Angiopoietin-2 overexpression in morris hepatoma results in increased tumor perfusion and induction of critical angiogenesis-promoting genes.
Monitoring of angiogenesis-relevant approaches with functional imaging and histomorphometric analyses is desirable to evaluate the biologic effects. In this study we wished to examine the complex effects of angiopoietin-2 (Ang-2) gene transfer in a rat hepatoma model.


METHODS
Using a bicistronic retroviral vector for Ang-2, Morris hepatoma (MH3924A) cell lines with Ang-2 expression were generated (Ang-2-MH3924A). In human umbilical vein endothelial cells (HUVECs) cocultured with Ang-2-MH3924A, the proliferative action with or without growth factors were determined. Furthermore, animal experiments were performed to measure effects on tumor growth and perfusion. Finally, tumors were examined by immunohistochemistry and DNA chip analysis.


RESULTS
Ang-2-expressing MH3924A enhanced basic fibroblast growth factor-mediated endothelial cell proliferation. Perfusion, as measured by H(2)(15)O PET, was increased in genetically modified tumors. Consistent with the increased perfusion, micro- and macrovascularization were increased. However, tumor growth was similar to wild-type MH3924A (WT-MH3924A). Proliferating cell nuclear antigen and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) staining revealed an increased number of positive cells, indicating a compensation of increased proliferation by enhanced apoptosis. DNA chip analysis showed an induction of angiogenesis-promoting genes, including crucial vascular growth factor receptors, as well as genes related to extracellular matrix (ECM), apoptosis, signal transduction, and oxidative stress.


CONCLUSIONS
Our results suggest that Ang-2 expression increases perfusion or vascularization, especially in interaction with the vascular growth factor system, without affecting tumor growth. Simultaneous, enhanced expression of genes for ECM, apoptosis, and signal transduction indicates Ang-2's versatile role in angiogenesis including its destabilizing function on ECM and endothelium.
Kunz
Pierre
P
Department of Anatomy and Cell Biology III, University of Heidelberg, Heidelberg, Germany.
Hoffend
Johannes
J
Altmann
Annette
A
Dimitrakopoulou-Strauss
Antonia
A
Koczan
Dirk
D
Eisenhut
Michael
M
Bonaterra
Gabriel A
GA
Dengler
Thomas J
TJ
Mier
Walter
W
Haberkorn
Uwe
U
Kinscherf
Ralf
R
D043925:Angiogenesis Inducing Agents / Q000378:metabolism; D042501:Angiogenic Proteins / Q000378:metabolism*; D042702:Angiopoietin-2 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001783:Blood Flow Velocity; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D015972:Gene Expression Regulation, Neoplastic; D008114:Liver Neoplasms, Experimental / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D043925:Angiogenesis Inducing Agents; D042501:Angiogenic Proteins; D042702:Angiopoietin-2; D011994:Recombinant Proteins