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Citation: Corti, A.; Anderluzzi, G.;
Curnis, F. Neuropilin-1 and Integrins
as Receptors for Chromogranin
A-Derived Peptides. Pharmaceutics
2022,14, 2555. https://doi.org/
10.3390/pharmaceutics14122555
Academic Editor: Tatiana
B. Tennikova
Received: 14 October 2022
Accepted: 18 November 2022
Published: 22 November 2022
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pharmaceutics
Review
Neuropilin-1 and Integrins as Receptors for Chromogranin
A-Derived Peptides
Angelo Corti 1, 2, * , Giulia Anderluzzi 2and Flavio Curnis 2, *
1Faculty of Medicine, UniversitàVita-Salute San Raffaele, 20132 Milan, Italy
2Tumor Biology and Vascular Targeting Unit, Division of Experimental Oncology,
IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy
*Correspondence: corti.angelo@hsr.it (A.C.); curnis.flavio@hsr.it (F.C.); Tel.: +39-02-26434802 (A.C.)
Abstract:
Human chromogranin A (CgA), a 439 residue-long member of the “granin” secretory
protein family, is the precursor of several peptides and polypeptides involved in the regulation of
the innate immunity, cardiovascular system, metabolism, angiogenesis, tissue repair, and tumor
growth. Despite the many biological activities observed in experimental and preclinical models
for CgA and its most investigated fragments (vasostatin-I and catestatin), limited information is
available on the receptor mechanisms underlying these effects. The interaction of vasostatin-1 with
membrane phospholipids and the binding of catestatin to nicotinic and b2-adrenergic receptors
have been proposed as important mechanisms for some of their effects on the cardiovascular and
sympathoadrenal systems. Recent studies have shown that neuropilin-1 and certain integrins may
also work as high-affinity receptors for CgA, vasostatin-1 and other fragments. In this case, we
review the results of these studies and discuss the structural requirements for the interactions of
CgA-related peptides with neuropilin-1 and integrins, their biological effects, their mechanisms, and
the potential exploitation of compounds that target these ligand-receptor systems for cancer diagnosis
and therapy. The results obtained so far suggest that integrins (particularly the integrin avb6) and
neuropilin-1 are important receptors that mediate relevant pathophysiological functions of CgA and
CgA fragments in angiogenesis, wound healing, and tumor growth, and that these interactions may
represent important targets for cancer imaging and therapy.
Keywords:
chromogranin A; vasostatin-1; catestatin; angiogenesis; tumor diagnosis; neuropilin-1
integrin avβ6; integrin avβ8
1. Introduction
Human chromogranin A (CgA), a member of the “granin” protein family, is a
439-residues
long protein present in the secretory vesicles of various normal and neoplastic neuro-
endocrine tissues and neurons, and exocytotically released into the blood stream upon cell
stimulation [1,2].
Abnormal levels of CgA, detected by immunoassay, are present in the blood of patients
with neuroendocrine tumors or with other diseases, such as cardiovascular, gastrointestinal,
renal, and inflammatory diseases [3].
CgA undergoes various post-translational modifications in different cells and tis-
sues, including phosphorylation, sulphation, glycosylation, and proteolytic cleavage [
2
,
4
].
Intra-granular and/or extra-cellular proteolytic enzymes, such as furin, cathepsin L, prohor-
mone convertase 1 and 2, thrombin and plasmin, can cleave the full-length CgA precursor
(CgA
1-439
) at different sites to generate various biologically active fragments involved in the
regulation of the innate immunity [
5
–
8
], cardiovascular system [
9
–
12
], metabolism [
13
–
15
],
angiogenesis [
16
–
18
], tissue repair [
19
] and tumor growth [
14
,
20
,
21
]. These fragments
include N-terminal large polypeptide fragments (e.g., CgA
1-373
) [
17
], as well as shorter frag-
ments, such as CgA
1-76
(vasostatin-1) [
9
], CgA
79-113
(vasoconstrictive-inhibitory factor) [
22
],
Pharmaceutics 2022,14, 2555. https://doi.org/10.3390/pharmaceutics14122555 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2022,14, 2555 2 of 14
CgA
1-113
(vasostatin-2) [
23
], CgA
250-301
(pancreastatin) [
13
], CgA
352-372
(catestatin) [
24
],
CgA
411-436
(serpinin) [
25
,
26
], and others [
3
,
14
]. The
in vitro
and
in vivo
assays used to
investigate the biological effects of all these fragments and their mechanisms are reviewed
in detail elsewhere [2,6,10,12,14,18,26].
Marie Hélène Metz-Boutigue and coll. were the first to demonstrate that vasostatin-1
and catestatin, two of the most investigated fragments, are endowed of antibacterial and
antifungal activities [
2
,
6
,
27
,
28
]. However, several studies have shown that these peptides
can also affect the physiology of mammalian cells and exert several regulatory functions
under physiological and pathological conditions. For example, catestatin and vasostatin-1
induce vasodilation [
2
,
9
,
29
]. In addition, catestatin inhibits nicotinic-cholinergic-stimulated
catecholamine secretion [
24
], promotes the release of histamine from rat mast cells and
stimulates monocyte chemotaxis [
30
]. Furthermore, vasostatin-1, catestatin, and full-
length CgA
1-439
reduce myocardial contractility and relaxation [
31
–
33
], counteract the
β
-adrenergic-stimulated positive inotropism, and regulate the coronary tone [
12
]. Addi-
tionally, CgA and vasostatin-1 can affect, in an opposite manner, the adhesion of cardiomy-
ocytes, keratinocytes, fibroblasts, and smooth muscle cells to proteins of the extracellular
matrix [
2
,
34
]. CgA and vasostatin-1 can also prevent the disassembly of vascular endothe-
lial cadherin-dependent adherens junctions [
35
], inhibit vascular leakage induced by tumor
necrosis factor-
α
[
35
], and exert angiogenic effects [
17
], whereas catestatin and CgA
1-373
promote angiogenesis [
16
,
17
]. In human microvascular endothelial cells, vasostatin-1 in-
hibits the expression of tumor necrosis factor-
α
–induced intercellular adhesion molecule-1,
the release of monocyte chemoattractant protein-1, and the relocation of high mobility
group box-1 [
36
]. Physiological concentrations of full-length CgA
1-439
, and vasostatin-1
may also have a regulatory role in wound healing [
19
] and tumor growth [
21
,
37
,
38
], and
exert several other biological effects in the regulation of metabolism and cardiovascular
system [14].
Despite the numerous activities reported for CgA, vasostatin-1, and catestatin, limited
information is available on the underlying receptors. Biochemical studies have shown that
vasostatin-1 can interact with phosphatidylserine and other membrane-relevant phospho-
lipids [
39
]. Furthermore, a mechanism involving the binding of vasostatin-1 to heparan
sulfate proteoglycans and phosphoinositide 3-kinase-dependent eNOS phosphorylation
has been observed in bovine aortic endothelial cells [
40
]. Other studies have shown that the
nicotinic acetylcholine receptor mediates the inhibitory effect of catestatin on the secretion
of catecholamines from chromaffin cells [
14
,
24
]. Catestatin can also act on the
β
2-adrenergic
receptor, as suggested by the results of a combination of experimental and computational
studies [
41
]. Interestingly, recent studies have shown that integrins and neuropilin-1 may
also act as important receptors for CgA, vasostatin-1, CgA
1-373
, and other fragments, in
endothelial cell biology, cardiovascular function, angiogenesis, wound healing, and tumor
growth. Here, we review the structural requirements for the interactions of CgA and
CgA-fragments with neuropilin-1 and integrins, their biological effects, their mechanisms,
and the potential use of compounds targeting these ligand-receptor interactions for cancer
diagnosis and therapy.
2. Neuropilin-1 as a Receptor for Chromogranin A-Derived Peptides
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 ad-
ministration of recombinant CgA
1-439
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 anti-
angiogenic 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 Q
76
K
77
peptide bond for
full activation [
17
]. Physiological concentrations of CgA
1-439
and vasostatin-1 inhibit, with
U-shaped dose response curves, the pro-angiogenic activity of fibroblast growth factor-2
Pharmaceutics 2022,14, 2555 3 of 14
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 CgA
1-439
depends on the induction of protease-nexin-1 in en-
dothelial 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 CgA
1-373
, have shown that cleavage of the R
373
–R
374
dibasic site of circu-
lating 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 CgA
1-439
and
vasostatin-1 (anti-angiogenic), the fragment CgA
1-373
can promote angiogenesis with a
bell-shaped dose-response curve and with a maximal activity at 0.2–1 nM, i.e., at concentra-
tions found in certain cancer patients [
17
]; thus, the full-length CgA
1-439
and its fragments
may form a balance of anti- and pro-angiogenic factors that can be finely regulated by
proteolytic cleavage at Q
76
and R
373
. Studies in murine models of lung carcinoma, mam-
mary adenocarcinoma, melanoma and fibrosarcoma have shown that circulating CgA can
be partially cleaved in tumors after the R
373
residue, and that the consequent exposure
of the PGPQLR
373
site is crucial for tumor progression [
37
]. A blockade of the exposed
PGPQLR
373
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
PGPQLR
373
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.
2.2. Mechanisms Underlying the Biological Effects of CgA Fragments in Angiogenesis and
Tumor Growth
The findings described above raise a series of questions. First, which proteases can
switch on this pro-angiogenic mechanism in tumors? Second, which receptor mediates
the biological activity of CgA
1-373
? Considering that thrombin and plasmin are known to
be activated in tumors, and that these enzymes can efficiently cleave the R
373
R
374
peptide
bond [
17
,
43
], both enzymes are good candidates for cleaving CgA in tumors. Although
other proteases might also be involved (discussed below), it is interesting to note that
full-length CgA
1-439
can induce, in endothelial cells, the production of protease-nexin 1
(a potent inhibitor of plasmin, plasminogen activators, and thrombin [
21
]), and that the
plasminogen activator inhibitor-1 inhibits CgA cleavage to CgA
1-373
by cultured endothelial
cells [
43
]. It is therefore tempting to speculate that changes in the relative levels of these
protease/anti-protease molecules in tumor lesions represent a major mechanism for the
regulation of the CgA-dependent angiogenic switch.
Regarding the second question, the results of biochemical studies suggest that CgA
1-373
can bind to neuropilin-1 (NRP-1) with high affinity (Kd = 3.49
±
0.73 nM), via its
C-terminal
PGPQLR
373
sequence [
37
]. The functional role of this ligand-receptor interaction is sug-
gested by the fact that the pro-angiogenic effects of this fragment are blocked by anti-
neuropilin-1 or anti-PGPQLR
373
antibodies. No interaction of full-length CgA and CgA
1-372
with neuropilin-1 occurs, indicating that the PGPQLR
373
binding site is cryptic in the full-
length precursor and that the C-terminal arginine residue (R
373
, which is absent in CgA
372
)
is necessary for the binding [37].
Studies on the topology of the NRP-1-binding site showed that CgA
1-373
and short
peptides containing the PGPQLR sequence interact with a pocket of the b1 domain of the
receptor, a site that recognizes peptides and polypeptides ending with the so-called C-end
Rule (CendR) motif (R/K-X-X-R/K, as in the prototypical CendR peptide RPARPAR). Re-
markably, this binding pocked can also accommodate and bind the C-terminal sequence of
VEGF
165
, a pro-angiogenic factor that contains a CendR motif (CD
K
PR
R
) [
44
,
45
]; thus,
Pharmaceutics 2022,14, 2555 4 of 14
CgA
1-373
, VEGF
165
, and even the short PGPQLR and RPARPAR peptides, all with a
C-terminal
arginine, compete for the same binding pocket of the b1 domain of NRP-1.
Although the PGPQLR
373
sequence cannot be fully considered a CendR motif, as it lacks
the first R/K residue of the consensus sequence, it is interesting to note this sequence, such
as the CendR motif, ends with an arginine that is crucial for NRP-1 recognition.
The importance of the C-terminal arginine of CgA
1-373
for neuropilin-1 recognition
is also supported by the results of molecular docking and molecular dynamics experi-
ments, performed with CgA
352-372
(catestatin) and CgA
352-373
(catestatin-R). Despite these
compounds differ only for the presence of an arginine (C-terminal sequence: PGPQL in
catestatin; PGPQLR in catestatin-R), a clear difference in the interaction with NRP-1 was ob-
served [
46
]. The interaction of catestatin-R with neuropilin-1 showed strong similarity with
that of the compound EG00229, a small inhibitor of NRP-1 that contains an arginine with a
free carboxyl group and whose structure in the complex with NRP-1 has been resolved by
crystallography studies. In both cases, complex formation is driven by salt bridges between
the guanidine moiety of the C-terminal arginine of the ligand and the carboxyl group of
an aspartate residue of neuropilin-1 (D
48
) [
46
]. Remarkably, despite the presence of other
positively charged arginine residues and amino-groups in catestatin-R, the best binding
mode was obtained with the interaction of the C-terminal R
373
of the ligand with D
48
of
the receptor.
Another important question raised by these findings concerns the possible involve-
ment of co-receptors. Experimental evidence showed that the pro-angiogenic activity of
CgA
1-373
in assays based on endothelial cell spheroids can be inhibited by mecamylamine
and
α
-bungarotoxin, two antagonists of nicotinic acetylcholine receptors [
37
]. Consider-
ing that (a) the nicotinic acetylcholine receptors are expressed on endothelial cells and are
known to contribute to the regulation of angiogenesis [
47
–
50
], and (b) catestatin (CgA
352-372
)
is known to bind nicotinic acetylcholine receptors [
51
–
53
], this class of receptors may rep-
resent important co-receptors for CgA
1-373
signaling in endothelial cells. It cannot be
excluded, however, that other receptor systems are also involved.
A final point that should be discussed concerns the issue of counterregulatory mech-
anisms. Experimental evidence suggests that R
373
is rapidly removed from CgA
1-373
by
plasma carboxypeptidases, when this fragment is released in circulation [
37
]. Given the
importance of R
373
for neuropilin-1 recognition, the cleavage of CgA
1-373
to form CgA
1-372
may represent an important mechanism to limit the CgA
1-373
activity at the site of its
production (for example, in cancer lesions) and to avoid systemic effects.
Thus, the results obtained so far support a model in which cleavage of the R
373
R
374
bond of circulating CgA, followed by neuropilin-1 engagement in tumors, and the sub-
sequent removal of R
373
in plasma, represent a sort of “off /on/off ” switch for the spatio-
temporal regulation of angiogenesis in tumor lesions (see Figure 1A for a schematic repre-
sentation of the model).
Interestingly, it is well known that pro-hormone convertases can cleave proteins at
dibasic sites (R/K-R/K), and that carboxypeptidase H/E remove the C-terminal R or K.
It is possible that also these enzymes are brought into play in the regulation of the CgA-
dependent angiogenic switch. Indeed, it is possible that a lower expression, or a reduced
activity, of carboxypeptidase H/E in tissues in which CgA
1-373
is overproduced (see below)
may contribute to activate the pro-angiogenic switch. On the other hand, the normal
expression/function of carboxypeptidase H/E in other tissues might have a role in the
generation of the anti-angiogenic vasostatin-1 fragment (by removal of K
77
after cleavage of
the K
77
/K
78
dibasic site) and CgA
1-372
(by removal of R
373
after cleavage of the R
373
/R
374
dibasic site), thereby promoting an anti-angiogenic effect, a hypothesis that deserves to
be tested.
Pharmaceutics 2022,14, 2555 5 of 14
Pharmaceutics 2022, 14, x FOR PEER REVIEW 5 of 15
Figure 1. Hypothetical model of the CgA-dependent “off/on/off” switch for the regulation of angio-
genesis in tumor and its inhibition by anti-PGPQLR antibodies. (A) Mechanisms of activation/deac-
tivation of the NRP-1 binding site of chromogranin A. According to this model cleavage of the
R373R374 peptide bond of full-length CgA (CgA1-439) leads to exposure of the PGPQLR sequence, a site
that can recognize the CendR-binding pocket of the b1 domain of neuropilin-1 (NRP-1) on endotal
cells. Removal of the R373 residue by carboxypeptidases causes loss of NRP-1 recognition [37]. (B)
Mechanism of anti-tumor activity of anti-PGPQLR antibodies. Cleavage of bloodborne full-length
CgA (CgA1-439) in tumors, e.g., by plasmin or thrombin, causes loss of anti-angiogenic CgA1-439 and
generates the pro-angiogenic CgA1-373 fragment, which may interact with NRP-1 and contribute to
promote angiogenesis and tumor growth. Antibodies against the NRP-1 binding site of CgA1-373
(anti-PGPQLR antibodies) block the CgA1-373/NRP-1 interaction and, consequently inhibit angiogen-
esis and tumor growth [37]. This schematic representation has been prepared using the BioRender
software.
Interestingly, it is well known that pro-hormone convertases can cleave proteins at
dibasic sites (R/K-R/K), and that carboxypeptidase H/E remove the C-terminal R or K. It
is possible that also these enzymes are brought into play in the regulation of the CgA-
Figure 1.
Hypothetical model of the CgA-dependent “off /on/off ” switch for the regulation of
angiogenesis in tumor and its inhibition by anti-PGPQLR antibodies. (
A
) Mechanisms of activa-
tion/deactivation of the NRP-1 binding site of chromogranin A. According to this model cleavage
of the R
373
R
374
peptide bond of full-length CgA (CgA
1-439
) leads to exposure of the PGPQLR se-
quence, a site that can recognize the CendR-binding pocket of the b1 domain of neuropilin-1 (NRP-1)
on endotal cells. Removal of the R
373
residue by carboxypeptidases causes loss of NRP-1 recogni-
tion [
37
]. (
B
) Mechanism of anti-tumor activity of anti-PGPQLR antibodies. Cleavage of bloodborne
full-length CgA (CgA
1-439
) in tumors, e.g., by plasmin or thrombin, causes loss of anti-angiogenic
CgA
1-439
and generates the pro-angiogenic CgA
1-373
fragment, which may interact with NRP-1 and
contribute to promote angiogenesis and tumor growth. Antibodies against the NRP-1 binding site of
CgA
1-373
(anti-PGPQLR antibodies) block the CgA
1-373
/NRP-1 interaction and, consequently inhibit
angiogenesis and tumor growth [
37
]. This schematic representation has been prepared using the
BioRender software.
Pharmaceutics 2022,14, 2555 6 of 14
2.3. Potential Therapeutic Applications of Compounds That Interfere with CgA Fragment/
Neuropilin-1 Interaction
The unbalanced production of anti- and pro-angiogenic factors in tumor tissues can
trigger aberrant angiogenesis and altered vascular morphology, which, in turn, may con-
tribute to tumor cell proliferation, invasion, trafficking and formation of metastases [
54
–
56
].
The studies performed in patients with multiple myeloma have shown that CgA is cleaved
into the proangiogenic form CgA
1-373
in the bone marrow and that, consequently, the ratio
of pro-/anti-angiogenic forms of CgA is higher in patients compared to healthy individu-
als [
43
]. Enhanced CgA cleavage correlated with increased levels of vascular endothelial
growth factor and fibroblast growth factor-2 in the bone marrow plasma, and with an
increased bone marrow microvascular density [
43
]. Studies on the mechanism of action
revealed that multiple myeloma and endothelial cells can promote CgA cleavage through
the activation of the plasminogen activator/plasmin system [43].
Other studies aimed at evaluating the extent and prognostic value of CgA cleavage
in patients with pancreatic ductal adenocarcinoma, an aggressive cancer arising from the
exocrine component of the pancreas [
57
–
59
], have shown that cleavage of the R
373
R
374
bond
and of other sites in the C-terminal region of circulating CgA is increased in these patients.
Remarkably, CgA cleavage predicts progression-free survival and overall survival in these
cancer patients [
38
]. Experimental evidence, obtained using various pre-clinical models of
pancreatic ductal adenocarcinoma, suggests that the plasminogen activator/plasmin system
has a role in CgA processing in this case, and that CgA cleavage has a functional role of in
the regulation of tumor vascular biology. Remarkably, anti-PGPQLR
373
antibodies capable
of blocking the binding of CgA
1-373
to neuropilin-1 can reduce the growth of pancreatic
ductal adenocarcinoma in mice, which implicates an important role of neuropilin-1 as
mediator of these effects [38].
As cleavage of plasma CgA in tumors and the consequent interaction with neuropilin-
1 may represent an important mechanism for the regulation of tumor vascular biology
and growth, the assessment of the extent of CgA fragmentation in cancer patients may
have a prognostic value, whereas the development of compounds that target and block
this ligand-receptor interaction (e.g., anti-PGPQLR
373
monoclonal antibodies) may have a
therapeutic value (see Figure 1B for a schematic representation of this concept).
2.4. Role of CgA Fragment/Neuropilin-1 Interactions in Cardiovascular Regulation
Global neuropilin-1 null mice develop severe cardiovascular abnormalities, indicating
that neuropilin-1 has also a crucial cardiovascular function [
60
]. In addition, the observation
that the selective knockout of neuropilin-1 in cardiomyocytes and vascular smooth muscle
cells leads to cardiomyopathy, increased propensity to heart failure, and reduced survival
after myocardial infarction, suggests a role for neuropilin-1 in the pathogenesis of cardio-
vascular diseases [
61
]. Based on these notions, and on the fact that CgA is the precursor of
various cardio-regulatory fragments, a recent study has investigated the possibility that the
fragment CgA
1-373
affects the myocardial performance by interacting with neuropilin-1 [
46
].
Hemodynamic assessment (performed using the Langendorff rat heart model) and studies
on the mechanism of action (performed using perfused hearts and cultured cardiomy-
ocytes) have shown that CgA
1-373
can elicit negative inotropism and vasodilation, whereas
no significant effects were observed with CgA
1-372
, which lacks the C-terminal arginine
necessary for neuropilin-1 recognition [
46
]. These effects were abolished by antibodies
directed against the PGPQLR
373
sequence of CgA
1-373
. Furthermore, ex vivo and
in vitro
studies showed that these biological effects are mediated by the endothelium and involve
neuropilin-1, Akt/NO/Erk1,2 activation and S-nitrosylation [
46
]. The effects elicited by
CgA
1-373
and the lack of activity observed with CgA
1-372
suggest that CgA
1-373
is a cardio-
regulatory factor and that the removal of its C-terminal arginine by carboxypeptidases may
work as an important switch for “turning off ” its cardio-regulatory activity.
Pharmaceutics 2022,14, 2555 7 of 14
3. Integrins as Receptors for CgA and CgA-Derived Peptides
3.1. The Interaction of CgA and CgA Fragments with Integrins
The first evidence for a role of integrins as receptors for full-length CgA and vasostatin-
1 comes from a study on wound healing in injured mice [
19
]. This study has shown that
CgA and vasostatin-1, at nanomolar concentrations, selectively interact with the integrin
α
v
β
6 (see Table 1), suggesting that both polypeptides are natural ligands of this integrin.
The integrin
α
v
β
6 is an epithelial-specific cell-surface receptor of vitronectin, tenascin,
fibronectin, and also of the latency associated protein of TGF
β
1 [
62
–
64
]. In general,
α
v
β
6
recognizes a site consisting of an arginine-glycine-aspartate (RGD) motif, followed by the
LXXL/I motif (RGDLXXL/I) [
65
,
66
]. The latter motif folds into one
α
-helical turn upon
binding to the receptor [
65
–
70
]. Interestingly, a short CgA-derived peptide comprising
the residues 39–63 (CgA
39-63
, FETL
RGDERIL
SILRHQNLLKELQD) is sufficient for high-
affinity binding and highly selective recognition of
α
v
β
6 (Ki: 15.5
±
3.2 nM, Table 1) [
71
].
This peptide exhibits a degenerate RGDLXXL/I motif, in which a glutamate residue (E
46
) is
present in place of the leucine downstream of the RGD sequence (RGDEXXL). Interestingly,
in this peptide both the RGD motif (CgA
43-45
) and the adjacent sequence (CgA
46-63
) are
crucial for
α
v
β
6-integrin binding affinity and selectivity, as suggested by the observation
that the replacement of RGD with RGE abrogates integrin recognition (Table 1), and the
deletion of even a part of the C-terminal sequence markedly reduces binding affinity
and selectivity [
19
]. The molecular determinants of
α
v
β
6 recognition by CgA
39-63
have
been elucidated by NMR, computational, and biochemical studies [
71
]. Homonuclear and
heteronuclear multidimensional NMR analyses of this peptide in physiological conditions
have shown that the region between residues E
46
and K
59
has an
α
-helical conformation,
while the RGD motif is relatively flexible; the first three turns of the
α
-helix are amphipathic,
with the hydrophilic aminoacid residues E
46
, R
47
, S
50
on one side and the hydrophobic
I
48
, L
49
, I
51
, L
52
on the opposite side [
71
]. The propensity of CgA
39-63
to form an
α
-helix
is consistent with the results of a previous NMR study on CgA
47-66
, an antifungal CgA-
derived peptide, showing all-helical conformation in trifluoroethanol, an
α
-helix-promoting
solvent [
72
]. Saturation transfer difference (STD) spectroscopy experiments, performed
with the extracellular region of human
α
v
β
6 and isotopically labeled (
13
C/
15
N) CgA
39-63,
have shown that the hydrophobic residues I
48
, L
49
, I
51
, and L
52
of the
α
-helix display the
strongest STD values (>75%) [
71
], suggesting that these aminoacids contribute to receptor
binding. Molecular docking experiments led to a model of receptor-ligand interactions that
is highly reminiscent of that proposed for the proTGFβ1/αvβ6 complex [71].
Table 1. Binding affinity of CgA-derived fragments for integrins.
Competitor Competitive Binding Assay to Integrins
(Ki, nM) aRef.
αvβ6αvβ8αvβ3αvβ5α5β1
CgA1-439 105 ±34 >2000 >2000 >2000 >2000 [19]
Vasostatin-1 74 ±30 >10,000 >10,000 >10,000 >10,000 [19]
CgA39-63 15.5 ±3.2 7663 ±1704 2192 ±690 3600 ±525 9206 ±1810 [71]
CgA39-63 (RGE) >50,000 >50,000 >50,000 >50,000 >50,000 [71]
CgA39-63 (RGDL) 1.6 ±0.3 8.5 ±3.7 1928 ±226 2405 ±592 924 ±198 [71]
CgA39-63 (RGDL)-Stapled 0.6 ±0.1 3.2 ±1.2 2453 ±426 2741 ±615 1310 ±389 [71]
a
Ki, equilibrium dissociation constant of the competitor (mean
±
SEM). The Ki values were determined by
competitive binding assay using an isoDGR-peroxidase conjugate as a probe for the integrin binding site [71].
No binding of CgA
39-63
has been observed to other integrins (such as
α
1
β
1,
α
6
β
4,
α
3
β
1,
α
9
β
1
α
6
β
7,
α
5
β
1,
α
v
β
3,
α
v
β
5, and
α
v
β
8) at low-nanomolar concentrations [
19
].
However, peptides of containing the CgA
39-63
region could recognize the integrin
α
v
β
3 and
other integrins of the RGD-family when used at high concentrations in the low-micromolar
range [
19
]. For example, competitive binding assays performed with purified integrins
showed that peptide CgA
39-63
can bind
α
v
β
6 and
α
v
β
3 with Ki values of 15.5 and
2192 nM
,
Pharmaceutics 2022,14, 2555 8 of 14
respectively (Table 1), indicating that this peptide can recognize both integrins, but with
markedly different affinities [71].
3.2. Role of CgA/Integrin Interactions in Wound Healing
The
α
v
β
6 integrin is barely expressed in normal adult tissues, whereas it is highly
expressed during wound healing, tissue remodeling, and embryogenesis [
73
,
74
]. This
integrin is involved in TGF
β
1 maturation, it regulates the expression of matrix metallo-
proteases and modulates keratinocyte adhesion, proliferation, and migration in wound
healing [
19
,
62
,
64
,
75
]. It is possible, therefore, that CgA and its fragments have also a role in
the regulation of the wound healing process, by interacting with this integrin. According
to this view, experimental data showed that local injection of recombinant CgA
1-439
, but
not of a CgA
1-439
mutant with RGE in place of RGD, can accelerate wound healing in
mice [
19
]. Immunohistochemical analysis of skin tissue sections obtained from injured
mice, showed that CgA, but not the RGE mutant, could induce keratinocyte proliferation
and thickening of epidermis, suggesting that CgA can regulate the keratinocytes phys-
iology and the process of wound healing through an RGD-dependent mechanism that
likely involves the
α
v
β
6-integrin. Interestingly, both CgA and
α
v
β
6 are expressed in
wound keratinocytes [
19
,
76
]. The fact that both ligand and receptor are expressed at injured
sites lends further support to the hypothesis that the CgA/
α
v
β
6 interaction may have a
pathophysiological role in this process.
Regarding the integrin
α
v
β
3, this cell-adhesion receptor is an important player in
endothelial cell biology and angiogenesis [
77
,
78
]. Although it is unlikely that this integrin
has a receptor function for the circulating CgA polypeptides, considering its micromolar
affinity, significant ligand-receptor interactions can possibly occur at sites where CgA is
produced and, therefore, where this protein is present at high concentrations, such as in the
microenvironment of wound keratinocytes and neuroendocrine secretory cells or in the
microenvironment of neuroendocrine tumors. Furthermore, this interaction might occur
on
α
v
β
3-positive endothelial cell after the interaction with other high-affinity binding sites,
i.e., through a sort of ligand-passing mechanism.
3.3. Potential Diagnostic and Therapeutic Applications of CgA-Derived Peptides That Interact with
Integrins in Cancer
The integrin
α
v
β
6 is overexpressed by several types of cancer cells, such as head and
neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, breast, colon, liver, and
ovarian cancers, and others [
73
,
74
,
79
–
83
]. This integrin modulates cancer cell invasion,
inhibits apoptosis, and, importantly, is involved in the maturation of TGF
β
1, a potent
immunosuppressive cytokine. Increased expression levels of
α
v
β
6 are prognostic indicators
of poor survival in patients with various types of tumors [
79
,
82
,
84
–
86
], and various ligands
of this integrin coupled to tumor imaging agents are currently being tested in cancer patients
for tumor imaging purposes [
87
–
91
]; thus, the development of CgA-derived peptides
capable of recognizing this integrin in tumors is of great experimental and clinical interest.
Following this line of thought, experimental work has been carried out to obtain new
peptides with higher affinity for
α
v
β
6-integrin, starting from CgA
39-63
as a lead compound.
The model of CgA
39-63
/
α
v
β
6 interactions, obtained by NMR and computational studies,
allowed to predict that restoring the canonical RGDLXXL motif by replacing the glutamate
(E) residue in the RGDERIL site of CgA
39-63
with a leucine (L) may increase its affinity
for
α
v
β
6. Intriguingly, the replacement of E
46
with L not only increased, as expected,
the binding affinity for
α
v
β
6, but, unexpectedly, also that for the integrin
α
v
β
8 (Table 1);
thus, the E
46
L replacement converted CgA
39-63
into a bi-selective ligand of both
α
v
β
6 and
α
v
β
8 integrins (Ki: 1.6
±
0.3 nM and 8.5
±
3.7 nM, respectively) integrins [
71
]. Chemical
“stapling” of the
α
-helix of the E
46
L-CgA
39-63
mutant, by side-chain-to-side-chain cross
linking with a triazole-bridge, further increased the affinity for both
α
v
β
6 and
α
v
β
8 (Ki:
0.6
±
0.1 nM and 3.2
±
1.2 nM, respectively) by stabilizing the
α
-helix [
71
]. Notably, the
α
v
β
8 integrin represents another cell-surface receptor expressed by various carcinoma
Pharmaceutics 2022,14, 2555 9 of 14
cells [
92
–
94
]; thus, the mutated/chemically stapled peptide (called peptide
5a
) represents
a strong bi-selective ligand for these integrins, which can be potentially exploited as a
tumor-homing ligand for delivering imaging and anticancer compounds to
α
v
β
6/
α
v
β
8
single- or double-positive tumors, such as oral and skin squamous cell carcinoma [
95
]
(see Figure 2for a schematic representation of this concept). This hypothesis is supported
by the results of a very recent study aimed at investigating the tumor-homing properties
of compounds consisting of peptide
5a
coupled with IRDye 800 CW (a near-infrared
fluorescent dye) or with
18
F-NOTA (a label for positron emission tomography) [
96
]. This
study showed that both conjugates can bind
α
v
β
6 and
α
v
β
8 with an affinity similar to that
of the free peptide and that they can selectively recognize various
α
v
β
6/
α
v
β
8 single- or
double-positive cancer cells, including cells from melanoma, pancreatic carcinoma, oral
mucosa, prostate, and bladder cancer. Furthermore, biodistribution studies, performed
with these conjugates in mice bearing orthotopic or subcutaneous
α
v
β
6-positive pancreatic
tumors, showed high target-specific uptake of fluorescence- and radio-labeled peptide by
tumors [
96
]. Tumor-specific uptake of the fluorescent conjugate was also observed in mice
bearing
α
v
β
8-positive prostate tumors [
96
], confirming the hypothesis that peptide
5a
can
home to αvβ6- and/or αvβ8-positive tumors.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 9 of 15
peptides capable of recognizing this integrin in tumors is of great experimental and clini-
cal interest. Following this line of thought, experimental work has been carried out to ob-
tain new peptides with higher affinity for αvβ6-integrin, starting from CgA39-63 as a lead
compound. The model of CgA39-63/αvβ6 interactions, obtained by NMR and computa-
tional studies, allowed to predict that restoring the canonical RGDLXXL motif by replac-
ing the glutamate (E) residue in the RGDERIL site of CgA39-63 with a leucine (L) may in-
crease its affinity for αvβ6. Intriguingly, the replacement of E46 with L not only increased,
as expected, the binding affinity for αvβ6, but, unexpectedly, also that for the integrin
αvβ8 (Table 1); thus, the E46L replacement converted CgA39-63 into a bi-selective ligand of
both αvβ6 and αvβ8 integrins (Ki: 1.6 ± 0.3 nM and 8.5 ± 3.7 nM, respectively) integrins
[71]. Chemical “stapling” of the α-helix of the E46L-CgA39-63 mutant, by side-chain-to-side-
chain cross linking with a triazole-bridge, further increased the affinity for both αvβ6 and
αvβ8 (Ki: 0.6 ± 0.1 nM and 3.2 ± 1.2 nM, respectively) by stabilizing the α-helix [71]. Nota-
bly, the αvβ8 integrin represents another cell-surface receptor expressed by various carci-
noma cells [92–94]; thus, the mutated/chemically stapled peptide (called peptide 5a) rep-
resents a strong bi-selective ligand for these integrins, which can be potentially exploited
as a tumor-homing ligand for delivering imaging and anticancer compounds to
αvβ6/αvβ8 single- or double-positive tumors, such as oral and skin squamous cell carci-
noma [95] (see Figure 2 for a schematic representation of this concept). This hypothesis is
supported by the results of a very recent study aimed at investigating the tumor-homing
properties of compounds consisting of peptide 5a coupled with IRDye 800 CW (a near-
infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography)
[96]. This study showed that both conjugates can bind αvβ6 and αvβ8 with an affinity
similar to that of the free peptide and that they can selectively recognize various
αvβ6/αvβ8 single- or double-positive cancer cells, including cells from melanoma, pan-
creatic carcinoma, oral mucosa, prostate, and bladder cancer. Furthermore, biodistribu-
tion studies, performed with these conjugates in mice bearing orthotopic or subcutaneous
αvβ6-positive pancreatic tumors, showed high target-specific uptake of fluorescence- and
radio-labeled peptide by tumors [96]. Tumor-specific uptake of the fluorescent conjugate
was also observed in mice bearing αvβ8-positive prostate tumors [96], confirming the hy-
pothesis that peptide 5a can home to αvβ6- and/or αvβ8-positive tumors.
Figure 2. Use of the CgA-derived peptide 5a (stapled) for delivering imaging or therapeutic com-
pounds to αvβ6/αvβ8 single- or double-positive tumors. The peptide 5a, derived from the region
38–63 of human CgA (originally published in [96]) is characterized by the sequence CFETLRGDL-
RILSILRX1QNLX2KELQ, where X1 and X2 are propargylglycine and azidolysine residues,
Figure 2.
Use of the CgA-derived peptide
5a
(stapled) for delivering imaging or therapeutic
compounds to
α
v
β
6/
α
v
β
8 single- or double-positive tumors. The peptide
5a
, derived from
the region 38–63 of human CgA (originally published in [
96
]) is characterized by the sequence
CFETLRGDLRILSILRX
1
QNLX
2
KELQ, where X
1
and X
2
are propargylglycine and azidolysine
residues, respectively, which form a triazole bridge after a click chemistry reaction, thereby in-
creasing the
α
-helix stability. Peptide
5a
can be exploited for delivering radioactive or fluorescent
imaging compounds to
α
v
β
6/
α
v
β
8 single- or double-positive tumors or for developing new ther-
apeutic tumor-homing agents. The image in the right panel shows the radiotracer uptake in a
mouse bearing a pancreatic tumor implanted subcutaneously (arrow), as assessed by PET/CT scan
(originally published in [96]).
Remarkably, both
α
v
β
6 and
α
v
β
8 integrins (which are upregulated in many tumors
and, in the case of
α
v
β
8, also in tumor infiltrating Treg cells) can activate the latency
associated peptide/TGF
β
complex, through interactions of integrins with the RGD sites of
the complex [
73
,
83
,
92
]. These interactions can lead to the local activation of TGF
β
in the
tumor microenvironment, a potent immunosuppressive mechanism that may contribute to
Pharmaceutics 2022,14, 2555 10 of 14
tumor progression. Interestingly,
in vitro
studies have shown that peptide
5a
can inhibit
the integrin-mediated TGF
β
activation [
96
]. Thus, in principle, the peptide
5a
can be used
not only as a ligand for delivering imaging or anticancer agents to
α
v
β
6/
α
v
β
8 single- or
double-positive tumors, but also as a tumor-homing inhibitor of these TGFβactivators.
Finally, considering the role of
α
v
β
6/
α
v
β
8-mediated TGF
β
activation in fibrosis [
97
]
the dual targeting capability of peptide
5a
might be also exploited in the development of
anti-fibrotic drugs. This is another hypothesis that deserves to be investigated.
4. Conclusions
The results obtained so far suggest that integrins (particularly the integrin
α
v
β
6) and
neuropilin-1 are important receptors that mediate relevant pathophysiological functions
of CgA and its fragments in angiogenesis, wound healing, and tumor growth. Experi-
mental evidence indicates that these interactions may also represent important targets for
cancer imaging and therapy. Although further work is necessary to clarify the receptor
mechanisms of CgA and its fragments in the regulation of cardiovascular homeostasis,
metabolism, and tumor growth, the results obtained so far highlight the complexity of
the “CgA system”, which consists of a multitude of CgA-derived peptides and various
receptors. The complexity of this system is even higher if we consider that full-length CgA
and some of its fragments show biphasic dose-response curves in angiogenesis assays, as
well as in cardio-regulatory and tumor pre-clinical models, likely because of the activation
of counterregulatory mechanisms at higher doses. These mechanisms are not clearly under-
stood and, therefore, their full elucidation remains a challenge. A third level of complexity
is related to the fact that CgA undergoes differential post-translational modifications in
different cells and tissues, such as glycosylation, sulfation, and phosphorylation. As most
of the studies carried out so far on the biological functions of CgA have been performed
with recombinant or synthetic peptides lacking these modifications, the impact of these
structural modifications on proteolytic cleavage, fragment generation, receptor recognition,
and biological activity, remains to be investigated.
Author Contributions:
A.C., G.A. and F.C. performed literature research, interpreted data, prepared
figures, and wrote and approved the submitted manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding:
This research was funded by Associazione Italiana per la Ricerca sul Cancro (AIRC) under
IG 2019–ID. 23470 project–P.I. Angelo Corti and by Fondazione AIRC 5 per Mille 2019 (ID 22737)
program, P.I. MC Bonini, Group Leader A. Corti.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest:
A.C. and F.C. are inventor of a patent on CgA-derived peptides and their use in
cancer imaging.
Abbreviations
Chromogranin A (CgA), neuropilin-1 (NRP-1), transforming growth factor-
β
1 (TGF
β
1).
References
1.
Taupenot, L.; Harper, K.L.; O’Connor, D.T. The chromogranin-secretogranin family. N. Engl. J. Med.
2003
,348, 1134–1149.
[CrossRef]
2.
Helle, K.B.; Corti, A.; Metz-Boutigue, M.H.; Tota, B. The endocrine role for chromogranin A: A prohormone for peptides with
regulatory properties. Cell Mol. Life Sci. 2007,64, 2863–2886. [CrossRef]
3.
Corti, A.; Marcucci, F.; Bachetti, T. Circulating chromogranin A and its fragments as diagnostic and prognostic disease markers.
Pflugers Arch. 2018,470, 199–210. [CrossRef]
Pharmaceutics 2022,14, 2555 11 of 14
4.
Gadroy, P.; Stridsberg, M.; Capon, C.; Michalski, J.C.; Strub, J.M.; Van Dorsselaer, A.; Aunis, D.; Metz-Boutigue, M.H. Phosphory-
lation and O-glycosylation sites of human chromogranin A (CGA79-439) from urine of patients with carcinoid tumors. J. Biol.
Chem. 1998,273, 34087–34097. [CrossRef]
5.
Metz-Boutigue, M.H.; Lugardon, K.; Goumon, Y.; Raffner, R.; Strub, J.M.; Aunis, D. Antibacterial and antifungal peptides derived
from chromogranins and proenkephalin-A. From structural to biological aspects. Adv. Exp. Med. Biol. 2000,482, 299–315.
6.
Shooshtarizadeh, P.; Zhang, D.; Chich, J.F.; Gasnier, C.; Schneider, F.; Haikel, Y.; Aunis, D.; Metz-Boutigue, M.H. The antimicrobial
peptides derived from chromogranin/secretogranin family, new actors of innate immunity. Regul. Pept.
2009
,165, 102–110.
[CrossRef]
7.
Briolat, J.; Wu, S.D.; Mahata, S.K.; Gonthier, B.; Bagnard, D.; Chasserot-Golaz, S.; Helle, K.B.; Aunis, D.; Metz-Boutigue, M.H.
New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A. Cell. Mol. Life Sci. CMLS
2005
,
62, 377–385. [CrossRef]
8.
Ioannidis, M.; Mahata, S.K.; van den Bogaart, G. The immunomodulatory functions of chromogranin A-derived peptide
pancreastatin. Peptides 2022,158, 170893. [CrossRef]
9.
Aardal, S.; Helle, K.B. The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its independence of
extracellular calcium in isolated segments of human blood vessels. Regul. Pept. 1992,41, 9–18. [CrossRef]
10.
Helle, K.B. The chromogranin A-derived peptides vasostatin-I and catestatin as regulatory peptides for cardiovascular functions.
Cardiovasc. Res. 2010,85, 9–16. [CrossRef]
11.
Tota, B.; Mazza, R.; Angelone, T.; Nullans, G.; Metz-Boutigue, M.H.; Aunis, D.; Helle, K.B. Peptides from the N-terminal domain of
chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart. Regul. Pept.
2003
,114, 123–130. [CrossRef]
12.
Tota, B.; Angelone, T.; Cerra, M.C. The surging role of Chromogranin A in cardiovascular homeostasis. Front. Chem.
2014
,2, 64.
[CrossRef]
13.
Tatemoto, K.; Efendic, S.; Mutt, V.; Makk, G.; Feistner, G.J.; Barchas, J.D. Pancreastatin, a novel pancreatic peptide that inhibits
insulin secretion. Nature 1986,324, 476–478. [CrossRef]
14.
Mahata, S.K.; Corti, A. Chromogranin A and its fragments in cardiovascular, immunometabolic, and cancer regulation. Ann. N. Y.
Acad. Sci. 2019,1455, 34–58. [CrossRef]
15.
Bandyopadhyay, G.K.; Mahata, S.K. Chromogranin A Regulation of Obesity and Peripheral Insulin Sensitivity. Front. Endocrinol.
2017,8, 20. [CrossRef]
16.
Theurl, M.; Schgoer, W.; Albrecht, K.; Jeschke, J.; Egger, M.; Beer, A.G.; Vasiljevic, D.; Rong, S.; Wolf, A.M.; Bahlmann, F.H.; et al.
The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism. Circ.
Res. 2010,107, 1326–1335. [CrossRef]
17.
Crippa, L.; Bianco, M.; Colombo, B.; Gasparri, A.M.; Ferrero, E.; Loh, Y.P.; Curnis, F.; Corti, A. A new chromogranin A-dependent
angiogenic switch activated by thrombin. Blood 2013,121, 392–402. [CrossRef]
18.
Helle, K.B.; Corti, A. Chromogranin A: A paradoxical player in angiogenesis and vascular biology. Cell. Mol. Life Sci.
2015
,78,
339–348. [CrossRef]
19.
Curnis, F.; Gasparri, A.; Longhi, R.; Colombo, B.; D’Alessio, S.; Pastorino, F.; Ponzoni, M.; Corti, A. Chromogranin A Binds to
αvβ6-Integrin and Promotes Wound Healing in Mice. Cell. Mol. Life Sci. 2012,69, 2791–2803. [CrossRef]
20.
Colombo, B.; Curnis, F.; Foglieni, C.; Monno, A.; Arrigoni, G.; Corti, A. Chromogranin a expression in neoplastic cells affects
tumor growth and morphogenesis in mouse models. Cancer Res. 2002,62, 941–946.
21.
Curnis, F.; Dallatomasina, A.; Bianco, M.; Gasparri, A.; Sacchi, A.; Colombo, B.; Fiocchi, M.; Perani, L.; Venturini, M.;
Tacchetti, C.; et al.
Regulation of tumor growth by circulating full-length chromogranin A. Oncotarget
2016
,7, 72716–72732.
[CrossRef]
22.
Salem, S.; Jankowski, V.; Asare, Y.; Liehn, E.; Welker, P.; Raya-Bermudez, A.; Pineda-Martos, C.; Rodriguez, M.; Munoz-Castaneda,
J.R.; Bruck, H.; et al. Identification of the Vasoconstriction-Inhibiting Factor (VIF), a Potent Endogenous Cofactor of Angiotensin
II Acting on the Angiotensin II Type 2 Receptor. Circulation 2015,131, 1426–1434. [CrossRef]
23.
Brekke, J.F.; Kirkeleit, J.; Lugardon, K.; Helle, K.B. Vasostatins. Dilators of bovine resistance arteries. Adv. Exp. Med. Biol.
2000
,
482, 239–246.
24.
Mahata, S.K.; O’Connor, D.T.; Mahata, M.; Yoo, S.H.; Taupenot, L.; Wu, H.; Gill, B.M.; Parmer, R.J. Novel autocrine feedback
control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist. J. Clin.
Investig. 1997,100, 1623–1633. [CrossRef]
25.
Koshimizu, H.; Cawley, N.X.; Kim, T.; Yergey, A.L.; Loh, Y.P. Serpinin: A novel chromogranin A-derived, secreted peptide
up-regulates protease nexin-1 expression and granule biogenesis in endocrine cells. Mol. Endocrinol.
2011
,25, 732–744. [CrossRef]
26.
Loh, Y.P.; Koshimizu, H.; Cawley, N.X.; Tota, B. Serpinins: Role in granule biogenesis, inhibition of cell death and cardiac function.
Curr. Med. Chem. 2012,19, 4086–4092. [CrossRef]
27.
Mancino, D.; Kharouf, N.; Scavello, F.; Helle, S.; Salloum-Yared, F.; Mutschler, A.; Mathieu, E.; Lavalle, P.; Metz-Boutigue, M.H.;
Haikel, Y. The Catestatin-Derived Peptides Are New Actors to Fight the Development of Oral Candidosis. Int. J. Mol. Sci.
2022
,
23, 2066. [CrossRef]
28.
Metz-Boutigue, M.H.; Goumon, Y.; Lugardon, K.; Strub, J.M.; Aunis, D. Antibacterial peptides are present in chromaffin cell
secretory granules. Cell. Mol. Neurobiol. 1998,18, 249–266. [CrossRef]
Pharmaceutics 2022,14, 2555 12 of 14
29.
Fung, M.M.; Salem, R.M.; Mehtani, P.; Thomas, B.; Lu, C.F.; Perez, B.; Rao, F.; Stridsberg, M.; Ziegler, M.G.; Mahata, S.K.; et al.
Direct vasoactive effects of the chromogranin A (CHGA) peptide catestatin in humans
in vivo
.Clin. Exp. Hypertens.
2010
,32,
278–287. [CrossRef]
30.
Kruger, P.G.; Mahata, S.K.; Helle, K.B. Catestatin (CgA344-364) stimulates rat mast cell release of histamine in a manner
comparable to mastoparan and other cationic charged neuropeptides. Regul. Pept. 2003,114, 29–35. [CrossRef]
31.
Pasqua, T.; Corti, A.; Gentile, S.; Pochini, L.; Bianco, M.; Metz-Boutigue, M.H.; Cerra, M.C.; Tota, B.; Angelone, T. Full-length
human Chromogranin-A cardioactivity: Myocardial, coronary and stimulus-induced processing evidence in normotensive and
hypertensive male rat hearts. Endocrinology 2013,154, 3353–3365. [CrossRef]
32.
Angelone, T.; Quintieri, A.M.; Brar, B.K.; Limchaiyawat, P.T.; Tota, B.; Mahata, S.K.; Cerra, M.C. The antihypertensive chromo-
granin a peptide catestatin acts as a novel endocrine/paracrine modulator of cardiac inotropism and lusitropism. Endocrinology
2008,149, 4780–4793. [CrossRef]
33.
Corti, A.; Mannarino, C.; Mazza, R.; Colombo, B.; Longhi, R.; Tota, B. Vasostatins exert negative inotropism in the working heart
of the frog. Ann. N. Y. Acad. Sci. 2002,971, 362–365. [CrossRef]
34.
Gasparri, A.; Sidoli, A.; Sanchez, L.P.; Longhi, R.; Siccardi, A.G.; Marchisio, P.C.; Corti, A. Chromogranin A fragments modulate
cell adhesion. Identification and characterization of a pro-adhesive domain. J. Biol. Chem. 1997,272, 20835–20843. [CrossRef]
35.
Ferrero, E.; Scabini, S.; Magni, E.; Foglieni, C.; Belloni, D.; Colombo, B.; Curnis, F.; Villa, A.; Ferrero, M.E.; Corti, A. Chromogranin
A protects vessels against tumor necrosis factor alpha-induced vascular leakage. FASEB J. 2004,18, 554–556. [CrossRef]
36.
Di Comite, G.; Rossi, C.M.; Marinosci, A.; Lolmede, K.; Baldissera, E.; Aiello, P.; Mueller, R.B.; Herrmann, M.; Voll, R.E.;
Rovere-Querini, P.; et al. Circulating chromogranin A reveals extra-articular involvement in patients with rheumatoid arthritis
and curbs TNF-alpha-elicited endothelial activation. J. Leukoc. Biol. 2009,85, 81–87. [CrossRef]
37.
Dallatomasina, A.; Gasparri, A.M.; Colombo, B.; Sacchi, A.; Bianco, M.; Daniele, T.; Esposito, A.; Pastorino, F.; Ponzoni, M.;
Marcucci, F.; et al. Spatiotemporal Regulation of Tumor Angiogenesis by Circulating Chromogranin A Cleavage and Neuropilin-1
Engagement. Cancer Res. 2019,79, 1925–1937. [CrossRef]
38.
Reni, M.; Andreasi, V.; Gasparri, A.M.; Dugnani, E.; Colombo, B.; Macchini, M.; Bianco, M.; Dallatomasina, A.; Citro, A.;
Assi, E.; et al
. Circulating Chromogranin A Is Cleaved Into Vasoregulatory Fragments in Patients with Pancreatic Ductal
Adenocarcinoma. Front. Oncol. 2020,10, 613582. [CrossRef]
39.
Blois, A.; Holmsen, H.; Martino, G.; Corti, A.; Metz-Boutigue, M.H.; Helle, K.B. Interactions of chromogranin A-derived
vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine. Regul. Pept. 2006,134,
30–37. [CrossRef]
40.
Ramella, R.; Boero, O.; Alloatti, G.; Angelone, T.; Levi, R.; Gallo, M.P. Vasostatin 1 activates eNOS in endothelial cells through a
proteoglycan-dependent mechanism. J. Cell. Biochem. 2010,110, 70–79. [CrossRef]
41.
Kiranmayi, M.; Chirasani, V.R.; Allu, P.K.; Subramanian, L.; Martelli, E.E.; Sahu, B.S.; Vishnuprabu, D.; Kumaragurubaran, R.;
Sharma, S.; Bodhini, D.; et al. Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in
Human Populations via Endothelial Nitric Oxide Pathway. Hypertension 2016,68, 334–347. [CrossRef]
42.
Belloni, D.; Scabini, S.; Foglieni, C.; Veschini, L.; Giazzon, A.; Colombo, B.; Fulgenzi, A.; Helle, K.B.; Ferrero, M.E.; Corti, A.; et al.
The vasostatin-I fragment of chromogranin A inhibits VEGF-induced endothelial cell proliferation and migration. FASEB J.
2007
,
21, 3052–3062. [CrossRef]
43.
Bianco, M.; Gasparri, A.M.; Colombo, B.; Curnis, F.; Girlanda, S.; Ponzoni, M.; Bertilaccio, M.T.; Calcinotto, A.; Sacchi, A.; Ferrero,
E.; et al. Chromogranin A is preferentially cleaved into pro-angiogenic peptides in the bone marrow of multiple myeloma patients.
Cancer Res. 2016,76, 1781–1791. [CrossRef]
44.
Teesalu, T.; Sugahara, K.N.; Kotamraju, V.R.; Ruoslahti, E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and
tissue penetration. Proc. Natl. Acad. Sci. USA 2009,106, 16157–16162. [CrossRef]
45. Teesalu, T.; Sugahara, K.N.; Ruoslahti, E. Tumor-penetrating peptides. Front. Oncol. 2013,3, 216. [CrossRef]
46.
Rocca, C.; Grande, F.; Granieri, M.C.; Colombo, B.; De Bartolo, A.; Giordano, F.; Rago, V.; Amodio, N.; Tota, B.; Cerra, M.C.; et al.
The chromogranin A1-373 fragment reveals how a single change in the protein sequence exerts strong cardioregulatory effects by
engaging neuropilin-1. Acta Physiol. 2021,231, e13570. [CrossRef]
47.
Pena, V.B.; Bonini, I.C.; Antollini, S.S.; Kobayashi, T.; Barrantes, F.J. Alpha 7-type acetylcholine receptor localization and its
modulation by nicotine and cholesterol in vascular endothelial cells. J. Cell. Biochem. 2011,112, 3276–3288. [CrossRef]
48.
Cooke, J.P.; Ghebremariam, Y.T. Endothelial nicotinic acetylcholine receptors and angiogenesis. Trends Cardiovasc. Med.
2008
,18,
247–253. [CrossRef]
49.
Arias, H.R.; Richards, V.E.; Ng, D.; Ghafoori, M.E.; Le, V.; Mousa, S.A. Role of non-neuronal nicotinic acetylcholine receptors in
angiogenesis. Int. J. Biochem. Cell. Biol. 2009,41, 1441–1451. [CrossRef]
50.
Heeschen, C.; Jang, J.J.; Weis, M.; Pathak, A.; Kaji, S.; Hu, R.S.; Tsao, P.S.; Johnson, F.L.; Cooke, J.P. Nicotine stimulates angiogenesis
and promotes tumor growth and atherosclerosis. Nat. Med. 2001,7, 833–839. [CrossRef]
51.
Taupenot, L.; Mahata, S.K.; Mahata, M.; Parmer, R.J.; O’Connor, D.T. Interaction of the catecholamine release-inhibitory peptide
catestatin (human chromogranin A(352–372)) with the chromaffin cell surface and Torpedo electroplax: Implications for nicotinic
cholinergic antagonism. Regul. Pept. 2000,95, 9–17. [CrossRef]
52.
Mahata, S.K.; Mahata, M.; Fung, M.M.; O’Connor, D.T. Catestatin: A multifunctional peptide from chromogranin A. Regul. Pept.
2010,162, 33–43. [CrossRef]
Pharmaceutics 2022,14, 2555 13 of 14
53.
Sahu, B.S.; Mohan, J.; Sahu, G.; Singh, P.K.; Sonawane, P.J.; Sasi, B.K.; Allu, P.K.; Maji, S.K.; Bera, A.K.; Senapati, S.; et al. Molecular
interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor. J. Cell.
Sci. 2012,125, 2323–2337. [CrossRef]
54. Folkman, J. Angiogenesis: An organizing principle for drug discovery? Nat. Rev. Drug Discov. 2007,6, 273–286. [CrossRef]
55.
Italiano, J.E., Jr.; Richardson, J.L.; Patel-Hett, S.; Battinelli, E.; Zaslavsky, A.; Short, S.; Ryeom, S.; Folkman, J.; Klement, G.L.
Angiogenesis is regulated by a novel mechanism: Pro- and antiangiogenic proteins are organized into separate platelet alpha
granules and differentially released. Blood 2008,111, 1227–1233. [CrossRef]
56. Ribatti, D. Endogenous inhibitors of angiogenesis: A historical review. Leuk. Res. 2009,33, 638–644. [CrossRef]
57.
Kleeff, J.; Korc, M.; Apte, M.; La Vecchia, C.; Johnson, C.D.; Biankin, A.V.; Neale, R.E.; Tempero, M.; Tuveson, D.A.;
Hruban, R.H.; et al. Pancreatic cancer. Nat. Rev. Dis. Prim. 2016,2, 16022. [CrossRef]
58.
Rawla, P.; Sunkara, T.; Gaduputi, V. Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors. World J. Oncol.
2019,10, 10–27. [CrossRef]
59.
Conroy, T.; Desseigne, F.; Ychou, M.; Bouche, O.; Guimbaud, R.; Becouarn, Y.; Adenis, A.; Raoul, J.L.; Gourgou-Bourgade, S.; de la
Fouchardiere, C.; et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N. Engl. J. Med.
2011
,364, 1817–1825.
[CrossRef]
60.
Kawasaki, T.; Kitsukawa, T.; Bekku, Y.; Matsuda, Y.; Sanbo, M.; Yagi, T.; Fujisawa, H. A requirement for neuropilin-1 in embryonic
vessel formation. Development 1999,126, 4895–4902. [CrossRef]
61.
Wang, Y.; Cao, Y.; Yamada, S.; Thirunavukkarasu, M.; Nin, V.; Joshi, M.; Rishi, M.T.; Bhattacharya, S.; Camacho-Pereira, J.;
Sharma, A.K.; et al. Cardiomyopathy and Worsened Ischemic Heart Failure in SM22-alpha Cre-Mediated Neuropilin-1 Null Mice:
Dysregulation of PGC1alpha and Mitochondrial Homeostasis. Arterioscler Thromb. Vasc. Biol. 2015,35, 1401–1412. [CrossRef]
62.
Thomas, G.J.; Nystrom, M.L.; Marshall, J.F. Alphavbeta6 integrin in wound healing and cancer of the oral cavity. J. Oral Pathol.
Med. 2006,35, 1–10. [CrossRef]
63.
Busk, M.; Pytela, R.; Sheppard, D. Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein. J. Biol. Chem.
1992,267, 5790–5796. [CrossRef]
64.
Bandyopadhyay, A.; Raghavan, S. Defining the role of integrin alphavbeta6 in cancer. Curr. Drug Targets
2009
,10, 645–652.
[CrossRef]
65.
Ozawa, A.; Sato, Y.; Imabayashi, T.; Uemura, T.; Takagi, J.; Sekiguchi, K. Molecular Basis of the Ligand Binding Specificity of
alphavbeta8 Integrin. J. Biol. Chem. 2016,291, 11551–11565. [CrossRef]
66.
Kraft, S.; Diefenbach, B.; Mehta, R.; Jonczyk, A.; Luckenbach, G.A.; Goodman, S.L. Definition of an unexpected ligand recognition
motif for alphav beta6 integrin. J. Biol. Chem. 1999,274, 1979–1985. [CrossRef]
67.
Dong, X.; Hudson, N.E.; Lu, C.; Springer, T.A. Structural determinants of integrin beta-subunit specificity for latent TGF-beta.
Nat. Struct. Mol. Biol. 2014,21, 1091–1096. [CrossRef]
68.
Dong, X.; Zhao, B.; Iacob, R.E.; Zhu, J.; Koksal, A.C.; Lu, C.; Engen, J.R.; Springer, T.A. Force interacts with macromolecular
structure in activation of TGF-beta. Nature 2017,542, 55–59. [CrossRef]
69.
Kotecha, A.; Wang, Q.; Dong, X.; Ilca, S.L.; Ondiviela, M.; Zihe, R.; Seago, J.; Charleston, B.; Fry, E.E.; Abrescia, N.G.A.; et al. Rules
of engagement between alphavbeta6 integrin and foot-and-mouth disease virus. Nat. Commun. 2017,8, 15408. [CrossRef]
70.
DiCara, D.; Rapisarda, C.; Sutcliffe, J.L.; Violette, S.M.; Weinreb, P.H.; Hart, I.R.; Howard, M.J.; Marshall, J.F. Structure-function
analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands. J. Biol. Chem. 2007,282, 9657–9665. [CrossRef]
71.
Nardelli, F.; Ghitti, M.; Quilici, G.; Gori, A.; Luo, Q.; Berardi, A.; Sacchi, A.; Monieri, M.; Bergamaschi, G.; Bermel, W.; et al. A
stapled chromogranin A-derived peptide is a potent dual ligand for integrins alphavbeta6 and alphavbeta8. Chem. Commun.
2019,55, 14777–14780. [CrossRef]
72.
Lugardon, K.; Chasserot-Golaz, S.; Kieffer, A.E.; Maget-Dana, R.; Nullans, G.; Kieffer, B.; Aunis, D.; Metz-Boutigue, M.H.
Structural and biological characterization of chromofungin, the antifungal chromogranin A-(47-66)-derived peptide. J. Biol. Chem.
2001,276, 35875–35882. [CrossRef]
73.
Koivisto, L.; Bi, J.; Hakkinen, L.; Larjava, H. Integrin alphavbeta6: Structure, function and role in health and disease. Int. J.
Biochem. Cell. Biol. 2018,99, 186–196. [CrossRef]
74.
Liu, H.; Wu, Y.; Wang, F.; Liu, Z. Molecular imaging of integrin alphavbeta6 expression in living subjects. Am. J. Nucl. Med. Mol.
Imaging 2014,4, 333–345.
75.
Koivisto, L.; Larjava, K.; Hakkinen, L.; Uitto, V.J.; Heino, J.; Larjava, H. Different integrins mediate cell spreading, haptotaxis and
lateral migration of HaCaT keratinocytes on fibronectin. Cell Adhes. Commun. 1999,7, 245–257. [CrossRef]
76.
Radek, K.A.; Lopez-Garcia, B.; Hupe, M.; Niesman, I.R.; Elias, P.M.; Taupenot, L.; Mahata, S.K.; O’Connor, D.T.; Gallo, R.L. The
neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury. J. Investig. Dermatol.
2008
,
128, 1525–1534. [CrossRef]
77.
Avraamides, C.J.; Garmy-Susini, B.; Varner, J.A. Integrins in angiogenesis and lymphangiogenesis. Nat. Rev. Cancer
2008
,8,
604–617. [CrossRef]
78.
Desgrosellier, J.S.; Cheresh, D.A. Integrins in cancer: Biological implications and therapeutic opportunities. Nat. Rev. Cancer
2010
,
10, 9–22. [CrossRef]
Pharmaceutics 2022,14, 2555 14 of 14
79.
Elayadi, A.N.; Samli, K.N.; Prudkin, L.; Liu, Y.H.; Bian, A.; Xie, X.J.; Wistuba, I.I.; Roth, J.A.; McGuire, M.J.; Brown, K.C. A peptide
selected by biopanning identifies the integrin alphavbeta6 as a prognostic biomarker for nonsmall cell lung cancer. Cancer Res.
2007,67, 5889–5895. [CrossRef]
80.
Moore, K.M.; Thomas, G.J.; Duffy, S.W.; Warwick, J.; Gabe, R.; Chou, P.; Ellis, I.O.; Green, A.R.; Haider, S.; Brouilette, K.; et al.
Therapeutic targeting of integrin alphavbeta6 in breast cancer. J. Natl. Cancer Inst. 2014,106. [CrossRef]
81. Sipos, B.; Hahn, D.; Carceller, A.; Piulats, J.; Hedderich, J.; Kalthoff, H.; Goodman, S.L.; Kosmahl, M.; Kloppel, G. Immunohisto-
chemical screening for beta6-integrin subunit expression in adenocarcinomas using a novel monoclonal antibody reveals strong
up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro. Histopathology 2004,45, 226–236. [CrossRef]
82. Reader, C.S.; Vallath, S.; Steele, C.W.; Haider, S.; Brentnall, A.; Desai, A.; Moore, K.M.; Jamieson, N.B.; Chang, D.; Bailey, P.; et al.
The integrin alphavbeta6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.
J. Pathol. 2019,249, 332–342. [CrossRef]
83. Niu, J.; Li, Z. The roles of integrin alphavbeta6 in cancer. Cancer Lett. 2017,403, 128–137. [CrossRef]
84.
Hazelbag, S.; Kenter, G.G.; Gorter, A.; Dreef, E.J.; Koopman, L.A.; Violette, S.M.; Weinreb, P.H.; Fleuren, G.J. Overexpression of
the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. J. Pathol.
2007
,212,
316–324. [CrossRef]
85.
Zhang, Z.Y.; Xu, K.S.; Wang, J.S.; Yang, G.Y.; Wang, W.; Wang, J.Y.; Niu, W.B.; Liu, E.Y.; Mi, Y.T.; Niu, J. Integrin alphanvbeta6 acts
as a prognostic indicator in gastric carcinoma. Clin. Oncol. 2008,20, 61–66. [CrossRef]
86.
Bates, R.C.; Bellovin, D.I.; Brown, C.; Maynard, E.; Wu, B.; Kawakatsu, H.; Sheppard, D.; Oettgen, P.; Mercurio, A.M. Transcrip-
tional activation of integrin beta6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive
colon carcinoma. J. Clin. Investig. 2005,115, 339–347. [CrossRef]
87.
Altmann, A.; Sauter, M.; Roesch, S.; Mier, W.; Warta, R.; Debus, J.; Dyckhoff, G.; Herold-Mende, C.; Haberkorn, U. Identification
of a Novel ITGalphavbeta6-Binding Peptide Using Protein Separation and Phage Display. Clin. Cancer Res.
2017
,23, 4170–4180.
[CrossRef]
88.
Roesch, S.; Lindner, T.; Sauter, M.; Loktev, A.; Flechsig, P.; Muller, M.; Mier, W.; Warta, R.; Dyckhoff, G.;
Herold-Mende, C.; et al
.
Comparison of the RGD Motif-Containing alphavbeta6 Integrin-Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Applica-
tion in HNSCC. J. Nucl. Med. 2018,59, 1679–1685. [CrossRef]
89.
Quigley, N.G.; Czech, N.; Sendt, W.; Notni, J. PET/CT imaging of pancreatic carcinoma targeting the “cancer integrin” alphavbeta6.
Eur. J. Nucl. Med. Mol. Imaging 2021,48, 4107–4108. [CrossRef]
90.
Kimura, R.H.; Wang, L.; Shen, B.; Huo, L.; Tummers, W.; Filipp, F.V.; Guo, H.H.; Haywood, T.; Abou-Elkacem, L.; Baratto, L.; et al.
Evaluation of integrin alphavbeta6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis. Nat. Commun.
2019,10, 4673. [CrossRef]
91.
Hausner, S.H.; Bold, R.J.; Cheuy, L.Y.; Chew, H.K.; Daly, M.E.; Davis, R.A.; Foster, C.C.; Kim, E.J.; Sutcliffe, J.L. Preclinical
Development and First-in-Human Imaging of the Integrin
α
v
β
6 with [18F]
α
v
β
6-Binding Peptide in Metastatic Carcinoma. Clin.
Cancer Res. 2019,25, 1206–1215. [CrossRef]
92.
McCarty, J.H. alphavbeta8 integrin adhesion and signaling pathways in development, physiology and disease. J. Cell Sci.
2020
,
133. [CrossRef]
93.
Takasaka, N.; Seed, R.I.; Cormier, A.; Bondesson, A.J.; Lou, J.; Elattma, A.; Ito, S.; Yanagisawa, H.; Hashimoto, M.; Ma, R.; et al.
Integrin alphavbeta8-expressing tumor cells evade host immunity by regulating TGF-beta activation in immune cells. JCI Insight
2018,3. [CrossRef]
94.
Jin, S.; Lee, W.C.; Aust, D.; Pilarsky, C.; Cordes, N. beta8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance. Mol.
Cancer Res. 2019,17, 2126–2138. [CrossRef]
95.
Ahmedah, H.T.; Patterson, L.H.; Shnyder, S.D.; Sheldrake, H.M. RGD-Binding Integrins in Head and Neck Cancers. Cancers
2017
,
9, 56. [CrossRef]
96.
Monieri, M.; Rainone, P.; Sacchi, A.; Gori, A.; Gasparri, A.M.; Coliva, A.; Citro, A.; Ferrara, B.; Policardi, M.; Valtorta, S.; et al.
A stapled chromogranin A-derived peptide homes in on tumors that express
α
v
β
6 or
α
v
β
8 integrins. Int. J. Biol. Sci.
2022
,19,
156–166.
97.
Conroy, K.P.; Kitto, L.J.; Henderson, N.C. Alphav integrins: Key regulators of tissue fibrosis. Cell Tissue Res.
2016
,365, 511–519.
[CrossRef]
