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other in the rat aortic ring (RAR) assay (Bianco et al. 2016 ) suggesting that CgA
and its fragments may form a balance of anti- and pro-angiogenic factors tightly
regulated by proteolysis.
6 Proteases Involved in CgA Fragmentation
and Angiogenesis Regulation
Considering that 50% of CgA is proteolytically processed in bovine chromaffin
cells (Metz-Boutigue et al. 1993 ), prohormone convertases and furin, capable of
cleaving dibasic residues, might be involved in the intra-granular processing of CgA
N-terminal and C-terminal regions (Metz-Boutigue et al. 1993 ; Koshimizu et al.
2010 ). However, also extracellular proteases might come into play for local angio-
genesis regulation. Interestingly, recent studies have shown that the R 373 -R 374 dibasic
site of CgA is efficiently cleaved by limited digestion with thrombin and plasmin
(Crippa et al. 2013 ; Bianco et al. 2016 ), which may tip the balance toward a pro-
angiogenic state in pathophysiological conditions characterized by their activation,
e.g. in wound healing and cancer. However, extensive digestion of CgA with throm-
bin may cause additional cleavage at R362 and R394 (Crippa et al. 2013 ).
Furthermore, prolonged digestion with plasmin may generate even more fragments
with unknown functions (Bianco et al. 2016 ). Thus, extensive proteolysis of CgA
and its fragments may represent other important regulatory mechanisms that deserve
further investigation. Cleavage by carboxypeptidases might also represent addi-
tional mechanisms for CgA regulation: interestingly, CgA1-78 is rapidly converted
to CgA1-76 when injected in mice (Crippa et al. 2013 ), suggesting that a carboxy-
peptidase B-like enzyme capable of removing the C-terminal dibasic residues of
CgA1-78 is likely present in circulation. As CgA1-76 is more potent than CgA1-
78 in the RAR assay, this enzyme might represent an important element for the
homeostatic regulation of blood vessels at the systemic level. All these enzymes
may contribute, therefore, to the generation and processing of the circulating frag-
ments of CgA in normal and pathological conditions.
Finally, it is important to mention that the dose-response curve of CgA and its
fragments in angiogenesis assays are U-shaped (Crippa et al. 2013 ). The mecha-
nism underlying this behavior is unknown.
7 Circulating CgA and Fragments in Health and Disease
Given the different, and sometimes opposite, functions of CgA and its fragments in
the regulation of vascular physiology, quantification of the circulating levels of
these molecules in health and disease represents an important issue. The normal
values of circulating CgA reported by different laboratories, as measured with
F. Curnis et al.