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Sympathetic Nervous System Modulation of Inflammation
and Remodeling in the Hypertensive Heart
Scott P. Levick, David B. Murray, Joseph S. Janicki, Gregory L. Brower
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
Abstract—Chronic activation of the sympathetic nervous system is a key component of cardiac hypertrophy and fibrosis.
However, previous studies have provided evidence that also implicate inflammatory cells, including mast cells (MCs),
in the development of cardiac fibrosis. The current study investigated the potential interaction of cardiac MCs with the
sympathetic nervous system. Eight-week– old male spontaneously hypertensive rats were sympathectomized to establish
the effect of the sympathetic nervous system on cardiac MC density, myocardial remodeling, and cytokine production
in the hypertensive heart. Age-matched Wistar Kyoto rats served as controls. Cardiac fibrosis and hypertension were
significantly attenuated and left ventricular mass normalized, whereas cardiac MC density was markedly increased in
sympathectomized spontaneously hypertensive rats. Sympathectomy normalized myocardial levels of interferon-␥,
interleukin 6, and interleukin 10, but had no effect on interleukin 4. The effects of norepinephrine and substance P on
isolated cardiac MC activation were investigated as potential mechanisms of interaction between the two. Only
substance P elicited MC degranulation. Substance P was also shown to induce the production of angiotensin II by a
mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes, and macrophages. These results
demonstrate the ability of neuropeptides to regulate inflammatory cell function, providing a potential mechanism by
which the sympathetic nervous system and afferent nerves may interact with inflammatory cells in the hypertensive heart. (Hypertension. 2010;55:270-276.)
Key Words: sympathectomy 䡲 fibrosis 䡲 mast cell 䡲 cytokines 䡲 inflammation
䡲 substance P 䡲 sympathetic nervous system
U
pregulation of the sympathetic nervous system (SNS) is
involved in numerous cardiovascular disease processes
and is responsible for alterations in normal myocardial
structure and function, including the induction of arrhythmias, alterations in contractile properties, and cardiac remodeling.1 In an experimental setting, there is evidence that the
SNS mediates hypertension-induced cardiac fibrosis and
hypertrophy through ␣- and ␤-adrenergic receptors, respectively.2 However, concomitant with elevated SNS activity,
induction of an inflammatory infiltrate into the heart also
occurs, which appears to mediate the development of
hypertension-induced cardiac fibrosis but not hypertrophy.
This inflammatory component includes mast cells (MC),3– 6
macrophages,7,8 and possibly T cells.9,10 MCs maintain a
close anatomic association with nerves,11,12 with sympathetic
neurons able to alter rat basophilic leukemia (RBL-2H3) MC
membrane resistance.13 In addition, other inflammatory cells
express adrenergic receptors, with modulation of cytokine
production being either enhanced or repressed by stimulation
of these receptors.14 Alternatively, we have shown that
cardiac MCs can also be activated by neuropeptides, such as
substance P,15 which is found mainly in afferent sensory
nerves. Given the prominent involvement of both the SNS
and inflammation in myocardial remodeling associated with
hypertension, we sought to determine whether SNS-mediated
regulation of inflammation is a mechanism involved in
myocardial remodeling secondary to hypertension and
whether afferent nerves also have the potential to regulate
inflammation in the hypertensive heart.
Methods
All of the in vivo experiments were performed using adult male
spontaneously hypertensive rats (SHRs; n⫽18) and Wistar Kyoto
rats (WKYs; n⫽13) housed under standard environmental conditions
and maintained on commercial rat chow and tap water ad libitum.
SHRs were chosen for this study because they have been documented to have elevated SNS activity.16 All of the studies conformed
to the principles of the National Institutes of Health Guide for the
Care and Use of Laboratory Animals. In addition, the protocol was
approved by our institution’s animal care and use committee. All of
the rats were anesthetized by IP injection of sodium pentobarbital
(50 mg/kg).
Experimental Protocol and
Sympathectomy Surgery
Rats were divided into 4 groups: (1) untreated WKYs; (2) sympathectomized WKYs (WKY⫹Symp); (3) untreated SHRs; and (4)
Received September 3, 2009; first decision September 27, 2009; revision accepted December 3, 2009.
From Cell Biology and Anatomy (S.P.L., J.S.J., G.L.B.), School of Medicine, University of South Carolina, Columbia, S.C.; School of Pharmacy
(D.B.M.), University of Mississippi, Oxford, Miss.
Correspondence to Joseph S. Janicki, Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC 29208. E-mail
[email protected]
© 2010 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.109.142042
270
Levick et al
Table.
Sympathetic Control of Inflammation in the Heart
271
Biometric Data
Group
BW, g
LV, mg
LV/BW, mg/g BW
Lung Weights, mg
SBP, mm Hg
DBP, mm Hg
MAP, mm Hg
WKY
328⫾28
786.8⫾54.0
2.24⫾0.11
1476⫾217
116⫾19
86⫾20
100⫾20
SHR
345⫾11
929.5⫾81.1*
2.70⫾0.24*
1445⫾122
229⫾26*
167⫾13*
193⫾17*
WKY⫹Symp
332⫾14
755.8⫾45.3
2.28⫾0.06
1303⫾179
123⫾20
98⫾17
109⫾19
SHR⫹Symp
421⫾18*†
715.8⫾26.9†
1.70⫾0.04*†
1511⫾154
147⫾16*†
116⫾11*†
130⫾13*†
All of the values are mean⫾SD. BW indicates body weight; LV, left ventricle; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean
arterial pressure.
*P⬍0.05 vs WKY.
†P⬍0.05 vs SHR.
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sympathectomized SHRs (SHR⫹Symp). Data reported herein for
untreated WKYs and SHRs have been published previously.6 Bilateral sympathectomy surgery was performed at 8 weeks of age, and
all of the rats were euthanized at 20 weeks of age. This time point
was chosen because it represents a steady state of hypertension, as
well as established left ventricular (LV) hypertrophy and fibrosis in
the SHR. The sympathectomy surgery was conducted by the attending veterinarian at Charles River Laboratories using the following
procedure. A ventral midline incision was made in the neck, allowing
the exposed omohyoid muscle to be retracted laterally to expose the
carotid artery internal and external branch regions. The superior
cervical ganglion lying within this bifurcation was carefully excised.
This procedure was repeated on the contralateral side. A successful
sympathectomy was evidenced by the occurrence of Horner’s
syndrome. Adrenalectomy was not performed in these animals
because the aim was to examine the specific interactions between the
SNS and MCs, not the role of circulating norepinephrine.
Hemodynamic Parameters
At the experimental end point, animals were anesthetized, and
measurements of systolic, diastolic, and mean arterial pressures were
made using a high fidelity Millar catheter inserted into the carotid
artery and advanced into the aortic arch. Catheter data were analyzed
using PVAN 3.5 software.
Histological Analysis
MC number and density were determined from pinacyanol erythrosinate–stained LV sections as described previously (please see the
online Data Supplement at http://hyper.ahajournals.org).6,17 LV interstitial collagen volume fraction was determined by analysis of
picrosirius red–stained sections as described previously (please see
the online Data Supplement).6
Cytokine Characterization
Interferon (IFN)-␥, interleukin (IL) 4, IL-6, and IL-10 were quantified from frozen LV tissue (please see the online Data Supplement)
to determine the effects of sympathectomy, using commercial
ELISA kits (Alpco Diagnostics).
[1.3 mmol/L], and phenol red; Hyclone) before undergoing
treatment.
Isolated Cardiac MC Studies
Cardiac MCs were obtained during the cardiac inflammatory cell
isolation procedure described above. Cardiac MC responses were
examined by incubating 4⫻103 MCs per treatment tube with
norepinephrine or substance P at specific concentrations: 0,
1⫻10⫺12, 1⫻10⫺9, 1⫻10⫺6, and 1⫻10⫺4 M. The samples were
incubated at 37°C for 20 minutes, and the posttreatment supernatants
and pellets were separated for subsequent analysis of histamine as a
marker of MC degranulation. The percentage of histamine release
was determined by dividing the histamine value from the supernatant
by total histamine (supernatant plus pellet). Histamine concentration
was measured using a Neogen Veratox Histamine ELISA.
Substance P–Mediated Production of Angiotensin II by
Isolated Cardiac Inflammatory Cells
Isolated cardiac inflammatory cells (2⫻105 per well) were maintained in DMEM containing 10% FBS and treated with substance P
(1⫻10⫺4 M) for 20 hours. The medium was collected and assayed for
angiotensin II using a commercial enzyme immunoassay kit
(SPIbio), which has cross-reactivities of 4%, 36%, and 33% with
angiotensin I, angiotensin III, and angiotensin 3-8, respectively.
Immunofluorescent Staining of
␣-Adrenergic Receptors
Please see the online Data Supplement for details.
Statistical Analysis
All of the grouped data were expressed as mean ⫾ SD or SEM, as
appropriate. Group comparisons were made by 1-way ANOVA
using SPSS 11.5 software (SPSS Inc). When a significant F test
(Pⱕ0.05) was obtained, intergroup comparisons were analyzed using
the Fisher protected least-significant difference post hoc testing.
Results
Biometrics
Cardiac Inflammatory Cell Isolation Procedure
Cardiac inflammatory cells were isolated as described previously.
Briefly, a thoracotomy exposed the intact pericardial sac. The tip of
a Teflon catheter sleeve, attached to a sterile syringe, was then
inserted into the pericardial sac, and the sac filled with Hanks’
balanced salt solution (7.4 pH) which was composed of the following: (1) Hanks’ calcium- and magnesium-free salt solution; (2)
HEPES (13 mmol/L); (3) 607 U/mL of deoxyribonuclease I (Sigma
Chemical Co); and (4) an antibiotic-antimycotic mixture of penicillin
G sodium, streptomycin sulfate, and amphotericin B (Gibco-BRL,
Life Technologies). The buffer was then aspirated into another sterile
syringe. This was repeated several times, resulting in the collection
of, predominantly, T cells, MCs, and monocyte/macrophages (Figure
S1, available in the online Data Supplement). The cells were pelleted
and resuspended in Hyclone buffer (Hanks’ balanced salt solution
containing magnesium sulfate [1.1 mmol/L], calcium chloride
15
The Table displays grouped biometric data. Although no
differences in body weight were observed between WKYs,
SHRs, and WKY⫹Symps, a significant increase was observed in sympathectomized SHRs. The Table also includes
LV weight and LV weight indexed to body weight as
measures of hypertrophy. SHRs developed significant LV
hypertrophy in comparison with WKYs by both measures,
with sympathectomy preventing this hypertrophic response.
There were no weight differences between WKYs and
WKY⫹Symp. Lung weights showed no significant differences. SHRs showed marked increases in systolic, diastolic,
and mean arterial pressures compared with WKYs. Sympathectomy in the SHRs caused a significant attenuation of the
February 2010
SHR
W
+S
Y
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+S
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Y+
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0
p
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m
R
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3.5
†
8
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10
S
4.5
Mast Cell Density (cells/mm2)
Hypertension
Collagen Volume Fraction (%)
272
Figure 2. Changes in LV MC density in WKYs (n⫽4), SHRs
(n⫽6), WKY⫹Symps (n⫽6), and SHR⫹Symps (n⫽4). All of the
values are mean⫾SD. *P⬍0.05 vs WKYs; †P⬍0.05 vs SHRs.
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
on MC number or density in WKYs (WKY⫹Symp:
84.2⫾12.1 cells per section).
Myocardial Cytokine Profile
100 µM
WKY+Symp
SHR+Symp
SHR+Ned
Figure 1. LV collagen volume fraction in WKYs (n⫽4), SHRs
(n⫽4), WKY⫹Symps (n⫽4), and SHR⫹Symps (n⫽4). All of the
values are mean⫾SEM. *P⬍0.05 vs WKYs; †P⬍0.05 vs SHRs.
blood pressure parameters, whereas sympathectomy had no
effect on blood pressure in the WKY group.
Collagen Volume Fraction
Collagen volume fraction results are displayed in Figure 1.
LV collagen volume fraction in the SHRs was markedly
increased in comparison with that in WKYs. However, this
increase was significantly attenuated in the sympathectomized SHR group. Collagen volume fraction was not affected
by sympathectomy in the WKYs.
Cardiac MC Number and Density
A significant increase in the number of MCs occurred within
the left ventricle of SHRs (WKY: 73.3⫾10.3 versus SHR:
105.7⫾14.8 cells per section; P⬍0.05). However, because of
the larger area of the SHR left ventricle, there was no
difference when expressed as MC density (Figure 2). In
contrast, SHRs that had undergone sympathectomy showed a
dramatic increase in both LV MC number and density
(SHR⫹Symp: 316.3⫾68.6 cells per section; P⬍0.05 versus
WKYs and SHRs). In contrast, sympathectomy had no effect
Elevated levels of IFN-␥ in the SHR (Figure 3A) were
normalized by sympathectomy. Sympathectomy had no effect
in the WKY. Although myocardial IL-4 levels were also
elevated in the SHR (Figure 3B), sympathectomy did not
change these levels. However, sympathectomy in the WKY
did cause a significant increase in IL-4. Myocardial IL-6
(Figure 3C) was found to be markedly lower in the SHR than
in the WKY, but was normalized by sympathectomy. Sympathectomy did not change IL-6 levels in the WKY. IL-10
was also found to be decreased in the SHR myocardium
(Figure 3D). Sympathectomy resulted in increased amounts
of IL-10 in both WKYs and SHRs.
Effect of SNS Products on Cardiac MC Activation
and Angiotensin II Production
Incubation of isolated cardiac MCs with norepinephrine at
concentrations of 1⫻10⫺12, 1⫻10⫺9, 1⫻10⫺6, and 1⫻10⫺4 M
did not induce degranulation (Figure 4A). In contrast, while no
effects of substance P were seen at lower concentrations,
1⫻10⫺4 M did stimulate cardiac MC degranulation (Figure
4B). Also, this concentration of substance P stimulated the
production of angiotensin II by a mixed population of isolated
cardiac inflammatory cells (Figure 4C).
Identification of ␣-Adrenergic Receptors on
Cardiac MCs
Isolated cardiac MCs did not possess ␣-adrenergic receptors
(Figure S2).
Discussion
Chronic elevation of SNS activity in the myocardium is a key
component of the altered signaling pathways that accompany
cardiac remodeling resulting from hypertension. Although
there is evidence that the SNS mediates cardiac fibrosis in
hypertension via ␣-adrenergic receptor stimulation,2 the exact
mechanisms have not been fully elucidated. However, it is
likely to be more complex than direct activation of
␣-adrenergic receptors on cardiac fibroblasts. To this end,
Levick et al
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p
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SH
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1000
Figure 3. Changes in LV levels of (A) IFN-␥, (B) IL-4, (C) IL-6, and (D) IL-10 in WKYs (n⫽4), SHRs (n⫽4), WKY⫹Symps (n⫽4), and
SHR⫹Symps (n⫽4). All of the values are mean⫾SEM. *P⬍0.05 vs WKYs; †P⬍0.05 vs SHRs.
inflammation is also an important component in the development of hypertension-induced cardiac fibrosis.3–5,7,8,18 More
specifically, we recently demonstrated a causal role for MCs
in regulating myocardial cytokines, macrophage recruitment,
and development of fibrosis in the hypertensive heart.6 Given
the critical roles of both SNS upregulation and MCs in
hypertension-induced myocardial remodeling, the current
study aimed to explore the effects of surgical sympathectomy
on cardiac MCs, as well as specific cytokines, in the hypertensive heart.
After sympathectomy in SHRs, we observed a dramatic
⬇5-fold increase in cardiac MC density. Although surgical
C
Concentration (M)
M
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removal of the superior cervical ganglion likely does not
result in complete denervation of the heart, nerve fibers from
this ganglion do project to the heart,19 and this striking change
in MC density clearly demonstrates an impact of its removal
on the heart. This same relationship is not present in the
normal heart, because such a change did not occur in
sympathectomized WKYs. Facoetti et al20 also found cardiac
MC density to be unchanged in normal Sprague-Dawley rats
after 10 weeks of chemical sympathectomy induced by
6-hydroxydopamine. These disparate effects on MC density
are likely indicative of underlying differences in the local
environment of normal versus hypertensive hearts. That
Angiotensin II Concentration
(pg/mL)
A
C
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Histamine Release (%)
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IL-6 (pg/mL)
C
273
4500
SH
IFN-γ (pg/mL)
B
*
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IL-4 (pg/mL)
A
Sympathetic Control of Inflammation in the Heart
Figure 4. Concentration-responses for histamine release by isolated cardiac MCs in response to norepinephrine (n⫽4; A) and substance P (n⫽4; B). Angiotensin II was produced in response to stimulation with substance P (1⫻10⫺4 M) by a mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes, and macrophages (C). P⬍0.05 vs control.
274
Hypertension
February 2010
Tryptase
PAR-2
MAST CELL
NK-1
Receptor
FIBROSIS
AFFERENT
NEURON
Sub P
Angiotensin II
NK-1
Receptor
T cell
FIBROBLAST
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NK-1
Receptor
AT 1
Receptors
p
α-adrenergic
receptor
Macrophage
EFFERENT
NEURON
Norepinephrine
Figure 5. Schematic depicting a possible series of events in the development of fibrosis in the hypertensive heart. Cardiac MC tryptase
may activate PAR-2 on afferent nerve fibers resulting in the release of substance P, which, in turn, acts on the neurokinin-1 (NK-1)
receptor to induce production of angiotensin II by inflammatory cells. Angiotensin II, in addition to activating fibroblasts, may also stimulate efferent nerves to produce norepinephrine, in turn further stimulating fibroblasts to produce collagen.
fibrosis was attenuated in sympathectomized SHRs despite
MC density being elevated suggests that either sympathectomy altered MCs to those of a nonfibrotic phenotype or,
alternatively, MCs signal the SNS, but can no longer induce
a profibrotic response from the damaged SNS. Interestingly,
in our isolated cardiac MC experiments, incubation with
norepinephrine failed to induce degranulation, and we were
unable to detect ␣-adrenergic receptors on cardiac MCs using
flow cytometry. This is consistent with a previous report that
norepinephrine was not the mechanism by which contact with
sympathetic neurons decreased membrane resistance in immortalized RBL-2H3 cells, a widely used model for MCs.13
However, some afferent nerve fibers projecting from the heart
travel through the superior cervical ganglia,21 and substance
P, which is primarily located in afferent nerve fibers, has been
demonstrated to be present in superior cervical ganglia.22
Substance P– containing nerves are associated with numerous
areas of the heart, including the ventricle, atria, valves, and
connective tissue lining.23 Here we showed that substance P
was a strong inducer of isolated cardiac MC degranulation,
consistent with what we have demonstrated previously15 and
suggestive of afferent activation of cardiac MCs.
Suzuki et al,24 using a coculture system, showed that
RBL-2H3 MCs activated sympathetic neurons after IgE
stimulation. Activation of sympathetic neurons did not occur
if RBL-2H3 cells were not present. The most obvious
mechanism by which MC activation of neurons could occur is
through release of MC tryptase acting on protease activated
receptor 2 (PAR-2). Steinhoff et al25 recently detected PAR-2
on a large number of neurons containing substance P, and
activation of these receptors by tryptase in dorsal horn slices
initiated the release of substance P. However, cardiac MCs
are also capable of producing renin and are assumed to be
involved in the local generation of angiotensin II in the
heart.12,26,27 Silver et al12 postulated that MC-generated angiotensin II could then activate nerves through angiotensin
receptors. We isolated a mixed population of inflammatory
cells, including lymphocytes, macrophages and MCs, from
rat hearts and found that incubation with substance P resulted
in angiotensin II production. Thus, while it appears that the
SNS is regulating cardiac MC density in sympathectomized
SHRs, MCs may in actuality be upstream of SNS activation
and, therefore, responding to an underlying stimulus (eg,
increased wall stress) by unsuccessfully attempting to upregulate SNS activity. That is, the increase in MC density
may be an attempt to achieve increased SNS activity that
cannot be elicited because of the sympathectomy. Therefore,
we speculate that a self-perpetuating relationship may exist
where cardiac MC tryptase could activate PAR-2 on afferent
nerve fibers causing the release of substance P, which, in turn,
Levick et al
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induces angiotensin II production by inflammatory cells that
then acts on fibroblasts and also sympathetic nerves to release
norepinephrine (Figure 5). Angiotensin II has been shown to
induce myocardial necrosis through the stimulation of sympathetic neurons.28,29 In fact, a similar situation has been
described in the rat knee joint, where it appears that sensory
nerve activation of MCs led to stimulation of the SNS and,
subsequently, plasma extravasion.30 Elaboration of norepinephrine by the SNS in response to stimulation by MCs
would explain how ␣-adrenergic receptors mediate fibrosis2
when cardiac MCs do not degranulate in response to norepinephrine in our study. This may also explain why sympathectomy does not result in increased cardiac MC density in the
normal heart where there is no underlying pathological
stimulus driving MCs to upregulate sympathetic activity.
With this clear SNS-MC interaction, we sought to determine the effect of sympathectomy on selected myocardial
cytokines that we have shown previously to be regulated,
either directly or indirectly, by MCs in the hypertensive
heart.6 Both IFN-␥ and IL-4 were elevated in the SHR, with
sympathectomy normalizing IFN-␥ but not IL-4. IFN-␥ is a
marker of T-helper 1 T-cell phenotype,31 and its elevation in
hypertension is consistent with the supposition advanced by
Yu et al9 that T-helper 1 cells may be profibrotic. Interestingly, although sympathectomy had no effect on IFN-␥ in the
WKY, sympathectomy increased IL-4 levels in the WKY.
Combined with our previous studies,6 it would appear that the
SNS has a role in regulating IL-4 in the normal heart, but that
role is shifted to MCs in the hypertensive heart. Although the
levels of IL-6 and IL-10 were lower in the SHR myocardium
compared with the WKY, sympathectomy returned them to
normal. What is intriguing about these results is that, with the
exception of IL-4, these changes are almost identical to what
we have observed previously in the SHR after MC stabilization.6 This similarity, together with the close spatial relationship between the nervous system and MCs,11,12 is more
evidence of an interplay between the SNS and MCs in the
regulation of the inflammatory process in the hypertensive
heart.
In addition to attenuating cardiac fibrosis, sympathectomy
in the SHR also prevented myocardial hypertrophy. However,
our previous finding that MC stabilization did not prevent
hypertrophy in the hypertensive heart indicates that MCs are
not involved in modulation of hypertrophy.6 Rather than
acting via inflammatory cells to induce hypertrophy, the SNS
most likely has direct effects on cardiomyocytes via
␤-adrenergic receptors. Although it cannot be ruled out that
the changes in these parameters and the assayed cytokines
were the result of the sympathectomy-related attenuation of
blood pressure, we believe that the following observations
support the argument that the changes in cytokines and
fibrosis were related to direct interactions between the SNS
and MCs. First, attenuation of hypertrophy by sympathectomy has been shown to be independent of systolic blood
pressure,2,32 and Perlini et al2 further demonstrated that
chemical sympathectomy in hypertensive rats prevented cardiac fibrosis independent of blood pressure. Second, the
increase in cardiac MC density was dramatic with sympathectomy in the SHR but not in the normal nonhypertensive
Sympathetic Control of Inflammation in the Heart
275
heart. Third, our in vitro studies demonstrate that substance P,
found in afferent nerves, can stimulate cardiac MC activation
and angiotensin II production from a mixed population of
cardiac inflammatory cells. Finally, the cytokine profile
reported herein after sympathectomy in the SHR is almost
identical to that after MC stabilization in the SHR, where
blood pressure was unaffected.6
Perspectives
The results of this study reiterate the importance of the SNS
in the development of myocardial remodeling and provide
evidence of a link between the neurohormonal and inflammatory paradigms of hypertension by demonstrating the
importance of SNS interactions with cardiac MCs and cytokine production. Furthermore, although a number of inflammatory cell types have been implicated in the development of
fibrosis,7,9,10 our findings are consistent with the hypothesis
that MCs not only trigger and orchestrate inflammatory
responses33 but are also responsible for cardiac fibrosis.
However, it is important to note that not all of the effects of
the SNS cardiac remodeling are mediated via MCs. Clearly
hypertrophy does not appear to involve SNS interactions with
MCs, but instead is likely the result of direct stimulation of
cardiomyocyte ␤–adrenergic receptors. The findings herein
indicate the need for further investigation regarding the
mechanisms mediating SNS activation, as well as inflammatory cell and afferent nerve interactions in the hypertensive
heart, with particular focus on the role of substance P.
Acknowledgments
We thank Purnima Jani for her excellent technical assistance.
Sources of Funding
This study was supported in part by an American Heart Association
postdoctoral fellowship 0825510E (to S.P.L.), as well as National
Heart, Lung, and Blood Institute grants R01-HL-62228 (to J.S.J.)
and R01-HL-073990 (to J.S.J.).
Disclosures
None.
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Sympathetic Nervous System Modulation of Inflammation and Remodeling in the
Hypertensive Heart
Scott P. Levick, David B. Murray, Joseph S. Janicki and Gregory L. Brower
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Hypertension. 2010;55:270-276; originally published online January 4, 2010;
doi: 10.1161/HYPERTENSIONAHA.109.142042
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ONLINE SUPPLEMENT
SYMPATHETIC NERVOUS SYSTEM MODULATION OF INFLAMMATION AND REMODELING IN THE HYPERTENSIVE HEART Scott P. Levick1, David B. Murray2, Joseph S. Janicki1 and Gregory L. Brower1 1
Cell Biology and Anatomy School of Medicine University of South Carolina Columbia, SC 29208, USA 2
School of Pharmacy University of Mississippi Oxford, MS 38677, USA Correspondence: Joseph S. Janicki, PhD Cell Biology and Anatomy School of Medicine University of South Carolina Columbia, SC 29208, USA Telephone (803) 733‐1536; fax (803) 733‐3153; email [email protected] Materials and Methods
Mast Cell Number and Density
At the end of the 12 week study period, anesthetized rats were euthanized by excision of the
heart, which was then rinsed in ice-cold saline prior to separating and weighing the LV plus
septum and RV. A slice of the LV from its equatorial region was fixed with Telly’s fixative (100
mL 70% ethanol; 5 mL glacial acetic acid; 10 mL formaldehyde) for histological analysis, while
the apical portion was snap-frozen in liquid nitrogen and stored at -80˚C for subsequent use. To
determine mast cell density, 5 µm thick paraffin embedded LV cross-sections were stained with
the mast cell specific stain, pinacynol erythrosinate.1 Mast cell counts from the entire LV section
were carried out in a blinded fashion at 400x magnification, with mast cell density determined by
dividing the total number of mast cells by the area of the respective LV cross-section as
determined from the digitized image (ImageQuant, Molecular Dynamics).
Collagen Volume Fraction
Paraffin-embedded LV cross-sections cut to 5 µm in thickness were incubated with
phosphomolybdic acid (0.2%) to reduce background before staining with picrosirius red (0.1%
Sirius Red F3BA in picric acid). Tissue sections were analysed in a blinded fashion using a
Biorad MRC-1024 confocal laser-scanning microscope with a 200x magnification. Twenty
representative fields per LV cross-section were imaged. Image analysis was performed using
Scion Image software (National Institutes of Health, USA). Briefly, a pixel count for the amount
of collagen was obtained for each field. This was then expressed as a percentage of the overall
number of pixels present in the field giving a percentage area of interstitial collagen. Perivascular
areas were excluded from the analysis.
Protein Extraction from LV tissue
100mg of LV tissue from each heart was maintained on ice and cut into pieces approximately 1
mm2. This tissue was then placed into the homogenization tubes and immersed in 800 µL of
PBS/protease inhibitor solution. Tissues were homogenized on ice before being sonicated. 50 µL
of 10 % Triton-X 100 was then added to each sample and vortexed. The samples were then
allowed to sit on ice for 30 minutes. Samples were again vortexed after 15 of the 30 minutes.
Following Triton-X treatment the samples were centrifuged @ 16,000 rpm (4°C) for 10 min and
the supernatant collected and frozen at -80°C.
Flow Cytometry
Isolated cells were washed and blocked against non-specific staining with PBS/1% BSA for 30
min before incubation for 45 min at room temperature with either AR32AA4 (MC; mouse
monoclonal, 1:100; BD Pharmingen), anti-CD4-PE (mouse monoclonal, 1:100; BD
Pharmingen), anti-CD8-PE (mouse monoclonal, 1:100; BD Pharmingen), anti-CD68
(macrophage; mouse monoclonal, 1:100; Serotec) and anti-α1 adrenergic receptor (rabbit
monoclonal, 1:500; Abcam). AR32AA4 and anti-CD68 were conjugated with Alexa Fluor 488
secondary antibody for the inflammatory cell profiling experiments (supplemental figure 1).
AR32AA4 was conjugated with PE secondary antibody for the α1 adrenergic receptor
experiments. α1 adrenergic receptor was conjugated with Alexa Fluor 488 secondary antibody.
Cell preparations were then analyzed using an Epics XL FACS system equipped with a 15mW
argon ion laser operating at 488nm and data analyzed with EXPO 32 software.
Reference List (1) Bensley SH. Pinacyanol erthrosinate as a stain for mast cells. Stain Technol. 1952;27:269‐273. 0.0%
0.0%
88 4%
88.4%
11 6%
11.6%
B
54.9%
0.0%
46.1%
0.1%
CD4+
A
Mast Cells
0.1%
0.0%
88.0%
11.9%
.9%
D
12.8%
0.1%
87.1%
0.0%
CD8+
C
CD68+
Figure S1. Representative flow cytometric analysis of the distribution of inflammatory cells
isolated from normal rat hearts using the isolation technique described in Methods. (A) mast
cells; (B) CD4+ T cells; (C) CD68+ macrophages; and (D) CD8+ T cells.
16.6 0.1
71.5 11.8
Figure S2. Flow cytometric analysis of α-adrenergic receptors and cardiac mast cells in the isolated cardiac
inflammatory cell preparation. The analysis indicates that cardiac mast cells do not possess α-adrenergic receptors.