytes to the ECM.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture
Primary culture of ventricular cardiac myocytes were prepared from neonatal rats as previously described.23 Briefly, heart ventricles were aseptically removed from neonatal Wistar rats, minced in calcium-free PBS, and digested with 0.025% trypsin-EDTA in PBS. The isolated cells were washed in DMEM (GIBCO Laboratories) containing 10% FBS and preplated onto plastic dishes for 1 hour to selectively remove fibroblasts. Nonadherent cells (enriched in cardiac myocytes) were collected and replated onto gelatin-coated culture dishes. They were cultured for 2 days in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 34 µmol/L streptomycin until they were confluent. They were starved for 24 hours before stimulation with VEGF.
The percentage of myocytes was estimated to be 
90%, as judged by
immunoperoxidase staining with a mouse anti-cardiac myosin heavy chain
monoclonal antibody (mAb) derived from the CMA-19 clone,24
followed by counterstaining with hematoxylin. Preparation of mouse
anti-cardiac myosin heavy chain mAb (CMA19) was previously
described.24 Contamination by endothelial
cells was estimated to be <1%, as judged by immunoperoxidase staining
with mouse monoclonal anti–factor VIII–related antigen antibody
(Z002; Zymed Laboratories) (data not shown). Therefore, we concluded
that the rest of the contaminating nonmuscle cells (10% of total
cells) mostly consisted of fibroblasts.
Analyses of Phosphotyrosine Content of KDR/Flk-1,
p125FAK, and Paxillin
The cells were treated with 22.8 pmol/L (1.0 ng/mL) recombinant
human VEGF (rhVEGF; Upstate Biotechnology, Inc) for various times as
indicated, and then they were frozen in liquid nitrogen and lysed on
ice with NP-40 buffer containing (in mmol/L) Tris/HCl 25 (pH 7.6),
NaCl 25, Na3VO4 1, sodium
pyrophosphate 10, EGTA 0.5, and phenylmethylsulfonyl fluoride
1; 10 nmol/L okadaic acid; and 1% NP-40. The cell lysates were
centrifuged, and the supernatants containing detergent-soluble
proteins were collected. Proteins were immunoprecipitated at 4°C
overnight with mouse anti-phosphotyrosine mAb (PY20; Transduction
Laboratories) and protein G–Sepharose (Pharmacia LKB). The
immunoprecipitates were subjected to SDS-PAGE and then transferred onto
polyvinylidene difluoride transfer membranes (NEN Research
Products). Polyvinylidene difluoride membranes were blocked
with 1% BSA in PBS and then incubated overnight at 4°C with rabbit
polyclonal anti-KDR/Flk-1 antibody (C20; Santa Cruz Biotechnology),
rabbit polyclonal anti-p125FAK
antibody,25 or mouse anti-paxillin mAb (349; Transduction
Laboratories). The preparation of rabbit polyclonal
anti-p125FAK antibody was previously
described.25 After incubation with alkaline
phosphatase–conjugated anti-rabbit or anti-mouse IgG antibody (both
antibodies from Vector Laboratories), the blots were developed with a
chemiluminescence detection kit (New England Biolabs). Tyrosine
phosphorylation of p125FAK was
confirmed by immunoprecipitation using
anti-p125FAK polyclonal antibody followed by
Western blotting with anti-phosphotyrosine mAb (4G10; Upstate
Biotechnology, Inc) or anti-p125FAK polyclonal
antibody. The increase in tyrosine phosphorylation of
KDR/Flk-1 was quantified by scanning densitometry. For analysis
of expression of VEGF receptors, total cell lysates in Laemmli sample
buffer were electrophoresed and immunoblotted using rabbit
polyclonal anti-KDR/Flk-1 antibody or rabbit polyclonal anti-Flt-1
antibody (C-17; Santa Cruz Biotechnology).
Kinase Assay of p125FAK
Cardiac myocytes were treated with rhVEGF (22.8 pmol/L) for
various times as indicated, and then they were frozen in liquid
nitrogen. Immune complex tyrosine kinase assays of
p125FAK were performed with a nonradioactive
isotope solid-phase ELISA kit using the exogenous substrate
poly(Glu-Tyr)15 26 27 (Universal Tyrosine Kinase Assay
Kit, Takara Shuzou Co Ltd) according to the manufacturer's
instructions. Briefly, the cell lysates were centrifuged and
the supernatants were immunoprecipitated with rabbit polyclonal
anti-p125FAK antibody. The immunoprecipitates
were incubated with ATP in the microtiter plate onto which
poly(Glu-Tyr) had been precoated. The amount of
phosphorylated poly(Glu-Tyr) was measured by ELISA
using a horseradish peroxidase–linked anti-phosphotyrosine (PY20)
antibody.
Analyses of the Effects of Tyrosine Kinase
Inhibitors on Phosphorylation of
p125FAK
Cells were preincubated for 30 minutes with or without either of
the tyrosine kinase inhibitors genistein28
(37 µmol/L) and tyrphostin29 (50 µmol/L)
(both from GIBCO-BRL), and then they were stimulated with 22.8 pmol/L
rhVEGF. The cell lysates were immunoprecipitated with mouse
anti-phosphotyrosine mAb (PY20) and subjected to Western
analysis using rabbit polyclonal
anti-p125FAK antibody. The increase in tyrosine
phosphorylation at the 125-kDa band was quantified by
scanning densitometry.
Analyses of Association of p125FAK With
p60c-src, GRB2, or Shc
After stimulation with 22.8 pmol/L rhVEGF for various times as
indicated, the cell lysates were immunoprecipitated with mouse
anti-v-Src mAb 2-17 (LA074; Quality Biotech), rabbit polyclonal
anti-GRB2 antibody (Santa Cruz Biotechnology), or mouse monoclonal
anti-Shc antibody (PG-797; Santa Cruz Biotechnology) along with protein
G–Sepharose and subjected to Western analysis using rabbit
polyclonal anti-p125FAK antibody.
Subcellular Fractionation
After stimulation with 22.8 pmol/L rhVEGF for various times as
indicated, cytosolic and membrane fractions were prepared from cardiac
myocytes according to modified methods previously
described.30 31 Briefly, cells were washed twice with PBS
and then harvested in a buffer containing 20 mmol/L HEPES and
250 mmol/L sucrose (pH 7.4) followed by centrifuging at
1000g for 3 minutes. The supernatant was discarded, and the
pellet was suspended in hypotonic Tris buffer containing (in
mmol/L) Tris (pH 7.5) 10, MgCl2 1,
phenylmethylsulfonyl fluoride 1, and
Na3VO4 1, and 100
µmol/L leupeptin. The pellet was homogenized by 80
strokes of Dounce homogenizer. The
homogenate was centrifuged at 1000g for
15 minutes to remove nuclei and debris. The supernatant was
centrifuged at 48 000g for 30 minutes, resulting in
a pellet, which was resuspended in hypotonic Tris buffer containing 1%
NP-40 and stored as a membrane fraction, and the supernatant, which was
recentrifuged at 246 000g for 90 minutes to remove
the microsome-rich fraction. The resulting supernatant was
stored as a cytosolic fraction. All of the above steps were carried out
at 4°C unless otherwise indicated. Protein estimation was
carried out using the BCA protein assay reagent (Pierce). The fractions
were suspended in Laemmli sample buffer and incubated at 37°C
for 30 minutes, and then their aliquots were subjected to
SDS-PAGE followed by immunoblotting with polyclonal
anti-p125FAK antibody or rabbit polyclonal
anti–rat Na,K-ATPase 
1 fusion protein
(Upstate Biotechnology, Inc).
Immunocytochemistry
To investigate whether subcellular distribution of
p125FAK in cardiac myocytes and fibroblasts is
altered in response to VEGF, we performed double staining for
p125FAK and cardiac myosin heavy chain.
rhVEGF-treated (60 minutes) or untreated cardiac myocytes were fixed in
acetone for 5 minutes at room temperature. The cells were incubated
first with rabbit polyclonal anti-p125FAK
antibody for 1 hour at 37°C and then incubated sequentially with
biotinylated anti-rabbit IgG (Vector Laboratories) for 1 hour at 37°C
and FITC-conjugated avidin D (Vector Laboratories) for 30 minutes at
37°C. The cells were then incubated sequentially with mouse
anti-cardiac myosin heavy chain mAb (CMA-19) for 1 hour at 37°C and
TRITC-conjugated anti-mouse IgG1 antibody (Chemicon International, Inc)
for 30 minutes at 37°C. To investigate the relationship between the
immunofluorescent staining of p125FAK and
focal adhesions in rhVEGF-treated (60 minutes) cells, we performed
double staining for p125FAK and vinculin.
rhVEGF-treated (60 minutes) cells were first stained with rabbit
polyclonal anti-p125FAK antibody and then were
stained sequentially with biotinylated anti-rabbit IgG and
FITC-conjugated avidin D as described above. The cells were then
incubated with mouse anti-vinculin mAb (V-4505; Sigma) for 1 hour at
37°C and TRITC-conjugated anti-mouse IgG1 antibody for 30 minutes at
37°C. The sections were examined and photographed under a
fluorescence microscope (Microphoto-FX, Nikon).
Cell-Substrate Adhesion Assays
Cell-substrate adhesion was measured using the electric
cell-substrate impedance sensing system (Applied BioPhysics, Inc)
previously reported by Giaever and Keese32 33 and Ghosh et
al.34 In this system, the cells were cultured on a small
gold electrode (area, 10–4
cm2) deposited on the bottom of tissue culture
vessels. A small alternating current signal (1 µA) at a frequency of
4000 Hz was passed between the small electrode and a larger counter
electrode (area, 10–1
cm2) placed at a distance. The voltage between
the small and large electrodes was monitored. As cell membranes have
very high impedance, the attachment of cells to the small electrode
blocks the current, forcing it to flow under the cells, causing
an increase in the impedance. Primary cultures of
ventricular cardiac myocytes were prepared as above.
Cardiac myocytes were seeded on gelatin-precoated electrodes and
cultured for 2 days in DMEM supplemented with 10% FBS, 50 U/mL
penicillin, and 34 µmol/L streptomycin. They were starved for 24
hours and then stimulated with rhVEGF (34.2 pmol/L). The resistance,
which reflects the extent of cell-substrate adhesion, was monitored as
described previously.35 36
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiac Myocytes Express KDR/Flk-1 and Flt-1
It has been shown that VEGF binds to 2 structurally related tyrosine kinase receptors, Flt-137 38 39 and KDR/Flk-1.40 41 42 43 We examined whether cardiac myocytes expressed these receptors by Western analysis. As shown in Figure 1
, KDR/Flk-1 and Flt-1 were
expressed in the cultured cardiac myocytes. To examine tyrosine
phosphorylation of VEGF receptors, the cell lysates
from rhVEGF-treated and untreated myocytes were immunoprecipitated with
anti-phosphotyrosine mAb (PY20) followed by Western blotting using
anti-KDR/Flk-1 or anti-Flt-1 antibodies. KDR/Flk-1 was significantly
tyrosine phosphorylated on VEGF stimulation, with
phosphorylation reaching a maximum level at 5 to 10
minutes with rhVEGF (Figure 2A),
whereas Flt-1 was not (data not shown). The increase in tyrosine
phosphorylation of KDR/Flk-1 was quantified by scanning
densitometry of the autoradiogram and expressed as a
ratio of the increase to the initial phosphorylation
before addition of rhVEGF. As shown in Figure 2B, tyrosine
phosphorylation of KDR/Flk-1 was significantly
increased at 5 to 10 minutes after rhVEGF treatment as compared with
the initial value.
|
|
VEGF Phosphorylates p125FAK and
Paxillin
To examine whether VEGF can stimulate tyrosine
phosphorylation of a focal adhesion-associated protein
tyrosine kinase p125FAK, quiescent rat cardiac
myocytes were treated with rhVEGF for various time periods and lysed.
The cell lysates from the treated myocytes were immunoprecipitated with
anti-p125FAK polyclonal antibody followed by
Western blotting with anti-phosphotyrosine mAb (4G10) or
anti-p125FAK polyclonal antibody. Conversely,
immunoprecipitates with anti-phosphotyrosine mAb (PY20) were also
analyzed by Western blotting with
anti-p125FAK antibody. As shown in Figure 3A and 3B (top panel), VEGF significantly
increased tyrosine phosphorylation of
p125FAK, which peaked at 5 to 10 minutes after
addition of rhVEGF. We confirmed that almost equal amounts of
p125FAK protein were electrophoresed in each
reaction (Figure 3A, bottom panel). We also investigated whether
VEGF caused tyrosine phosphorylation of paxillin,
another focal adhesion-associated protein, which interacts with several
proteins, including p125FAK, members of the src
family of tyrosine kinases, the transforming protein v-crk, and the
cytoskeletal protein vinculin. Immunoprecipitates with
anti-phosphotyrosine mAb (PY20) from the stimulated cardiac myocytes
were analyzed by Western blotting with anti-paxillin mAb. As
shown in Figure 3B (bottom panel), VEGF also caused a
significant increase in the tyrosine phosphorylation of
paxillin. The tyrosine phosphorylation of paxillin
reached a maximum level at 5 to 10 minutes after addition of VEGF and
decreased subsequently.
|
VEGF Stimulates Protein Tyrosine Kinase Activity of
p125FAK
To examine whether the catalytic activity of
p125FAK is stimulated by VEGF, kinase activity of
p125FAK immunoprecipitates from untreated or
rhVEGF-treated cardiac myocytes was assayed using poly(Glu-Tyr) as
substrate. As shown in Figure 4, VEGF
induced activation of p125FAK as early as 2
minutes, peaking at 5 minutes, which paralleled its tyrosine
phosphorylation.
|
Tyrosine Kinase Inhibitors Partially Inhibit
VEGF-Induced Activation of p125FAK
Next, to examine whether VEGF-induced tyrosine
phosphorylation of p125FAK was
dependent on tyrosine kinase activity, the cardiac myocytes were
pretreated with or without either of the tyrosine kinase
inhibitors genistein (37 µmol/L) and tyrphostin
(50 µmol/L) before addition of VEGF.
Representative results of 1 of 3 independent
experiments are shown in Figure 5.
Both genistein and tyrphostin at least partially inhibited VEGF-induced
increase in tyrosine phosphorylation of
p125FAK at 5 minutes.
|
VEGF Induces Association of p125FAK With Shc, GRB2,
and p60c-src
To determine whether c-Src plays a role in VEGF-induced tyrosine
phosphorylation of p125FAK,
immunoprecipitates with anti-v-Src mAb were subjected to SDS-PAGE
followed by immunoblotting with
anti-p125FAK antibody. As shown in Figure 6a, VEGF caused a significant increase in
the association of p60c-src with
p125FAK, which peaked at 5 to 10 minutes after
addition of VEGF. To determine whether the VEGF-activated
p125FAK signaling complexes contained other known
Src homology (SH) 2–containing proteins, such as the SH2/SH3 adapter
proteins GRB2 and Shc, we examined the VEGF-induced association of
p125FAK with these adapter proteins.
Immunoprecipitates with either anti-GRB2 polyclonal antibody or
anti-Shc mAb from VEGF-stimulated cardiac myocytes were subjected to
SDS-PAGE followed by immunoblotting with
anti-p125FAK polyclonal antibody. As shown in
Figure 6b and 6c, VEGF caused a significant increase in the
association of Shc and GRB2 with p125FAK. The
association peaked at 5 to 10 minutes after addition of VEGF.
|
VEGF Induces Subcellular Translocation of p125FAK in
Cardiac Myocytes and Fibroblasts
It has been shown that cell adhesion to ECM, such as fibronectin,
through integrins causes increased tyrosine
phosphorylation of p125FAK as
well as accumulation of p125FAK in focal
adhesions.13 44 45 46 We examined the effects of VEGF on
subcellular localization of p125FAK in cardiac
myocytes and nonmuscle cells (which mostly consisted of fibroblasts).
To distinguish cardiac myocytes from nonmuscle cells, we performed
double staining for cardiac myosin heavy chain and
p125FAK by
immunofluorescence. Figures 7B and 7D show that myofibrils of cardiac
myocytes were strongly stained, making it easy to distinguish cardiac
myocytes from fibroblasts. As shown in Figure 7A, p125FAK predominantly localized in the
perinuclear region in nonstimulated cardiac myocytes. Only weak
fluorescent dots of p125FAK were seen in
some nonstimulated fibroblasts (Figure 7A, arrows).
Fluorescent dots of p125FAK, which mostly
localized in the central regions at a higher density, appeared to
scatter to the peripheral cytoplasm in cardiac myocytes
stimulated with rhVEGF for 60 minutes (Figure 7C, arrowheads).
In fibroblasts stimulated with VEGF for 60 minutes, fluorescent
dots of p125FAK were observed to be concentrated
in the patchy arrowhead-like structures at the peripheral
cytoplasm reminiscent of focal adhesions (Figure 7C, arrow). To
compare the localization of p125FAK staining to
focal adhesions in VEGF-treated cells, we performed double staining for
p125FAK (Figure 7E) and vinculin (Figure 7F) by immunofluorescence.
Fluorescent dots of p125FAK showed
localization similar to that of vinculin in cardiac myocytes (Figure 7E and 7F, arrowheads), which suggests that
p125FAK was localized to focal adhesions in
response to VEGF, although the rest of the
p125FAK remained in cytoplasm. In fibroblasts,
the patchy arrowhead-like fluorescent dots of
p125FAK were clearly observed with higher
resolution (Figure 7E, arrow). Incubation of cardiac myocytes
with control nonimmune rabbit serum yielded no significant staining
(data not shown).
|
Next, to confirm that VEGF induces subcellular translocation of
p125FAK, we analyzed
p125FAK content in subcellular fractions by
Western analysis with and without VEGF stimulation. As shown in
Figure 8, the amount of
p125FAK in the membrane fraction was increased in
response to rhVEGF (Figure 8b, top panel), whereas that in the
cytosolic fraction remained almost unchanged (Figure 8a). We
confirmed that almost equal amounts of the membrane fraction were
electrophoresed in each reaction by Western analysis using
anti–Na,K ATPase 1 subunit antibody (Figure 8b, lower panel).
|
VEGF Strengthens Adhesion of Cardiac Myocytes to the ECM
To assess the effects of VEGF on the extent of cell-substrate
adhesion, we monitored changes in cell-substrate resistance after
addition of rhVEGF, using an electric cell-substrate impedance sensor.
Typical results are shown in Figure 9A.
The extent of cell-substrate adhesion is expressed as "normalized"
resistance, which was defined as a ratio of the resistance to the
initial value before addition of rhVEGF. VEGF induced a significant
increase in normalized resistance, indicating strengthening of the
cell-substrate adhesion at 3 hours after addition of rhVEGF (Figure 9B).
|
. To simplify the comparison, ordinate represents normalized
resistance defined as a fraction of the initial resistance before
addition of rhVEGF. B, Comparison of percentage increase in normalized
resistance at 3 hours after addition of rhVEGF, VEGF(+), with control,
VEGF(–). Results shown are mean±SD from 4 independent
experiments. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The biological effects of VEGF are mediated by specific cell surface receptors. It has been shown that VEGF binds with high affinity to 2 structurally related tyrosine kinase receptors, Flt-137 38 39 and KDR/Flk-1.40 41 42 43 Studies on signal transduction from Flt-1 and KDR showed that these 2 receptor tyrosine kinases have different signal transduction properties.47 48 It has been reported that KDR-expressing cells showed striking changes in cell morphology, actin reorganization and membrane ruffling, chemotaxis, and mitogenicity on VEGF stimulation, whereas Flt-1–expressing cells lacked such responses in a cultured human umbilical vein endothelial cell population.47 We confirmed by Western blot analysis that KDR/Flk-1, as well as Flt-1, was expressed in the cultured cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation, whereas Flt-1 was not. Regarding the capacity for autophosphorylation, KDR has been reported to undergo autophosphorylation much more efficiently than Flt-1 in response to VEGF, on the basis of a comparison between Flt-1–transfected porcine aortic endothelial cells and KDR-transfected ones.47 By analogy, it is possible that detectable tyrosine phosphorylation was not induced for Flt-1 protein in response to VEGF in cardiac myocytes at least at the concentration used in this study, although KDR/Flk-1 protein was efficiently tyrosine phosphorylated. It might also in part reflect the difference in the sensitivity between the anti-KDR/Flk-1 antibody and the anti-Flt-1 antibody used in the present study. These results suggest that the VEGF-induced signaling pathway may be more dependent on KDR/Flk-1 than Flt-1 or that it requires formation of heterodimeric complexes between KDR and Flt-1. To clarify which of the 2 receptor tyrosine kinases (or both) mediates VEGF-induced activation in cardiac myocytes, further investigation is needed.
p125FAK is a widely expressed nonreceptor protein tyrosine kinase that localizes to focal adhesion structures. p125FAK is thought to be one of the key elements in the signal transduction pathway underlying changes in cell behavior induced by diverse stimuli, including integrin engagement; oncogenic transformation; and mitogenic neuropeptides such as bombesin, endothelin, vasopressin, angiotensin, platelet-derived growth factor, and lysophosphatidic acid.14 15 17 18 19 20 21 49 It was shown that tyrosine-phosphorylated pp125FAK directly interacts with pp60c-src and pp59fyn as one of their major substrates.27 50 Indeed, tyrosine phosphorylation of p125FAK by Src-family kinases has been shown to be directly correlated with increased protein tyrosine kinase activity, which is an important step in the formation of an active signaling complex.15 Paxillin is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the ECM, which has also been demonstrated to be one of the major substrates of pp60c-src in Rous sarcoma virus–transformed cells as p125FAK.51 It becomes tyrosine phosphorylated concomitantly with p125FAK in response to multiple stimuli, including integrin-mediated cell adhesion, several neuropeptide growth factors, and platelet-derived growth factor.52 53 54
In the present study, we have shown that VEGF stimulated tyrosine phosphorylation and activation of p125FAK in cultured cardiac myocytes, peaking at 2 to 10 minutes. Although the time courses of the change in tyrosine phosphorylation and the activation of the kinase activity were similar, the extent of p125FAK activation seemed to be smaller than that expected from the increase in its tyrosine phosphorylation. The kinase activity assayed with exogenous substrate poly(Glu-Tyr) reflected only the ability to phosphorylate downstream substrates, including other kinases. On the other hand, tyrosine phosphorylation of p125FAK results not only from autophosphorylation but also from phosphorylation by other upstream protein kinases, including Src family kinases.27 50 The contribution from the latter might be relatively large compared with that from the former. This may be the reason for the discrepancy between the extent of its activation measured with the exogenous substrate and tyrosine phosphorylation.
We also showed that VEGF also stimulated tyrosine phosphorylation of paxillin, and VEGF-induced tyrosine phosphorylation of p125FAK was inhibited at least partially by the tyrosine kinase inhibitors genistein and tyrphostin. Moreover, VEGF caused increased association of p125FAK with pp60c-src concomitantly with increased tyrosine phosphorylation of p125FAK. Recently, Abedi and Zachary22 have reported that VEGF induces tyrosine phosphorylation of p125FAK and paxillin in endothelial cells, suggesting that they are components in a VEGF-stimulated signaling pathway. Our results indicate that tyrosine phosphorylation of p125FAK and paxillin is also a part of the signal transduction by VEGF in cardiac myocytes as in endothelial cells, resulting in organization of the cytoskeleton. Tyrosine kinases, especially p60c-src, might take some part in VEGF-induced tyrosine phosphorylation and activation of p125FAK in cardiac myocytes.
Transduction of various mitogenic signals from the cell membrane to the nucleus involves the adapter proteins Shc and GRB2, which mediate activation of the Ras/MAPK pathway.55 56 57 Shc is an immediate substrate of receptor tyrosine kinase activity and serves to physically link activated receptors to downstream signaling components.58 59 GRB2 is a ubiquitously expressed-24 kDa mammalian protein, which directly binds autophosphorylated tyrosine kinase receptors as well as phosphorylated Shc proteins and p125FAK through its SH2 domain.55 56 60 61 It is well known that activation of insulin receptor results in interaction of the GRB2-Sos complex with insulin receptor substrate-1 and Shc via the SH2 domain of GRB2.59 62 The tyrosine kinase–Shc–GRB2–Sos pathway was shown also to be involved in signal transduction from Gq protein–coupled angiotensin II receptor leading to activation of p21ras in cardiac myocytes.63 It has also been demonstrated that adhesion of fibroblasts to fibronectin promotes SH2-domain–mediated association of GRB2 and p60c-src with p125FAK in vivo, resulting in activation of MAPK.60 In the present study, we demonstrated VEGF-induced association of p125FAK with GRB2 and Shc, occurring concomitantly with increased tyrosine phosphorylation of p125FAK. This strongly suggests that 2 ubiquitously expressed adapter proteins, GRB2 and Shc, play a role in VEGF-induced signal transduction involving the MAPK pathway in cardiac myocytes, as we previously reported.10
Furthermore, the immunocytochemical study demonstrated that VEGF stimulation significantly altered the subcellular localization of p125FAK from the perinuclear region to the peripheral cytoplasm in cardiac myocytes and increased accumulation of p125FAK in the patchy arrowhead-like structures at the peripheral cytoplasm (ie, focal adhesions) in cardiac myocytes as well as in fibroblasts. In addition, we confirmed quantitatively by Western analysis that the amount of p125FAK in the membrane-rich fraction significantly increased in response to VEGF. These results indicate that p125FAK translocates to focal adhesions in response to VEGF. Taken together, VEGF causes activation as well as subcellular translocation of p125FAK to focal adhesions, where it works. Activation and accumulation of p125FAK in focal adhesions strongly suggests that adhesive interaction between cardiac myocytes and ECM may be strengthened in response to VEGF. Using an electric cell-substrate impedance sensor, we confirmed that VEGF induced significant increase in adhesion between cardiac myocytes and ECM. Whether this may reflect one of the cardiac adaptive responses in vivo in situations such as acute ischemia, in which cardiac myocytes are exposed to high levels of VEGF, is unknown and remains to be clarified.
|
Acknowledgments |
|---|
This work was supported by a grant for cardiomyopathy from the Ministry of Health and Welfare, Japan; a grant for scientific research from the Ministry of Education, Science and Culture, Japan; a grant from Sankyo Foundation of Life Science; Japan Heart Foundation-Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease; and a grant from the Study Group of Molecular Cardiology. We thank Kaori Takahashi for excellent technical assistance.
|
Footnotes |
|---|
This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received June 8, 1998; accepted February 11, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983–985.
2.
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara
N. Vascular endothelial growth factor is a secreted
angiogenic mitogen. Science. 1989;246:1306–1309.
3.
Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder
J, Connolly DT. Vascular permeability factor, an
endothelial cell mitogen related to PDGF.
Science. 1989;246:1309–1312.
4. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843–845.[Medline] [Order article via Infotrieve]
5. Conolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfina JJ, Diegel NR, Leimgruber RM, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest. 1989;84:1470–1478.
6.
Goldberg MA, Schneider TJ. Similarities between the
oxygen-sensing mechanisms regulating the expression of vascular
endothelial growth factor and erythropoietin.
J Biol Chem. 1994;269:4355–4359.
7.
Levy AP, Levy NS, Loscalzo J, Calderone A, Takahashi
N, Yeo K-T, Koren G, Colucci WS, Goldberg MA. Regulation of vascular
endothelial growth factor in cardiac myocytes.
Circ Res. 1995;76:758–766.
8. Ladoux A, Frelin C. Hypoxia is strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem Biophys Res Commun. 1993;195:1005–1010.[Medline] [Order article via Infotrieve]
9. Seko Y, Imai Y, Suzuki S, Kamijukkoku S, Hayasaki K, Sakomura Y, Tobe K, Kadowaki T, Maekawa H, Takahashi N, Yazaki Y. Serum levels of vascular endothelial growth factor in patients with acute myocardial infarction undergoing reperfusion therapy. Clin Sci. 1997;92:453–454.
10. Seko Y, Takahashi N, Tobe K, Ueki K, Kadowaki T, Yazaki Y. Vascular endothelial growth factor (VEGF) activates Raf-1, mitogen-activated protein (MAP) kinases, and S6 kinase (p90rsk) in cultured rat cardiac myocytes. J Cell Physiol. 1998;175:239–246.[Medline] [Order article via Infotrieve]
11. Hynes RO. Integrins: versatility, modulation and signaling in cell adhesion. Cell. 1992;69:11–25.[Medline] [Order article via Infotrieve]
12. Hynes RO, Lander AD. Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons. Cell. 1992;68:303–322.[Medline] [Order article via Infotrieve]
13.
Miyamoto S, Akiyama SK, Yamada KM. Synergistic roles
for receptor occupancy and aggregation in integrin transmembrane
function. Science. 1995;267:883–885.
14.
Kornberg L, Earp HS, Parsons JT, Schaller M, Juliano
RL. Cell adhesion or integrin clustering increases
phosphorylation of a focal adhesion-associated tyrosine
kinase. J Biol Chem. 1992;267:23439–23442.
15. Guan JL, Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by cellular adhesion and oncogenic transformation. Nature. 1992;358:690–692.[Medline] [Order article via Infotrieve]
16.
Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds
AB, Parsons JT. pp125FAK: a structurally
distinctive protein-tyrosine kinase associated with focal adhesions.
Proc Natl Acad Sci U S A. 1992;89:5192–5196.
17. Zachary I, Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992;71:891–894.[Medline] [Order article via Infotrieve]
18.
Zachary I, Sinnett-Smith J, Rozengurt E. Bombesin,
vasopressin, and endothelin stimulation of tyrosine
phosphorylation in Swiss 3T3 cells: identification of a
novel tyrosine kinase as a major substrate. J Biol
Chem. 1992;267:19031–19034.
19. Polte TR, Naftilan AJ, Hanks SK. Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II. J Cell Biochem. 1994;55:106–119.[Medline] [Order article via Infotrieve]
20. Kumagai N, Morii N, Fujisawa K, Yoshimasa T, Nakao K, Narumiya S. Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells. FEBS Lett. 1993;329:273–276.[Medline] [Order article via Infotrieve]
21.
Chen HC, Guan JL. Stimulation of phosphatidylinositol
3'-kinase association with focal adhesion kinase by
platelet-derived growth factor. J Biol Chem. 1994;269:31229–31233.
22.
Abedi H, Zachary I. Vascular
endothelial growth factor stimulates tyrosine
phosphorylation and recruitment to new focal adhesions
of focal adhesion kinase and paxillin in endothelial
cells. J Biol Chem. 1997;272:15442–15451.
23.
Simpson P, Savion S. Differentiation of rat myocytes in
single cell cultures with and without proliferating nonmyocardial
cells: cross-striations, ultrastructure, and chronotropic response to
isoproterenol. Circ Res. 1982;50:101–116.
24. Yazaki Y, Tsuchimochi H, Kuro-o M, Kurabayashi M, Isobe M, Ueda S, Nagai R, Takaku F. Distribution of myosin isozymes in human atrial and ventricular myocardium: comparison in normal and overloaded heart. Eur Heart J. 1984;5(suppl F):103–110.
25. Tobe K, Sabe H, Yamamoto T, Yamauchi T, Asai S, Kaburagi Y, Tamemoto H, Ueki K, Kimura H, Akanuma Y, Yazaki Y, Hanafusa H, Kadowaki T. Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins. Mol Cell Biol. 1996;16:4765–4772.[Abstract]
26. Braun S, Ghany MA, Lettieri JA, Racker E. Partial purification and characterization of protein tyrosine kinases from normal tissues. Arch Biochem Biophys. 1986;247:424–432.[Medline] [Order article via Infotrieve]
27.
Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines
RR, Parsons JT. Autophosphorylation of the focal
adhesion kinase, pp125FAK, directs SH2-dependent
binding of pp60src. Mol Cell Biol. 1994;14:1680–1688.
28.
Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S,
Itoh N, Shibuya M, Fukami Y. Genistein, a specific
inhibitor of tyrosine-specific protein kinases.
J Biol Chem. 1987;262:5592–5595.
29. Gazit A, Yaish P, Gilon C, Levitzki A. Tyrphostins, I: synthesis and biological activity of protein tyrosine kinase inhibitors. J Med Chem. 1989;32:2344–2352.[Medline] [Order article via Infotrieve]
30.
Yuan S, Sunahara FA, Sen AK. Tumor-promoting phorbol
esters inhibit cardiac functions and induce redistribution of protein
kinase C in perfused beating rat heart. Circ Res. 1987;61:372–378.
31. Limas CJ, Limas C, Goldenberg IF. Intracellular distribution of adrenoreceptors in the failing human myocardium. Am Heart J. 1989;117:1310–1316.[Medline] [Order article via Infotrieve]
32. Giaever I, Keese CR. A morphological biosensor for mammalian cells. Nature. 1993;366:591–592.[Medline] [Order article via Infotrieve]
33.
Giaever I, Keese CR. Micromotion of mammalian cells
measured electrically. Proc Natl Acad Sci U S A.. 1991;88:7896–7900.
34. Ghosh P, Keese CR, Giaever I. Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells. Biophys J. 1993;64:1602–1609.[Medline] [Order article via Infotrieve]
35.
Noiri E, Hu Y, Bahou WF, Keese CR, Giaever I,
Goligorsky MS. Permissive role of nitric oxide in endothelin-induced
migration of endothelial cells. J Biol
Chem. 1997;272:1747–1752.
36. Moy AB, Engelenhoven JV, Bodmer J, Kamath J, Keese C, Giaever I, Shasby S, Shasby DM. Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. J Clin Invest. 1996;97:1020–1027.[Medline] [Order article via Infotrieve]
37. Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A, Matsuhime H, Sato M. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene. 1990;5:519–524.[Medline] [Order article via Infotrieve]
38.
De Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N,
Williams LT. The fms-like tyrosine kinase, a receptor for vascular
endothelial growth factor. Science. 1992;255:989–991.
39.
Peters KG, De Vries C, Williams LT. Vascular
endothelial growth factor receptor expression during
embryogenesis and tissue repair suggests a role in
endothelial differentiation and blood vessel growth.
Proc Natl Acad Sci U S A.. 1993;90:8915–8919.
40.
Matthews W, Jordan CT, Gavin M, Jenkins NA, Copeland
NG, Lemischka IR. A receptor tyrosine kinase cDNA isolated from a
population of enriched primitive hematopoietic cells and exhibiting
close genetic linkage to c-kit. Proc Natl Acad Sci U S A. 1991;88:9026–9030.
41. Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, Bohlen P. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun. 1992;187:1579–1586.[Medline] [Order article via Infotrieve]
42. Millauer B, Wizigmann-Voos S, Schnurch H, Martinez R, Moller NP, Risau W, Ullrich A. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell. 1993;72:835–846.[Medline] [Order article via Infotrieve]
43. Oelrichs RB, Reid HH, Bernard O, Ziemiecki A, Wilks AF. NYK/FLK-1: a putative receptor protein tyrosine kinase isolated from E10 embryonic neuroepithelium is expressed in endothelial cells of the developing embryo. Oncogene. 1993;8:11–18.[Medline] [Order article via Infotrieve]
44.
Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo
PD, Akiyama SK, Yamada KM. Integrin function: molecular hierarchies of
cytoskeletal and signaling molecules. J Cell Biol. 1995;131:791–729.
45.
Schaller MD, Otey CA, Hildebrand JD, Parsons JT. Focal
adhesion kinase and paxillin bind to peptides mimicking beta integrin
cytoplasmic domains. J Cell Biol. 1995;130:1181–1187.
46. Lewis JM, Schwartz MA. Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants. Mol Biol Cell. 1995;6:151–160.[Abstract]
47.
Waltenberger J, Claesson-Welsh L, Siegbahn A,
Shibuya M, Heldin CH. Different signal transduction properties of KDR
and Flt1, two receptors for vascular endothelial growth
factor. J Biol Chem. 1994;269:26988–26995.
48. Seetharam L, Gotoh N, Maru Y, Neufeld G, Yamaguchi S, Shibuya M. A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene. 1995;10:135–147.[Medline] [Order article via Infotrieve]
49.
Hanks SK, Calalb MB, Harper MC, Patel SK. Focal
adhesion protein-tyrosine kinase phosphorylated in
response to cell attachment to fibronectin. Proc Natl Acad Sci
U S A. 1992;89:8487–8491.
50.
Cobb BS, Schaller MD, Leu TH, Parsons JT. Stable
association of pp60src and
pp59fyn with the focal adhesion-associated
protein tyrosine kinase, pp125FAK. Mol Cell
Biol. 1994;14:147–155.
51.
Glenney JR, Zokas L. Novel tyrosine kinase substrates
from Rous sarcoma virus-transformed cells are present in the
membrane skeleton. J Cell Biol. 1989;108:2401–2408.
52.
Burridge K, Turner CE, Romer LH. Tyrosine
phosphorylation of paxillin and
pp125FAK accompanies cell adhesion to
extracellular matrix: a role in cytoskeletal assembly. J
Cell Biol. 1992;119:893–903.
53.
Zachary I, Sinnett-Smith J, Turner CE, Rozengurt E.
Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine
phosphorylation of the focal adhesion-associated
protein paxillin in Swiss 3T3 cells. J Biol Chem. 1993;268:22060–22065.
54.
Rankin S, Rozengurt E. Platelet-derived growth
factor modulation of focal adhesion kinase
(p125FAK) and paxillin tyrosine
phosphorylation in Swiss 3T3 cells: bell-shaped dose
response and cross-talk with bombesin. J Biol Chem. 1994;269:704–710.
55. Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, Schlessinger J, Pawson T. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992;360:689–692.[Medline] [Order article via Infotrieve]
56. Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, Skoinik EY, Bar-Sagi D, Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992;70:431–442.[Medline] [Order article via Infotrieve]
57. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993;363:45–51.[Medline] [Order article via Infotrieve]
58. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, Nicoletti I, Grignani F, Pawson T, Pelicci PG. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992;70:83–104.
59.
Myers MG, Wang L-M, Sun XJ, Zhang Y, Yenush L,
Schlessinger J, Pierce JH, White MF. Role of IRS-1–GRB-2 complexes in
insulin signaling. Mol Cell Biol. 1994;14:3577–3587.
60. Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994;372:786–791.[Medline] [Order article via Infotrieve]
61.
Kharbanda S, Saleem A, Yuan Z, Emoto Y, Prasad KVS.
Stimulation of human monocytes with macrophage
colony-stimulating factor induces a Grb2-mediated association of the
focal adhesion kinase pp125FAK and dynamin.
Proc Natl Acad Sci U S A. 1995;92:6132–6136.
62.
Skolnik EY, Batzer A, Li N, Lee CH, Lowenstein E,
Mohammadi M, Margolis B, Schlessinger J. The function of GRB2 in
linking the insulin receptor to Ras signaling pathways.
Science. 1993;260:1953–1955.
63. Sadoshima J, Izumo S. The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. EMBO J. 1996;15:775–787.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  R. Madonna and R. De Caterina VEGF receptor switching in heart development and disease Cardiovasc Res, October 1, 2009; 84(1): 4 - 6. [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhou, K. Bourcy, and Y. J. Kang Copper-induced regression of cardiomyocyte hypertrophy is associated with enhanced vascular endothelial growth factor receptor-1 signalling pathway Cardiovasc Res, October 1, 2009; 84(1): 54 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Hausenloy and D. M. Yellon Cardioprotective growth factors Cardiovasc Res, July 15, 2009; 83(2): 179 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Walsh, D. R. Ampasala, J. Hatfield, R. Vander Heide, S. Suer, A. K. Rishi, and M. D. Basson Transforming Growth Factor-{beta} Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms Am. J. Pathol., August 1, 2008; 173(2): 385 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Saban, J. M. Backer, M. V. Backer, J. Maier, B. Fowler, C. A. Davis, C. Simpson, X.-R. Wu, L. Birder, M. R. Freeman, et al. VEGF receptors and neuropilins are expressed in the urothelial and neuronal cells in normal mouse urinary bladder and are upregulated in inflammation Am J Physiol Renal Physiol, July 1, 2008; 295(1): F60 - F72. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sarkar, D. Chakroborty, U. R. Chowdhury, P. S. Dasgupta, and S. Basu Dopamine Increases the Efficacy of Anticancer Drugs in Breast and Colon Cancer Preclinical Models Clin. Cancer Res., April 15, 2008; 14(8): 2502 - 2510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Bahary, K. Goishi, C. Stuckenholz, G. Weber, J. LeBlanc, C. A. Schafer, S. S. Berman, M. Klagsbrun, and L. I. Zon Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish Blood, November 15, 2007; 110(10): 3627 - 3636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shimojo, S. Jesmin, S. Zaedi, T. Otsuki, S. Maeda, N. Yamaguchi, K. Aonuma, Y. Hattori, and T. Miyauchi Contributory role of VEGF overexpression in endothelin-1-induced cardiomyocyte hypertrophy Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H474 - H481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Thamilselvan, D. H. Craig, and M. D. Basson FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway FASEB J, June 1, 2007; 21(8): 1730 - 1741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Chang, C. A. Lemmon, D. Park, and L. H. Romer FAK Potentiates Rac1 Activation and Localization to Matrix Adhesion Sites: A Role for betaPIX Mol. Biol. Cell, January 1, 2007; 18(1): 253 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Rafiq, M. A. Kolpakov, M. Abdelfettah, D. N. Streblow, A. Hassid, L. J. Dell'Italia, and A. Sabri Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis J. Biol. Chem., July 14, 2006; 281(28): 19781 - 19792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Eiras, P. Fernandez, R. Pineiro, M. J. Iglesias, J. R. Gonzalez-Juanatey, and F. Lago Doxazosin induces activation of GADD153 and cleavage of focal adhesion kinase in cardiomyocytes en route to apoptosis Cardiovasc Res, July 1, 2006; 71(1): 118 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Samarel Costameres, focal adhesions, and cardiomyocyte mechanotransduction Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2291 - H2301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. C. Sasso, D. Torella, O. Carbonara, G. M. Ellison, M. Torella, M. Scardone, C. Marra, R. Nasti, R. Marfella, D. Cozzolino, et al. Increased Vascular Endothelial Growth Factor Expression But Impaired Vascular Endothelial Growth Factor Receptor Signaling in the Myocardium of Type 2 Diabetic Patients With Chronic Coronary Heart Disease J. Am. Coll. Cardiol., September 6, 2005; 46(5): 827 - 834. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-s. Yoon, S. Uchida, O. Masuo, M. Cejna, J.-S. Park, H.-c. Gwon, R. Kirchmair, F. Bahlman, D. Walter, C. Curry, et al. Progressive Attenuation of Myocardial Vascular Endothelial Growth Factor Expression Is a Seminal Event in Diabetic Cardiomyopathy: Restoration of Microvascular Homeostasis and Recovery of Cardiac Function in Diabetic Cardiomyopathy After Replenishment of Local Vascular Endothelial Growth Factor Circulation, April 26, 2005; 111(16): 2073 - 2085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Le Boeuf, F. Houle, and J. Huot Regulation of Vascular Endothelial Growth Factor Receptor 2-mediated Phosphorylation of Focal Adhesion Kinase by Heat Shock Protein 90 and Src Kinase Activities J. Biol. Chem., September 10, 2004; 279(37): 39175 - 39185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Saffitz and A. G. Kleber Effects of Mechanical Forces and Mediators of Hypertrophy on Remodeling of Gap Junctions in the Heart Circ. Res., March 19, 2004; 94(5): 585 - 591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A.U. Heilmann, T. Attmann, A. Thiem, E. Haffner, F. Beyersdorf, and G. Lutter Gene therapy in cardiac surgery: intramyocardial injection of naked plasmid DNA for chronic myocardial ischemia Eur. J. Cardiothorac. Surg., November 1, 2003; 24(5): 785 - 793. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H Wu, M. Guo, S. Y Yuan, and H. J Granger Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor J. Physiol., November 1, 2003; 552(3): 691 - 699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Germani, A. Di Carlo, A. Mangoni, S. Straino, C. Giacinti, P. Turrini, P. Biglioli, and M. C. Capogrossi Vascular Endothelial Growth Factor Modulates Skeletal Myoblast Function Am. J. Pathol., October 1, 2003; 163(4): 1417 - 1428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A.U. Heilmann, T. Attmann, P. von Samson, H. Gobel, D. Marme, F. Beyersdorf, and G. Lutter Transmyocardial laser revascularization combined with vascular endothelial growth factor121 (VEGF121) gene therapy for chronic myocardial ischemia - do the effects really add up? Eur. J. Cardiothorac. Surg., January 1, 2003; 23(1): 74 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Melendez, S. Welch, E. Schaefer, C. S. Moravec, S. Avraham, H. Avraham, and M. A. Sussman Activation of pyk2/Related Focal Adhesion Tyrosine Kinase and Focal Adhesion Kinase in Cardiac Remodeling J. Biol. Chem., November 15, 2002; 277(47): 45203 - 45210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. P. Eliceiri, X. S. Puente, J. D. Hood, D. G. Stupack, D. D. Schlaepfer, X. Z. Huang, D. Sheppard, and D. A. Cheresh Src-mediated coupling of focal adhesion kinase to integrin {alpha}v{beta}5 in vascular endothelial growth factor signaling J. Cell Biol., April 1, 2002; 157(1): 149 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Ross and T. K. Borg Integrins and the Myocardium Circ. Res., June 8, 2001; 88(11): 1112 - 1119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Pham, A. E. Harpf, R. S. Keller, H. T. Vu, S.-Y. Shai, J. C. Loftus, and R. S. Ross Striated muscle-specific beta 1D-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2916 - H2926. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sahni and C. W. Francis Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation Blood, December 1, 2000; 96(12): 3772 - 3778. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rousseau, F. Houle, H. Kotanides, L. Witte, J. Waltenberger, J. Landry, and J. Huot Vascular Endothelial Growth Factor (VEGF)-driven Actin-based Motility Is Mediated by VEGFR2 and Requires Concerted Activation of Stress-activated Protein Kinase 2 (SAPK2/p38) and Geldanamycin-sensitive Phosphorylation of Focal Adhesion Kinase J. Biol. Chem., March 31, 2000; 275(14): 10661 - 10672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. P. Eliceiri, X. S. Puente, J. D. Hood, D. G. Stupack, D. D. Schlaepfer, X. Z. Huang, D. Sheppard, and D. A. Cheresh Src-mediated coupling of focal adhesion kinase to integrin {alpha}v{beta}5 in vascular endothelial growth factor signaling J. Cell Biol., April 1, 2002; 157(1): 149 - 160. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||