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(Circulation. 1996;94:3281-3290.)
© 1996 American Heart Association, Inc.
Articles |
Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion
the Departments of Medicine (Cardiology) (Y.T., S.T., D.C., M.K., S.T.R., J.P., J.R.H., J.M.I.), Biomedical Research (Y.T., S.T., D.C., M.K., S.T.R., J.P., J.R.H., J.F.S., J.M.I.), and Surgery (J.F.S.), St Elizabeth's Medical Center of Boston, Tufts University School of Medicine, Boston, Mass.
Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center of Boston, 736 Cambridge St, Boston, MA 02135. E-mail jisner.opal.tufts.edu.
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Abstract |
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Background Striated muscle has been shown to be capable of taking up and expressing foreign genes transferred in the form of naked plasmid DNA, although typically with a low level of gene expression. In the case of genes that encode secreted proteins, however, low transfection efficiency may not preclude bioactivity of the secreted gene product. Accordingly, we investigated the hypothesis that intramuscular (IM) gene therapy with naked plasmid DNA encoding vascular endothelial growth factor (VEGF) could augment collateral development and tissue perfusion in an animal model of hindlimb ischemia.
Methods and Results Ten days after ischemia was induced in one rabbit hindlimb, 500 µg of phVEGF165, or the reporter gene LacZ, was injected IM into the ischemic hindlimb muscles. Thirty days later, angiographically recognizable collateral vessels and histologically identifiable capillaries were increased in VEGF transfectants compared with controls. This augmented vascularity improved perfusion to the ischemic limb, documented by a superior calf blood pressure ratio for phVEGF165 (0.85±0.05) versus controls (0.64±0.05, P<.01), improved blood flow in the ischemic limb (measured with an intra-arterial Doppler wire) at rest (phVEGF165=21.3±3.9 mL/min, control=14.6±1.6 mL/min, P<.01) and after a vasodilator (phVEGF165=54.2±12.0 mL/min, control=37.3±8.9 mL/min, P<.01) and increased microspheres in the adductor (phVEGF165=4.3±1.6 mL·min-1·100 g of tissue-1, control=2.9±1.2 mL·min-1·100 g of tissue-1, P<.05) and gastrocnemius (phVEGF165=3.9±1.0 mL·min-1·100 g of tissue-1, control=2.8±1.4 mL·min-1·100 g of tissue-1, P<.05) muscles of the ischemic limb.
Conclusions Ischemic skeletal muscle represents a promising target for gene therapy with naked plasmid DNA. IM transfection of genes encoding angiogenic cytokines, particularly those that are naturally secreted by intact cells, may constitute an alternative treatment strategy for patients with extensive peripheral vascular disease in whom the use of intravascular catheter–based gene transfer is compromised and/or prohibited.
Key Words: growth substances • collateral circulation • genes • muscles
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Introduction |
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VEGF,1 also known as vascular permeability factor,2 is an endothelial cell–specific mitogen that has been shown to stimulate the development of collateral arteries in animal models of peripheral3 4 5 6 and myocardial7 8 9 ischemia. This strategy has been termed "therapeutic angiogenesis."
Because no recombinant protein formulation of any of the three principal VEGF isoforms10 is currently approved or available for human clinical application, we investigated arterial gene transfer of VEGF as an alternative strategy for accomplishing therapeutic angiogenesis in patients with limb ischemia.11 Administration of VEGF in this fashion is particularly appealing because the first exon of the VEGF gene includes a signal sequence that permits the protein to be naturally secreted from intact cells.1 Previous studies from our laboratory12 13 suggested that arterial gene transfer of cDNA encoding for a secreted protein could potentially yield meaningful biological outcomes despite a low transfection efficiency. Indeed, preclinical animal studies of phVEGF165 applied to the hydrogel polymer coating of an angioplasty balloon14 augmented collateral vessel development when delivered percutaneously to the iliac artery of rabbits in which the femoral artery had been excised to cause unilateral hindlimb ischemia.15 Preliminary clinical application of this strategy to patients with critical ischemia of the distal portion of one lower extremity,11 in which gene transfer is performed in a residual patent artery of the proximal limb, has demonstrated angiographic and histological evidence of angiogenesis.16
Despite these encouraging findings, evaluation of candidates for phVEGF165 gene therapy has exposed certain potential limitations of arterial gene transfer, particularly for lower extremity ischemia. By definition, arterial gene transfer requires access to a satisfactory arterial donor site in the lower extremity circulation. In patients with critical limb ischemia, several factors may conspire to compromise such access. Lower extremity vascular disease is often so extensive that conventional sites for arterial puncture cannot be accessed percutaneously. Arterial sites that are patent may be nevertheless diffusely diseased by atherosclerosis.17 Even in the absence of a thickened neointima, extensive calcific deposits at the intimal/medial interface ("Monckeberg's disease"18 ) may limit gene transfer to the smooth muscle cells of the arterial media and/or make the vessel so brittle that balloon inflation fractures the calcified vessel,19 leading to unpredictable abrupt vessel closure; this complication may be devastating if the involved artery is the major donor of preexisting collaterals or the only patent vessel supplying the ischemic limb. Even if arterial access is possible in such patients, it is often limited to the uppermost portion of the limb, 
60 cm from sites in the distal limb at which ischemia and/or necrosis is most profound. Because recent studies have demonstrated evidence of paracrine-mediated endothelial cell upregulation of VEGF receptors by conditioned media of hypoxic muscle,20 there may be a tactical advantage to positioning the putative sites of constitutive VEGF synthesis in closer proximity to the ischemic focus.
IM gene transfer, which was pioneered by Wolff and colleagues,21 22 23 represents a less invasive alternative to arterial transfection. Striated muscle has been shown to take up and express foreign genes transferred in the form of "naked" plasmid DNA, ie, DNA unassociated with viral or other adjunctive vectors. IM gene transfer of naked plasmid DNA would be potentially advantageous because it obviates immunological concerns associated with adenoviral vectors.24 Because naked plasmid DNA injected IM remains in a nonreplicative, unintegrated, circular form,21 this strategy also is unlikely to be complicated by insertional mutagenesis.
Although IM gene transfer of naked DNA would thus appear to address the technical limitations of arterial gene transfer in particular and certain safety issues relevant to cardiovascular gene therapy in general, the magnitude of gene expression resulting from IM transfection has been a subject of further concern. Wolff et al23 documented reporter gene expression for as long as 19 months after IM transfection with naked DNA but concluded that the use of this approach for treatment of Duchenne's myopathy did not achieve satisfactory levels of dystrophin.25 Subsequent investigators have been more optimistic when naked DNA was administered IM for use as a vaccine.26 27 28 29 30 In the case of phVEGF165, we considered that the secreted features of the gene product might permit a level of gene expression sufficient to achieve therapeutic angiogenesis. Accordingly, the present study was undertaken to test the hypothesis that IM injection of naked plasmid DNA encoding the 165–amino acid isoform of VEGF could augment collateral development and tissue perfusion in the setting of experimentally induced hindlimb ischemia.
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Methods |
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Plasmids
Complementary DNA clones for recombinant human VEGF165, isolated from cDNA libraries prepared from HL-60 leukemia cells, were assembled into a simple eukaryotic expression plasmid that uses the 736-bp CMV promoter/enhancer to drive VEGF expression. Downstream from the VEGF cDNA is an SV40 polyadenylation sequence. Also included in this plasmid is a fragment containing the SV40 origin of replication that includes the 72-bp repeat, but this sequence is not functionally relevant (for autonomous replication) in the absence of SV40 T antigen. These fragments occur in the pUC118 vector, which includes an Escherichia coli origin of replication and the ß-lactamase gene for ampicillin resistance. The biological activity of VEGF secreted from cells transfected with this construct (phVEGF165) was previously confirmed by evidence that media conditioned by transfected human 293 cells promoted the proliferation of capillary endothelial cells.1
The plasmid pGSVLacZ (courtesy of Dr Claire Bonnerot) containing a nuclear targeted ß-galactosidase sequence coupled to the SV40 early promoter31 was used for the control transfection experiments. To specifically evaluate transfection efficiency, a promoter-matched reporter plasmid, pCMVß (Clontech), that encodes ß-galactosidase under control of CMV promoter/enhancer was used. Finally, pCMVluc, encoding firefly luciferase under control of CMV promoter/enhancer, was used for chemiluminescent detection of transgene expression in transfected and remote tissues.
Animal Model
New Zealand White rabbits with surgically induced unilateral hindlimb vascular insufficiency3 32 were used for all experiments. All protocols were approved by the Institutional Animal Care and Use Committee. Fifty-seven male New Zealand White rabbits (mean weight, 3 kg) were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg). Briefly, the femoral artery was completely excised from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates into the saphenous and popliteal arteries. Prior studies have documented that this surgical procedure produces severe limb ischemia, including reduced TcO233 ; increased femoral venous lactate; and skeletal muscle necrosis.33
IM Gene Transfer
Thirty-one rabbits were used specifically to study the effects of IM gene therapy on hindlimb ischemia. An interval of 10 days was permitted for postoperative recovery, including development of spontaneous collateral vessels, after which the rabbits were returned to the catheterization laboratory. After completion of the baseline physiological measurements (vide infra), five different sites in three major thigh muscles received direct injections with plasmid DNA with the use of a 3-mL syringe and a 27-gauge needle advanced through a small skin incision. For each injection, the tip of the needle was inserted typically to a depth of 3 to 5 mm in the adductor (two sites), medial large (two sites), and semimembranous (one site) muscle. Relative to the site of vessel "occlusion" and "reconstitution," these muscles are located between the origin of the excised femoral artery and the origin of the popliteal (reentry) artery, ie, in the avascular area created by femoral artery resection. To avoid through-and-through needle penetration of the injected muscles and to ensure that there is no leakage of injectate from the puncture site, each injection was performed slowly (
5 seconds) under direct visualization. This technique was used to administer 500 µg of phVEGF165 (n=16) or 500 µg of pGSVLacZ (n=15). This dose of plasmid DNA was determined to be optimal based on preliminary dose-finding studies in this animal model. After the completion of five injections (100 µg/0.5 mL at each of five sites, for a total of 500 µg/2.5 mL for each animal), the skin was then closed with 4-0 nylon sutures.
Anatomic Assessment
Selective Angiography
Selective internal iliac angiography was performed as previously described3 with an automated angiographic injector (Medrad) programmed to reproducibly deliver a total of 5 mL of nonionic contrast media (Isovue-370; Squibb Diagnostics) at a flow rate of 1 mL/s through a 3F infusion catheter (Tracker-18; Target Therapeutic) positioned in the internal iliac artery; the interspace between the seventh lumbar and the first sacral vertebrae was used as a constant reference site for positioning of the catheter tip.
Morphometric angiographic analysis of collateral vessel development was performed using a grid overlay that comprised 2.5-mm-diameter circles arranged in rows spaced 5 mm apart. This overlay was placed over the 4-second angiogram recorded at the level of the medial thigh. The number of contrast-opacified arteries crossing over circles and the total number of circles encompassing the medial thigh area were counted in a single blind fashion. An angiographic score was calculated for each film as the number of circles crossed by opacified arteries divided by the total number of circles in the defined area of the ischemic thigh.
Capillary Density and Capillary-to-Myocyte Ratio
Vascular density was also evaluated at the capillary level using light microscopic sections taken from the ischemic hindlimb.3 Transverse tissue sections harvested from the adductor muscle of the ischemic limb when the animals were killed (day 30) were embedded in O.C.T. compound (Miles), snap-frozen in liquid nitrogen, and cut into 5-µm-thick sections. Tissue sections were stained for alkaline phosphatase with an indoxyl-tetrazolium method to detect capillary endothelial cells as previously described34 and then were counterstained with eosin. A total of 20 randomly selected fields from one muscle section were examined under a x20 objective to determine the mean number of capillaries/mm2 (capillary density); the numbers of myofibers in each field were also counted to calculate the capillary-to-myocyte ratio.
Physiological Assessment
Calf Blood Pressure
The diminutive size of the distal arteries of the rabbit ischemic hindlimb precludes serial in vivo measurement. Accordingly, the pulse of the posterior tibial artery was identified with a Doppler probe, and the systolic blood pressure in both limbs was determined with standard techniques.3 The calf blood pressure ratio was defined for each rabbit as the ratio of systolic pressure of the ischemic limb to that of the normal limb.
In Vivo Measurement of Resting and Maximum Blood Flow
Blood flow was quantified in vivo before selective internal iliac angiography on days 0 and 30 with a 0.018-in Doppler guide wire (Cardiometrics) as previously described.35 The wire was advanced via the 3F infusion catheter positioned at the origin of the common iliac artery to the proximal segment of the internal iliac artery supplying the ischemic limb. APV was recorded at rest; maximum APV was recorded after bolus injection of 2 mg of papaverine. After the completion of identical measurements in the normal limb, selective internal iliac angiography was performed as described above.
Angiographic luminal diameter of the iliac artery was determined with an automated edge-detection system (Quantum 2000I; QCS) as previously described.35 The luminal diameter was measured at the site of the Doppler sample volume (5 mm distal to the wire tip). Cross-sectional area was calculated assuming a circular lumen.
Doppler-derived flow was calculated as QD=(
d2/4)(0.5xAPV), where QD is Doppler-derived time-averaged flow, d is vessel diameter, and APV is time average of the spectral peak velocity. The mean velocity was estimated as 0.5xAPV by assuming a time-averaged parabolic velocity profile across the vessel. The Doppler-derived flow calculated in this fashion has been shown to correlate with flow measurements determined with electromagnetic flowmeters both in vitro and in vivo.35 Because 2 mg of papaverine had no effect on vessel diameter,35 the diameter measurements from the angiogram recorded immediately before the Doppler measurements were used to calculate both rest and maximum flow.
Regional Blood Flow to Limb Muscles
Regional perfusion of the ischemic hindlimb muscles in the resting state was determined with colored microspheres (15-µm diameter).36 After the completion of the invasive measurements described above, 3x106 of Dye-Trak colored microspheres (Triton Technology) were injected through a 3F Teflon catheter into the left ventricle via the common carotid artery. To collect blood samples for reference blood flow, a second catheter, which was connected to a syringe withdrawal pump (Sage 351; Orion Research), was inserted into the distal abdominal aorta via the common carotid artery. Through this catheter, the blood sample was withdrawn beginning 10 seconds before microsphere injection and then continued for 3 minutes at a rate of 1.2 mL/min. The animals were then killed, and tissue samples (weight, 2 g) of two different muscles (transfected adductor muscle from the thigh and nontransfected gastrocnemius muscle) of each hindlimb (ischemic and nonischemic) were retrieved. The tissue samples and reference blood samples were digested with potassium hydroxide, after which microspheres were retrieved through vacuum filtering. After dyes were extracted from microspheres with dimethylformamide, the photometric absorption of each sample was determined with a spectrophotometer (model 8452A; Hewlett Packard). Regional blood flow to muscle was calculated as follows: Tissue Blood Flow=(Withdrawal Rate/Tissue Weight)x(ODtissue/ODreference blood), where OD is optical density.
Transfection Efficiency
The efficiency of transgene expression was evaluated morphometrically in four additional rabbits with the use of a promoter-matched reporter plasmid, pCMVß, administered IM into the muscle of both the ischemic and normal hindlimbs. The transfected muscles were harvested 5 days later, fixed in 1% paraformaldehyde, and incubated with X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactosidase chromogen (Sigma Chemical Co) for 2 hours at 37°C17 ; previous studies in our laboratory have indicated that such limited incubation is sufficient to permit staining of skeletal myocytes transfected with pCMVß while obviating background staining due to endogenous ß-galactosidase activity in nontransfected control muscles (Y. Tsurumi and T. Couffinhal, unpublished data). Harvested tissues were then paraffin embedded, sectioned, and counterstained with hematoxylin and eosin. Five sections from each sample were randomly selected, and the numbers of positive and total myocytes in five low-power fields distributed among an area of skeletal muscle cross section measuring 10 mm in diameter were counted manually for each specimen.
In addition, chemiluminescent analysis of Luciferase activity was performed in four additional rabbits transfected with the promoter-matched reporter plasmid, pCMVluc. At 5 days after transfection, tissue samples from transfected and contralateral adductor muscles, as well as distal organs, including brain, heart, lung, liver, spleen, and testis, were homogenized and prepared for chemiluminescent assay with a commercially available kit (Promega) as previously described.37 Light emission for each sample was counted for 5 seconds with a LUMAT luminometer (Berthold). RLU reading was corrected for protein content of each tissue homogenate determined with a BioRad protein assay kit.
Analysis of VEGF Gene Expression
RT-PCR
Gene expression was evaluated at the mRNA level with RT-PCR in 10 additional male New Zealand White rabbits with hindlimb ischemia that had been killed at 3, 7, 14, and 30 days after transfection with phVEGF165 (n=2 at each time point) or pGSVLacZ (n=2 at 7 days after transfection). In the 4 rabbits killed at 3 and 7 days after transfection, remote tissues (eg, gastrocnemius muscle in the same limb; adductor muscle of contralateral limb, brain, heart, lung, liver, spleen, and testis) were also retrieved for analysis of human VEGF mRNA. To ensure specificity and avoid amplification of endogenous rabbit VEGF, each primer was selected from a region that is not conserved between the two species. Sequences of primers used were 5'-GAGGGCAGAATCATCACGAAGT-3' (from exon 2: sense) and 5'-TGAGAGATCTGGTTCCCGAAAC-3' (from exon 8: antisense). The size of the PCR product for VEGF165 was 531 bp. RT-PCR products were analyzed by 2% agarose gel electrophoresis.
Immunohistochemistry
Expression of VEGF protein after IM gene transfer was evaluated by immunostaining. Transfected adductor muscles were harvested at 5 days after IM gene transfer, fixed with methanol, and paraffin embedded. To block endogenous peroxidase activity and nonspecific binding, 5-µm sections were incubated in 3% hydrogen peroxidase and then pretreated with 10% normal horse serum. Specimens were incubated with monoclonal anti-human VEGF165 antibody (Sigma) (20 µg/mL) at 4°C overnight. Bound primary antibody was detected with the avidin-biotin-immunoperoxidase method (Signet). Nonimmune normal mouse IgG was used to confirm specificity. Muscle samples transfected with pGSVLacZ were used as negative controls. In several rabbits, proliferative activity in the ischemic limb muscle at 10 days after induction of ischemia (ie, corresponding to the time of IM gene transfer) was evaluated through immunohistochemical staining of PCNA as previously described.38
Statistical Analysis
Results are expressed as mean±SD. Statistical significance was evaluated with Student's t test. A value of P<.05 was interpreted to denote statistical significance.
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Anatomic Assessment
Angiography
Representative angiograms recorded from both control and VEGF-treated animals at day 30 are shown in Fig 1
. In control animals, collateral artery development in the medial thigh typically appeared unchanged or progressed only slightly in serial angiograms recorded at days 0 and 30 (Fig 1A and 1B). In contrast, in the VEGF-transfected group, marked progression of the collateral artery was observed between days 0 and 30 (Fig 1C and 1D). At baseline (day 0), there were no significant differences in angiographic score between groups (VEGF=0.45±0.09, control=0.42±0.08; P=NS). By day 30, however, the angiographic score of VEGF-transfected animals was significantly improved compared with that of control (VEGF=0.83±0.06, control=0.55±0.07; P<.01) (Fig 2A).
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Capillary Density and Capillary-to-Myocyte Ratio
A favorable effect of IM phVEGF165 gene transfer upon revascularization was also apparent at the capillary level (Figs 2B and 3
). The adductor muscle of ischemic limbs was histologically examined at day 30. Capillary density was increased among VEGF-transfected rabbits versus controls (VEGF=258±27/mm2, control=195±23/mm2; P<.01). Analysis of capillary-to-myocyte ratio yielded similar results (VEGF=0.77±0.09, control=0.53±0.07; P<.01).
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Physiological Assessment
Calf Blood Pressure Ratio
Reduction of the hemodynamic deficit in the ischemic limb after IM VEGF transfection was confirmed through measurement of calf blood pressure (Fig 2C). At 10 days after induction of ischemia (immediately before transfection), calf blood pressure ratio was virtually identical in the two groups (VEGF=0.36±0.04, control=0.34±0.05; P=NS). By day 30 after transfection, blood pressure ratio for the VEGF-transfected group was significantly higher than that for the controls (VEGF=0.85±0.05, control=0.64±0.05; P<.01).
Intra-arterial Doppler Guide Wire Measurements
Resting blood flow and papaverine-stimulated blood flow in the ischemic limb were similar in both groups at day 0 (rest flow: VEGF=14.6±1.7, control=13.6±2.0 mL/min, P=NS; hyperemic flow: VEGF=32.0±3.4 mL/min, control=29.7±3.7 mL/min, P=NS) (Table 1). On day 30, VEGF-transfected animals developed significantly enhanced flow in the ischemic limb, both at rest (VEGF=21.3±3.9 mL/min, control=14.6±1.6 mL/min; P<.01) and after papaverine (VEGF=54.2±12.0 mL/min, control=37.3±8.9 mL/min; P<.01). Baseline and hyperemic flows in the nonischemic limb were similar in both groups at day 0 as well as at day 30.
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Regional Blood Flow to Limb Muscle
Regional blood flow to ischemic limb muscles was analyzed in the resting state with colored microspheres (Table 2). Regional blood flow to the transfected thigh (adductor) muscle (VEGF=4.3±1.6 mL·min-1·100 g of tissue-1, control=2.9±1.2 mL·min-1·100 g of tissue-1; P<.05) as well as distal lower limb (gastrocnemius) muscle (VEGF=3.9±1.0 mL·min-1·100 g of tissue-1, control=2.8±1.4 mL·min-1·100 g of tissue-1; P<.05) were 1.5-fold greater in VEGF-transfected animals. No differences were observed in regional flow among nonischemic muscles of both groups (flow to adductor: VEGF=5.2±1.5 mL·min-1·100 g of tissue-1, control=4.9±1.6 mL·min-1·100 g of tissue-1, P=NS; flow to gastrocnemius: VEGF=4.4±1.0 mL·min-1·100 g of tissue-1, control=4.6±1.2 mL·min-1·100 g of tissue-1, P=NS).
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Histopathology of Muscle in Ischemic Limb
Examination of sections of muscle harvested from the ischemic limb disclosed myocyte necrosis and regenerating myotubes accompanied by mononuclear cell infiltrates (Fig 4A). PCNA immunostaining performed on the tissue sections retrieved 10 days after induction of ischemia (ie, corresponding to the time of IM gene transfer) revealed PCNA-positive cells, which is consistent with myocyte regeneration at this time point (Fig 4B).
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Transfection Efficiency
Efficiency of transgene expression was evaluated morphometrically in four additional rabbits in which both ischemic and nonischemic limb muscles were transfected with pCMVß (500 µg each). In ischemic limb muscles transfected with pCMVß, evidence of successful transfection, indicated by dark-blue staining, was observed in 2.2±1.2% of skeletal myocytes. In contrast, transfection efficiency was significantly less in nonischemic muscles transfected with the same reporter gene (0.3±0.2%, P<.01 versus ischemic muscle; Fig 5A and 5B). Chemiluminescent detection of luciferase activity 5 days after IM transfection with pCMVluc was limited to transfected muscle tissue homogenates only (1.56±0.32x104 RLU/mg of protein). No significant luciferase activity was detected in distal organs, including contralateral muscles, brain, heart, lung, liver, spleen, and testis.
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Analysis of VEGF Gene Expression
RT-PCR
To confirm human VEGF gene expression in the transfected rabbit limb muscle in vivo, we analyzed transfected muscles for the presence of human VEGF mRNA by RT-PCR. As indicated above, to ensure the specificity of RT-PCR for human VEGF mRNA resulting from successful transfection (versus endogenous rabbit VEGF mRNA), primers that we used were selected from a region that is not conserved between the two species. The presence of human VEGF mRNA was detected in adductor muscles from day 3 to day 14 after gene transfection. No human VEGF mRNA was detected in distal tissues, including gastrocnemius muscle in the same limb and adductor muscle of contralateral limb, brain, heart, lung, liver, spleen, and testis. Rabbit adductor muscles injected with pGSVLacZ gene were consistently negative for human VEGF mRNA (Fig 6A and 6B).
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Immunohistochemical Staining
At 5 days after transfection, evidence of gene expression at the protein level was seen in skeletal myocytes; the morphology of the positively immunostained cells suggested that these were typically immature muscle cells (Fig 7). Positive staining could not be localized to infiltrating monocytes, nor was there evidence of noncellular staining in the extracellular matrix. Finally, no positive staining was observed in either specimens retrieved from LacZ-transfected muscles or nontransfected limbs.
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Discussion |
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The results of the present study document the successful transfer and expression of phVEGF165 in skeletal muscles of the ischemic limb, with evidence of increased collateral vessel development, and consequent amelioration in hemodynamic and physiological deficits induced by ischemia. Previous studies have shown that such improvements in perfusion are associated with improved performance of skeletal muscle groups in the treated limb.39 This study is the first to demonstrate the feasibility of the IM gene transfer with naked DNA encoding VEGF for therapeutic angiogenesis in particular and the first to show bioactivity of naked DNA after IM transfection for cardiovascular gene therapy in general. From a clinical standpoint, these findings suggest that IM transfection represents a suitable alternative to arterial gene transfer of phVEGF165 in patients with proximal obstruction of the lower extremity vasculature that precludes catheter access.
The gene transfer strategy that we used is predicated on the seminal work of Wolff and associates,21 22 23 who first demonstrated the feasibility of direct IM gene transfer of naked DNA with a variety of reporter genes. Direct injection of nonviral, covalently closed circular plasmid DNA into skeletal muscle was shown to produce evidence of gene expression in skeletal myocytes for as long as several months. Both Ascadi et al40 and Danko et al41 reported long-term expression of the dystrophin gene in animal models of muscular dystrophy. With the possible exception of DNA vaccines,26 29 30 42 however, the limiting factor associated with the use of naked DNA for direct IM gene transfer has been the level of gene expression required for phenotypic modulation.21 43 44 45
Many of the previously successful applications of IM gene transfer were achieved in young (4- to 6-week-old) mice, in which transgene uptake appears to be highest.46 The present study suggests that the use of IM transfer of naked DNA is neither age nor species specific. VEGF expression was sufficient to yield augmented collateral vessel development associated with improved pressure, flow, and perfusion in adult rabbits with hindlimb ischemia. The increase in angiographically apparent vessels and capillary density has been previously shown to result from VEGF-induced increase in endothelial cell proliferation.47 Consequently, the magnitudes of improvement in calf blood pressure, blood flow (measured by intravascular Doppler analysis), and tissue perfusion (measured by an increase in microsphere distribution to the ischemic thigh and calf muscles) were all statistically significant compared with controls.
Several factors likely contributed to the success of phVEGF165 IM gene transfer. First, plasmid DNA was delivered in a relatively large volume of fluid, directly into the target muscle; the injection was performed slowly to prevent fluid loss from the epimysium. This approach may have allowed for more uniform distribution of the transgene. Wolff et al22 demonstrated that higher and less variable levels of gene expression could be achieved by injecting a larger rather than a smaller volume of plasmid. Similarly, Davis et al48 showed that preinjection of muscles with a relatively large volume of hypertonic sucrose facilitated more uniform distribution and less variable expression of reporter genes.
Second, the skeletal muscle that was the site of gene transfer was ischemic. Recent work by Takeshita et al49 has shown that the transfection efficiency of IM gene transfer is augmented more than fivefold when the injected muscle is ischemic. This finding may be the result of the skeletal muscle regeneration, including stem cell (myoblast) proliferation. Vitadello et al50 reported an 80-fold increase in chloramphenicol acetyltransferase activity after transfection of regenerating versus control muscle. Consistent with this concept, Danko et al44 found that bupivacaine, which produced myonecrosis followed by satellite cell (muscle stem cell) proliferation and myotube formation 1 and 3 days later, may be used to enhance the expression of naked DNA injected IM into striated muscles.
Interestingly, the histological features of muscle retrieved from the ischemic limbs of animals used for the present series of experiments were similar in many respects to findings reported previously for bupivacaine-treated muscle, including myonecrosis, mononuclear cell infiltration, muscle stem cell proliferation, and myotube formation.51 Evidence of myocyte regeneration was in fact demonstrable in the present experiments at the time of IM plasmid administration; this finding may be related to the nearly sevenfold-higher transfection efficiency suggested by morphometric analysis of ß-galactosidase gene expression. Taken together, it is likely that ischemic myonecrosis, a predictable feature of hindlimb ischemia in this animal model,33 led to spontaneous muscle regeneration and, coincidentally, augmented uptake of the transgene.
The ischemic milieu of the transfected hindlimb muscle further contributes to the success of therapeutic angiogenesis, independent of transgene uptake, by modulating VEGF receptor expression. Recent work from our laboratory has demonstrated paracrine induction of the kinase domain receptor (KDR) (13-fold increase in KDR/cell) in endothelial cells exposed to media conditioned by hypoxic myoblasts.20 This finding presumably not only acts to amplify the impact of any given concentration of VEGF but also potentially accounts for the site-specific nature of the angiogenic response in this animal model.5
A third factor contributing to the results achieved with IM transfection of naked DNA in the present study is the secreted nature of the gene product. Previous work with reporter genes12 13 has indicated that low efficiency of transfection with genes encoding for secreted proteins may achieve biologically meaningful effects not realized by transfection with genes encoding for proteins that remain intracellular. Thus, even if VEGF gene expression is limited to a relatively small number of cells, the paracrine effects of the secreted gene product may be sufficient to achieve a meaningful biological effect.
Expression of the human VEGF transgene was detected by RT-PCR for as long as 14 days after gene transfer in the present study. It is assumed, although not yet documented, that expression at the protein level is limited to a similar time frame. This relatively brief duration of transgene expression is likely related to the CMV promoter used in the phVEGF165 plasmid construct. Previous work by Wolff et al21 23 and others44 46 demonstrated that naked DNA constructs that include the RSV promoter appear to express for considerably longer periods of time, although the level of expression obtained may be lower than that obtained with CMV. The addition of selected introns and/or regulatory sequences in the 3' noncoding region of CMV constructs, however, may augment both the magnitude and temporal stability of transgene expression.52 53
Use of a skeletal muscle–specific promoter represents another potential consideration for enhancing the efficiency of IM gene transfer. It is not clear, however, that this strategy would necessarily augment gene expression for the application described in the present series of experiments. Buttrick et al,54 for example, showed that cardiac-specific expression from the rat myosin heavy-chain gene was 20-fold less active than expression from RSV regulatory sequences. Likewise, Vincent et al55 documented similar levels of expression for a pRSV-CAT construct and an MCK-CAT construct (containing the promoter and enhancer of the rabbit creatine phosphokinase [MM] gene) in transfection of rat cardiac and skeletal muscles. More recently, 198.3 chicken skeletal actin promoter (-198 to -1 and +1 to 313 fragment) was found to be 1% as active as RSV-LTR or CMV promoters in direct IM injection of rat heart (C.K. Vincent and C. Walsh, unpublished observations, 1993). Therefore, constitutive viral regulatory sequences (versus tissue-specific muscle promoter) may be optimal even for IM expression of exogenous genes due to their inherently higher activity. It is possible, however, that application of the modifications cited above52 53 to the CMV vector used here might yield more robust gene expression.
The duration of gene expression in the present series of experiments was nevertheless sufficient to permit augmented collateral vessel development. This is consistent with the time course of collateral development reported previously in this animal model after the administration of the recombinant protein, in which maximal endothelial cell proliferation was observed within 5 days of VEGF therapy.47 Provided that a satisfactory clinical benefit has been realized, early cessation of gene expression may represent a safety feature of the strategy proposed in this report, in that the recipient is not exposed indefinitely to increased levels of the transgene product.
In the present study, VEGF mRNA expression was limited to the site of IM injection, consistent with the absence of luciferase expression in organs remote from the site of gene transfer. From a safety standpoint, localized gene expression might obviate concern regarding the effects of VEGF on proliferative retinopathy in diabetic patients56 and growth and development of latent tumors.57 58 59 Furthermore, it is conceivable, although yet unproved, that continuous, local production of VEGF resulting from the transgene may have greater bioactivity than larger or multiple smaller doses or recombinant protein administered by any route (eg, intra-arterial, intravenous, intramuscular).
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Acknowledgments |
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This work was supported in part by an Academic Award in Vascular Medicine (HL-02824) and grant HL-40518 from the Heart, Lung, and Blood Institute, National Institutes of Health.
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Presented in abstract form at the Annual Scientific Sessions of the American College of Cardiology, Orlando, Fla, March 27, 1996.
Received April 18, 1996; revision received July 31, 1996; accepted August 7, 1996.
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1. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989;246:1306-1309.
2. Connolly DT, Hewelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel RN, Leimgruber RS, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest. 1989;84:1470-1478.
3. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis: a single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hindlimb model. J Clin Invest. 1994;93:662-670.
4.
Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor. Circulation. 1995;91:2802-2809.
5. Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Site-specific therapeutic angiogenesis following systemic administration of vascular endothelial growth factor. J Vasc Surg. 1995;21:314-325.[Medline] [Order article via Infotrieve]
6. Takeshita S, Pu L-Q, Zheng L, Ferrara N, Stein LA, Sniderman AD, Isner JM, Symes JF. Vascular endothelial growth factor induces dose-dependent revascularization in a rabbit model of persistent limb ischemia. Circulation. 1994;90(suppl II):II-228-II-234.
7.
Banai S, Jaklitsch MT, Shou M, Lazarous DF, Scheinowitz M, Biro S, Epstein SE, Unger EF. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. 1994;89:2183-2189.
8. Pearlman JD, Hibberd MG, Chuang ML, Harada K, Lopez JJ, Gladston SR, Friedman M, Sellke FW, Simons M. Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nat Med. 1995;1:1085-1089.[Medline] [Order article via Infotrieve]
9. Hariawala M, Horowitz JR, Esakof D, Sheriff DD, Walter DH, Chaudhry GM, Desai V, Keyt B, Isner JM. Vascular endothelial growth factor accelerates collateral development in porcine hearts. J Surg Res.1996;63:77-82.
10.
Tischer E, Mitchell R, Hartmann T, Silva M, Gospodarowicz D, Fiddes J, Abraham J. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J Biol Chem. 1991;266:11947-11954.
11.
Isner JM, Walsh K, Symes JF, Piezcek A, Takeshita S, Lowry J, Rosenfield K, Weir L, Brogi E, Jurayj D. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. Circulation. 1995;91:2687-2692.
12.
Losordo DW, Pickering JG, Takeshita S, Leclerc G, Gal D, Weir L, Kearney M, Jekanowski J, Isner JM. Use of the rabbit ear artery to serially assess foreign protein secretion after site specific arterial gene transfer in vivo: evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression. Circulation. 1994;89:785-792.
13. Takeshita S, Losordo DW, Kearney M, Isner JM. Time course of recombinant protein secretion following liposome-mediated gene transfer in a rabbit arterial organ culture model. Lab Invest. 1994;71:387-391.[Medline] [Order article via Infotrieve]
14. Riessen R, Rahimizadeh H, Blessing E, Takeshita S, Barry JJ, Isner JM. Arterial gene transfer using pure DNA applied directly to a hydrogel-coated angioplasty balloon. Hum Gene Ther. 1993;4:749-758.[Medline] [Order article via Infotrieve]
15. Takeshita S, Zheng LP, Asahara T, Riessen R, Brogi E, Ferrara N, Symes JF, Isner JM. In vivo evidence of enhanced angiogenesis following direct arterial gene transfer of the plasmid encoding vascular endothelial growth factor. Circulation. 1993;88(suppl I):I-476. Abstract.
16. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes J. Early report: clinical evidence of angiogenesis following arterial gene transfer of phVEGF165. Lancet.1996;348:370-374.
17. Feldman LJ, Steg PG, Zheng LP, Chen D, Kearney M, McGarr SE, Barry JD, Dedieu JF, Perricaudet M, Isner JM. Low-efficiency of percutaneous adenovirus-mediated arterial gene transfer in the atherosclerotic rabbit. J Clin Invest. 1995;95:2662-2671.
18. Lachman AS, Spray TL, Kerwin DM, Shugoll GI, Roberts WC. Medial calcinosis of Monckeberg. Am J Med. 1977;63:615-622.[Medline] [Order article via Infotrieve]
19.
Fitzgerald PJ, Ports TA, Yock PG. Contribution of localized calcium deposits to dissection after angioplasty: an observational study using intravascular ultrasound. Circulation. 1992;86:64-70.
20. Brogi E, Schatteman G, Wu T, Kim EA, Varticovski L, Keyt B, Isner JM. Hypoxia-induced paracrine regulation of VEGF receptor expression. J Clin Invest. 1996;97:469-476.[Medline] [Order article via Infotrieve]
21.
Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. Direct gene transfer into mouse muscle in vivo. Science. 1990;247:1465-1468.
22. Wolff JA, Williams P, Acsadi G, Jiao S, Jani A, Chong W. Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques. 1991;11:474-485.[Medline] [Order article via Infotrieve]
23.
Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet. 1992;1:363-369.
24.
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994;91:4407-4411.
25.
Acsadi G, Jani A, Massie B, Simoneau M, Holland P, Blaschuk K, Karpati G. A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity. Hum Mol Genet. 1994;3:579-584.
26.
Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, Gramkowski SH, Deck RR, DeWitt CM, Friedman A, Hawe LA, Leander KR, Martinez D, Perry HC, Shiver JW, Montgomery DL, Liu MA. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993;259:1745-1749.
27. Tang DJ, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992;356:152-154.[Medline] [Order article via Infotrieve]
28. Hsu C-H, Chua K-Y, Tao M-H, Lai Y-L, Wu H-D, Huang S-K, Hsieh K-H. Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway hyperresponsiveness in vivo by genetic immunization. Nat Med. 1996;2:540-544.[Medline] [Order article via Infotrieve]
29.
McDonnell WM, Askari FK. DNA vaccines. N Engl J Med. 1996;334:42-45.
30. Donnelly JJ, Friedman A, Maretinez D, Montgomery DL, Shiver JW, Motzel SL, Ulmer JB, Liu MA. Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus. Nat Med. 1995;1:583-587.[Medline] [Order article via Infotrieve]
31.
Bonnerot C, Rocancourt D, Briand P, Grimber G. A b-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc Natl Acad Sci U S A. 1987;84:6795-6799.
32.
Pu LQ, Sniderman AD, Brassard R, Lachapelle KJ, Graham AM, Lisbona R, Symes JF. Enhanced revascularization of the ischemic limb by means of angiogenic therapy. Circulation. 1993;88:208-215.
33. Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg. 1992;16:181-191.[Medline] [Order article via Infotrieve]
34. Ziada AM, Hudlicka O, Tyler KR, Wright AJ. The effect of long-term vasodilation on capillary growth and performance in rabbit heart and skeletal muscle. Cardiovasc Res. 1984;18:724-732.[Medline] [Order article via Infotrieve]
35.
Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol. 1993;265:H586-H592.
36.
Kowallik P, Schulz R, Guth BD, Schade A, Paffhausen W, Gross R, Heusch G. Measurement of regional myocardial blood flow with multiple colored microspheres. Circulation. 1991;83:974-982.
37. Leclerc G, Gal D, Takeshita S, Nikol S, Weir L, Isner JM. Percutaneous arterial gene transfer in a rabbit model: efficiency in normal and balloon-dilated atherosclerotic arteries. J Clin Invest. 1992;90:936-944.
38. Majesky MW, Lindner V, Twardzik DR, Schwartz SM. Production of transforming growth factor-ß1 during repair of arterial injury. J Clin Invest. 1992;90:1582-1592.
39. Walder CE, Errett CJ, Ogez J, Heinshon H, Bunting S, Lindquist P, Ferrara N, Thomas GR. Vascular endothelial growth factor (VEGF) improves blood flow and function in a chronic ischemic hindlimb model. J Cardiovasc Pharmacol. 1996;27:91-98.[Medline] [Order article via Infotrieve]
40. Ascadi G, Dickson G, Love DR, Jani A, Walsh FS, Gurusinghe A, Wolff JA, Davies KE. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nat Med. 1991;352:815-818.
41.
Danko I, Fritz JD, Latendresse JS, Herweuer H, Schultz E, Wolff JA. Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection. Hum Mol Genet. 1993;2:2055-2061.
42.
Conry RM, LoBuglio AF, Kantor J, Schlom J, Loechel F, Moore SE, Sumerel LA, Barlow DL, Abrams S, Curiel DT. Immune response to a carcinoembryonic antigen polynucleotide vaccine. Cancer Res. 1994;54:1164-1168.
43. Ledley FD. Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum Gene Ther. 1995;6:1129-1144.[Medline] [Order article via Infotrieve]
44. Danko I, Fritz JD, Jiao S, Hogan K, Latendresse JS, Wolff JA. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Ther. 1994;1:114-121.[Medline] [Order article via Infotrieve]
45. Jiao S, Williams P, Berg RK. Direct gene transfer into nonhuman primate myofibers in vivo. Hum Gene Ther. 1992;3:21-33.[Medline] [Order article via Infotrieve]
46. Wells DJ, Goldspink G. Age and sex influence expression of plasmid DNA directly injected into mouse skeletal muscle. FEBS Lett. 1992;305:203-205.[Medline] [Order article via Infotrieve]
47. Takeshita S, Rossow ST, Kearney M, Zheng LP, Bauters C, Bunting S, Ferrara N, Symes JF, Isner JM. Time course of increased cellular proliferation in collateral arteries following administration of vascular endothelial growth factor in a rabbit model of lower limb vascular insufficiency. Am J Pathol. 1995;147:1649-1660.[Abstract]
48. Davis HL, Whalen RG, Demeneix BA. Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum Gene Ther. 1993;4:151-159.[Medline] [Order article via Infotrieve]
49. Takeshita S, Isshiki T, Sato T. Increased expression of direct gene transfer into skeletal muscles observed following acute ischemic injury in rats. Lab Invest. 1996;75:487-502.[Medline] [Order article via Infotrieve]
50. Vitadello M, Schiaffino M, Picard A, Scarpa M, Schiaffino S. Gene transfer in regenerating muscle. Hum Gene Ther. 1994;5:11-18.[Medline] [Order article via Infotrieve]
51. Hall-Craggs ECB. Early ultrastructural changes in skeletal muscle exposed to the local anaesthetic bupivacaine (Marcaine). Br J Exp Pathol. 1980;61:139-149.[Medline] [Order article via Infotrieve]
52.
Coleman ME, DeMayo F, Yin KC, Lee HM, Geske R, Montgomery C, Schwartz RJ. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem. 1995;270:12109-12116.
53. Manthorpe M, Cornefert-Jensen F, Hartikka J, Felgner J, Rundell A, Margalith M, Dwarki V. Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum Gene Ther. 1993;4:419-431.[Medline] [Order article via Infotrieve]
54.
Buttrick PM, Kass A, Kitsis RN, Kaplan ML, Leinwand LA. Behavior of genes directly injected into the rat heart in vivo. Circ Res. 1992;70:193-198.
55.
Vincent CK, Gualberto A, Patel CV, Walsh K. Different regulatory sequences control creatine-kinase-M gene expression in directly injected skeletal and cardiac muscle. Mol Cel Biol. 1993;13:1264-1272.
56.
Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, Pasquale LR, Theme H, Iwamoto MA, Park JE, Nguyen MD, Aiello LM, Ferrara N, King GL. Vascular endothelial growth factor in ocular fluids of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994;331:1480-1487.
57. Kim KJ, Li B, Winer J, Armanini M, Billett N, Phillips HS, Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature. 1993;362:841-844.[Medline] [Order article via Infotrieve]
58. Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature. 1992;359:845-848.[Medline] [Order article via Infotrieve]
59. Brown LF, Berse B, Jackman RW, Tognazzi K, Guidi AJ, Dvorak HF, Senger DR, Connolly JL, Schnitt SJ. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol. 1995;26:86-91.[Medline] [Order article via Infotrieve]
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
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Z. S. Kyriakides, P. Petinakis, L. Kaklamanis, E. Sbarouni, P. Karayannakos, D. Iliopoulos, I. Dontas, and D. Th. Kremastinos Intramuscular administration of estrogen may promote angiogenesis and perfusion in a rabbit model of chronic limb ischemia Cardiovasc Res, February 16, 2001; 49(3): 626 - 633. [Abstract] [Full Text] [PDF]
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 C. Partovian, S. Adnot, B. Raffestin, V. Louzier, M. Levame, I. M. Mavier, P. Lemarchand, and S. Eddahibi Adenovirus-Mediated Lung Vascular Endothelial Growth Factor Overexpression Protects against Hypoxic Pulmonary Hypertension in Rats Am. J. Respir. Cell Mol. Biol., December 1, 2000; 23(6): 762 - 771. [Abstract] [Full Text]
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J. K. Chae, I. Kim, S. T. Lim, M. J. Chung, W. H. Kim, H. G. Kim, J. K. Ko, and G. Y. Koh Coadministration of Angiopoietin-1 and Vascular Endothelial Growth Factor Enhances Collateral Vascularization Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20(12): 2573 - 2578. [Abstract] [Full Text] [PDF]
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J. Duan, T. Murohara, H. Ikeda, K.-i. Sasaki, S. Shintani, T. Akita, T. Shimada, and T. Imaizumi Hyperhomocysteinemia Impairs Angiogenesis in Response to Hindlimb Ischemia Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20(12): 2579 - 2585. [Abstract] [Full Text] [PDF]
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 S. L. Meyerson, C. L. Skelly, M. A. Curi, and L. B. Schwartz Gene Therapy for Cardiovascular Disease Seminars in Cardiothoracic and Vascular Anesthesia, November 1, 2000; 4(4): 289 - 300. [Abstract] [PDF]
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K. A. Vincent, K.-G. Shyu, Y. Luo, M. Magner, R. A. Tio, C. Jiang, M. A. Goldberg, G. Y. Akita, R. J. Gregory, and J. M. Isner Angiogenesis Is Induced in a Rabbit Model of Hindlimb Ischemia by Naked DNA Encoding an HIF-1{alpha}/VP16 Hybrid Transcription Factor Circulation, October 31, 2000; 102(18): 2255 - 2261. [Abstract] [Full Text] [PDF]
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P.B.J Burton, V.J Owen, S Hafizi, P.J.R Barton, G Carr-White, T Koh, A De Souza, M.H Yacoub, and J.R Pepper Vascular endothelial growth factor release following coronary artery bypass surgery: extracorporeal circulation versus 'beating heart' surgery Eur. Heart J., October 2, 2000; 21(20): 1708 - 1713. [Abstract] [PDF]
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 E. Tanaka, N. Hattan, K. Ando, H. Ueno, Y. Sugio, M. U. Mohammed, S. A. Voltchikhina, and H. Mori Amelioration of microvascular myocardial ischemia by gene transfer of vascular endothelial growth factor in rabbits J. Thorac. Cardiovasc. Surg., October 1, 2000; 120(4): 720 - 728. [Abstract] [Full Text] [PDF]
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 K. Amann and E. Ritz Microvascular disease--the Cinderella of uraemic heart disease Nephrol. Dial. Transplant., October 1, 2000; 15(10): 1493 - 1503. [Abstract] [Full Text] [PDF]
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M. Simons, R. O. Bonow, N. A. Chronos, D. J. Cohen, F. J. Giordano, H. K. Hammond, R. J. Laham, W. Li, M. Pike, F. W. Sellke, et al. Clinical Trials in Coronary Angiogenesis: Issues, Problems, Consensus : An Expert Panel Summary Circulation, September 12, 2000; 102 (11): e73 - e86. [Abstract] [Full Text] [PDF]
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C. Patterson and M. S. Runge Therapeutic Myocardial Angiogenesis Via Vascular Endothelial Growth Factor Gene Therapy : Moving on Down the Road Circulation, August 29, 2000; 102(9): 940 - 942. [Full Text] [PDF]
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P. R. Vale, D. W. Losordo, C. E. Milliken, M. Maysky, D. D. Esakof, J. F. Symes, and J. M. Isner Left Ventricular Electromechanical Mapping to Assess Efficacy of phVEGF165 Gene Transfer for Therapeutic Angiogenesis in Chronic Myocardial Ischemia Circulation, August 29, 2000; 102(9): 965 - 974. [Abstract] [Full Text] [PDF]
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R. J. Lee, M. L. Springer, W. E. Blanco-Bose, R. Shaw, P. C. Ursell, and H. M. Blau VEGF Gene Delivery to Myocardium : Deleterious Effects of Unregulated Expression Circulation, August 22, 2000; 102(8): 898 - 901. [Abstract] [Full Text] [PDF]
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L. H. W. Gowdak, L. Poliakova, X. Wang, I. Kovesdi, K. W. Fishbein, A. Zacheo, R. Palumbo, S. Straino, C. Emanueli, M. Marrocco-Trischitta, et al. Adenovirus-Mediated VEGF121 Gene Transfer Stimulates Angiogenesis in Normoperfused Skeletal Muscle and Preserves Tissue Perfusion After Induction of Ischemia Circulation, August 1, 2000; 102(5): 565 - 571. [Abstract] [Full Text] [PDF]
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I. Baumgartner, G. Rauh, A. Pieczek, D. Wuensch, M. Magner, M. Kearney, R. Schainfeld, and J. M. Isner Lower-Extremity Edema Associated with Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Ann Intern Med, June 6, 2000; 132(11): 880 - 884. [Abstract] [Full Text] [PDF]
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T. O'Brien Adenoviral Vectors and Gene Transfer to the Blood Vessel Wall Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1414 - 1416. [Full Text] [PDF]
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R. C. Hendel, T. D. Henry, K. Rocha-Singh, J. M. Isner, D. J. Kereiakes, F. J. Giordano, M. Simons, and R. O. Bonow Effect of Intracoronary Recombinant Human Vascular Endothelial Growth Factor on Myocardial Perfusion : Evidence for a Dose-Dependent Effect Circulation, January 18, 2000; 101(2): 118 - 121. [Abstract] [Full Text] [PDF]
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 J. F. Symes, D. W. Losordo, P. R. Vale, K. G. Lathi, D. D. Esakof, M. Mayskiy, and J. M. Isner Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease Ann. Thorac. Surg., September 1, 1999; 68(3): 830 - 836. [Abstract] [Full Text] [PDF]
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P. R. Vale, D. W. Losordo, T. Tkebuchava, D. Chen, C. E. Milliken, and J. M. Isner Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping J. Am. Coll. Cardiol., July 1, 1999; 34(1): 246 - 254. [Abstract] [Full Text] [PDF]
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A. Bouloumie, V. B. Schini-Kerth, and R. Busse Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells Cardiovasc Res, March 1, 1999; 41(3): 773 - 780. [Abstract] [Full Text] [PDF]
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I. J. Kullo, R. D. Simari, and R. S. Schwartz Vascular Gene Transfer : From Bench to Bedside Arterioscler. Thromb. Vasc. Biol., February 1, 1999; 19(2): 196 - 207. [Full Text] [PDF]
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K.-G. Shyu, O. Manor, M. Magner, G. D. Yancopoulos, and J. M. Isner Direct Intramuscular Injection of Plasmid DNA Encoding Angiopoietin-1 but not Angiopoietin-2 Augments Revascularization in the Rabbit Ischemic Hindlimb Circulation, November 10, 1998; 98(19): 2081 - 2087. [Abstract] [Full Text] [PDF]
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S. Takeshita, T. Isshiki, M. Ochiai, K. Eto, H. Mori, E. Tanaka, K. Umetani, and T. Sato Endothelium-Dependent Relaxation of Collateral Microvessels After Intramuscular Gene Transfer of Vascular Endothelial Growth Factor in a Rat Model of Hindlimb Ischemia Circulation, September 29, 1998; 98(13): 1261 - 1263. [Abstract] [Full Text] [PDF]
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P. Carmeliet, L. Moons, and D. Collen Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis Cardiovasc Res, July 1, 1998; 39(1): 8 - 33. [Abstract] [Full Text] [PDF]
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 V. Ollivier, S. Bentolila, J. Chabbat, J. Hakim, and D. de Prost Tissue Factor-Dependent Vascular Endothelial Growth Factor Production by Human Fibroblasts in Response to Activated Factor VII Blood, April 15, 1998; 91(8): 2698 - 2703. [Abstract] [Full Text] [PDF]
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I. Baumgartner, A. Pieczek, O. Manor, R. Blair, M. Kearney, K. Walsh, and J. M. Isner Constitutive Expression of phVEGF165 After Intramuscular Gene Transfer Promotes Collateral Vessel Development in Patients With Critical Limb Ischemia Circulation, March 31, 1998; 97(12): 1114 - 1123. [Abstract] [Full Text] [PDF]
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J. Waltenberger Modulation of Growth Factor Action : Implications for the Treatment of Cardiovascular Diseases Circulation, December 2, 1997; 96(11): 4083 - 4094. [Abstract] [Full Text]
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L. J Feldman and G. Steg Optimal techniques for arterial gene transfer Cardiovasc Res, September 1, 1997; 35(3): 391 - 404. [Abstract] [Full Text] [PDF]
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G. Melillo, M. Scoccianti, I. Kovesdi, J. Safi Jr, T. Riccioni, and M. C Capogrossi Gene therapy for collateral vessel development Cardiovasc Res, September 1, 1997; 35(3): 480 - 489. [Abstract] [Full Text] [PDF]
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B. S Lewis, M. Y Flugelman, A. Weisz, I. Keren-Tal, and W. Schaper Angiogenesis by gene therapy: a new horizon for myocardial revascularization? Cardiovasc Res, September 1, 1997; 35(3): 490 - 497. [Abstract] [Full Text] [PDF]
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M. W. Majesky A Little VEGF Goes a Long Way: Therapeutic Angiogenesis by Direct Injection of Vascular Endothelial Growth Factor–Encoding Plasmid DNA Circulation, December 15, 1996; 94(12): 3062 - 3064. [Full Text]
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J. C. Fox and J. L. Swain Angiogenic Gene Therapy: A Leg to Stand On? Circulation, December 15, 1996; 94(12): 3065 - 3066. [Full Text]
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H. Su, R. Lu, and Y. W. Kan Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart PNAS, December 5, 2000; 97(25): 13801 - 13806. [Abstract] [Full Text] [PDF]
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 S. C. FRANCIS, M. K. RAIZADA, A. A. MANGI, L. G. MELO, V. J. DZAU, P. R. VALE, J. M. ISNER, D. W. LOSORDO, J. CHAO, M. J. KATOVICH, et al. Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb? Physiol Genomics, December 21, 2001; 7(2): 79 - 94. [Abstract] [Full Text] [PDF]
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