© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Microangiographic assessment of collateral vessel formation following direct gene transfer of vascular endothelial growth factor in rats
aDepartment of Medicine (Cardiology), Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi, Tokyo 173, Japan
bDepartment of Physiology, Tokai University School of Medicine, Isehara, Japan
cCentral Research Laboratory, Hitachi Ltd., Tokyo, Japan
* Corresponding author. Tel.: +81-3-3964-1211 (ext. 1580); fax: +81-3-5375-1308; e-mail: stangiol@med.teikyo-u.ac.jp
Received 22 January 1997; accepted 26 May 1997
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: The development of collateral microvessels following therapeutic angiogenesis with vascular endothelial growth factor (VEGF) was investigated using a new system of microangiography that employs monochromatic synchrotron radiation (SR) and a high definition video system to visualize arteries with a spatial resolution of 30 µm. Methods: Ischemia was induced in the hindlimb of 20 rats by excision of the femoral artery, followed by transfection of the plasmid (400 µg) encoding VEGF or β-galactosidase (control) into limb muscles. Microangiography was used to assess the development of collaterals in the ischemic limb four weeks after treatment. Results: Gene transfer of VEGF produced morphologically similar, but significantly more extensive, collateral networks at the microvascular level as compared with the naturally occurring collateral arteries in the control animals (angiographic score: 0.88±0.08 versus 0.54±0.05, p<0.01). No adverse vascular effects such as hemangiomas and/or arteriovenous (AV) fistulae were observed following VEGF treatment. The vasodilator effect of papaverine was evident in relatively large vessels in both groups. At the microvascular level (diameter <100 µm), however, papaverine induced significant vasodilation in the VEGF-treated animals, and almost no vasodilation in the controls. Conclusions: SR microangiography allowed us to assess the development of small collateral arteries following VEGF-gene transfer. The information obtained may provide new insights regarding the collateral microcirculation and therapeutic angiogenesis.
KEYWORDS Angiogenesis; Collateral circulation; Endothelial cells; Growth factors; Microvessels; Rat
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Despite major advances in techniques of surgical and percutaneous revascularization, there are limited options for treating patients with vascular obstructive disease of the lower extremities [1]. Conventional drug therapy lacks proven benefit. Procedures such as percutaneous revascularization may not be feasible for those with extensive vascular obstruction. Surgical therapy is complicated by variable morbidity and mortality, and its success depends on the long-term patency of the graft.
Recent investigations in animal models indicate the feasibility of using angiogenic growth factors to augment the development of collateral arteries. We [2, 3]and others [4]have shown that the endothelial cell (EC) specific mitogen, vascular endothelial growth factor (VEGF) [5, 6], is a potent agent for augmenting the development of collateral vessels in the lower extremity and in the coronary circulation. This new approach to the treatment of vascular insufficiency has been termed therapeutic angiogenesis [2].
The first clinical trial of therapeutic angiogenesis, which is currently underway in the United States, has confirmed the feasibility of using VEGF for treating patients with severe ischemia of a limb [7]. Gene transfer of VEGF was performed in a residual patent artery of the proximal limb, and the blood flow in the limb was successfully increased via the collateral vessels. This improvement in blood flow was documented by intravascular Doppler analysis and magnetic resonance imaging. In some cases, however, contrast angiography failed to reveal any new collaterals. Since improvement in collateral-dependent flow typically results from the proliferation of vessels less than 180 µm in diameter [8–10], it is possible that conventional systems of angiography, which cannot visualize arteries <200 µm [11, 12], fail to display the full extent of collateral formation, leading to an underestimation of the angiogenic potential of VEGF.
Mori et al. developed a new system of angiography, synchrotron radiation (SR) microangiography, which uses monochromatic SR as an X-ray source and a high definition video camera coupled with a fluorescent screen as a detector, with a resulting resolution limit of 30 µm [11, 12]. Using this microangiography system, we recently demonstrated small collateral arteries with a diameter <100 µm in ischemic limbs of the rat [13]. The current study in rats used the same system of microangiography to visualize the collateral microvessels following the gene transfer of VEGF in therapeutic angiogenesis.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Animal model
We investigated the development of small collateral vessels following therapeutic angiogenesis with VEGF using the model of rat limb ischemia described previously [13, 14]. These investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1985). Male Wistar rats weighing 300–350 g (Charles River Japan, Yokohama, Japan) were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg) (Nembutal, Abbott Laboratories, North Chicago, IL). To excise the left femoral artery and induce ischemia in the limb, an incision was made that extended inferiorly from the left inguinal ligament to a point just proximal to the knee. The femoral artery was then completely excised from its proximal origin to the point distally where it bifurcates into the saphenous and popliteal arteries. A previous study performed in our laboratory has confirmed that this procedure induces significant limb ischemia that leads to atrophy of the skeletal muscles [14]. After completing gene transfer into limb muscles (see below), the skin incision was closed in layers. The animals were housed for 4 weeks until they were assessed microangiographically.
2.2 Plasmids
Complementary DNA (cDNA) clones for recombinant human VEGF165, which were isolated from cDNA libraries prepared from HL60 leukemia cells, were assembled into a simple eukaryotic expression plasmid that utilizes the 736 bp cytomegalovirus (CMV) promoter/enhancer to drive the expression of VEGF. An SV40 polyadenylation sequence is downstream from the VEGF cDNA. These fragments occur in the pUC118 vector, which includes an Escherichia coli origin of replication, and the β-lactamase gene for ampicillin resistance. The biological activity of VEGF165 secreted from the cells transfected with this construct (phVEGF165) was previously confirmed by evidence that media conditioned by transfected human 293 cells promotes the proliferation of capillary ECs [6]. A promoter-matched reporter plasmid pCMVβ (Clontech, Palp Alto, CA), which encodes β-galactosidase under the control of a CMV promoter, was used in the control transfection experiments.
2.3 Direct gene transfer of VEGF into limb muscles
Twenty rats were transfected with either the VEGF (N=10) or β-galactosidase (N = 10) genes to investigate the development of microvessels following VEGF gene therapy. The plasmid (400 µg) was dissolved in sterile saline (final volume of 1.0 mL) and was directly injected into the limb muscles using a syringe with a 27-gauge needle (Terumo, Tokyo, Japan) as previously described [14, 15]. DNA was injected into five different sites of three major thigh muscles in each animal: the adductor (2 sites), quadriceps (2 sites), and semimembranous (1 site). To avoid the "through-and-through" penetration of the injected muscles by the needle, and to ensure that the solution would not leak out of the puncture site, each injection was administered slowly for 5 s under direct visualization. After the administration of 5 injections (80 µg/0.2 mL per site for a total of 400 µg/1.0 mL per animal), the incision was closed in layers.
2.4 Microangiography
Microangiography was performed as previously described [13]. Briefly, at 4 weeks after transfection, the animals were again anesthetized with sodium pentobarbital and an incision was made in the thigh of the contralateral non-ischemic limb. The femoral artery was exposed, and a 24-gauge catheter (Surflo, Terumo) was cannulated with its tip positioned just above the aortic bifurcation in the lower abdominal aorta. Angiography was performed with the use of an automated injector (Medrad, Pittsburgh, PA) that was programmed to reproducibly deliver 2 mL of non-ionic contrast media (Iomeprol, Eisai, Tokyo). Serial images of the ischemic limb vessels were then recorded using the microangiography system described below.
Morphometric angiographic analysis of collateral vessel development was performed using a method described previously [2]with minor modifications. A composite of 2-mm2 grids was placed over the medial thigh area of the angiogram. The total number of grid intersections as well as the total number of intersections crossed by a contrast-opacified artery were counted individually by a single observer blinded to the treatment regimen. An angiographic score was calculated for each film as the ratio of grid intersections crossed by opacified arteries divided by the total number of grid intersections in the medial thigh.
2.5 Imaging system
Angiographic procedures were performed at the Photon Factory in the National Laboratory for High Energy Physics in Tsukuba, Japan. Details of this imaging system were previously described [11–13]. In brief, SR was derived from a storage ring of positrons with an energy of 2.5 GeV and an average beam current of 310 mA. This SR beam with a wide energy band was monochromatized and magnified by an asymmetrically-cut silicon crystal that was placed in front of the animal. The final photon density of the SR beam after monochromatization and magnification was estimated to be 1–2x109 photons/mm2/s, approximating that obtained from a conventional X-ray source (8–10x108 photons/mm2/s), with a 15–80 keV energy band. This high photon flux of SR resulted in an adequate signal-to-background ratio (300–1000:1) at the detector even after the X-ray beam had passed through the tissue. Images of the limb arteries were formed on a fluorescent screen (HR3, Fuji Medical Systems, Tokyo), scanned by a high-definition camera (HarpiconTM, Hitachi, Tokyo), and stored as digital images (1024x1024, 12-bit) by computer [16]. The "avalanche-type" of high-definition camera used here was approximately 30 times more sensitive than a conventional charge-coupled device (CCD) in terms of quantum efficiency; that is, in its conversion ratio of photons to electrons. The combination of SR and this high-definition video system resulted in a spatial resolution of 30 µm.
2.6 Microvascular reactivity studies
Microvascular reactivity of the collaterals was evaluated in selected animals (5 control and 5 VEGF-treated animals). Fresh stock solutions of papaverine hydrochloride (Sigma, St. Louis, MO) were prepared in saline immediately before each experiment. Papaverine (0.4 mg/kg) was infused intraarterially via the catheter positioned just above the aortic bifurcation. Since each animal underwent repeated angiography, care was taken to reestablish the basal hindlimb blood flow before obtaining each angiogram. We allowed a 10 min interval between angiograms. Ten minutes after the first angiogram, papaverine was infused intraarterially as a bolus through the catheter. The second angiogram was then recorded. After two angiograms were obtained, the animals were killed with an overdose of pentobarbital.
Preliminary studies performed in our laboratory have confirmed that the dose of papaverine used in the present study does not affect the systemic blood pressure and/or heart rate of the rat. As a reference, the average systolic blood pressure was 125.7±3.5 mmHg and the average heart rate was 357.0±9.8 beats per minute prior to papaverine administration. Following papaverine administration, the average systolic blood pressure was 123.0±3.3 mmHg and the average heart rate was 352.0±6.9 beats per minute (N = 3).
2.7 Statistics
Results are expressed as mean±SEM. Statistical significance was evaluated using unpaired Student's t test for comparisons between two means. A level of p<0.05 was considered statistically significant.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Development of microvessels after direct gene transfer of VEGF
SR microangiography revealed small collateral artery networks with a diameter less than 100 µm in the control and the VEGF-treated animals (Fig. 1; note that the imaging field of each angiogram in this figure is only 20 mmx20 mm). Most of these collateral vessels originated from the hypogastric trunk of the ipsilateral iliac artery and traversed the entire thigh with fine arterial anastomoses.
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The collateral network consisted of arteries that exhibited two distinct morphologies. The first group of collateral vessels appeared relatively linear, and morphologically resembled the normal vessels (A and C, large and small arrows). The second group of collaterals exhibited an undulating appearance, and typically lacked side branches (open arrows). Although morphologic evaluation of these collaterals showed no apparent differences between the control and VEGF-treated animals, the development of collateral arteries appeared to be more extensive in the VEGF-treated animals. Quantitative analysis of collateral vessels showed that the angiographic score in the VEGF-treated group was significantly higher than in the control group (0.88±0.08 versus 0.54±0.05, p<0.01). No untoward effects related to angiogenesis such as the development of hemangiomas and/or of arteriovenous (AV) fistulae were observed following VEGF treatment.
3.2 Reactivity of collateral vessels to administration of papaverine following direct gene transfer of VEGF
To evaluate the reactivity of collateral vessels, papaverine was administered into the iliac artery of the ischemic limb (Fig. 1). In control animals, the administration of papaverine induced significant vasodilation in relatively large vessels having a linear appearance (A and B, large arrows). However, little or no vasodilator effect was apparent in vessels <100 µm in diameter (small arrows) and/or those with an undulating appearance (open arrows).
In VEGF-treated animals, papaverine achieved a similar vasodilator effect in relatively large vessels with a linear appearance (C and D, large arrows) as that observed in control animals. In contrast to controls, the vasodilator effect of papaverine was also evident in some of the smaller vessels with a diameter <100 µm (small arrows). However, papaverine had almost no vasodilator effect on vessels with an undulating appearance (open arrows).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Previous studies have shown that the administration of recombinant angiogenic growth factors [2, 4]or genes [15, 17]enhances the formation of collateral vessels following experimental vascular occlusion. Although these studies documented by use of angiography an increase in the number of collateral vessels, they failed to demonstrate the full extent of the collateral networks, especially of vessels <200 µm in diameter, due to the low resolution of the angiography systems used. Visualization of such small collaterals is important since these vessels contribute directly to the perfusion of the ischemic tissues.
Mori et al. reported that the SR microangiography system may be used to visualize small arteries with a resolution limit of 30 µm [11, 12]. With this microangiography system, we recently documented the development of naturally occurring collateral arteries in the ischemic limb of rats [13]. The diameter of those arteries is well below the resolution limit of conventional angiography systems. Using this microangiography system, we now successfully visualized a network of small collateral arteries following therapeutic angiogenesis with VEGF. The development of collateral networks was significantly more extensive in the VEGF-treated animals than in the controls. Furthermore, collateral microvessels in the VEGF-treated animals exhibited no apparent morphologic differences as compared with the naturally occurring collaterals in the control animals. Considering the recent report by Isner et al. in which one patient who received VEGF-gene therapy developed transient hemangioma of the skin [7], the lack of pathological angiogenesis in our study appears to provide important information regarding the use of VEGF in therapeutic angiogenesis. However, the current investigation was performed 4 weeks following the VEGF-gene transfer. Tsurumi and Isner recently reported that VEGF gene expression following direct intramuscular transfection is limited to <4 weeks [15]. Thus, it is possible that, when we performed microangiography at 4 weeks, no transfected VEGF-gene was expressed in the limb. Further investigation is required to determine whether pathological angiogenesis may occur in the presence of certain levels of VEGF expression at earlier points following transfection.
A previous study using animal models of limb ischemia indicates that the collateral blood flow to the ischemic limb after the administration of papaverine is significantly higher in the VEGF-treated than in the control animals [18]. The current study documented such vasomotor responses of the collaterals at the microvascular level using SR angiography. In relatively large vessels, the vasodilator effect of papaverine was evident both in the VEGF-treated and the control animals. At the microvascular level (diameter <100 µm), however, papaverine induced almost no vasodilation in the controls. This differing vasodilator response to papaverine among the vessels of differing sizes could be due to a difference in the number of smooth muscle cells (SMCs) constituting the tunica media of each vessel. Since papaverine is a direct SMC relaxant, it seems reasonable that the larger collateral vessels that possess a larger number of SMCs would show a greater response than the smaller vessels.
Although the collateral microvessels (diameter <100 µm) in control animals did not show vasodilation following stimulation with papaverine, some portion of the collaterals in the VEGF-treated animals showed vasodilation. We recently reported that the proliferation of SMCs in the collateral vessels is significantly augmented by the administration of VEGF [10]although the direct mitogenic effect of VEGF is limited to ECs [5, 6]. The improved vasodilator response to papaverine observed in the VEGF-treated animals could be due to an increase in the number of SMCs following VEGF-gene therapy. Another possible explanation for the lack of effect of papaverine in the control animals may be that their microvessels are already dilated at rest due to severe ischemia so that papaverine would not induce additional vasodilation. In contrast, papaverine may induce vasodilation in the VEGF-treated animals because the vessels of these animals are not fully dilated at rest due to the increased number of collaterals.
We also observed differing vasomotor responses among collaterals with differing morphologies, regardless of the size of the arteries and/or the treatment strategies. Linear collateral vessels showed vasodilation in response to papaverine, while undulating collaterals showed almost no vasodilation. We recently documented with the use of microangiography the presence of undulating microvessels in ischemic rat limbs [13]. Since undulating vessels were not found in the normal rat limbs, these undulating collaterals may be newly formed or may result from a remodeling of the pre-existing vessels following the induction of limb ischemia, and may thus possess functionally different, and/or fewer, SMCs.
In this study, intramuscular gene transfer was achieved without the use of transfection vehicles such as liposomes or viral vectors. The feasibility of such a "naked" approach has been established previously in studies employing reporter genes [19]. Indeed, in our previous experience, all rat limb muscles transfected with the luciferase gene (98/98, 100%) expressed luciferase activity [14]. However, transfection of the lacZ gene revealed that <1% of the total myocytes in the ischemic limbs were successfully transfected, consistent with recently published data for the rabbit ischemic limb [15]. The current study demonstrated that such small number of transfected myocytes was sufficient to augment the formation of collateral microvessels in this animal model of limb ischemia. There are at least three possible explanations for this success of VEGF-gene therapy. First, and most importantly, the secreted nature of the gene product (i.e., VEGF protein) may have facilitated the effects of gene transfer. We previously demonstrated, for example, that transfection of the rabbit arterial tissue with the plasmid pXGH5 encoding human growth hormone (hGH) resulted in physiologic levels of hGH secretion despite immunohistochemical evidence of successful transfection in <1% of the total arterial cells [20]. Second, the ischemic milieu of transfected muscles may have resulted in an upregulation of EC receptors for VEGF. A recent report by Brogi et al., for example, showed that KDR receptor in ECs increased 13-fold when exposed to the media conditioned by hypoxic myoblasts [21]. Lastly, the angiogenic effect of VEGF may have been facilitated in the skeletal muscles by the inherently well-vascularized nature of this tissue. Thus, the success of gene therapy is not solely a function of transfection efficiency. A combination of favorable conditions would be required.
4.1 Study limitations
The primary limitation of this study is that SR is currently available only at a limited number of institutions. This greatly restricts the use of SR microangiography at present. However, the demand for SR is increasing in various fields of research including medicine. For example, several groups are currently using SR to develop methods of intravenous coronary angiography for clinical application [22]. The demand for SR will likely increase its availability in the future. A second limitation is that the development of the collateral arteries was not quantitated precisely. This was mainly due to the inability of the currently available system of quantitative angiography to evaluate the size and/or the morphology of the microvessels. Third, because the expression of transfected VEGF-gene is limited to 2–3 weeks [15], the relationship between the in situ expression of the transfected VEGF and the degree of collateral artery development was not assessed. We previously reported that the extent of collateral artery development following intraarterial bolus of VEGF protein (500 or 1000 µg) did not depend upon the dose administered, but was inversely related to the vasculopenia present at the pretreatment baseline [2]. Additional study is required to determine whether this relationship may be observed at the microvascular level following VEGF-gene therapy. Finally, the precise mechanism for the differing vasomotor responses observed among collaterals of differing morphologies remains to be determined. It is possible that vasoactive modulators such as the NO produced by pericytes and/or SMCs are involved in these responses.
4.2 Conclusions
We used SR microangiography to visualize networks of small collateral arteries that developed following therapeutic angiogenesis with VEGF. SR microangiography appears to be a powerful means of assessing the development of small collateral arteries following VEGF treatment, and may help to provide new insights regarding the collateral microcirculation and therapeutic angiogenesis.
Time for primary review 34 days.
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Acknowledgements |
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We are grateful to Genentech, Inc. for providing phVEGF165. This work was supported in part by a Grant-in-Aid for Scientific Research (07670809 to Dr. Takeshita; 07557060, 07807073 to Dr. Mori) from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan; by a grant from the Ryoichi Naito Foundation for Medical Research, Osaka, Japan (to Dr. Takeshita), and by a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo, Japan (to Dr. Takeshita). This project was conducted as a Joint Research Program of the National Laboratory for High Energy Physics, Tsukuba, Japan (93G241, 95G287).
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References |
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