VEGF-mediated elevated intracellular calcium and angiogenesis in human microvascular endothelial cells in vitro are inhibited by dominant negative TRPC6.
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.
Hamdollah Zadeh
M A
MA
Bristol Heart Institute, Department of Physiology and Pharmacology, School of Veterinary Sciences, University of Bristol, United Kingdom.
Glass
Catherine A
CA
Magnussen
Anette
A
Hancox
Jules C
JC
Bates
David O
DO
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D050052:TRPC Cation Channels / Q000096:biosynthesis* / Q000235:genetics; D000074287:TRPC6 Cation Channel; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology*
D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D002118:Calcium
Circ Res. 2006 Jun 23;98(12):1520-7
16690880
Cell Calcium. 2006 Jul;40(1):29-40
16697039
Cardiovasc Res. 2007 Jan 1;73(1):111-9
17129578
Nat Cell Biol. 2007 Jun;9(6):636-45
17486119
Am J Physiol Heart Circ Physiol. 2000 Oct;279(4):H1625-34
11009449
Am J Physiol Cell Physiol. 2001 May;280(5):C1184-92
11287332
Nat Rev Neurosci. 2001 Jun;2(6):387-96
11389472
Growth Factors. 2001;19(1):35-48
11678208
Endothelium. 2003;10(1):5-15
12699072
Vascul Pharmacol. 2002 Nov;39(4-5):225-37
12747962
Am J Physiol Heart Circ Physiol. 2004 Mar;286(3):H1015-26
14551041
J Biol Chem. 2004 Apr 2;279(14):13696-704
14736881
Am J Pathol. 1991 Jan;138(1):213-21
1987767
Am J Physiol. 1996 Jan;270(1 Pt 2):H411-5
8769777
Diabetologia. 1997 May;40(5):528-32
9165220
Am J Physiol. 1997 Aug;273(2 Pt 2):H687-94
9277485
Nature. 1999 Jan 21;397(6716):259-63
9930701
Am J Physiol. 1999 Feb;276(2 Pt 2):H535-42
9950855
Am J Physiol Lung Cell Mol Physiol. 2004 Dec;287(6):L1303-13
15347566
Circ Res. 2005 Jun 24;96(12):1282-90
15920022
J Biol Chem. 2005 Aug 19;280(33):29559-69
15972814
Pflugers Arch. 2005 Oct;451(1):99-104
15924237
Pflugers Arch. 2005 Oct;451(1):131-42
15988589
Arterioscler Thromb Vasc Biol. 2006 Aug;26(8):1768-76
16763162