Cardiovascular Research

Cardiovascular Research 1999 44(2):294-302; doi:10.1016/S0008-6363(99)00203-5
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis

Daisy F Lazarous^*, Matie Shou, Jonathan A Stiber, Everett Hodge, Venugopal Thirumurti, Lino Gonçalves and Ellis F Unger

Experimental Physiology and Pharmacology Section, Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA

^* Corresponding author. Present address: The Johns Hopkins University School of Medicine, Division of Cardiology, A1 East, Johns Hopkins Bayview Medical Center, 4940 Eastern Avenue, Baltimore, MD 21224, USA. Tel.: +1-410-550-7035; fax: +1-410-550-1183 dlazarou{at}welch.jhu.edu

Received 22 October 1998; accepted 16 June 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Angiogenic peptides like VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) have entered clinical trials for coronary artery disease. Attempts are being made to devise clinically relevant means of delivery and to effect site-specific delivery of these peptides to the cardiac tissue, in order to limit systemic side-effects. We characterized the response of the pericardium to delivery of a replication-deficient adenovirus carrying the cDNA for AdCMV.VEGF₁₆₅, and assessed the effect of pericardial VEGF₁₆₅ on myocardial collateral development in a canine model of progressive coronary occlusion. Methods: Ameroid constrictors were placed on the proximal left circumflex coronary artery of mongrel dogs. Ten days later, 6x10⁹ pfu AdCMV.VEGF₁₆₅ (n=9), AdRSV.β-gal (n=9), or saline (n=7) were injected through an indwelling pericardial catheter. Transfection efficiency was assessed by X-gal staining. Pericardial and serum VEGF levels were measured serially by ELISA. Maximal myocardial collateral perfusion was quantified with radiolabeled or fluorescent microspheres 28 days after treatment. Results: In AdRSV.β-gal-treated dogs, there was extensive β-gal staining in the pericardium and epicardium, with minimal β-gal staining in the mid-myocardium and endocardium. Pericardial delivery of AdCMV.VEGF₁₆₅ resulted in sustained (8–14 day) pericardial transgene expression, with VEGF levels peaking 3 days after infection (>200 ng/ml) and decreasing thereafter. There was no detectable increase in serum VEGF levels. Maximal collateral perfusion, a principal correlate of collateral development and angiogenesis, was equivalent in all groups. Conclusion: Adenoviral-mediated gene transfer is capable of inducing sustained VEGF₁₆₅ expression in the pericardium; however, locally targeted pericardial VEGF delivery failed to improve myocardial collateral perfusion in this model.

KEYWORDS Collateral circulation; Coronary circulation; Gene therapy; Growth factors; Ischemia

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) is a secreted, glycosylated, dimeric protein that is structurally related to platelet-derived growth factor (PDGF) [1,2]. VEGF is an endothelial cell-specific mitogen that has been identified as a hypoxia-inducible angiogenic factor [3] and a potential tumor angiogenesis factor in vivo [4]. VEGF has been associated with pathological retinal neovascularization [5,6] and intra-ocular expression of VEGF has been correlated with retinal neovascularization [7]. In experimental models of limb ischemia, VEGF has been demonstrated to promote angiogenesis and collateral development [8–12]. VEGF has also shown promise for myocardial angiogenesis [13–15]; however, parenteral administration of VEGF has been associated with dose-limiting hypotension [15,16] and acceleration of injury-induced arterial neointimal accumulation [16], suggesting that local targeting of the growth factor may be essential for clinical use.

Adenoviral-mediated gene transfer has been used successfully to transfer genes to mammalian tissues, and preliminary studies have demonstrated the feasibility of efficient gene transfer to the pericardial sac. [17]. In an attempt to target VEGF protein delivery to cardiac tissues and to limit systemic side-effects, we employed a second-generation adenovirus for transfer of the gene encoding human VEGF to cardiac cells in vivo. An E1a-deleted replication-deficient recombinant adenoviral vector was utilized in which the gene encoding human VEGF₁₆₅ was under the control of the immediate early cytomegalovirus promoter. We exploited the sequestered environment of the pericardial space for site-specific gene transfer, a strategy that might preclude unwanted viral dissemination and enable the vector to evade immune surveillance, thereby prolonging transgene expression. The immediate proximity of the epicardial vasculature and developing collateral circulation to the pericardial space and pericardial fluid suggested that this mode of VEGF delivery might lead to enhancement of myocardial angiogenesis while limiting systemic effects.

Using a β-gal marker gene, our first goal was to determine the anatomic distribution of transgene expression after intra-pericardial virus administration. Second, we sought to characterize the magnitude and time course of VEGF₁₆₅ transgene expression and to determine the distribution of adenovirus in body fluids. Our third goal was to determine whether intrapericardial VEGF gene transfer was capable of improving myocardial collateral perfusion in a well-characterized canine model of progressive coronary occlusion.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The experimental protocol was approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals [18] and NIH issuance 3040-2: Animal Care and Use in the Intramural Program.

2.1 Surgical instrumentation
Thirty-two mongrel dogs of either sex were obtained from Haycock Kennels, Quakertown, PA. Dogs were anesthetized with acepromazine, 0.2 mg/kg i.m., thiopental sodium, 15 mg/kg i.v., and inhaled methoxyflurane. A left thoracotomy was performed using sterile technique, as previously described [13,16]. An ameroid constrictor (Research Instruments and Manufacturing, Corvallis, OR) was fitted on the proximal left circumflex coronary artery (LCx) before the origin of the first marginal branch. A silastic catheter was positioned in the left atrial appendage for microsphere injections to assess collateral perfusion. Multiple side holes were fashioned in the distal 2 cm of a 6.6-Fr silastic end hole catheter (Bard Access Systems, Salt Lake City, UT), and the catheter was secured in the pericardial space. The pericardium was carefully approximated, and the chest was closed in layers. The termini of the two catheters were positioned in the subcutaneous tissue of the back, the skin was closed, and the dogs were allowed to recover.

2.2 Integrity of the pericardium
Ten days after surgery, the pericardium and catheter were tested for leakage in a pilot dog by injecting 2 ml of contrast agent (Hypaque) into the pericardial space under fluoroscopic guidance. The dye flowed freely in the pericardial space, and no leak was visualized.

2.3 Transduction efficiency of pericardial gene transfer
In one dog, 6x10⁹ pfu AdRSV.β-gal was instilled into the pericardial cavity. This gene encodes nuclear-targeted bacterial β-galactosidase. Myocardium, pericardium, lung, intestine, kidney, gonad, brain, liver, and spleen were harvested 72 h after transfection, and bacterial β-galactosidase activity was assessed by 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal) staining. The tissue was embedded in paraffin and 5-µm sections were counter-stained with Nucleofast red for microscopic analysis.

2.4 Gene expression: serum and pericardial VEGF levels
To establish the ability of transduced tissue to secrete VEGF and to assess the time course of protein secretion, recombinant VEGF protein production was quantified by ELISA in six animals (three VEGF-treated, three controls). Samples of pericardial fluid (withdrawn from the indwelling catheter) and serum samples (forelimb venous puncture) were collected serially: before treatment, and on post-treatment days 1, 3, 5, 7, 10, 14, and 21. Samples were centrifuged at 4°C for 10 min; the supernatant and serum were assayed for human VEGF using a solid-phase ELISA kit according to the manufacturer's instructions (catalog no. DVE00, R&D Systems, Inc., Minneapolis, MN).

2.5 In vitro techniques: biological activity of the secreted VEGF
In order to confirm that the expressed human VEGF was biologically active in canine tissue, we tested the mitogenic activity of the expressed human VEGF on canine endothelial cells in vitro. Endothelial cells were harvested from the saphenous veins of dogs and expanded in culture. The endothelial cells were infected with AdCMV.VEGF₁₆₅ or AdRSV.β-gal at a multiplicity of infection (moi) of 100 and the supernatant was collected 3 days later. The protein product was quantified in the supernatant of both AdCMV.VEGF₁₆₅ and AdRSV.β-gal-infected cells with a solid-phase ELISA kit as above. Two milliliters conditioned media was obtained from 1x10⁶ cells infected 3 days earlier. This was placed on canine endothelial cells previously growth-arrested in 0.5% serum. Absolute cell numbers from triplicate cultures were determined 3 days after exposure to the conditioned media.

2.6 Quantification of E1a-deleted adenovirus in body fluids
Viral titer was quantified in urine, feces, and pericardial aspirates to validate confinement of the adenovirus to the pericardial space. Serial samples were collected from an animal pretreatment and 1, 2, 3, and 6 days after pericardial instillation of 6x10⁹ pfu AdCMV.VEGF₁₆₅. Detection and quantification of adenovirus in the samples was done using 293 cells (TCID₅₀ assay, i.e. tissue culture infectious dose 50%). In the presence of E1a-deleted adenovirus, 293 cells (embryonic human kidney cells transformed by stable adenovirus E1a gene insertion) exhibit typical adenoviral cytopathic effects within 72–96 h [19] characterized by clumping, detachment, cell lysis, and intra-nuclear basophilic or amphophilic inclusion bodies. Ad5 CMV-LacZ inoculated into normal canine urine served as the positive control; the negative control consisted of a ten-fold dilution of normal canine urine.

2.7 Treatment groups
Dogs were randomized to 6x10⁹ pfu AdCMV.VEGF₁₆₅, AdRSV.β-gal or saline, delivered through the indwelling pericardial catheter 10 days after ameroid placement. Selection of the dose was based on our pilot experiments and previous studies [20]. Aliquots of stock AdCMV.VEGF₁₆₅ and AdRSV.β-gal (6x) (generously supplied by GenVec, Inc., Rockville, MD), 10¹⁰ pfu (volume 1 ml) were stored at –80°C until used. The viral solution was diluted in 3 ml serum-free EBM medium (Clonetics, San Diego, CA) immediately prior to administration. Using sterile technique, the viral solution was injected transcutaneously through the infusion port of the pericardial catheter, followed by 2 ml of EBM medium.

2.8 Analysis of organ toxicity
Using aseptic technique, an 18-gauge needle was inserted into the subcutaneous port of the pericardial catheter on a regular basis to quantify and remove, if necessary, accumulated pericardial fluid. If less than 20 ml of pericardial fluid could be withdrawn, it was returned to the pericardial space. Larger effusions were evacuated to prevent pericardial tamponade. Routine hematologic and biochemical studies were performed weekly on all animals.

2.9 Histology of the heart
Cardiac histological analysis was carried out in 16 animals (saline=6, β-gal=4, and VEGF=6). Transmural sections of the heart at the mid ventricular level were embedded in paraffin (3–5-µm sections) and stained with H&E. The sections were reviewed by a veterinary pathologist who was naïve to treatment assignment. The presence of myocardial fibrosis, inflammation, and increased vascularity were noted.

2.10 Hemodynamic measurements and quantification of collateral perfusion
Regional myocardial blood flow was quantified using the reference sample technique 28 days after treatment [21]. All microsphere blood flow studies were performed in the conscious state during maximal coronary vasodilatation. The dogs were lightly sedated with diazepam 1–2 mg/kg administered through the left atrial catheter. Using local lidocaine anesthesia, a 5F catheter (Cordis Corp.) was inserted into the femoral artery for measurement of arterial pressure and withdrawal of arterial reference samples. Arterial pressure and the electrocardiogram were recorded continuously. Maximal coronary vasodilatation was induced by infusing chromonar 8 mg/kg (Hoechst-Roussel Pharmaceuticals) into the left atrial catheter over 30 min as we have done previously [13,16]. After infusion of chromonar, approximately 3x10⁶ radiolabeled microspheres (15 µm) were injected into the left atrial catheter. In a subset of animals, perfusion measurements were made using fluorescent microspheres (Interactive Medical Technology) [22]. Microsphere perfusion measurements were performed in duplicate with two labels (⁴⁶Ce and ⁹⁵Nb for radiolabeled microspheres; dual-labeled red, orange or violet dyes for fluorescent microspheres), and the results were averaged. The dogs were heparinized (5000 Units i.v.), killed with an overdose of sodium pentobarbital and KCl, and the myocardium was perfusion-fixed with 10% buffered formaldehyde at physiologic pressure. The heart was cut into 7-mm transaxial slices and examined macroscopically for the presence of infarcts or patchy areas of fibrosis. For radiolabeled microsphere analyses, the two central left ventricular slices were divided into 16 wedges and ranked with respect to perfusion. The four wedges with the highest perfusion were selected to represent the normal zone (NZ); the four wedges with the most compromised perfusion were selected to represent the collateral zone (CZ) as we have done previously [13,16]. For fluorescent microsphere analyses, only the central slice was analyzed. Using a dual wavelength detector system, intact microspheres from tissue digests were characterized with respect to dye ratio and counted using a coulter system. Tissue digestion and fluorescent microsphere analyses were carried out by Interactive Medical Technology using standard techniques.

Mean NZ and CZ perfusion were calculated from the duplicate perfusion measurements, and maximal collateral perfusion was expressed in relative terms as the CZ/NZ perfusion ratio as we have done previously [13,16]. The CZ/NZ ratio was defined prospectively as the primary study endpoint. Data are expressed as mean±S.E.M.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Twenty-six of 32 dogs completed the studies. Five dogs died suddenly, 2–15 days after ameroid placement, presumably from sudden coronary occlusion or ameroid-induced coronary artery spasm. Two of five dogs expired before initial gene transfer. Of the three animals that died after gene transfer, one had received AdCMV.VEGF₁₆₅ and two had received AdRSV.β-gal. Autopsies failed to show an obvious cause of death and death was attributed to arrhythmia from sudden coronary occlusion. A sixth dog (AdCMV.VEGF₁₆₅-treated) died 5 days after randomization; autopsy revealed 250 ml of fluid in the pericardial space and death was attributed to cardiac tamponade. There was discordance between the two measurements of myocardial perfusion in one dog, and it was excluded from the analysis prior to breaking the study code. Thus, perfusion measurements are reported for 25 dogs. One additional dog was used for a determination of in vivo β-gal transfection efficiency.

3.1 In vitro VEGF production and biological activity
VEGF was detected in the supernatant of AdCMV.VEGF₁₆₅ cells but not in the cells infected with AdRSV.β-gal. Peak VEGF levels of approximately 1 µg/million cells/day were observed between days 3 and 5 after infection, tapering off thereafter (Fig. 1). The expressed human VEGF induced a two-fold increase in proliferation of canine endothelial cells, indicating that the VEGF was biologically active, and that its activity was conserved between species (data not shown).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 In vitro VEGF production. VEGF was detected in the supernatant of AdCMV.VEGF165 cells but not in the cells infected with AdRSV.β-gal. Peak VEGF levels of approximately 1 µg/million cells/day were produced 3–5 days after infection. The expressed human VEGF induced a two-fold increase in proliferation of canine endothelial cells, indicating that the biological activity was conserved between species.

 
3.2 Pericardial transfection efficiency
There was extensive β-gal expression in the parietal pericardium (Fig. 2a) and epicardium (Fig. 2b). Reporter gene transfer was almost entirely restricted to pericardial and epicardial cells, with scattered sparse expression in the mid-myocardium, and no detectable staining of the endocardium.

View larger version (90K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Expression of β-galactosidase after intrapericardial instillation of 6x109 pfu AdRSV.β-gal. The heart was harvested 72 h post-treatment and X-gal histochemical staining was performed. Panel A: reporter gene transfer was almost entirely restricted to the parietal pericardial cells, and panel B: visceral pericardial cells (epicardium). There was scattered scanty expression in the mid-myocardium, and no detectable staining of the endocardium. Distant tissues were free of β-gal staining.

 
3.3 Pericardial and serum VEGF levels
Pre-treatment VEGF levels were negligible in both virus-treated groups. Transgene expression was evident 24 h after viral infection, and peak pericardial VEGF levels were recorded on day 3 in AdCMV.VEGF-treated dogs (>200 ng/ml). VEGF levels diminished to 15 ng/ml by day 8, and tapered off gradually thereafter, such that the total duration of the response was 14 days (Fig. 3). Serum VEGF levels did not increase from pretreatment values. Neither pericardial nor serum VEGF levels were elevated in AdRSV.β-gal treated animals.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Line plot of pericardial and serum VEGF levels from six animals (three AdCMV.VEGF-treated and three AdRSV.β-gal-treated). Transgene expression was evident 24 h after viral infection, with the measurable increase in [VEGF] persisting for 14 days. Pretreatment VEGF levels were negligible in both virus-treated groups. The VEGF concentration in pericardial fluid peaked on day 3 in AdCMV.VEGF-treated dogs (>200 ng/ml). VEGF levels diminished to 15 ng/ml by day 8, and tapered off gradually thereafter. VEGF levels were not elevated in AdRSV.β-gal-treated animals. Serum VEGF concentrations remained at pretreatment levels in all groups.

 
3.4 Detection of recombinant adenovirus in body fluids
There was no detectable shedding of virus in the fecal or urine samples. At 6 days, the pericardial aspirate remained infectious and contained 5x10²/ml TCID₅₀ virus, still capable of replication in 293 cells.

3.5 Analysis of organ toxicity
All AdCMV.VEGF₁₆₅-treated animals developed significant pericardial effusions, 55–180 ml in volume. One AdCMV.VEGF₁₆₅-treated animal expired from pericardial tamponade with a 250-ml hemorrhagic inflammatory effusion (elevated polymorphonuclear leucocyte count). The aspirated fluid was cultured in all cases and found to be free of bacterial infection. None of the AdRSV.β-gal-treated animals had significant effusions (>20 ml).

3.6 Collateral conductance and CZ/NZ perfusion ratios
Microsphere perfusion determinations, performed in duplicate, showed excellent intra-animal agreement. In the normally-perfused territory of the left anterior descending coronary artery, maximal coronary conductance was similar in the three treatment groups on day 28 (Table 1). Maximal collateral conductance in the territory of the occluded LCx was similar in all groups, and the CZ/NZ ratios were virtually identical in the three treatment groups. For the critical comparison between the AdRSV.β-gal and AdCMV.VEGF₁₆₅ groups, mean CZ/NZ ratios were 0.36±0.03 and 0.36±0.04, respectively.

View this table: [in this window] [in a new window]   Table 1 Myocardial perfusion and the CZ/NZ perfusion ratioa

 
3.7 Histological analysis
No cytopathic effects were grossly evident in the pericardium or surrounding lungs at the time of tissue harvest, i.e. structures were relatively free of adhesions. X-gal staining of distant organs did not reveal β-gal expression in any tissue.

Histological analysis of the heart revealed no difference between treatment groups in the degree of vascularity, fibrosis or inflammation or thrombosis. Microscopic angiomas or hemangiomas were not noted.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
VEGF is one of several angiogenic heparin-binding growth factors reported to promote collateral development in animal models of myocardial and limb ischemia [8–15]. In previous experiments in a canine model, we showed that VEGF enhanced coronary collateral development when administered repeatedly into collateral-dependent myocardium [13]. VEGF has also been reported to improve coronary collateral function in pigs when infused chronically near the point of coronary occlusion [14]. Enhancement of coronary collateral perfusion has not been attained, however, following relatively brief periods of VEGF treatment [15,16]. Moreover, intracoronary [15] and systemic arterial [16] administration of the growth factor has been associated with severe dose-limiting side effects. VEGF is a potent vascular permeability factor [2] and endothelium-dependent vasodilator [23], and causes severe hypotension when administered parenterally [15,16]. VEGF has also shown the potential to induce neointimal accumulation after vascular injury [16]. Thus, although VEGF appears to have the potential to promote myocardial angiogenesis, specific local targeting and/or extended peptide delivery may be essential to induce myocardial angiogenesis in the absence of toxicity [15,16]. For these reasons, we considered gene transfer as a means to bring about localized myocardial targeting of VEGF, with extended delivery of protein to maximize therapeutic effect.

Arterial gene transfer is currently under investigation as a means to promote therapeutic angiogenesis in ischemic limbs, utilizing a hydrogel polymer-coated balloon to establish direct contact of naked VEGF plasmid cDNA with the arterial wall [24,25]. This technique has important limitations due to the inherent inefficiency of direct arterial plasmid gene transfer, a problem that is magnified in atherosclerotic arteries. Moreover, in at least one investigation in which this technique has been successful, a generalized drug effect was observed, [26] suggesting that limited, locally targeted therapy may not be attainable with this method.

Several investigators have demonstrated that second generation adenoviral vectors function efficiently as gene delivery vehicles in vivo [27,28], superior in efficiency to plasmid DNA injection [29]. Successful intramyocardial injection of adenoviral vectors has been demonstrated in mammals [20,32]. Recently, intracoronary injection of a recombinant adenovirus expressing human FGF-5 was shown to increase perfusion and contractile function in the ischemic porcine heart [30]. Intracoronary gene delivery appears promising, although the potential for viral dissemination to non-target tissues poses an important concern. Viral receptors are found on the surface of a variety of cell types, and generalized dissemination of angiogenic genes could lead to undesirable neovascularization in sites such as the retina or occult tumors. Host immune responses to foreign adenoviral antigens pose an additional barrier for gene therapy, limiting the duration of gene expression [31].

We evaluated pericardial VEGF gene transfer as a means to target the myocardium and avoid the potential problems associated with systemic adenoviral dissemination. E1a-deleted replication-deficient adenovirus with the gene encoding VEGF₁₆₅ was delivered to the pericardial space. We hypothesized that the pericardium might provide an abundant surface area for gene transfer as well as an inherent means for viral containment. Furthermore, the limited pericardial volume of distribution would tend to favor gene transfer by maximizing the concentration of the virus, and would serve to maintain the concentration of the expressed VEGF gene product at a relatively high level. In essence, the pericardium might function as a paracrine organ, producing high localized VEGF levels in direct apposition to the epicardial arteries and developing collaterals, leading to myocardial angiogenesis.

The pericardial space has been evaluated previously as a reservoir for angiogenic growth factor delivery [32,33]. Pericardial injection of basic fibroblast growth factor has been reported to enhance myocardial angiogenesis [36] and limit infarct size after coronary embolization [37]. Woody et al. have reported gene transfer to the pericardium [17]. Thus, these studies, taken together, suggest that pericardial gene transfer might provide an effective means to induce myocardial angiogenesis.

In the present study, we demonstrated that gene transfer to non-replicating pericardial and epicardial cells using recombinant adenovirus vectors resulted in acceptable gene targeting efficiency and expression of the VEGF transgene over a 10–14-day period. Although this strategy was highly effective in directing pericardial VEGF expression, the pericardial VEGF so produced was ineffective in promoting collateral development. There are a number of potential explanations for the apparent lack of effect.

4.1 Adequacy of VEGF production
It is worthwhile to contrast the calculated mass of VEGF produced by gene transfer in the present experiment to the quantities of the VEGF peptide administered in previous studies in which it was found to be effective. We observed peak steady state VEGF levels of approximately 200 ng/ml in a pericardial fluid volume of roughly 200 ml: an estimated mass of approximately 40 µg. Considering that the pericardial VEGF concentration decreased to 15 ng/ml by day 8, the total quantity of VEGF produced in the present investigation, integrated over the 2-week period of expression, was probably an order of magnitude less than the cumulative injected dose we previously found to be effective (45 µg/dayx20 doses=900 µg) [13]. On the other hand, the quantity of VEGF produced in this experiment significantly exceeded the dose used by Harada et al. (0.06 µg/dayx28 days=1.68 µg) [14].

4.2 Direction of VEGF delivery across the vascular wall
The presence of VEGF in the pericardial space presents the growth factor to the abluminal (rather than the luminal) side of the vasculature. Though this unconventional mode of VEGF administration might be responsible for its lack of effect in this experiment, Harada et al. [14] found perivascular infusion of VEGF (mixed with heparin) to be effective, a method that also delivered VEGF to the abluminal aspect of the vasculature. Two key differences between the present and previous investigations should be underscored: Harada et al. [14] used a longer duration of infusion (4 weeks, although VEGF potency was reduced by a factor of 2.5 at the end of their study), and VEGF and heparin were co-administered in their study. VEGF is a heparin-binding growth factor, and the interactions between VEGF and heparin have not been well-characterized in vivo [34,35].

4.3 Preservation of VEGF activity across species
We considered the possibility that human VEGF₁₆₅ was not active in canine cells. In the present study, the expressed human VEGF₁₆₅ induced proliferation of canine endothelial cells in vitro. Moreover, we previously found human VEGF₁₆₅ to be effective on canine endothelial cells in vitro [13].

4.4 Role of inflammation
It is plausible that adenoviral-related inflammation had a negative influence on collateral development in this study. We found, however, that collateral function was not depressed in AdRSV.β-gal-treated animals (relative to saline-treated controls), suggesting that this was not the case. Animals treated with the β-gal virus did not develop hemodynamically significant pericardial effusions, but large effusions were encountered consistently in AdCMV.VEGF-treated dogs, requiring drainage to avert hemodynamic compromise. Leucocyte counts were generally elevated in these aspirates, indicative of an inflammatory response. Thus, it appears that a specific biological effect of VEGF was responsible for the sizable and hemodynamically significant effusions characteristic of these dogs. VEGF-induced vascular hyperpermeability may have played an important role in this regard [2]. The requirement to tap the effusions subsided by day 15 post-transfection, coinciding with waning VEGF expression.

Recently it has been reported that the plasmid for VEGF injected in the border zone of myocardial infarct in rats created angiomas that did not contribute to regional myocardial blood flow [36]. VEGF gene delivery to non-ischemic muscle in mice led to an increase in vascular channels and hemangiomas, associated with local high serum VEGF levels [37]. Thus, there is concern that over expression/high doses of growth factors may have deleterious effects on both ischemic and non-ischemic tissue. We did not observe angiomas or hemangiomas in this study.

In summary, adenoviral-mediated gene transfer is capable of inducing sustained VEGF₁₆₅ expression in the pericardium. Pericardial VEGF levels in the 200 ng/ml range were achieved in the absence of an increase in systemic VEGF levels. Despite appreciable local VEGF production, however, myocardial collateral perfusion was not improved in this model. These data add to a growing number of studies on the effects of VEGF on myocardial angiogenesis – a biological effect of VEGF has been demonstrated in some but not all of these investigations. Additional studies are needed to better characterize the response of the coronary collateral circulation to VEGF. The optimal VEGF dose, the timing, duration, and route of VEGF delivery, and the potential role of heparin co-administration are important issues that merit further study. Moreover, it remains to be determined whether pericardial gene transfer of alternative forms of VEGF or other angiogenic growth factors might foster collateral growth.

Time for primary review 32 days.

	Acknowledgements

 
The authors thank V. Hampshire, J. Bacher, and their staffs for providing veterinary care, GenVec, Inc. for supplying the recombinant adenoviruses, and Sanjay Shah for technical assistance.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Leung D.W, Cachianes G, Kuang W.J, Goeddel D.V, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (1989) 246:1306–1309.[Abstract/Free Full Text]
2. Keck P.J, Hauser S.D, Krivi G, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science (1989) 246:1309–1312.[Abstract/Free Full Text]
3. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature (1992) 359:843–845.[CrossRef][Medline]
4. Plate K, Breier G, Weich H.A, Risau W. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature (1992) 359:845–848.[CrossRef][Medline]
5. Aiello L.P, Pierce E.A, Foley E.D, et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA (1995) 92:10457–10461.[Abstract/Free Full Text]
6. Stone J, Itin A, Alon T, et al. Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. J Neurosci (1995) 15:4738–4747.[Abstract]
7. Pe’er J, Shweiki D, Itin A, et al. Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases. Lab Invest (1995) 72:638–645.[Web of Science][Medline]
8. Bauters C, Asahara T, Zheng L.P, et al. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol (1994) 267:H1263–1271.[Web of Science][Medline]
9. Takeshita S, Zheng L.P, Brogi E, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest (1994) 93:662–670.[Web of Science][Medline]
10. Takeshita S, Pu L.Q, Stein L.A, et al. Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation (1994) 90:II228–II234.[Medline]
11. Asahara T, Bauters C, Zheng L.P, et al. Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation (1995) 92:II365–II371.[Medline]
12. Bauters C, Asahara T, Zheng L.P, et al. Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg (1995) 21:314–324.[CrossRef][Web of Science][Medline]
13. Banai S, Jaklitsch M.T, Shou M, et al. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation (1994) 89:2183–2189.[Abstract/Free Full Text]
14. Harada K, Friedman M, Lopez J.J, et al. Vascular endothelial growth factor administration in chronic myocardial ischemia. Am J Physiol (1996) 270:H1791–H1802.[Medline]
15. Hariawala M.D, Horowitz J.J, Esakof D, et al. VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts. J Surg Res (1996) 63:77–82.[CrossRef][Web of Science][Medline]
16. Lazarous D.F, Shou M, Scheinowitz M, et al. Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury. Circulation (1996) 94:1074–1082.[Abstract/Free Full Text]
17. Woody M, Mehdi K, Zipes D.P, et al. High efficiency adenovirus-mediated pericardial gene transfer in vivo [abstract]. J Am Coll Cardiol. (1996) 27(Suppl_A):31A.
18. National Research Council. Guide for the care and use of laboratory animals. (1996) Washington, DC: National Academy Press.
19. Dulbecco E, Ginsberg A, eds. Virology. (1980) New York: Harper and Row. 881.
20. Guzman R.J, Lemarchand P, Crystal R.G, Epstein S.E, Finkel T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circ Res (1993) 73:1202–1207.[Abstract/Free Full Text]
21. Heymann M.A, Payne B.D, Hoffman J.I.E, Rudolph A.M. Blood flow measurements with radionuclide-labelled particles. Prog Cardiovasc Dis. (1977) 20:55–79.[CrossRef][Web of Science][Medline]
22. Austin G.E, Tuvlin M.B, Martino-Salzman D, Hunter R, Justicz A.G. Determination of regional myocardial blood flow using fluorescent microspheres. Am J Cardiovasc Pathol (1993) 4:352–357.[Medline]
23. Ku D.D, Zaleski J.K, Liu S, Brock T.A. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol (1993) 265:H586–H592.[Web of Science][Medline]
24. Isner J.M, Walsh K, Symes J, et al. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease [news]. Circulation (1995) 91:2687–2692.[Free Full Text]
25. Isner J.M, Pieczek A, Schainfeld R, et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet (1996) 348:370–374.[CrossRef][Web of Science][Medline]
26. Asahara T, Chen D, Kearney M, et al. Accelerated re-endothelialization and reduced neointimal thickening following catheter transfer of phVEGF₁₆₅ [abstract]. J Am Coll Cardiol (1996) 27(Suppl A):1A.[Medline]
27. Rade J.J, Schulick A.H, Virmani R, Dichek D.A. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nat Med (1996) 2:293–298.[CrossRef][Web of Science][Medline]
28. Muhlhauser J, Merrill M.J, Pili R, et al. VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo. Circ Res (1995) 77:1077–1086.[Abstract/Free Full Text]
29. French B.A, Mazur W, Geske R.S, Bolli R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors. Circulation (1994) 90:2414–2424.[Abstract/Free Full Text]
30. Giordano F.J, Ping P, McKirnan M.D, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med (1996) 2:534–539.[CrossRef][Web of Science][Medline]
31. Gilgenkrantz H, Duboc D, Juillard V, et al. Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response. Hum Gene Ther (1995) 6:1265–1274.[Web of Science][Medline]
32. Landau C, Jacobs A.K, Haudenschild C.C. Intrapericardial basic fibroblast growth factor induces myocardial angiogenesis in a rabbit model of chronic ischemia. Am Heart J (1995) 129:924–931.[CrossRef][Web of Science][Medline]
33. Uchida Y, Yanagisawa-Miwa A, Nakamura F, et al. Angiogenic therapy of acute myocardial infarction by intrapericardial injection of basic fibroblast growth factor and heparin sulfate: An experimental study. Am Heart J (1995) 130:1182–1188.[CrossRef][Web of Science][Medline]
34. Cohen T, Gitay-Goren H, Sharon R. VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J Biol Chem (1995) 270:11322–11326.[Abstract/Free Full Text]
35. Terman B, Khandke L, Dougher-Vermazan M, et al. VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor. Growth Factors (1994) 11:187–195.[Web of Science][Medline]
36. Schwarz E.R, Spealman M.T, Patterson M, et al. Effect of intra myocardial injection of DNA expressing vascular endothelial growth factor in myocardial infarct tissue in the rat heart: angiogenesis and angioma formation. Circulation (1998) 456(Suppl 1):2397.
37. Springer M.L, Chen A.S, Kraft P.E, Bednarski M, Blau H.M. VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell (1998) 2:549–558.[CrossRef][Web of Science][Medline]

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