Angiostatic mechanisms of endogenous angiogenesis inhibitors

Angiogenesis, the process of sprouting of new capillaries from the existing blood vessels, is essential for tumor growth and metastasis. Angiostatin was discovered as the first specific endogenous angiogenesis inhibitor nearly 10 years ago. The structure of angiostatin consists of the first three/four kringle (K1-3/K1-4) domains of plasminogen. However, plasminogen contains five kringle structures and it has been shown that kringle 5 (K5) displays at least as potent inhibitory activity as angiostatin in suppression of endothelial cell growth.

This thesis work describes the discovery of kringle 1-5 (K1-5) as one of the most potent plasminogenderived angiogenesis inhibitors. It specifically inhibits endothelial cell growth in vitro and angiogenesis in vivo. In a mouse tumor model, K1-5 potently inhibits primary tumor growth at the dose of which angiostatin is inactive. Further mechanistic studies show that K1- 5 specifically induces endothelial apoptosis by binding to endothelial cell surface F0F1-ATP synthase, and activating caspases-3, -8, and -9. In vivo experiments show that endothelial apoptosis is essential for the angiostatic activity of K1-5.

In addition to K1-5, we found that adiponectin, a secreted adipocytokine, potently inhibits angiogenesis and tumor growth. Similar to K1-5, adiponectin-induced endothelial apoptosis involves activation of procaspases. Thus, our findings demonstrate that induction of endothelial apoptosis is a common pathway for these endogenous inhibitors to specifically inhibit angiogenesis.

In the effort of standardizing our in vitro assay systems, primary bovine capillary endothelial (BCE) cells were immortalized using human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase nucleoriboprotein complex. Surprisingly we have found that the average telomere lengths in the hTERT-transfected cells are shorter than those found in primary pre-senescent cells, indicating that immortalization of these bovine endothelial cells must be mediated by alternative mechanisms.

Our results indicate that one of the alternative mechanisms might include inactivation of p16 by hTERT-induced DNA methylation. This thesis work provides important information for developing therapeutic antiangiogenic agents in the treatment of angiogenesis- dependent diseases such as cancer and metastasis.

List of scientific papers

I. Cao R, Wu HL, Veitonmaki N, Linden P, Farnebo J, Shi GY, Cao Y (1999). Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc Natl Acad Sci U S A. 96(10): 5728-33.
https://pubmed.ncbi.nlm.nih.gov/10318952

II. Veitonmaki N, Cao R, Wu HL, Moser T, Li B, Pizzo S, Zhivotovsky B, Cao Y (2003). Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for K1-5-induced antiangiogenesis. [Submitted]

III. Brakenhielm E, Veitonmaki N, Cao R, Zhivotovsky B, Funahashi T, Cao Y (2003). Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial apoptosis. [Submitted]

IV. Veitonmaki N, Fuxe J, Hultdin M, Roos G, Pettersson RF, Cao Y (2003). Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb. FASEB J. 17(6): 764-6.
https://pubmed.ncbi.nlm.nih.gov/12586745

V. Veitonmaki N, Fuxe J, Hultdin M, Roos G, Pettersson RF, Cao Y (2003). Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb. FASEB J.