From the Section of Cardiovascular Sciences, The Methodist Hospital and
The DeBakey Heart Center, Department of Medicine, the Speros P. Martel
Laboratory, Section of Leukocyte Biology, Department of Pediatrics, and Texas
Children's Hospital, Baylor College of Medicine, Houston, Tex.
Correspondence to Mark L. Entman, MD, Department of Medicine, Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, M/S F-602, Houston, TX 77030-3498. E-mail mentman{at}bcm.tmc.edu
Methods and Results—Using a canine model of myocardial
ischemia and reperfusion, we demonstrated a striking increase
of mast cell numbers during the healing phase of a myocardial
infarction. Mast cell numbers started increasing after 72 hours of
reperfusion, showing maximum accumulation in areas of collagen
deposition (12.0±2.6-fold increase; P<0.01) and
proliferating cell nuclear antigen (PCNA) expression. The majority of
proliferating cells were identified as
Conclusions—Mast cells increase in number in areas of collagen
deposition and PCNA expression after myocardial ischemia. The
data provide evidence of mast cell precursor infiltration into the
areas of cellular injury. SCF is induced in a subset of
macrophages infiltrating the healing myocardium. We
suggest an important role for SCF in promoting chemotaxis and growth of
mast cell precursors in the healing heart.
Immunohistochemistry and Histology
Molecular Cloning
RNA Isolation
Nuclease Protection Assay
Statistical Analysis
Mast Cells Accumulate in Areas of Collagen Deposition and PCNA
Expression
Most PCNA-Positive Cells Are Identified as Myofibroblasts and
Endothelial Cells
Absence of PCNA-Positive Mast Cells in the Healing
Myocardium
Cloning of Canine SCF
SCF mRNA Is Induced in the Ischemic and Reperfused
Myocardium
SCF Protein Localization in the Ischemic and
Reperfused Myocardium
Evidence for the Presence of Mast Cell Precursors in the
Healing Heart
Mast Cells Accumulate in Areas of Cell Proliferation and
Collagen Deposition
Cellular Identity of Proliferating Cells
Mast Cells Do Not Appear to Proliferate: Mast Cell Precursors
Infiltrate the Ischemic Heart
SCF Is Induced in Areas of Healing
A Subset of Macrophages Is the Main Source of
SCF
Significance of Hypothesized Cellular Interactions in Cardiac
Healing
Received December 17, 1997;
revision received February 13, 1998;
accepted March 18, 1998.
© 1998 American Heart Association, Inc.
Basic Science Reports
Stem Cell Factor Induction Is Associated With Mast Cell Accumulation After Canine Myocardial Ischemia and Reperfusion
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Myocardial infarction is
associated with an intense inflammatory reaction leading to healing and
scar formation. Because mast cells are a significant source of
fibrogenic factors, we investigated mast cell accumulation and
regulation of stem cell factor (SCF), a potent growth and tactic factor
for mast cells, in the healing myocardium. 
-smooth muscle
actin–positive myofibroblasts or factor VIII–positive
endothelial cells. Mast cells did not appear to
proliferate. Using a nuclease protection assay, we demonstrated
induction of SCF mRNA within 72 hours of reperfusion.
Immunohistochemical studies demonstrated that a subset of
macrophages was the source of SCF immunoreactivity in the
infarcted myocardium. SCF protein was not found in
endothelial cells and myofibroblasts. Intravascular
tryptase–positive, FITC-avidin–positive, CD11b-negative mast cell
precursors were noted in the area of healing and in the cardiac lymph
after 48 to 72 hours of reperfusion.
Key Words: cells • collagen • myocardial infarction • reperfusion • growth substances
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Mast cells are
recognized effector cells in allergic inflammatory responses. Recent
findings indicate that mast cells can influence biological processes
through the production of cytokines and growth
factors.1 The suggestion that mast cells may
participate in the fibrotic process is supported by studies
demonstrating that mast cells are capable of producing a wide variety
of mediators, such as histamine, tryptase,
TNF-,1 bFGF,2 and
TGF-ß,3 which can modulate fibroblast
phenotypic characteristics and extracellular matrix synthesis. The
association of inflammation with myocardial infarction has been
recognized for more than a century4 and is
properly considered part of the healing process. Our laboratory has
concentrated on characterizing the biological basis for reperfusion
injury in a canine model of myocardial
infarction.5 6 In the companion
article,7 we present evidence suggesting that
the cardiac mast cell is the primary source of preformed TNF- in the
canine heart and, through degranulation and TNF- release, initiates
a cytokine cascade involving IL-6 induction in infiltrating
mononuclear cells and subsequent intercellular adhesion molecule-1
induction in cardiac myocytes. The present study was designed to
investigate cardiac mast cells during the healing phase of an
experimental canine myocardial infarction. We found that during the
healing phase, mast cells were markedly increased in number in the
ischemic segments of canine myocardium,
accumulating in areas of collagen deposition and cell proliferation. We
present the first in vivo demonstration of mast cell precursor
influx into an evolving tissue injury. In addition, we present the
first evidence for induction of SCF,8 a potent
tactic and growth factor for mast cells, in a subset of infiltrating
macrophages after myocardial ischemia. We postulate
that SCF may attract circulating mast cell precursors in the healing
myocardium and promote their maturation and growth.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Ischemia/Reperfusion Protocols
Healthy mongrel dogs (15 to 25 kg) of either sex were surgically
instrumented as previously described9 with a
hydraulic occluder secured around the circumflex coronary
artery and a cannula placed in the cardiac lymph duct. After surgery,
the animals were allowed to recover for 72 hours before occlusion.
Ischemia-reperfusion protocols were performed in awake animals
as described.9 Coronary artery occlusion
was achieved by inflating the coronary cuff occluder until mean
flow in the coronary vessel was zero as determined by the
Doppler flow probe. At the end of 1 hour, the cuff was deflated and
the myocardium was reperfused. Reperfusion intervals ranged
from 1 hour to 7 days. During the experiment, the cardiac lymph was
collected. The samples were centrifuged, and the supernatant
was collected and immediately frozen at -80°C. The pelleted cells
were fixed in Carnoy's fixative and used for
histological studies. After reperfusion, hearts were
stopped by the rapid intravenous infusion of 30 mEq of KCl
and removed from the chest for sectioning. Tissue samples were isolated
from infarcted or normally perfused myocardium on the basis
of visual inspection. Myocardial segments were fixed in 10% buffered
formalin, Carnoy's fixative, Mota's fixative, or B*5 fixative for
histological analysis or were immediately
frozen, homogenized, and processed for RNA extraction.
Duplicate adjacent samples were also processed for blood flow
determinations with radiolabeled microspheres as previously
described.5 9 The presence of a myocardial
infarct was based on light-microscopic examination of
hematoxylin-eosin–stained tissue sections by findings of contraction
bands, "wavy fibers," interstitial edema, and
neutrophil infiltration, all in segments displaying markedly reduced
blood flow during the ischemic period. For experiments lasting
24 hours after the start of the ischemic insult, the presence
of histological elements characteristic of myocyte
necrosis and fibrosis was added to the required criteria. Samples
described as ischemic were all from areas in which
ischemic blood flow was <25%. Samples of control tissues had
normal blood flow during coronary occlusion. Data from 25
experiments, all showing evidence of ischemia based on blood
flow determinations and light-microscopic examination, were
analyzed (n=5 for 1 hour of ischemia and 3 hours of
reperfusion, n=4 for 1 and 24 hours, n=5 for 1 and 72 hours, n=6 for 1
and 120 hours, and n=5 for 1 and 144 hours).
For histological studies, sections were fixed in
10% phosphate buffered formalin, Carnoy's fixative, or B*5 fixative
and embedded in paraffin. Sequential 3- to 5-µm sections were cut by
microtomy. Immunostaining was performed with the ELITE
rabbit or mouse kit (Vector Laboratories). The following primary
antibodies were used for immunohistochemistry: mouse monoclonal
antibody to CD11b MY904 (a clone from the American Type Culture
Collection), mouse monoclonal antibody to human -smooth muscle actin
(Sigma), monoclonal mouse anti-PCNA antibody (clone PC10) (Dako),
rabbit anti-human polyclonal antibody to factor VIII (Dako), monoclonal
anti–smooth muscle myosin antibody10 (kindly
donated by Dr C.L. Seidel, Baylor College of Medicine, Houston, Tex),
monoclonal anti–canine SCF antibody11
(generously donated by Dr H.J. Deeg, University of Washington,
Seattle), and monoclonal anti-macrophage antibody
AM-3K12 (a generous gift from Dr K. Takahashi,
Kumamoto University, Japan). Fluorescent immunohistochemistry
was done with appropriate rhodamine- or Cy3-labeled secondary
antibodies (Sigma). Appropriate positive and negative controls were
used for each antibody. Dual immunohistochemistry was performed by
combining peroxidase-based immunostaining for PCNA with
fluorescent immunohistochemistry for -smooth muscle actin or
factor VIII with rhodamine-labeled secondary antibodies. A similar
technique was used for SCF/-smooth muscle actin and SCF/factor VIII
immunostaining. Fluorescent labeling of mast
cells with FITC-avidin was performed as previously
described.13 The histochemical staining technique
for tryptase was performed as previously described by Caughey et
al.14 Quantitative analysis of mast cell
numbers in canine heart was performed by counting FITC-labeled mast
cells in sections from control and ischemic areas. Mast cells
from 10 random fields (x200) were counted, and at least 2 different
sections from 2 different tissue samples from each experiment were used
for quantitative analysis. Samples labeled as ischemic
had evidence of coronary ischemia based on
histological analysis and blood flow
quantification by use of radiolabeled microspheres. For studies
on mast cell accumulation in areas of collagen deposition, sections
from ischemic samples were stained for tryptase. At least 15
random fields (x400) were photographed, and the number of
tryptase-positive mast cells was counted in each field. Sections were
then incubated overnight in 10% Tween 20 to erase the tryptase stain.
Subsequently, picrosirius red staining was performed to visualize
collagen fibers. The same fields were photographed again and on the
basis of the presence of fibrotic tissue were classified as fibrotic
(where myocytes were fully replaced by scar), partially fibrotic (where
myocytes were partially replaced by fibrotic tissue), and nonfibrotic
(where no evidence of scar formation was present). The mast cell
numbers in the fibrotic and partially fibrotic areas were normalized on
the basis of the corresponding numbers for nonfibrotic areas
(nonfibrotic=1). Mast cell numbers were statistically analyzed
by Student's t test (two-tailed) for 5 different
experiments of myocardial ischemia, with reperfusion intervals
ranging from 96 to 144 hours. Cell proliferation studies were performed
by use of immunohistochemistry with the antibody to PCNA. Sections from
canine bowel were used as positive controls. To correlate mast cell
numbers with the number of proliferating cells, sections were stained
with FITC-avidin and photographed. The number of FITC-avidin–positive
mast cells was counted in each field. Subsequently, sections were
immunostained for PCNA, and the numbers of PCNA-positive
cells in the same fields were counted. Correlation studies between the
number of mast cells and the number of PCNA-positive nuclei were
performed for 5 experiments, with reperfusion intervals ranging from 96
to 144 hours.
A cDNA clone for canine SCF was prepared by RT with RNA
extracted from the ischemic and reperfused
myocardium. RT-PCR was performed with the antisense primer
5'-TTGCAACATACT TATTTCATTATCC-3' and the sense primer
5'-ATGAAGAAGA CACAAACTTGGATTA-3'. The nucleotide
sequence of the primers was based on the published sequence for canine
SCF15 and corresponded to base pairs 1 through 25
and 770 through 795 of the sequence. RT protocols were performed with 5
µg of total RNA. After first-strand synthesis, primed with the
antisense primer, aliquots of the RT reaction were amplified with 5 U
Taq DNA polymerase (Promega Corp) for 30 cycles of 93°C, 1
minute; 55°C, 2 minutes; and 72°C, 3 minutes. The resulting 795-bp
fragment was purified, cloned in the PCR vector (Invitrogen), and
sequenced.
RNA isolation from myocardial segments was performed by the acid
guanidinium–phenol-chloroform procedure. RNA (30 µg) was
electrophoresed in 1% agarose gels containing formaldehyde, then
transferred to a nylon membrane (Gene Screen Plus, New England Nuclear)
by standard procedures.
SCF mRNA expression in samples from the ischemic and
reperfused myocardium was studied with an RNAse protection
assay. The vector containing the 795-bp canine SCF insert was
linearized by restriction digestion of the unique EcoRV site
in the polylinker at the 5' end of the insert. Antisense
32P-labeled transcripts were synthesized at
37°C in a reaction containing 1 µg of linearized template DNA;
1 mmol/L each of ATP, GTP, and UTP; 70 µCi
[-32P]CTP (400 Ci/mmol); 10 U Sp6 RNA
polymerase; and standard transcription buffer. The probe was purified
by 2 rounds of precipitation with ammonium acetate and ethanol. RNAse
protection was performed with the RNase protection kit
(Boehringer Mannheim) or the Riboquant kit (Pharmingen)
according to the manufacturer's instructions. Briefly, 30 µg of
total RNA was hybridized with 300 000 cpm
32P-labeled riboprobe overnight at 50°C
followed by RNAse digestion. As a control reaction, the riboprobe was
hybridized with yeast tRNA followed by RNAse digestion. The protected
fragments were resolved on a 6% denaturing polyacrylamide gel
and quantified with a GS-363 molecular imager system (Biorad). Size
determinations of protected fragments were made by comparison to the
full-length riboprobe and a DNA sequencing ladder. The predicted sizes
of the full-length riboprobe and the protected fragment are 893 and 795
nucleotides, respectively. The riboprobe contains some
linker sequences that are not protected. Loading of RNA was monitored
with ethidium bromide staining as well as by probing of the nylon
membranes with canine GAPDH.
ANOVA followed by Student's t test, corrected for
multiple comparisons (Bonferroni), was used to test the significance of
differences between mean values. Mean values are given with SEM.
Correlation studies were performed for 5 consecutive experiments of
myocardial infarction, with reperfusion intervals ranging from 120 to
144 hours. For this and all earlier time periods, immunohistochemical
experiments were performed in at least 5 different experiments from
each time point. The results described were consistently
reproduced in all experiments.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Mast Cells Increase in Number After an Experimental Canine
Myocardial Infarction
Staining with FITC-labeled avidin (Figure 1
) demonstrated a 3.39-fold increase of
mast cell numbers in the ischemic sections after experiments of
1 hour of ischemia and 7 days of reperfusion,
compared with control sections from the same experiments
(P<0.01, n=5) (Figure 2). The
increase in mast cell numbers was first noted after 72 hours of
reperfusion (1.65-fold; P<0.05, n=5). We demonstrated the
presence of the mast cell–specific proteinase tryptase in the
cytoplasm of these cells (Figure 3a and 3b).
View larger version (122K):
[in a new window]
Figure 1. Mast cell number increase after myocardial
ischemia and reperfusion. A, Control canine
myocardium stained with FITC-avidin to identify mast cells,
located predominantly along arterioles (a) and venules (v) (x100). B,
Ischemic section of canine myocardium after 1 hour
of ischemia and 7 days of reperfusion stained with FITC-avidin.
Section shows area of scar formation (bottom) where myocytes have been
replaced by fibrotic tissue and area where myocyte architecture is
relatively preserved. A striking accumulation of mast cells is noted in
healing area (x160).
View larger version (41K):
[in a new window]
Figure 2. Time course of mast cell numbers after
myocardial ischemia/reperfusion. Note statistically significant
increase in number of mast cells in ischemic areas after 72
hours (1.69-fold increase, P<0.05), 5 days (3.3-fold,
P<0.01), and 7 days (3.39-fold, P<0.01)
of reperfusion vs respective numbers in control areas from same
experiment by ANOVA followed by t test with Bonferroni
correction. For shorter reperfusion intervals, mast cell numbers in
control and ischemic sections were comparable. Numbers of
animals used for statistical analysis: n=5 for 3 hours, n=4 for
24 hours, n=5 for 72 hours, n=6 for 5 days, and n=5 for 7 days of
reperfusion. *P<0.05, **P<0.01.
View larger version (157K):
[in a new window]
Figure 3. Mast cells accumulate in areas of collagen
deposition and cell proliferation. a, c, and e, Serial 5-µm sections
from totally fibrotic area of ischemic section of canine
myocardium after 1 hour of coronary occlusion and 7
days of reperfusion; b, d, and f, serial sections from border zone
containing fibrotic area (right) and area with relatively intact
myocardium (left). Sections were stained for tryptase to
identify mast cells (a, b), with picrosirius red to demonstrate
collagen (c, d), and immunostained for PCNA (e, f) to label
nuclei of proliferating cells. Note that tryptase-positive mast cells
are located in areas with marked collagen deposition and PCNA
expression, whereas the relatively intact area in b, d, and f does not
show evidence of mast cell accumulation (x400). These observations are
representative of 11 consecutive experiments of
myocardial infarction with reperfusion intervals ranging from 120 to
144 hours.
Serial sections from tissue samples obtained from
ischemia/reperfusion experiments were stained for tryptase to
identify mast cells (Figure 3a
and 3b), for collagen with picrosirius
red (Figure 3c and 3d), and immunohistochemically for PCNA (Figure 3e and 3f) to identify proliferating cells. Mast cells appeared to
accumulate in areas of cell proliferation and collagen deposition
(Figure 3). Dual staining by the histochemical staining technique for
tryptase followed by staining for collagen demonstrated that mast cells
accumulated in areas with evidence of fibrosis (Figure 4a through 4d). Figure 4e demonstrates
that mast cell numbers in fibrotic areas in which myocytes were fully
replaced by scar were markedly higher than the numbers from areas of
the same section that showed intact myocardium
(12.0±2.6-fold increase; P<0.01, n=5). Furthermore,
partially fibrotic areas had significantly higher mast cell numbers
than nonfibrotic areas (6.1±2.2-fold increase; P<0.05,
n=5). Further experiments combining FITC-avidin staining with
immunostaining for PCNA (Figure 5A and 5B) showed an
excellent correlation between the numbers of tryptase-positive cells
and PCNA-positive nuclei in the same field (r=0.75,
P<0.001) (Figure 5C).
View larger version (119K):
[in a new window]
Figure 4. Mast cells accumulate in areas of
collagen deposition. a through d, Sections from ischemic areas
of canine myocardium after 1 hour of ischemia and 7
days of reperfusion were stained for tryptase to identify and count
mast cells (b, d). Subsequently, tryptase stain was erased with Tween,
and sections were stained with picrosirius red to demonstrate collagen
deposition (a, c). Note presence of significant numbers of mast cells
in areas with collagen deposition (x400). Five consecutive experiments
were used for statistical analysis (e). Note that mast cell
numbers were markedly higher in areas demonstrating total or partial
fibrosis (fibrotic, 12.0±2.6-fold increase, **P<0.01
vs normal areas, n=5; partially fibrotic, 6.1±2.2 fold increase,
*P<0.05 vs normal areas, n=5).
View larger version (48K):
[in a new window]
Figure 5. Mast cells accumulate in areas of PCNA expression.
Sections from ischemic samples of canine myocardium
after 1 hour of ischemia and 7 days of reperfusion were stained
with FITC-avidin (B). Fifteen random fields were photographed and mast
cell numbers counted. Sections were then immunostained with
a monoclonal antibody to PCNA (Dako, clone PC10), and same fields were
photographed after eosin counterstaining (A) (x160). An excellent
correlation was found (r=0.75, P<0.001)
between mast cell numbers and PCNA-positive nuclei (C).
The number of PCNA-positive cells in the healing scar peaked
after 3 to 5 days of reperfusion. The majority of these cells
demonstrated cytoplasmic staining with an antibody to -smooth muscle
actin (Figure 6) but were smooth muscle
myosin–negative (Figure 7). Other
proliferating cells were identified as factor VIII–positive
endothelial cells (Figure 8).
View larger version (98K):
[in a new window]
Figure 6. Many proliferating cells are identified as
-smooth muscle actin–positive myofibroblasts. Sections from
experiment of 1 hour ischemia and 5 days reperfusion were
immunostained for -smooth muscle actin with a
rhodamine-labeled secondary antibody (A, C) followed by
immunohistochemistry for PCNA (B, D) with a peroxidase-based system
(DAB without nickel). Many -smooth muscle actin–expressing cells
(A, arrowheads) demonstrated evidence of proliferation as indicated by
PCNA expression (B, arrowheads). Some cells not expressing -smooth
muscle actin also showed PCNA-positive nuclei (arrows). In contrast,
-smooth muscle actin–positive cells in control areas were located
predominantly in media of vessels (C) and did not demonstrate PCNA
expression (D) (x1000).
View larger version (118K):
[in a new window]
Figure 7. -Smooth muscle actin–positive, smooth
muscle myosin–negative myofibroblasts in postischemic
myocardium. Sections of canine heart after 1 hour of
ischemia and 5 days of reperfusion immunostained
for -smooth muscle actin (A, B, C) and smooth muscle myosin (D) with
a rhodamine-labeled secondary antibody. A, Control area demonstrating
-smooth muscle actin immunoreactivity mainly in media of vessels. B,
Ischemic area showing numerous -smooth muscle
actin–positive cells in healing area. C and D, Serial sections from
experiment of 1 hour ischemia and 5 days reperfusion
immunostained for -smooth muscle actin (C) and smooth
muscle myosin (D). Note that majority of -smooth muscle
actin–positive cells in healing myocardium do not express
smooth muscle myosin (x100).
View larger version (110K):
[in a new window]
Figure 8. Endothelial cell proliferation in
ischemic and reperfused myocardium. Sections from
ischemic areas of canine myocardium after 1 hour of
ischemia and 5 days of reperfusion. Dual immunohistochemistry
combining peroxidase-based staining for PCNA (a) and
fluorescent immunostaining for factor VIII (b)
shows PCNA expression (arrowheads) in some endothelial
cells in ischemic myocardium (arrows)
(x1000).
Dual staining combining immunostaining for PCNA
and FITC-avidin labeling (Figures 3 and 5) did not demonstrate
significant numbers of PCNA-positive mast cells (<1%).
A specific canine clone for SCF was obtained by use of
RT-PCR techniques encoding nucleotides 1 to 795 of the
previously published sequence for canine SCF15
(data not shown).
Expression of SCF mRNA was studied in the ischemic
and reperfused myocardial segments with a nuclease protection assay
(Figure 9). Low levels of constitutive
SCF mRNA expression were noted in several experiments. Significant
upregulation of SCF mRNA occurred in the ischemic and
reperfused samples after reperfusion intervals of 72 to 120 hours. No
significant induction of SCF mRNA was noted with shorter reperfusion
intervals (3 to 48 hours of reperfusion).
View larger version (46K):
[in a new window]
Figure 9. SCF mRNA induction. A nuclease protection assay
was used to investigate SCF mRNA regulation in ischemic and
reperfused myocardium. Note significant induction of SCF
mRNA in ischemic samples of experiments undergoing
coronary occlusion after 72 to 96 hours of reperfusion.
Induction of SCF mRNA was not seen in experiments with shorter
reperfusion intervals. In some experiments, low constitutive expression
of SCF mRNA was noted. Blood flows were determined with radiolabeled
microspheres. Similar findings were observed in 5 consecutive
experiments of myocardial ischemia with reperfusion intervals
ranging from 72 to 144 hours.
Immunohistochemical studies using a monoclonal antibody to
canine SCF were performed to localize SCF protein in the healing
myocardium. Sections of canine liver were used as a
positive control and showed staining of the bile ducts in accordance
with previous reports16 (Figure 10a). In the ischemic and
reperfused heart, a significant increase of SCF immunoreactive cells
was identified within 96 hours of reperfusion (Figure 10b). Serial
sections were immunostained for SCF and the
macrophage-specific antibody AM-3K, demonstrating that
the source of SCF is a subset of macrophages (Figure 11). Dual immunohistochemistry did not
show SCF immunoreactivity in factor VIII–positive
endothelial cells or -smooth muscle actin–positive
myofibroblasts (data not shown).
View larger version (121K):
[in a new window]
Figure 10. SCF immunoreactivity in ischemic
myocardium. Immunohistochemistry for SCF in canine liver
(a) and postischemic heart (b). Significant number of SCF
immunoreactive cells with mononuclear cell characteristics was noted in
ischemic myocardium after 1 hour of
ischemia and 5 days of reperfusion (b). Liver was used as a
positive control and demonstrated staining of bile ducts as previously
demonstrated (a) (x400).
View larger version (133K):
[in a new window]
Figure 11. SCF expression by a subset of
macrophages. Serial sections from ischemic areas of
canine heart after 1 hour of coronary occlusion and 5 days of
reperfusion were immunostained for
macrophage-specific antibody AM-3K (a) and for SCF (b).
A subset of macrophages (arrows) was shown to be source of
immunoreactive SCF in healing myocardium (x400).
Staining for tryptase identified several intravascular
tryptase-positive cells in the healing heart after 72 to 144 hours of
reperfusion (Figure 12a and 12b).
Studies using the cells isolated from the postischemic
cardiac lymph demonstrated the presence of occasional metachromatic
tryptase-positive cells that appeared only after 48 to 72 hours of
reperfusion (12c and 12d). No tryptase-positive cells were present
in the preischemic cardiac lymph. Furthermore, dual
staining demonstrated the presence of a number of intravascular
FITC-avidin–positive cells that did not stain for the basophil marker
CD11b (Figure 13A through 13C). These
findings suggested the attraction of mast cell precursors to the
healing myocardium. The presence of these cells in the
cardiac lymph demonstrates that these mast cell precursors can migrate
through the venular endothelium and enter the
tissue.
View larger version (93K):
[in a new window]
Figure 12. Mast cell precursors in healing
myocardium and postischemic cardiac lymph.
Staining for tryptase sections from ischemic areas of canine
heart after 1 hour of ischemia and 7 days of reperfusion
demonstrated intravascular tryptase-positive cells (arrowheads) (a, b).
Staining of cells isolated from cardiac lymph for toluidine blue (c)
and tryptase (d) showed presence of metachromatic tryptase-positive
cells (arrowheads) within 48 to 72 hours of reperfusion. Magnification
x150 (a), x1000 (b, c, d).
View larger version (63K):
[in a new window]
Figure 13. CD11b-negative, FITC-avidin–positive
intravascular cells in healing canine heart. Sections from
ischemic areas after 1 hour of coronary occlusion and 7
days of reperfusion were immunostained for monoclonal
antibody to canine CD11b MY904 with a Cy3-labeled secondary antibody
(C). Section was subsequently stained with FITC-avidin (B),
photographed, and stained with eosin (A). Note intravascular
FITC-avidin–positive cell (arrowhead), which does not express
basophilic marker CD11b. CD11b-positive cells are identified with
arrows (x1000).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Myocardial ischemia and reperfusion are followed by an
acute inflammatory response associated with leukocyte infiltration.
Tissue repair follows, with removal of cellular and tissue debris by
phagocytes, repair and remodeling of extracellular matrix by connective
tissue cells, and new vessel formation. In this report, we provide
evidence that mast cell accumulation occurs in healing areas of an
experimental myocardial infarction. Our findings indicate that
macrophage-derived SCF is induced in the
postischemic heart and may be responsible for chemotaxis of
mast cell precursors and mast cell growth in the
myocardium. The presence of mast cells in the heart has
been described in several species: in recent reports, human heart mast
cells have been isolated and characterized.17
Although the role of cardiac mast cells remains unclear, the discovery
that mast cells are an important source of cytokines and growth
factors suggests new ways in which mast cell activation may be involved
in myocardial inflammation and repair.
Our data demonstrate significant accumulation of
FITC-avidin–positive, tryptase-positive mast cells in the
ischemic and reperfused segments of the canine
myocardium. Mast cells start accumulating after 72 hours of
reperfusion (Figure 2) and are concentrated predominantly in areas of
collagen synthesis (Figures 3 and 4) and cell proliferation (Figures 3 and 5). Several studies have suggested that mast cells and their
mediators may play an active role in wound healing and
fibrosis.18 19 Mast cell degranulation
products have been demonstrated to induce
fibroblast18 19 as well as
endothelial cell
proliferation20 21 and
angiogenesis.22 When activated mast cells
were cocultured with fibroblasts, they were found to increase collagen
synthesis and stimulate fibroblast proliferation, indicating a direct
involvement of mast cells in the fibrotic
process.18 Studies of specific mast cell
mediators have provided information on their effects in fibroblast
activation and proliferation. Histamine has been shown to stimulate
fibroblast growth and collagen synthesis in
vitro,23 24 an effect mediated by
H2 receptors. Tryptase, the most abundant of the
proteases found in mast cell granules, is a potent mitogen for
fibroblasts.25 Nanomolar concentrations of dog
tryptase strongly stimulate thymidine incorporation in Chinese hamster
lung and rat-1 fibroblasts and markedly potentiate DNA synthesis
stimulated by epidermal growth factor and bFGF.25
Recent studies demonstrated that tryptase directly stimulates
fibroblast chemotaxis and induces procollagen mRNA
synthesis.26 Furthermore, mast cells have
recently been recognized to be important sources of
bFGF2 and TGF-ß,3 factors
that can regulate fibroblast growth and modulate extracellular matrix
metabolism. Mast cell products may also have a role in
metalloproteinase activation and subsequent matrix degradation, an
important component of wound healing. Cleutjens and
colleagues27 demonstrated increased MMP-1, MMP-2,
and MMP-9 activity in the rat myocardium after myocardial
infarction. They suggested that posttranslational activation of latent
collagenase (MMP-1) played a greater role in the
wound-healing response than transcription of collagenase
mRNA. Recently, canine mast cells have been found to secrete a 92-kDa
gelatinase28 (MMP-9), which was extracellularly
activated by chymase. This provided the first demonstration of
a cell that activates an MMP it secretes by cosecreting an
activating enzyme. These findings suggest that mast cells may have an
important role in myocardial remodeling and fibrosis after myocardial
ischemia.
Dual immunohistochemical studies identified a significant
number of proliferating cells as -smooth muscle
actin–positive myofibroblasts (Figure 6). These phenotypically altered
fibroblasts develop several ultrastructural and biochemical features of
smooth muscle cells, including the expression of -smooth muscle
actin. Recent studies29 30 suggest the importance
of -smooth muscle actin–expressing myofibroblasts in wound healing
because of their ability to alter the contractile properties of the
affected tissues, to manifest heightened collagen gene expression, and
to elaborate cytokines. TGF-ß is a significant factor in
mediating the development of myofibroblastic
features.31 Willems and
colleagues32 have previously identified
nonvascular spindle-shaped -smooth muscle actin–positive cells in
healing myocardial scars. These cells were shown to be the main source
of type I and type III procollagen mRNA in the infarcted
myocardium.33 Our studies clearly
established the myofibroblast-like phenotype of these cells,
which do not express smooth muscle myosin (Figures 6 and 7). Other
proliferating cells were identified as factor VIII–positive
endothelial cells (Figure 8), suggesting active
angiogenesis in the healing myocardium. Mast cell–derived
TGF-ß and bFGF may have a role in mediating the fibroblast phenotypic
modulation and the angiogenic process.
Our findings failed to demonstrate significant mast cell
proliferation in the ischemic myocardium. Although
the contribution of cell proliferation cannot be ruled out, we believe
that chemotaxis of circulating mast cell precursors in the healing
myocardium may be the predominant mechanism responsible for
mast cell accumulation in the ischemic myocardium.
Mast cells originate from CD34+ stem
cells34 in the bone marrow and circulate as
immature precursor cells in the peripheral
blood.35 However, information on the phenotypic
characteristics of these precursor cells is scarce. Rodewald and
colleagues36 recently identified a cell
population in murine fetal blood that fulfills the criteria of
progenitor mastocytes. It is defined by the phenotype Thy-1
(lo) c-kit (hi), contains cytoplasmic granules, and expresses RNAs
encoding mast cell–associated proteases but lacks expression of the
high-affinity IgE receptor.36 Our studies
identified intravascular cells expressing the mast cell–specific
protease tryptase in sections from the ischemic and reperfused
myocardium (Figure 12a and 12b). Furthermore,
tryptase-positive metachromatic cells could be identified in the
postischemic cardiac lymph as early as 48 hours after
reperfusion (Figure 12c and 12d). The expression of tryptase, a
specific mast cell marker not found in other cell types, by these
intravascular cells suggests that they represent mast cell
precursors infiltrating the healing myocardium. Further
experiments demonstrated the presence of intravascular
FITC-avidin–positive cells that do not express the basophil marker
CD11b (Figure 13). These findings support our hypothesis, identifying
cells with phenotypic characteristics of mast cell progenitors in the
healing heart.
The factors responsible for mast cell recruitment in areas
of fibrosis have yet to be defined. In vitro studies have implicated
SCF as a potent mast cell chemoattractant that stimulates directional
motility of both mucosal- and connective tissue–type mast
cells.37 38 39 SCF along with the anaphylatoxins
C3a and C5a40 are the only chemotactic factors
shown to induce migration of human mast cells. Recently, several
angiogenic factors (platelet-derived growth factor-AB, vascular
endothelial growth factor, bFGF) have also been
demonstrated to promote murine mast cell chemotaxis in
vitro.41 Galli and
colleagues42 showed that subcutaneous
administration of recombinant human SCF to baboons produced a striking
expansion of the mast cell population, which was reversed when
administration of the cytokine was discontinued. These
experiments provided the first evidence that a specific
cytokine can regulate mast cell development in vivo. However,
the significance of this finding in the fibrotic process remains
unclear. We present evidence demonstrating significant SCF mRNA
upregulation in ischemic segments of the canine
myocardium. SCF mRNA induction was first noted after 1 hour
of ischemia and 72 hours of reperfusion (Figure 9); at the same
time point, an increase in mast cell numbers is noted in the
ischemic myocardium. Anaphylatoxins, such as C5a,
cannot represent an important migratory stimulus for mast
cells, because they are generated in the injured heart early after
myocardial necrosis43 and are not active after 3
to 6 hours. In addition to being a mast cell chemoattractant, SCF
critically regulates the maturation and survival of mast cells by
suppressing mast cell apoptosis,44
enhancing mast cell maturation,45 and inducing
mast cell adhesion to fibronectin.46 SCF is also
an important factor affecting mast cell activation and histamine
release.47 Furthermore, SCF is capable of
upregulating TNF- mRNA in canine mastocytoma
cells.48 All these actions may be important in
promoting mast cell growth and activity after myocardial
ischemia. Recent observations suggest that the ability of SCF
to support certain stages of mast cell differentiation is influenced by
interactions with specific cofactors, such as IL-3, IL-4, and
IL-10.49 Experiments from our laboratory have
demonstrated induction of IL-10 mRNA in the infarcted
myocardium, peaking at 72 to 96 hours of
reperfusion.50 IL-10 may be important in
costimulating and sustaining SCF-dependent mast cell accumulation.
SCF can be produced by stimulated fibroblasts and
endothelial cells.51 52 Recently,
bone marrow macrophages were demonstrated to produce
SCF.53 It seemed reasonable to hypothesize that
cytokine-stimulated fibroblasts or endothelial
cells have the potential to secrete SCF and may promote the recruitment
of mast cells in areas of fibrosis. However, immunohistochemical
studies using a monoclonal antibody to canine SCF produced a surprise:
SCF immunoreactivity was localized predominantly in a small subset of
macrophages infiltrating the healing myocardium
identified by immunohistochemistry with the
macrophage-specific antibody AM-3K (Figures 10 and 11).
SCF protein was not found in -smooth muscle actin–positive
myofibroblasts or factor VIII–positive endothelial
cells.
Previous studies from our laboratory suggest that the potent
mononuclear cell chemoattractant MCP-1 is induced in the
ischemic and reperfused
myocardium.54 MCP-1 appears to be an
important factor in promoting mononuclear cell influx after 3 hours of
reperfusion.43 It is interesting to speculate
that under the influence of a variety of factors, mononuclear cells
mature to macrophages, which may have a significant role in
scar formation through the production of growth factors. It
appears that a small subset of macrophages is responsible for
production of SCF, promoting mast cell accumulation in the
healing myocardium. Mast cell secretagogues may be
important in mediating the phenotypic modulation and proliferation of
fibroblasts and endothelial cells in the healing
myocardium.
Selected Abbreviations and Acronyms
bFGF
=
basic fibroblast growth factor
IL
=
interleukin
MMP
=
matrix metalloproteinase
PCNA
=
proliferating cell nuclear antigen
PCR
=
polymerase chain reaction
RT
=
reverse transcription
SCF
=
stem cell factor
TGF-ß
=
transforming growth factor-ß
TNF-
=
tumor necrosis factor-
Acknowledgments
This work was supported by NIH grant HL-42550. Dr Alan
Burns is a recipient of a grant from the Methodist Hospital Foundation.
Merry L. Lindsey is supported by Baylor College of Medicine Graduate
Program in Cardiovascular Sciences training grant
HL-07816. The authors wish to thank Sharon Malinowski and Concepcion
Mata for their editorial assistance with the manuscript and Evelyn
Brown, Peggy Jackson, Gary Liedtke, and Alida Evans for their
outstanding technical assistance.
Footnotes
Guest editor for this article was Benedict R. Lucchesi, MD, PhD, University of Michigan, Ann Arbor.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
,
initiating the cytokine cascade in experimental canine
myocardial ischemia/reperfusion. Circulation. 1998;98:699–710.
-smooth muscle actin-positive cells in healing human myocardial
scars. Am J Pathol. 1994;145:868–875.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  H. Higuchi, M. Hara, K. Yamamoto, T. Miyamoto, M. Kinoshita, T. Yamada, K. Uchiyama, and A. Matsumori Mast Cells Play a Critical Role in the Pathogenesis of Viral Myocarditis Circulation, July 22, 2008; 118(4): 363 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Fazel, L. Chen, D. Angoulvant, S.-H. Li, R. D. Weisel, A. Keating, and R.-K. Li Activation of c-kit is necessary for mobilization of reparative bone marrow progenitor cells in response to cardiac injury FASEB J, March 1, 2008; 22(3): 930 - 940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Thakker, N. G. Frangogiannis, M. Bujak, P. Zymek, J. W. Gaubatz, A. K. Reddy, G. Taffet, L. H. Michael, M. L. Entman, and C. M. Ballantyne Effects of diet-induced obesity on inflammation and remodeling after myocardial infarction Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2504 - H2514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 |