Journal: Molecular Biology of the Cell

Article Title: Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane

doi: 10.1091/mbc.E13-07-0418

Figure Lengend Snippet: Cellular and in vivo actions of VEGF165-GFP. (A) The effect of conditioned medium (CM) on Ca 2+ responses of HUVECs. Medium collected at 20°C (4 h) from COS-7 cells transfected for 24 h with empty vector, VEGF165-GFP, untagged VEGF165, or the GlycM form was added to HUVECs preloaded with Fura-2 for single-cell [Ca 2+ ] i measurements. Cells were also treated with the EGF receptor tyrosine kinase inhibitor AG1478 (1 μM) for 30 min to eliminate the effect of any EGF potentially secreted. Although VEGF165-GFP shows a smaller response than untagged VEGF165, it is still active, whereas the medium collected from GlycM cells is less effective. (B) VEGF165-GFP or control GFP sequences were cloned into a lentiviral backbone, and the resulting lentiviruses were injected into the left and right cerebral cortices of P0 rat pups, respectively. Pups were killed at P10, and fixed brain slices were immunostained with antibodies specific for GFP and RECA-1 to visualize virus-infected cells and cortical microvasculature, respectively. Low-magnification epifluorescence images demonstrate an intense, focal vasculogenic reaction around VEGF-GFP injection sites in contrast to control GFP injections. Scale bars, 100 μm. Note that the cells expressing VEGF165-GFP are not the endothelial cells that proliferate in response to VEGF. (B) Quantifications of microvessel density based on high-magnification confocal images randomly captured at VEGF165-GFP and GFP injection sites show that both the number of vessels and the average RECA-1–positive area/image is significantly higher after VEGF165-GFP injections than after control GFP injections, indicating strong biological activity of the fusion protein (30 pairs of images from three independent brains, **** p < 0.0001).

Article Snippet: Images were acquired with a Nikon Eclipse 80i epifluorescence microscope using optimized filter sets and for quantification with a Zeiss LSM510 META confocal laser scanning microscope.

Techniques: In Vivo, Transfection, Plasmid Preparation, Control, Clone Assay, Injection, Virus, Infection, Expressing, Activity Assay