VEGF‐Mediated Elevated Intracellular Calcium and Angiogenesis in Human Microvascular Endothelial Cells In Vitro are Inhibited by Dominant Negative TRPC6

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.