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(Circulation Research. 1997;81:932-939.)
© 1997 American Heart Association, Inc.

Articles

Vascular Endothelial Growth Factor Increases the Mitogenic Response to Fibroblast Growth Factor-2 in Vascular Smooth Muscle Cells In Vivo via Expression of fms-Like Tyrosine Kinase-1

Leslie L. Couper, Shane R. Bryant, Jens Eldrup-Jørgensen, Carl E. Bredenberg, , Volkhard Lindner

From the Maine Medical Center Research Institute, South Portland, Me.

Correspondence to Volkhard Lindner, MD, PhD, Maine Medical Center Research Institute, Suite 8, 125 John Roberts Rd, South Portland, ME 04106. E-mail lindnv{at}mail.mmc.org

	Abstract

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Abstract Vascular endothelial growth factor (VEGF) has traditionally been considered an endothelial cell–specific factor inducing angiogenesis and vascular permeability in vivo. In the present study, expression of VEGF and its receptors, fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase-1 (flt-1), was examined in rat carotid arteries after balloon injury. Although VEGF and flk-1 were not detectable, high levels of flt-1 mRNA and protein were expressed by smooth muscle cells (SMCs) in the neointima, as demonstrated by en face in situ hybridization and Western blotting. Intimal SMC proliferation in chronically denuded rat carotid arteries was unaffected by intraluminal infusion of VEGF, whereas fibroblast growth factor (FGF)-2 increased the number of replicating SMCs 4-fold. Pretreatment with VEGF doubled the mitogenic response to infused FGF-2 by increasing SMC replication in deeper layers of the intima. VEGF increased the permeability of chronically denuded vessels to plasma proteins but had no effect on the uptake of locally infused biotinylated FGF-2. These findings demonstrate that vascular SMCs express functional flt-1 receptors after arterial injury and that VEGF has synergistic effects with FGF-2 on SMC proliferation. These effects are likely to be mediated by a VEGF-mediated increase in permeability as well as a direct interaction between the VEGF and FGF signaling pathways.

Key Words: intima • vascular endothelial growth factor • fibroblast growth factor • permeability

	Introduction

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Vascular permeability factor (VPF) was originally described as a protein secreted by tumors that caused vascular leakage.¹ The same protein was later cloned independently by several groups and named VEGF^2–4 because of its mitogenic effects on cultured endothelial cells. The protein is made by many cell types, including a variety of tumor cells, folliculostellate cells, epithelial cells of renal glomeruli, and podocytes.^1,5–7 As suggested by the names, VEGF/VPF has at least two functions. First, it is involved and possibly primarily responsible for angiogenesis in wound healing and a variety of pathologies, such as diabetic retinopathy, rheumatoid arthritis, and solid tumor growth.^1,8,9 During embryonic vascular development, VEGF is an essential factor, and mutation of a single allele causes lethality in mouse embryos at days 11 and 12.^10,11 During vasculogenesis in the embryo, expression of VEGF correlates spatially and temporally with endothelial cell proliferation.^12,13 Second, VEGF/VPF is also an important enhancer of endothelial permeability, being 50 000 times more potent than histamine.¹⁴ The persistent expression of VEGF by epithelial cells near fenestrated endothelium has led to the hypothesis that this growth factor is involved in the maintenance of fenestrae in endothelium.¹²

There are two well-described high-affinity receptors for VEGF, flt-1 and flk-1 (KDR).^15,16 Mitogenicity of VEGF has been reported to require the flk-1 receptor.¹⁷ For flt-1, Kendall and Thomas¹⁸ have described a splice variant that gives rise to a soluble and truncated form. This splice variant serves as an endogenous inhibitor of VEGF. Recently, a third receptor binding VEGF₁₆₅ with high affinity has been described.^19,20 These receptors are tyrosine kinases and are thought to be expressed exclusively on endothelial cells, although VEGF effects have also been observed in a variety of other cell types.^21–27 More recently, flt-1 and KDR were also reported on uterine SMCs.²⁸

We have recently studied the role of VEGF in the carotid arteries and aortas of rats and found that migrating and proliferating endothelial cells at the wound edge express high levels of flt-1,²⁹ but no expression of flk-1 was detectable. After VEGF administration, increased permeability was seen at sites of flt-1 expression, but VEGF had no effect on endothelial proliferation. In the present study, we examined the expression of VEGF and its receptors in SMCs after balloon catheter injury and studied the effect of VEGF on SMC proliferation and permeability. In addition, the combination of VEGF and FGF-2 on SMC replication was also examined in this model.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arterial Injury Model
All animal studies were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (400 g, 3 to 4 months old) were purchased from Taconic, Germantown, NY. All surgical procedures were carried out under general anesthesia by intraperitoneal injection of xylazine (2.2 mg/kg AnaSed, Lloyd Laboratories) and ketamine (50 mg/kg body wt Ketaset, Aveco Co, Inc). The left and right common carotid artery and the aorta were denuded with a 2F balloon catheter as recently described.³⁰ In all experiments, deendothelialized segments of arteries were identified by intravenous injection of Evans blue (0.3 mL of 5% solution in saline) 5 minutes before they were killed for study. Animals were perfusion-fixed with phosphate (0.1 mol/L, pH 7.4)–buffered 4% paraformaldehyde for in situ hybridization. For Western and Northern blotting, animals were perfused with ice-cold lactated Ringer's solution to remove blood and plasma proteins; perfusion was followed by excision and removal of adventitial tissue. The vessels were then snap-frozen in liquid nitrogen. Rats were killed at the indicated times after injury.

Growth Factor Infusion Experiments
For all experiments involving growth factor infusions, five groups of rats (n=5 per group) had the left common carotid artery denuded with a 2F balloon catheter via the right femoral artery. Five weeks later, when SMC replication had decreased to levels near those seen in normal vessels, a catheter was inserted into the left external carotid artery with the tip reaching into the common carotid artery. A temporary ligature holding the catheter in position and at the same time interrupting flow was placed just proximal to the carotid bifurcation. In the control group, 150 µL of vehicle containing 0.1% BSA in PBS was infused. The catheter and ligature were removed 15 minutes later. In the VEGF-treated and FGF-2–treated groups, 10 µg human recombinant VEGF (165 amino acid form, kindly provided by Genentech, Inc, South San Francisco, Calif) in 150 µL vehicle or 5 µg human recombinant FGF-2 (kindly provided by Synergen, Boulder, Colo) in 150 µL vehicle was infused in the same manner. In another group of rats, a 15-minute VEGF infusion (10 µg in 150 µL vehicle) was followed by a 15-minute FGF-2 infusion (5 µg in 150 µL vehicle). In an additional group of animals, a 15-minute FGF-2 infusion (5 µg in 150 µL vehicle) was followed by a 15-minute VEGF infusion (10 µg in 150 µL vehicle). All animals were injected subcutaneously with two doses of the thymidine analogue BrdU (6 mg per injection, Boehringer Mannheim) given 26 and 40 hours after the infusion. The rats were killed within 3 hours after the last BrdU injection with an overdose of pentobarbital. Five minutes before killing, Evans blue (0.5 mL of a 5% solution in PBS) was injected into the tail vein. Perfusion/fixation was then performed with phosphate (0.1 mol/L, pH 7.4)–buffered 4% paraformaldehyde.

SMC Replication
After perfusion/fixation, the proximal two thirds of the carotid arteries was excised, leaving behind the distal segment that had been in contact with the infusion catheter. The denuded segment identified by the Evans blue staining was embedded in paraffin and sectioned. The SMC replication indexes in the media and in the intima were determined by staining sections with a mouse monoclonal antibody against BrdU (1:200 dilution, Cappel). Before application of the antibody, the sections were treated with pepsin (0.1 mg/mL in 0.1N HCl) for 30 minutes at 37°C, washed with distilled water, and incubated in 1.5N HCl at 37°C for 15 minutes. The sections were rinsed in distilled water, washed twice for 5 minutes in 0.1 mol/L Borax buffer (sodium tetraborate, pH 8.5), and washed with Tris-buffered saline (0.8% NaCl and 25 mmol/L Tris, pH 7.6) for 5 minutes before application of the anti-BrdU antibody (1 hour at 37°C). After three washes with Tris-buffered saline (5 minutes each), the sections were incubated for 30 minutes at room temperature with biotinylated horse anti-mouse IgG (1:1000 dilution, Vector). Subsequent steps were carried out as previously described.³¹

The number of total and stained nuclei was counted separately for the media and intima, and the BrdU labeling indexes [(stained nuclei/total nuclei)x100] were calculated for both the media and the intima. In addition, the number of BrdU-labeled SMCs present in the two intimal SMC layers closest to the lumen was also counted separately. Four sections spaced 100 µm apart were analyzed per animal.

Western Blotting
Left and right common carotid arteries were harvested from rats at the indicated time points after balloon injury to prepare vessel wall extract samples. The endothelium was removed from uninjured carotid arteries by balloon denudation immediately before snap freezing. Two independent sets of samples were analyzed (two times, 2 rats per time point), and representative immunoblots are shown. The vessels were pulverized in liquid nitrogen using mortar and pestle and resuspended in buffer containing 20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl₂, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1.5 µg/mL pepstatin A, 40 µmol/L calpain inhibitor I (Boehringer Mannheim), 1 mmol/L Na₃VO₄, and 1 mmol/L NaF. A protein extract was generated by freeze-thawing the suspension three times and then removing the insoluble material by centrifugation at 14 000 rpm for 10 minutes at 4°C. The protein concentrations were determined by the Coomassie protein assay (Pierce). Twenty micrograms of extract per lane was loaded onto SDS-polyacrylamide gels (10%), followed by electrophoresis. Prestained molecular weight standards (Bio-Rad) were run on each gel. Proteins were electroblotted onto nitrocellulose (BA 85, Schleicher & Schuell), stained with primary antibodies and a horseradish peroxidase–coupled secondary antibody, and detected with an enhanced chemiluminescence detection system (ECL, Amersham Corp). A polyclonal rabbit antibody against flt-1 (Santa Cruz) was used at a 1:1000 dilution. Bands were visualized on film, and the intensity was quantified by densitometry using a scanner and image analysis software (NIH image 1.60) for Macintosh computers. Equal protein loading of the samples was further verified by evaluation of Coomassie-stained gels.

Vascular Permeability and Uptake of FGF-2
Balloon catheter denudation of the left carotid artery via the femoral artery was performed as described above. Four weeks later, VEGF (10 µg in 150 µL vehicle) was infused locally into the left carotid artery of one group of rats for 15 minutes, followed by infusion of 5 µg biotin-labeled FGF-2 in 150 µL vehicle (EZ-Link NHS-LC-Biotin, Pierce). In a second group of rats, a 15-minute vehicle infusion was followed by a 15-minute infusion of biotinylated FGF-2 (5 µg in 150 µL vehicle). After the infusion catheter had been removed and flow was reestablished, 10 mg of nonimmune rabbit IgG and Evans blue (0.5 mL, 5% in PBS) were injected into the tail vein. Five minutes later, the animals were killed with an overdose of pentobarbital followed by perfusion of 100 mL of lactated Ringer's solution via the abdominal aorta. The perfusate was allowed to drain from the renal vein. The denuded segment of the left carotid (Evans blue–stained) and the right carotid artery were excised and processed for Western blotting as described above. Ten micrograms from each sample was run on 12% SDS-polyacrylamide gels and transferred to nitrocellulose. To quantify the amount of rabbit IgG present in the samples, membranes were incubated with peroxidase-labeled anti-rabbit antibody as described above. The same samples were also run on 15% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes, which were then incubated with the avidin-biotin-peroxidase complex (ABC Elite Kit, Vector) to detect the biotinylated FGF-2. The ECL detection system (Amersham) was used, and the intensity of the bands on the film was quantified by densitometry using a scanner and image analysis software (NIH image 1.60).

Northern Blot Analysis
Common carotid arteries were harvested from unmanipulated rats (normal carotid with endothelium), from rats with carotid arteries denuded with a balloon catheter immediately before killing (normal carotid without endothelium), and from rats at 6 hours and at 3, 7, 14, and 28 days after balloon injury (3 or 4 animals per time point). Vessels were stripped of periadventitial fat and connective tissue in PBS at 4°C and were then snap-frozen in liquid nitrogen. Frozen arterial tissue was ground to a fine powder under liquid nitrogen, and total cellular RNA was prepared by acid guanidinium thiocyanate extraction.³² Agarose gel electrophoresis of RNA (15 µg total RNA per lane) and transfer to nylon membranes (Zeta Probe, Bio-Rad Laboratories) were carried out as previously described.³³ After transfer, RNA blots were exposed to shortwave UV light both to cross-link RNA to the membrane and to visualize the major ribosomal RNA bands. The blot was hybridized using cDNA probes labeled with [³²P]dCTP by random primer extension (Amersham), washed at 65°C in two changes of 0.045 mol/L NaCl/0.0045 mol/L sodium citrate (pH 7.0)/0.1% SDS for 15 minutes each, and then exposed to Kodak X-Ark5 film at -70°C.

In Situ Hybridization and cDNA Probes
In situ hybridization was carried out on en face preparations of vessel segments and on cultured rat aortic SMCs grown on glass slides, as recently described.³⁴ Mouse cDNAs for VEGF (300 bp), flt-1 (extracellular domain, 2200 bp), and flk-1 (1200 bp) were kindly provided by Dr Werner Risau (Max-Planck-Institut, Bad Nauheim, Germany).^12,35 A 972-bp rat cDNA encoding the tyrosine kinase domain of flt-1 was cloned into pCRII (Invitrogen) by RT-PCR using 5'TAAGAAATCACCCACCTG3' and 5'GAGGGGGATGTAGTCTTT3' as primers.³⁶ The identity of the sequence was verified by restriction digest analysis and sequencing. To examine VEGF expression, 25 specimens were hybridized with antisense and 8 with sense probes. Twenty-three specimens were probed with antisense and 9 with sense probes for flk-1. For flt-1 expression, 63 en face preparations were hybridized with the antisense extracellular domain probe and 18 with sense probes. An additional 12 specimens were hybridized with the antisense rat flt-1 tyrosine kinase domain probe. After in situ hybridization, the slides were coated with autoradiographic emulsion (Kodak, NTB2), exposed for 3 to 6 weeks, and then developed (Kodak, D-19). Preparations were observed under the light microscope using dark-field illumination.

Statistics
ANOVA was used to determine whether significant differences between the means of treatment groups were present (P<.05). Multiple comparisons between groups were then performed using Scheffé's test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
VEGF and VEGF Receptor Expression
The technique of in situ hybridization carried out on en face preparations of rat carotid arteries and aortas was used to examine mRNA expression by SMCs present on the luminal surface.³⁴ This method enabled us to examine the entire population of cells on the luminal surface at times of rapid intimal SMC proliferation (5 to 8 days after balloon injury), at times of decreasing replication (14 days), and at times when SMC replication had returned to near normal levels such as seen in uninjured vessels (4 to 6 weeks after denudation).

We were unable to detect expression of VEGF mRNA in SMCs by in situ hybridization in injured rat arteries. Similarly, expression of mRNA for the VEGF receptor flk-1 was not detectable in SMCs at any time after arterial injury.

The murine cDNA encoding the extracellular domain of flt-1 was used to generate a ³⁵S-UTP–labeled antisense probe that showed strong hybridization to proliferating SMCs present on the luminal surface of the intima at 7 days after balloon denudation (Fig 1A). A strong hybridization signal with the same probe was also seen over SMCs at 2 and 4 weeks after injury (Fig 1B and 1C), whereas the sense probe revealed low background hybridization (Fig 1E). In cultured rat aortic SMCs grown from explants, only some cells showed expression of flt-1 mRNA, and compared with the in vivo samples, the hybridization signal was much less intense (Fig 1D). Since soluble forms of the flt-1 receptor lacking the intracellular domain have been reported,¹⁸ we wanted to rule out the possibility that only the soluble form of flt-1 was expressed by these SMCs. The rat cDNA encoding most of the tyrosine kinase domain was cloned by RT-PCR, and the antisense riboprobe was used for en face in situ hybridization. Similar to the extracellular domain probe, high levels of mRNA expression were detected in SMCs at 7 days (Fig 1f) and at 14 and 28 days (data not shown) after balloon catheter denudation. Using the tyrosine kinase domain probe on cultured SMCs, lower levels of flt-1 expression were seen in some SMCs (Fig 1G). Northern blots of total RNA isolated from carotid arteries at various times after injury were hybridized with both flt-1 probes. These blots showed only very low levels of the flt-1 transcript at time points 3 days after injury, whereas the transcript was readily detectable in RNA isolated from rat lung (data not shown).

View larger version (127K): [in this window] [in a new window]   Figure 1. Expression of VEGF receptor flt-1 on en face preparations of rat arteries. A, In situ hybridization of 7-day balloon-injured rat carotid artery hybridized with [35S]UTP-labeled antisense riboprobe of the flt-1 extracellular domain shows high levels of expression in SMCs that have migrated onto the luminal surface. B and C, The same probe shows flt-1 expression in SMCs on the luminal surface of carotid arteries at 2 weeks (B) and 4 weeks (C) after balloon injury. D, Cultured rat SMCs revealed only low levels of hybridization with the labeled antisense extracellular domain probe in some cells. E, The corresponding sense probe showed low levels of background hybridization over SMCs at 7 days after denudation. F and G, When the antisense tyrosine kinase domain probe of flt-1 was used, a strong signal was seen in SMCs from 7-day balloon-injured vessels (F), whereas some cultured SMCs showed only low expression levels (G).

The presence of flt-1 receptor protein in the carotid artery was then examined by Western blot analysis (Fig 2). Samples from normal carotid arteries with endothelium revealed a major band with an approximate molecular mass of 200 kD. Samples with the endothelium removed with a balloon catheter showed a complete loss of this band, indicating that flt-1 is present in endothelial cells but not SMCs of normal vessels. At 3 days after balloon injury (when SMCs in the media are rapidly proliferating), very little flt-1 protein was detectable. Deendothelialized vessels at 7 days after injury showed the reappearance of the 200-kD flt-1 band. At this time point, SMCs are accumulating in the neointima. With a highly cellular intimal lesion present at 2 weeks after denudation, a further increase in expression of this protein was evident. Aortic samples at 7 days after denudation with regenerating endothelium that we have previously reported to express flt-1²⁹ served as a positive control. This sample showed the same flt-1 protein band. In cultured rat SMCs grown from aortic explants, the major flt-1 band was of slightly higher molecular mass. The antibody also recognized one protein with a lower molecular mass in the vessel wall samples.

View larger version (50K): [in this window] [in a new window]   Figure 2. Western blot analysis of extracts from normal rat carotid arteries with endothelium (+EC) and without endothelium (-EC), extracts at various time points after balloon injury, and cultured rat aortic SMCs (RASMC). Equal amounts of protein were loaded in each lane. The blot was stained with an flt-1 antibody with molecular mass markers indicated on the left. The major band with an approximate molecular mass of 200 kD was completely absent from samples of normal vessels without endothelium. Note the increasing expression of flt-1 at 7 and 14 days after balloon injury in the absence of endothelium.

VEGF and SMC Proliferation
After it was established that SMCs in the intima express flt-1 receptors after balloon injury, we examined whether VEGF would stimulate SMC proliferation. Rat carotid arteries were denuded with a balloon catheter via the femoral artery. Four weeks later (when spontaneous SMC proliferation in the neointima had returned to very low levels), the left carotid artery was exposed to 10 µg of VEGF for 15 minutes by intraluminal infusion with temporary interruption of flow. Labeling of replicating SMCs was achieved by BrdU injection at 26 and 40 hours after the growth factor infusion. Compared with vehicle infusion in the control group, VEGF infusion had no significant effect on the intimal SMC replication index (Fig 3A). In a separate group of animals, 5 µg of FGF-2 was then infused in an identical manner, and the replication index increased from 4.5±1.2% (mean±SEM) in the control group to 14.8±3.0% (mean±SEM) in the group with FGF-2 treatment. However, this was not a statistically significant difference, although the absolute number of BrdU-labeled intimal SMCs was significantly increased (Fig 3B).

View larger version (13K): [in this window] [in a new window]   Figure 3. Intimal SMC proliferation in denuded rat carotid arteries in response to intraluminal infusion of VEGF and FGF-2. Groups of 5 rats received either vehicle, 10 µg VEGF, 5 µg FGF-2, 10 µg VEGF followed by 5 µg FGF-2 (VEGF+FGF-2), or 5 µg FGF-2 followed by 10 µg VEGF (FGF-2+VEGF). Horizontal bars with asterisk indicate significant difference (*P<.05) between groups. Data represent mean±SEM. A, Intimal SMC replication index determined by BrdU staining. Note that pretreatment with VEGF increased the FGF-2 response 2-fold. B, Total number of BrdU-labeled SMCs in the intima. C, Number of BrdU-labeled SMCs in deeper layers of the intima.

SMCs on the luminal surface of chronically denuded arteries are known to form a "pseudoendothelium" that has cobblestone appearance, as assessed by scanning electron microscopy.³⁷ It is possible that the presence of this pseudoendothelium represents a permeability barrier limiting the availability of infused FGF-2 to those SMCs closer to the luminal surface. We had previously shown that endothelial cells expressing high levels of flt-1 receptors respond to VEGF with an increase in permeability. These considerations prompted us to investigate the effect of FGF-2 infusion after pretreatment with VEGF. The 15-minute intraluminal infusion of VEGF was therefore followed by infusion of FGF-2 for 15 minutes. This treatment led to a significant increase in intimal SMC replication (27.9±2.7%) compared with vehicle, VEGF, and FGF-2 treatments alone (Fig 3A). This replication index was nearly twice as high as the one achieved with FGF-2 alone. To further determine whether this synergistic effect of VEGF was dependent on pretreatment with VEGF, we reversed the order of growth factor infusion. A 15-minute intraluminal infusion of FGF-2 followed by a 15-minute infusion of VEGF also led to an increase in both the BrdU index and the total number of BrdU-labeled intimal SMCs compared with infusion with FGF-2 alone (Fig 3A and 3B). However, this increase was not statistically significant.

If the synergistic effect of VEGF and FGF-2 on SMC proliferation was the result of increased availability of FGF-2 to SMCs in deeper layers of the intima, then one would expect to see more BrdU-labeled SMCs in deeper layers of the intimal lesion. The total number of BrdU-positive intimal SMCs in these deeper layers, ie, those below the two most luminal layers, was therefore quantified (Fig 3C). The group that had received FGF-2 after pretreatment with VEGF had significantly more labeled intimal SMCs in deeper layers compared with the group infused with FGF-2 alone. No significant increase in the response to FGF-2 was seen, however, when VEGF was administered after the FGF-2 infusion (Fig 3C).

VEGF and Permeability in Chronically Denuded Vessels
To address the issue of vascular permeability in a more direct way, we performed balloon catheter denudation of the left carotid artery via the femoral artery, and 4 weeks later, we infused VEGF as described above. Fifteen minutes later, 5 µg of biotinylated FGF-2 was infused for 15 minutes, followed by intravenous injection of nonimmune rabbit IgG (10 mg). The rabbit IgG was allowed to circulate for 5 minutes before the denuded left carotid artery and the unmanipulated right carotid artery were harvested for Western blot analysis and quantification by densitometry. In control animals, infusion of biotinylated FGF-2 was preceded by a 15-minute vehicle infusion. A representative Western blot for 2 animals of each group is shown in Fig 4. Vessels treated with VEGF contained an increased amount of rabbit IgG (2-fold). Interestingly, the contralateral normal carotid artery of VEGF-treated rats also contained more rabbit IgG.

View larger version (55K): [in this window] [in a new window]   Figure 4. Influx of immunoglobulin into denuded rat carotid arteries in response to VEGF stimulation. Four weeks after balloon catheter injury of the left carotid artery, 10 µg VEGF or vehicle was infused into the vessel for 15 minutes. Fifteen minutes after the infusion, rabbit IgG was injected and was allowed to circulate for 5 minutes. For each animal, extracts from the normal right carotid artery and the denuded left carotid artery were analyzed by Western blotting with an antibody against rabbit IgG. Representative samples from 2 rats infused with VEGF and 2 rats infused with vehicle are shown. Note that both normal and denuded carotid arteries contained more rabbit IgG in VEGF-treated rats.

VEGF and Uptake of Infused FGF-2 by the Vessel Wall
If VEGF was affecting vascular permeability in the denuded vessels, then there was the possibility that a larger percentage of the infused FGF-2 would become bound to binding sites within the vessel wall, leading to an increase in subsequent SMC proliferation.³⁸ Using the same samples obtained for the permeability studies with rabbit IgG, we therefore quantified the amount of infused biotinylated FGF-2 present in the carotid artery samples from rats treated with VEGF or vehicle. The amount of labeled FGF-2 present in the denuded left carotid artery was very similar in VEGF- or vehicle-treated rats (Fig 5), indicating that VEGF did not increase the uptake of FGF-2. The amount of biotinylated FGF-2 present in the contralateral normal carotid artery was much smaller and required longer exposure times to be detectable.

View larger version (52K): [in this window] [in a new window]   Figure 5. Uptake of biotinylated FGF-2 in the denuded rat carotid artery with and without pretreatment of VEGF (10 µg). Four weeks after balloon catheter injury of the left carotid artery, 10 µg VEGF or vehicle was infused into the vessel for 15 minutes, followed by a 15-minute infusion of biotinylated FGF-2 (5 µg). The labeled FGF-2 present in the denuded left carotid artery and in the contralateral normal (right) carotid artery was detected by Western blotting using an avidin-biotin-peroxidase reaction. Representative samples from 2 rats pretreated with VEGF and 2 rats pretreated with vehicle are shown, with no obvious difference in the content of labeled FGF-2. Note that the normal right carotid artery contained much less labeled FGF-2.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
VEGF/VPF has been considered an endothelium-specific factor that induces angiogenesis and microvascular hyperpermeability.^1–4 More recently, VEGF receptors have been found to be expressed by a number of different cell types in vitro, including pericytes,²² pancreatic duct cells,²⁶ islets of Langerhans,²⁷ monocytes,²¹ uterine SMCs,²⁸ retinal pigment epithelial cells,²⁵ and osteoblasts.²⁴ With the exception of embryonic development, reports of VEGF receptor expression in vivo on cell types other than endothelial cells have not been as convincing, and as a consequence, functional in vivo data are restricted to endothelial cells. In a recent publication,²⁹ we have demonstrated that endothelial wounding in the rat aorta and carotid artery induces high-level expression of flt-1 in the migrating and proliferating endothelial cells along the wound edge. These cells with high levels of flt-1 expression responded to infused VEGF with an increase in permeability but not proliferation. In the present study, we used the same rat arterial injury model and demonstrated the presence of flt-1 mRNA and protein in vascular SMCs. Our data indicate that in normal vessels flt-1 is present only in the endothelium, since Western blot analysis showed a complete loss of the flt-1 band in vessels from which the endothelium had been removed completely by balloon catheterization 1.5 hours before harvest. It was interesting to note that significant amounts of flt-1 immunoreactivity were not seen in the vessel wall until 7 days after balloon injury, when SMCs accumulate on the luminal surface in the neointima. It should be pointed out that only the deendothelialized portion of the vessels was examined; thus, we can rule out endothelium as a source of the flt-1 in the samples. The data indicate further that flt-1 expression in SMCs does not correlate solely with proliferation, since at 3 days after injury the SMC replication index in the media is high and SMCs are still absent from the intima,³⁹ yet the immunoblot showed very little protein at that time. En face in situ hybridization revealed high levels of flt-1 mRNA expression in SMCs on the luminal surface, but no flt-1 mRNA expression was detectable in SMCs deeper in the vessel wall by in situ hybridization performed on cross sections (data not shown). Northern blot analysis of RNA from the entire vessel wall revealed only very low levels of the flt-1 transcript at time points when an intimal lesion was present. Together, these findings indicate that flt-1 expression might be limited to SMCs present on the luminal surface, and this raises the question whether the exposure to blood flow and shear stress is playing a role in flt-1 induction.

In the present study, we used a local delivery method to investigate the functions of VEGF and FGF-2 in carotid arteries. There are several advantages to this approach. Only relatively small amounts of growth factors are necessary to achieve sufficient local concentrations that can be maintained for extended periods of time. There is no mechanical trauma to the vessel, with the exception of the area at the bifurcation, which was excluded from our analyses. Our data demonstrate that intimal SMCs are capable of responding to FGF-2 in the absence of trauma, as shown by the 4-fold increase in the number of replicating SMCs. This result indicates that in the presence of plasma proteins FGF-2 is sufficient to induce SMC replication and that mechanical trauma and platelet deposition are not required for this process. Our data disagree with those recently published by Koyama and Reidy,⁴⁰ who reported that intimal SMCs are not stimulated by exogenous FGF-2. Compared with our studies, the authors administered a 12-fold larger dose of FGF-2 given by intravenous injection and used a longer BrdU labeling period to measure SMC replication. Considering the rapid clearance of systemically injected FGF-2,^38,41 the most likely explanation for the differing results is that local concentrations of FGF-2 achieved by systemic intravenous injection are insufficient to stimulate intimal SMC proliferation, especially since the pseudoendothelium formed by SMCs could further limit the availability of the mitogen.

The mitogenic effect of VEGF has been shown to require the flk-1 receptor,¹⁷ and it was therefore not surprising that VEGF alone had no effect on SMC proliferation, since no flk-1 expression was detectable in these cells. It was unexpected that VEGF increased the mitogenic response to a subsequent infusion of FGF-2 2-fold. However, when FGF-2 was administered before VEGF, the synergistic effect was less pronounced compared with infusion of FGF-2 alone. One explanation for this finding is that VEGF enhances the permeability of the vessel wall, thereby increasing the availability of FGF-2 to SMCs deeper in the intima. The elevated levels of rabbit IgG in the vessel wall of VEGF-treated arteries and the increased number of replicating SMCs in deeper layers of the intima would support this explanation. The studies using the biotinylated FGF-2, on the other hand, indicate that the total amount of infused FGF-2 present in the arterial wall was similar. Unfortunately, attempts to explore the spatial distribution of the infused biotinylated FGF-2 by immunohistochemistry on cross sections did not lead to conclusive results. If the number of BrdU-positive SMCs in deeper layers of the intima is compared with the total number of replicating intimal SMCs, then it becomes evident that the combined treatment of VEGF plus FGF-2 also gives rise to the highest number of replicating SMCs in the remainder of the vessel wall, ie, the surface layer. This can probably not be explained simply by an effect of VEGF on permeability. Instead, this would argue for synergy of the signaling pathways via flt-1 and FGF receptor-1, which is the dominant FGF receptor on SMCs.³⁴ We have observed a similar synergistic effect of VEGF and FGF-2 in vitro in one rat aortic SMC line expressing low levels of flt-1 (data not shown). Another example for VEGF and FGF-2 interaction has been reported by Guerrin et al.²⁵ Guerrin et al have demonstrated that VEGF is an autocrine growth factor for retinal pigment epithelial cells, and overexpression of VEGF in these cells caused an increase in the mitogenic response to FGF-2, suggesting cross regulation of VEGF and FGF signal transduction pathways.²⁵

There is also the possibility that the synergistic effects of VEGF and FGF-2 on SMC proliferation are indirect effects mediated by soluble factors released by VEGF. As an example, a recent report demonstrated that the mitogenic effect of VEGF on coronary venular endothelium is mediated by nitric oxide.⁴² It is unlikely, however, that nitric oxide is responsible for the increased mitogenic response to FGF-2, since it is a well-known inhibitor of SMC proliferation.⁴³ The possibility that VEGF releases other soluble factors affecting FGF-2–stimulated SMC proliferation still exists. This would be supported by the fact that we were unable to detect expression of flt-1 mRNA in deeper layers of the intima using in situ hybridization on cross sections but that an increase in FGF-2–induced SMC proliferation in this location was seen after VEGF administration.

In summary, the present study demonstrates that vascular SMCs express functional flt-1 receptors after arterial injury. Furthermore, VEGF has synergistic effects with FGF-2 on SMC proliferation in vivo. These effects are likely to be mediated by a VEGF-mediated increase in permeability as well as a direct interaction between the VEGF and FGF signaling pathways.

	Selected Abbreviations and Acronyms

 

BrdU = 5-bromo-2'-deoxyuridine FGF = fibroblast growth factor flk-1 = fetal liver kinase-1 flt-1 = fms-like tyrosine kinase-1 RT-PCR = reverse-transcriptase polymerase chain reaction SMC = smooth muscle cell VEGF = vascular endothelial growth factor VPF = vascular permeability factor

	Acknowledgments

 
This study was supported by a Grant-in-Aid from the American Heart Association and with funds contributed by the American Heart Association, Maine Affiliate, Inc (Dr Lindner).

Received June 20, 1997; accepted September 5, 1997.

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