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(Circulation. 2001;103:1887.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Increased Vascular Endothelial Growth Factor 165 Binding to Kinase Insert Domain–Containing Receptor After Infection of Human Endothelial Cells by Recombinant Adenovirus Encoding the Vegf₁₆₅ Gene

Anat Weisz, PhD; Belly Koren, MSc; Tzafra Cohen, PhD; Gera Neufeld, PhD; Tamar Kleinberger, PhD; Basil S. Lewis, MD, FRCP; Moshe Y. Flugelman, MD

From the Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Lady Davis Carmel Medical Center (A.W., B.K., B.S.L., M.Y.F.); the Unit of Molecular Microbiology (T.K.), Bruce Rappaport School of Medicine; and the Faculty of Biology (T.C., G.N.), Technion-IIT, Haifa, Israel.

Correspondence to Moshe Y. Flugelman, MD, Department of Cardiology, Lady Davis Carmel Medical Center, 7 Michal St, Haifa, Israel 34632. E-mail myf{at}tx.technion.ac.il

	Abstract

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Background—The angiogenic effect of vascular endothelial growth factor (VEGF₁₆₅) is mediated mainly through the high-affinity tyrosine kinase receptor VEGF-R2 (KDR/flk-1). This study examined the effects of VEGF overexpression by primary human endothelial cells (ECs), which do not express VEGF under physiological conditions, on cell proliferation, VEGF binding to the kinase insert domain–containing receptor (KDR), and KDR expression.

Methods and Results—Human primary ECs and SMCs were infected by recombinant adenoviral vector encoding VEGF₁₆₅ (rAdVEGF). Proliferation rate, bromodeoxyuridine incorporation, ¹²⁵I-labeled VEGF₁₆₅ binding to the KDR receptor, and KDR expression were tested in the infected cells and in cells supplemented with VEGF protein. Enhanced proliferation and a significant increase in ¹²⁵I-VEGF₁₆₅ binding to the KDR receptor were induced by rAdVEGF infection of ECs (autocrine effect) as well as by addition of recombinant VEGF₁₆₅ to noninfected cells. Infection of ECs by rAdVEGF led to posttranscriptional upregulation of the KDR receptor, whereas KDR mRNA expression levels remained unchanged. Similar effects were observed with supplemented recombinant VEGF₁₆₅ to noninfected ECs; nevertheless, this phenomenon occurred only with high VEGF₁₆₅ concentrations (10 ng/mL).

Conclusions—The effect of VEGF₁₆₅ on proliferation and upregulation of KDR receptor expression demonstrated an autocrine phenomenon of EC sensitization. The fact that high concentrations of VEGF may be achieved in vivo by local continuous overexpression of VEGF₁₆₅ by gene transfer emphasizes the potential advantage of gene transfer over protein supplementation for therapeutic angiogenesis.

Key Words: genes • cells • viruses

	Introduction

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Vascular endothelial growth factor (VEGF) is a key protein regulating angiogenesis under physiological and pathological conditions.^{1 2 3 4 5} VEGF is a homodimeric glycoprotein identifiable as 5 isoforms (206, 189, 165, 145, and 121 amino acids), generated by alternative splicing from a single gene.⁶ The human VEGF₁₆₅ form was shown to be angiogenic and is secreted from various cells as a soluble factor.^{7 8} Local administration of VEGF₁₆₅ enhanced reendothelialization in denuded arteries in vivo⁹ and increased the formation of new blood vessels in ischemic myocardium and limbs.^{10 11 12} Vegf₁₆₅ gene transfer to vascular tissue was shown to improve blood flow in ischemic limbs and myocardium.^{13 14 15} The use of Vegf₁₆₅ gene transfer to induce therapeutic angiogenesis is currently the focus of several animal and human studies.^{16 17 18 19} Therapeutic angiogenesis has the potential to improve symptoms in patients with ischemic syndromes who are not candidates for mechanical revascularization procedures.^{15 20 21 22 23}

VEGF₁₆₅ binds to the high-affinity tyrosine kinase receptors VEGF-R1 (flt-1) and VEGF-R2 (KDR/flk-1), which are expressed almost exclusively in endothelial cells (ECs).^{24 25 26} In addition, VEGF₁₆₅ binds to the recently identified neuropilin-1 and neuropilin-2 receptors, which are also VEGF₁₆₅ receptors expressed by ECs.²⁷ Activation of the kinase insert domain–containing receptor (KDR) represents a major step in VEGF₁₆₅-initiated angiogenesis. The expression of this receptor is temporally and spatially correlated with VEGF expression during development. Studies using a dominant-negative mutation of flk-1 (the mouse homologue of the KDR receptor) have shown that this receptor plays a major role as a regulator of tumor angiogenesis.^{28 29} Both VEGF₁₆₅ and KDR expression are upregulated by hypoxemia.^{30 31 32 33}

To explore the changes in receptor expression induced by VEGF₁₆₅ overexpression in vascular cells, we developed a recombinant adenoviral vector encoding VEGF₁₆₅ and infected human vascular cells with the recombinant adenovirus. The present study examined VEGF₁₆₅ binding to the KDR receptors in primary human ECs and smooth muscle cells (SMCs) after infection by recombinant adenoviral vector encoding VEGF₁₆₅.

	Methods

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Preparation of Adenoviral Vectors
The recombinant adenoviral vectors expressing the human VEGF₁₆₅ (rAdVEGF) or the bacterial ß-galactosidase (rAdlacZ) genes were constructed by cloning and a homologous recombination procedure in the 293 cell line.³⁴ A 600-bp BamHI fragment containing the human VEGF₁₆₅ cDNA, including the signal sequence for secretion (gift of Dr J. Abraham, Scios Nova, Mountain View, Calif) and the 3700-bp HindIII-BamHI fragment containing the bacterial ß-galactosidase gene were inserted separately into pCA3 plasmid, under the control of the constitutive cytomegalovirus (CMV) immediate early promoter. This expression plasmid also contained the left arm (16%) of the Ad5 genome, with a deletion in the E1 region into which hCMV was inserted.³⁵ The pCA3 plasmid containing the VEGF₁₆₅ or the ß-galactosidase genes was cotransfected with the pJM17 plasmid³⁶ into 293 cells. Homologous recombination between the expression plasmid and pJM17 after transfection replaced the E1 region with the expression cassette from the pCA3 plasmid. Plaque formation occurred between 2 and 4 weeks after cotransfection. Individual plaques were isolated, and the viral extracts were amplified by infection of 293 cells. The titer of each viral stock was determined by plaque assay in 293 cells, and the titers ranged at 10¹⁰ pfu/mL. The presence of the cDNA insert was confirmed by X-gal staining of infected cells for ß-galactosidase expression and Western analysis of growth medium for VEGF secretion.

Cell Culture
ECs were isolated from human saphenous veins (HSVECs) and cultured on gelatin-coated dishes in M-199 medium (Beit Haemek) supplemented with 20% human serum, 100 µg/mL heparin (Sigma), and 2 ng/mL basic fibroblast growth factor. Human ECs were identified by immunohistochemistry using anti–von Willebrand factor–specific antibodies (Zymed). SMCs were cultured by explant outgrowth from human saphenous veins (HSVSMCs) and left internal mammary arteries (HLSMCs). Cells were cultured in DMEM (Beit Haemek) supplemented with 10% FCS (Beit Haemek). SMCs were identified by antibodies specific to -smooth muscle actin (Zymed). The 293 cell line was cultured in DMEM containing 10% FCS.

Cell Proliferation Assay
ECs and SMCs (passages 5 to 11) were seeded 24 hours before adenoviral infection at 30% confluence (5x10⁴ cells/35 mm and 1x10⁵ cells/35 mm, respectively) in medium containing 2% serum and 2 ng/mL basic fibroblast growth factor. The cells were infected (10³ pfu/cell) in serum-free medium with rAdVEGF or rAdlacZ as a control adenoviral vector. Two additional control groups were used: uninfected cells, which were grown in the presence of 2 or 10 ng/mL baculovirus-derived recombinant human VEGF₁₆₅ (added twice daily),³⁷ and a control group of untreated cells. After 90 minutes of exposure to adenoviral vectors at 37°C, serum-containing medium was added, and 16 to 18 hours later, the medium was substituted with M199 containing 7% human serum (HSVECs) or DMEM with 2% FCS (HSVSMCs, HLSMCs). Two, 5, 7, and 12 days later, the cells were trypsinized and counted in triplicate by hemacytometer. Cell proliferation was also measured quantitatively by bromodeoxyuridine (BrdU) immunohistochemistry assay according to the manufacturer’s recommendation (Zymed).

Northern Analysis for KDR RNA
Total RNA (10 to 20 µg) isolated³⁸ from HSVECs was fractionated on a 1.2% formaldehyde-agarose gel and transferred to nytrane filters (MSI). The probe, a 791-bp fragment containing the KDR gene, was labeled with ³²P by random priming.³⁹ After hybridization, filters were washed and autoradiographed on Kodak BS-XAR film at -80°C. The radioactivity intensity was measured after the films had been photographed and analyzed with an Electrophoresis Documentation and Analysis System (EDAS 12, Kodak). Differences in RNA loading amounts were calculated according to the 28S band intensity detected in the ethidium bromide–stained gel.

Western Blot Analysis for VEGF₁₆₅
ECs and SMCs were infected as described above. At days 2, 5, 7, and 12 after infection, the supernatants were collected to test for VEGF₁₆₅ secretion. Secreted VEGF₁₆₅ was extracted from supernatant proteins through a heparin-sepharose column (Pharmacia). Extracted samples were loaded on 12.5% SDS-polyacrylamide gel under reducing conditions and electroblotted onto nitrocellulose (Schleicher & Schuell). A 1:700 dilution of rabbit polyclonal antibody against the amino-terminal epitope (1 to 20 amino acids) of VEGF₁₆₅ (Santa Cruz) was used. After exposure to a peroxidase-conjugate secondary antibody (Sigma), blots were developed with ECL reagents (Sigma) and autoradiographed.

³⁵S-Methionine Labeling and KDR Immunoprecipitation
Three days after infection of ECs by adenoviral vectors, the growth medium was replaced with 2 mL DMEM, high glucose, without methionine. After 2 hours of incubation at 37°C, the medium was replaced by 2.5 mL DMEM (without methionine) and 10 µg/mL ³⁵S-methionine. After overnight incubation, the cells were lysed with 1 mL lysis buffer containing 1% nonident P-40, 0.5% deoxycholate, and protease inhibitors. The lysates were centrifuged briefly to remove insoluble debris, and 45 µL of anti-KDR agarose-conjugated antibody (SC-504AC, Santa Cruz) was incubated for 6 hours at room temperature. After 3 washes with cold PBS (5 minutes each), the KDR receptors were eluted by 3 minutes of boiling in sample buffer and separated on a 6% SDS-polyacrylamide gel.

Iodination of VEGF₁₆₅ and Receptor Cross-Linking
Iodination of 5 µg of human recombinant VEGF₁₆₅ was performed by the chloramine T method. Free iodine was separated from ¹²⁵I-VEGF₁₆₅ by heparin-sepharose affinity chromatography. The specific activity of ¹²⁵I-VEGF₁₆₅ was 0.5 to 1.5x10⁵ cpm/ng.²⁶ HSVECs and HSVSMCs were grown in 60- or 35-mm dishes. The cells were washed with cold PBS, and the binding of ¹²⁵I-labeled VEGF₁₆₅ was carried out in binding buffer containing DMEM, 25 mmol/L HEPES (pH 7.4), 1 µg/mL heparin, and 0.1% gelatin for 2 hours at 4°C. In cross-linking experiments, the washed cell layer was incubated with disuccinimidyl suberate, followed by SDS-polyacrylamide gel electrophoresis, chromatography of cell lysates, and autoradiography.²⁶ The gels were stained with Coomassie blue to assess the amounts of loaded protein. The intensity of the bands was evaluated by densitometry (EDAS 120, Kodak) and adjusted to protein amounts detected by scanning of the stained gels. The effect of suramin on ¹²⁵I-VEGF₁₆₅ binding to the receptors was examined by addition of 0.2 mmol/L suramin (Sigma) to the growth medium after adenoviral infection.

	Results

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Effect of rAdVEGF on EC and SMC Proliferation
EC infection with rAdVEGF led to a significant increase in EC number compared with the control groups (rAdlacZ-infected and uninfected cells) (P<0.0001 for days 2, 5, 7, and 12 by ANOVA). The enhanced EC proliferation was similar to the mitogenic effect observed by addition of 2 ng/mL recombinant VEGF₁₆₅ to uninfected EC growth medium (Figure 1). Similar findings were observed with BrdU incorporation for identification of DNA-synthesizing cells. Enhanced BrdU incorporation rate (up to 5-fold) compared with the control group was detected 5 days after Vegf₁₆₅ gene transfer by recombinant adenovirus (data not shown). In contrast, overexpression of VEGF₁₆₅ after rAdVEGF infection in SMCs did not confer a growth advantage over the control groups of rAdlacZ-infected and uninfected cells and had no effect on BrdU incorporation.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of rAdVEGF infection on EC and SMC proliferation rate. HSVECs (5x104 cells/35 mm) and HSVSMCs (105 cells/35 mm) were seeded 24 hours before infection with adenoviral vectors. Cells infected (103 pfu/cell) with rAdVEGF (solid bars) or with rAdlacZ (hatched bars) and uninfected control cells (open bars) were cultured for 12 days. Uninfected ECs were grown in presence of recombinant human VEGF165 (10 ng/mL) (shaded bars). Cell proliferation was measured by cell counting at different time points: 2, 5, 7, and 12 days after adenoviral infection. Results are expressed as mean±SEM.

VEGF₁₆₅ Expression by Infected ECs and SMCs
Secreted VEGF was detected in conditioned medium samples collected from both human ECs and SMCs infected by rAdVEGF. Two specific bands were detected, 19 and 22 kDa in size, which represent the glycosylated and nonglycosylated forms of VEGF₁₆₅ monomer (Figure 2). These bands exhibited a pattern similar to that of the purified recombinant VEGF₁₆₅ protein, which was used as positive control. In contrast, no VEGF₁₆₅ production was detected in control cells infected by rAdlacZ or uninfected cells. The production of VEGF₁₆₅ protein was detected 2 days after infection and persisted up to 12 days after infection (Figure 2).

View larger version (42K): [in this window] [in a new window]   Figure 2. VEGF165 expression by rAdVEGF-infected ECs and SMCs (Western blotting). ECs and SMCs were infected as described before. Recombinant VEGF165 expression and secretion were examined in supernatants (1 mL). Extracted samples (50 µL) from different days after infection (bottom) were separated on 12.5% SDS-polyacrylamide gel under reducing conditions. Lane A, Control, uninfected cells; B, cells infected with rAdlacZ; and C, cells infected with rAdVEGF.

¹²⁵I-VEGF₁₆₅ Binding to KDR Receptor
KDR receptor expression was examined by affinity cross-linking analysis at 2 different time points (2 and 5 days) after adenovirus infection of HSVECs. The ¹²⁵I-VEGF₁₆₅ binding pattern of HSVECs was similar to the recognized pattern found in human umbilical vein ECs (HUVECs).²⁶ Two specific major bands were detected: the upper band (220 kDa), which represents the KDR receptor, and the lower band, which was recently identified as the neuropilin receptor (Figure 3).²⁷ rAdVEGF infection of ECs led to enhanced ¹²⁵I-VEGF₁₆₅ binding to its specific receptors. A 3-fold increase in VEGF₁₆₅ binding to the KDR receptor was detected 2 days after infection (Figure 3A), and further augmentation (6-fold) at day 5, compared with the control groups (Figure 3B). Similar results were obtained by the addition of conditioned medium collected from rAdVEGF-infected ECs as well as addition of 10 ng/mL recombinant VEGF protein to noninfected cells (6.4- and 7.2-fold increase in KDR binding, respectively, Figure 3B, lanes 4 and 6). Addition of a lower concentration of recombinant VEGF (2 ng/mL) to noninfected ECs, however, did not induce increased KDR binding (Figure 3B, lane 4). In contrast to rAdVEGF-infected cells, ¹²⁵I-VEGF₁₆₅ binding was not changed by infection with the adenoviral control vector (rAdlacZ).

View larger version (40K): [in this window] [in a new window]   Figure 3. Binding and cross-linking of 125I-VEGF to specific receptors expressed on HSVECs. VEGF receptor expression in ECs was detected at day 2 (A) and day 5 (B) after adenoviral infection. Cells were incubated with 125I-VEGF (1 or 5 ng/mL as in A). Binding specificity was detected by competition in presence of 1.5 µg/mL unlabeled recombinant VEGF165 (marked as + lanes at top of A). Lanes in both panels: 1, control uninfected cells; 2, cells infected with rAdlacZ; 3, cells infected with rAdVEGF; 4, uninfected ECs supplemented with recombinant VEGF165 (2 ng/mL); 5, uninfected ECs supplemented with recombinant VEGF165 (10 ng/mL); and 6, uninfected ECs supplemented with conditioned medium collected from rAdVEGF-infected cells. Table at bottom presents KDR and neuropilin band intensity (5 ng/mL 125I-VEGF–treated cells) in relative values by densitometry and after standardization for protein quantity loaded on gel.

In accordance with the increased KDR binding, an increased binding of ¹²⁵I-VEGF₁₆₅ to the neuropilins was observed (4- to 5-fold increase). Both neuropilin receptors (1 and 2) specifically bind the VEGF₁₆₅ isomer. Because there is a size similarity between neuropilin 1 and 2, we assume that the increased binding observed relates to both receptors.

The binding and cross-linking assay was performed separately with 2 different concentrations of labeled protein, 1 and 5 ng/mL of ¹²⁵I-VEGF₁₆₅ (Figure 3A). A concentration of 1 ng/mL ¹²⁵I-VEGF₁₆₅ was found to saturate KDR receptors in both control groups (cells infected with rAdlacZ and uninfected cells), because binding intensity did not increase with higher concentration (5 ng/mL) of labeled protein. Nonetheless, rAdVEGF-infected cells exhibited a 3-fold increased binding in the presence of 5 ng/mL ¹²⁵I-VEGF₁₆₅ compared with binding at a concentration of 1 ng/mL. To prove specificity of ¹²⁵I-VEGF₁₆₅ binding, 1 µg/mL unlabeled VEGF was added to the assay. An excess of VEGF eliminated binding because of competition of the unlabeled ligand (Figure 3A). These phenomena indicate that KDR binding of VEGF₁₆₅ was enhanced in ECs overexpressing VEGF₁₆₅ after gene transfer.

To test whether ECs overexpressing VEGF are activated by an autocrine mechanism, we used suramin, a polyanionic drug known to inhibit growth factor–receptor interaction, to limit the external effect of VEGF. Addition of 0.2 mmol/L suramin to EC growth medium after infection by rAdVEGF led to a moderate (40%) decrease in ¹²⁵I-VEGF binding intensity (data not shown). Suramin inhibited VEGF binding both to KDR receptors and to neuropilins. Furthermore, addition of suramin to ECs that were supplemented with 2 ng/mL exogenous VEGF₁₆₅ eliminated the basal levels of VEGF binding (data not shown). Because the suramin concentration used was subcytotoxic to the endothelial cells, no higher concentrations were tested. These results imply that an autocrine mechanism is responsible for the increased proliferation observed in cells overexpressing VEGF. Because a high concentration of VEGF₁₆₅ would be needed in vivo to induce KDR upregulation for induction of the angiogenic switch and because supplemented VEGF is subjected to degradation and dilution in tissue fluids, we conclude that an autocrine activation after gene transfer is superior to protein supplementation.

KDR Expression by Infected ECs
The effect of VEGF₁₆₅ overexpression by rAdVEGF-infected endothelial cells on KDR gene expression was examined by Northern analysis. Levels of KDR mRNA expressed by untreated HSVECs at baseline remained unchanged after rAdVEGF infection and the addition of 10 g/mL recombinant VEGF protein (Figure 4). To examine the effect of VEGF overexpression on KDR protein expression, an immunoprecipitation assay was performed. A significant increase in KDR protein levels was detected in rAdVEGF-infected ECs (Figure 5, lane 3), in uninfected cells supplemented with exogenous recombinant VEGF protein (lane 4), and in uninfected ECs supplemented with rAdVEGF-infected EC conditioned medium (lane 5). From these experiments, we concluded that increased expression of KDR was due to posttranscriptional regulation.

View larger version (61K): [in this window] [in a new window]   Figure 4. KDR mRNA expression in ECs after adenoviral infection (Northern analysis). Total RNA was extracted 7 days after EC infection by recombinant adenoviral vectors. Expression of KDR and control 28S mRNA is presented. Lanes: 1, uninfected cells; 2, cells infected with rAdlacZ; 3, cells infected with rAdVEGF; and 4, uninfected cells grown in presence of 10 ng/mL recombinant VEGF165. Each lane was loaded with 10 µg total RNA.

View larger version (19K): [in this window] [in a new window]   Figure 5. Immunoprecipitation of KDR receptor. ECs were infected by adenoviral vectors. Four days after infection, cell labeling was performed with 35S-methionine–containing medium. Lanes: 1, uninfected cells; 2, cells infected with rAdlacZ; 3, cells infected with rAdVEGF; 4, uninfected cells grown in presence of 10 ng/mL recombinant VEGF165; 5, uninfected cell supplemented with AdVEGF-infected EC conditioned medium; and 6, positive control, porcine aortic ECs constitutively expressing KDR.

	Discussion

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The major findings of this study were that VEGF overexpression by ECs resulted in autocrine-mediated enhanced proliferation, augmented KDR expression, and increased VEGF₁₆₅ binding to the KDR and neuropilin receptors.

Upregulation of flk1/KDR (VEGFR-2) receptor levels was reported in other experimental systems. Shen et al⁴⁰ showed that VEGF induced upregulation of KDR mRNA and protein expression directly in bovine adrenal cortex endothelial cells via the activation of the receptor’s tyrosine kinase in vitro. Induction of receptor gene transcription was observed in this study, whereas in our experiments, changes were detected in KDR protein levels only. Upregulation of VEGFR-2 protein levels was implicated in mouse cerebral slice cell culture by recombinant VEGF₁₆₅; this effect was inhibited by preincubation with a neutralizing anti-VEGF₁₆₅ antibody.⁴¹ VEGFR-2 upregulation was also demonstrated in avian embryo after transduction by retroviral vectors by 2 groups: in the developing chick limb bud after recombinant quail vegf₁₂₂ gene transfer and in the chorioallantoic membrane by the addition of recombinant human VEGF₁₂₁.^{42 43} These reports focused on induction of VEGFR-2 protein expression. Unlike the previous reports, Brogi et al³² reported no effect on KDR expression in HUVECs by high concentrations of VEGF (100 ng/mL). The conflicting findings of all the cited reports may reflect differences in experimental models, endothelial cell phenotype, and activity of various VEGF preparations.

The exact role of neuropilins in angiogenesis is still unclear. Early data show that semaphorines bind to neuropilins; semaphorine 3 was shown to inhibit EC migration.⁴⁴ Neuropilin-1 mutant mice were shown to exhibit various types of vascular defects, indicating a crucial role for this receptor in the developing embryo⁴⁵ ; still, the physiological consequences of VEGF binding to neuropilin receptors are yet to be fully explored. Our findings may provide a clue to the role of neuropilin, because binding of VEGF was increased in parallel to the increase in KDR binding, but our data cannot determine whether these changes have a synergistic or inhibitory effect. The role of other isoforms of VEGF in KDR activation and binding to neuropilins can be inferred from differences in their properties.⁴⁶ VEGF₁₂₁ is a secreted isoform that lacks the heparin-binding domain; it does not bind to neuropilins.⁴⁷ VEGF₁₈₉ is a nonsecreted isoform and therefore is not expected to produce an autocrine effect in ECs.

The sensitization phenomenon of ECs overexpressing VEGF can be ascribed to the fact that human ECs do not produce VEGF₁₆₅ under physiological conditions. Upregulation of the KDR receptor due to VEGF overexpression represents a positive feedback mechanism that is physiologically important in the process of angiogenesis.

Therapeutic Implications
Upregulation of KDR receptor binding in infected cells may represent a potential advantage of gene transfer over protein treatment. Although direct application of VEGF₁₆₅ protein may be sufficient to induce angiogenesis in animal models and ischemic tissues in humans, its short half-life in the serum and the high concentration needed for upregulation of its receptor may hamper its in vivo efficiency.⁴⁸ The addition of suramin blocked KDR binding in ECs supplemented with 2 ng/mL VEGF, whereas only a moderate reduction was observed in rAdVEGF-infected ECs. The increased binding in our studies may explain the therapeutic effects observed after vegf gene transfer by plasmid vector despite the low efficiency of plasmid gene transfer in vivo.^{10 15} Because KDR upregulation is triggered by VEGF, a process requiring several days, it is reasonable to assume that by the time KDR is upregulated, the concentration of supplemented VEGF will be very low. Gene transfer confers the advantage of an autocrine, prolonged effect on the cell and should produce increased protein expression through a longer time period.

	Acknowledgments

 
This research was supported by a grant from the German Federal Ministry of Education, Science, Research, and Technology (BMBF) and the Israeli Ministry of Science (MOS).

Received June 2, 2000; revision received October 15, 2000; accepted October 23, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Brown LF, Detmar M, Claffey K, et al. Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine. EXS. 1997;79:233–269.[Medline] [Order article via Infotrieve]

2. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435–439.[Medline] [Order article via Infotrieve]

3. Ferrara N, Carver-Moor K, Chen H. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439–442.[Medline] [Order article via Infotrieve]

4. Hanahan D. Signaling vascular morphogenesis and maintenance. Science. 1997;277:48–50.[Free Full Text]

5. Risau W. Mechanisms of angiogenesis. Nature. 1997;386:671–674.[Medline] [Order article via Infotrieve]

6. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J Biol Chem. 1991;266:11947–11954.[Abstract/Free Full Text]

7. Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived stellate cells. Proc Natl Acad Sci U S A. 1989;86:7311–7315.[Abstract/Free Full Text]

8. Leung DW, Cachians G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic factor. Science. 1989;246:1306–1309.[Abstract/Free Full Text]

9. Van Belle E, Maillard L, Tio FO, et al. Accelerated endothelialization by local delivery of recombinant human vascular endothelial growth factor reduces in-stent intimal formation. Biochem Biophys Res Commun. 1997;235:311–316.[Medline] [Order article via Infotrieve]

10. Baumgartner I, Pieczek A, Manor O, et al. Constitutive expression of phVEGF₁₆₅ after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation. 1998;97:1114–1123.[Abstract/Free Full Text]

11. Takeshita S, Pu LQ, Stein LA, 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(suppl II):II-228–II-34.

12. Tsurumi Y, Kearney M, Chen DF, et al. Treatment of acute limb ischemia by intramuscular injection of vascular endothelial growth factor gene. Circulation. 1997;96:382–388.

13. Isner JM, Pieczek A, Schainfeld R, et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Cardiovasc Res. 1996;348:370–374.

14. Tsurumi Y, Takeshita S, Chen D, et al. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation. 1996;94:3281–3290.[Abstract/Free Full Text]

15. Losordo DW, Vale RR, Symes JF, et al. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF₁₆₅ as sole therapy for myocardial ischemia. Circulation. 1998;98:2800–2804.[Abstract/Free Full Text]

16. Bauters C, Asahara T, Zheng LP, et al. Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg. 1995;21:314–324.[Medline] [Order article via Infotrieve]

17. Engler DA. Use of vascular endothelial growth factor for therapeutic angiogenesis. Circulation. 1996;94:1496–1498.[Free Full Text]

18. Muhlhauser J, Merrill MJ, 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]

19. Takeshita S, Zheng LP, 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.

20. Flugelman MY. Vascular gene therapy. Adv Exp Med Biol. 1995;382:269–277.[Medline] [Order article via Infotrieve]

21. Lewis BS, Flugelman MY, Weisz A, et al. Angiogenesis by gene therapy: a new horizon for myocardial revascularization? Cardiovasc Res. 1997;35:490–497.[Abstract/Free Full Text]

22. Pickering JG, Takeshita S, Feldman L, et al. Vascular applications of human gene therapy. Vasc Med. 1998;135:357–364.

23. Schumacher B, Pecher P, von Specht BU, et al. Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease [see comments]. Circulation. 1998;97:645–650.[Abstract/Free Full Text]

24. Devries C, Escobedo JA, Ueno H, et al. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science. 1992;255:989–991.[Abstract/Free Full Text]

25. Terman B, Dougher-Vermazen M, Carrion ME, et al. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun. 1992;187:1579–1586.[Medline] [Order article via Infotrieve]

26. Vaisman N, Gospodarowicz D, Neufeld G. Characterization of the receptors for vascular endothelial growth factor. J Biol Chem. 1990;265:19461–19466.[Abstract/Free Full Text]

27. Soker S, Takashima S, Miao HQ, et al. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform specific receptor for vascular endothelial growth factor. Cell. 1998;92:735–745.[Medline] [Order article via Infotrieve]

28. Millauer B, Shawver LK, Plate KH, et al. Glioblastoma growth inhibited in vivo by a dominant negative Flk-1 mutant. Nature. 1994;367:576–579.[Medline] [Order article via Infotrieve]

29. Rockwell P, Neufeld G, Glassman A, et al. In vitro neutralization of vascular endothelial growth factor activation of FLK-1 by a monoclonal antibody. Mol Cell Diff. 1995;3:91–109.

30. Namiki A, Brogi E, Kearney M, et al. Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells. J Biol Chem. 1994;90:649–652.

31. Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia-initiated angiogenesis. Nature. 1992;359:843–845.[Medline] [Order article via Infotrieve]

32. Brogi E, Schatteman G, Wu T, et al. Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. J Clin Invest. 1996;97:469–476.[Medline] [Order article via Infotrieve]

33. Waltenberger J, Mayr U, Pentz S, et al. Functional upregulation of the endothelial growth factor receptor KDR by hypoxia. Circulation. 1996;94:1647–1654.[Abstract/Free Full Text]

34. Graham FL, Smiley J, Russell WC, et al. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36:59–74.[Abstract/Free Full Text]

35. Bett AJ, Haddara W, Prevec L, et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A. 1994;91:8802–8806.[Abstract/Free Full Text]

36. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988;163:614–617.[Medline] [Order article via Infotrieve]

37. Cohen T, Gitay Goren H, Neufeld G, et al. High levels of biologically active vascular endothelial growth factor (VEGF) are produced by the baculovirus expression system. Growth Factors. 1992;7:131–138.[Medline] [Order article via Infotrieve]

38. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]

39. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983;132:6–13.[Medline] [Order article via Infotrieve]

40. Shen B-Q, Lee DY, Gerber HP, et al. Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. J Biol Chem. 1998;273:29979–29985.[Abstract/Free Full Text]

41. Kremer C, Breier G, Risau W, et al. Up-regulation of flk-1/vascular endothelial growth factor receptor 2 by its ligand in a cerebral slice culture system. Cancer Res. 1997;57:3852–3859.[Abstract/Free Full Text]

42. Flamme I, von Reutern M, Drexler HC, et al. Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation. Dev Biol. 1995;171:399–414.[Medline] [Order article via Infotrieve]

43. Wilting J, Birkenhager R, Eichmann A, et al. VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane. Dev Biol. 1996;176:76–85.[Medline] [Order article via Infotrieve]

44. Miao H-Q, Soker S, Feiner L, et al. Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165. J Cell Biol. 1999;146:233–241.[Abstract/Free Full Text]

45. Kawasaki T, Kitsukawa T, Bekku Y, et al. Requirement for neuropilin-1 in embryonic vessel formation. Development. 1999;126:4895–4902.[Abstract]

46. Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell. 1993;4:1317–1326.[Abstract]

47. Gitay-Goren H, Cohen T, Tessler S, et al. Selective binding of VEGF121 to one of the three vascular endothelial growth receptors of vascular endothelial cells. J Biol Chem. 1996;8:5519–5523.

48. Folkman J. Therapeutic angiogenesis in ischemic limbs [editorial; comment]. Circulation. 1998;97:1108–1110. [Free Full Text]

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