Expression of VEGF Receptors in Arteries After Endothelial Injury and Lack of Increased Endothelial Regrowth in Response to VEGF
Vascular endothelial growth factor (VEGF) is an endothelial cell–specific factor with angiogenic effects in vivo and mitogenic effects in vitro. Administration of VEGF has been reported to stimulate endothelial growth in denuded arteries and new blood vessel formation in models of induced tissue ischemia. In the present study, expression of VEGF and its receptors flk-1 and flt-1 was determined in injured aortas and carotid arteries of rats and mice. Neither VEGF nor flk-1 mRNA was detectable in vascular cells. mRNA levels for flt-1 were dramatically upregulated at the leading edge of a growing endothelial monolayer in vivo; however, these cells did not demonstrate increased replication after VEGF infusion. Furthermore, all doses and treatment protocols of VEGF failed to promote reendothelialization in denuded arteries. At sites of flt-1 expression, VEGF increased permeability. These areas revealed a loss of endothelial contacts at the ultrastructural level. These findings suggest that VEGF is not a direct mitogen for large-vessel endothelium in vivo and that VEGF may play a role in abolishing contact inhibition, which may be a prerequisite for endothelial proliferation.
- Received March 29, 1996.
- Revision received June 18, 1996.
VEGF,1 2 3 also known as vascular permeability factor,4 has been shown to be an EC-specific mitogen in vitro3 and angiogenic in vivo.5 6 Another property of this factor is its ability to increase vascular permeability.7 Two receptors, flt-1 and flk-1 (the latter of which is the mouse homologue of human KDR), have been shown to bind VEGF with high affinity.8 9 10 Expression of flt-1 by ECs has been documented during mouse development as well as in adult tissue,9 11 and binding sites for radiolabeled VEGF colocalized with factor VIII–like immunoreactivity in rat vessels.12
The ability to stimulate new blood vessel formation and endothelial growth has prompted the use of VEGF as a therapeutic agent in models of decreased limb perfusion13 14 and reendothelialization of large vessels.15 Takeshita and coworkers13 16 reported increased perfusion and blood vessel formation in the rabbit hindlimb in response to VEGF injection after ligation of the femoral artery. Furthermore, an increase in endothelial regrowth and a reduction in intimal lesion formation in the balloon-injured carotid arteries of rabbits and rats have been recently reported after administration of VEGF.15 17
In contrast to these reports, we and others have been unable to elicit a mitogenic response in either ECs in vitro or bovine aortic ECs in vivo (unpublished data). These findings led us to study the expression of VEGF and its receptors flt-1 and flk-1 in the injured aorta and carotid arteries of rats and mice. Furthermore, we tested the effects of recombinant VEGF on endothelial replication and regrowth with a variety of doses and different treatment protocols. In the absence of any stimulatory effect on endothelial growth, we present evidence that infused VEGF increases endothelial permeability at sites of flt-1 expression.
A total of 107 male Sprague-Dawley rats (weight, 320 to 400 g; 3 to 4 months old) and 20 FVB mice (30 g) were used in the studies (Bantin & Kingman, Edmonds, Wash). All surgical procedures were performed under general anesthesia by intraperitoneal injection of xylazine (2.2 mg/kg body weight; AnaSed, Lloyd Laboratories) and ketamine (50 mg/kg body weight; Ketaset, Aveco Co, Inc). Animals were preanesthetized by ether injection into the tail vein. In mice, the aorta was partially denuded with a flexible wire as described.18 In rats, the left carotid artery and the aorta were denuded with a 2F balloon catheter as described.19 For studying the wounded endothelium at early time points (up to 48 hours after denudation), the aortic endothelium was only partially denuded by passing an uninflated balloon catheter along the aorta. Deendothelialized segments of arteries were identified by intravenous injection of Evans blue dye (0.3 mL of a 5% solution in saline) 10 minutes before the animals were killed. Animals were perfusion-fixed with phosphate-buffered (0.1 mol/L, pH 7.4) 4% p-formaldehyde. For in situ hybridization studies, the animals were killed at the indicated times after injury (between 24 hours and 6 weeks). All animal studies were performed in accordance with institutional guidelines and were approved by the animal care committee at the University of Washington.
Human recombinant VEGF (165–amino acid form) for all experiments was kindly provided by Dr Stuart Bunting (Genentech, Inc).
In Situ Hybridization and Northern Blotting
In situ hybridization was carried out on en face preparations of vessel segments as recently described.20 Mouse cDNAs for VEGF (300 bp), flt-1 (extracellular domain, 2200 bp), and flk-1 (1200 bp) were kindly provided by Dr Werner Risau (Bad Nauheim, Germany).21 22 For VEGF, 29 specimens were hybridized with antisense and 8 with sense probes. For flk-1, 30 specimens were probed with antisense and 10 with sense probes. For flt-1, 63 en face preparations were hybridized with antisense and 18 with sense probes. 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 by dark-field light microscopy. Northern blot analysis was performed on RNA isolated from rat carotid arteries at various times after balloon catheter injury.23
Stimulation of Endothelial Replication
The purpose of the following experiment was to test whether VEGF could restimulate EC replication at a wound edge after these cells became quiescent. Groups of rats had their left carotid arteries denuded by balloon catheterization as described.19 Six weeks later, when endothelial regrowth from the carotid bifurcation and aortic arch had slowed dramatically, doses of 10, 100, and 500 μg VEGF were tested for their ability to restimulate endothelial replication at the wound edge. The growth factor was diluted in saline and infused at a constant rate into the aortic arch via the right axillary artery during a 6-hour period. Control animals received infusions of the saline vehicle in an identical fashion. At 30, 38, and 46 hours after the start of the infusion, all animals were injected with [3H]thymidine (50 μCi/100 g body weight, 6.7 mCi·mmol−1·L−1; New England Nuclear). After injection of Evans blue dye (5% in 0.5 mL saline), all rats were killed and perfusion-fixed 47 hours after the start of growth factor treatment. Tissues of proximal and distal carotid arteries that contained the EC–smooth muscle cell interfaces (leading edge of the endothelium) were processed for en face preparation according to the procedure described by Schwartz and Benditt.24 The endothelium was identified by en face immunocytochemical staining with an antibody to factor VIII–related antigen as described.25 For autoradiography, the preparations were coated with photographic emulsion (Kodak NTB2) and exposed for 2 weeks. The slides were then developed, stained with hematoxylin, and mounted under coverslips. For measuring EC proliferation (ie, [3H]thymidine index), the numbers of all ECs and of labeled ECs were counted microscopically (with a square reticule in a 10× eyepiece and a 40× objective). Quantitation of replicating ECs was restricted to the area within 1 mm of the interface with the luminal smooth muscle cells, equivalent to four reticule fields deep into the endothelial monolayer.
In another experiment we tested whether VEGF could accelerate the replication of actively proliferating endothelium. This was done by injecting VEGF 1 mg IV into rats that had had their entire thoracic aortas deendothelialized by balloon catheter denudation 7 days before injection. Pulse labeling of replicating cells was performed 23 hours after VEGF administration by injection of the thymidine analogue BrdU (25 mg/kg body weight IP; Boehringer Mannheim) 1 hour before the animals were killed. Immunostaining of en face preparations with an antibody against BrdU was carried out as described.20 Quantitation of replication along the lateral leading edges of regenerating endothelium from intercostal arteries was carried out by counting the numbers of labeled and total ECs that were within 0.25 mm of the leading edge. This procedure was performed along a 10-mm segment of thoracic aorta, and replication was expressed as the percent BrdU replication index (labeled cells divided by total cells×100). Control animals that had received the saline vehicle were treated in an identical fashion. Segments of thoracic aorta from VEGF and control groups were also processed for transmission electron microscopy as described below.
The purpose of the experiments described below was to determine whether administration of VEGF after balloon denudation would lead to an increase in reendothelialized area.
Groups of rats had the endothelium of the left common carotid artery removed by balloon catheterization as previously described.19 One group of rats received three intravenous injections of VEGF per week (10 μg per injection) starting 2 weeks after denudation of the carotid artery, and control animals received vehicle alone (10 mmol/L sodium citrate and 126 mmol/L NaCl, pH 6.0) in an identical way. Injections were given for a total of 6 weeks. The animals were killed 8 weeks after injury and the tissue processed as described below.
In another experiment VEGF was delivered by continuous infusion into the jugular vein. Osmotic infusion pumps (Alzet 2 ml2, Alza Corp) were implanted subcutaneously 2 weeks after balloon catheter denudation of the left carotid artery. VEGF was delivered for 2 weeks at a rate of 2.5 μg/h, and control animals received vehicle only in an identical manner. The animals were killed 4 weeks after balloon catheter denudation (2 weeks of VEGF or vehicle infusion). Endothelialized areas were identified by injection of Evans blue dye. Endothelial outgrowth from the carotid bifurcation and the aortic arch was measured macroscopically with a ruler.
In an additional experiment we investigated whether a bolus injection of VEGF given at the time of denudation would result in increased regrowth of endothelium 8 days later. Two groups of rats had the entire thoracic aorta denuded of endothelium with a balloon catheter, and 1 mg VEGF or vehicle was infused into the aortic arch via the left carotid artery over a 3-minute period. After 8 days the rats were injected with Evans blue dye and perfusion-fixed with 4% p-formaldehyde, and the entire thoracic aorta was excised. The vessels were cut open longitudinally on the ventral side, mounted under a coverslip, and photographed. The photographs were digitized and the images analyzed with a computer (NIH Image 1.55). The total surface area and the endothelialized area (in intercostal arteries) of a 4-cm-long segment of the thoracic aorta was measured, and reendothelialization was expressed as a percentage of total surface area.
Transmission Electron Microscopy
Aortic segments from rats that had received 1 mg VEGF or vehicle 24 hours before they were killed (8 days after aortic denudation) were immersion-fixed in phosphate-buffered glutaraldehyde/p-formaldehyde (2%:2%, vol/vol). Further tissue processing for transmission electron microscopy was carried out as described.26 Thin sections cut perpendicular to the leading edge of endothelium were examined by electron microscopy.
Student's t test was used to compare treatment and control groups. ANOVA followed by Scheffe's F test was used to compare cell replication data for the animals treated with vehicle or 10, 100, or 500 μg VEGF (Fig 2⇓). Data were considered significantly different if P<.05.
Expression of VEGF and VEGF Receptors
Recently we developed a technique for in situ hybridization of en face preparations of rat arteries,20 which enabled us to examine mRNA expression in the entire cell population on the luminal surface. Wounding of the aortic and carotid endothelium was achieved by partial denudation with an uninflated balloon catheter, which allowed analysis of gene expression at various times after denudation.
We were unable to detect expression of VEGF mRNA in ECs or smooth muscle cells by in situ hybridization in injured rat arteries. Northern blot analysis of total RNA isolated from rat carotid arteries at various times after balloon injury also failed to show any transcripts for VEGF (data not shown). Similarly, expression of VEGF receptor flk-1 mRNA was not detectable at any time after injury in rat arteries, by neither in situ hybridization nor Northern blotting. The possibility that the absence of hybridization of mouse VEGF and flk-1 antisense probes was due to insufficient cross-hybridization with rat mRNA was ruled out by applying the same probes to injured mouse aortas, which also showed no hybridization (data not shown).
En face preparations of normal endothelium from the uninjured aorta and carotid artery showed either no hybridization or only low levels of hybridization with the radiolabeled antisense probe for flt-1 (Fig 1a⇓). Within 24 hours after wounding, however, high levels of flt-1 mRNA were expressed by those ECs near the wound edge (Fig 1b⇓). mRNA for flt-1 continued to be expressed by leading-edge endothelium undergoing replication and migration at 2 and 8 days after denudation (Fig 1c⇓). Recently regenerated endothelium away from the leading edge no longer expressed flt-1 mRNA (Fig 1d⇓). Specimens probed with the corresponding sense probe of flt-1 showed no hybridization (Fig 1e⇓).
Effect of VEGF on Endothelial Replication
After it was established that injured endothelium expressed receptors for VEGF, we tested whether VEGF administration would stimulate endothelial replication. These experiments were performed on the rat carotid artery, since endothelial regeneration in this vessel is incomplete and usually stops within 6 weeks after balloon catheter denudation. The ability of VEGF to restimulate the leading edge of the endothelial sheet was studied in groups of rats 6 weeks after balloon denudation of the carotid artery. None of the three different doses of VEGF (10, 100, and 500 μg) that were infused intra-arterially over 6 hours caused a significant increase in EC replication near the leading edge (Fig 2⇓).
We also tested whether infused VEGF could increase proliferation in the endothelium that was still undergoing replication. For this experiment, groups of rats had their thoracic aortas denuded of endothelium 7 days before administration of 1 mg VEGF. Cell replication in the aortic endothelium was determined 24 hours later by pulse labeling with BrdU. Compared with control animals, rats injected with VEGF showed no increase in endothelial replication (Fig 3⇓).
VEGF and Endothelial Outgrowth
In subsequent experiments, we tested whether repeated injections of VEGF could promote endothelial outgrowth onto the denuded surface. In one experiment, 10-μg injections of VEGF were given three times per week for 6 weeks. The treatment period was started when spontaneous endothelial regeneration began to decrease, ie, 2 weeks after denudation. When total endothelial regrowth from the carotid bifurcation and aortic arch was measured 8 weeks after injury, no significant difference was found between animals treated with vehicle or those treated with VEGF (Fig 4⇓). To confirm this result, VEGF was also administered by continuous intravenous infusion with osmotic pumps. VEGF infusion into these animals was started 2 weeks after denudation, and endothelial outgrowth was measured 2 weeks later. This regimen also failed to stimulate endothelial regrowth in denuded vessels (Fig 5⇓).
In an additional experiment, we determined whether a 1-mg VEGF injection administered via the carotid artery at the time of aortic denudation would lead to increased reendothelialization 8 days later. The percentage of reendothelialized surface area in the thoracic aorta was not significantly different between control rats and rats that had received VEGF (Fig 6a and 6b⇓⇓).
VEGF and Vascular Permeability
Aortas obtained from rats injected with 1 mg VEGF 24 hours before they were killed (8 days after denudation) revealed diffusely increased Evans blue staining in areas close to the leading edge where flt-1 expression was known to occur (Fig 7a and 7b⇓⇓). The aorta from the animal in Fig 7a⇓ revealed “speckled” blue staining near the wound edge. In another animal, vascular permeability after VEGF infusion had increased so dramatically that the endothelialized areas in the periphery of intercostal arteries were no longer able to exclude the Evans blue dye–albumin complex. This lack of exclusion led to an apparent reduction in white areas and also caused their borders to appear irregular and poorly defined (Fig 7b⇓). Control animals injected with vehicle, however, always showed a sharp demarcation between white areas of regenerated endothelium and blue areas of denuded surface (Fig 7c and 7d⇓⇓). Since these findings were suggestive of increased endothelial permeability at these sites, we carried out an ultrastructural analysis with transmission electron microscopy. Sections through the leading-edge endothelium showed gaps between ECs in VEGF-treated animals (Fig 8a⇓). Junctions between ECs in vehicle-treated rats, however, appeared normal (Fig 8b⇓).
Recent studies have suggested that VEGF stimulates not only angiogenesis but also endothelial regrowth in denuded arteries.13 14 15 17 27 28 These data are important, since VEGF, unlike other factors such as FGF-2 (bFGF), stimulates only endothelial and no other cell type in the artery wall. Thus, VEGF could be used to selectively stimulate EC growth in vivo without inducing unwanted cell proliferation in blood vessels; for this reason, VEGF is now being used in clinical trials for therapeutic angiogenesis.
In previous studies on EC regrowth in vivo, we observed that even potent mitogenic factors have no effect on intact, confluent endothelium but are able to stimulate those cells adjacent to a wound edge.25 This variation in response may be explained by contact inhibition; ie, cells at the wound are released from an inhibitory signal. In addition, in vivo only those cells that adjoin the wound express the relevant receptors that allow them to respond to the mitogenic signal. For example, in an injured artery only those ECs that adjoin the wound and are not within the intact monolayer strongly express the receptor for FGF-1,20 and administration of FGF-2 stimulates just these cells at the wound edge and not those within the intact monolayer. The same is true for urokinase-type plasminogen activator receptor, which is important in allowing ECs to migrate.29 Only those cells that directly abut the wound express mRNA for this receptor,29 which presumably then allows such cells to migrate into the denuded area.
On the basis of the aforementioned findings, we undertook to determine whether ECs in an injured artery express VEGF receptors in a similar manner. Our in situ hybridization data showed that neither VEGF ligand nor the receptor flk-1 was expressed by the endothelium in proximity or at a distance from the wound (data not shown) but that ECs adjacent to the wound did express flt-1 mRNA. This pattern of flt-1 expression predicts that only ECs adjacent to the wound edge will respond to VEGF. We were therefore surprised that in all of our studies, VEGF did not stimulate EC replication either at the wound edge or within the intact monolayer. None of the doses of VEGF infused over 6 hours (10, 100, or 500 μg) caused a significant increase in replication. In addition, in a separate experiment, one injection of 1 mg VEGF was given to the animals 8 days after injury; likewise, no increase in replication was observed. We know that rat endothelium can be stimulated under these conditions, since infusion of FGF-2 was able to stimulate endothelial replication at the wound edge by ≈20-fold.25
An alternative approach to measuring cell replication is based on whether administration of growth factor can affect the absolute amount of endothelial outgrowth. Therefore, we addressed this issue by measuring endothelial outgrowth after administering VEGF for a prolonged period. Continuous VEGF infusion for 2 weeks had no effect on total endothelial outgrowth, and in an additional study in which VEGF was given for 6 weeks, no increase in endothelial outgrowth was detected. Finally, because one large dose of VEGF given immediately after balloon injury has been reported to stimulate EC regrowth in rats,15 we evaluated the effect of one VEGF injection. In this study, one 1-mg injection of VEGF was given immediately after aortic balloon catheter denudation, and EC regrowth in the intercostal ostia was measured after 8 days. No significant changes were observed in either cell replication rate at the wound edge over 24 hours (data not shown) or area repopulated by endothelial outgrowth after 8 days. It should be pointed out that in this study, we opted to examine regrowth in the aorta, although there are multiple sites for endothelial regrowth. In addition, early after injury quantitation of cell outgrowth at these sites is highly reproducible and possibly more sensitive than in the carotid artery.
We conclude that VEGF is not a direct mitogen for large-vessel endothelium and is unable to stimulate EC outgrowth in denuded rat arteries. These data do not agree with those of Asahara et al,15 who showed that one dose of VEGF given immediately after injury caused a significant increase in EC regrowth over 14 days. The dose and route of administration of any mitogen are problematic, since parameters such as pharmacokinetics and local availability are not well understood. For these reasons we tried four different doses via the most direct intra-arterial route (10, 100, and 500 μg and 1 mg) and in three different ways (multiple intravenous injections for 6 weeks, continuous infusion for 2 weeks, and one dose immediately after surgery) for VEGF administration. Despite these efforts, we were unable to influence the rate of EC regrowth. VEGF, however, is considered to be angiogenic, and one important issue is that our study focused on EC replication and regrowth in large arteries and not angiogenesis per se. Not all factors that are angiogenic stimulate endothelial regrowth, and one conclusion is that VEGF may belong to this category of growth factors. The possibility that the VEGF used in this study was biologically inactive can be discounted, because there was a response to this factor in the rat endothelium permeability assay. Reidy and Schwartz30 studied the effects of endotoxin in rat aortic endothelium in vivo and found that endotoxin infusion actually increased endothelial replication. We think that effects due to potential endotoxin contamination can therefore be ruled out.
One possible explanation for our results comes from the observations of Keyt and coworkers,31 who recently found that there are different determinants on VEGF for binding to its flk-1 and flt-1 receptors. These authors demonstrated that VEGF binding to the flt-1 receptor was unrelated to mitogenesis induction and proliferation.31 Binding to the flk-1 receptor, however, exhibited full activity with respect to EC proliferation. These data are supported by our findings, in that we detected flt-1 and not flk-1 expression and that no cell replication was observed after VEGF administration.
An interesting finding of the present study was that endothelium near the leading edge showed a speckled pattern of Evans blue dye staining 24 hours after VEGF injection. Normally after in vivo administration, Evans blue dye binds to albumin and acts as a permeability marker, so that those zones without endothelium stain blue and those with endothelium remain white. The patchy blue staining over leading-edge endothelium could mean either that these areas were only partially covered by endothelium or that the endothelium was “leaky.” We believe that the first possibility can be excluded, since the pattern and extent of endothelialized zones were identical in VEGF- and vehicle-treated animals (Fig 6⇑). VEGF has been well documented to cause changes in permeability and indeed was first recognized by Dvorak's laboratory3 7 32 33 as a stimulator of permeability (hence the name vascular permeability factor). Electron microscopy of the endothelium showed gaps between ECs in these areas, which were not observed in control animals. Thus, we believe that the blue zones in intact, regrown endothelium represent sites of increased permeability due to disruption of endothelial junctions. Release from contact inhibition may be a requirement of ECs before they can respond to a mitogenic stimulus via cell division, and one possibility is that VEGF binding to its flt-1 receptor plays a role in releasing these cells from contact inhibition. This hypothesis is supported by our earlier findings obtained from in vivo studies with FGF-2,25 wherein significant and widespread increases in endothelial replication after FGF-2 infusion were seen only along the leading edges of endothelial sheets and not within the confluent, quiescent monolayers away from the leading edge. One explanation may be that cells at the leading edge are not contact inhibited, and ECs that abut the wound edge are known to have poorly formed junctions.34 35 These results may be summarized in a unifying hypothesis: VEGF is important in the disruption of contact inhibition, which in turn may be a prerequisite for other endothelial mitogens to induce cell proliferation. The fact that permeability was seen only in VEGF-treated animals demonstrates that the VEGF was biologically active and that the lack of mitogenicity was not due to infusion of inactive VEGF.
In summary, we report that VEGF receptor flt-1 is markedly upregulated in the endothelium near the wound edge but that VEGF infusion failed to increase endothelial replication at these sites. Colocalization of receptor expression and increased permeability in response to VEGF suggests a role for this factor in cellular events that are not directly related to mitogenicity. At present, there is no ready explanation as to why our data do not agree with some other published data; however, our data suggest that the role of VEGF as an EC mitogen may not be as simple as first postulated.
Selected Abbreviations and Acronyms
|FGF||=||fibroblast growth factor|
|flk-1||=||fetal liver kinase 1|
|flt-1||=||fms-like tyrosine kinase 1|
|VEGF||=||vascular endothelial growth factor|
This work was supported by a Grant-in-Aid from the American Heart Association (AHA) and with funds contributed in part by the AHA, Alaska Affiliate, Inc. The generous gift of recombinant human VEGF from Genentech, Inc, is greatly appreciated.
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