CgA in hypertension

O'Connor's group was the first to report that circulating levels of CgA are associated with hypertension. In 1984, they reported a normal plasma CgA level of 2.64 nM, which was increased by ~12‐fold (32.99 nM) in pheochromocytoma patients,59 as well as ~1.5‐fold in hypertensive patients (4.04 nM).60 His group also reported that plasma CgA levels are increased in both essential (primary) hypertension (4.05 versus 2.64 nM) and secondary hypertension (3.93 versus 2.64 nM).61 Since blocking sympathetic outflow by guanabenz resulted in reduced plasma CgA, it was suggested that plasma CgA is, at least in part, regulated by sympathetic nerve, and essential hypertension could represent an increased sympathoadrenal activity.61 Subsequently, increased CgA content was reported both in the adrenal gland62, 63 and plasma.63

Vasodilatory and hypotensive effects of CST

The hypotensive effects of CST (bovine CgA_344–364) were first identified in pithed rats.83 Intravenous injection of bCST resulted in the reduction of pressor responses to the activation of sympathetic outflow by electrical stimulation. bCST also blunted neuropeptide Y agonist–induced hypertension. The vasodepressor effects of bCST appeared to be mediated by massive release of histamine (21‐fold increase) from mast cells, which is consistent with the vasodilatory effects of histamine.92 Using primary pleural and peritoneal cell populations containing 5% of mast cells, it was reported that bCST acts directly on mast cells to release histamine. The potency and efficacy of bCST were better evaluated when compared with the wasp venom peptide mastoparan.93 In human mast cell line LAD2, hCST induced migration of peripheral blood–derived mast cells to migrate, and caused degranulation and release of leukotriene C₄ and prostaglandins D₂ and E₂;94 hCST also caused increased intracellular Ca²⁺ mobilization and induced the production of proinflammatory cytokines/chemokines, such as granulocyte‐macrophage colony–stimulating factor, chemokine C‐C motif ligand 2 (CCL2, also known as MCP1), chemokine C‐C motif ligand 3 (CCL3, also known as MIP1A), and chemokine C‐C motif ligand 4 (CCL4, also known as MIP1B).94 The vasodilatory effect of bCST was subsequently tested on human dorsal hand vein (to avoid systemic counterregulation) using hCST after pharmacologic vasoconstriction with phenylephrine, which showed dose‐dependent vasodilation, especially in female subjects;86 histamine‐induced vasodilation is believed to be mediated through increased production of nitric oxide. In rodents, the hypotensive effects of hCST were reported both in monogenic (hypertensive and hyperadrenergic Chga‐KO mice) and polygenic (blood pressure high mice) models of hypertension. In the monogenic model, hCST exerted its hypotensive effects by inhibiting the secretion of catecholamines33 (Fig. 2).

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Figure 2

Schematic diagram showing the inhibition of catecholamine secretion and regulation of cardiovascular functions by CST. Acetylcholine released from preganglionic sympathetic endings at the splanchnic–adrenal synapse binds to nicotinic acetylcholine receptors and induces influx of Na⁺ inside chromaffin cells resulting in the depolarization of the cell membrane and opening of voltage‐gated Ca²⁺ channels. Increased cytosolic Ca²⁺ concentration stimulates the transcription of Chga and induces exocytotic release of the secretory cocktail containing catecholamines, CST, ATP, chromogranins, and neuropeptides. Exocytotically released CST inhibits further release of catecholamines by an autocrine/paracrine mechanism; decreases blood pressure and improves BRS and HRV by inhibiting catecholamine secretion; causes vasodilation by stimulating massive release of histamine; and decreases inotropy and lusitropy by activating the β₂‐AR‐G_i/o‐protein–PI‐3K–AKT–NO–cGMP signaling pathway. Ach, acetylcholine; BRS, baroreflex sensitivity; CA, catecholamine; CBP, CREB binding protein; CgA, chromogranin A protein; CREB, cAMP‐responsive element–binding protein; DA, dopamine; ERK, extracellular signal–regulated kinase; GA, Golgi apparatus; HRV, heart rate variability; MC, mitochondria; NE, norepinephrine; NTS, nucleus tractus solitarius; PKC, protein kinase C; PSNS, peripheral sympathetic nervous system; RER, rough endoplasmic reticulum; SNS, sympathetic nervous system; Tyr, tyrosine.

Effects of CST on BRS and HRV

Consistent with diminished BRS in hypertensive subjects, hypertensive Chga‐KO mice also display diminished BRS.46 Intravenous administration of hCST increased BRS in hypertensive Chga‐KO mice, where both the reflex bradycardia caused by phenylephrine‐induced hypertension and the reflex tachycardia caused by sodium nitroprusside–induced hypotension were abolished or attenuated.46 These findings indicate that hCST resets the entire autonomic nervous reflex arc to restore normal cardiovascular function.46 Like BRS, hypertensive subjects display decreased HRV; and hypertensive Chga‐KO mice also exhibit decreased HRV.88 Intraperitoneal administration of hCST decreased total power (TP: ms2), which comprises low‐frequency power (LF: 0.4–1.5 Hz), high‐frequency power (HF: 1.5–4.0 Hz), and frequencies outside of low LF and HF power components, in the frequency domain of HRV spectra. hCST also improved each of the time domain parameters, such as mean difference in R–R between consecutive beats in ms (N–N), standard deviation of N–N in ms (SDNN), coefficient of variation of N–N (CVNN), % of consecutive beats more than 7 ms different in R–R (NN7%), reflecting cardiac parasympathetic tone, and root mean square of standard deviation of N–N in ms (RMSSD), which were decreased in Chga‐KO mice.88 Thus, hCST improves both BRS and HRV in Chga‐KO mice, which represents a monogenic model of rodent hypertension (Fig. 2).

Central effects of CST

CgA is ubiquitously expressed in mouse,30, 95 rat,96, 97, 98 and sheep99 central nervous systems. More precisely, CgA is expressed in the cell bodies of most anatomically defined inhibitory, excitatory, and aminergic cell groups of the brain and spinal cord. Barosensitive, bulbospinal neurons in the rostral ventrolateral medulla (RVLM) provide the major tonic excitatory drive to the sympathetic neurons that maintain arterial pressure.100 Microinjection of a pharmacological dose of CST (rat CgA_367–387; 50 nL, 1 mM dissolved in PBS) into the RVLM of vagotomized normotensive rat resulted in sympathoactivation of up to 22% with an increase in arterial blood pressure of up to 35 mmHg. rCST also increased sympathetic BRS with concomitant attenuation of somatosympathetic reflex, implicating rCST as a major player in the regulation of adaptive reflexes. The caudal ventrolateral medulla (CVLM), residing caudal to the RVLM, is the major source of the tonic GABAergic inhibition of presympathetic RVLM neurons. CVLM neurons inhibit immediate changes in blood pressure via the baroreflex and tonically blunt the activity of the presympathetic RVLM neurons by baroreceptor‐independent mechanisms. The later effects play an important role in determining the long‐term level of sympathetic vasomotor tone and blood pressure.100 Microinjection of rCST into the CVLM neurons of vagotomized normotensive rats caused excitation of CVLM neurons resulting in decreased mean arterial blood pressure (by ~23%) and attenuation of sympathetic baroreflex (by ~64%).101 By contrast, in spontaneously hypertensive (SHR) rats, infusion of hCST into the pyramidal neurons of the central amygdala exerted profound decrease in arterial blood pressure (by 65 mmHg).102 Like CVLM, central amygdaloid neurons are also GABAergic, send projections to RVLM, and inhibit excitatory outputs from RVLM with consequent decreases in arterial blood pressure. It thus appears that centrally CST acts as an excitatory peptide as opposed to its inhibitory action in the periphery.

The role of CST in cardioprotection

The first indication that CST acts as a cardioprotective peptide came from a Langendorff‐perfused rat heart preparation study, where hCST inhibited both inotropy and lusitropy under basal and stimulated (β‐adrenergic and endothelin) conditions.36 These cardioprotective effects were shown to be mediated by the activation of the β₂‐AR‐G_i/o protein–NO–cGMP pathways. In rat papillary muscles and isolated cardiomyocytes, hCST exerted antiadrenergic effects through the endothelial PI3K–AKT–eNOS pathway.103 It was later shown that hCST provides cardioprotection by stimulating phosphodiesterase type‐2 and NO‐dependent S‐nitrosylation.39 SHR rats mimic human CHF and display decreased Frank–Starling response.104 In both normotensive Wistar–Kyoto (WKY) and SHR rats, hCST caused a significant increase of the Frank–Starling response as evidenced by hCST‐induced increase in left ventricular pressure (LVP) and the maximal rate of LV contraction (LV dP/dt max) with greater effects in SHR rats. hCST also resulted in decreased myocardial relaxation of both WKY and SHR rats as evidenced by CST‐induced decrease in the maximal rate of LV relaxation (LV dP/dt min). A positive correlation was reported between hCST‐induced improvement in myocardial mechanical response and protein S‐nitrosylation. Thus, aged HF patients suffering from ventricular dilation or diminished autonomic heart modulation coupled with increased afterload and impaired Frank–Starling response could receive benefits from hCST.

hCST decreased I/R‐induced infarct size by ~41%, which is comparable to the cardioprotection (~44% decrease in infarct size) achieved through a postconditioning mechanism.105 Although data for attenuated I/R injury for hCST may seem promising, the data are based on experimental animal models that may not reflect human disease and previous clinical studies in the field have all had negative outcomes. Exogenous hCST (75 nM) at the onset of reperfusion resulted in attenuated postischemic rise of diastolic LVP (an index of contracture) and improved postischemic recovery of developed LVP. In addition, in isolated cardiomyocytes, hCST caused profound increase (by 65%) in cell viability after simulated I/R.105 Subsequently, it was shown that cardioprotection by hCST in early reperfusion stage was achieved by the activation of mitochondrial ATP‐sensitive K⁺  (mitoK_ATP) channels, ROS signaling, and the prevention of mitochondrial permeability transition pore opening via upstream PI‐3K–AKT and PKC signaling106 (Fig. 3). In isolated rat cardiomyocytes undergoing simulated I/R, hCST increased cell survival 65% and decreased cell contracture of I/R cardiomyocytes. The maintenance of mitochondrial membrane potential in I/R cardiomyocytes and increased phosphorylation of AKT at S473, GSK3β at S9, PLB at T17, and eNOS at S1179 have been shown to play important roles in cardioprotection induced by hCST107 (Fig. 3).

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Figure 3

Schematic diagram showing the signaling pathways used by CST to provide cardioprotection. CST‐induced cardioprotection begins by the activation of G protein–coupled or cytokine receptors and the consequent recruitment of signaling pathways: (1) the reperfusion injury salvage kinase (RISK) pathway, including PI‐3K–AKT, ERK1/2, and the downstream target glycogen synthase kinase 3 beta (GSK‐3β); and (2) the PKG pathways. These salvage pathways activate downstream mediators, such as endothelial nitric oxide synthase (eNOS), GSK‐3β, hexokinase II (HKII), protein kinase C‐epsilon (PKCε), the mitochondrial ATP–dependent potassium channel (KATP) with consequent inhibition of mitochondrial permeability transition pore (MPTP). PDK1, pyruvate dehydrogenase kinase 1; ROS, reactive oxygen species.

Effects of human variants of CST on rodent cardioprotection

Five single nucleotide polymorphisms have been discovered in the CST expressing region of CHGA in different human populations, namely, Gly364Ser (rs9658667), Pro370Leu (rs965868), Arg374Gln (rs9658669),32 Tyr363Tyr (rs9658666), and Gly367Val (rs200576557).108, 109 The cardiotropic actions of WT‐CST, Gly364Ser‐CST, and Pro370Leu‐CST were tested in isolated, Langendorff‐perfused rat heart preparation, demonstrating a dose‐dependent (11–200 nM) decrease in LVP (index of contractile activity), rate pressure product (index of cardiac work), and both positive and negative LV dP/dt (index of maximal rate of left ventricular contraction and relaxation, respectively), and increased coronary pressure by WT‐CST. Gly364Ser‐CST was ineffective on basal cardiac performance, and Pro370Leu‐CST induced only negative inotropic activity. Human CST variants also differed in the potencies with which they counteracted the positive inotropic and lusitropic effects of β‐adrenergic stimulation by ISO, with WT‐CST > Gly364Ser‐CST > Pro370Leu‐CST (against ISO‐induced positive inotropism) and Gly364Ser‐CST > WT‐CST > Pro370Leu‐CST (against ISO‐induced positive lusitropism).36

Effects of PST on hepatic glucose metabolism

pPST has been shown to inhibit insulin‐stimulated glycogen synthesis in primary hepatocytes153 and activates glycogenolysis in the rat liver, which implicates a direct anti‐insulin effect on liver metabolism.154, 155 pPST decreases the phosphorylation of insulin receptor substrate 2 at tyrosine residues through the activation of conventional PKC, resulting in the activation of gluconeogenesis. Consistent with the anti‐insulin action, hPST also increases the production of NO with subsequent attenuated phosphorylation of protein kinase B (AKT), forkhead box protein O1 (FOXO1), and reduced matured sterol regulatory element‐binding transcription factor 1c (SREBP1c),45 thereby promoting gluconeogenesis (Fig. 5).

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Figure 5

Schematic diagram showing the PST inhibition of gluconeogenesis in hepatocytes. PST initiates a GTP‐binding protein–linked signaling cascade, which results in the activation of diacylglycerol (DAG) and calcium‐dependent conventional PKC (cPKC), thereby attenuating IRS–PI3K–PDK1–AKT signaling pathway. In addition, the stimulation of the cGMP–NOS pathway also dampens this signaling pathway by nitrosylation of IRS. Suppression of this pathway by PST allows FoxO1 and SREBP1c to stimulate the expression of gluconeogenic genes, Pck1 (also known as Pepck) and G6pc (also known as G6Pase), and thus prevents insulin action.

Effects of PST on lipid metabolism

In addition to pronounced effects on glucose metabolism, PST also modulates lipid metabolism. PST (rat CgA_263–314) decreases insulin‐stimulated synthesis of lipids in rat adipocytes.156 This finding is consistent with the hPST‐dependent increased expression of hepatic lipogenic genes in Chga‐KO mice, including Srebp1c, peroxisome proliferator‐activated receptor‐gamma (Pparg), and glycerol‐3‐phosphate acetyltransferase (Gpat).45 rPST has also been shown to stimulate the release of glycerol and free fatty acids from rat adipocytes, which is completely inhibited by insulin.156 In humans, hPST augments free fatty acid efflux into the circulation, resulting in an overall spillover of ~4.5‐fold, which is consistent with the reported lipolytic action of rPST,156 confirming the anti‐insulin effects of PST.

Role of full‐length CgA and vasostatin‐1 in tumor angiogenesis and growth

It is well known that the unbalanced production of anti‐ and proangiogenic factors in tumors can lead to vascular abnormalities that contribute to tumor cell proliferation, invasion, trafficking, and metastasis formation.171, 172, 173 CgA may be one of the factors that contributes to regulate tumor vascular biology.76, 165 For example, recent studies have shown that full‐length CgA (hCgA_1–439) can function as an antiangiogenic factor, with a functional antiangiogenic site in the C‐terminal region 410–439 and a latent (or less active) site in the N‐terminal region 1–76 (vasostatin‐1), which requires proteolytic cleavage for full activation.41 Full‐length CgA and vasostatin‐1 can inhibit endothelial cell migration, motility, invasion, and capillary‐like structure formation induced by vascular endothelial growth factor (VEGF) or fibroblast growth factor‐2 (FGF2), two important proangiogenic factors.41, 174 Vasostatin‐1 can also inhibit the nuclear translocation of hypoxia inducible factor‐1α, a master regulator of angiogenesis, in endothelial cells.175 These proteins can enhance the endothelial barrier function and reduce vascular leakage and trafficking of tumor cells through the endothelium.176, 177

Studies performed in mice have shown that endogenous murine CgA or exogenous recombinant hCgA_1–439, but not the fragments lacking the C‐terminal region, can inhibit tumor growth in models of nonneuroendocrine solid‐tumors, including fibrosarcoma, mammary adenocarcinoma, Lewis lung carcinoma, and primary and metastatic melanoma.178 The inhibition of tumor growth is associated with reduction of microvessel density and blood flow in neoplastic tissues. Also, low‐dose vasostatin‐1 can delay tumor growth and reduce microvessel density in murine models of solid tumors.41 In these models, neutralization of endogenous murine CgA with antibodies against its C‐terminal region enhances tumor growth rate, whereas systemic administration of low amounts of hCgA_1–439 reduces tumor growth.178 It appears, therefore, that circulating full‐length CgA and vasostatin‐1 can work as physiological inhibitors of tumor growth.

Mechanistically, full‐length CgA induces the production of protease nexin‐1 in endothelial cells, a serine protease inhibitor (serpin) that can inhibit endothelial cell migration, capillary tube formation, and angiogenesis, independently of its serpin activity179, 180 Given that the neutralization of protease nexin‐1 with specific antibodies can suppress the antiangiogenic and antitumor effects of recombinant hCgA_1–439, it is possible that the induction of protease nexin‐1 by CgA, as observed in animal models, is an important mechanism underlying its antiangiogenic and antitumor activities (Fig. 6). Furthermore, considering that protease nexin‐1 is also a potent inhibitor of plasmin and thrombin,181 two proteolytic enzymes often present in tumors, and that CgA can be cleaved by these enzymes,41, 170 this inhibitor might also serve to prevent the local cleavage of CgA, thereby preserving its antiangiogenic activity (Fig. 6). In view of the inhibitory activity of circulating full‐length CgA on angiogenesis and tumor growth, and considering that cleavage of its C‐terminal region markedly reduces its activity, it is reasonable to hypothesize that pathophysiological changes of circulating levels of full‐length CgA and/or its fragmentation may contribute to the regulation of disease progression in patients with non‐neuroendocrine tumors.

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Figure 6

Hypothetical model of the CgA‐dependent angiogenic switch. According to this model, in normal conditions, full‐length CgA_1–439 exerts antiangiogenic effects by inducing protease nexin‐1 (PN‐1), an antiangiogenic protein. Since PN‐1 is also a potent inhibitor of plasmin and thrombin, this protein can also prevent CgA cleavage by these enzymes and preserve its antiangiogenic activity. In damaged tissues or tumors, the balance of protease/antiprotease activity is altered leading to CgA cleavage and formation of fragments, for example, CgA_1–373, capable of interacting with neuropilin‐1 on endothelial cells (via its PGPQLR sequence) and stimulating angiogenesis (see text).

CgA typically affects tumor growth with a U‐shaped dose–response curve.178 This implies that a marked increase of its concentration may paradoxically result in a reduced effect. For this reason, the effects of CgA on neuroendocrine tumors could be different from those observed in murine models of nonneuroendocrine tumors (and difficult to predict), considering the high concentration that CgA can reach in the tumor microenvironment because of the secretory activity of neuroendocrine cancer cells. Furthermore, one cannot exclude the possibility that the tumor‐derived CgA might be fragmented in a different manner, compared with CgA released in the blood by the neuroendocrine system. Thus, although CgA is a widely used serological and histopathological marker for neuroendocrine tumor diagnosis and follow‐up, development of appropriate models is necessary to address the functional role of CgA and its fragments in patients with such tumors.

Role of CgA_1–373 in tumor angiogenesis and growth

Recent studies have shown that a human CgA fragment encompassing residues 1–373 (hCgA_1–373; thus lacking the C‐terminal region) promotes angiogenesis by inducing the release of FGF2 from endothelial cells.35, 41, 182 Notably, hCgA_1–373, hCgA_1–76, and hCgA_1–439 can counterbalance the activity of each other in angiogenesis assays,183 suggesting that these proteins may provide a balance of pro‐ and anti‐angiogenic activities tightly regulated by proteolysis. The transition from anti‐ to pro‐angiogenic forms, which can be induced by limited digestion of CgA with plasmin or thrombin,41, 183 occurs in the bone marrow of multiple myeloma patients and correlates with increased tumor microvessel density.170

Studies aimed at elucidating the proangiogenic mechanisms of hCgA_1–373 and its role in tumor progression have shown that cleavage of the R₃₇₃R₃₇₄ dibasic site of CgA leads to the exposure of the PGPQLR₃₇₃ epitope (100% conserved in mouse and human CgA), and that this is crucial for tumor progression in various murine models of nonneuroendocrine tumors.184 Indeed, blockade of the PGPQLR site of murine CgA with specific antibodies, unable to recognize full‐length CgA, inhibits tumor growth in various experimental models.184 Blockade of the PGPQLR site with antibodies induces changes in the tumor vascular bed and blood flow in murine fibrosarcomas, suggesting that circulating CgA might work as an “off/on” switch for tumor angiogenesis, when cleaved at the PGPQLR site. Thus, fragmentation of circulating CgA in tumors by local proteases may contribute to “switch on” angiogenesis, and changes in the balance of protease/antiprotease molecules in tumor tissues might represent major mechanisms for regulating the CgA activity (Fig. 6).

Mechanistic studies have shown that hCgA_1–373, but not hCgA_1–439, can bind neuropilin‐1 with good affinity (K _d = 3.49 ± 0.73 nM), suggesting that this protein is an important receptor for hCgA_1–373. In particular, the PGPQLR site of hCgA_1–373, but not that of hCgA_1–439, can bind the VEGF binding‐site of neuropilin‐1.184 It appears, therefore, that the PGPQLR binding site is cryptic in the full‐length molecule or that this region undergoes profound conformational changes upon cleavage of CgA at the R₃₇₃R₃₇₄ dibasic site. No binding to neuropilin‐1 occurs with hCgA_1–372, a fragment lacking R₃₇₃, suggesting that the C‐terminal arginine of PGPQLR site (R₃₇₃) is crucial for binding184 (Fig. 6). Considering that the interaction between fragmented CgA and neuropilin‐1 is important for tumor growth, the PGPQLR site may be a potential therapeutic target.

Neuropilin‐1 is an important coreceptor of various protein factors that regulate vascular permeability and angiogenesis, such as semaphorins and VEGF. The C‐terminal sequence of VEGF (CDKPRR), which contains an R/K‐X‐X‐R/K C‐end rule motif (CendR), can recognize a pocket in the b1 domain of neuropilin‐1, a site that can also accommodate other peptides containing the CendR motif;185, 186 in addition, the PGPQLR site of hCgA_1–373 can interact with this pocket of neuropilin‐1.184 Of note, while both hCgA_1–373 and VEGF can bind neuropilin‐1 with similar affinity, only VEGF can bind neuropilin‐2.184

It is possible that hCgA_1–373 engages other coreceptors for signaling. The hCgA_352–372 sequence (catestatin), for example, is known to bind the acetylcholine nicotinic receptor (nAChRs).85, 187, 188 Considering that this class of receptors is expressed on endothelial cells and known to regulate angiogenesis,189, 190, 191, 192 nAChRs might be potential targets. This view is supported by the observation that mecamylamine and α‐bungarotoxin, two nAChRs antagonists, can inhibit the proangiogenic activity of hCgA_1–373 in a human umbilical vein endothelial cell (HUVEC) spheroid assay.184 Thus, these receptors, in addition to neuropilin‐1, might play a role in the proangiogenic activity of hCgA_1–373.

The R₃₇₃ residue of hCgA_1–373 can be rapidly removed by a plasma carboxypeptidase,184 resulting in the loss of neuropilin‐1 binding and proangiogenic activity and gain of antiangiogenic activity. Interestingly, subnanomolar concentrations of hCgA_1–372 (antiangiogenic) can inhibit the activity of subnanomolar levels of hCgA_1–373 (proangiogenic), but not that of VEGF and FGF2.184 Thus, the transition of hCgA_1–373 to hCgA_1–372 may be a specific counter‐regulatory mechanism that serves to limit the activity of hCgA_1–373 at the site of production in tumors. Another potential counter‐regulatory mechanism induced by an excess of hCgA_1–373 could be related to its biphasic behavior, as suggested by its bell‐shaped dose–response curve observed in angiogenesis assays.41, 178, 184 The mechanisms underlying this behavior are unknown.

In summary, the cleavage of the R₃₇₃R₃₇₄ site of CgA in tumors and the subsequent removal of R₃₇₃ in plasma may provide an important on/off switch for spatiotemporal regulation of angiogenesis in tumors and normal tissues. The interaction of the PGPQLR site with neuropilin‐1 might be, therefore, an important target for angiogenesis inhibition and tumor growth suppression.

Role of full‐length CgA and vasostatin‐1 in tumor cell trafficking

Cancer progression involves the seeding of malignant cells in circulation and the colonization of distant organs. Circulating neoplastic cells can also reinfiltrate the tumor of origin in a process referred to as tumor self‐seeding.177 A study performed in murine models of solid tumors has shown that a modest increase of circulating CgA, as obtained in mice after the administration of omeprazole (a proton pump inhibitor) or low‐dose recombinant hCgA_1–439, can reduce tumor self‐seeding and dissemination.177 In particular, the study showed that hCgA_1–439 can inhibit shedding of cancer cells in circulation, tumor reinfiltration by circulating cells, and lung colonization in murine models of mouse mammary adenocarcinomas and melanomas. Mechanistic studies showed that hCgA_1–439 can reduce endothelial cell gap formation and vascular leakage induced by tumor‐derived factors in tumors, as well as the transendothelial migration of cancer cells.176, 177 Thus, circulating full‐length CgA, besides reducing angiogenesis in tumors, can enhance endothelial barrier function in both tumors and normal tissues thereby inhibiting the trafficking of tumor cells via tumor–to–blood and blood–to–tumor/normal tissue routes (i.e., metastasis and tumor self‐seeding processes).176, 177 Similar effects have been reported for vasostatin‐1.177

A role for circulating CgA in cancer cell trafficking has also been investigated in chronic lymphocytic leukemia (CLL), a hematological cancer characterized by the progressive accumulation of CD5⁺ leukemic B cells in peripheral blood, lymphoid tissues, and bone marrow, and by the continuous trafficking of these cells among these tissue compartments.193 Physiologically relevant doses of recombinant hCgA_1–439 have been shown to reduce the bone marrow/blood ratio of leukemic cells in the Eµ‐TCL1 murine transgenic CCL model, and decrease bone marrow, kidney, and lung infiltration in Rag2 ^−/− γc (Il2rg)^−/− mice engrafted with human MEC1 CLL cells.170 This treatment also reduced the loss of body weight associated with disease progression and improved animal motility. hCgA_1–439 was found to enhance the integrity of the endothelial barrier and to reduce the transendothelial migration of MEC1 cells with a bimodal dose–response curve. Vasostatin‐1, but not hCgA_1–373, could inhibit CLL progression in the xenograft model, suggesting that the N‐terminal domain contains a site activated by proteolytic cleavage, and that the C‐terminal region is crucial for CgA activity.183 It is therefore possible that CgA and its fragments contribute to regulate leukemic cell trafficking and tissue infiltration in patients with CLL.

This work was supported by a Grant from the Department of Veterans Affairs (I01BX000323 to S.K.M.) and by the Associazione Italiana per la Ricerca sul Cancro (AIRC‐5 × 1000 and AIRC‐IG‐19220 to A.C.).