© 1999 American Heart Association, Inc.
Original Contributions |
The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-
and Protein Kinase C-
Presented in part at the Annual Meeting of the American Society of Nephrology, San Diego, California, November 5–7, 1995.
From the Franz Volhard Clinic and Max Delbrück Center, Virchow Klinikum, Humboldt University, Berlin, Germany.
Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Str 50, 13122 Berlin, Germany. E-mail haller{at}orion.rz.mdc-berlin.de
Abstract—The heparin-binding protein vascular
endothelial growth factor (VEGF) is a highly specific
growth factor for endothelial cells. VEGF binds to
specific tyrosine kinase receptors, which mediate intracellular
signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular
calcium [Ca2+]i regulation and
[Ca2+]i-dependent messenger systems; and (2)
these mechanisms are important for VEGF's proliferative effects.
[Ca2+]i was measured in human umbilical vein
endothelial cells using fura-2 and fluo-3. Protein
kinase C (PKC) activity was measured by histone-like pseudosubstrate
phosphorylation. PKC isoform distribution was observed
with confocal microscopy and Western blot. Inhibition of PKC isoforms
was assessed by specific antisense oligonucleotides
(ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient
increase in [Ca2+]i followed by a sustained
elevation. The sustained [Ca2+]i plateau was
abolished by EGTA. Pertussis toxin also abolished the plateau phase,
whereas the initial peak was not affected. The PKC isoforms 
, 
,
, and 
were identified in endothelial cells. VEGF
induced a translocation of PKC- and PKC- toward the nucleus and
the perinuclear area, whereas cellular distribution of PKC- and
PKC- was not influenced. Cell exposure to TPA led to a
down-regulation of PKC- and reduced the proliferative effect of
VEGF. VEGF-induced endothelial cell proliferation also
was reduced by the PKC inhibitors staurosporine
and calphostin C. Specific down-regulation of PKC- and PKC- with
antisense ODN reduced the proliferative effect of VEGF significantly.
Our data show that VEGF induces initial and sustained Ca2+
influx. VEGF leads to the translocation of the
[Ca2+]i-sensitive PKC isoform and the
atypical PKC isoform . Antisense ODN for these PKC isoforms block
VEGF-induced proliferation. These findings suggest that PKC isoforms
and are important for VEGF's angiogenic effects.
Key Words: VEGF • protein kinase C • isoforms • endothelial cells • cytosolic calcium • cell proliferation
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