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Circulation Research. 1995;76:758-766

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(Circulation Research. 1995;76:758-766.)
© 1995 American Heart Association, Inc.

Articles

Regulation of Vascular Endothelial Growth Factor in Cardiac Myocytes

Andrew P. Levy, Nina S. Levy, Joseph Loscalzo, Angelino Calderone, Nobuyuki Takahashi, Kiang-Teck Yeo, Gideon Koren, Wilson S. Colucci, Mark A. Goldberg

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.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
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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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 1Down, 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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  		Figure 1. Diagrams and photographs show results of RNase protection assay (RPA) done to quantify vascular endothelial growth factor (VEGF) mRNA isoforms. mRNA was isolated from neonatal rat cardiac myocytes grown at 1% or 21% O2 for 24 hours, and RPA was performed as described in "Materials and Methods." Top left, Exon structure and differential splicing of VEGF isoforms. VEGF121, VEGF165, VEGF189, VEGF206, and VEGF23 correspond to isoforms containing 121, 165, 189, 206, and 23 amino acids, respectively. Top right, Diagram describes probes used to detect the 23–, 121–, 165–, and 189–amino acid mRNA VEGF isoforms. *Only fragments unique to a given VEGF isoform are included. #Transcripts corresponding to VEGF206 would also protect a 209-bp fragment. However, no mRNA for VEGF206 was found by polymerase chain reaction analysis. Therefore, in this assay the 209-bp band corresponds solely to VEGF189. Bottom, Representative RPA shows specific protected fragments of expected size. U3 mRNA was used to normalize the samples. p indicates undigested probe; N, RNA from normoxic cells; H, RNA from hypoxic cells; and t, tRNA.

Probes were then designed that would enable the detection of the distinct VPF/VEGF isoforms by RPA (Fig 1Up, 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 1Up, 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 1Up, 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 {approx}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 1Up (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.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
VPF/VEGF mRNA Is Increased With Hypoxia and Respiration Inhibition in Cardiac Myocytes
As shown in Fig 2ADown, 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 2BDown 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 2CDown 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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  		Figure 2. Northern blot analyses, each representative of several experiments, of hypoxic cardiac myocytes, are shown. The experiment represented in panel A was repeated five times, that in panel B was repeated three times, and that in panel C was repeated two times. A, Time course is shown of induction of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) mRNA isolated from rat neonatal primary cardiac myocyte cultures grown at 1% or 21% O2, normalized to ß-actin. B, Northern blot analysis shown for VPF/VEGF mRNA from W1 cells grown at 1% or 21% O2 for 24 hours, normalized to ß-actin. C, Northern blot analysis shown for VPF/VEGF mRNA from adult rat ventricular myocytes grown at 1% or 21% O2 for 24 hours, normalized to ß-actin.

Fig 3Down 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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  		Figure 3. Northern blot analysis of amobarbital-treated neonatal cardiac myocytes is shown. This experiment was repeated five times. Cells were treated with amobarbital as described in "Materials and Methods." Time shown refers to the period after the last amobarbital treatment. Vascular endothelial growth factor (VEGF) mRNA is normalized to 18S rRNA.

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 1Up, 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 1Up, 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 4Down, 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 {approx}70 pmol/L.



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  		Figure 4. Bar graph shows results of immunofluorometric assay of conditioned media from neonatal rat cardiac myocytes. Values shown represent the percentage increase of immunoreactive vascular endothelial growth factor (VEGF) in the conditioned media of hypoxic cardiac myocytes compared with parallel cultures of normoxic cardiac myocytes. All samples, once they were concentrated, had measurable levels of VEGF in the assay. Samples were done in duplicate. The experiment was repeated twice with different preparations of myocytes and produced similar results. The concentration of VEGF in the conditioned media of myocytes grown at 1% O2 for 24 hours was 70 pmol/L.

Cardiac Myocyte–Conditioned Medium Stimulates [3H]Thymidine Incorporation Into Microvascular Endothelial Cells
Fig 5Down 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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  		Figure 5. Bar graph shows [3H]thymidine incorporation in microvascular endothelial cells. The assay was performed as described in "Materials and Methods." All values are expressed as percent change from the value obtained when endothelial cells were incubated with fresh medium (containing 5% fetal bovine serum [FBS] for the primary cardiac myocyte experiment or 10% FBS for the W1 experiment). Data shown represent the mean±SEM for four separate experiments comparing 1% O2, 21% O2, and CoCl2, each of which was performed in triplicate. Statistical analysis showed significant differences for 1% O2 compared with 21% O2 (P<.01) and for CoCl2 compared with 21% O2 (P<.01). Data shown for the W1 cell line and rat cardiac myocytes treated with MnCl2 are the means of two separate experiments, each performed in triplicate. bFGF indicates basic fibroblast growth factor; CM, conditioned medium.

Regulation of VPF/VEGF mRNA by Activated Second Messenger Systems
As shown in Fig 6Down (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 6Down, 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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  		Figure 6. Top, Bar graph shows induction of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) mRNA by second messengers under normoxic conditions. All conditions described represent the mean of at least four replicate samples. RNA was harvested 3 hours after the cells were treated with the various agents. VPF/VEGF mRNA was analyzed by RNase protection assay, and the results were normalized to U3 mRNA using a Molecular Dynamics phosphorimager. Phorbol 12-myristate 13-acetate (PMA) was used at 100 ng/mL. Forskolin and dibutyryl-cAMP (Bt2-cAMP) were used at 100 µmol/L. Veratridine was used at 150 µmol/L. Data are expressed as fold induction (mean±SEM) compared with cells grown at 21% O2. *P<.01. Bottom, Northern blot analysis shows induction of VPF/VEGF mRNA expression by veratridine. 18S rRNA is used as control. Veratridine (150 µmol/L) was incubated with the myocytes for 3, 6, or 24 hours.

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 7Down).



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  		Figure 7. Results of RNase protection assay (RPA) demonstrating that H7 inhibits the hypoxic induction of vascular endothelial growth factor (VEGF) mRNA. H7 (20 µmol/L) was added to cells just before the beginning of the hypoxic incubation. This representative RPA was repeated seven times with four different preparations of neonatal cardiac myocytes. Percentage inhibition of the hypoxic induction by H7 (53±7%) was calculated by using the VEGF/U3 ratio as follows: 100-100{[(1% O2+H7) ratio-(21% O2+H7) ratio]/(1% O2 ratio-21% O2 ratio)}.

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 8Down, 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 5Up).



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  		Figure 8. Northern blot analyses of transition metal induction of vascular endothelial growth factor (VEGF) mRNA in primary cardiac myocyte cultures. The experiment was repeated three times with a representative sample shown for the CoCl2 dose response (top) and the MnCl2 dose response (bottom). All mRNA was harvested at 24 hours. VEGF mRNA is normalized to ß-actin or 18S rRNA.

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.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {approx}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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