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Intrabody-based strategies for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis¹

²Correspondence: Section of Cardiology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1045, USA. E-mail: dsane{at}wfubmc.edu

SPECIFIC AIMS

We hypothesized that down-regulation of vascular endothelial growth factor receptor-2 (KDR) expression with an intracellular antibody ("intrabody") would inhibit the angiogenic potential of endothelial cells. We examined the effect of the intrabody on KDR cell surface expression, cell viability, proliferation, and tube formation in HUVEC.

PRINCIPAL FINDINGS

1. Plasmid-mediated expression of a tethered intrabody to KDR reduces HUVEC expression of KDR
A single chain antibody to KDR (p3S5, from Imclone Systems) was engineered to have a hemagglutinin antigen (HA) tag to enhance intrabody detection. In a second construct, an endoplasmic reticulum retention signal (KDEL) was added at the carboxyl terminus. The cDNA constructs, designated p3S5-HA and p3S5-HAK, respectively, were expressed using the pIRES-2 vector, which allows for bicistronic expression of the gene of interest and enhanced green fluorescence protein (EGFP). HUVECs were transfected with p3S5-HA and p3S5-HAK, and surface KDR expression was monitored by flow cytometry analysis. Cells were analyzed by EGFP expression so that successfully transfected cells could be distinguished from cells that were not transfected. As shown in Fig. 1 , cells that expressed the tethered intrabody (containing KDEL) had a significant reduction in cell surface expression of KDR.

View larger version (28K): [in this window] [in a new window]   Figure 1. Decreased KDR expression in p3S5-HAK-transfected HUVECs. HUVECs grown in 100 mm tissue culture plates were transfected with 16 µg of plasmid DNA; 48 h later, the cells were stained with anti-KDR and PE-conjugated mouse anti-IgG, followed by flow cytometry analysis. A) Negative control (nontransfected cells with no primary antibody), B) lipofectamine only (no plasmid cDNA), demonstrating baseline KDR expression, C) pIRES2-EGFP control transfected HUVECs, D) p3S5-HA transfected HUVECs, and E) p3S5-HAK transfected HUVECs. The percentage of successfully transfected cells that are KDR (–) is tabulated in the column on the right. The mean of 3 experiments ± SE is shown.

2. Intrabody expression produces a specific inhibition of proliferation in response to VEGF
After transfection with p3S5-HA or p3S5-HAK, cells were sorted according to those expressing (+) or not expressing (–) EGFP. A [³H]-thymidine incorporation assay was performed on EGFP-positive cells in response to VEGF₁₆₅ (15 ng/mL) or bFGF (10 ng/mL). Cells expressing the tethered (p3S5-HAK) intrabody had reduced [³H]-thymidine incorporation (*P<0.025) in response to VEGF, but not in response to bFGF. Furthermore, p3S5-HA cells did not exhibit a reduced proliferative response to VEGF or bFGF. These results demonstrate that the ER retention signal is necessary for the intrabody effect and that the effect is restricted to VEGF-KDR signaling.

3. Expression of tethered intrabody by adenoviral infection results in HUVEC apoptosis
cDNAs for the intrabody constructs were also expressed using a recombinant adenovirus Tet-off system (Clontech) and designated Ad-HA and Ad-HAK. Infection with Ad-HAK, but not Ad-HA or Ad-LacZ produced progressive HUVEC death, with only 7.8 ± 1.3% of the cells remained viable at 48 h. In the presence of doxycycline (100 ng/mL), the apoptosis induced by AD-HAK was completely inhibited. This concentration of doxycycline also completely blocked intrabody expression.

The effect of Ad-HAK infection was analyzed using time-lapsed videomicroscopy (Fig. 2 ). In the absence of doxycycline, 89.8% of cells developed apoptosis 48 h postinfection. No cells undergoing apoptosis were detected in HUVECs that were infected with Ad-HAK in the presence of doxycycline. In addition to the morphological characteristics seen by light microscopy, apoptosis of HUVEC infected with Ad-HAK was verified by demonstrating nuclear fragmentation of DAPI-stained cells and by identification of the activated form of caspase-3.

View larger version (91K): [in this window] [in a new window]   Figure 2. Examples of apoptotic cells. HUVEC were infected with Ad-HAK, then apoptosis was analyzed by time-lapsed videomicroscopy. Frames are shown from immediately postinfection ("starting") and at 24 and 48 h postinfection. In the absence of doxycycline, the 89.8% cells have undergone apoptosis at 48 h postinfection.

The apoptosis induced by Ad-HAK was specific to HUVEC and not observed with SMC cells, MCF-7, or BT474 despite the 90% successful infection rate in these cells. These cells had little or no KDR detectable by flow cytometry.

4. Intrabody expression inhibits tube formation
HUVECs infected with Ad-HAK failed to form tubes when grown on ECMatrix. Tube formation was restored in the presence of doxycycline (100 ng/mL). Infection with Ad-HA or Ad-LacZ had minimal or no effect on tube formation. Combined infection with Ad-HAK + Ad-GFP behaved similarly to infection with Ad-HAK alone, indicating that viral infection alone is not adequate to disrupt tube formation.

CONCLUSIONS AND SIGNIFICANCE

We show that an intrabody with a carboxyl-terminal ER retention signal (KDEL) is highly effective in reducing endothelial cell surface expression of VEGFR-2 (KDR). With the plasmid-based strategy, we demonstrated that >70% of the transfected endothelial cells had no detectable surface KDR and had a greatly reduced mitogenic response to VEGF₁₆₅. With the adenoviral system, which resulted in much higher efficiency of expression, we found that the intrabody induced apoptosis and inhibited tube formation. Intrabody expression was essential for these effects as confirmed by control experiments using doxycycline and adenoviruses expressing irrelevant gene products. These effects were KDR dependent, as cells that do not express KDR were not sensitive to the KDR intrabody.

The KDR receptor is an attractive anti-angiogenesis target, since it is expressed almost exclusively on activated endothelial cells, in contrast to Flt-1, which is expressed on both proliferating and quiescent endothelial cells. The VEGF-KDR interaction is an important factor in promoting endothelial cell survival by inhibiting apoptosis. A variety of anti-apoptotic signals, including protein kinase phosphatidylinositol 3 kinase (PI3K/Akt), are involved in the endothelial cell survival promoted by VEGF.

The intrabody–KDEL construct was designed to exploit the ER receptor-mediated retention system, binding to both KDR and the retention receptors, thereby preventing the transport of the KDR-intrabody complex to the cell surface. Only a small percentage (27.9%) of receptors were able to elude this trapping mechanism. It is notable that neither p3S5-HA transfection nor Ad-HA infection was effective in reducing KDR expression, consistent with prior observations that tethering with KDEL greatly enhances receptor trapping within the ER. Thus, the expression and receptor binding of intrabody alone are not able to significantly affect the trafficking of the KDR to the cell membrane. We could not detect secreted antibody rebound to neighboring cells and no intrabody was detected in the culture medium of p3S5-HAK-transfected or Ad-HAK-infected cells. Furthermore, the EGFP-negative HUVEC cells derived from the population that were transfected with the p3S5-HAK intrabody vector had [³H]-thymidine incorporation equal to those transfected with the vector. Thus, the inhibition of receptor expression probably occurred by trapping of the KDR within the ER by the KDEL-tagged intrabody.

The intrabody strategy has several potential advantages over previous approaches to inhibiting the VEGF-KDR pathway. By acting intracellularly, the intrabody can evade immune responses that are elicited by extracellular antibodies, thereby avoiding the formation of neutralizing antibodies. The intrabody can disrupt signaling induced by VEGF binding to intracellular KDR, the "internal autocrine loop," a process that would be difficult to inhibit by other strategies.

In summary, we have shown that a tethered intrabody to KDR decreases cell surface expression of the receptor, resulting in endothelial cell apoptosis. HUVEC expressing the intrabody are unable to form tubes, demonstrating an anti-angiogenic effect. The expression of an intrabody to KDR, especially using the adenoviral strategy, appears to be a promising method for inhibiting tumor angiogenesis.

View larger version (44K): [in this window] [in a new window]   Figure 3. Human endothelial cells express large amount of KDR (trapezoid) on the surface and can form tubes on EHS tumor gel matrix. After the tethered intrabody (bucket) to KDR is expressed in the endoplasmic reticulum (ER) of the endothelial cells, it binds to the newly synthesized KDR and blocks the receptor transport to the cell surface. This leads to decreased surface expression of KDR, the induction of endothelial cell apoptosis and the inhibition of tube formation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0942fje; doi: 10.1096/fj.02-0942fje

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This Article Full Text (PDF) All Versions of this Article: 17/12/1733 02-0942fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WHEELER, Y. Y. Articles by SANE, D. C. Search for Related Content PubMed PubMed Citation Articles by WHEELER, Y. Y. Articles by SANE, D. C.