Antiangiogenic AAV2 gene therapy with a truncated form of soluble VEGFR-2 reduces the growth of choroidal neovascularization in mice after intravitreal injection.

Pathological angiogenesis related to neovascularization in the eye is mediated through vascular endothelial growth factors (VEGFs) and their receptors. Ocular neovascular-related diseases are mainly treated with anti-VEGF agents. In this study we evaluated the efficacy and safety of novel gene therapy using adeno associated virus 2 vector expressing a truncated form of soluble VEGF receptor-2 fused to the Fc-part of human IgG1 (AAV2-sVEGFR-2-Fc) to inhibit ocular neovascularization in laser induced choroidal neovascularization (CNV) in mice. The biological activity of sVEGFR-2-Fc was determined in vitro. It was shown that sVEGFR-2-Fc secreted from ARPE-19 cells was able to bind to VEGF-A165 and reduce VEGF-A165 induced cell growth and survival. A single intravitreal injection (IVT) of AAV2-sVEGFR-2-Fc (1 µl, 4.7 × 1012 vg/ml) one-month prior laser photocoagulation did not cause any changes in the retinal morphology and significantly suppressed fluorescein leakage at 7, 14, 21 and 28 days post-lasering compared to controls. Macrophage infiltration was observed after the injection of both AAV2-sVEGFR-2-Fc and PBS. Our findings indicate that AAV2 mediated gene delivery of the sVEGFR-2-Fc efficiently reduces formation of CNV and could be developed to a therapeutic tool for the treatment of retinal diseases associated with neovascularization.

Novel Designed Proteolytically Resistant VEGF-B186R127S Promotes Angiogenesis in Mouse Heart by Recruiting Endothelial Progenitor Cells.

Background: Previous studies have indicated that vascular endothelial growth factor B186 (VEGF-B186) supports coronary vascular growth in normal and ischemic myocardium. However, previous studies also indicated that induction of ventricular arrhythmias is a severe side effect preventing the use of VEGF-B186 in cardiac gene therapy, possibly mediated by binding to neuropilin 1 (NRP1). We have designed a novel VEGF-B186 variant, VEGF-B186R127S, which is resistant to proteolytic processing and unable to bind to NRP1. Here, we studied its effects on mouse heart to explore the mechanism of VEGF-B186-induced vascular growth along with its effects on cardiac performance. Methods: Following the characterization of VEGF-B186R127S, we performed ultrasound-guided adenoviral VEGF-B186R127S gene transfers into the murine heart. Vascular growth and heart functions were analyzed using immunohistochemistry, RT-PCR, electrocardiogram and ultrasound examinations. Endothelial progenitor cells (EPCs) were isolated from the circulating blood and characterized. Also, in vitro experiments were carried out in cardiac endothelial cells with adenoviral vectors. Results: The proteolytically resistant VEGF-B186R127S significantly induced vascular growth in mouse heart. Interestingly, VEGF-B186R127S gene transfer increased the number of circulating EPCs that secreted VEGF-A. Other proangiogenic factors were also present in plasma and heart tissue after the VEGF-B186R127S gene transfer. Importantly, VEGF-B186R127S gene transfer did not cause any side effects, such as arrhythmias. Conclusion: VEGF-B186R127S induces vascular growth in mouse heart by recruiting EPCs. VEGF-B186R127S is a novel therapeutic agent for cardiac therapeutic angiogenesis to rescue myocardial tissue after an ischemic insult.

Adenoviral VEGF-B186R127S gene transfer induces angiogenesis and improves perfusion in ischemic heart.

Vascular endothelial growth factor B (VEGF-B) is an interesting therapeutic candidate for coronary artery disease. However, it can also cause ventricular arrhythmias, potentially preventing its use in clinics. We cloned VEGF-B isoforms with different receptor binding profiles to clarify the roles of VEGFR-1 and Nrp-1 in angiogenesis and to see if angiogenic properties can be maintained while avoiding side effects. VEGF-B constructs were studied in vivo using adenovirus (Ad)-mediated intramyocardial gene transfers into the normoxic and ischemic porcine heart (n = 51). It was found that the unprocessed isoform VEGF-B186R127S is as efficient angiogenic growth factor as the native VEGF-B186 in normoxic and ischemic heart. In addition, AdVEGF-B186R127S increased myocardial perfusion reserve by 22% in ischemic heart without any side effects. AdVEGF-B127 (VEGFR-1 and Nrp-1 ligand) and AdVEGF-B109 (VEGFR-1 ligand) did not induce angiogenesis. Thus, VEGF-B186 is angiogenic only before its proteolytic processing to VEGF-B127. Only the VEGF-B186 C-terminal fragment was associated with arrhythmias.

Axon Guidance-Related Factor FLRT3 Regulates VEGF-Signaling and Endothelial Cell Function.

Vascular endothelial growth factors (VEGFs) are key mediators of endothelial cell (EC) function in angiogenesis. Emerging knowledge also supports the involvement of axon guidance-related factors in the regulation of angiogenesis and vascular patterning. In the current study, we demonstrate that fibronectin and leucine-rich transmembrane protein-3 (FLRT3), an axon guidance-related factor connected to the regulation of neuronal cell outgrowth and morphogenesis but not to VEGF-signaling, was upregulated in ECs after VEGF binding to VEGFR2. We found that FLRT3 exhibited a transcriptionally paused phenotype in non-stimulated human umbilical vein ECs. After VEGF-stimulation its nascent RNA and mRNA-levels were rapidly upregulated suggesting that the regulation of FLRT3 expression is mainly occurring at the level of transcriptional elongation. Blockage of FLRT3 by siRNA decreased survival of ECs and their arrangement into capillary-like structures but enhanced cell migration and wound closure in wound healing assay. Bifunctional role of FLRT3 in repulsive vs. adhesive cell signaling has been already detected during embryogenesis and neuronal growth, and depends on its interactions either with UNC5B or another FLRT3 expressed by adjacent cells. In conclusion, our findings demonstrate that besides regulating neuronal cell outgrowth and morphogenesis, FLRT3 has a novel role in ECs via regulating VEGF-stimulated EC-survival, migration, and tube formation. Thus, FLRT3 becomes a new member of the axon guidance-related factors which participate in the VEGF-signaling and regulation of the EC functions.

Adenoviral intramyocardial VEGF-DΔNΔC gene transfer increases myocardial perfusion reserve in refractory angina patients: a phase I/IIa study with 1-year follow-up.

AIMS: We evaluated for the first time the effects of angiogenic and lymphangiogenic AdVEGF-DΔNΔC gene therapy in patients with refractory angina. METHODS AND RESULTS: Thirty patients were randomized to AdVEGF-DΔNΔC (AdVEGF-D) or placebo (control) groups. Electromechanical NOGA mapping and radiowater PET were used to identify hibernating viable myocardium where treatment was targeted. Safety, severity of symptoms, quality of life, lipoprotein(a) [Lp(a)] and routine clinical chemistry were measured. Myocardial perfusion reserve (MPR) was assessed with radiowater PET at baseline and after 3- and 12-months follow-up. Treatment was well tolerated. Myocardial perfusion reserve increased significantly in the treated area in the AdVEGF-D group compared with baseline (1.00 ± 0.36) at 3 months (1.31 ± 0.46, P = 0.045) and 12 months (1.44 ± 0.48, P = 0.009) whereas MPR in the reference area tended to decrease (2.05 ± 0.69, 1.76 ± 0.62, and 1.87 ± 0.69; baseline, 3 and 12 months, respectively, P = 0.551). Myocardial perfusion reserve in the control group showed no significant change from baseline to 3 and 12 months (1.26 ± 0.37, 1.57 ± 0.55, and 1.48 ± 0.48; respectively, P = 0.690). No major changes were found in clinical chemistry but anti-adenovirus antibodies increased in 54% of the treated patients compared with baseline. AdVEGF-D patients in the highest Lp(a) tertile at baseline showed the best response to therapy (MPR 0.94 ± 0.32 and 1.76 ± 0.41 baseline and 12 months, respectively, P = 0.023). CONCLUSION: AdVEGF-DΔNΔC gene therapy was safe, feasible, and well tolerated. Myocardial perfusion increased at 1 year in the treated areas with impaired MPR at baseline. Plasma Lp(a) may be a potential biomarker to identify patients that may have the greatest benefit with this therapy.

Snake venom VEGF Vammin induces a highly efficient angiogenic response in skeletal muscle via VEGFR-2/NRP specific signaling.

Vascular Endothelial Growth Factors (VEGFs) are promising molecules for the treatment of ischemic diseases by pro-angiogenic therapy. Snake venom VEGFs are a novel subgroup with unique receptor binding profiles and as such are potential new therapeutic agents. We determined the ligand-receptor interactions, gene regulation and angiogenic properties of Vipera ammodytes venom VEGF, Vammin, and compared it to the canonical angiogenic factor VEGF-A to evaluate the use of Vammin for therapeutic angiogenesis. Vammin efficiently induced VEGFR-2 mediated proliferation and expression of genes associated with proliferation, migration and angiogenesis. VEGF-A165 and especially VEGF-A109 induced less pronounced effects. Vammin regulates a number of signaling pathways by inducing the expression of NR4A family nuclear receptors and regulators of calcium signaling and MAP kinase pathways. Interestingly, MARC1, which encodes an enzyme discovered to catalyze reduction of nitrate to NO, was identified as a novel VEGFR-2 regulated gene. In rabbit skeletal muscle adenoviral delivery of Vammin induced prominent angiogenic responses. Both the vector dose and the co-receptor binding of the ligand were critical parameters controlling the type of angiogenic response from sprouting angiogenesis to vessel enlargement. Vammin induced VEGFR-2/NRP-1 mediated signaling more effectively than VEGF-A, consequently it is a promising candidate for development of pro-angiogenic therapies.

Differential regulation of angiogenic cellular processes and claudin-5 by histamine and VEGF via PI3K-signaling, transcription factor SNAI2 and interleukin-8.

AIMS: Histamine and vascular endothelial growth factor A (VEGF) are central regulators in vascular pathologies. Their gene regulation leading to vascular remodeling has remained obscure. In this study, EC regulation mechanisms of histamine and VEGF were compared by RNA sequencing of primary endothelial cells (ECs), functional in vitro assays and in vivo permeability mice model. METHODS AND RESULTS: By RNA sequencing, similar transcriptional alterations of genes involved in activation of primary ECs, cell proliferation and adhesion were observed between histamine and VEGF. Seventy-six commonly regulated genes were found, representing ~53% of all VEGF-regulated transcripts and ~26% of all histamine-regulated transcripts. Both factors regulated tight junction formation and expression of pro-angiogenic transcription factors (TFs) affecting EC survival, migration and tube formation. Novel claudin-5 upstream regulatory genes were identified. VEGF was demonstrated to regulate expression of SNAI2, whereas pro-angiogenic TFs NR4A1, MYCN and RCAN1 were regulated by both histamine and VEGF. Claudin-5 was shown to be regulated VEGFR2/PI3K-Akt dependently by VEGF and PI3K-Akt independently by histamine. Interleukin-8 was shown to downregulate claudin-5 by histamine. Additionally, SNAI2, NR4A1 and MYCN were shown to mediate EC survival, migration and tube formation and to regulate expression of claudin-5. Further systemic delivery of VEGF and histamine was shown to induce a fast vascular hyperpermeability response in intact vasculature of C57/Bl6 mice followed by regulation of NR4A1 and MYCN. CONCLUSIONS: Our study identifies novel claudin-5 upstream regulatory genes of histamine and VEGF that induce cellular angiogenic processes. Our results increase knowledge of angiogenic EC phenotype and provide novel treatment targets for vascular pathologies.

Slit2 modifies VEGF-induced angiogenic responses in rabbit skeletal muscle via reduced eNOS activity.

AIMS: Slit2 is a possible modulator of VEGF-induced angiogenesis, but its effects have not been tested on large animal models. We studied the effect of Slit2 on therapeutic angiogenesis induced by VEGF receptor 2 (VEGFR2) ligands Vammin and VEGF-D(ΔNΔC) in vivo in rabbit skeletal muscles. The Slit2 target genes were also studied by RNA sequencing in endothelial cells. METHODS AND RESULTS: Adenoviral intramuscular gene transfers were performed into New Zealand White rabbit hindlimbs. Confocal and multiphoton microscopes were used for blood vessel imaging. Signaling experiments and gene expression analyses were performed to study mechanisms of Slit2 action. Slit2 decreased VEGFR2-mediated vascular permeability. Slit2 also reduced VEGFR2-mediated increase in blood perfusion and capillary enlargement, whereas sprouting of the capillaries was increased. Slit2 gene transfer alone did not have any effects on vascular functions or morphology. VEGFR2 activation was not affected by Slit2, but eNOS phosphorylation was diminished. The transcriptome profiling showed Slit2 down-regulating angiogenesis-related genes such as Nuclear receptor subfamily 4 group A member 1 (NR4A1) and Stanniocalcin-1 (STC-1) as well as genes related to endothelial cell migration and vascular permeability. CONCLUSION: Combining Slit2 with VEGFs adjusts VEGFR2-mediated angiogenic effects into a more physiological direction. This possibly allows the use of higher VEGF vector doses to achieve a more widespread vector and VEGF distribution in the target tissues leading to a better therapeutic outcome while reducing excess vascular permeability.

The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features.

BACKGROUND: Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2). METHODS: Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability. RESULTS: Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding. CONCLUSIONS: VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability. GENERAL SIGNIFICANCE: This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties.

Preclinical safety, toxicology, and biodistribution study of adenoviral gene therapy with sVEGFR-2 and sVEGFR-3 combined with chemotherapy for ovarian cancer.

Abstract Antiangiogenic and antilymphangiogenic gene therapy with soluble vascular endothelial growth factor receptor-2 (VEGFR-2) and soluble VEGFR-3 in combination with chemotherapy is a potential new treatment for ovarian carcinoma. We evaluated the safety, toxicology, and biodistribution of intravenous AdsVEGFR-2 and AdsVEGFR-3 combined with chemotherapy in healthy rats (n=90) before entering a clinical setting. The study groups were: AdLacZ and AdLacZ with chemotherapy as control groups, low dose AdsVEGFR-2 and AdsVEGFR-3, high dose AdsVEGFR-2 and AdsVEGFR-3, combination of low dose AdsVEGFR-2 and AdsVEGFR-3 with chemotherapy, combination of high dose AdsVEGFR-2 and AdVEGFR-3 with chemotherapy, and chemotherapy only. The follow-up time was 4 weeks. Safety and toxicology were assessed by monitoring the clinical status of the animals and by histological, hematological, and clinical chemistry parameters. For the biodistribution studies, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used. Low dose (2×10(10) vp) AdsVEGFR-2 and AdsVEGFR-3 gene therapy was well tolerated, even when gene therapy was combined with chemotherapy. Notably, only transient elevation of liver enzymes and mild regenerative changes were seen in liver after the gene transfer in the groups that received high doses (2×10(11) vp) of AdsVEGFR-2 and AdsVEGFR-3 with or without chemotherapy. No life-threatening adverse effects were noticed in any of the treatment groups. The highest protein concentration of soluble VEGFR-2 (sVEGFR-2) in circulation was seen 1 week after the gene transfer. The combination of chemotherapy to gene therapy seemed to prolong the time of detectable transgene protein at least 1 week in the circulation. The expression of AdsVEGFR-2 and AdsVEGFR-3 transgenes was mainly seen in the liver and spleen as detected by qRT-PCR. According to these results, AdsVEGFR-2 and AdsVEGFR-3 gene therapy combined with chemotherapy is safe and can be brought to clinical testing in ovarian cancer patients.

Vascular endothelial growth factor (VEGF)-D stimulates VEGF-A, stanniocalcin-1, and neuropilin-2 and has potent angiogenic effects.

OBJECTIVE: The mature form of human vascular endothelial growth factor-D (hVEGF-D(ΔNΔC)) is an efficient angiogenic factor, but its full mechanism of action has remained unclear. We studied the effects of hVEGF-D(ΔNΔC) in endothelial cells using gene array, signaling, cell culture, and in vivo gene transfer techniques. METHODS AND RESULTS: Concomitant with the angiogenic and proliferative responses, hVEGF-D(ΔNΔC) enhanced the phosphorylation of VEGF receptor-2, Akt, and endothelial nitric oxide synthase. Gene arrays, quantitative reverse transcription-polymerase chain reaction, and Western blot revealed increases in VEGF-A, stanniocalcin-1 (STC1), and neuropilin (NRP) 2 expression by hVEGF-D(ΔNΔC) stimulation, whereas induction with hVEGF-A(165) altered the expression of STC1 and NRP1, another coreceptor for VEGFs. The effects of hVEGF-D(ΔNΔC) were seen only under high-serum conditions, whereas for hVEGF-A(165), the strongest response was observed under low-serum conditions. The hVEGF-D(ΔNΔC)-induced upregulation of STC1 and NRP2 was also evident in vivo in mouse skeletal muscle treated with hVEGF-D(ΔNΔC) by adenoviral gene delivery. The importance of NRP2 in hVEGF-D(ΔNΔC) signaling was further studied with NRP2 small interfering RNA and NRP antagonist, which were able to block hVEGF-D(ΔNΔC)-induced survival of endothelial cells. CONCLUSIONS: In this study, the importance of serum and upregulation of NRP2 and STC1 for VEGF-D(ΔNΔC) effects were demonstrated. Better knowledge of VEGF-D(ΔNΔC) signaling and regulation is valuable for the development of efficient and safe VEGF-D(ΔNΔC)-based therapeutic applications for cardiovascular diseases.

A laboratory-scale device for the straightforward production of uniform, small sized cell microcapsules with long-term cell viability.

Microencapsulated and genetically engineered cells may be used for prolonged delivery of therapeutically active proteins. The objective of this study was to develop a simple, inexpensive and flexible laboratory-scale device for the production of cell microcapsules, especially capsules of small diameter (<300 µm). Many microencapsulation devices are expensive, difficult to assemble and to use, and often more suitable for large-scale experiments. However, the simplicity and low price of the encapsulation system should not limit the quality of capsules and reproducibility of the process: for successful in vitro and in vivo experiments it is important to be able to produce uniform, spherical microcapsules without deformities with high reproducibility. In addition, an advantage of the present procedure compared to other similar, co-axial laminar gas flow systems is the possibility to produce also small microcapsules, less than 200 µm in diameter, with narrow size distribution. First, design, optimization and reproducibility testing of this custom-built device were carried out. Second, microencapsulated retinal pigment epithelial cells (ARPE-19) capable of secreting soluble vascular endothelial growth factor receptor 1 (sVEGFR1) were engineered. The cells remained viable in alginate-poly-L-lysine-alginate microcapsules and secreted sVEGFR1 for prolonged periods.

Structural determinants of vascular endothelial growth factor-D receptor binding and specificity.

Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function.

A 96-well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells.

BACKGROUND: Baculovirus expression vector system (BEVS) has become a standard in recombinant protein production and virus-like particle preparation for numerous applications. FINDINGS: We describe here protocols which adapt baculovirus generation into 96-well format. CONCLUSION: The established methodology allows simple baculovirus generation, fast virus titering within 18 h and efficient recombinant protein production in a high-throughput format. Furthermore, the produced baculovirus vectors are compatible with gene expression in vertebrate cells in vitro and in vivo.

Novel vascular endothelial growth factor D variants with increased biological activity.

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D(DeltaNDeltaC), the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D(DeltaNDeltaC). The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D(DeltaNDeltaC). More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D(DeltaNDeltaC) and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D(DeltaNDeltaC). The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D(DeltaNDeltaC) described here are potential agents for therapeutic applications, where induction of vascular formation is required.

Antiangiogenic gene therapy with soluble VEGFR-1, -2, and -3 reduces the growth of solid human ovarian carcinoma in mice.

We studied antiangiogenic and antilymphangiogenic effects of sVEGFR-1 (sFlt-1), sVEGFR-2 (sFlk-1/KDR), and sVEGFR-3 (sFlt-4) gene transfers and their combinations in intraperitoneal ovarian cancer xenograft mice (Balb/c-Anu, n = 55). Gene therapy was initiated when the presence of sizable tumors was confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously via tail vein as follows: AdLacZ as a control (group I), AdsFlt-1 (group II), AdsKDR (group III), AdsFlt-4 (group IV) and two combination groups of AdsFlt-1 and AdsFlt-4 (group V) and AdsFlt-1, AdsKDR, and AdsFlt-4 (group VI). Antitumor effectiveness was assessed by sequential MRI, immunohistochemistry, microvessel density, overall tumor growth, and survival time. In combination group VI, intraperitoneal tumors were significantly smaller than in the control group at the end of the follow-up (P < 0.001). Furthermore, in group VI the microvessel density (microvessels/mm(2)) in tumor tissue and the total area of tumors covered by microvessels were significantly smaller than in the controls. One mouse in group V was cured. The combined antiangiogenic gene therapy with soluble VEGFRs reduced tumor growth, tumor vascularity, and ascites formation in ovarian cancer xenografts. The results suggest that the combined antiangiogenic gene therapy is a potential approach for the treatment of ovarian cancer patients.

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo.

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.