The recombinant replication-deficient adenovirus encoding dominant negative form of IκB under control of the cytomegalovirus (CMV) promoter, hereafter referred to as AddnIκB, contains a hemagglutinin (HA)-tagged super-repressor IκB. This super-repressor IκB has serine-to-alanine mutations in residues 32 and 36, which inhibit its phosphorylation and proteosome-mediated degradation 14. Virus was grown on HEK293 cells and purified in Hepes/sucrose buffer, pH 8.0, according to conventional double CsCl gradient centrifugation methods, and the number of viral particles was calculated from the optical density at 260 nm (OD₂₆₀). AdLacZ, which contains the E. coli β-galactosidase gene, was grown and purified as described above and used as a control virus. Standard plaque assays were performed to determine the viral particles (vp)/plaque forming unit ratio, which were found to be 15 for both viruses.

For the transduction experiments, HUVECs were plated at 12,500 cells/cm² in 25 cm²-tissue culture flasks (Costar, Cambridge, MA, USA) for western blotting, or in 6-well tissue culture plates (Costar) for flow cytometric analysis and real time RT-PCR, and cultured overnight before starting the experiments. The various viral vectors diluted in Dulbecco's modified Eagle's medium without FCS were added to the HUVECs and incubated for 90 minutes at 37°C. The medium was then replaced by normal endothelial cell culture medium and cells were incubated for another 24 hours to allow transgene production. In the case of competition experiments, cells were incubated with RGD-protein (50 μg/ml), recombinant knob5 (20 μg/ml), or both for 30 minutes at 4°C prior to the addition of viruses.

HUVECs were infected with AddnIκB, AddnIκB-PEG-RGD or AddnIκB-PEG-RAD (3,000 vp/cell) as described. After another 24 hours of culturing, cells were detached from the surface by trypsin/EDTA treatment, lysed in cell culture lysis reagent (Promega Corporation, Madison, WI, USA) and sonicated twice for five seconds. After centrifugation for ten minutes at 10,000g, cleared cell lysates were collected and protein content was determined using the Bradford protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA), using bovine serum albumin as the standard. Samples were then mixed 1:1 with 2 × SDS sample buffer, boiled for 5 minutes, and 30 μg was loaded on SDS-PAGE 10% acrylamide gels followed by blotting to nitrocellulose membranes (Bio-Rad Laboratories). The dnIκB protein was detected using a rabbit anti-HA-tag antibody (sc805; Santa Cruz Biotechnology, Santa Cruz, CA, USA), while both endogenous IκB and dnIκB were detected using a rabbit anti-IκB antibody (sc847; Santa Cruz Biotechnology). Blots were blocked in blocking buffer (5% non-fat drymilk in PBS) for two hours, incubated for one hour with primary antibody diluted in blocking buffer 1:200 (sc805) or 1:250 (sc847) and subsequently with horseradish peroxidase-conjugated swine anti-rabbit antibody (Dako, Glostrup, Denmark) diluted in blocking buffer 1:2,000. Detection was performed using ECL detection reagents (Amersham Corp., Arlington Heigths, IL, USA) according to the manufacturer's protocol.

HUVECs were infected with AddnIκB and AddnIκB-PEG-RGD at 7,500 vp/cell as described. After another 24 hours of culturing, cells were activated with 100 ng/ml TNF-α (Boehringer, Ingelheim, Germany), or left resting. Total RNA was isolated 24hours after activation using the Absolutely RNA Microprep Kit (Stratagene, Amsterdam, The Netherlands) according to the protocol of the manufacturer. RNA yield (OD₂₆₀) and purity (OD₂₆₀/₂₈₀) was measured using a ND-1000 UV- Vis Spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). One μg total cellular RNA was subsequently used for the synthesis of first strand cDNA using SuperScript III RNase H^- Reverse Transcriptase (Invitrogen, Breda, The Netherlands) in 20 μl final volume containing 250 ng random hexamers (Promega) and 40 units RNase OUT inhibitor (Invitrogen). After RT-reaction, cDNA was diluted with distilled water to 100 μl. The following exons overlapping primers and minor groove binder (MGB) probes used for real time RT-PCR were purchased as Assay-on-Demand from Applied Biosystems (Nieuwekerk a/d IJssel, The Netherlands): housekeeping gene GAPDH (assay ID Hs99999905_m1), endothelial cell marker CD31 (PECAM-1 (platelet endothelial cell adhesion molecule 1), Hs00169777_m1), E-selectin (Hs00174057_m1), VCAM-1 (Hs00365486_m1), ICAM-1 (Hs00164932_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), Hs00173626_m1 (hVEGF-A), and Hs00176096 (hTie-2). The final concentration of primers and MGB probes in TaqMan PCR MasterMix (Applied Biosystems, Foster City, CA, USA) for each gene was 900 nM and 250 nM, respectively. As a control, RNA samples not subjected to reverse transcriptase were analyzed to exclude unspecific signals arising from genomic DNA. Those samples consistently showed no amplification signals.

TaqMan real time RT-PCR was performed in an ABI PRISM 7900HT Sequence Detector (Applied Biosystems). Amplification was performed using the following cycling conditions: 2 minutes at 50°C, 10 minutes at 95°C, and 40 to 45 two-step cycles of 15 seconds at 95°C and 60 s at 60°C. Triplicate real time RT-PCR analyses were executed for each sample, and the obtained threshold cycle (Ct) values were averaged. According to the comparative Ct method described in the ABI manual, gene expression was normalized to the expression of the housekeeping gene GAPDH, yielding the ΔCt value. The average ΔCt value obtained from resting HUVECs was then subtracted from the average ΔCt value of each corresponding sample subjected to TNF-α stimulation, yielding the ΔΔCt value. The gene expression level, normalized to the housekeeping gene, and relative to the control sample, was calculated by 2^-ΔΔCt. Data were normalized to untreated, non-activated control HUVECs arbitrarily set at 1.

In our preliminary analysis, we used CD31 as a housekeeping gene since it is constitutively expressed in HUVECs and its expression is NF-κB-independent (JM Kuldo and G Molema, unpublished data. The outcome for all genes studied remained the same as when GAPDH was used as the housekeeping gene. We therefore regarded GAPDH as a good housekeeping gene for use in the experimental conditions used in this study.

HUVECs were infected with AddnIκB (7,500 vp/cell), AdLacZ (7,500 vp/cell) and AddnIκB-PEG-RGD at different amounts of vp/cell as described. After another 24 hours of culturing, cells were activated with 100 ng/ml TNF-α (Boehringer) or left resting. Cells were detached from the surface by trypsin/EDTA 4 hours after activation and resuspended in PBS with 5% FCS. Cells were subsequently centrifuged at 200 × g, after which the cell pellets were incubated for 1 hour at 37°C with 100 μl of primary antibody. The following primary antibodies were used: mouse anti-human ICAM (5/3-2.1, kindly provided by Dr MA Gimbrone Jr, Boston, MA, USA), mouse anti-human CD31 (M0823, Dako) to detect CD31 as a standard marker for endothelial cells, and mouse anti-rat ICAM-1 (1A29, kindly provided from Dr M Miyasaka, Osaka Univ., Osaka, Japan) as an iso-type control. After washing, cells were incubated for one hour with 100 μl rat anti-mouse F(ab')₂-FITC (F0313, Dako). After extensive washing, cells were fixed with 0.5% formalin in PBS. Flow cytometric analysis was performed within 24 hours after fixation using a Coulter Epics-Elite flow cytometer (Coulter Electronics, Hialeah, FL, USA). Data were analyzed using Winlist (version 3D; verity Software House, Topsham, ME, USA) and WinMDI (version 2.8; The Scripps Research Institute, La Jolla, CA, USA) software.

Statistical significance of differences was evaluated by means of the two-sided Student's t test, assuming equal variances. Differences were considered to be significant when p < 0.05.

For the functional validation of the virus itself, we first infected HUVECs with AddnIκB. Western blotting experiments showed that the transgene was successfully expressed upon infection. Furthermore, pre-incubation with recombinant knob strongly inhibited the transduction of AddnIκB while not affecting the expression level of endogenous IκB (Figure 1a). Neither non-infected nor AdLacZ-infected HUVECs expressed the transgene (data not shown). Several genes that are characteristic for the inflammatory responses in endothelial cells contain functional NF-κB binding sites in their promoter regions, leading to enhanced transcription upon NF-κB activation 13. We therefore determined the pharmacological effects of dnIκB transgene expression in HUVECs by analysis of mRNA levels of typical cell adhesion molecules, cytokines and some other angiogenesis-related genes in HUVECs upon TNF-α stimulation (Figure 1b). TNF-α stimulation enhanced mRNA levels of all genes investigated in untreated HUVECs, ranging from 3,778-fold for E-selectin to 3.1-fold for VEGF-A, except for the mRNA level of CD31, the expression of which was non-responsive to TNF-α stimulation (Table 1). This transcriptional induction was completely abolished in dnIκB expressing HUVECs activated with TNF-α. In contrast, transduction with the control virus AdLacZ did not affect the TNF-α induced up-regulation of cell adhesion molecules and the angiogenesis-related genes encoding VEGF-A and Tie-2. In AdLacZ infected HUVECs, IL-6 and IL-8 mRNA levels exhibited higher and lower increases, respectively, upon TNF-α stimulation compared to uninfected HUVECs, which may be a result of viral infection per se. Yet, TNF-α driven increases in mRNA levels of these genes was completely abolished in dnIκB expressing HUVECs. The effect of AddnIκB or AdLacZ infection per se on basal mRNA expression in the absence of TNF-α was within 20% for all genes investigated. This implies that viral infection does not influence basal expression under the conditions studied and, furthermore, that the observed non-responsiveness of dnIκB expressing HUVECs to an inflammatory stimulus was due to NF-κB blockade, and not due to viral infection itself. In all conditions, >80% of the dnIκB expressing HUVECs remained viable, as assessed microscopically as well as by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay (data not shown).

To further confirm the inhibitory effects of the transgene on expression levels of NF-κB driven proteins, we determined the expression level of the transmembrane protein ICAM-1 (Figure 2), mRNA levels of which were shown to be silenced by the transgene (Figure 1b). TNF-α stimulation markedly induced the expression of ICAM-1 protein on the membrane of the endothelial cells. This expression was completely inhibited in HUVECs infected with AddnIκB prior to TNF-α stimulation, while no inhibitory effect was observed after pre-infection with control virus (AdLacZ). In contrast, the constitutively expressed endothelial gene CD31 was not affected by dnIκB (data not shown), thereby corroborating other observations that CD31 expression is NF-κB independent (JM Kuldo and G Molema, unpublished data). From these data, we concluded that the transgene employed could be functionally expressed in the primary endothelial cells without compromising cell viability.

We next confirmed the change in the entry pathway of RGD-retargeted adenovirus into HUVECs from a CAR-dependent to an αvβ3 integrin-dependent mode. Western blotting analysis of HA-tagged dnIκB (Figure 3) demonstrated that non-modified AddnIκB exhibited efficient transduction upon infection to HUVECs. The presence of exogenously added RGD-protein did not affect this transduction, suggesting that the entry pathway of non-modified virus is exclusively CAR-dependent. On the other hand, the transduction of HUVECs by AddnIκB-PEG-RGD was significantly inhibited by the presence of RGD-protein but not by recombinant knob5, while AddnIkB-PEG-RAD showed no transduction at all. These results strongly suggest that RGD modification successfully endowed adenovirus with αv integrin specific infectivity to endothelial cells, and that peptide modification per se was not responsible for directing the tropism of the virus.

To study whether chemically modified AddnIκB exerted therapeutic potential for interference of inflammatory and angiogenic processes, we evaluated mRNA levels of the same set of genes investigated to study functionality of the non-modified virus.

Figure 4 shows that TNF-α driven expression of all pro-inflammatory and pro-angiogenic genes was completely abolished in HUVECs infected with the RGD-PEG modified virus. Moreover, AddnIκB-PEG-RGD exhibited a similar inhibitory effect on gene expression as the non-modified virus (Figure 1b).

mRNA data demonstrating that the chemically modified RGD-PEG-adenovirus could transfer functionally active dnIκB gene into endothelial cells were confirmed by the analysis of ICAM-1 protein expression (Figure 5). The larger the number of viral particles of AddnIκB-PEG-RGD used for the infection, the higher the percentage of ICAM-1dim cells (cells that do not express significant levels of ICAM-1 protein) upon TNF-α activation, ranging from 6% for HUVECs transduced at 1.5 × 10³ vp/cell to 27% for HUVECs transduced at the highest number of viral particles, 15 × 10³ vp/cell. AddnIκB-PEG-RAD did not show any significant inhibitory effect on ICAM-1 protein expression upon TNF-α stimulation (data not shown), which is in line with the absence of dnIκB protein expression in cells exposed to this control virus (see western blot analysis shown in Figure 3).