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Section I: Research Areas<br />
Molecular rendering of<br />
tumor vascularization<br />
Angiogenesis<br />
Angiogenesis is the formation of new blood vessels from pre-existing larger blood vessels. It is required<br />
during embryogenesis, and defects in or interference with this process have severe consequences<br />
for embryonic development. In adult organisms, angiogenesis plays a key role in normal physiological<br />
processes such as wound healing and inflammation, and in mammalian placental development.<br />
The process of angiogenesis can also be hijacked by tumors, which require a supply of oxygen and<br />
nutrients to support the high metabolic rate of tumor cells.<br />
VEGF Receptor Signaling<br />
Vascular endothelial growth factor receptors (VEGFR) are a receptor tyrosine kinase family composed of<br />
seven extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail<br />
containing an active kinase domain. VEGFR1 plays an important role in EC function and normal vascular<br />
development, as well as in hematopoietic function. VEGFR2 is a major receptor for VEGF-induced signaling<br />
in endothelial cells. Upon ligand binding, VEGFR2 undergoes autophosphorylation and becomes<br />
activated. Signaling from VEGFR2 is necessary for the execution of VEGF-stimulated proliferation,<br />
chemotaxis, and sprouting, as well as survival of cultured ECs in vitro and in vivo. VEGFR3 expression is<br />
largely restricted to adult lymphatic endothelium and is thought to control lymphangiogenesis.<br />
VEGFR phosphorylation<br />
at tyrosine residues<br />
activates downstream<br />
signaling cascades that<br />
support angiogenesis.<br />
Tyr801<br />
Tyr951<br />
Tyr966<br />
Tyr1008<br />
Tyr1054<br />
Tyr1059<br />
Tyr1175<br />
Tyr1214<br />
VEGF<br />
lg-like<br />
domains<br />
P<br />
Akt<br />
P TSAd<br />
P<br />
P PLCγ<br />
P<br />
Cbl<br />
P<br />
P PLCγ Cbl<br />
P Shb<br />
P Grb2 Gab1<br />
SHP2<br />
Src<br />
PI3K<br />
PLCγ<br />
PI3K<br />
Src<br />
chapter 06: Development and differentiation<br />
Vascular permeability,<br />
cell migration<br />
Endothelial cell migration<br />
Differentiation, tubulogenesis<br />
Cell proliferation, angiogenesis<br />
Focal adhesion formation,<br />
cell migration<br />
Cell migration, lamellipodia<br />
formation, capillary formation<br />
Kinase domain<br />
Autophosphorylation<br />
Angiogenesis Signaling<br />
When angiogenesis is stimulated, pro-angiogenic growth factors such as VEGF, PDGF, FGF, and TGF<br />
are released. These growth factors bind their cognate receptors on endothelial cells (EC) within preexisting<br />
vessels. This triggers a signaling cascade that activates several signaling pathways such as<br />
PI3K/Akt, Erk1/2, Smad, and Notch and results in EC proliferation and migration. New blood vessel<br />
formation occurs as ECs use matrix metalloproteases (MMPs) and integrins to digest extracellular<br />
matrix and migrate into new territory where they lengthen and form tubes.<br />
VEGF treatment results in phosphorylation<br />
of VEGFR-2 at Tyr1175.<br />
PathScan ® Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA Kit #7335:<br />
Treatment of HUVEC with VEGF stimulates phosphorylation of VEGFR-2 at<br />
Tyr1175, detected by #7335, but does not affect the level of total VEGFR-2<br />
protein detected by PathScan ® Total VEGFR-2 Sandwich ELISA kit #7340.<br />
Absorbance at 450 nm is shown in the top figure, while the corresponding<br />
western blot using Phospho-VEGFR-2 (Tyr1175) Rabbit mAb #2478 (right<br />
panel) or VEGFR-2 Rabbit mAb (7340-55B11) (left panel), is shown in the<br />
bottom figure.<br />
Untreated<br />
VEGF-treated<br />
Absorbance 450nm<br />
3.5<br />
3.0<br />
2.5<br />
2.0<br />
1.5<br />
1.0<br />
0.5<br />
0<br />
kDa<br />
200<br />
140<br />
Total VEGFR-2<br />
Phospho-VEGFR-2<br />
(Tyr1175)<br />
– + – + VEGF<br />
TPA induces expression of MMP-9, a protease that digests<br />
extracellular matrix and is associated with tumor angiogenesis.<br />
MMP-9 (D6O3H) XP ® Rabbit mAb #13667:<br />
IHC analysis of paraffin-embedded human breast<br />
carcinoma (A) using #13667. WB analysis of<br />
concentrated, serum-free cultured medium from<br />
U-2 OS cells, untreated (-) or treated with TPA<br />
#4174 (200 nM, 48 hr; +) (B), using #13667.<br />
A<br />
B<br />
kDa<br />
200<br />
140<br />
100<br />
80<br />
60<br />
50<br />
40<br />
30<br />
– +<br />
MMP-9<br />
TPA<br />
Neuropilin-2<br />
In addition to neuronal guidance, the single pass, transmembrane glycoprotein neuropilin-2<br />
is a co-receptor for VEGFR2 and VEGFR3, and plays a role in the development of vascular<br />
and lymphatic systems through binding VEGF 165 and VEGF145.<br />
Neuropilin-2 is expressed in the<br />
developing mouse embryo.<br />
Neuropilin-2 (D39A5) XP ® Rabbit mAb #3366: Confocal<br />
IF analysis of E14.5 mouse embryo using #3366 (green).<br />
Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye).<br />
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166 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.<br />
www.cellsignal.com/cstangiogenesis<br />
167
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