Pyry I. Toivanen

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.

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.

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-DΔNΔC) 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-DΔNΔC, the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-DΔNΔC. The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-DΔNΔC. More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-DΔNΔC 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-DΔNΔC. 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-DΔNΔC described here are potential agents for therapeutic applications, where induction of vascular formation is required.

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.

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&Dgr;N&Dgr;C) is an efficient angiogenic factor, but its full mechanism of action has remained unclear. We studied the effects of hVEGF-D&Dgr;N&Dgr;C 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&Dgr;N&Dgr;C 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&Dgr;N&Dgr;C stimulation, whereas induction with hVEGF-A165 altered the expression of STC1 and NRP1, another coreceptor for VEGFs. The effects of hVEGF-D&Dgr;N&Dgr;C were seen only under high-serum conditions, whereas for hVEGF-A165, the strongest response was observed under low-serum conditions. The hVEGF-D&Dgr;N&Dgr;C-induced upregulation of STC1 and NRP2 was also evident in vivo in mouse skeletal muscle treated with hVEGF-D&Dgr;N&Dgr;C by adenoviral gene delivery. The importance of NRP2 in hVEGF-D&Dgr;N&Dgr;C signaling was further studied with NRP2 small interfering RNA and NRP antagonist, which were able to block hVEGF-D&Dgr;N&Dgr;C-induced survival of endothelial cells. Conclusion—In this study, the importance of serum and upregulation of NRP2 and STC1 for VEGF-D&Dgr;N&Dgr;C effects were demonstrated. Better knowledge of VEGF-D&Dgr;N&Dgr;C signaling and regulation is valuable for the development of efficient and safe VEGF-D&Dgr;N&Dgr;C-based therapeutic applications for cardiovascular diseases.

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

BackgroundBaculovirus expression vector system (BEVS) has become a standard in recombinant protein production and virus-like particle preparation for numerous applications.FindingsWe describe here protocols which adapt baculovirus generation into 96-well format.ConclusionThe 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.

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.

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

Abstract 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.

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.