Journal of the American College of Cardiology
© 2006 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 48, No. 11, 2006
ISSN 0735-1097/06/$32.00
doi:10.1016/j.jacc.2006.07.060
The Role of Platelet-Derived Growth
Factor Signaling in Healing Myocardial Infarcts
Pawel Zymek, MD,* Marcin Bujak, MD,* Khaled Chatila, MD,* Anna Cieslak, MS,*
Geeta Thakker, PHD,† Mark L. Entman, MD, FACC,* Nikolaos G. Frangogiannis, MD, FACC*
Houston, Texas
This study sought to examine the role of platelet-derived growth factor (PDGF) signaling in
healing myocardial infarcts.
BACKGROUND Platelet-derived growth factor isoforms exert potent fibrogenic effects through interactions
with PDGF receptor (PDGFR)-␣ and PDGFR-␤. In addition, PDGFR-␤ signaling
mediates coating of developing vessels with mural cells, leading to the formation of a mature
vasculature. We hypothesized that PDGFR activation may regulate fibrosis and vascular
maturation in healing myocardial infarcts.
METHODS
Mice undergoing reperfused infarction protocols were injected daily with a neutralizing
anti–PDGFR-␤ antibody (APB5), an anti-PDGFR-␣ antibody (APA5), or control immunoglobulin G, and were killed after 7 days of reperfusion.
RESULTS
The PDGF-B, PDGFR-␣, and PDGFR-␤ mRNA expression was induced in reperfused
mouse infarcts. Perivascular cells expressing phosphorylated PDGFR-␤ were identified in the
infarct after 7 days of reperfusion, indicating activation of the PDGF-BB/PDGFR-␤
pathway. The PDGFR-␤ blockade resulted in impaired maturation of the infarct vasculature,
enhanced capillary density, and formation of dilated uncoated vessels. Defective vascular
maturation in antibody-treated mice was associated with increased and prolonged extravasation of red blood cells and monocyte/macrophages, suggesting increased permeability. These
defects resulted in decreased collagen content in the healing infarct. In contrast, PDGFR-␣
inhibition did not affect vascular maturation, but significantly decreased collagen deposition
in the infarct.
CONCLUSIONS Platelet-derived growth factor signaling critically regulates postinfarction repair. Both
PDGFR-␤– and PDGFR-␣–mediated pathways promote collagen deposition in the infarct.
Activation of PDGF-B/PDGFR-␤ is also involved in recruitment of mural cells by
neovessels, regulating maturation of the infarct vasculature. Acquisition of a mural coat and
maturation of the vasculature promotes resolution of inflammation and stabilization of the
scar. (J Am Coll Cardiol 2006;48:2315–23) © 2006 by the American College of Cardiology
Foundation
OBJECTIVES
Infarct healing is regulated by a well-orchestrated inflammatory response that ultimately leads to formation of a scar
(1,2). Myocardial necrosis results in marked induction of
inflammatory mediators and recruitment of leukocytes in
the infarcted myocardium. Activated neutrophils and
mononuclear cells secrete proteolytic enzymes and debride
the infarcted area from dead cells and extracellular matrix
fragments. The inflammatory phase is followed by the
proliferative phase of healing, when fibroblasts accumulate
in the infarct granulation tissue and deposit extracellular
matrix proteins (3). During the proliferative phase the
wound is highly cellular and metabolically active and requires a rich vascular supply to provide the proliferating cells
with oxygen and nutrients. Thus, formation of infarct
neovessels is a critical part of the healing process. As the
wound matures, resolution of the inflammatory infiltrate
occurs and the highly vascular granulation tissue is replaced
From the *Section of Cardiovascular Sciences and †Section of Atherosclerosis, the
DeBakey Heart Center, Baylor College of Medicine, and the Methodist Hospital,
Houston, Texas. Supported by National Institutes of Health grants R01 HL-76246
(to Dr. Frangogiannis) and P01 HL-42550 (to Drs. Entman and Frangogiannis), the
Methodist Hospital Research Institute, and a grant-in-aid from the American Heart
Association Texas affiliate (to Dr. Frangogiannis).
Manuscript received June 1, 2006; revised manuscript received July 13, 2006,
accepted July 17, 2006.
by collagen-rich scar. The infarct vasculature undergoes
dynamic changes: infarct angiogenesis is suppressed, and
many infarct neovessels acquire a muscular coat (4,5) while
uncoated vessels regress. As a result, the mature scar
contains few capillaries and a large number of coated
arterioles (5). Resolution of the inflammatory response and
maturation of the neovasculature may be important steps in
infarct healing, necessary to prevent uncontrolled angiogenesis and expansion of granulation tissue formation.
Platelet-derived growth factor (PDGF) signaling regulates events critical to fibrous tissue deposition and angiogenesis through interactions with its 2 PDGF receptors
(PDGFR) ␣ and ␤. Activation of both PDGFR-␣ and
PDGFR-␤ pathways stimulates fibroblast migration, proliferation, and activation (6 – 8). In addition, PDGF-BB/
PDGFR-␤ interactions play a crucial role for investment of
developing vessels with pericytes, a key process in vascular
maturation (9,10). Endothelial cells form vascular tubes and
direct the recruitment of mural cell precursors by secreting
PDGF-BB (11), whereas PDGFR-␤ is expressed by pericytes and smooth muscle cells. Although the critical significance of PDGFR-␤–mediated pericyte coating in embryonic vascular development has been established (12,13), the
importance of vascular maturation and acquisition of a
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Abbreviations and Acronyms
APA
⫽ anti–PDGFR-␣ antibody
APB
⫽ anti–PDGFR-␤ antibody
IgG
⫽ immunoglobulin G
PCR
⫽ polymerase chain reaction
PDGF ⫽ platelet-derived growth factor
PDGFR ⫽ platelet-derived growth factor receptor
SMA
⫽ smooth muscle actin
muscular coat in healing wounds has not been investigated.
In the retina, investment of the vessels with mural cells
marks the end of the plasticity window and leads to vascular
stabilization (14) and decreased angiogenic potential. We
hypothesized that activation of the PDGFR-␤ pathway in
the infarcted myocardium is crucial for maturation of infarct
neovessels and acquisition of a muscular coat. Thus, recruitment of mural cells by infarct neovessels may be important
in mediating suppression of the angiogenic process and
resolution of the inflammatory infiltrate during wound
maturation.
Our study examined the effects of disrupted PDGFR-␤
and PDGFR-␣ signaling, through systemic injection of
neutralizing antibodies (15), on infarct angiogenesis, wound
maturation, and scar formation in reperfused murine infarcts. The PDGFR-␤ inhibition resulted in impaired
pericyte coating, formation of a chaotic and tortuous infarct
vasculature, increased capillary density, prolonged extravasation of blood cells, and impaired collagen deposition in
the infarct. In contrast, PDGFR-␣ inhibition had no effect
on vascular maturation but significantly attenuated collagen
deposition in the infarcted myocardium. Acquisition of a
muscular coat through PDGF-BB/PDGFR-␤ interactions
is critical for stabilization of the infarct neovessels and
resolution of the postinfarction inflammatory response.
METHODS
Murine ischemia/reperfusion protocols. All animal protocols were approved by the Baylor College of Medicine
Institutional Review Board. Wild-type C57/BL/6 mice, 8
to 12 weeks of age (purchased from Charles River, Wilmington, Massachusetts), were used for myocardial infarction experiments. Mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (10 ␮g/g). A
closed-chest mouse model of reperfused myocardial infarction was used as previously described (16,17) to avoid the
confounding effects of surgical trauma and inflammation.
The left anterior descending coronary artery was occluded
for 1 h and then reperfused for 6 h to 7 days. At the end of
the experiment, the chest was opened and the heart was
immediately excised, fixed in zinc formalin, and embedded
in paraffin for histological studies, or snap frozen and stored
at ⫺80°C for RNA isolation. Sham animals were prepared
identically without undergoing coronary occlusion/
reperfusion. Animals used for histology underwent 72-h
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and 7-day reperfusion protocols (5 animals per group). Mice
used for RNA extraction underwent 6 h, 24 h, 48 h, 72 h,
and 7 days of reperfusion (3 animals per group).
Immunohistochemistry and quantitative histology. Sections were cut at 3 ␮m and stained immunohistochemically
with the following antibodies: anti-␣ smooth muscle actin
(␣-SMA) (Sigma, St. Louis, Missouri), rat anti-mouse
macrophage antibody clone F4/80 (Research Diagnostics
Inc., Flanders New Jersey), rat anti-neutrophil antibody
(Serotec, Raleigh, North Carolina), anti-phosphorylated
PDGFR-␤ antibody (Upstate Biotechnology, Charlottesville, Virginia), and rat anti-mouse CD31 antibody (Pharmingen, San Diego, California). Staining was performed
using a peroxidase-based technique with the Vectastain
ELITE rat or goat kit (Vector Labs, Burlingame, California). Negative control procedures were performed with
omission of the primary antibodies. The Mouse-on-Mouse
kit (Vector) was used for ␣-SMA immunohistochemistry.
For CD31 staining, sections were pretreated with trypsin
and staining was performed using the Tyramide Signal
Amplification kit (Perkin Elmer, Boston, Massachusetts).
Collagen staining was performed using picrosirius red.
Quantitative assessment of macrophage and neutrophil
density was performed by counting the number of F4/80
positive cells and immunoreactive neutrophils respectively
in the infarcted area (18). Macrophage and neutrophil
density was expressed in cells/mm2. Infarct microvascular
density was assessed by counting the number of CD31stained vascular profiles in infarcted murine hearts. To
assess the acquisition of a muscular coat by infarct vessels,
the density of coated vessels in the infarct was measured by
using a section stained for ␣-SMA. The density of uncoated
vessels was assessed by subtracting the density of coated
vessels from the total vascular density in the infarct. The
percentage of the infarcted area stained for collagen with
picrosirius red (collagen percent staining) was quantitatively
assessed in infarcts after 7 days of reperfusion using Image
Pro software (Image Pro Plus, version 4.5, Media Cybernetics Inc., Silver Spring, Maryland) as previously described
(19).
RNA extraction and quantitative real-time polymerase
chain reaction (PCR). Total RNA was extracted from
frozen hearts using a commercially available acid-phenol
reagent (TRIzol, Invitrogen, Carlsbad, California). Total
RNA was treated with DNase to remove any genomic
contamination as described by the manufacturer (DNAfree, Ambion, Austin, Texas). First-strand cDNA was
synthesized using SuperScript II reverse transcriptase and
random hexamer primers as described in the manufacturer’s
protocol (Invitrogen). Relative standard curve method was
used to measure the expression levels of PDGF-BB, -AA,
PDGFR-␣, and PDGFR-␤. TaqMan primers and probes
for PDGF-BB, PDGF-AA, PDGFR-␣, and PDGFR-␤
were obtained from Applied Biosystems (Foster City, California). Real-time PCR was performed and analyzed with
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Figure 1. The microvascular network in reperfused mouse infarcts is identified by CD31 immunohistochemistry. (A) CD31 staining of control heart labels
the myocardial vasculature. (B) Serial section with omission of the primary antibody serves as a negative control. (C) After 72 h of reperfusion, infarct
granulation tissue contains many CD31-positive endothelial cells (arrowheads). (D) After 7 days of reperfusion, the infarcted area contains relatively few
capillaries (arrows) and a significant number of coated vessels (arrowheads). Counterstained with eosin (magnification 200⫻).
1:10 diluted cDNA according to the manufacturer’s instructions on an ABI Prism 7000 Sequence Detection System.
Target gene expression was normalized to an internal
control, ribosomal protein S3 (RPS3). The RPS3 was
measured using SYBR Green chemistry and the relative
standard curve method. At the end of the PCR cycle,
Figure 2. The ␣-smooth muscle actin (SMA) immunohistochemistry in reperfused murine infarcts. (A) In control murine hearts, ␣-SMA staining
identifies the vascular media (arrowheads). (B) Serial section stained with omission of the primary antibody serves as a negative control. (C) After 72 h
of reperfusion, ␣-SMA immunohistochemistry is predominantly localized in spindle-shaped myofibroblasts (arrows). Relatively few coated vessels are noted
(arrowheads). (D) After 7 days of reperfusion, the infarct vasculature matures through the recruitment of mural cells by infarct neovessels (arrowheads).
Counterstained with eosin (magnification 200⫻).
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Figure 3. Assessment of mRNA expression of platelet-derived growth factor receptor (PDGFR)-␣ (A), platelet-derived growth factor (PDGF)-A chain
(B), PDGFR-␤ (C), and PDGF-B chain (D) in mouse infarcts using quantitative real-time polymerase chain reaction. (A) The PDGFR-␣ mRNA
expression in the heart peaked after 6 h of reperfusion (**p ⬍ 0.01 vs. sham). (B) The PDGF-A mRNA expression also increased after 6 h of reperfusion;
however, the difference with sham hearts did not reach statistical significance (p ⫽ 0.08). (C and D) The PDGFR-␤ (C) and PDGF-B (D) mRNA levels
show a similar time course, peaking after 7 days of reperfusion (*p ⬍ 0.05 vs. sham, **p ⬍ 0.01 vs. sham).
dissociation curve analysis was performed to ascertain the
amplification of a single PCR product. Sequence of the
murine primers is as follows: RPS3 forward (5=ATCAGAGAGTTGACCGCAGTTG-3=), RPS3 reverse (5=-AATGAACCGAAGCACACCATAG-3=).
PDGFR-␣ and PDGFR-␤ inhibition in mouse infarcts. To examine the effects of PDGFR-␤ inhibition on
myocardial infarct healing, mice undergoing coronary occlusion/reperfusion received daily intraperitoneal injections
(200 ␮g/day, the first dose was administered after 24 h of
reperfusion) with the neutralizing rat anti-mouse PDGFR-␤
antibody (APB) 5, the rat anti-mouse PDGFR-␣ antibody
(APA) 5 (both from eBioscience, San Diego, California), or rat
immunoglobulin G (IgG) (Sigma) (APA5, n ⫽ 7; APB5, n ⫽
7; rat IgG, n ⫽ 14). Selection of the dose was based on
previously published studies using these antibodies in vivo to
inhibit PDGFR signaling in mouse models (15,20,21). After 7
days of reperfusion the animals were killed, and the hearts were
fixed in zinc formalin (Z-fix; Anatech, Battle Creek, Michigan) and embedded in paraffin for histological studies.
Statistical analysis. Statistical analysis was performed using
1-way ANOVA followed by t test corrected for multiple
comparisons (Student-Newman-Keuls). Data were expressed
as mean values ⫾ SEM. Statistical significance was set at 0.05.
Figure 4. Activation of the platelet-derived growth factor receptor (PDGFR)-␤ signaling pathway in healing murine myocardial infarcts was detected using
immunohistochemical staining with an antibody to phosphorylated PDGFR-␤ (pPDGFR-␤). (A) Noninfarcted areas showed minimal staining for
pPDGFR-␤. (B) After 7 days of reperfusion, perivascular and mononuclear-like cells (arrowheads) with intense immunoreactivity to pPDGFR-␤ were
found in the infarcted myocardium. (C) Negative control with omission of the primary antibody shows no staining. Counterstained with eosin
(magnification 200⫻).
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Figure 5. (A) Platelet-derived growth factor receptor (PDGFR)-␣ and PDGFR-␤ inhibition decreased collagen deposition in the infarcted myocardium.
(A) After 7 days of reperfusion, Sirius red staining identified a well-organized collagen network in the infarcted heart from animals treated with control
rat immunoglobulin (Ig)G. (B) Mice treated with anti–PDGFR-␣ antibody (APA5) had attenuated collagen deposition in the infarcted myocardium. (C)
Mice injected with anti–PDGFR-␤ antibody (APB5) showed decreased and disorganized collagen deposition. Note the presence of dilated vessels (arrows).
(D) Quantitative assessment of collagen deposition in the infarcted heart (*p ⬍ 0.05 vs. IgG control group). Magnification 400⫻.
RESULTS
Vascular maturation in healing mouse infarcts. CD31
immunohistochemistry identified the vascular network of
the mouse heart, specifically labeling endothelial cells (Figs.
1A and 1B), whereas ␣-SMA immunoreactivity was localized in smooth muscle cells (Figs. 2A and 2B). After 72 h
of reperfusion, dead cardiomyocytes in the infarcted area
were replaced with granulation tissue that contained an
extensive capillary network (Fig. 1C). At the same time
point, ␣-SMA immunohistochemistry labeled a large number of myofibroblasts and occasional vessels coated with
smooth muscle cells (Fig. 2C). As the wound matured,
more infarct vessels acquired a muscular coat, and after 7
days of reperfusion (Fig. 1D) the scar contained a significant
number of mature, coated neovessels (Fig. 2D). Acquisition
of a muscular coat confers stability and indicates maturation
of the vasculature.
Expression of PDGF-A, PDGFR-␣, PDGF-B, and
PDGFR-␤ in healing infarcts. The PDGFR-␣ mRNA
expression was markedly induced in the infarcted myocardium after 6 h of reperfusion (p ⬍ 0.01, corrected for
multiple comparisons) and decreased after 24 h of reperfusion (Fig. 3A). Although PDGF-A mRNA expression also
peaked after 6 h of reperfusion, the difference between
PDGF-A mRNA expression in infarcted and sham animals
was not statistically significant (Fig. 3B). In contrast,
PDGFR- ␤ and PDGF-B mRNA showed late up-
regulation, peaking after 7 days of reperfusion (Figs. 3C and
3D).
Phosphorylated PDGFR-␤ localization in the infarcted
heart. Many perivascular cells in the healing infarct showed
expression of phosphorylated PDGFR-␤, suggesting activation of the PDGF-B/PDGFR-␤ signaling pathway (Fig
4B). In contrast, minimal phosphorylated PDGFR-␤ immunoreactivity was noted in the noninfarcted heart (Fig.
4A).
PDGFR-␤ and PDGFR-␣ neutralization inhibit collagen deposition in the healing infarct. After 7 days of
reperfusion, mouse infarcts showed replacement of the dead
cardiomyocytes with collagen-based scar. Healing infarcts
in antibody-treated animals showed decreased matrix deposition in the infarcted myocardium (Fig. 5). The
PDGFR-␣ neutralization resulted in significantly diminished collagen content in the infarcted area (anti–
PDGFR-␣: 12.45 ⫾ 0.48, n ⫽ 7; vs. rat IgG: 20.34 ⫾ 1.94,
n ⫽ 14; p ⬍ 0.05, corrected for multiple comparisons). The
PDGFR-␤ inhibition also attenuated collagen deposition in
the infarcted area (anti–PDGFR-␤: 13.46 ⫾ 1.88, n ⫽ 7;
vs. rat IgG: 20.34 ⫾ 1.94, n ⫽ 14, p ⬍ 0.05, corrected for
multiple comparisons).
PDGFR-␤ but not PDGFR-␣ neutralization results in
impaired maturation of the infarct vasculature. The
PDGFR-␤ inhibition impaired vascular maturation and
increased microvascular density in healing infarcts (anti–
PDGFR-␤: 471.6 ⫾ 73.8 vessels/mm2 vs. IgG: 267.7 ⫾
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Figure 6. Platelet-derived growth factor receptor (PDGFR)-␤ inhibition resulted in impaired vascular maturation and enhanced capillary density in
the healing infarct. (A) CD31 immunohistochemistry identified the infarct vasculature in immunoglobulin IgG-treated mice (magnification 200⫻).
(B) Numerous coated vessels were noted in the infarct after 7 days of reperfusion (arrows). (C to F) The PDGFR-␤ inhibition with the neutralizing
antibody APB5 resulted in impaired vascular maturation and in formation of dilated, disorganized, irregularly-shaped vascular structures (arrows).
(C, magnification 200⫻; D to F, magnification 400⫻). (G to I) Quantitative analysis of total microvascular density (G), density of uncoated vessels
(H), and density of coated vessels (I) in the infarct. The PDGFR-␤ inhibition significantly increased microvascular density (**p ⬍ 0.01 vs.
IgG-treated animals) (G) and the number of uncoated vessels (**p ⬍ 0.01 vs. IgG-injected mice) (H), but decreased the density of coated vessels
(I) in the infarcted area (*p ⬍ 0.05 vs. IgG-treated mice). In contrast, PDGFR-␣ inhibition did not affect vascular maturation. Other abbreviations
as in Figure 5.
13.98 vessels/mm2, p ⬍ 0.01, corrected for multiple
comparisons). Anti–PDGFR-␤ treatment resulted in
formation of a disorganized, “chaotic” vasculature comprised of irregular uncoated vessels (Figs. 6C to 6F). The
PDGFR-␤ antibody-treated mice had a significantly
lower density of coated vessels (anti–PDGFR-␤: 70.02 ⫾
6.75 vessels/mm2 vs. IgG: 117.5 ⫾ 15.7 vessels/mm2,
p ⬍ 0.05, corrected for multiple comparisons) and a
higher number of uncoated vessels compared with IgGinjected animals (anti–PDGFR-␤: 401.61 ⫾ 68.94 vessels/mm2 vs. IgG: 150.16 ⫾ 21.83 vessels/mm2, p ⬍
0.01, corrected for multiple comparisons). In contrast,
PDGFR-␣ inhibition did not affect microvessel formation in the healing infarct (anti–PDGFR-␣: 216.77 ⫾
15.2 vessels/mm2, p ⫽ NS, corrected for multiple comparisons) (Fig. 6).
Effects of PDGFR-␣ and PDGFR-␤ inhibition on
leukocyte infiltration in the healing infarct. We have
previously shown that reperfused mouse infarcts show an
intense but transient inflammatory reaction leading to
formation of a mature scar after 7 to 14 days of reperfusion
(17). After 7 days of reperfusion, murine myocardial scars
contained large amounts of matrix (Fig. 4A) and relatively
low numbers of leukocytes. The PDGFR-␤ inhibition
resulted in prolonged extravasation of blood cells in the
healing infarct. After 7 days of reperfusion, anti-PDGFR␤–treated mice, but not IgG-injected control mice, showed
hemorrhagic areas containing extravasated red blood cells
(Figs. 7A and 7B). In addition, PDGFR-␤ neutralization
resulted in increased macrophage density in the infarcted
area compared with IgG-treated animals (Fig. 7D). The
PDGFR-␣ inhibition also increased macrophage density
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Figure 7. Hemorrhagic foci in PDGFR-␤ antibody-treated infarcts. (A) After 7 days of reperfusion, immunoglobulin IgG-treated mice show a well-formed
wound with significant deposition of extracellular matrix and no red blood cell extravasation. (B) The PDGFR-␤ inhibition with the neutralizing antibody
APB5 resulted in extensive extravasation of red blood cells in the infarct (arrows). (C) In contrast, PDGFR-␣ inhibition with the neutralizing antibody
APA5 did not induce hemorrhagic areas in the infarct. (D) Quantitative analysis of macrophage density in the infarcted myocardium. The PDGFR-␣ and
PDGFR-␤ inhibition resulted in enhanced macrophage density in the infarcted myocardium (**p ⬍ 0.01 vs. IgG-treated animals). (E) In contrast,
neutrophil density was low in the mature scar after 7 days of reperfusion, and not significantly different between groups (magnification 400⫻). Abbreviations
as in Figure 5.
but did not induce hemorrhagic infiltrates in the scar. In
contrast, neutrophil density after 7 days of reperfusion was
low in both antibody-treated and control animals (Fig. 7E).
DISCUSSION
PDGF signaling modulates fibrosis and angiogenesis.
Platelet-derived growth factors are a family of disulfidelinked dimeric proteins encoded by four genes, PDGF-A,
-B, -C, and -D, that signal by binding to homodimers or
heterodimers of the 2 PDGF receptor proteins,
PDGFR-␣ and PDGFR-␤. The PDGF isoforms mediate fibrogenic actions and play an important role in
regulation of angiogenesis. PDGFR-␣ and PDGFR-␤
activation stimulates fibroblast migration and gene expression (6); ligands of these receptors are known inducers of cardiac fibrosis. Cardiac overexpression of the
PDGFR-␣ ligand PDGF-C induced fibrosis, collagen
deposition, vascular defects, and myocardial hypertrophy
(22). In addition, transgenic overexpression of the active
core domain of PDGF-D, a PDGFR-␤ ligand, resulted
in cardiac fibrosis followed by dilated cardiomyopathy
and subsequent cardiac failure. On the other hand,
extensive evidence supports the crucial role of PDGFR␤–mediated interactions in recruitment of mural cells by
newly formed vessels (23–25). Genetic disruption of
PDGF-B or PDGFR-␤ in mice results in virtually
identical phenotypes, leading to the development of
microvascular leakage, lethal hemorrhage, and edema in
late embryogenesis (26,27); these defects are caused by
a severe deficit in pericyte investment (12). Thus,
PDGFR-␤ signaling plays an important role in stabilizing the vasculature through acquisition of a mural coat.
PDGFR-␤ signaling critically regulates maturation of
the infarct vasculature. Our study explored for the first
time the functional role of PDGF signaling in healing
myocardial infarcts. We showed that both PDGF receptors
exert important and distinct actions in regulation of the
cellular events associated with formation of the scar. Although both receptors mediate fibrogenic effects (Fig. 5),
PDGFR-␤ signaling also plays a unique and crucial role in
vascular maturation by regulating coating of infarct microvessels with mural cells (Fig. 6). The PDGFR-␤ inhibition through injection of the neutralizing antibody APB5
(15) critically impaired vascular maturation in healing infarcts. The APB5 injection resulted in defective investment
of infarct microvessels with mural cells, increased capillary
density, and formation of tortuous and dilated vascular
structures (Fig. 6). The role of PDGFR-␤ signaling in
healing infarcts likely involves a paracrine interaction between endothelial and mural cells: endothelial cells secrete
PDGF-B, whereas pericytes and smooth muscle cells express PDGFR-␤ (28). The importance of the endothelial
cell as a source of PDGF-B is illustrated by the severe
pericyte deficiency noted in mice with endothelium-specific
ablation of PDGF-B (29,30). In contrast, selective
PDGF-B disruption in hematopoietic cells has no obvious
effects on the vasculature (31).
The dilated and tortuous vasculature noted in APB5treated infarcts suggests an important role for PDGFR-␤
signaling in regulating vessel size and shape. Hellstrom et al.
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(13) showed that PDGFB- and PDGFR-␤– deficient embryos show increased and variable capillary diameter with
high- and low-diameter segments. In addition, pericyte
coating inhibits endothelial proliferation and suppresses
angiogenesis (32). Our findings suggest that PDGFR-␤–
mediated mural cell recruitment may be a crucial step in
maturation of the infarct vasculature and in suppression of
the angiogenic process in the mature scar.
PDGFR-␣ signaling does not regulate vascular maturation but promotes collagen deposition. In contrast,
PDGFR-␣ inhibition did not affect vascular maturation but
significantly decreased collagen deposition in the infarcted
myocardium (Fig. 5). This finding is consistent with the
established role of PDGFR-␣ ligands (such as PDGF-AA
and PDGF-C) in the development of myocardial fibrosis.
PDGF-AA potently stimulates cardiac fibroblast proliferation inducing cell division to the same extent and with the
same kinetics as PDGF-BB (33); PDGF-AA may also
mediate, at least in part, the fibrogenic actions of angiotensin II in the heart (34). In addition, PDGF-C induces
fibroblast proliferation (35) and promotes cardiac fibrosis
when overexpressed in transgenic mice (22).
PDGFR-␤ signaling stabilizes the infarct vasculature
promoting maturation of the scar and resolution of
inflammation. Resolution of inflammation is critical for
effective cardiac repair after myocardial infarction. Chemokine and cytokine expression is markedly but transiently
induced in the infarcted myocardium (17); suppression of
inflammatory gene synthesis is followed by resolution of the
inflammatory infiltrate. We have recently identified
thrombospondin-1 mediated transforming growth factor-␤
activation as a crucial mechanism for suppression of the
inflammatory response (36) in healing myocardial infarcts.
Thrombospondin-1 ⫺/⫺ infarcts showed increased and
prolonged inflammation and expansion of the inflammatory
process into the noninfarcted myocardium; this resulted in
adverse postinfarction remodeling. Thus, timely suppression
of the inflammatory response is critical for optimal repair.
Our current study identifies another important mechanism
with a key role for resolution of inflammation. Disruption of
PDGFR-␤ signaling results in prolonged red blood cell and
monocyte/macrophage extravasation in the infarcted myocardium (Fig. 7), suggesting that PDGFR-␤–mediated
vascular maturation is critical for resolution of the inflammatory process. The pro-inflammatory effects of PDGFR-␤
inhibition lead to formation of a more cellular wound, with
lower collagen content. In addition, inhibition of the direct
stimulatory effects of PDGFR-␤ signaling on fibroblast
migration, proliferation, and activation (37,38) may further
suppress fibrous tissue deposition in the healing infarct.
Impaired maturation of the infarct and decreased collagen
content may decrease tensile strength, adversely affecting
postinfarction remodeling.
Study limitations. Antibody neutralization is a valuable
tool for temporary inhibition of the biological effects of
PDGF. However, this strategy has significant limitations.
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The exact dose of neutralizing antibody needed to inhibit a
particular biological response cannot be predicted. In addition, the delivery of the antibody to the reperfused myocardium may vary among experiments. We did not quantitatively evaluate the effectiveness of the anti-PDGF
antibodies in inhibition of PDGF signaling in the infarcts.
Although these antibodies have been extensively used for in
vivo neutralization of PDGF-mediated effects (15,20,21) in
murine models, the dose we used may have resulted in
partial inhibition of PDGF signaling. In addition, our study
did not quantitatively assess PDGF-A and -B chain protein
levels in the infarcted heart and did not identify the cell
types expressing PDGFR-␤ in the infarct.
Conclusions. The PDGF-mediated pathways play crucial
but distinct roles in regulating postinfarction repair. Both
PDGFR-␤ and PDGFR-␣ signaling pathways mediate
fibrogenic responses, probably through direct effects on
fibroblasts. However, activation of the PDGF-B/
PDGFR-␤ pathway is also involved in recruitment of mural
cells by infarct neovessels, critically regulating acquisition of
a mural coat by the vasculature. Coating and maturation of
the vasculature is an important step in suppression of the
angiogenic process and promotes resolution of inflammation by preventing extravasation of blood cells into the
mature scar. Disruption of PDGFR-␤ signaling results in
formation of a defective infarct vasculature, impaired maturation of the scar, prolonged inflammation, and decreased
collagen content in the wound. Defective scar formation
may adversely affect function of the infarcted heart by
enhancing left ventricular remodeling.
Reprint requests and correspondence: Dr. Nikolaos G. Frangogiannis, Section of Cardiovascular Sciences, One Baylor Plaza,
M/S F-602, Baylor College of Medicine, Houston, Texas 77030.
E-mail: [email protected].
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