Abstract
Objective: Vascular endothelial growth factor (VEGF)-induced vascular permeability has been shown to be dependent on calcium influx, possibly through a transient receptor potential cation channel (TRPC)-mediated cation channel with properties of the TRPC3/6/7 subfamily. To investigate further the involvement of this subfamily, we determined the effects of dominant negative TRPC6 overexpression on VEGF-mediated changes of human microvascular endothelial cell (HMVEC) calcium, proliferation, migration, and sprouting. Methods: Cytoplasmic calcium concentration was estimated by fura-2 fluorescence spectrophotometry, migration by Boyden chamber assay, sprouting by immunofluorescence imaging of stimulated endothelial cells, and proliferation by flow cytometry. Results: Overexpression of a dominant negative TRPC6 construct in HMVECs inhibited the VEGF-mediated increases in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild-type TRPC6 construct increased the proliferation and migration of HMVECs. Conclusions: TRPC6 is an obligatory component of cation channels required for the VEGF-mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis.
Acknowledgements
This work was funded by the Wellcome Trust (grant no. 77589, AMH-Z), The Richard Bright VEGF Research Trust (grant no. 07/PG01, CAG, AM), and the British Heart Foundation (BS/06/005, DOB; PG/07/026, DOB, JCH).