Continuous treatment of bestatin induces anti-angiogenic property in endothelial cells.
CD13/aminopeptidase-N (CD13/APN) is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. A previous study demonstrated that angiogenesis is suppressed under the presence of high concentrations of aminopeptidase antagonists. However, the mechanisms underlying the inhibition of morphogenesis by aminopeptidase antagonists have not been elucidated. In this study, we have for the first time examined the effects of continuous treatment of therapeutic dose of aminopeptidase antagonists on vascular endothelial capillary-like tube formation. In the antagonists tested, only bestatin significantly interfered in the capillary tube formation of primary endothelial cells (EC) after treatment for 72 h. Aminopeptidase analysis revealed that inhibitory activity of bestatin was not specific for CD13/APN, and the other inhibitors lacking anti-angiogenic properties also inhibit cell-surface aminopeptidase activity as well or more potently than bestatin, suggesting that the angiogenesis-inhibitory effect of bestatin was not due to inhibition of CD13/APN activity at this concentration. To elucidate the influence of continuous treatment of bestatin on endothelial cells, we performed microarray analysis and revealed that 72-h treatment of a pharmacokinetic dose of bestatin modulated the several angiogenesis-related genes including vascular endothelial growth factor (VEGF). Northern blot analysis indicated that modulation of the VEGF gene became obvious after 48 h of treatment. Furthermore, knockdown of the VEGF gene by siRNA remarkably suppressed capillary tube formation and required a higher concentration of exogenous VEGF to reverse the capillary formation ability. These data suggested that bestatin decreases a reactivity of EC to angiogenesis stimuli, and it can be achieved by the regulation of angiogenesis-related gene expression.
Mishima
Yuji
Y
Cancer Chemotherapy Center, Clinical Chemotherapy, Japanese Foundation for Cancer Research, 3-10-6, Ariake, Koto-ku, Tokyo, 135-8550, Japan.
Terui
Yasuhito
Y
Sugimura
Natsuhiko
N
Matsumoto-Mishima
Yuko
Y
Rokudai
Akiko
A
Kuniyoshi
Ryoko
R
Hatake
Kiyohiko
K
D000626:Aminopeptidases / Q000032:analysis; D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D002196:Capillaries / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000254:growth & development; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007231:Infant, Newborn; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D020411:Oligonucleotide Array Sequence Analysis; D012867:Skin / Q000166:cytology; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000187:drug effects / Q000235:genetics
D043925:Angiogenesis Inducing Agents; D042461:Vascular Endothelial Growth Factor A; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex