A new chromogranin A–dependent angiogenic switch activated by thrombin

Abstract

• Circulating chromogranin A and its fragments form a balance of anti-and proangiogenic factors regulated by thrombin-dependent cleavage.• The alteration of this balance could provide a new mechanism for triggering angiogenesis in cancer and other pathophysiologic conditions.Angiogenesis, the formation of blood vessels from pre-existing vasculature, is regulated by a complex interplay of anti and proangiogenic factors. We found that physiologic levels of circulating chromogranin A (CgA), a protein secreted by the neuroendocrine system, can inhibit angiogenesis in various in vitro and in vivo experimental models. Structure-activity studies showed that a functional antiangiogenic site is located in the C-terminal region, whereas a latent anti-angiogenic site, activated by cleavage of Q76-K77 bond, is present in the N-terminal domain. Cleavage of CgA by thrombin abrogated its anti-angiogenic activity and generated fragments (lacking the C-terminal region) endowed of potent proangiogenic activity. Hematologic studies showed that biologically relevant levels of forms of full-length CgA and CgA1-76 (anti-angiogenic) and lower levels of fragments lacking the C-terminal region (proangiogenic) are present in circulation in healthy subjects. Blood coagulation caused, in a thrombin-dependent manner, almost complete conversion of CgA into fragments lacking the C-terminal region. These results suggest that the CgA-related circulating polypeptides form a balance of anti and proangiogenic factors tightlyregulated byproteolysis. Thrombin-induced alteration of this balance could provide a novel mechanism for triggering angiogenesis in pathophysiologic conditions characterized by prothrombin activation. (Blood. 2013;121(2):392-402) IntroductionAngiogenesis, the process of formation of new blood vessels from pre-existing vessels, is tightly regulated by the coordinated action of anti and proangiogenic factors. [1][2][3] When this balance is disturbed, the result is either excessive or insufficient angiogenesis. Altered angiogenesis, causing excessive or insufficient blood vessel growth, is a common denominator underlying many pathologic conditions, including cardiovascular diseases, macular degeneration, skin diseases, diabetic ulcers, stroke, rheumatoid arthritis, cancer, and many others. [1][2][3] A growing body of evidence suggests that angiogenesis can be regulated by peptides derived from proteins released by neurons and neuroendocrine cells. For example, we have previously shown the recombinant fragment 1-78 of human chromogranin A (CgA), a 439-residue long protein stored in the secretory granules of many endocrine cells, neurons, and cardiomyocytes 4-6 can inhibit angiogenesis in experimental models. 7 Other investigators have shown that a synthetic fragment encompassing residues 352-372 (CgA352-372) can promote angiogenesis. 8 Studies on the mechanism of action have shown that CgA1-78 (also called vasostatin-1) can inhibit endothelial cell proliferation, migration, and invasion induced by vascular endothelial growth factor...

Chromogranin A Is Preferentially Cleaved into Proangiogenic Peptides in the Bone Marrow of Multiple Myeloma Patients

“…Hematological studies have shown that biologically relevant levels of full-length CgA 1-439 and fragment CgA 1-76 plus lower levels of other fragments lacking the Cterminal region are present in circulation in normal subjects. Blood coagulation causes, in a thrombin-dependent manner, conversion of CgA 1-439 into the proangiogenic fragment CgA 1-373 (15). Thus, CgA-related circulating polypeptides form a balance of anti-and proangiogenic factors tightly regulated by N-and C-terminal proteolysis.…”

“…This fragment can also inhibit the nuclear translocation of hypoxia inducible factor (HIF)-1a, a master regulator of angiogenesis, in endothelial cells (22). However, the fragments CgA 1-373 and CgA can induce the release of FGF2 from endothelial cells (15,19). Hematological studies have shown that biologically relevant levels of full-length CgA 1-439 and fragment CgA 1-76 plus lower levels of other fragments lacking the Cterminal region are present in circulation in normal subjects.…”

“…Accordingly, full-length CgA 1-439 and the N-terminal fragment CgA 1-76 (called vasostatin-1) inhibit angiogenesis in various angiogenesis assays, whereas the fragment CgA 1-373 can stimulate angiogenesis (15). Mechanistic studies have shown that fulllength CgA and the N-terminal fragment vasostatin-1 can inhibit endothelial cell migration, motility, sprouting, invasion, and capillary-like structure formation induced by VEGF, a potent proangiogenic factor, as well as the proangiogenic activity of basic fibroblast growth factor (FGF2), another important factor involved in the regulation of angiogenesis (15,21). This fragment can also inhibit the nuclear translocation of hypoxia inducible factor (HIF)-1a, a master regulator of angiogenesis, in endothelial cells (22).…”

“…Tissue-specific intragranular and extracellular proteolytic processing of CgA leads to production of various bioactive peptides involved in the regulation of angiogenesis, metabolism, and cardiovascular system (18). Regarding angiogenesis, it has been recently shown that CgA contains: (i) a functional antiangiogenic site in the C-terminal region 410-439; (ii) a latent antiangiogenic site in the Nterminal region 1 to 76, and (iii) a latent proangiogenic site in the region 352-372 (15,19,20). These sites are activated by proteolytic cleavage of Q76-K77 and R373-R374 bonds, respectively.…”

“…Among the wide range of angiogenesis regulators so far discovered, recent studies have shown that chromogranin A (CgA) may have an important role in the regulation of angiogenesis (15,16). CgA is a glycosylated, sulfated, and phosphorylated protein, 439 residue-long, stored in the secretory vesicles of many neuroendocrine cells and neurons (17), and exocytotically released in circulation together with the costored hormones, to reach 0.5 to 1 nmol/L levels in normal conditions (18).…”