*2.1. Biological Effects of CgA and Its Fragments in Angiogenesis and Tumor Growth*

Studies in various pre-clinical models of solid tumors have shown that systemic administration of recombinant CgA1-439 to tumor-bearing mice enhances the endothelial barrier function, inhibits tumor neo-vascularization, and reduces tumor growth [17,21,35]. In vitro studies have shown that CgA1-439 is an anti-angiogenic molecule and that an antiangiogenic site is located in the region 410–439 (i.e., the C-terminal region of the full-length protein). A latent (or less active) site is also present in the N-terminal region 1–76, this site requiring vasostatin-1 liberation by proteolytic cleavage of the Q76K77 peptide bond for full activation [17]. Physiological concentrations of CgA1-439 and vasostatin-1 inhibit, with U-shaped dose response curves, the pro-angiogenic activity of fibroblast growth factor-2

and vascular endothelial growth factor, two important proteins involved in angiogenesis regulation [17,42]. Studies on the mechanism of action have shown that the anti-angiogenic and anti-tumor activity of CgA1-439 depends on the induction of protease-nexin-1 in endothelial cells, a serine protease inhibitor endowed with potent anti-angiogenic activity [21]. Furthermore, a recent study, aimed at elucidating the pro-angiogenic mechanisms triggered by the fragment CgA1-373, have shown that cleavage of the R373–R374 dibasic site of circulating CgA in tumors and the subsequent engagement of neuropilin-1 by the fragment are crucial mechanisms for the spatio-temporal regulation of angiogenesis in cancer lesions and, consequently, for the regulation of tumor growth [37]. Opposite to CgA1-439 and vasostatin-1 (anti-angiogenic), the fragment CgA1-373 can promote angiogenesis with a bell-shaped dose-response curve and with a maximal activity at 0.2–1 nM, i.e., at concentrations found in certain cancer patients [17]; thus, the full-length CgA1-439 and its fragments may form a balance of anti- and pro-angiogenic factors that can be finely regulated by proteolytic cleavage at Q76 and R373. Studies in murine models of lung carcinoma, mammary adenocarcinoma, melanoma and fibrosarcoma have shown that circulating CgA can be partially cleaved in tumors after the R373 residue, and that the consequent exposure of the PGPQLR373 site is crucial for tumor progression [37]. A blockade of the exposed PGPQLR373 site with specific polyclonal and monoclonal antibodies (unable to recognize the CgA precursor) reduced tumor vascular bed, blood flow, and tumor growth [37]. These findings suggest that cleavage of CgA by proteases and the subsequent exposure of the PGPQLR373 site may contribute to regulate the vascular physiology in tumor tissues [37]. Given that no CgA was produced by cancer cells in the models studied, it is very likely that these fragments were generated by cleavage of bloodborne CgA in tumor lesions. It appears, therefore, that CgA molecules present in the blood can work as an "*off*/*on*" switch for the activation of angiogenesis in tumors after local cleavage.
