Role for substance p-based nociceptive signaling in progenitor cell activation and angiogenesis during ischemia in mice and in human subjects.

Abstract

Pain triggers a homeostatic alarm reaction to injury. It remains unknown, however, whether nociceptive signaling activated by ischemia is relevant for progenitor cells (PC) release from bone marrow. To this end, we investigated the role of the neuropeptide substance P (SP) and cognate neurokinin 1 (NK1) nociceptor in PC activation and angiogenesis during ischemia in mice and in human subjects.
The mouse bone marrow contains sensory fibers and PC that express SP. Moreover, SP-induced migration provides enrichment for PC that express NK1 and promote reparative angiogenesis after transplantation in a mouse model of limb ischemia. Acute myocardial infarction and limb ischemia increase SP levels in peripheral blood, decrease SP levels in bone marrow, and stimulate the mobilization of NK1-expressing PC, with these effects being abrogated by systemic administration of the opioid receptor agonist morphine. Moreover, bone marrow reconstitution with NK1-knockout cells results in depressed PC mobilization, delayed blood flow recovery, and reduced neovascularization after ischemia. We next asked whether SP is instrumental to PC mobilization and homing in patients with ischemia. Human PC express NK1, and SP-induced migration provides enrichment for proangiogenic PC. Patients with acute myocardial infarction show high circulating levels of SP and NK1-positive cells that coexpress PC antigens, such as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC are abundant in infarcted hearts but not in hearts that developed an infarct after transplantation.
Our data highlight the role of SP in reparative neovascularization. Nociceptive signaling may represent a novel target of regenerative medicine.

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Role for Substance P–Based Nociceptive Signaling in
Progenitor Cell Activation and Angiogenesis During
Ischemia in Mice and in Human Subjects
Silvia Amadesi, PhD*; Carlotta Reni, MSc*; Rajesh Katare, MD; Marco Meloni, PhD;
Atsuhiko Oikawa, PhD; Antonio P. Beltrami, MD, PhD; Elisa Avolio, MSc;
Daniela Cesselli, MD, PhD; Orazio Fortunato, PhD; Gaia Spinetti, PhD;
Raimondo Ascione, MD, ChM, FRCS, FETCS; Elisa Cangiano, MD; Marco Valgimigli, MD, PhD;
Stephen P. Hunt, PhD, FMedSci; Costanza Emanueli, PhD; Paolo Madeddu, MD
Background—Pain triggers a homeostatic alarm reaction to injury. It remains unknown, however, whether nociceptive
signaling activated by ischemia is relevant for progenitor cells (PC) release from bone marrow. To this end, we
investigated the role of the neuropeptide substance P (SP) and cognate neurokinin 1 (NK1) nociceptor in PC activation
and angiogenesis during ischemia in mice and in human subjects.
Methods and Results—The mouse bone marrow contains sensory fibers and PC that express SP. Moreover, SP-induced
migration provides enrichment for PC that express NK1 and promote reparative angiogenesis after transplantation in a
mouse model of limb ischemia. Acute myocardial infarction and limb ischemia increase SP levels in peripheral blood,
decrease SP levels in bone marrow, and stimulate the mobilization of NK1-expressing PC, with these effects being
abrogated by systemic administration of the opioid receptor agonist morphine. Moreover, bone marrow reconstitution
with NK1-knockout cells results in depressed PC mobilization, delayed blood flow recovery, and reduced neovascu-
larization after ischemia. We next asked whether SP is instrumental to PC mobilization and homing in patients with
ischemia. Human PC express NK1, and SP-induced migration provides enrichment for proangiogenic PC. Patients with
acute myocardial infarction show high circulating levels of SP and NK1-positive cells that coexpress PC antigens, such
as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC are abundant in infarcted hearts but not in hearts that
developed an infarct after transplantation.
Conclusions—Our data highlight the role of SP in reparative neovascularization. Nociceptive signaling may represent a
novel target of regenerative medicine. (Circulation. 2012;125:1774-1786.)
Key Words: limb ischemia ? myocardial infarction ? neovascularization ? stem cells
T
paracrine interaction with stromal cells of the endosteal and
vascular niches.1Tissue injury, such as acute myocardial
infarction and limb ischemia, disrupts the retaining microen-
vironment, leading to forced PC egress into the circulation.
Concurrently, the local release of cytokines, chemokines,
growth factors, and neurohormones attracts circulating cells
to the injury site.2–5Pain is an essential component of the
alarm reaction to tissue damage. However, the contribution of
rafficking of progenitor cells (PC) from bone marrow to
the peripheral blood is tightly regulated by physical and
nociceptive reflexes in PC mobilization during ischemia
remains largely unexplored.
Clinical Perspective on p 1786
Sympathetic and primary afferent sensory fibers innervate
the heart and peripheral tissues and are also expressed in bone
and bone marrow (reviewed by Nance and Sanders6). After
injury or thermal/chemical stimulation, sensory fibers release
neuropeptides, such as substance P (SP) and calcitonin
gene-related peptide (CGRP), from central terminals proj-
Received August 7, 2011; accepted February 27, 2012.
From the Laboratories of Experimental Cardiovascular Medicine (S.A., C.R., R.K., A.O., P.M.), Vascular Pathology and Regeneration (M.M., C.E.),
and Cardiac Surgery and Translational Research (R.A.), Bristol Heart Institute, University of Bristol, Bristol, UK; IRCCS MultiMedica, Milan, Italy (O.F.,
G.S.); Interdepartmental Center for Regenerative Medicine, University of Udine, Udine, Italy (A.P.B., E.A., D.C.); University of Ferrara, Ferrara, Italy
(E.C., M.V.); and University College London, London, UK (S.P.H.).
*The first 2 authors contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.
111.089763/-/DC1.
Correspondence to Paolo Madeddu, MD, Experimental Cardiovascular Medicine, Regenerative Medicine Section, Bristol Heart Institute, School of
Clinical Sciences, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol BS2 8HW, United Kingdom. E-mail
madeddu@yahoo.com
© 2012 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.111.089763
1774

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ecting to distinct brain stem levels, thus contributing to pain
perception and pain-induced reactions. In addition, neuropep-
tides released from peripheral terminals of sensory neurons
induce neurogenic inflammation, angiogenesis, and wound
healing.2,7Neuropeptides can also enter the systemic circu-
lation, reaching distant organs where they regulate additional
cellular responses. SP mediates its effects by preferentially
binding and activating the tachykinin receptor neurokinin 1
(NK1), whereas CGRP acts on the calcitonin receptor-like
receptor (CRLR), which is associated with and functionally
regulated by the receptor activity-modifying protein 1
(RAMP-1).8
Growing evidence indicates the implication of bone mar-
row sympathetic fibers in the regulation of PC proliferation
and mobilization.9–11However, the role of nociceptors has
not been investigated thoroughly.12A recent study showed
that induction of corneal ulcers increases peripheral blood
levels of SP, which in turn contributes to wound healing
through the mobilization of mesenchymal stem cells from the
bone marrow.2This seminal work suggests that noxious
stimuli could activate cell mobilization through local nerves
and neuropeptides from the circulation.
The present study investigates whether SP-based signaling
modulates the mobilization of proangiogenic PC, thereby
contributing to postischemic tissue healing. Our results newly
show that ischemia induces reactive cellular responses in both
animals and humans through the activation of SP release from
peripheral nociceptors and modulation of SP content in bone
marrow. This signaling mechanism is important for proper
revascularization in animal models of ischemia.
Methods
Expanded methods are provided in the online-only Data Supplement.
Animal Studies
Seven- to 8-week old male CD1 and C57BL/6 mice (both from
Harlan) and transgenic mice expressing an “enhanced” green fluo-
rescent protein cDNA under the control of a chicken ?-actin
promoter and cytomegalovirus enhancer (Jackson Laboratory) were
used. Furthermore, bone marrow reconstitution experiments were
performed with the use of NK1-knockout (NK1-KO) or wild-
type littermate mice as donors and sublethally irradiated wild-type
mice as recipients. NK1-KO mice were generated by inserting a
cassette containing an internal ribosome entry site and the LacZ
coding sequence together with a neomycin resistance gene in exon 1
of the NK1 gene.13
All experimental procedures were performed in accordance with
the Guide for the Care and Use of Laboratory Animals (Institute of
Laboratory Animal Resources, 1996) and with approval of the
British Home Office and the University of Bristol.
Myocardial Infarction Model
Myocardial infarction was induced by occlusion of the left anterior
descending coronary artery.14Twenty-four hours later, peripheral
blood and bone marrow were collected for assessment of neuropep-
tide levels, immunohistochemistry, and flow cytometry analysis of
cell antigenic profiles.
Limb Ischemia Model
Operative unilateral limb ischemia was induced as described previ-
ously.15Mice were then allocated to specific experimental protocols
(see below). Blood flow recovery was measured by laser Doppler
flowmetry (Moor Instruments, UK) immediately after induction of
ischemia and 3, 7, 14, and 21 days thereafter. Then mice were
euthanized, and the adductor muscles were collected for assessment
of capillary and arteriole density.
Effect of Morphine on SP Release and PC
Mobilization After Limb Ischemia
CD1 mice received morphine (20 mg/kg IP)1610 minutes before and
12 and 24 hours after induction of ischemia, whereas controls
received vehicle. Peripheral blood and bone marrow were collected
immediately before (time 0) and 1, 3, 12, 24, and 48 hours after
induction of ischemia (n?3 per time point) for measurement of SP
levels (EIA, Cayman Chemical) and flow cytometry assessment of
NK1-expressing PC.
Effect of Bone Marrow Reconstitution With
NK1-KO Cells on PC Mobilization and
Reparative Angiogenesis
Wild-type mice were sublethally irradiated and then randomly
assigned to receive 1?106marrow cells from NK1-KO or wild-type
mice through the tail vein (n?12 in each group). Eight weeks later,
chimerism was verified on peripheral blood cells of recipient mice by
assessing the expression of NK1 by polymerase chain reaction and
LacZ transgene by ?-galactosidase assay (Calbiochem, UK). Then
mice were submitted to limb ischemia, and perfusion recovery was
monitored until 21 days after ischemia. Peripheral blood samples
were collected from the tail vein at day 3 to assess PC mobilization.
At euthanasia, the adductor muscles were harvested after perfusion
fixation for analysis of capillaries and arterioles. Cryosections of
femurs were stained with a ?-galactosidase antibody (AbD Serotec,
UK) to confirm chimerism.
Effect of Exogenous SP on PC Mobilization
To demonstrate the direct effect of neuropeptide on mobilization,
CD1 mice were injected with SP (5 nmol/L per kilogram IV;
Bachem, UK),13followed by collection of peripheral blood at 1, 3,
12, 24, and 48 hours (n?3 per time point) for measurement of SP and
PC levels.
Transplantation of NK1-Enriched Cells in a
Mouse Limb Ischemia Model
We next investigated whether the fraction of bone marrow cells that
is functionally responsive to neuropeptides possesses proangiogenic
activity in vivo. To this aim, bone marrow cells of enhanced green
fluorescent protein–transgenic mice were submitted to a migration
assay with the use of SP or CGRP as chemoattractant. The cells
migrating to the lower chamber of the migration system (ie, SP- or
CGRP-migrated cells [SPmigand CGRPmigcells, respectively]) or
the cells migrating spontaneously in the presence of vehicle were
harvested and, within 3 hours, transplanted into 3 different sites of
the left adductor muscle of C57Bl/6 mice at the occasion of limb
ischemia induction (1?105cells per 30 ?L phosphate-buffered
saline per mouse). Control animals were injected with vehicle. Blood
flow recovery was monitored until 21 days. At euthanasia, the
adductor muscles were collected for analysis of neovascularization.
Immunostaining Procedures
Immunohistochemistry
Bones were fixed with 4% paraformaldehyde for 24 hours at 4°C and
decalcified. Sections of 15-?m thickness were mounted on poly-L-
lysine–coated slides and processed for immunostaining.17The im-
munosignal was amplified with the use of a tyramide signal
amplification kit according
instructions (PerkinElmer).
The capillary and arteriolar densities of ischemic limb muscles
were assessed with the use of isolectin B4 (Invitrogen) and ?-smooth
muscle actin (Sigma) staining as reported.15Counts from 30 micro-
scopic fields were averaged and expressed as the number of
capillaries and arterioles per square-millimeter section.
tothemanufacturer’s
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Immunocytochemistry
Bone marrow cells were depleted from lineage-positive cells, and a
cytospin from single-cell suspension was processed for immuno-
staining. Slides were mounted in Vectashield mounting medium
containing 4?,6-diamidino-2-phenylindole (DAPI) and observed with
a confocal microscope with a ?63 objective.
Cells and Cell Culture
Mouse Bone Marrow Cell Isolation
Bone marrow cells were depleted of mature hematopoietic cells by
magnetic cell sorting with the use of a lineage cell depletion kit and
a cocktail of lineage marker antibodies (MACS, Miltenyi Biotec) and
then processed for immunocytochemistry or in vitro functional
assays.18
Primary Culture of Mouse Sensory Neurons
Dorsal root ganglia (DRG) from mouse thoracic and lumbar spinal
cords were cultured for 48 hours in Dulbecco’s modified Eagle’s
medium containing 5% fetal bovine serum and 5% horse serum. 100
U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L glu-
tamine.19DRG were then cultured overnight in 0.25% fetal bovine
serum and 0.25% horse serum and next DRG and DRG conditioned
media were used in migration assays.
In Vitro Assays
Migration
Cell migration was assessed with the use of transwell cell culture
inserts with 3- to 5-?m pore size filters as described.20Briefly,
freshly isolated cells were plated in the upper compartment, and the
test compound, DRG or conditioned medium was placed in the lower
compartment. After 16 hours, cells from the upper (nonmigrated
cells) and lower compartments (migrated cells) were collected and
processed for flow cytometry analysis with the use of AccuCheck
counting beads (Invitrogen) for absolute and reproducible quantifi-
cation of cell numbers. Migration-induced enrichment of antigeni-
cally defined populations was expressed as the ratio of migrated to
nonmigrated cells, followed by normalization to control (vehicle).
This double normalization allows for direct control of changes in the
antigenic profile that may have occurred during the migration assay.
Furthermore, we know from pilot experiments that the attractant per
se does not alter the cell antigenic characteristics. In selected
experiments, cells were pretreated with CGRP and SP receptor
antagonists for 30 minutes.
In Vitro Angiogenesis Assay
Migrated and nonmigrated cell fractions were cocultured on Matrigel
with human umbilical vein endothelial cells for 16 hours at 37°C.
Network formation was quantified by counting the number of
branches per view field with the use of Image Pro-Plus software
(Media Cybernetics). Each condition was performed with 6 biolog-
ical replicates, and the assay was repeated 3 times. Counts of
migrated cells were normalized by counts of respective nonmigrated
cells.15
Flow Cytometry
Cells were stained with primary and secondary antibodies and then
analyzed with the use of a FACS Canto II equipped with FACS Diva
software (BD Biosciences).20In the text and figures, we will refer to
cells expressing different markers by stating the cluster of differen-
tiation marker (eg, CD117/c-Kit) followed by positive or negative in
superscript format (eg, c-Kit?).
Western Blot Analysis
Protein extracts and immunoblot analyses were performed as de-
scribed.21Briefly, lineage-negative cells were starved for 3 hours in
low serum medium and then treated with SP (100 nmol/L, 15
minutes in RPMI medium). Protein extracts from cell lysates were
separated by sodium dodecyl sulfate polyacrylamide gel electropho-
resis and processed for Western blotting with the use of phospho-Akt
(Ser473) and total Akt antibodies (Cell Signaling Technology) and a
horseradish peroxidase–conjugated anti-rabbit secondary antibody
(Sigma).
Reagents
SP, CGRP, and ?-CGRP8–37, a CGRP receptor antagonist, were
from Bachem. The NK1 antagonist RP67580 was from Tocris
Bioscience. LY294002, a phosphoinositide-3 kinase antagonist, was
from Calbiochem. Lineage markers and primary and secondary
antibodies used for immunostaining are reported in Table I in the
online-only Data Supplement.
Human Studies
Experiments on human samples complied with the principles stated
in the Declaration of Helsinki and were covered by institutional
ethical approval. Subjects gave written informed consent to sample
collection.
Peripheral blood was obtained for assessment of neuropeptide
receptor–expressing cells from patients with acute myocardial in-
farction and age-matched controls with similar risk factors but no
evidence of coronary artery disease who participated in an observa-
tional clinical trial on the prognostic value of circulating PC at
IRCCSMultiMedica,Milan,Italy(http://www.clinicaltrials.gov; iden-
tifier: NCT01271309) (Table II in the online-only Data Supplement).
Moreover, migration assays followed by flow cytometry character-
ization of migrated cells were performed on peripheral blood
mononuclear cells from 6 healthy subjects (average age, 30 years)
and bone marrow mononuclear cells from 4 patients participating in
the Bristol Heart Institute cell therapy trial TransACT1 (http://
www.controlled-trials.com; identifier: ISRCTN65630838/Trans-
ACT) (Table III in the online-only Data Supplement).
Finally, human heart explants were collected from patients (n?9)
who underwent cardiac transplantation 4 to 13 days after acute
myocardial infarction at the University Hospital of Udine, Udine,
Italy (Table IV in the online-only Data Supplement). Three of these
patients had received a second transplantation for an infarct of the
graft, which led in 2 cases to cardiogenic shock. Samples from
infarcted area, border, and distant myocardium were obtained for
immunohistochemistry of SP, NK1, CD34, and CD45 on 5-?m-thick
paraffin-embedded sections. Control specimens from comparable
areas were sampled from explanted hearts that were judged not
suitable for cardiac transplantation (n?5).
Statistical Analysis
Results are presented as mean?SEM. If data failed to pass normality
and equal variance tests, a nonparametric analysis was applied, and
results are expressed as median with 5 to 95 percentile distribution.
Multiple groups were compared by parametric ANOVA, followed by
Bonferroni t test, or nonparametric ANOVA on ranks, followed by
Tukey pairwise comparison or Dunnett test for multiple comparisons
against a single control group. Analysis of the effect of bone marrow
transplantation on postischemic blood flow recovery was performed
with repeated-measures ANOVA followed by Bonferroni multiple
comparison. Comparison of 2 groups was performed by paired or
unpaired Student t test or Mann-Whitney rank sum test. P?0.05
was considered significant. Stated n values represent biological
replicates.
Results
Sensory Neuropeptidergic Neurons Are Present in
Mouse Bone Marrow
Using the panneuronal marker Protein Gene Product 9.5
(PGP 9.5), we showed the presence of nerve fibers in the
periosteum and endosteum and also in marrow perivascular
areas of femur epiphysis (Figure I in the online-only Data
Supplement). We also found that a subset of these fibers in
trabecular bone and marrow is positive for nociceptor mark-
ers, such as SP (Figure 1), CGRP, and transient receptor
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potential cation channel subfamily V member 1 (TRPV1)
(Figure I in the online-only Data Supplement).
We then analyzed the expression of SP and CGRP recep-
tors on cytospin preparations of mouse bone marrow cells.
We found that lineage-negative (Lin?) cells express the SP
receptor NK1 at the plasma membrane and also within
cytoplasmic vesicles (Figure 2A). Similarly, we found that
bone marrow cells express the CGRP receptors RAMP-1 and
CRLR (Figure 2A through 2C). The immunoreactive signal
was not detected when primary antibodies were omitted
(Figure 2A and 2D). We further confirmed and quantified the
percentage of cells expressing SP and CGRP receptors by
flow cytometry. We found that freshly isolated bone marrow
cells abundantly express NK1 (86?1%), RAMP-1 (50?2%),
and CRLR (77?2%) (Figure 2B through 2D). Moreover,
neuropeptide receptor–positive cells coexpress markers for
hematopoietic PC. Of the NK1?cells (Figure 2B), 40?2%
were Lin?, 28?2% expressed c-Kit (the receptor for stem
cell factor), and 8?1% expressed Sca-1 (typical markers for
hematopoietic PC). Of the RAMP-1?(Figure 2C) and
CRLR?cells (Figure 2D), 45?2% and 68?2% were Lin?,
22?1% and 29?1% expressed c-Kit, and 6?1% and 5?1%
expressed Sca-1, respectively. Moreover, 15?3% of the
NK1?, 15?2% of the RAMP-1?, and 52?9% of CRLR?
cells also expressed CXCR4 (data not shown). Within the
Lin?Sca-1?c-Kit?PC population, 98?1% expressed NK1,
89?1% RAMP-1, and 91?3% CRLR. Nonhematopoietic
cells identified as c-Kit?CD45?cells also expressed NK1
(62?4%), RAMP-1 (65?7%), and CRLR (73?7%) (data not
shown). Thus, neuropeptides and their receptors are abun-
dantly expressed in mouse bone marrow cells.
SP and CGRP Exert Chemoattractant Activity on
Bone Marrow PC
We next assessed whether neuropeptides regulate bone mar-
row cells motility. Using the transwell migration assay, we
found that SP and CGRP (100–1000 nmol/L) exert a che-
moattractant action on Lin?cells (Figure 3A). These effects
were reduced in cells treated 30 minutes in advance with the
NK1 antagonist RP675880 (75% reduction of SP response;
n?3; P?0.005) or the CGRP antagonist CGRP8–37(76%
reduction of CGRP 100 nmol/L response; n?4; P?0.001).
We also found that stimulation of Lin?cells with SP for 15
minutes increases the phosphorylation of Akt, and this is
prevented by the PI3K antagonist LY294002 (Figure 3B, top
and left). Moreover, PI3K inhibition prevented PC migration
induced by SP (Figure 3B, right). SP and CGRP (both at 100
nmol/L) increased cAMP production in Lin?PC (SP,
1.5?0.3-fold; CGRP, 1.8?0.5-fold; n?3).
We next investigated whether neuropeptides released by
nerve terminals exert a retaining action on bone marrow cells.
To this aim, we used primary cultures of sensory neurons
isolated from mouse DRG or conditioned medium in a
migration assay on bone marrow cells. DRG and DRG
conditioned medium induced the migration of bone marrow
cells and the enrichment of c-Kit?Sca-1?PC within the
migrated fraction (Figure 3C and 3D). The chemoattractant
effect induced by DRG was reduced by NK1 and CGRP
antagonists (Figure 3E).
Ischemia Increases Peripheral Blood Levels
of SP and Induces the Mobilization of
NK1-Expressing Cells
Next we evaluated the mobilization of NK1- and CGRP-
expressing cells in relation to changes of SP and CGRP levels
in mouse models of acute myocardial infarction and limb
ischemia. Circulating levels of SP were increased by 5-fold
24 hours after myocardial infarction compared with controls
(236?80 versus 48?19 pg/mL, respectively; n?6 per group;
P?0.05), whereas no difference between groups was detected
in bone marrow levels of SP (P?0.35).
Analysis of distinct cell populations showed that myocar-
dial infarction increases the abundance of granulocytes in
Figure 1. Trabecular bone and bone
marrow contain nerve fibers that express
SP. A through D, Representative fluores-
cence images of SP-positive nerve fibers
(green fluorescence) traveling within both
the bone spicules (A through D; arrow-
heads) and the marrow parenchyma (C
and D; arrows). Bone edges are indi-
cated by dashed lines, and hematopoi-
etic cells are decorated by anti-CD45
antibody (red fluorescence). Nuclei are
shown by the blue fluorescence of 4?,
6-diamidino-2-phenylindole.
Bars?20 ?m.
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peripheral blood while reducing them in bone marrow (Figure
II in the online-only Data Supplement). Similarly, myocardial
infarction induced a 1.8-fold increase in CD45?c-Kit?NK1?
granulocytes in peripheral blood (12 657?1532 versus
6819?827 cells per 100 ?L in controls; n?6 per group;
P?0.01) and a reduction of their relative abundance in bone
marrow (41.0?0.6% versus 52.0?1.0% in controls; P?0.01).
The abundance of total and CD45?c-Kit?NK1?lymphocytes/
monocytes was not altered in both peripheral blood and bone
marrow (data not shown). Moreover, myocardial infarction did
not induce any change in circulating CGRP and in the abun-
dance of cell populations expressing CGRP receptors (data not
shown).
Limb ischemia caused opposite changes in the levels of SP
in bone marrow (Figure 4A) and peripheral blood (Figure
4B), resulting in the modification of SP gradient between the
2 compartments. This was associated with an increased
abundance of Sca-1?NK1?(Figure 4F) and Sca-1?c-
Kit?NK1?granulocytes (3-fold; data not shown) in periph-
eral blood from 24 hours after ischemia, whereas the change
in c-Kit?NK1?granulocytes was not significant (Figure 4E).
Nociceptive signaling is modulated by opioid receptors at
the level of the central nervous system and primary afferent
neurons. We next investigated whether opioid-induced anal-
gesia interferes with the release of SP and the mobilization of
nociceptor-expressing cells in mice with limb ischemia.
Morphine inhibited the increase of SP in peripheral blood
(Figure 4D) and blunted the decrease of SP in bone marrow
(Figure 4C), thus nullifying the SP gradient between the 2
compartments. Moreover, we found that morphine remark-
ably attenuated the mobilization of NK1-expressing PC after
ischemia (Figure 4G and 4H).
Figure 2. Bone marrow progenitor cells express substance P and calcitonin gene-related peptide receptors. A, Immunostaining of
lineage-negative (Lin?) cells (selected with the use of magnetic beads and a cocktail of antibodies against committed hematopoietic
cells) expressing neurokinin 1 (NK1) (a), receptor activity-modifying protein 1 (RAMP-1) (b), and calcitonin receptor-like receptor (CRLR)
(c) (green). Nuclei are stained with 4?, 6-diamidino-2-phenylindole (blue). Immunoreactivity was not detected when primary antibodies
were omitted (negative control, d). B through D, Flow cytometry confirms the expression of neuropeptide receptors in progenitor cells.
Typical scatterplots and bar graphs show the analyzed data. A substantial fraction of NK1?, RAMP-1?, and CRLR?cells are Lin?and
express the progenitor cell markers c-Kit (c-Kit?) and Sca-1 (Sca-1?).
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In addition, we showed that systemic injection of SP per se,
in the absence of ischemia, temporarily increases SP in
peripheral blood and concomitantly induces the mobilization
of Sca-1?c-Kit?NK1?cells (Figure III in the online-only Data
Supplement).
Role of NK1-Expressing Bone Marrow Cells in
Postischemic Healing
To investigate the relevance of NK1-expressing cells in the
reparative process after ischemia, sublethally irradiated
mice were randomly assigned to bone marrow replacement
with cells from NK1-KO or wild-type mice (n?12 in each
group). Eight weeks later, mice were subjected to unilat-
eral limb ischemia (Figure 5A). Mice transplanted with
NK1-KO cells showed reduced peripheral blood levels of
c-Kit?granulocytes compared with controls transplanted
with wild-type cells (Figure 5B). Moreover, repeated-
measures ANOVA showed that blood flow recovery is
delayed in the former group (Figure 5C). In agreement
with this finding, NK1-KO cell recipients showed reduced
reparative neovascularization at the capillary and arteriolar
levels compared with mice replaced with wild-type bone
marrow cells (Figure 5D).
Neuropeptide-Induced Migration Enriches PC
Able to Promote Reperfusion of Ischemic Limbs
To determine whether cells responsive to neuropeptide-
induced chemoattraction are endowed with reparative activ-
ity, we transplanted mouse bone marrow cells that migrate
toward SP (SPmigcells) or CGRP (CGRPmigcells) into the
adductor muscle of mice with unilateral limb ischemia. Cells
migrating toward vehicle (Vehmigcells) were used as con-
trols. Flow cytometry analysis of cells, before transplantation,
indicated that migration itself provides enrichment for c-Kit?
PC, and this effect is enhanced when migration is stimulated
by SP and CGRP (Figure 6A). Using laser Doppler flowmet-
ry, we found that mice transplanted with SPmigand CGRPmig
cells have improved blood flow recovery at 3 weeks after
ischemia compared with mice injected with vehicle (no cells)
or Vehmigcells (Figure 6B and 6C). Analysis of ischemic
adductor vascularization showed an effect of cell transplan-
tation on arteriolar density in SPmigand Vehmigcell groups
compared with vehicle (Figure 6D).
Figure 3. Substance P (SP) and calcitonin gene-related peptide (CGRP) exert chemoattractant effects on mouse bone marrow cells. A,
SP and CGRP induce migration of lineage-negative (Lin?) bone marrow (BM) cells. Data are expressed as fold increase of vehicle (veh).
B, SP (100 nmol/L for 15 minutes) induces phosphorylation/activation of Akt in Lin?cells, which is prevented by the phosphoinositide-3
kinase antagonist LY 294002 (LY) (15 ?mol/L). *P?0.05 vs control; n?6 to 9. LY 294002 pretreatment also prevents SP-induced pro-
genitor cell migration. *P?0.05 vs control; #P?0.05 vs SP only; n?3. C and D, Primary culture of mouse dorsal root ganglia (DRG) and
respective conditioned medium (CM) induces cell migration (C) leading to an enrichment of Lin?c-Kit?Sca-1?PC in the migrated frac-
tion (D). E, The migratory effect induced by DRG is reduced by antagonists for CGRP (CGRP8–37, 1000 nmol/L) and NK1 (RP67580,
100 nmol/L).*P?0.05 vs vehicle; n?6; #P?0.05 vs DRG alone; n?5.
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Human PC Express Neuropeptide Receptors
Circulating CD34?PC from healthy subjects express neuro-
peptide receptors (Figure 7A) (gating strategies reported in
Figure IV in the online-only Data Supplement) and migrate
toward SP and CGRP (?1.6- and 1.2-fold increase compared
with vehicle), and this migratory activity is inhibited by NK1
and CGRP antagonism (Figure 7B). Moreover, neuropeptide-
induced migration provides enrichment for CD34?CXCR4?
(Figure 7C) and CD34?KDR?PC (Figure 7D) coexpressing
NK1 and RAMP-1. In particular, NK1 expression is in-
creased in the CD34?CXCR4?and CD34?KDR?cell sub-
fractions migrating toward SP. Likewise, CGRP induces a
distinct enrichment of RAMP-1?cells but does not enrich
cells expressing CRLR. Interestingly, CD34?KDR?NK1?
and CD34?KDR?RAMP-1?PC are attracted by both ago-
nists. Furthermore, PC selected by SP- and CGRP-induced
migration are able to enhance human umbilical vein endothe-
lial cell branch formation on Matrigel compared with nonmi-
grated cells, thus indicating the in vitro proangiogenic activity
of cells that are functionally responsive to neuropeptides
(Figure 7E).
We next asked whether human bone marrow cells also
express neuropeptide receptors. Flow cytometry analysis
of freshly collected bone marrow from patients with
chronic myocardial ischemia showed the abundance of
neuropeptide receptors on CD34?cells, which are
75?15% NK1?, 70?20% RAMP-1?, and 75?20%
CRLR?. Moreover, of CXCR4?cells, 73?6% are NK1?,
55?15% RAMP-1?, and 56?22% CRLR?(n?4). We
also found that SP and CGRP induce a migratory effect on
human bone marrow mononuclear cells (1.3- and 1.2-fold
increase, respectively, versus vehicle) (Figure V in the
online-only Data Supplement).
Figure 4. Limb ischemia induces pro-
genitor cell mobilization, which is inhib-
ited by morphine. A and B, Bar graphs
show time course of changes in sub-
stance P (SP) levels in bone marrow
(BM) (A) and peripheral blood (PB) (B)
after induction of unilateral limb ische-
mia. C and D, Morphine blunts the SP
decrease in bone marrow (C) and inhib-
its the SP increase in peripheral blood
(D). E through H, Moreover, limb ische-
mia induces the release of PC express-
ing the neurokinin 1 (NK1) receptor (E
and F), and this effect is inhibited by
morphine (G and H). ANOVA followed by
Dunnett multiple comparison test;
*P?0.05, **P?0.01 vs time 0; #P?0.05,
##P?0.01 vs corresponding time point in
mice without morphine; n?6 different
mice per time point.
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SP Modulates PC Mobilization and Homing in
Patients With Acute Myocardial Infarction
Finally, we investigated the implication of the SP signaling
pathway in PC mobilization and homing in patients with
myocardial infarction. We found higher circulating SP levels
in myocardial infarction patients (142?32 pg/mL; n?23)
compared with healthy controls (46?6 pg/mL; n?19;
P?0.001) (Figure 8A), whereas CGRP did not differ between
the 2 groups (30.5?10.9 versus 14.7?2.0 pg/mL; P?0.11).
This was associated with a remarkable increase in the relative
abundance of cells expressing NK1 and RAMP-1 within the
CD34?CXCR4?and CD34?KDR?PC fractions (Figure 8B
and 8C) as well as in the proportion of NK1?CD34?
CXCR4?and NK1?CD34?CXCR4?cells in total mononu-
clear cells (0.010?0.003% versus 0.003?0.001% in controls;
P?0.03; n?10 in both groups; and 0.006?0.001% versus
0.004?0.001% in controls; P?0.05; n?10 in both groups,
respectively). The expression of CRLR was unchanged after
acute myocardial infarction.
We next analyzed hearts of patients that were transplanted
after acute myocardial infarction (n?6). Ventricular frag-
ments obtained from 5 explanted normal hearts that were
judged not to be suitable for cardiac transplantation were
employed as controls. SP could be identified by immunohis-
tochemistry particularly in the perivascular interstitium of the
border zone (Figure 8D) and remote zone of infarcted hearts
(Figure 8E) compared with controls (Figure 8F). Quantita-
tively, a significantly larger volume fraction of the region
bordering the infarct was immunoreactive for this neurotrans-
mitter (Figure 8G). We next investigated, on the same
samples, the presence of NK1?cells expressing the endothe-
lial and hematopoietic stem cell marker CD34. Moreover,
CD45 was employed to label cells of clear hematopoietic
origin. Both CD45?(Figure 8H) and CD45?cells (Figure 8I)
coexpressing NK1 and CD34 were identified. Quantitatively,
although CD34?NK1?CD45?cell density was significantly
higher in myocardium distant from the myocardial injury,
CD34?NK1R?CD45?cells were significantly more frequent
in the border zone compared with controls.
Finally, to verify whether denervated hearts had an impair-
ment in the recruitment of CD34?NK1R?cells in response to
injury, we compared hearts of patients undergoing cardiac
transplantation after an acute infarct with hearts of patients
retransplanted after an infarct of the graft (n?3). In agree-
ment with our hypothesis, both CD34?NK1R?CD45?and
CD34?NK1R?CD45?cells were less abundant in this latter
class of patients (Figure 8J).
Figure 5. Bone marrow (BM) reconstitution with neurokinin 1 knockout (NK1-KO) cells inhibits reparative angiogenesis and perfusion
recovery after limb ischemia. A, Schematic representation of the study protocol, consisting of BM reconstitution followed by induction
of limb ischemia (LI). IHC indicates immunohistochemistry. B, Bar graph showing the levels of peripheral blood (PB) PC at 3 days after
ischemia in mice that had their marrow reconstituted with wild-type (WT) or NK1-KO cells. C, Representative images of laser Doppler
flowmetry captured at 3 weeks after induction of ischemia and line graph showing the time course of blood flow recovery in the 2
groups. D, Capillary and arteriolar density in ischemic adductor muscles; endothelial cells are stained with lectin (green) and smooth
muscle cells with ?-smooth muscle actin (?-SMA) (red). Groups were compared by unpaired Student t test or Mann-Whitney rank sum
test as appropriate. Analysis of blood flow recovery was performed by repeated-measures ANOVA followed by Bonferroni comparison;
*P?0.05, ***P?0.001; n?9 per group.
Amadesi et alNociception Triggers Stem Cell Mobilization
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Discussion
In this study, we identified the presence of primary nocicep-
tive sensory fibers in bone marrow. We also found that bone
marrow PC express NK1, the preferential receptor of SP, and
migrate in response to SP stimulation. Ischemic injury re-
markably increases SP levels in peripheral blood and induces
the mobilization of proangiogenic PC, with these responses
being abrogated by the opioid agonist morphine. Moreover,
genetic disruption of the NK1 receptor in bone marrow cells
results in defective PC mobilization, reduced reparative
angiogenesis, and delayed postischemic recovery. NK1 re-
ceptor–expressing cells are abundant in human infarcted
hearts but not in denervated hearts that suffered an infarct
after transplantation. Hence, we conclude that the SP/NK1
duo is implicated in postischemic reparative response.
Primary Sensory Neurons Innervate Mouse Bone
Marrow, and Bone Marrow PC Express SP and
CGRP Receptors
The presence of sympathetic and sensory nerve fibers in
bones and marrow of rodents has been reported previ-
ously.22–24This study further identifies the intramedullary
distribution of peptidergic primary sensory fibers, which were
recognized by costaining for SP, CGRP, and TRPV1, a
marker for capsaicin-sensitive fibers implicated in neurogenic
inflammation and pain25(Figure I in the online-only Data
Supplement). Multicolor fluorescence microscopy confirmed
the distribution of SP-positive fibers in bone marrow paren-
chyma, thus providing an anatomic basis for the existence of
neurogeniccontrolofPChomeostasis(Figure1).Thisconceptis
strengthened by the other finding that bone marrow cells express
neuropeptide receptors.26–29Using flow cytometry, we newly
document that NK1, CRLR, and RAMP-1 receptors are partic-
ularly abundant in mouse c-Kit?PC (Figure 2) and human
CD34?PC(FigureVintheonline-onlyDataSupplement).Both
populationshavebeenreportedtoparticipateinthepostischemic
regenerative process.4,30Moreover, we report for the first time
the ability of bone marrow cells to respond to neuropeptide
stimulation in migration assays (Figures 3 and 7) as well as in
vivo after intravenous administration of SP (Figure III in the
online-only Data Supplement) or change in the SP gradient
between bone marrow and peripheral blood after ischemia
(Figures 4 and 8). Interestingly, PC expressing distinct neuro-
peptide receptors show competence to respond to both SP and
CGRP,suggestingcomplementary/synergisticattractionbythe2
agonists. Moreover, because neuropeptide receptor–expressing
cells coexpress chemokine receptors, such as CXCR4, neuroen-
docrine mechanisms may integrate the chemokine-mediated
mechanism of PC mobilization and recruitment.31The 2 mech-
anisms share common postreceptor signaling pathways. In fact,
similartoSDF-1,whichistheligandofCXCR4,SPinducesAkt
phosphorylation in bone marrow PC. Inhibition of phosphoino-
sitide-3 kinase contrasts the stimulatory effects of SP on Akt
phosphorylation and PC migration.
Opioid Analgesia Abrogates the Mobilization of
NK1-Expressing Cells After Ischemia
Pain is a typical symptom of acute ischemia and an essential
component of the alert response to injury. Moreover, it is well
known that opioid receptors and the SP receptor NK1 coexist
Figure 6. Substance P (SP)– and calci-
tonin gene-related peptide (CGRP)–en-
riched bone marrow progenitor cells
stimulate the recovery of ischemic limbs.
A, SP (SPmigcells) and CGRP (CGRPmig
cells) induce migration and enrichment
of progenitor cells that express the stem
cell factor receptor c-Kit. Enrichment is
expressed as ratio between c-Kit?
migrated and nonmigrated cells. B and
C, SP and CGRP migrated cells improve
the limb blood flow recovery at 3 weeks
from ischemia induction compared with
vehicle (veh). Original representative
images (B) and average values (C) are
shown. Isch indicates ischemia; contr,
control. D, Histological analysis of angio-
genesis with the use of immunostaining
to visualize capillaries (isolectin B4;
green) and arterioles (?-smooth muscle
actin; red). Nuclei are stained with 4?,6-
diamidino-2-phenylindole (blue). Bar
graph summarizes arteriolar density
data. ANOVA followed by Bonferroni
comparison test; *P?0.05, **P?0.01;
n?8 to 10.
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April 10, 2012

Page 10

and functionally interact in somatic and visceral sensory
neurons, spinal cord projection and interneurons, midbrain,
and cortex. Opioid receptors and neuropeptides like SP are
synthesized in the DRG and transported along intra-axonal
microtubules into central and peripheral processes of the
primary afferent neuron. At the terminals, opioid receptors
are incorporated into the neuronal membrane and become
functional receptors. Activation of mature opioid receptors by
endogenous ligands or systemically administered agonists
potently inhibits SP release induced by peripheral noxious
stimuli through coupling to G proteins that suppress cAMP-
dependent Ca2?or Na?currents.32
After acute injury, nociceptors and opioid receptors may
also cooperate in the fine tuning of reparative responses. It was
reported previously that morphine delays wound closure, re-
duces the number of circulating endothelial PC, and impairs
reparative angiogenesis in mice.16However, whether ischemic
pain participates in mobilization of PC through the SP/NK1 duo
has not been considered previously. We here report that acute
myocardial infarction and limb ischemia increase the levels of
circulating SP in mice and human patients, and this effect is
associated with an augmented abundance of circulating NK1-
expressing PC. Importantly, morphine abrogated the SP gradient
between bone marrow and peripheral blood and the mobilization
of NK1?PC (Figure 4), thus confirming the intertwined link
between ischemic injury, SP signaling, and PC egress. However,
further studies are warranted to address the role of pain afferents
or efferents in this process.
The NK1 Receptor Is Fundamental for PC
Mobilization and Reparative Response
After Ischemia
Previous studies from Mishima et al33have shown that limb
ischemia increases the expression of pro-CGRP mRNA and
of CGRP protein in the lumbar DRG. In CGRP knockout
mice, they observed impaired blood flow recovery from
ischemia and decreased capillary density. Likewise, SP has
been implicated in reparative angiogenesis, regulation of
hematopoiesis, and recruitment of mesenchymal stem
cells.2,7,34To dissect reparative actions that could be ascribed
to either local stimulation of angiogenesis or recruitment of
proangiogenic cells by SP, we studied the postischemic
recovery of mice whose bone marrow had been reconstituted
with NK1-KO cells. Results indicate that the lack of NK1
receptor results in impaired PC mobilization, defective an-
giogenesis, and delayed perfusion recovery (Figure 5). There-
Figure 7. Neuropeptide receptors are expressed in circulating human progenitor cells. A, CD34?progenitor cells from human peripheral
blood (hPB) express neuropeptide receptors. NK1 indicates neurokinin 1; CRLR, calcitonin receptor-like receptor; and RAMP-1, receptor
activity-modifying protein 1. B, Substance P (SP) and calcitonin gene-related peptide (CGRP) induce migration of hPB mononuclear cells
(MNC), with this effect inhibited by antagonists for NK1 (RP67580, RP, 100 nmol/L) and CGRP (CGRP8–37, 1000 nmol/L). C and D, Neuro-
peptides induce enrichment of hPB MNC expressing CD34/CXCR4 (C) and CD34/KDR (D) and coexpressing SP (NK1) and CGRP (RAMP-1)
receptors. E, Circulating progenitor cells migrating toward SP and CGRP induce branch formation by human umbilical vein endothelial cells
in a Matrigel angiogenesis assay. *P?0.05 vs no treatment or vehicle; n?6. #P?0.05 vs SP or CGRP treatment plus vehicle; n?6.
Amadesi et al Nociception Triggers Stem Cell Mobilization
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fore, an operative NK1 receptor is required for bone marrow
cells to elicit reparative responses.
The spectrum of cells released after an ischemic event is
heterogeneous and comprises inflammatory and regenerative
subpopulations. We used a functional enrichment method
based on in vitro migration to verify whether neuropeptide-
responsive cells belong to the class of regenerative cells.20
We found that human SPmigand CGRPmigcells enhance the
formation of new branches of cocultured human umbilical vein
endothelial cells (Figure 7). Moreover, mice transplanted with
SP-responsive PC show increased arteriogenesis and improved
blood flow recovery of the ischemic limb compared with
vehicle-injected mice receiving injection of vehicle (Figure 6).
Homing of NK1-Expressing Cells in Infarcted
Human Hearts
We report the presence of SP and NK1?CD34?CD45?PC
in the remote zone of infarcted human hearts (Figure 8).
These cells are remarkably less abundant in posttransplant
infarcted hearts, suggesting that denervation may have
hampered PC recruitment. These data need to be confirmed
in a larger series of transplanted hearts and in models of
cardiac denervation. Moreover, we observed that NK1?
CD34?CD45?cells are more abundant in the border of
infarcted human myocardium and mainly localized in
vascular-like structures. It is not clear whether NK1?
CD34?CD45?cells are resident vascular cells or derive
Figure 8. Activation of substance P (SP)–based nociceptive signaling after acute myocardial infarction (aMI) is associated with mobili-
zation and homing of neurokinin 1 (NK1)–expressing progenitor cells. A, aMI increases peripheral blood (PB) levels of SP; *P?0.05, aMI
vs controls (n?23 and 19, respectively). B and C, aMI increases PB levels of PC positive for CD34/CXCR4 (B) and CD34/KDR (C) that
coexpress NK1 and receptor activity-modifying protein 1 (RAMP-1) receptors. *P?0.05, **P?0.01, ***P?0.001 vs controls; n?10 per
group. CRLR indicates calcitonin receptor-like receptor. D through F, Representative microscopy images of SP staining in human myo-
cardium at the level of infarct border zone (D) and remote zone (E). Normal myocardium shows low levels of SP expression (F). G, Bar
graph showing the expression of SP in hearts of aMI patients (n?6) and healthy controls (n?5). IHC indicates immunohistochemistry. H
and I, Abundance of NK1-expressing cells coexpressing CD34 and CD45 (H) or CD34 only (I). Bar graphs show the abundance of
NK1?CD34?CD45?cells (H) and NK1?CD34?CD45?cells (I) in hearts of aMI patients (n?6) and healthy controls (n?5). *P?0.05 vs
healthy, #P?0.05 vs border zone. J, Levels of SP and abundance of NK1?CD34?CD45?cells and NK1?CD34?CD45?cells in hearts
of transplanted (n?6) and retransplanted aMI patients (n?3). *P?0.05 vs transplanted.
1784 Circulation
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Page 12

from nonhematopoietic PC, which incorporate in peri-
infarct neovascularization.
Clinical Impact
The present study identifies a novel regulatory mechanism
triggered by ischemic injury and involving the release of SP
from peripheral tissues into the circulation. This neural reflex
also results in reduction of SP levels in bone marrow, at least
after limb ischemia. The creation of a SP gradient between
the 2 compartments facilitates the egress of NK1-expressing
cells from bone marrow into the circulation, which is abro-
gated by opioid receptor stimulation. Moreover, disruption of
NK1 on bone marrow cells jeopardizes reparative responses
after ischemia.
These new findings may have important clinical implica-
tions. Dysfunction of the neurogenic mechanism triggered by
ischemic injury may contribute to the impairment of postis-
chemic repair during aging or degenerative diseases (eg,
diabetes mellitus).35–37Furthermore, pharmacological control
of pain could be detrimental. In 1928, Sir James MacKenzie
suggested treating cardiac patients with bed rest, morphine,
and chloroform until unconsciousness ensued.38Eighty-three
years later, the American College of Cardiology/American
Heart Association guidelines continue to recommend intra-
venous morphine as a class IC indication for patients with
suspected acute coronary syndromes whose pain is not
relieved after nitroglycerin or whose symptoms recur.39
However, results from the CRUSADE Quality Improvement
Initiative showed that morphine is associated with higher
mortality in patients with acute coronary syndrome after risk
and treatment adjustment.40Whether this detrimental effect is
attributable to suppression of nociceptor-mediated PC release
remains unknown.
In conclusion, our study opens the path to further mecha-
nistic investigation of the role of nociceptive signaling in the
regulation of PC mobilization from the perspective of finding
new therapeutic targets compatible with pain relief and
cardiovascular repair.
Acknowledgments
The authors wish to acknowledge the assistance of Dr Andrew
Herman and the University of Bristol Faculty of the Medical and
Veterinary Sciences Flow Cytometry Facility.
Sources of Funding
This study was supported by a grant of the EU-FP7 “Resolve” and by
British Heart Foundation grants for “Bone marrow dysfunction alters
vascular homeostasis in diabetes.” Costanza Emanueli is BHF senior
basic research fellow and Carlotta Reni is supported by a Medical
Research Council PhD studentship.
Disclosures
None.
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CLINICAL PERSPECTIVE
Pain and inflammation are generally thought of as medical problems. Treatment of these defense responses is routine in
patients with myocardial and peripheral ischemia. However, blocking a defense can be harmful. It has been shown that
taking nonsteroidal anti-inflammatory drugs can increase a person’s risk of having a heart attack or stroke. Furthermore,
morphine has been associated with higher mortality in patients with acute coronary syndrome. The present study provides
novel insight into the role of the pain mediator substance P in vascular regeneration by bone marrow–derived stem cells.
After ischemic injury, substance P is released from central terminals projecting to distinct brain stem centers, thus
contributing to pain perception and pain-induced reactions, as well as from sensory fibers innervating the myocardium,
leading to local neurogenic inflammation. In the present study, we show that substance P also contributes to mobilize stem
cells from the bone marrow and to recruit them to the infarcted heart. Bone marrow cells attracted by substance P are able
to promote neovascularization, thereby accelerating the healing of ischemic tissues. Conversely, genetic abrogation of
substance P signaling or pharmacological inhibition of substance P release by morphine results in attenuation of both stem
cell mobilization and reparative vascularization in models of ischemia. These new findings may have important clinical
implications for tailoring new regenerative treatments based on stem cell recruitment by pain mediators. Nonetheless, the
nociceptive signaling is also used in other biological contexts in which pain is not operant. Therefore, additional work is
warranted to refine new therapeutic strategies compatible with pain relief and cardiovascular repair.
1786Circulation
April 10, 2012