Human Vascular Endothelial Growth factor-D Research Articles

Efficacy and Safety of Clinical-Grade Human Vascular Endothelial Growth Factor-DΔNΔC Gene Therapy Containing Residual Replication-Competent Adenoviruses.

Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 1010 viral particles (vp): <10 RCA (VEGF-D L-RCA1), 10-100 RCA (VEGF-D H-RCA2), and 100-200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (PCR) RCA assay in a preclinical rabbit model (n = 21). β-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 1011 vp. Efficacy of the products was tested by the combination of contrast pulse sequencing ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution, and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood cell count and body temperature as well as using quantitative real-time PCR with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral GT products may not be as important major safety issue as previously anticipated.

Abstract LB-327: Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D.

Abstract Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor called vascular endothelial growth factor-D (VEGF-D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors, and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF-D, as well as a specific inhibitory activity against the binding of human VEGF-D to VEGFR-3. In addition, cVE199 was found to inhibit the biological activity of VEGF-D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK-N-DZ, abundantly expressed VEGF-D, an in vivo efficacy study was performed using a xenograft model of SK-N-DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK-N-DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF-D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti-VEGF-D monoclonal antibody, cVE199, with specific reactivity against human VEGF-D prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF-D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti-metastasis therapeutic antibody in neuroblastoma treatment. Citation Format: Kenji Kashima, Miho Watanabe, Yasuko Satoh, Junichi Hata, Nobuya Ishii, Yuko Aoki. Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2013-LB-327

Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor‐D

Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF-D, as well as a specific inhibitory activity against the binding of human VEGF-D to VEGFR-3. In addition, cVE199 was found to inhibit the biological activity of VEGF-D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK-N-DZ, abundantly expressed VEGF-D, an in vivo efficacy study was performed using a xenograft model of SK-N-DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK-N-DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF-D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti-VEGF-D monoclonal antibody, cVE199, with specific reactivity against human VEGF-D, prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF-D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti-metastasis therapeutic antibody in neuroblastoma treatment.

Vascular endothelial growth factor-D: signaling mechanisms, biology, and clinical relevance

Vascular endothelial growth factor-D (VEGF-D) is a secreted glycoprotein that promotes growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis), and can induce remodeling of large lymphatics. VEGF-D enhances solid tumor growth and metastatic spread in animal models of cancer, and in some human cancers VEGF-D correlates with metastatic spread, poor patient outcome, and, potentially, with resistance to anti-angiogenic drugs. Hence, VEGF-D signaling is a potential target for novel anti-cancer therapeutics designed to enhance anti-angiogenic approaches and to restrict metastasis. In the cardiovascular system, delivery of VEGF-D in animal models enhanced angiogenesis and tissue perfusion, findings which have led to a range of clinical trials testing this protein for therapeutic angiogenesis in cardiovascular diseases. Despite these experimental and clinical developments, our knowledge of the signaling mechanisms driven by VEGF-D is still evolving—here we explore the biology of VEGF-D, its signaling mechanisms, and the clinical relevance of this growth factor.

Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies

Vascular endothelial growth factor-D (VEGF-D), a secreted angiogenic and lymphangiogenic glycoprotein, enhances tumor growth and metastasis in animal models, and its expression correlates with metastasis and poor patient outcome in some cancers – it is therefore considered a target for novel anti-cancer therapeutics. The definition of the structure of the complex of VEGF-D bound to its receptors would be beneficial for design of inhibitors of VEGF-D signaling aimed at restricting the growth and spread of cancer. In addition, there is interest in using VEGF-D protein for therapeutic angiogenesis and lymphangiogenesis in the settings of cardiovascular diseases and lymphedema, respectively. However, VEGF-D has proven difficult to express and purify in a highly bioactive form due to a tendency to exist as monomers rather than bioactive dimers. Here we describe a protocol for expression and purification of mature human VEGF-D, and a mutant thereof with reduced glycosylation, potentially suitable for preclinical therapeutic and structural studies, respectively. The degree of glycosylation in mature VEGF-D was reduced by eliminating one of the two N-glycosylation sites, and expressing the protein in Lec3.2.8.1 cells which had reduced glycosylation capacity. Mature VEGF-D and the glycosylation mutant were each enriched for the biologically active dimeric form by optimizing the separation of dimer from monomer via gel filtration, followed by conversion of remaining monomers to dimers via treatment with cysteine. The glycosylation mutant of VEGF-D intended for structural studies preserved all the cysteine residues of mature VEGF-D, in contrast to previous structural studies, exhibited comparable receptor binding to mature VEGF-D and might facilitate structural studies of the VEGF-D/VEGFR-3 complex.

Vascular Endothelial Growth Factor (VEGF)-D Stimulates VEGF-A, Stanniocalcin-1, and Neuropilin-2 and Has Potent Angiogenic Effects

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. 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. 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.

Vascular endothelial growth factor D promoting the growth of lymphatic vessel in gastric carcinoma

To investigate the relationship between vascular endothelial growth factor D (VEGF-D) and metastasis of lymphatic vessel in gastric carcinoma. The human VEGF-D cDNA was amplified from total RNA isolated from human normal gastric tissue, then it was inserted into T-A clone plasmid and subcloned into pEGFP eukaryotic expression vector. After the full-length sequence expected was confirmed by enzymatic digestion and sequencing,the human gastric carcinoma cell line SGC7901, which expressed a low level of VEGF-D, was transfected with the pEGFP/VEGF-D expression vector. Stable SGC7901 clones with high expression of VEGF-D were selected in vitro with G418, which were then combined and subcutaneously injected into nude mice to observe the density and morphology of lymphatic vessel. The outcomes were later compared with those of SGC7901 cells transfected with null vector(pEGFP) by immunostaining with a specific antibody LYVE-1. The average weight of tumors in the pEGFP group (1.13+/-0.40) g at day 35, was significantly lower than that in the pEGFP/VEGF-D group (2.24+/-0.82)g (P<0.05). The lymphatic vessel density (LVD) in the pEGFP group (2.89+/-1.32) was significantly lower than that in the pEGFP/VEGF-D group (5.74+/-1.30)(P<0.01). There were dilated functional lymphatic vessels around the tumor margin. VEGF-D may promote the growth and metastasis of tumor in gastric carcinoma by increasing the growth of lymphatic vessels.

Adenovirus-Mediated Gene Transfer of Human Vascular Endothelial Growth Factor-D Induces Transient Angiogenic Effects in Mouse Hind Limb Muscle

We evaluated the therapeutic potential of adenovirus (Ad)-mediated human vascular endothelial growth factor-D (hVEGF-D) gene delivery in mice. Hind limbs of hypercholesterolemic mice ( n = 120) were injected with AdhVEGF-D, AdhVEGF-A, control AdLacZ (all at 1x10(11)viral particles) or saline. Animals were killed at 4, 7, 14, 28, and 42 days. Newly formed vessels were characterized for their quantity, sprouting, angiogenic versus lymphangiogenic phenotype, and arterial versus venous phenotype by endothelial enzymes markers, pericyte coverage, and electron microscopy. Perfusion was measured by power Doppler ultrasound and edema by magnetic resonance imaging (MRI). AdhVEGF-D induced significant formation of new blood vessels, which featured lumenal enlargement, branching, and sprouting. Branching originated mainly from arterioles. The highest vessel density was present on days 4-7 and the effect lasted up to 28 days. Endothelial marker enzyme activity indicated the predominance of arterial capillaries and arterioles. Forty percent of the neovessels were positive for desmin, indicating that VEGF-D increased pericyte coverage. However, branching vessels were highly positive for smooth muscle actin pericyte marker but negative for desmin. Maximal perfusion was measured during the first week after AdhVEGF-D gene transfer. Ultrastructural analysis showed endothelial cells enriched with vesiculo-vacuolar organelles and cytoplasmic protrusions. Modest lymphangiogenic activity was also detected, which could contribute to the relatively low level of edema detected by MRI. In conclusions, AdhVEGF-D has a strong angiogenic effect and a modest lymphangiogenic effect in mouse skeletal muscle. VEGF-D also increases the presence of pericytes/smooth muscle cells in neovessels. AdhVEGF-D is a potential new agent for the induction of therapeutic vascular growth in skeletal muscle.

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis.

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.

The Specificity of Receptor Binding by Vascular Endothelial Growth Factor-D Is Different in Mouse and Man

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.

Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.