© 1999 American Heart Association, Inc.
Vascular Biology |
Expression of the Angiogenic Protein, Platelet-Derived Endothelial Cell Growth Factor, in Coronary Atherosclerotic Plaques
In Vivo Correlation of Lesional Microvessel Density and Constrictive Vascular Remodeling
From the Department of Internal Medicine II (M.C.I., E.G.-S., A.H., S.R.-M., I.K., H.D.G., I.M.L.), Division of Cardiology, University of Vienna, Vienna, Austria, and the Scripps Research Institute (R.R.S.), La Jolla, Calif.
Correspondence to Irene M. Lang, MD, Department of Internal Medicine II, Division of Cardiology, University of Vienna, Austria, Währinger Gürtel 18-20, 1090 Vienna, Austria. E-mail irene.lang{at}univie.ac.at
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Abstract |
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Abstract—Recent information indicates that platelet-derived endothelial cell growth factor (PD-ECGF), a 45-kDa angiogenic protein, is expressed in the endothelium of various tissues and that its level of expression is correlated with the number of microvessels in human tumors. Because the formation of neovessels is also thought to play a role in atherosclerotic vascular remodeling, we analyzed PD-ECGF expression in fresh, coronary plaque tissues obtained by directional coronary atherectomy. Specimens from 31 patients were collected and analyzed by reverse transcription–polymerase chain reaction, histochemical staining, immunohistochemistry, and in situ hybridization with the use of PD-ECGF–specific primers and probes. Lesional vascular remodeling was assessed by intravascular ultrasound. PD-ECGF immunoreactivity and mRNA were found in plaque macrophages, endothelial cells of plaque neovessels, and stellate smooth muscle cells of 20 atherectomy specimens (64.5%). PD-ECGF immunoreactivity was correlated with the number of lesional microvessels and mast cells. Double-staining experiments revealed a close spatial proximity of PD-ECGF–positive cells and mast cells. Furthermore, the numbers of microvessels and mast cells were significantly higher in lesions lacking compensatory enlargement. The data indicate that PD-ECGF is expressed within cells of the atherosclerotic plaque and may be involved in driving angiogenesis in concert with mast cells.
Key Words: atherosclerosis • angiogenesis • endothelium • platelet-derived factors • growth substances
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Introduction |
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Atherosclerosis is the principal cause of heart attack, stroke, and peripheral vascular disease and is responsible for 50% of all mortality in the United States, Europe, and Japan.1 The vascular lesions result from an excessive, inflammatory-fibroproliferative response of the endothelium and smooth muscle cells (SMCs) of the arterial wall to various forms of insult.1 The inner parts of the walls of large blood vessels normally do not contain intrinsic vasculature. In conditions such as atherosclerosis or thrombosis, intimal angiogenesis occurs as part of an adaptive change recently addressed as vascular remodeling.2 Histopathologic studies3 4 and intracoronary ultrasound imaging have shown that human coronary arteries enlarge parallel to the formation of atherosclerotic plaque and preserve the lumen area until plaque volume exceeds the compensatory mechanism.5 One current hypothesis of neovascularization in atherosclerosis is that the new, small vessels arise from adventitial vasa vasorum.6 Several factors that stimulate the proliferation of endothelial cells (ECs) in vitro have been shown to induce angiogenesis in vivo7 and include wide-spectrum multifunctional mitogens (eg, the fibroblast growth factors) as well as factors with distinct specificities for vascular ECs (eg, platelet-derived endothelial cell growth factor [PD-ECGF]). The expression of fibroblast growth factors, PDGFs AA and BB, and their receptors has been investigated in atherectomy tissue and has been correlated with lesion activity.8 9 10 However, the role of lesional angiogenesis has remained unclear.
PD-ECGF is a 45-kDa nonglycosylated, single-chain polypeptide that stimulates growth and chemotaxis of ECs in vitro and of angiogenesis in vivo.11 PD-ECGF has been shown to catalyze the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine, thus reducing thymidine levels that would otherwise be inhibitory to EC growth. Although PD-ECGF has not been detected in the normal aorta12 or myocardium,13 PD-ECGF has been recognized within various human tumors and tumor cell lines,14 as well as in mononuclear cells,15 macrophages, keratinocytes, glial cells, and epithelial cells and within the endothelium of the breast, brain, and placenta. Despite the reported lack of PD-ECGF in the cardiovascular system, experiments were designed to examine PD-ECGF expression in coronary atherosclerosis and its relationship to vascular remodeling at the lesion site.
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Methods |
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Patient Characteristics
Coronary atherectomy specimens were obtained from 31 consecutive patients (the Table
; mean±SD age
58.2±8.8 years; range 41 to 74 years) undergoing directional
coronary atherectomy (DCA) at the University of Vienna General
Hospital between 1995 and 1997. All specimens were derived from
primary, single lesions in the left anterior descending
coronary artery (n=10, 32.2%), circumflex coronary
artery (n=5, 16.1%), or right coronary artery (n=16, 51.6%).
Ischemia in the target vessel–dependent myocardial area was
documented by exercise stress test and 201Tl
myocardial scintigrams. At the time of the intervention, all patients
were in clinically stable Canadian Cardiovascular
Society class 0 through 4 (the Table). Conventional
coronary angiography and quantitative coronary
angiography were performed before and after DCA (mean preprocedural
target stenosis by quantitative coronary angiography
was 67.6±11.3%). The intravascular ultrasound imaging system
consisted of an imaging catheter (Ultracross 2.9F), carrying a 30-MHz
transducer, and an intravascular ultrasound imaging console (Clear View
Ultra, Boston Scientific Corp). Before DCA, the imaging catheter was
introduced over a 0.014-in. coronary guiding catheter, and
coaxiality of the imaging catheter in the region of interest was
controlled by x-ray fluoroscopy. Imaging was performed by automated
pullback at 0.5 mm/s against the direction of blood flow, which is
indicated by the arrows in Figure 1. To obtain a numerical measure for
vascular remodeling, vessel cross-sectional area in the
stenotic segment was subtracted from the average vessel
cross-sectional area at the proximal and distal reference segments and
divided by the average vessel cross-sectional area at the proximal and
distal reference segments. Compensatory enlargement (delineated
"Pos" in the Table) was considered to be present when
the vessel cross-sectional area at the lesion site was larger than that
at the reference site (Figure 1A), ie, when the ratio was >0.
Inadequate compensatory enlargement (delineated "Neg" in the
Table) was considered to be present when the vessel
cross-sectional area at the lesion site was smaller than that at the
reference site (Figure 1B), ie, when the ratio was 
0. The
study was approved by the investigational review board of the
University of Vienna, Austria.
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Tissue Processing
DCA specimens were flushed from the instrument chamber with
saline solution and divided into 2 equivalent parts. The first half was
immediately immersed in LN2, and the second half
was fixed in 7.5% buffered formalin for 24 hours. Normal
transplant-donor coronary arteries and atherosclerotic arteries
from heart transplant recipients served as controls. Formalin-fixed
tissues were processed with the Miles Scientific Tissue-Tek VIP
(Schoeller Pharma) and embedded in paraffin. Serial 3-µm
sections were stained with hematoxylin and eosin to assess general
histology. A modified trichrome stain was performed for collagen and
fibrin localization.16 Thrombus was identified by the
presence of inflammatory cells, erythrocytes, and aggregated
platelets caught within a fibrin meshwork and strands of
proliferating fibroblasts, whereas the smooth, extracellular
distribution and red-purple color in the modified trichrome stain
identified fibrin. Vessel medial and adventitial tissues were retrieved
from 12 and 10 patient samples, respectively (38.7% and 32.2%), which
is in good agreement with published reports.17
Immunohistochemistry
The indirect avidin-biotin–horseradish peroxidase method was
used. Bound antibodies were detected with a biotinylated goat
anti-mouse or anti-rabbit secondary antibody (Zymed) and
aminoethylcarbazole as the substrate reagent. Monospecific IgG1
antibodies and a preimmune rabbit antiserum were used as controls for
monoclonal and polyclonal antibodies, respectively. Breast cancer
tissue was used for antibody optimization. A protein
A–Sepharose–purified rabbit polyclonal antiserum against PD-ECGF
(used at a 1:200 dilution in 0.1% normal goat serum) kindly provided
by Dr Carl Henrik Heldin, Ludwig Institute for Cancer Research
Biomedical Center, Uppsala, Sweden,18 was used.
Because nonspecific background staining was obtained with this
antibody, the data were confirmed with a mouse monoclonal anti–PD-ECGF
antibody (10 µg/mL, thymidine
phosphorylase/PD-ECGF/gliostatin Ab-1 clone P-GF.44C;
Neomarkers, Union City, Calif). Furthermore, mouse monoclonal antibody
against CD68 (16 µg/mL; clone KP1, DAKO, Glostrup, Denmark), mouse
monoclonal antibody against SM 
-actin (clone 1A4, DAKO, used at 10
µg/mL), mouse monoclonal antibody against von Willebrand
factor (clone F8/86, DAKO, used at a 1:150 dilution), and mouse
monoclonal antibody against mast cell tryptase (MAB122, Chemicon
International Inc, Temecula, Calif, used at 10 µg/mL) were
used.
Immunohistochemical double staining served to colocalize PD-ECGF–expressing cells and mast cells. For this purpose, anti–PD-ECGF was used in the first step with alkaline phosphatase and nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt as the color substrate (Zymed). Anti-tryptase was used in the second step with aminoethylcarbazole. Gill's hematoxylin was used as the counterstain.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Tissues frozen in LN2 were thawed in
guanidine thiocyanate followed by total RNA extraction according to the
method of Chomczynski and Sacchi.19 RNA (1 µg) was
reverse-transcribed (Boehringer Mannheim Co) with
oligo-(dT)15 primers. The cDNA was amplified by
using PD-ECGF–specific primers, forward PD-ECGF primer
5'-gcctgagcgaagcggacatc-3' and reverse PD-ECGF primer
5'-catctgctctgggctctgga-3', resulting in a 377-bp product (fragment
between nucleotides 143 to 519 of PD-ECGF
cDNA11 ) that was analyzed on ethidium
bromide–stained agarose gels.
Riboprobe Preparation
A full-length PD-ECGF cDNA (kindly provided by Dr C.H. Heldin,
Ludwig Institute for Cancer Research Biomedical Center,
Uppsala, Sweden) was cut with EcoRI-PstI
and cloned into pGEM-3Z (Promega, Madison, Wis), yielding a 1363-bp
template. Sense and antisense riboprobes were prepared with
digoxigenin-labeled UTP by in vitro transcription with SP6 and T7 RNA
polymerase (Promega), respectively.
In Situ Hybridization
Nonradioactive in situ hybridization was performed on 3- to
5-µm paraffin sections. In brief, sections were mounted onto
Superfrost/plus slides (Fisher) and dried at 60°C overnight to
improve adhesion. After deparaffinization in xylene and rehydration in
graded alcohols, the slides were fixed in 4%
paraformaldehyde. The slides were washed in 0.5x SSC
(one 10-minute wash; 1x SSC is 150 mmol/L NaCl and 15 mmol/L
sodium citrate, pH 7.0) and treated with protease K solution (20
µg/mL for 20 minutes at room temperature). The slides were
incubated with prehybridization solution [50% (wt/vol) formamide, 0.3
mol/L NaCl, and 20 mmol/L Tris-HCl, pH 8.0; 5 mmol/L EDTA,
0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% BSA, 10%
(wt/vol) dextran sulfate, and 10 mmol/L DTT] for 1 hour at 42°C
in a humidified chamber. Hybridizations were started by adding 0.5
µg/mL digoxigenin-labeled riboprobe in 20 µL of prehybridization
buffer containing 2.5 mg/mL tRNA (48°C, overnight). The next day, the
slides were incubated with RNase A for 30 minutes and washed for 2
hours in 0.1x SSC and 50% formamide at 48°C, followed by incubation
with 10% goat serum and an alkaline phosphatase–labeled
anti-digoxigenin Fab fragment (Boehringer Mannheim, 1:1000 in
0.1% goat serum and Tris-buffered saline at room temperature). Signal
was developed using nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt as the
substrate and Gill's hematoxylin as the counterstain. Parallel
sections were analyzed by using a sense probe as the control
for nonspecific hybridization.
Computer-Assisted, Quantitative Histological
Evaluation of Atherectomy Specimens
All atherectomy specimens were digitized at full size by using a
slide scanner (Nikon 6.0 35-mm-film scanner, LS-20, Nikon Corp). Images
were processed and the color contrast enhanced with the Adobe PhotoShop
3.0 software package (Adobe Systems Inc). Measurement of thrombus area
expressed as percent of total area was performed on trichrome-stained
specimens by computer-based planimetry (National Institutes of Health
Image 1.61/ppc; the Table
). In the trichrome staining technique,
erythrocytes stain yellow, fibrin stains red, collagen stains green,
and elastic fibers stain dark blue. Three independent observers who
were blinded to the patients' identities manually counted the number
of lesional mast cells, small vessels, and capillaries. The numbers
were divided by the respective specimen area in millimeters
squared.
Statistical Evaluation of Data
ANOVA and correlation analysis were used. A value of
P<0.05 was considered statistically significant.
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Results |
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Histological Characterization of Lesions
The primary, single lesions encountered in the present study qualified as complicated lesions according to Stary et al,20 with moderate inflammation and variable amounts of thrombus, fibrin, macrophages, extracellular lipid deposits, and fibrosis (example shown in Figure 3a
). The majority of
patient samples (83.8%) contained thrombus. Fibrin was present in
24 specimens (77.4%). Overall there were only 2 specimens devoid of
both thrombus and fibrin.
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Identification of PD-ECGF in Coronary Atherosclerotic
Plaques
We used RT-PCR as a sensitive screening assay to determine whether
PD-ECGF was expressed in atherosclerotic coronary vessels and
coronary atherectomy specimens. By using PD-ECGF–specific
primers, a 377-bp band (positive control in Figure 2; lane denoted +) was identified in 13
of 20 atherectomy samples. To verify the specificity of the reaction,
bands of the respective size were isolated and the PD-ECGF sequence
confirmed by sequence analysis. To ensure the success of the RT
reaction, control experiments were performed with primers specific for
glyceraldehyde-3-phosphate dehydrogenase (data not
shown).
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Identification of PD-ECGF–Expressing Cells in Coronary
Atherosclerotic Lesions From Patients With Severe Coronary
Heart Disease and in Intimal-Medial Cuts Obtained During DCA
Light microscopic analysis of sections of whole
atherosclerotic coronary arteries revealed PD-ECGF mRNA
predominantly within microvessels of the plaque shoulder (Figure 3, panels a through c). As a first step,
a more thorough analysis of microvessels within atherectomy
specimens (Figure 4a) was performed by
probing parallel sections with monoclonal antibodies directed against
von Willebrand factor (Figure 4b), antisense PD-ECGF
(Figure 4c), monoclonal anti–PD-ECGF (Figure 4d), sense
PD-ECGF (Figure 4e), and isotype control antibody (Figure 4f). The data demonstrated PD-ECGF mRNA and immunoreactivity
within ECs of intimal neovessels. To examine a PD-ECGF–dependent
mechanism of angiogenesis involving tissue mast cells (tryptase stain
of a parallel section is shown in Figure 4g), double-staining
experiments were performed with the use of antibodies directed against
PD-ECGF, followed by anti-tryptase. These experiments confirmed
colocalization of PD-ECGF–positive ECs and tryptase-positive cells
(Figure 4h).
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Second, PD-ECGF signal was detected in plaque macrophages
(Figure 3d) compared with the sense control (Figure 3e).
A thorough analysis of parallel sections of an atherectomy
specimen (shown in Figure 4a) with antisense PD-ECGF (Figure 4i), monoclonal anti–PD-ECGF (Figure 4j), CD68 (Figure 4k), and a monospecific IgG control (Figure 4l) confirmed
PD-ECGF–positive cells of the monocyte-macrophage lineage.
Parallel analysis of areas with stellate SMCs (Figure 4m) revealed PD-ECGF immunoreactivity (Figure 4o) within
SM -actin–positive cells (Figure 4n), whereas anti–von
Willebrand factor staining was negative in these areas (Figure 4p). PD-ECGF mRNA was lacking in medial SMCs of the vessel wall
(Figure 3b) but was present in stellate SMCs of
neointimal areas (data not shown).
Statistical Evaluation of PD-ECGF Immunoreactivity Status and
Clinical as Well as Morphometric Data
There was a trend toward greater mass of material retrieved from
lesions undergoing compensatory enlargement (P=0.06; cf
specimen areas in the Table). Staining of 31 consecutive
atherectomy specimens demonstrated PD-ECGF immunoreactivity in 21
specimens (the Table). Although all patients were in a stable
Canadian Cardiovascular Society class at the time of
the intervention in this study, acute coronary syndromes had
occurred in 11 patients within 1 and 21 days before DCA (patients
identified by bold type in the Table). Statistical
analysis of the clinical data in relation to the tissue
analysis revealed that there was a significantly more severe
angina score in patients from whom specimens with positive PD-ECGF
immunoreactivity status had been recovered (P=0.02, the
Table). Samples with positive PD-ECGF immunoreactivity status
had more plaque neovessels (P=0.03) than did samples with a
negative PD-ECGF immunoreactivity status. Furthermore, mast cell counts
were correlated with the number of lesional neovessels
(r=0.78, P<0.05), and mast cell counts per
square millimeter of specimen area were higher in specimens with a
positive PD-ECGF immunoreactivity status (P=0.007, the
Table>). Mast cells were absent in specimens where PD-ECGF
immunoreactivity was absent (the Table). No relationship was
found between minimal luminal diameter as assessed by quantitative
coronary angiography and PD-ECGF immunoreactivity status. On
the basis of data suggesting an interrelationship between vascular
thrombus and mast cell recruitment,21 further
analyses were directed at the statistical correlation of
atherectomy thrombus and number of mast cells. Figure 4a
represents a typical coronary atherectomy specimen with
10% thrombus (4.53±3.49%; mean mast cell counts 11.96±16.6,
n=26). In 5 specimens, relative thrombus area was >10%
(34.97±18.57%), and the mean mast cell count was 11.14±8.56. No
statistical relationship between mast cell count per square millimeter
of area and thrombus area was found.
Analysis of Neovessel Density in Relation to Vascular
Remodeling in Atherectomy Specimens
To elucidate the relevance of lesional expression of the EC
mitogen PD-ECGF, the numbers of lesional microvessels per square
millimeter of specimen area were statistically related to vascular
remodeling as assessed by intravascular ultrasound before specimen
retrieval (the Table). Numbers of both lesional microvessels and
mast cells were statistically higher in vessels demonstrating a lack of
compensatory enlargement (P=0.02 and P=0.04,
respectively).
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Discussion |
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The present study demonstrates expression of the potent EC-specific angiogenesis stimulator PD-ECGF/thymidine phosphorylase in coronary atherosclerosis. The data show that microvessels of the plaque shoulder are predominant sites for PD-ECGF expression. The data further imply that the number of microvessels is a critical determinant of vascular remodeling.
It has been recognized that rupture-related plaque progression due to luminal thrombosis and plaque hemorrhage through fragile, newly formed vessels at the base of advanced plaques are important mechanisms underlying acute coronary syndromes.22 Recent studies using coronary angioscopy have indicated that it takes at least 1 month after an ischemic coronary event for target lesions to heal.23 Therefore, the degree of PD-ECGF and growth factor expression in coronary plaques may be correlated histologically with both the active plaque causing an ischemic coronary syndrome and the repair phase of the individual lesion. As in various tumors,24 25 a significant correlation of PD-ECGF immunoreactivity and microvessel density was observed in coronary atherosclerosis. However, the small area that is cut by the DCA catheter may not reflect the biology of the whole vessel segment. Because of intrinsic differences in cut lesions undergoing compensatory enlargement and lesions undergoing shrinkage, there would also not be the same degree of sampling error applicable to both extremes of vascular remodeling. Therefore, the data correlating microvessel numbers and vascular remodeling must be evaluated with caution. Nevertheless, a positive correlation between high numbers of lesional microvessels and PD-ECGF immunoreactivity and between PD-ECGF immunoreactivity and angina score suggests a positive relationship between the expression of angiogenic factors, the degree of neovascularization, and lesion severity. These data are in accord with studies correlating clinical scores, neovascularization,26 and the expression of PDGF-AA, -AB, their receptors,9 and acidic and basic fibroblast growth factors.8 In contrast, recent studies of vascular endothelial growth factor expression in SMCs of atherosclerotic coronary arteries have failed to demonstrate a correlation between vascular endothelial growth factor immunostaining and the extent of vasa vasorum, raising speculations about a possible EC repair effect of this growth factor.27 Although evidence for this function is still lacking, PD-ECGF has been the subject of similar hypotheses.11 Like vascular endothelial growth factor, regulation of PD-ECGF expression in atherosclerosis may be mediated by hypoxia.28
One further finding was the colocalization of PD-ECGF–positive ECs and
mast cells (Figure 4h). Tissue mast cells are generally
concentrated around small blood vessels and lymphatics29
and newly forming microvasculature,30 and they accumulate
at the site of an atheromatous erosion or rupture, eg,
in the plaque shoulder.31 Previous in vitro data have
suggested a recruiting function of endothelial PD-ECGF
on mast cells,32 and heparin released from these cells may
stimulate capillary EC migration.33 34 Increased numbers
of mast cells have been found in auricular thrombosis, suggesting
attraction of mast cells through components within the
thrombus.21 However, in our study mast cell numbers were
independent of thrombus (the Table).
The present study demonstrates that neovascularization is associated with shrinkage of the diseased vessel segment. As an explanation, neovessel fragility could entail intramural bleeding with subsequent scarring. On the other hand, organizing neovascularization could follow thrombosis, secondary to the loss of cross-sectional and luminal vessel areas. Furthermore, mast cell products, eg, serotonin, and platelet products, eg, serotonin and transforming growth factor-ß, could mediate vasoconstriction and vascular shrinkage by promoting perivascular fibrosis.35 Because the lack of compensatory enlargement is an important contributing factor for the loss of luminal diameter in about one fourth of primary coronary lesions,5 the data suggest that lesional neovessels promote vascular occlusions. Angiogenic growth factors in atherosclerosis, eg, PD-ECGF, are involved in the process of vascular remodeling and could be targets for therapeutic inhibition to prevent vessel shrinkage.
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Acknowledgments |
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The work was supported by a grant from the Austrian Science Foundation P10559 MED, "Vermächtnis Josefine Hirtl Zur Förderung Der Medizinischen Forschung," and "Anton Dreher Gedächtnisstiftung" of the University of Vienna, Vienna, Austria (to I.M.L.).
Received June 4, 1998; accepted March 3, 1999.
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