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

Abstract

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.

Efficient Regulation of VEGF Expression by Promoter-Targeted Lentiviral shRNAs Based on Epigenetic Mechanism

“…To answer this question, we injected LV-856, LV-451, and LV-GFP control vector to mouse hindlimbs which were made ischemic at the time of the injection. 11 ELISA analysis from the skeletal muscles showed the same effect as was seen in C166 cells (Figure 2a). Also, TaqMan RT-PCR showed a similar profile of VEGF-A mRNA expression from skeletal muscles as was seen in C166 cells (Figure 2b).…”

Regulation of endometrial vascular remodelling: role of the vascular endothelial growth factor family and the angiopoietin–TIE signalling system

“…Increases in blood and lymph vessel size have previously been observed in muscle treated with adenovirus expressing VEGFC or VEGFD (Rissanen et al 2003, Kholova et al 2007) and in regenerating skin treated with transfected tumour cells expressing VEGFC (Goldman et al 2005). VEGFC and VEGFD bind and activate VEGFR2 and VEGFR3, which in the adult are found predominantly on blood and lymphatic endothelial cells, respectively.…”

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

“…15 For more details, please see the supplemental materials, available online at http://atvb.ahajournals.org.…”

“…This is a routinely used model in which AdhVEGF-D ⌬N⌬C has been shown to have an angiogenic effect from day 4 until day 28 after gene delivery. 15 Animals were euthanized at day 5, and the transgene expression was analyzed by VEGF-D ELISA (Supplemental Table IV). STC1 and NRP2 were shown to be upregulated at the mRNA level in mouse skeletal muscle ( Figure 3E).…”

“…5,12 Mature proteolytically processed form of human VEGF-D (hVEGF-D ⌬N⌬C ) has been shown to be an effective tool in vivo, inducing capillary enlargement and vascular permeability with an efficiency similar to that of hVEGF-A 165 . [13][14][15] This is probably due to the binding of hVEGF-D ⌬N⌬C to VEGFR-2, the main receptor mediating angiogenic effects of VEGFs. 3,4 However, because of the relatively low efficiency of recombinant hVEGF-D ⌬N⌬C to activate VEGFR-2 in cell cultures 16 the mechanism of action has remained unclear.…”