© 1999 American Heart Association, Inc.
Original Contributions |
Immunohistochemical Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor in Atherosclerotic Intimas of Human Coronary Arteries
From the Department of Pathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Correspondence to Katsuo Sueishi, MD, PhD, Department of Pathology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail sueishi{at}pathol1.med.kyushu-u.ac.jp
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Abstract |
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Abstract—Neovascularization is well known to occur in human atherosclerotic plaques; however, its pathophysiological roles, mechanisms, and stimuli in atherogenesis still remain unclear. In this study, 525 tissue blocks of coronary artery tissue obtained at autopsy from 48 patients ranging in age from 20 to 93 years old (mean±SD, 71±15 years) were immunohistochemically examined for vascular endothelial growth factor (VEGF) expression in the atherosclerotic intimas. The atherosclerotic lesions were histopathologically classified into types I through VI, as proposed by the American Heart Association Committee, and the numbers of intimal blood vessels and VEGF-positive cells were then morphometrically counted in sections that were immunohistochemically examined with anti-CD34 and human VEGF antibodies, respectively. The more the atherosclerotic lesion type advanced, the more often the lesion contained intimal blood vessels, which were expressed as percentages of the intimal section with intimal microvessels, viz, diffuse intimal thickening (DIT): 0% (0/111); type I, 31% (32/104); II, 42% (10/24); III, 66% (77/117); IV, 72% (48/67); V, 79% (70/89); and VI, 100% (13/13), P<0.0001. The number of VEGF-positive cells per intimal section was also positively correlated with the number of intimal blood vessels (P<0.0001). The VEGF-positive cells were scattered in the fibrous caps as well as the shoulders and deeper areas of the plaques, and the double-immunostaining method revealed that the VEGF-positive cells were largely spindle-shaped, smooth muscle cells with some macrophage-derived foam cells. These findings thus suggest the possibility that the VEGF expressed by the smooth muscle cells and foamy macrophages in the atherosclerotic intimas can act as a local and endogenous regulator of endothelial cell functions, including intimal neovascularization, in atherosclerotic lesions of human coronary arteries.
Key Words: vascular endothelial growth factor • intimal neovascularization • human coronary artery • smooth muscle cells • macrophages
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Introduction |
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Intimal neovascularization has been observed in the atherosclerotic plaques of humans, including the coronary arteries, for a long time, and several investigators have recently refocused much attention to its pathophysiological roles, not only as a function of intimal thickness but also in atherosclerotic progression, including the development of such complications as intimal hemorrhage, plaque rupture, the formation of occlusive thrombus, and regression.1 2 3 We4 recently reported that the newly formed blood vessels were ubiquitously observed in the atherosclerotic intimas of human coronary arteries, and, despite the diversities of their distribution and density in the atherosclerotic plaques, the vascular density in atherosclerotic intimas is well correlated with the degree of luminal stenosis. In addition, these newly formed blood vessels were frequently associated with such chronic inflammatory cell infiltrates and the formation of granulation tissue, such as markers of the chronic inflammation/repair process, which thus suggest active atherogenesis. However, the mechanisms of intimal neovascularization, in particular the major stimuli of neovascularization in the atherosclerotic intima, and its pathophysiological roles in atherogenesis still remain unresolved.
Vascular endothelial growth factor (VEGF)/vascular permeability factor is a multifunctional cytokine for endothelial cells expressing VEGF receptor-1 (flt-1) and 2 (flk-1/KDR),5 6 7 including endothelial cell–specific mitogen,8 the increasing activity in vascular permeability,9 integrin expression,10 and the modulation of endothelial expression of fibrinolysis- and coagulation-related agents such as plasminogen activators,11 von Willebrand factor (vWF)12 and plasminogen activator inhibitor-1.11 Recent in vitro and in vivo studies have revealed the interaction between VEGF isoform(s) and its receptors to be the most important angiogenic event not only in mammalian embryogenesis but also in the physiological and pathological angiogenesis in adults.13 VEGF also induces enhanced tissue factor expression14 and the migration of monocytes expressing VEGF receptor-1.15 Couffinhal et al16 recently reported that VEGF is often overexpressed mainly by smooth muscle cells (SMCs) and partly by T lymphocytes in human coronary plaques retrieved by directional atherectomy, and this finding has suggested that VEGF function is involved in the maintenance and repair of the luminal endothelium in addition to its role in promoting intimal angiogenesis. However, there has yet to be a report systematically examining VEGF expression and its correlation with atherosclerotic lesion types found in human coronary arteries.
The purpose of the present study was to examine by light microscopy and immunohistochemistry human coronary arteries obtained from autopsy cases, to define the relation between the coronary atherosclerotic lesion type based on the American Heart Association (AHA) classification17 18 19 and intimal neovascularization, and then to clarify the pathophysiological role of VEGF expression in intimal neovascularization based on the following points: (1) the immunohistochemical distribution of VEGF protein in the diverse atherosclerotic lesions, (2) the species of VEGF-positive cells, and (3) the relationship between the degree of neovascularization and VEGF expression in the atherosclerotic intimas.
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Methods |
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Subjects and Light Microscopic Examination
Within 16 hours of death, hearts were obtained at autopsy from 48 Japanese patients, 27 men and 21 women, ranging in age from 20 to 93 years old (mean±SD, 71±15 years) at Kyushu University Hospital. One patient had a history of a previus myocardial infarction while the others had no history of coronary arterial diseases.
The coronary arteries were cannulated, washed with 0.1 mol/L PBS (pH 7.4), and perfused with 1 L of freshly prepared 4% (wt/vol) paraformaldehyde in 0.1 mol/L sodium phosphate (pH 7.4) at 100 mm Hg. Next, the heart was immersed in 4% paraformaldehyde for at least 24 hours at 4°C. The right coronary artery and left anterior descending coronary artery were dissected free from the surface of the heart, cut perpendicular to the long axis (from the proximal to distal segment) at 3-mm intervals, and then embedded in paraffin. Two hundred forty-nine blocks from the right coronary artery and 276 from the left anterior descending coronary artery were cut into 3-µm-thick serial sections.
Each section was stained with hematoxylin and eosin, elastica–van Gieson's, and Masson's trichrome stains. In accordance with the definitions proposed by the Committee on Vascular Lesions of the Council on Arteriosclerosis, AHA,17 18 19 the atherosclerotic lesion type of each section was carefully classified by 2 investigators simultaneously using a double-headed light microscope.
Antibodies
Rabbit anti-human VEGF IgG was prepared by immunizing the
rabbits with synthetic peptide corresponding to the 20–amino acid,
NH2-terminal region of human mature VEGF, as
reported previously,20 and then affinity-purified
to specific IgG by column chromatography with protein
A–Sepharose (Pharmacia Fine Chemicals) and VEGF antigen
coupled–Sepharose (Pharmacia). The cell species–specific antibodies
used were as follows: vWF (Dakopatts A/S) and CD34 (Novocastra
Laboratories Ltd) mainly for the endothelial cells and
partly for the hematopoietic cells on the cell surface; HHF35 (Dako)
for the SMCs; HAM56 (Enzo Diagnostics, Inc) for the
monocytes or macrophages; UCHL-1 (Dako A/S) for CD45RO antigen,
mainly for T lymphocytes and partly for the macrophage marker;
and MX-PanB (Kyowa Medicus) for B lymphocytes. In this study, we
generally used mouse monoclonal anti-CD34 antibody as an
endothelial cell marker, because the
endothelial cells lining the newly formed blood vessels
were more frequently positive for CD34 than for vWF; in fact, some
newly formed intimal blood vessels, especially those located in the
cell-rich intimas, were immunohistochemically negative for vWF.
Biotinylated rabbit anti-IgG for mouse or goat IgG was obtained from
Nichirei Co.
Immunohistochemistry
An immunohistochemical examination was performed using the
standard avidin-biotin-peroxidase complex technique as described
previously.21 In brief, the sections were
deparaffinized and incubated with 10% normal goat or rabbit serum for
20 minutes to minimize the nonspecific binding of the primary antibody.
Then they were incubated with the primary rabbit anti-human VEGF IgG (5
µg/mL) or mouse monoclonal anti-human CD34 (1:100), vWF (1:1600),
HHF35 (1:100), HAM56 (1:100), UCHL 1 (1:100), and MX-PanB (1:100)
antibodies overnight at 4°C in a moisture chamber. All of the
following steps were separated by 3 washes with PBS for 5 minutes. The
sections were then incubated with the appropriate secondary antibody
for 30 minutes at room temperature. To inhibit any
endogenous peroxidase activity, the sections were incubated
with 0.3% (wt/vol) H2O2 in
absolute methanol for 30 minutes. Thereafter, the sections were
incubated with peroxidase-labeled streptavidin (Histofine SAB-PO kit)
for 30 minutes. Visualization of a positive reaction was developed with
a peroxidase substrate solution containing 0.02% (wt/vol)
H2O2 and 0.1% (wt/vol)
3,3'-diaminobenzidine tetrahydrochloride (Merck) in PBS to give the
reaction product a brown color, and then the sections were
counterstained with hematoxylin or methyl green.
To identify the VEGF-positive cell species, a double immunohistochemical examination was carried out according to the previously reported method.22 After the first color reaction was developed with a 3,3'-diaminobenzidine tetrahydrochloride–peroxidase reaction, the sections were washed with PBS 3 times and incubated with biotin solution (Vector Laboratories) for 30 minutes to block the remaining streptavidin residue. After being blocked with nonimmune serum, the secondary antibodies for the second antigen were applied in the same way as for the first antigen. The sections were then incubated with avidin-labeled alkaline phosphatase (Dakopatts A/S). The red reaction product was developed by using alkaline phosphatase substrate kit 1 (Vector Laboratories). The sections were lightly counterstained with hematoxylin.
The specificity of the anti-VEGF antibody was confirmed by preabsorption of the antibody with a VEGF peptide used as the antigen at a 10-fold concentration and by Western blot analysis, as reported previously.20 Nonimmune mouse and rabbit IgGs were also used instead of the respective primary antibody as other negative controls. For the immunohistochemical positive controls, the tissue blocks retrieved from human placenta and tonsilar tissue were used for VEGF and CD34, and vWF, HAM56, UCHL-1, and MX-PanB immunostaining, respectively.
Morphometric Study
Using a morphometric analyzer (Cosmozone-1S image
analyzer, Nikon), we measured the percentage of luminal
stenosis of each section as reported
previously.4 In brief, the area of the lumen (S1)
and the inside area of the internal elastic lamina (S2) were measured,
and then the percentage of luminal stenosis
[(S2-S1)/S2x100] was calculated. The total number of intimal blood
vessels, which were lined with CD34-positive
endothelial cells, was counted under a light microscope
at high-power magnification (x200) according to the criteria of
Weidner et al.23 The number of VEGF-positive
cells in the intima was counted for each section, and then a numerical
grade was assigned as follows, according to the number of VEGF-positive
cells: -, no staining; ±, 1 to 9; 1+, 10 to 29; 2+, 30 to 99; and 3+,
100 in each section of coronary artery. The densities of the
blood vessels or VEGF-positive cells per unit area of the
neointima were also calculated. All counts were performed
by 2 investigators using a double-headed light microscope.
Statistical Analysis
The results are presented as mean±SD unless otherwise
stated. The data were statistically analyzed by ANOVA, unpaired
Student's t test, and 
2 test.
Pearson's correlation coefficient was also calculated to
analyze the statistical correlation between age and the
incidence of atherosclerotic lesion types. A value of
P<0.05 was considered statistically significant.
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Results |
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Histopathology of Coronary Arteries
Five hundred twenty-five coronary arterial specimens were carefully classified according to the type of atherosclerotic lesion, and the degree of luminal stenosis of each cross section was measured as described in Methods. Twenty-one percent (111/525) of the specimens examined were classified as diffuse intimal thickening (DIT); 24% (128/525) as types I and II, which are early lesions; and 32% (169/525) as types IV through VI, which are advanced lesions. The type III "bridging lesions" between early and advanced lesions comprised 22% (117 of 525 specimens). Among them, the percentage of DIT lesions decreased (P<0.05, Pearson's method), whereas the type V lesions increased inversely (P<0.001, Pearson's method) as age advanced. In type III and IV lesions, the accumulation of macrophage-derived foam cells and T lymphocytes was frequently seen around the atherosclerotic lipid cores, especially in the fibrous cap and shoulder regions. Type VI lesions were found in 2% of the specimens (13/525) in this study, and 1 section was associated with luminal thrombus organization and recanalization, 1 section with 2 sites of atheroma rupture and intramural hemorrhage, 2 with a small luminal thrombus, and 9 with intimal hematoma or hemorrhage.
As the atherosclerotic lesions progressed from early type I to advanced type VI, luminal stenosis correlatively advanced. However, the statistical difference between both early type I and II lesions and between advanced type IV and V lesions was not significant. In addition, luminal stenosis in the coronary arteries with DIT only, which was characterized by concentric intimal thickening due to proliferation of SMCs and matrix but without either any apparent lipid deposition or foam cell appearance, ranged from 7% to 43% (23±9.6%) and was also significantly less than that in all other types of atherosclerosis lesions.
Intimal Neovascularization and Atherosclerotic Lesion Type
To define the relation between intimal neovascularization and the
coronary atherosclerotic lesion types, all specimens were
analyzed for the presence of intimal blood vessels labeled with
CD34 as an endothelial marker. No intimal
neovascularization was found in any coronary arteries with DIT
only, even though they showed a considerable degree of luminal
stenosis as described above. On the other hand, all
coronary arteries of type VI lesions were shown to have a newly
formed blood vessel in the atherosclerotic intimas. From type I to type
VI, the more the atherosclerotic lesions advanced, the more often the
neointimas contained newly formed blood vessels, viz, DIT,
0% (0/111); type I, 31% (32/104); II, 42% (10/24); III, 66%
(77/117); IV, 72% (48/67); V, 79% (70/89); and VI, 100% (13/13), as
indicated in Figure 1
(P<0.0001, ANOVA).
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Interestingly, while examining serial sections with type I or II lesions, we found that the neointimal blood vessels frequently communicated with the adventitial vasa vasorum through the media and were also distributed in the deeper intima. As the atherosclerotic lesions progressed to type III or IV, newly formed blood vessels were often seen around the atheromatous plaque, especially in the fibrous caps and shoulders of the plaques, where foam cell accumulation and T-lymphocytic infiltration generally coexisted. However, in type V lesions, which mostly demonstrated intimal calcification even though intimal blood vessels were often still observed, blood vessel density was decreased. The blood vessels observed in such regions were mainly located in the deeper plaque and partly in the shoulder or fibrous cap, where foam cells and T lymphocytes were rarely seen. A few B lymphocytes were scattered throughout all atherosclerotic lesion types.
Localization of VEGF-Positive Cells and Their Cell Species
The immunohistochemical expression and distribution of VEGF in
human coronary arteries were examined in 525 blocks of
coronary arteries by using anti-VEGF antibody. VEGF-positive
cells were detected in 68 of 111 sections (61%) of DIT and in 354 of
414 (86%) atherosclerotic sections corresponding to types I through
VI. The percentage of VEGF-positive blocks was as follows: type I, 65%
(68/104); type II, 75% (18/24); type III, 93% (109/117); type IV,
91% (61/67); type V, 95% (85/89); and type VI, 100% (13/13).
VEGF-positive cells were more commonly observed in the advanced lesions
(types IV through VI) than in the early lesions (types I and II;
P<0.0001, 2). In the
coronary artery lesions corresponding to DIT and type I, almost
all positive VEGF immunoreactions were restricted to the medial as well
as the spindle-shaped SMCs in the deeper portions of the intima (Figure 2D). In the coronary arteries
with type II through VI lesions, however, the distribution of
VEGF-positive immunoreactions was different from that in DIT and type I
lesions, and the medial SMCs were weakly immunopositive for VEGF. In
contrast, many spindle-shaped SMCs and some round or polygonal cells,
which were largely composed of macrophages, occasionally showed
a foamy cytoplasm. These cells were located in the fibrous plaque,
shoulder, and cap regions, where the newly formed blood vessels were
frequently found (Figures 3B and 4B
through 4D), and showed strong immunopositivity
for VEGF (Figures 3B through 3D, 4D, 4F, and 4G). Therefore, the
VEGF-positive cells in atherosclerotic intimas of human
coronary arteries were mostly SMCs and partly
macrophage-derived foam cells. In addition, the SMCs
surrounding the newly formed intimal microvessels and the adventitial
vasa vasorum were intensely positive for VEGF, although some
endothelial cells in these blood vessels were also
positive for VEGF (Figure 2D). However, no extracellular deposits of
VEGF were revealed in the atherosclerotic intimas (Figure 4D and 4F), even in cell-rich plaques
(Figure 3B and 3C). Although T lymphocytes were frequently found in
areas populated with VEGF-positive cells, we could not clearly identify
the source of the VEGF positivity in the cytoplasm of the T lymphocytes
(Figure 4D), even by examining serial sections. In addition, B
lymphocytes were also negative for VEGF.
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Negative controls for VEGF immunohistochemistry, with the use of either
nonimmunized rabbit IgG (5 µg/mL) or rabbit anti-human VEGF IgG (5
µg/mL) preabsorbed with a VEGF peptide as the antigen at a 10-fold
concentration instead of the primary rabbit anti-human VEGF IgG, showed
no apparent immunopositivity (Figures 3E
and 4H).
Relationship Between the Degree of VEGF
Immunostaining and the Number of Intimal
Vessels
All atherosclerotic coronary arterial sections
of type I through VI lesions (414/525) were analyzed regarding
the relationship between the degree of VEGF-positive cells and the
number of intimal vessels. One hundred thirteen of 164 (69%) sections
without intimal neovascularization and 241 of 250 (96%) sections with
intimal neovascularization possessed VEGF-positive cells. The total
numbers of intimal blood vessels and VEGF-positive cells in the intima
were morphometrically counted in each section, and the number of
VEGF-positive cells per whole intima was semiquantitatively classified
into 4 grades as described in Methods. As shown in Figure 5, the degree of VEGF-positive cell
occurrence in the atherosclerotic intimas was positively correlated
with the number of intimal blood vessels (P<0.0001, ANOVA).
We also calculated the density of the blood vessels and the
VEGF-positive cells per unit area of neointima, but no
statistically significant correlation was found.
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This study systematically examined newly formed blood vessels in 525 tissue specimens of coronary arteries retrieved from 48 Japanese autopsy cases in an attempt to clarify the topographic occurrence of neovascularization and the expression of VEGF protein, which has been thought to be a principal angiogenic factor in both physiological and pathological states. We categorized the types of histopathological alterations of coronary arteries, including the adaptive intimal thickening, according to the AHA classification.17 18 19 The above findings thus indicate that: (1) The newly formed blood vessels were ubiquitously distributed in the atherosclerotic intimas, and the prevalence of intimal sections containing microvessels increased as the lesion type progressed from type I to VI. No microvessels could be seen in DIT, which is generally accepted as adaptive intimal thickening only. (2) The VEGF-positive cells were also more commonly observed in the advanced lesions, and thus VEGF-positive cell number was well correlated with the degree of intimal vascularity. (3) The VEGF-positive cells consisted largely of spindle-shaped SMCs and lesser amounts of foamy macrophages. (4) These VEGF-positive cells were widely scattered in the atherosclerotic plaques but were most commonly located in the shoulder region and fibrous cap, particularly in atherosclerotic lesion types III and IV, where lymphocytic infiltration and the formation of granulation-like tissue were frequently associated.
In this study, to examine the relationship between the degree of neovascularization and VEGF expression in heterogeneous atherosclerotic lesions of human coronary arteries, we classified the atherosclerotic lesion type of each section according to AHA guidelines.17 18 19 This classification is useful for globally determining the histological gradations and constitutional characteristics of coronary atherosclerosis. In fact, the luminal stenosis of the coronary arteries examined did correlate well with the atherosclerotic lesion type. In addition, intimal neovascularization became more frequent as the atherosclerotic lesion type advanced. These findings thus support those of our previous study4 in which we reported that the density of intimal neovascularization increased with decreasing luminal size and probably with increasing relative intimal thickness as well. In contrast, the formation of intimal microvessels was affected by such histological characteristics as the presence of a chronic inflammatory infiltrate and granulation tissue, which are assumed to be markers of the chronic active inflammatory process in atherogenesis.
Angiogenesis in the adult human has been considered to be fundamentally quiescent because of the negative balance of function between angiogenic and angiostatic factors.24 Recent in vitro and in vivo studies have revealed that the transformation of endothelial cells to an angiogenic phenotype is induced by a large number of angiogenic factors, such as VEGF, basic fibroblast growth factor (bFGF), hepatocyte growth factor, interleukin (IL)-8, and others,25 but the interaction between the VEGF isoform(s) and its receptor types 1 and 2 appears to be mainly responsible for angiogenesis. This process occurs not only in embryogenesis26 27 28 29 30 (and is also known as vasculogenesis) but also during tumor growth as a trigger of the "angiogenic switch,"31 proliferative diabetic retinopathies,32 and the inflammation-repair process.33 VEGF receptors 1 and 2 have been revealed to be constitutively expressed by cultured endothelial cells34; however, controversy remains regarding the in vivo expression of these receptors in normal and injured blood vessels and the functional diversity in signal transduction elicited by the binding of VEGF to each receptor. Lindner and Reidy35 reported that receptor 1 (flt-1) is upregulated at the leading edge of growing endothelium on the de-endothelialized surface of balloon-injured rat arteries and thus suggested that this receptor bound with VEGF plays a role in increased vascular permeability. In contrast, receptor 2 (flk-1/KDR) has been postulated to be related to endothelial proliferation, and thus, overstimulation of this receptor may be a key event in tumor angiogenesis, but not in normal, quiescent tissue.36 37 bFGF may participate in the modulation of in vitro angiogenesis synergistically with VEGF,38 39 partly through a function of bFGF-binding protein.40 However, the angiogenic mechanism(s) and stimuli in atherosclerotic intimas remain to be elucidated in future investigations.
This study clearly demonstrated VEGF expression to be widely distributed in both diffusely thickened and atherosclerotic intimas: the more often that VEGF-positive cells were observed, the more the atherosclerotic lesion type progressed. In addition, the number of newly formed microvessels was positively correlated with the degree of VEGF-positive cell occurrence in atherosclerotic intimas. Together with our previous observation that introducing human VEGF cDNA165 into the rabbit carotid artery by a hemagglutinating virus of the Japan liposome system, VEGF overexpression in the arterial wall in vivo induces an angiomatoid proliferation of endothelial cells. This proliferation is accompanied by increased vascular permeability and "fresh" and old hemorrhages, which are very similar to the complicated microvascular networks observed in human coronary arteries.41 These findings thus suggest that VEGF, which is widely expressed in atherosclerotic intimas, can participate not only in angiogenic promotion but also in the functional regulation of endothelial cells in injured coronary arteries as a multifunctional cytokine. VEGF protein, however, has also been noted in the deeper intimal and medial layer of DIT lesions where no newly formed microvessels were noted. Therefore, VEGF expressed in both the adaptive and atherosclerotic coronary intima may also participate in the integral maintenance and functional regulation of endothelial cells lining the luminal surface and adventitial blood vessels, as proposed in part by Couffinhal et al,16 who examined directional atherectomy specimens retrieved from patients with coronary insufficiency.
In all types of atherosclerotic lesion, the major cell type displaying
a VEGF-positive reaction was the SMC not only in the medial layer but
also in the intima, especially in the fibrous caps of
atheromatous or fibroatheromatous
plaques (atherosclerotic lesion types III through V). These findings
thus support the previous in vitro and in vivo
data16 42 in which SMCs mainly were found to be
responsible for VEGF expression in the vascular wall. In addition, some
macrophages were convincingly immunopositive for VEGF. These
VEGF-positive macrophages were also generally distributed in
foam cell lesions (type II) and the fibrous caps and shoulders of both
atheromatous and fibroatheromatous
plaques. VEGF-positive cells also occasionally appeared close to the
newly formed microvessels (Figure 3B). These lesions were frequently
associated with T-lymphocytic infiltration, thus suggesting an active
inflammatory process in atherogenesis. Intimal angiogenesis thus seems
to be a histopathological marker of the active phase of atherogenesis.
Couffinhal et al16 reported that CD45RO-positive
lymphocytes and extracellular matrix in atherectomy specimens were also
VEGF-positive, whereas the macrophages were negative. Their
findings conflict with ours, and these discrepancies may be due to the
following reasons: (1) The samples examined were different, viz,
directional atherectomy specimens in the study of Couffinhal et al and
autopsy materials in ours. (2) CD45RO antigen is largely expressed by T
lymphocytes but also to a lesser extent by macrophages. Thus,
it may be possible that the CD45RO- and VEGF-positive cells were
macrophages, some of which were revealed to be VEGF-positive in
the present study. (3) It is difficult to convincingly distinguish
immunohistochemical positivity from false positivity in the
extracellular matrix. In fact, we noticed occasional weak staining in
the matrixes of hyalinized intimas as well as in cell-rich
atherosclerotic lesions, but we could not definitively establish such
weak staining to be truly positive compared with negative controls.
Regarding extracellular VEGF deposition, we also could not clearly
demonstrate this in the current study. The isoforms of VEGF longer than
VEGF165, however, have been thought to be sequestered in the
extracellular matrix through their high heparin-binding affinity. These
matrix-associated VEGF forms may participate in the modulation of
endothelial cell function by being released not only in
a soluble form with heparin or heparitinase but also as a proteolytic
fragment with plasmin.43 44 Additional studies
will be necessary to elucidate the extracellular distribution of VEGF
isoforms in atherosclerotic intimas. Further examination is thus
necessary to clarify whether T lymphocytes, a major cellular
constituent in atherosclerotic lesions, and the extracellular matrix
are possible VEGF-producing cells and a reservoir of functional VEGF,
respectively. Together with the previous in vitro study, which reported
that T lymphocytes can also synthesize and release
VEGF,45 these points are important for
comprehending the pathophysiological significance
of VEGF function in atherogenesis, especially the mechanisms of its
regression and the occurrence of such complications as intraplaque
hemorrhage and plaque rupture leading to occlusive thrombus
formation.
The mechanisms of VEGF overexpression in the atherosclerotic intima
remain unexplained in this study. Other recent in vitro investigations,
however, have shown that hypoxia,39 46
growth factors (including bFGF,38
platelet-derived growth factor,47 and
transforming growth factor-ß48), and
cytokines (such as tumor necrosis factor [TNF]-
and
IL-1ß),49 all of which have been shown to
participate in atherogenesis,50 stimulate SMCs,
macrophages and others to upregulate VEGF expression. In
addition, several potential binding sites for the transcriptional
factors activator protein (AP)-1, AP-2, and
Sp-151 and hypoxia regulatory
elements52 53 have been identified in the VEGF
gene promoter and in the 5' and 3' regions of the VEGF gene,
respectively. AP-1 activity is also assumed to participate in the
enhancement of VEGF expression induced not only by the proinflammatory
cytokine TNF- but also by hypoxia in tumor
cells.54 TNF- can increase Sp-1–mediated VEGF
expression also.55 Therefore, hypoxia in
the deeper56 and cell-rich sites of
atherosclerotic intimas57 and proinflammatory
cytokines such as TNF- and IL-1ß may also play a role in
VEGF overexpression in atherosclerotic intimas.
In summary, the intimal neovascularization in atherosclerotic intimas represents an essential response to arterial injury and also is an important event during arterial remodeling in the atherosclerotic process. In addition, overexpression of VEGF by SMCs and macrophages in atherosclerotic intimas may act as a local endogenous regulator of endothelial cell function in atherosclerotic lesions of human coronary arteries.
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Acknowledgments |
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The authors thank Hiroshi Fujii for his excellent technical assistance, Akiko Ogata for assisting in the manuscript preparation, and Dr Brian T. Quinn for proofreading this manuscript.
Received February 28, 1998; accepted June 16, 1998.
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