© 1995 American Heart Association, Inc.
Articles |
Regulation of Vascular Endothelial Growth Factor in Cardiac Myocytes
From the Cardiology Division (A.P.L., N.S.L., A.C., N.T., G.K., W.C.) and the Hematology-Oncology Division (M.A.G.), Department of Medicine, Brigham and Women's Hospital and Harvard Medical School; the Department of Pathology, Beth Israel Hospital and Harvard Medical School (K.-T.Y.); and the Whitaker Cardiovascular Institute and Cardiology Division, Evans Department of Medicine, Boston University School of Medicine (J.L.), Boston, Mass.
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Abstract Collateral blood vessels supplement normal coronary blood flow and coronary blood flow compromised by coronary artery disease, thereby protecting the myocardium from ischemia. Collateral vessel formation is the result of angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a secreted mitogen specific for endothelial cells and an extremely potent angiogenic factor. In the present study, VPF/VEGF mRNA and protein were demonstrated to be markedly stimulated in primary rat cardiac myocytes in vitro in response to reduction of the oxygen tension to 1% or inhibition of the electron transport chain. Four isoforms of VPF/VEGF were coordinately regulated by hypoxia, including a novel isoform not previously described. Phorbol ester and the depolarizing agent veratridine, stimulators of protein kinase C and calcium influx, respectively, were found to markedly increase VPF/VEGF mRNA expression in cardiac myocytes. Forskolin, a potent stimulator of adenylate cyclase, produced a small but significant increase in VPF/VEGF mRNA expression in the cardiac myocytes. However, only H7, an inhibitor of protein kinase C, inhibited the hypoxic induction of VPF/VEGF mRNA; inhibitors of calcium influx and the calcium-calmodulin–dependent protein kinase II as well as inhibition of protein kinase A did not block the hypoxic induction of VPF/VEGF mRNA. This suggests that more than one signal transduction pathway is involved in regulating VPF/VEGF expression. The sensor that regulates the expression of hypoxia-responsive genes has been proposed to be a heme protein. Consistent with this model, transition metals initiate a genetic program similar to hypoxia. In the present study, the transition metals cobalt and manganese increased VPF/VEGF mRNA in cardiac myocytes in vitro and myocardial tissue in vivo, providing evidence that a similar sensor may regulate VPF/VEGF in the cardiac myocyte. These data suggest a novel mechanism by which VPF/VEGF induction contributes to collateral vessel formation in ischemic myocardium and also suggest strategies to increase VPF/VEGF production in vivo.
Key Words: neovascularization • coronary disease • cell hypoxia • cobalt • manganese
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Coronary collateral vessels may supplement normal and compromised coronary blood flow, thereby protecting the myocardium from ischemia.1 2 The degree of collateral vessel formation is a major determinant of the extent of necrosis after coronary artery occlusion.3 Collateral blood flow can be so efficient as to completely prevent myocardial infarction when a coronary artery is occluded.4 Schaper et al5 first demonstrated that angiogenesis contributes to collateral blood vessel formation. Subsequently, numerous angiogenic factors have been identified in myocardial tissue.6 However, the regulation of these factors by stimuli that induce collateral formation,7 8 9 such as progressive coronary artery stenosis and left ventricular hypertrophy, is poorly understood. These diverse stimuli for collateral formation may both result in a change in the steady state oxygen tension in the cardiac myocyte as a result of either decreased oxygen supply, as in the case of progressive stenosis, or increased demand, as in the case of hypertrophy.
Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor (VPF), is a 45-kD homodimeric protein. VPF/VEGF functions as an endothelial cell–specific mitogen and a potent angiogenic factor.10 11 12 13 14 15 16 17 Multiple isoforms of VPF/VEGF, generated by differential mRNA splicing, have been described with similar bioactivities but different bioavailabilities.18 19 Recently, VPF/VEGF has been demonstrated to be induced by hypoxia in several cell types in vitro,20 21 22 including rat cardiac myocytes,22 and by hypoxia/ischemia in vivo.22 23 24 25 These observations support the hypothesis that hypoxia induces VPF/VEGF mRNA in cardiac myocytes, leading to increased VPF/VEGF protein production and secretion and thereby contributing to ischemia-mediated angiogenesis in vivo.
We sought to elucidate the mechanism of the hypoxic induction of VPF/VEGF by several means. We investigated the regulation of VPF/VEGF mRNA expression, protein production, secretion, and bioactivity in vitro in primary neonatal rat cardiac myocytes in response to inhibition of the electron transport chain or reduction of the oxygen tension to 1%. In addition, because modulators of calcium ion flux and protein kinase C activity have been reported to be influenced by hypoxia26 27 28 29 30 as well as to regulate VPF/VEGF mRNA in several cell types,19 22 31 we studied the effect of modulation of these second messengers on the hypoxic induction of VPF/VEGF mRNA in rat cardiac myocytes. Furthermore, we investigated similarities between VPF/VEGF regulation and the regulation of other hypoxia-inducible genes.21 Specifically, we examined the effects of transition metals known to stimulate other hypoxia-inducible genes21 on VPF/VEGF expression in rat neonatal cardiac myocytes. Finally, we have begun to determine the significance of our cell culture findings by studying the effects of the transition metals on VPF/VEGF mRNA expression in vivo.
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Materials and Methods |
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Chemicals
Amobarbital sodium was reconstituted in distilled H2O (Eli Lilly) or 0.1N NaOH (Sigma Chemical Co) at a concentration of 0.4 mol/L and used on the same day. W7 and H7 (Calbiochem) were prepared as 10 mmol/L stock solutions in distilled H2O and stored at 4°C. KT-5720 (Calbiochem) was prepared as a 1 mg/mL stock solution in dimethyl sulfoxide (DMSO). Dibutyryl-cAMP and forskolin (Calbiochem) were prepared as 5 mg/mL stock solutions in ethanol and DMSO, respectively. All of the following chemicals were obtained from Sigma: verapamil and diltiazam were prepared as a 1 mmol/L stock solution in distilled H2O; veratridine was prepared as a 20 mmol/L stock solution in 1 mmol/L acetic acid; phorbol 12-myristate 13-acetate (PMA) was prepared as a 3 mmol/L stock solution in DMSO; and the calcium ionophore A23187 was prepared as a 2.5 mmol/L stock solution in 100% ethanol. Cobalt chloride and manganese chloride were prepared as 100 mmol/L stock solutions in distilled H2O.
Cardiac Myocyte Cell Culture
Primary neonatal rat cardiac myocytes were prepared according to
published procedures32 with the following modifications.
After collagenase digestion and preplating, the cells were plated at a
density of 107 cells per 10-cm dish (Falcon) in medium
(hereafter referred to as growth medium) consisting of DMEM (1000 mg
glucose/L, Sigma), 1 mmol/L L-glutamine, 100 U/mL
penicillin (Sigma), and 100 µg/mL streptomycin (Sigma), supplemented
with 10% fetal bovine serum (FBS, Sigma). After 24 hours the medium
was replenished, and the cells were incubated for an additional 24
hours before experimentation. The human fetal cardiac myocyte line
W133 was a gift from Dr A. Ahmed-Ansari (Emory University,
Atlanta, Ga). W1 cells were grown in 40% basal medium Eagle (BME,
GIBCO/BRL), 40% F-10 medium (GIBCO), 10% horse serum, 5% FBS, 5%
human AB serum (all sera were obtained from Sigma as heat-inactivated
preparations), 10 µg/mL insulin, 1 mmol/L L-glutamine,
and 50 µg/mL gentamycin (GIBCO). Adult rat ventricular myocytes,
prepared as described by Berger et al,34 were a gift from
Dr Ralph Kelly (Brigham and Women's Hospital, Boston, Mass). In brief,
the rat adult ventricular myocytes were plated at a density of
2x106 cells per 10-cm dish (Falcon) in growth
medium as described above for the neonatal myocytes. The adult myocytes
were used for experiments on the same day they were plated.
Experiments examining the effects of the protein kinase inhibitors W7, KT-5720, and H7 on hypoxic (1% O2) and normoxic (21% O2) VPF/VEGF mRNA expression in neonatal rat cardiac myocytes used a 3-hour incubation in the appropriate experimental conditions. Experiments were performed on at least four replicate samples. VPF/VEGF mRNA was analyzed by RNase protection assay, and the results were normalized to U3 snRNA by using a Molecular Dynamics Phosphorimager. The protein kinase inhibitors W7, KT-5720, and H7 were used at final concentrations of 10, 10, and 20 µmol/L, respectively.
Bovine Capillary Endothelial Cell Culture
Bovine capillary endothelial (BCE) cells derived from bovine
adrenal cortex were the generous gift of Dr J. Folkman (Children's
Hospital, Boston, Mass). BCE cells were maintained in DMEM with 10%
calf serum, 3 ng/mL human recombinant basic fibroblast growth factor, 1
mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin (all from Sigma). The cells were grown on gelatin-coated
dishes at 37°C in a humidified atmosphere of 10% CO2 and
90% air and were passaged weekly at a ratio of 1:6. All experiments
were performed on cells passaged 
15.
Hypoxia
Before hypoxic exposure, cell medium was replaced with growth
medium containing 0.2% FBS, and the cells were then incubated in a
triple gas incubator (Tabai Espec Corp) under an atmosphere of 94%
N2, 5% CO2, and 1%
O2 for 24 hours. For bioactivity assays using the neonatal
rat cardiac myocyte–conditioned media, the cell medium was removed
after 24 hours of hypoxia and replaced with fresh growth medium
containing 0.2% FBS. The cells were incubated for an additional 2
hours under normoxic conditions. This was done to deplete the
conditioned media of transforming growth factor–ß (TGF-ß), which
has been shown to inhibit endothelial cell proliferation35
in two-dimensional systems at concentrations as low as 300 pg/mL. After
24 hours we determined that the concentration of active TGF-ß in the
neonatal rat cardiac myocyte culture media was 2 to 4 mg/mL by
titration in the mink lung bioassay.36 Of note, Madri and
colleagues37 38 have shown that in three-dimensional
collagen gels TGF-ß does not inhibit endothelial cell growth and
actually promotes tube formation. Furthermore, Pepper et
al39 have shown that VPF/VEGF and TGF-ß are synergistic
in in vitro models of angiogenesis. The W1 cell line does not make
active TGF-ß; therefore, the medium used for bioactivity assays was
conditioned for 24 hours under the relevant growth conditions.
Respiration Inhibition
This procedure was modeled after that of Esumi et
al,40 who studied the effect of amobarbital on NADH levels
in adult rat cardiac myocytes. For all treatments the medium consisted
of Ringer's solution, pH 7.4,40 containing 2% FBS at
37°C. Amobarbital was added to the medium at a final concentration of
10 mmol/L, and the pH was adjusted to 7.4 with 1N HCl. The cells were
preincubated in medium without amobarbital for 30 minutes. Addition of
amobarbital to the cells for 10 minutes was followed by three washes
with Ringer's solution. The washing procedure lasted for up to 5
minutes. The cells were allowed to recover for 15 minutes in fresh
medium and then treated twice more with amobarbital in the same manner.
After the final washes, the cells were incubated in growth medium
containing 0.2% FBS for 1, 3, or 6 hours at 37°C under 5%
CO2. Experiments with rotenone or cyanide were performed in
an identical repetitive fashion with 0.2 µmol/L rotenone or 1.5
mmol/L potassium cyanide replacing the amobarbital.
Three brief treatments were used because ischemia simulated by chemical agents was reported to induce hypercontracture in cardiac myocytes after periods of continuous exposure >20 minutes.40 We observed that after each 10-minute exposure to amobarbital followed by washout, the cardiac myocytes regained contractility within several minutes. In addition, there was no change in cell viability as assessed by trypan blue dye exclusion between amobarbital-treated cells and control cells 1 hour after this treatment.
Northern Blot
Total RNA was prepared from cultured cells or
tissues41 and electrophoresed on 1% agarose gels
containing formaldehyde according to published
procedures.42 Murine VPF/VEGF cDNA (a gift from Dr Kevin
Claffey, Beth Israel Hospital, Boston, Mass), human VPF/VEGF cDNA (a
gift from Dr Judith Abraham, Scios Nova, Inc), or rat cDNA generated by
polymerase chain reaction (PCR) using primers described below were used
to generate [32P]dCTP-labeled probes by the random primer
method.43 VPF/VEGF mRNA was normalized to U3 (an abundant
snRNA involved in rRNA processing), ß-actin, or 18S rRNA and
quantified by using a Molecular Dynamics phosphorimager or a LKB 2202
UltraScan Densitometer (LKB).
cDNA Synthesis, PCR, and Cloning
cDNA synthesis and PCR reactions necessary for generation of the
RNase protection assay (RPA) probes described below were performed
according to the protocol outlined in a first-strand cDNA synthesis kit
obtained from Pharmacia. Five micrograms of total RNA from hypoxic
neonatal rat cardiac myocytes was used as template in a 15-µL,
randomly primed, cDNA synthesis reaction. The entire cDNA reaction was
used in a subsequent PCR reaction (50 µL) and amplified under the
following conditions: denaturation at 94°C for 5 minutes (1 time);
50°C hybridization for 30 seconds, 72°C extension for 30 seconds,
94°C denaturation for 45 seconds (35 times); and 50°C hybridization
for 30 seconds, 72°C extension for 5 minutes, soak at 4°C (1 time).
All PCR products were gel-purified and blunt-end–cloned into the
Bluescript vector (Stratagene) and then sequenced to confirm their
identities.
Probe Construction for RPA
Initially, primers complementary to exon 1 (sense, 5'-CCA TGA
ACT TTC TGC TCT CTT G-3') and exon 8 (antisense, 5'-GGT GAG AGG TCT AGT
TCC CGA-3') of rat VPF/VEGF were designed that would enable the
detection of the four previously described distinct VPF/VEGF isoforms
by PCR (Fig 1
, top left). Analysis of the PCR products
by agarose gel electrophoresis using these primers revealed bands of
the predicted size for VPF/VEGF121, VPF/VEGF165, and VPF/VEGF189. In
addition, a smaller band was identified that we named VPF/VEGF23. This
band was gel-purified, cloned, and sequenced and was determined to be
the result of exon 1 spliced to exon 8. We found no evidence of
VPF/VEGF206 by PCR analysis.
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Probes were then designed that would enable the detection of the
distinct VPF/VEGF isoforms by RPA (Fig 1
, top right). Probe A, designed
to detect VPF/VEGF165 and VPF/VEGF189, was constructed using PCR with
primers complementary to rat VPF/VEGF exon 5 (sense, 5'-CCG AAT TCA CCA
AAG AAA GAT AGA ACA AAG-3') and exon 8 (antisense, 5'-GGT GAG AGG TCT
AGT TCC CGA-3'). A 239-bp cDNA sequence containing exons 5, 7, and 8 of
VPF/VEGF was amplified, gel-purified, cloned, and sequenced.
Hybridization of VPF/VEGF165 mRNA to probe A results in full-length
protection of probe A. mRNA for VPF/VEGF189, which differs from that of
VPF/VEGF165 by the addition of part of exon 6, only partially
hybridizes to probe A, resulting in the protection of two smaller
fragments, 209 bp and 30 bp. The 209-bp fragment corresponds to exons 7
and 8 and is specific for VPF/VEGF189. (VPF/VEGF206 mRNA would also
protect a 209-bp fragment. However, we found no mRNA corresponding to
this isoform by PCR analysis. Therefore, the 209-bp fragment is
specific for VPF/VEGF189 in neonatal cardiac myocytes.)
Probe B, designed to detect VPF/VEGF121 mRNA, was made using the same
primers complementary to exon 5 and 8 described for probe A. A 107-bp
cDNA sequence containing exons 5 and 8 was amplified, gel-purified,
cloned, and sequenced. Hybridization of VPF/VEGF121 mRNA to probe B
results in full-length protection of probe B (Fig 1, top right). Probe
C, designed to detect the novel isoform VPF/VEGF23, was made using
primers complementary to exon 1 (sense, 5'-CCA TGA ACT TTC TGC TCT CTT
G-3') and exon 8 (antisense, 5'-GGT GAG AGG TCT AGT TCC CGA-3'). A
143-bp cDNA sequence containing exons 1 and 8 of VPF/VEGF was
amplified, gel-purified, cloned, and sequenced. Hybridization of
VPF/VEGF23 mRNA results in full-length protection of probe C (Fig 1,
top right).
A probe for U3 was a gift from Dr E.J. Neer (Brigham and Women's
Hospital, Boston, Mass). Full-length protection of this probe results
in a doublet of 
140 bp.
RPA
Linearized plasmids were used to generate RNA probes according
to published procedures44 with the following
modifications. Because of the abundance of the U3 RNA in the cells, the
U3 probe was labeled with 3 µL [32P]CTP (800 Ci/mmol,
New England Nuclear) and 3 µL of 40 µmol/L unlabeled CTP in a
20-µL reaction volume. All other probes were labeled with 10 µL
[32P]CTP and no unlabeled nucleotide. Ten micrograms of
total cellular RNA was hybridized to 3x105 cpm of VPF/VEGF
probe and 1.5x105 cpm of U3 probe and incubated at 55°C
overnight. The mixture was digested with 40 µg/mL RNase A (USB) and
0.65 µg/mL RNase T1 (USB) for 30 minutes at 30°C. After
digestion with proteinase K, the products were extracted with
phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol,
and immediately centrifuged at 4°C (preincubation on ice or at
-20°C before centrifugation resulted in the formation of a large
precipitate). The products were electrophoresed through a denaturing
polyacrylamide gel and quantified by use of a Molecular Dynamics
phosphorimager or a LKB 2202 UltraScan densitometer. Fig 1 (bottom)
shows the data obtained from a representative RPA.
Immunofluorometric Assay of VPF/VEGF
A sensitive two-site time-resolved immunofluorometric assay as
previously described45 46 was modified to quantify
VPF/VEGF in rat cardiac myocyte–conditioned media. The capture
antibodies were affinity-purified rabbit IgG developed against the
C-terminal peptide of VPF/VEGF; the detector antibodies were
affinity-purified rabbit IgG developed against the N-terminal region of
mouse VPF/VEGF and were a generous gift from Dr Janice Nagy (Beth
Israel Hospital, Boston, Mass). Because recombinant mouse VPF/VEGF was
not available, we arbitrarily chose to use purified recombinant human
VPF/VEGF (R&D Systems) to calibrate this assay. Results are thus
expressed as human VPF/VEGF equivalents in picomoles per liter.
Conditioned medium was concentrated 5- to 10-fold by using a
Centriprep-10 concentrator (Amicon) before the VPF/VEGF assay.
[3H]Thymidine Incorporation Into Microvascular
Endothelial Cells
BCE cells were plated in DMEM with 1% FBS at a density of 2000
cells per well in a 96-well plate precoated with gelatin. After
overnight incubation, conditioned medium supplemented with 5% FBS was
added to the BCE cells and incubated for 18 hours, after which time
[3H]thymidine (50 to 80 Ci/mmol; New England Nuclear) in
1% FBS was added to the cells at a concentration of 5 µCi/mL. After
3 hours, the cells were washed with PBS (10 mmol/L sodium phosphate and
0.15 mol/L sodium chloride, pH 7.4) and incubated with 5%
trichloroacetic acid for 30 minutes at 4°C. The cells were washed
twice with ice-cold water and lysed in 0.4N NaOH for 30 minutes at room
temperature. The lysate (200 µL) was added to 4 mL scintillation
fluid and counted for 1 minute in a Beckman scintillation counter.
Animal Experiments
Male Sprague-Dawley rats (Charles River Laboratories)
weighing 200 to 225 g were used according to approved animal studies
protocols. Cobalt and manganese were given as 1-mL subcutaneous
injections of a 75 mmol/L solution, and ventricular tissue was
harvested for Northern blot analysis at 6 hours.
Statistical Analyses
Where indicated, data are presented as mean±SEM. Error
resulting from the computation of two or more data points, each of
which has an error associated with it, was calculated dependent upon
the arithmetic relation between the terms containing the
errors.47 Student's unpaired t test was used
to assess differences between two groups.
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Results |
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VPF/VEGF mRNA Is Increased With Hypoxia and Respiration Inhibition in Cardiac Myocytes
As shown in Fig 2A
, in neonatal rat cardiac
myocytes VPF/VEGF mRNA is induced within 3 hours after exposure to 1%
O2, and there is a 25.0±11.4-fold
(P<.01) induction of the mRNA by 24 hours as measured by
phosphorimaging analysis. Because the primary neonatal cardiac
myocyte preparations are only 80% to 90% pure, we cannot completely
exclude the possibility of contributions made by smooth muscle cells
and fibroblastic cells to this induction. However, further evidence
that cardiac myocytes make VPF/VEGF comes from experiments using the
human fetal cardiac myocyte cell line W1. This transformed cell line
provides a homogeneous population of cells of human cardiac myocyte
origin. The W1 cell line has been extensively characterized and shown
to closely resemble fetal cardiac myocytes in culture with regard to
various antigenic determinants.33 Fig 2B demonstrates that
VPF/VEGF mRNA is similarly upregulated in the W1 cells in response to
hypoxia. Additional proof that VPF/VEGF is derived from cardiac
myocytes comes from analysis of preparations of adult rat
ventricular myocytes that have been previously demonstrated to be 98%
pure.34 Fig 2C shows a Northern blot analysis of these
cells, cultured under conditions of normoxia or hypoxia for 24 hours.
Again, VPF/VEGF mRNA is markedly upregulated in response to
hypoxia.
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Fig 3 demonstrates that VPF/VEGF mRNA is induced
5.3±2.2-fold (P<.01) at 1 hour after three 10-minute
treatments with amobarbital, a respiratory inhibitor that acts at
complex I of the electron transport chain (ETC).48 Similar
results were obtained with rotenone, a respiratory inhibitor that acts
at the same site of the ETC as amobarbital. A distal inhibitor of the
ETC, potassium cyanide, did not increase VPF/VEGF mRNA levels (data not
shown).
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VPF/VEGF mRNA Isoforms Are Coordinately Regulated by Hypoxia
Several different isoforms of VPF/VEGF have been described that
result from alternative mRNA splicing (Fig 1, top left). To determine
whether differential regulation of VPF/VEGF isoforms by hypoxia could
account for increased VPF/VEGF secretion, PCR analysis was used to
determine which isoforms of VPF/VEGF were present in cardiac
myocytes. We identified VPF/VEGF121, VPF/VEGF165, and VPF/VEGF189 but
not VPF/VEGF206 in the cardiac myocytes (data not shown). In addition,
we identified a novel isoform of VPF/VEGF that is the result of
splicing of exon 1 to exon 8. This transcript should encode a 23–amino
acid signal-like peptide comprising 22 amino acids encoded by exon 1
and an additional methionine encoded by exon 8. Specific probes were
developed to quantify the abundance of all the VPF/VEGF isoforms
present in cardiac myocytes (Fig 1, top right). Normalization for
probe length allowed for quantification of the relative abundance of
the different isoforms in the VPF/VEGF pool. VEGF121, VEGF165, VEGF23,
and VEGF189 contributed 45%, 32%, 18%, and 5%, respectively, to the
VEGF mRNA pool. The relative abundance of the different isoforms did
not change appreciably after 24 hours of hypoxia because all the
isoforms were increased to the same degree.
VPF/VEGF Immunoreactive Protein Is Present in the Conditioned Media
of Hypoxic Cardiac Myocytes
A sandwich immunofluorometric assay using peptide antisera against
the N-terminal and C-terminal regions of rat VPF/VEGF was used to
quantify VPF/VEGF in the conditioned media from neonatal rat cardiac
myocytes. As shown in Fig 4, at 24 hours there was an
increase of approximately sevenfold to eightfold in the concentration
of VPF/VEGF in the medium of hypoxic (1% O2) cardiac
myocytes compared with that of normoxic (21% O2) cardiac
myocytes. The concentration of VPF/VEGF in the myocyte-conditioned
medium after 24 hours of hypoxia was 70 pmol/L.
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Cardiac Myocyte–Conditioned Medium Stimulates
[3H]Thymidine Incorporation Into Microvascular
Endothelial Cells
Fig 5 demonstrates statistically significant
stimulation of [3H]thymidine incorporation into
endothelial cells after incubation with the 2-hour conditioned medium
of cardiac myocytes grown at 1% O2 for the previous 24
hours. This conditioned medium does not stimulate
[3H]thymidine incorporation in BALB/c 3T3 cells, a point
of significant distinction between fibroblast growth factor and
VPF/VEGF. In addition, the quantitative immunofluorometric assay
demonstrated that the concentration of VPF/VEGF in this conditioned
medium used to stimulate [3H]thymidine incorporation into
endothelial cells was five times greater in myocytes that had been
previously grown at 1% O2 than in those grown at 21%
O2. Furthermore, the conditioned medium from the W1 cells
grown in 1% O2 for 24 hours stimulates endothelial cell
proliferation to a much greater degree than does medium from W1 cells
grown in 21% O2.
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Regulation of VPF/VEGF mRNA by Activated Second Messenger
Systems
As shown in Fig 6 (top), the regulation of
VPF/VEGF mRNA by activated second messenger systems was studied by RPA
and quantified by phosphorimaging analysis and densitometry. In
21% O2, 100 nmol/L PMA was found to stimulate
VPF/VEGF mRNA 4.7±0.4-fold (P<.01) at 3 hours of treatment
in the neonatal rat cardiac myocytes compared with untreated cells.
This is in agreement with the results of other
researchers.19 22 31 Dibutyryl-cAMP (100 µmol/L) did not
induce (0.9±0.03-fold) VPF/VEGF mRNA at 3 hours of treatment in the
neonatal rat cardiac myocytes compared with untreated cells, differing
from observations previously made with PC12 cells31 and
osteoblastic cells,49 whereas forskolin (100 µmol/L)
induced a small increase in VEGF mRNA (1.4±0.01-fold,
P<.01). Calcium influx stimulated by the depolarizing agent
veratridine (Fig 6, bottom) produced a 6.0±2.0-fold
(P<.01) increase in VPF/VEGF mRNA by 3 hours of treatment.
Similar data were obtained with the calcium ionophore A23187, in
agreement with results reported by Claffey et al31 in PC12
cells. The effect of veratridine could be inhibited with the L-type
calcium channel blocker verapamil (data not shown).
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Inhibition of calcium influx with the chelators EGTA and EDTA and
the calcium channel blockers verapamil and diltiazem had no significant
effect on the hypoxic induction of VPF/VEGF mRNA. In addition, W7, an
inhibitor of calcium-calmodulin–dependent protein kinase
II,50 had no significant effect on the hypoxic levels of
VPF/VEGF mRNA (0.9±0.1-fold change, P>.05), although it
did stimulate normoxic levels of VPF/VEGF mRNA by a modest but
significant amount (2.1±0.3-fold increase [P<.01]).
KT-5720, an inhibitor of protein kinase
A,51 moderately increased the levels of VPF/VEGF mRNA in
both hypoxic (1.8±0.3-fold, P<.01) and normoxic conditions
(1.7±0.1-fold, P<.01). However, H7, an inhibitor of
protein kinase C,50 significantly inhibited the hypoxic
induction of VPF/VEGF mRNA (0.53±0.07-fold reduction,
P<.01) at 3 hours of hypoxia (Fig 7).
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Transition Metals Stimulate VPF/VEGF Production
Like VPF/VEGF, expression of the erythropoietin (Epo) gene
is upregulated by hypoxia.52 Epo is also induced by the
transition metals cobalt and manganese. We recently demonstrated in two
tumor cell lines that cobalt also stimulates VPF/VEGF.21
We therefore investigated whether transition metals stimulated VPF/VEGF
in cardiac myocytes. As shown in Fig 8, cobalt and
manganese stimulated VPF/VEGF mRNA 34.0±22.0-fold (P<.01)
and 10.8±1.1-fold (P<.01), respectively, as determined by
RPA and phosphorimaging analysis. Furthermore, the 2-hour
conditioned medium from rat cardiac myocytes (depleted of TGF-ß as
described in "Materials and Methods") increased stimulation of
[3H]thymidine incorporation into endothelial cells (Fig 5).
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In Vivo Induction of VPF/VEGF mRNA by Cobalt
To examine whether VPF/VEGF mRNA can be induced in myocardium in
vivo, we gave adult rats a single subcutaneous injection of
CoCl2. There was a 2.0±0.2-fold (P<.01)
increase in VPF/VEGF mRNA (compared with sham injection) in ventricular
tissue 6 hours after the injection, as assessed by RPA
analysis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data demonstrate that VPF/VEGF, a secreted endothelial cell–specific mitogen and extremely potent angiogenic factor, is stimulated in cardiac myocytes in response to hypoxia. VPF/VEGF mRNA expression increased 25.0±11.4-fold in neonatal cardiac myocytes grown under hypoxic conditions. All isoforms of VPF/VEGF were coordinately upregulated at the mRNA level as demonstrated by RPA. Significantly more VPF/VEGF immunoreactive protein was present in the conditioned media of cardiac myocytes grown under hypoxic conditions. In addition, the conditioned media from the cardiac myocytes stimulated [3H]thymidine incorporation into microvascular endothelial cells but not BALB/c 3T3 cells, strongly supporting the contention that VPF/VEGF is the mitogen responsible for this activity.
The molecular mechanism by which hypoxia is sensed by the cardiac myocyte and in turn stimulates VPF/VEGF production is unknown. Putative oxygen sensors have been recently described in the carotid body53 54 and pulmonary vascular system.55 However, the identity of the oxygen sensor(s) remains elusive. Archer et al55 recently demonstrated that proximal inhibitors of the ETC such as amobarbital, rotenone, and antimycin A could all simulate hypoxia in the pulmonary vascular bed but that distal inhibitors of the ETC such as azide and cyanide did not mimic hypoxia. We report a similar phenomenon with regard to the induction of VPF/VEGF mRNA, whereby amobarbital and rotenone increase VPF/VEGF mRNA but cyanide does not. Archer et al proposed that the redox state of the cell, as well as the level of activated oxygen species formed in the proximal portion of the ETC, may play an important role in oxygen sensing. However, precisely how specific induction of gene expression might be achieved through such a redox mechanism is still unclear.
The time courses of the induction of VPF/VEGF mRNA differ in these two models of hypoxia. Amobarbital induction was maximal 1 hour after the third treatment, whereas there was no apparent increase in VPF/VEGF mRNA until 3 hours after the cells were placed in the hypoxia incubator. However, this does not necessarily mean that the two pathways for VPF/VEGF mRNA induction are different. Chemical agents mimic hypoxia rapidly (within minutes) by inhibiting the ETC. On the other hand, it may take up to 90 minutes to achieve a significant reduction in the oxygen tension in the tissue culture medium and the cellular milieu in the hypoxia incubator. In addition, the chemical hypoxia protocol involves repetitive 10-minute treatments, whereas the hypoxia experiments involve a continuous incubation under hypoxic conditions. Treatment of the cells with a 10-fold lower dose of amobarbital for longer periods of time did produce a sustained increase in VPF/VEGF mRNA (data not shown).
Hypoxia has been shown to increase calcium influx and membrane-bound protein kinase C in a variety of cell types.26 27 28 29 30 These same second messengers have been shown to stimulate VPF/VEGF mRNA in several cell types.19 22 31 We extend these observations to the myocyte and demonstrate by several different means that calcium influx can stimulate VPF/VEGF mRNA. The stimulation of VPF/VEGF by veratridine, a depolarizing agent, is blocked by the calcium channel blocker verapamil. Depolarization as a stimulus for VPF/VEGF is intriguing because depolarization is observed to occur in excitable cells in response to hypoxia and ischemia.54 56 57 58 However, calcium channel blockade and the calcium-calmodulin–dependent protein kinase inhibitor W7 fail to inhibit the hypoxic induction of VPF/VEGF. This suggests that agents that induce VPF/VEGF mRNA by increasing calcium influx are unlikely to do so through the same pathway as hypoxia. On the other hand, H7, an inhibitor of protein kinase C and protein kinase A, does partially inhibit the hypoxic induction of VPF/VEGF. The effect of H7 suggests that protein kinase C is involved in the hypoxic stimulation of VPF/VEGF. Although H7 can also inhibit protein kinase A, it seems unlikely that protein kinase A mediates this effect because (1) membrane-bound protein kinase C levels increase27 but cAMP levels do not change59 with hypoxia in cardiac myocytes; (2) PMA stimulates VPF/VEGF mRNA in cardiac myocytes, whereas dibutyryl-cAMP does not; and (3) KT-5720, a protein kinase A inhibitor, does not inhibit the hypoxic induction of VPF/VEGF. Nonetheless, we cannot rule out the possibility that H7 acts in some other manner.
The stimulation of VPF/VEGF by transition metals provides evidence for a common oxygen-sensing system regulating the expression of VPF/VEGF and Epo. It has been hypothesized that the ability of the transition metals cobalt and manganese to stimulate Epo production is due to their effect on an oxygen-sensing heme protein whose activity changes depending on the presence or absence of oxygen binding to the heme moiety.52 Cobalt and manganese can substitute for iron in the protoporphyrin ring, but the resulting metal protoporphyrins bind oxygen either poorly or not at all, thus locking the protein in a deoxy conformation and simulating the hypoxic state. It is of interest that Webster et al59 recently showed that c-jun and c-fos mRNA are also induced by hypoxia in rat cardiac myocytes. We have recently demonstrated that c-jun and c-fos mRNA are also induced by hypoxia and cobalt in a human hepatoma cell line.21 Because there are three AP-1 binding sites in the 5' flanking region of VPF/VEGF,19 these findings, taken together, suggest that the transcription factor AP-1, composed of members of the jun and fos families, may play a regulatory role in VPF/VEGF induction by hypoxia and the transition metals.
If the pathway for transition metal induction of VPF/VEGF is the same as hypoxia, H7 would be expected to inhibit the increase in VPF/VEGF seen with the metals. Three hours after the addition of CoCl2, there is only a 1.7±0.12-fold (P<.01) increase in VPF/VEGF mRNA. H7 did not significantly diminish this response at 3 hours (1.7±0.06). Similar results were seen with MnCl2. We have not investigated the effect of a more prolonged coincubation of H7 with the metals. However, we have not seen as great an effect on the inhibition of the hypoxic induction by H7 at later time points (6 to 24 hours) (authors' unpublished data, 1994). This lack of an effect at later time points may be explained by inactivation of H7 or by the existence of multiple mechanisms to increase VPF/VEGF mRNA with hypoxia, as is the case for the tyrosine hydroxylase gene60 (ie, increased transcriptional rate and increased mRNA stability). We cannot conclude from these studies that hypoxia and transition metals act by different pathways, except to say that hypoxia and transition metals appear to have different time courses for the induction of VPF/VEGF.
Four isoforms of VPF/VEGF have been described that differ in their
bioavailability.18 Expression studies in human embryonic
kidney cells showed that VPF/VEGF206 and VPF/VEGF189 remained cell
associated and were not secreted into the medium. VPF/VEGF165 was
partially secreted, and VPF/VEGF121 was entirely secreted. We reasoned
that hypoxia may increase VPF/VEGF bioavailability by differentially
upregulating the VPF/VEGF121 or VPF/VEGF165 isoforms, which appear to
be secreted to a greater extent than the larger isoforms. Analysis by
RPA showed that VPF/VEGF189, VPF/VDGF165, and VPF/VEGF121 are all
induced equivalently. Therefore, differential splicing is not a
mechanism of hypoxic upregulation of VPF/VEGF. In the course of our
study we found a previously unidentified isoform of VPF/VEGF that we
call VPF/VEGF23. This alternative splice product results from joining
of exon 1 to exon 8 and encodes a signallike peptide of 23 amino acids.
Analysis by RPA showed that this isoform is also upregulated by hypoxia
and accounts for 20% of the total VPF/VEGF mRNA. We have also found
this isoform to be present in vivo in ventricular tissue by RPA. In
vitro transcription and translation of VPF/VEGF23 demonstrates a
specific product of the predicted molecular weight (2.5 kD) (authors'
unpublished data, 1994). The potential biological activity of
VPF/VEGF23 is currently under investigation.
Manipulation of VPF/VEGF levels in vivo can modulate the angiogenic process. Administration of purified VPF/VEGF has been demonstrated to accelerate collateral blood vessel formation in several models of ischemia involving the rat hind limb61 and the epicardial coronary vessels.62 An alternative approach may be to augment VPF/VEGF production in situ in areas of ischemia by simulating more severe degrees of hypoxia through the use of the transition metals or by taking advantage of the alterations in membrane potential and calcium flux in areas of hypoxia/ischemia.
|
Acknowledgments |
|---|
This study was supported by grants from the National Institutes of Health (T32-HL-07604 to Dr Andrew Levy, DK-45098 to Dr Goldberg, HL-46005 to Dr Koren, CA-58845 to Dr Yeo, 1F32-HL-08838-01 to Dr Nina Levy, HL-47416 and HL-48743 to Dr Loscalzo, and HL-42539 to Dr Colucci); a research career development award from the National Institutes of Health to Dr Loscalzo (JL-02273); a merit review award from the Veterans Administration to Dr Loscalzo; an American Heart Association Established Investigator Award to Dr Colucci; and an American Heart Association Established Investigator Award and Grant-in-Aid to Dr Goldberg. We wish to thank Dr Harold Dvorak for his advice and support of this work as well as for his critical review of the manuscript.
|
Footnotes |
|---|
Reprint requests to Dr Mark A. Goldberg, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115.
Received August 12, 1994; accepted December 28, 1994.
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References |
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 P.B.J Burton, V.J Owen, S Hafizi, P.J.R Barton, G Carr-White, T Koh, A De Souza, M.H Yacoub, and J.R Pepper Vascular endothelial growth factor release following coronary artery bypass surgery: extracorporeal circulation versus 'beating heart' surgery Eur. Heart J., October 2, 2000; 21(20): 1708 - 1713. [Abstract] [PDF]
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 M. E. Olah and F. L. Roudabush Down-Regulation of Vascular Endothelial Growth Factor Expression after A2A Adenosine Receptor Activation in PC12 Pheochromocytoma Cells J. Pharmacol. Exp. Ther., June 1, 2000; 293(3): 779 - 787. [Abstract] [Full Text]
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 S. H. Lee, P. L. Wolf, R. Escudero, R. Deutsch, S. W. Jamieson, and P. A. Thistlethwaite Early Expression of Angiogenesis Factors in Acute Myocardial Ischemia and Infarction N. Engl. J. Med., March 2, 2000; 342(9): 626 - 633. [Abstract] [Full Text] [PDF]
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M. Clauss and W. Schaper Vascular Endothelial Growth Factor : A Jack-of-All-Trades or a Nonspecific Stress Gene? Circ. Res., February 18, 2000; 86(3): 251 - 252. [Full Text] [PDF]
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 M.A. Breider, A.W. Gough, J.R. Haskins, G. Sobocinski, and F.A. De La Iglesia Troglitazone-Induced Heart and Adipose Tissue Cell Proliferation in Mice Toxicol Pathol, September 1, 1999; 27(5): 545 - 552. [Abstract] [PDF]
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A. Schultz, L. Lavie, I. Hochberg, R. Beyar, T. Stone, K. Skorecki, P. Lavie, A. Roguin, and A. P. Levy Interindividual Heterogeneity in the Hypoxic Regulation of VEGF : Significance for the Development of the Coronary Artery Collateral Circulation Circulation, August 3, 1999; 100(5): 547 - 552. [Abstract] [Full Text] [PDF]
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J.-W. Gu, A. L. Brady, V. Anand, M. C. Moore, W. C. Kelly, and T. H. Adair Adenosine upregulates VEGF expression in cultured myocardial vascular smooth muscle cells Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H595 - H602. [Abstract] [Full Text] [PDF]
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J. R Kersten, P. S Pagel, W. M Chilian, and D. C Warltier Multifactorial basis for coronary collateralization: a complex adaptive response to ischemia Cardiovasc Res, July 1, 1999; 43(1): 44 - 57. [Abstract] [Full Text] [PDF]
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N. Takahashi, Y. Seko, E. Noiri, K. Tobe, T. Kadowaki, H. Sabe, and Y. Yazaki Vascular Endothelial Growth Factor Induces Activation and Subcellular Translocation of Focal Adhesion Kinase (p125FAK) in Cultured Rat Cardiac Myocytes Circ. Res., May 28, 1999; 84(10): 1194 - 1202. [Abstract] [Full Text] [PDF]
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H. Matsui, Y. Ihara, Y. Fujio, K. Kunisada, S. Akira, T. Kishimoto, and K. Yamauchi-Takihara Induction of interleukin (IL)-6 by hypoxia is mediated by nuclear factor (NF)-{kappa}B and NF-IL6 in cardiac myocytes Cardiovasc Res, April 1, 1999; 42(1): 104 - 112. [Abstract] [Full Text] [PDF]
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R. J Tomanek, N. Hu, B. Phan, and E. B Clark Rate of coronary vascularization during embryonic chicken development is influenced by the rate of myocardial growth Cardiovasc Res, March 1, 1999; 41(3): 663 - 671. [Abstract] [Full Text] [PDF]
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 M. J. Acarregui, S. T. Penisten, K. L. Goss, K. Ramirez, and J. M. Snyder Vascular Endothelial Growth Factor Gene Expression in Human Fetal Lung In Vitro Am. J. Respir. Cell Mol. Biol., January 1, 1999; 20(1): 14 - 23. [Abstract] [Full Text]
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 H. H. Marti and W. Risau Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors PNAS, December 22, 1998; 95(26): 15809 - 15814. [Abstract] [Full Text] [PDF]
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F. Koudssi, J. E. Lopez, S. Villegas, and C. S. Long Cardiac Fibroblasts Arrest at the G1/S Restriction Point in Response to Interleukin (IL)-1beta . EVIDENCE FOR IL-1beta -INDUCED HYPOPHOSPHORYLATION OF THE RETINOBLASTOMA PROTEIN J. Biol. Chem., October 2, 1998; 273(40): 25796 - 25803. [Abstract] [Full Text] [PDF]
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L. Zhang, A. Hach, and C. Wang Molecular Mechanism Governing Heme Signaling in Yeast: a Higher-Order Complex Mediates Heme Regulation of the Transcriptional Activator HAP1 Mol. Cell. Biol., July 1, 1998; 18(7): 3819 - 3828. [Abstract] [Full Text]
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N. S. Levy, S. Chung, H. Furneaux, and A. P. Levy Hypoxic Stabilization of Vascular Endothelial Growth Factor mRNA by the RNA-binding Protein HuR J. Biol. Chem., March 13, 1998; 273(11): 6417 - 6423. [Abstract] [Full Text] [PDF]
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 K. K. Graven, R. J. McDonald, and H. W. Farber Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase Am J Physiol Cell Physiol, February 1, 1998; 274(2): C347 - C355. [Abstract] [Full Text] [PDF]
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Y. Kim, R. Y. Imdad, A. H. Stephenson, R. S. Sprague, and A. J. Lonigro Vascular Endothelial Growth Factor mRNA in Pericytes Is Upregulated by Phorbol Myristate Acetate Hypertension, January 1, 1998; 31(1): 511 - 515. [Abstract] [Full Text] [PDF]
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 Z. Luo, M. Diaco, T. Murohara, N. Ferrara, J. M. Isner, and J. F. Symes Vascular Endothelial Growth Factor Attenuates Myocardial Ischemia-Reperfusion Injury Ann. Thorac. Surg., October 1, 1997; 64(4): 993 - 998. [Abstract] [Full Text]
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D. S. Winlaw Angiogenesis in the Pathobiology and Treatment of Vascular and Malignant Diseases Ann. Thorac. Surg., October 1, 1997; 64(4): 1204 - 1211. [Abstract] [Full Text]
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 T. Hayashi, K. Abe, H. Suzuki, and Y. Itoyama Rapid Induction of Vascular Endothelial Growth Factor Gene Expression After Transient Middle Cerebral Artery Occlusion in Rats Stroke, October 1, 1997; 28(10): 2039 - 2044. [Abstract] [Full Text]
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B. S Lewis, M. Y Flugelman, A. Weisz, I. Keren-Tal, and W. Schaper Angiogenesis by gene therapy: a new horizon for myocardial revascularization? Cardiovasc Res, September 1, 1997; 35(3): 490 - 497. [Abstract] [Full Text] [PDF]
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P. H. Maxwell, G. U. Dachs, J. M. Gleadle, L. G. Nicholls, A. L. Harris, I. J. Stratford, O. Hankinson, C. W. Pugh, and P. J. Ratcliffe Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth PNAS, July 22, 1997; 94(15): 8104 - 8109. [Abstract] [Full Text] [PDF]
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 C. Y. Cheung Vascular Endothelial Growth Factor: Possible Role in Fetal Development and Placental Function Reproductive Sciences, July 1, 1997; 4(4): 169 - 177. [Abstract] [PDF]
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T. Horiuchi and P. F. Weller Expression of Vascular Endothelial Growth Factor by Human Eosinophils: Upregulation by Granulocyte Macrophage Colony-stimulating Factor and Interleukin-5 Am. J. Respir. Cell Mol. Biol., July 1, 1997; 17(1): 70 - 77. [Abstract] [Full Text]
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 M. S. Pepper Manipulating Angiogenesis: From Basic Science to the Bedside Arterioscler Thromb Vasc Biol, April 1, 1997; 17(4): 605 - 619. [Abstract] [Full Text]
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J.-W. Gu, D. Santiago, Y. Olowe, and J. Weinberger Basic Fibroblast Growth Factor as a Biochemical Marker of Exercise-Induced Ischemia Circulation, March 4, 1997; 95(5): 1165 - 1168. [Abstract] [Full Text]
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W. Schaper and W. D. Ito Molecular Mechanisms of Coronary Collateral Vessel Growth Circ. Res., November 1, 1996; 79(5): 911 - 919. [Full Text]
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A. P. Levy, N. S. Levy, and M. A. Goldberg Hypoxia-inducible Protein Binding to Vascular Endothelial Growth Factor mRNA and Its Modulation by the von Hippel-Lindau Protein J. Biol. Chem., October 11, 1996; 271(41): 25492 - 25497. [Abstract] [Full Text] [PDF]
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Z. Kovacs, K. Ikezaki, K. Samoto, T. Inamura, M. Fukui, T. Kawamata, and S. P. Finklestein VEGF and flt: Expression Time Kinetics in Rat Brain Infarct Stroke, October 1, 1996; 27(10): 1865 - 1873. [Abstract] [Full Text]
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M. Fujita, M. Ikemoto, M. Kishishita, H. Otani, R. Nohara, T. Tanaka, S.-i. Tamaki, A. Yamazato, and S. Sasayama Elevated Basic Fibroblast Growth Factor in Pericardial Fluid of Patients With Unstable Angina Circulation, August 15, 1996; 94(4): 610 - 613. [Abstract] [Full Text]
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A. P. Levy, N. S. Levy, and M. A. Goldberg Post-transcriptional Regulation of Vascular Endothelial Growth Factor by Hypoxia J. Biol. Chem., February 2, 1996; 271(5): 2746 - 2753. [Abstract] [Full Text] [PDF]
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S. Pal, K. Datta, R. Khosravi-Far, and D. Mukhopadhyay Role of Protein Kinase Czeta in Ras-mediated Transcriptional Activation of Vascular Permeability Factor/Vascular Endothelial Growth Factor Expression J. Biol. Chem., January 19, 2001; 276(4): 2395 - 2403. [Abstract] [Full Text] [PDF]
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M. Kimura, Y. Mizukami, T. Miura, K. Fujimoto, S. Kobayashi, and M. Matsuzaki Orphan G Protein-coupled Receptor, GPR41, Induces Apoptosis via a p53/Bax Pathway during Ischemic Hypoxia and Reoxygenation J. Biol. Chem., July 6, 2001; 276(28): 26453 - 26460. [Abstract] [Full Text] [PDF]
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T. Shimizu, M. Yamato, Y. Isoi, T. Akutsu, T. Setomaru, K. Abe, A. Kikuchi, M. Umezu, and T. Okano Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces Circ. Res., February 22, 2002; 90 (3): e40 - e48. [Abstract] [Full Text] [PDF]
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 C. JIANG, H. LU, K. A. VINCENT, S. SHANKARA, A. J. BELANGER, S. H. CHENG, G. Y. AKITA, R. A. KELLY, M. A. GOLDBERG, and R. J. GREGORY Gene expression profiles in human cardiac cells subjected to hypoxia or expressing a hybrid form of HIF-1{alpha} Physiol Genomics, February 11, 2002; 8(1): 23 - 32. [Abstract] [Full Text] [PDF]
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