© 1995 American Heart Association, Inc.
Articles |
Immunohistochemical Demonstration of 15-Lipoxygenase in Transplant Coronary Artery Disease
From the Departments of Medicine, Division of Cardiology (S.R., P.J.C.), Cardiothoracic Surgery (R.E.M.), and Pathology (C.C.M., V.D.D.), Columbia University College of Physicians and Surgeons, New York, NY, and Syntex Discovery Research (E.S.), Palo Alto, Calif.
Correspondence to Paul J. Cannon, MD, Department of Medicine, Division of Cardiology, Columbia University College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032.
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Abstract |
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Abstract 15-Lipoxygenase (15-LO) catalyzes the oxygenation of arachidonic and linoleic acids and has been implicated in the oxidative modification of low-density lipoproteins (LDL). 15-LO mRNA and protein have previously been demonstrated in macrophages of rabbit and human atherosclerotic lesions. The purpose of this study was to investigate whether 15-LO is also present in the accelerated form of coronary artery disease that can complicate cardiac transplantation (TCAD). Immunohistochemical analysis of coronary arteries with TCAD was carried out by using a rabbit polyclonal antibody raised against human recombinant 15-LO and an avidin-biotin-immunoperoxidase system. Normal coronary and pulmonary arteries showed no immunostaining for 15-LO. Two different types of TCAD were observed. One type consisted of concentric intimal proliferation of smooth muscle cells, without lipid or calcium deposits. No immunoreactivity for 15-LO was present in these lesions. The second type of graft arteriosclerosis consisted of complex atheromatous lesions, containing myointimal cells, lipid-laden foam cells, fragmented internal elastic laminae, and calcifications. 15-LO immunostaining of myointimal cells, lipid-laden foam cells, and endothelial cells was consistently present in these atheromatous lesions. The majority of the myointimal and foam cells positive for 15-LO were recognized by antisera to
–smooth muscle actin; the
others were identified as macrophages. The results indicate that 15-LO
expression is present in endothelial, myointimal, and foam cells in
complex atheromatous lesions of TCAD, and suggest that 15-LO may play a
role in the pathogenesis of this form of the disease.
Key Words: 15-lipoxygenase • graft arteriosclerosis • transplantation • lipid oxidation • coronary artery disease
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Introduction |
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Cardiac transplantation has been demonstrated to be effective therapy for intractable heart failure, with 1-year and 5-year survival rates of 80% and 65%, respectively.1 Long-term survival has been limited, however, by rejection and by the development of graft arteriosclerosis. Coronary artery disease in the transplanted heart (TCAD) is the leading cause of death after the first posttransplant year and the most frequent cause of long-term cardiac allograft failure.2 The lesions of TCAD tend to be widespread and diffuse, and exhibit less tendency to focality than lesions of atherosclerosis in the native vasculature.3 4 5 6 Thus, once significant coronary vascular disease is apparent, therapeutic options other than retransplantation are minimal.
The pathogenesis of graft arteriosclerosis is unclear. Established risk factors for the development of TCAD include multiple episodes of rejection,7 cytomegalovirus infection,8 and the development of serum antibodies directed against donor HLA alloantigens within the first posttransplant year.9 The role of hyperlipidemia as a risk factor is uncertain even though serum cholesterol and triglyceride levels tend to be increased in cardiac transplant recipients.10 Some studies indicate that hypercholesterolemia is a significant risk factor,11 12 whereas others do not confirm this association.7 Increased lipoprotein(a) level has also been reported to be an independent risk factor.13 The possible contribution of lipid and lipoprotein peroxidation to the risk of developing TCAD has not been extensively investigated, despite pathological studies indicating that lipid-rich, complex atheromatous lesions are often present in long-term cardiac transplant survivors.3 4 5 6
Increasing evidence has accumulated that the oxidative modification of low-density lipoproteins (LDL) may play a role in the pathogenesis of atherosclerosis.14 15 16 Oxidative modification of LDL in vitro by endothelial cells, smooth muscle cells, or monocytes/macrophages changes the lipid and protein moieties of the lipoprotein so that the oxidatively modified LDL is no longer recognized by the receptor for native LDL but is recognized by the scavenger receptor on monocytes/macrophages and is taken up to form lipid-laden foam cells.15 16 Oxidatively modified LDL has been found in atherosclerotic lesions in humans and experimental animal models.17 18 19 20 Antioxidant therapy in the latter has been shown to retard lesion development significantly.21 22 23 24
Additional evidence has implicated cellular lipoxygenases, particularly 15-lipoxygenase (15-LO), in the oxidative modification of LDL and potentially in the process of atherogenesis.14 15 16 25 26 27 28 29 30 Lipoxygenases are a family of enzymes that insert molecular oxygen into polyenoic fatty acids such as arachidonic acid or linoleic acid at specific carbon atoms to form hydroperoxy derivatives.26 Products of the reaction of 15-LO with arachidonic acid and linoleic acid are 15-hydroperoxyeicosatetraenoic acid and 13-hydroperoxyoctadecadienoic acid, respectively. In vivo, these reactive compounds are reduced to hydroxy acids or are converted to other compounds that have a variety of biological actions.26 Work by several groups has implicated 15-LO in the oxidative modification of LDL. Native LDL can be oxidized in vitro by lipoxygenases, including 15-LO.27 28 30 Oxidative modification of LDL by endothelial cells or monocytes/macrophages is reduced by incubation of the cells with lipoxygenase inhibitors.25 29 Recently, using in situ hybridization and immunohistochemistry, 15-LO was localized to macrophages in atherosclerotic lesions from human arteries and Watanabe rabbits; these atherosclerotic lesions also contained the scavenger receptor, lipid, and epitopes of oxidized LDL apolipoprotein.18 19
In addition to foam cells, T lymphocytes are found in the lesions of atherosclerosis and in those of TCAD.31 The cytokine interleukin-4 (IL-4), a product of the TH2 subset of helper T lymphocytes, was reported to be unique among a panel of cytokines in its ability to stimulate the expression of 15-LO in human monocytes.32 IL-4 levels have also been reported to be higher in the coronary sinus blood than in arterial blood in patients after transplantation, despite levels of immunosuppressive therapy that were sufficient to prevent clinical episodes of rejection.33 Whether IL-4 induces 15-LO expression in the coronary arteries of transplanted hearts is unknown. Accordingly, the objective of the present study was to determine, using immunohistochemistry, whether 15-LO is present in the vascular lesions of TCAD.
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Methods |
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Patients
Coronary arteries were obtained from the explanted cardiac grafts of 7 patients undergoing retransplantation for TCAD and from the native hearts of 5 patients undergoing transplantation for ischemic heart disease (n=3), hypertrophic cardiomyopathy (n=1), and valvular heart disease (n=1). Some arteries were cut fresh, snap-frozen, and stored at -70°C; others were fixed in 10% buffered formalin and embedded in paraffin.
Antibodies
The source of each antibody used and the optimal working
dilution are summarized in Table 1
. HAM-56 (Enzo
Diagnostics) is an affinity-purified monoclonal mouse antibody that
reacts with two different cell types: fixed tissue macrophages and a
subpopulation of endothelial cells, particularly those lining
capillaries and small blood vessels.34 It was used to
identify macrophages. A polyclonal rabbit antibody anti–human von
Willebrand factor (Dako Corp) was used as a specific endothelial cell
marker.35 Smooth muscle cells were identified with a mouse
monoclonal antibody (Bio Genex Laboratories) directed against
–smooth muscle actin.36 The Sepharose-G–purified
rabbit antibody against human recombinant 15-LO has been shown to
specifically react with 15-LO from human reticulocytes, leukocytes,
respiratory epithelial cells, and tissue
macrophages.18 37 38 The antibody does not cross-react
with the human platelet 12-LO, as evidenced by negative immunostaining
of platelet-rich blood clots; neither did it appear to cross-react with
the leukocyte-type 12-LO recently reported to be present in human
adrenal tissue,39 because negative staining was also seen
in studies of normal adrenal glands.
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Immunohistochemistry
The avidin-biotin-immunoperoxidase system was used on both
acetone-fixed frozen sections and formalin-fixed, paraffin-embedded
tissue sections. Serial sections (4 to 6 µm) of the latter were
deparaffinized with xylene, rehydrated with serial alcohol baths, and
then washed with PBS. Frozen sections were air dried and fixed in
acetone for 5 minutes. Thereafter, the procedure was identical for
frozen and fixed tissue. Endogenous peroxidase was inactivated with 3%
hydrogen peroxide in ethanol for 30 minutes, and nonspecific antibody
binding was suppressed with 20% goat or horse serum in PBS for 30
minutes. Sections were incubated in a humidified chamber overnight at
4°C with anti–15-LO antibody or for 1 hour at room temperature with
the other antibodies. With intervening washes in PBS, sections were
then incubated for 30 minutes at room temperature with a 1:100 dilution
of a biotinylated secondary antibody. A goat anti-rabbit IgG (Vector
Laboratories) was used for 15-LO and anti–von Willebrand factor, and a
horse anti-mouse IgG (Vector) was used for HAM-56 and –smooth
muscle actin. The avidin-biotin-immunoperoxidase complex (ABC Elite),
diluted 1:100 in PBS, was then applied for 30 minutes at room
temperature. Slides were then incubated with a 0.1 mol/L solution of
3,3'-diaminobenzidine (Sigma Chemical Co) in 0.05 mol/L TRIS buffer, to
which had been added 0.5 mL 3% hydrogen peroxide, to yield a brown
reaction product. Sections were then washed in tap water,
counterstained with hematoxylin, dehydrated in sequential alcohols, and
mounted with coverslips. As negative controls, rabbit or horse IgG was
substituted, at identical concentrations, for the primary
antibody/antiserum. Bronchial epithelia from formalin-fixed,
paraffin-embedded or acetone-fixed, frozen lung specimens were positive
controls. Normal adrenal glands and sections of human thrombi were used
as additional controls to exclude cross-reactivity with platelet or
leukocyte-type 12-LO.
To study the effect of antigen unmasking on formalin-fixed tissue, we carried out immunohistochemistry twice in selected sections, before and after treatment with microwave heating. In brief, deparaffinized rehydrated sections were placed in a plastic container filled with sodium citrate buffer. The jar was irradiated for 15 minutes in a household microwave oven at 90% power output. The sections were then allowed to cool for 30 minutes at room temperature before being processed for immunohistochemistry.
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Results |
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A total of 26 coronary artery segments were available for analysis: 17 specimens from 7 patients with TCAD, 7 from 3 patients with atherosclerosis of native arteries, and 2 without histological evidence of disease. Table 2
depicts some clinical
characteristics of the 7 patients with TCAD. The results obtained on
formalin-fixed, paraffin-embedded tissue sections were the same as
those obtained on acetone-fixed frozen sections in terms of the
distribution of immunostaining, its intensity, and the amount of
nonspecific staining. Normal lung tissue, which was used as a positive
control, demonstrated a consistent, strong 15-LO immunoreactivity,
localized to ciliated cells of the bronchial epithelium (Fig 1) as well as to alveolar macrophages (data not
shown).
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Normal Blood Vessels
Normal coronary arteries demonstrated no detectable 15-LO
expression (Fig 2A). There was also no 15-LO
immunoreactivity apparent in branches of the pulmonary artery or in
pulmonary arterioles (Fig 2B). Microwave oven treatment of the
sections, a technique recently developed to improve antigen retrieval
in formalin-fixed tissues, did not bring out any 15-LO staining in
these cases.
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TCAD
Histologically, two types of TCAD were observed (Fig 3). The first type, present in 4 of the 7 patients
undergoing retransplantation, consisted of concentric intimal
proliferation of spindle-shaped mesenchymal cells overlying a mostly
intact internal elastic lamina (Fig 3A and 3B). Intracellular or
extracellular lipids or calcifications were not apparent in these
lesions. There was no demonstrable 15-LO expression in this type of
graft arteriosclerosis, even when the concentric proliferation of
myointimal cells was sufficient to produce luminal narrowing (Fig 4); neither did 15-LO staining become apparent when the
sections were subjected to microwaving before the application of the
anti–15-LO antibody.
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The second type of TCAD, noted in 3 of the 7 patients undergoing
retransplantation, consisted of lesions characteristic of complex
atheromatous plaques, with lipid-filled foam cells in the thickened
intima, a fragmented internal elastic lamina, extracellular lipid
deposits, patchy calcifications, and neovascularization within the
lesions (Fig 3C
and 3D). Immunostaining for 15-LO was uniformly
apparent in these "atheromatous" lesions of TCAD (Figs 5 and 6). 15-LO immunoreactivity appeared
uniformly to be in the cytoplasm of three phenotypically different cell
populations: spindle-shaped mesenchymal cells, foam cells, and
endothelial cells lining the vessel lumen (Figs 5 and 6B).
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The 15-LO staining was prominent in spindle-shaped cells present
throughout the intima (Figs 5, 6A, and 6B). These cells were recognized
by a monoclonal antibody to –smooth muscle actin, establishing
their origin from smooth muscle cells (Fig 6C and 6D). Macrophages
present in the thickened intima (identified with HAM-56) also
exhibited prominent 15-LO immunoreactivity (Fig 6E and 6F).
Lipid-filled foam cells in the intimal lesions were also stained
prominently with the anti–15-LO antibody (Figs 5 and 6B).
Surprisingly, the majority of these 15-LO–positive foam cells appeared
to be of smooth muscle cell origin (Fig 6D), and the remainder were
identified as macrophages (Fig 6F). In contrast to the positive 15-LO
staining of spindle cells and foam cells derived from smooth muscle
cells in the lesions, no 15-LO immunostaining was apparent in smooth
muscle cells of the media of the coronary arteries.
In the complex atheromatous lesions of TCAD, 15-LO immunostaining of
endothelial cells was also apparent (Figs 5 and 6B). The intensity of
endothelial staining varied significantly from one arterial region to
another, but in some regions it was quite striking. 15-LO
immunoreactivity was also apparent in cells lining neovessels that had
formed within some of the atheromatous lesions (Fig 7).
Immunostaining for 15-LO was not apparent in small intramyocardial
coronary vessels (data not shown).
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Atherosclerosis
Of the 7 specimens of atherosclerotic lesions of native coronary
arteries, 2 had minimal disease characterized by slight intimal
thickening with occasional foam cells. The remaining 5 arterial
segments had complex plaques with foam cells, lipid deposits,
calcifications, and disruption of the internal elastic membrane. The
minimal lesions did not show 15-LO immunostaining (data not shown). In
contrast, the arterial segments with advanced atherosclerotic lesions
demonstrated widespread 15-LO immunostaining (Fig 8).
Immunoreactivity for 15-LO was present in macrophages, spindle
cells, foam cells, and, to a variable extent, luminal endothelial
cells. In some advanced atherosclerotic lesions, 15-LO staining of
intimal neovessels was also apparent.
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Discussion |
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The aim of the present study was to investigate whether 15-LO is present in TCAD. Human 15-LO is present in abundance in reticulocytes, eosinophils, and human lung, especially bronchial epithelium.38 Using an antibody specific for human 15-LO, immunostaining was observed in bronchial epithelium (positive control), as reported above.38 However, 15-LO immunoreactivity was not observed in pulmonary arteries and arterioles or in normal coronary arteries.
In the sections of coronary arteries obtained from transplanted hearts, two histological forms of graft arteriosclerosis were observed. The first was characterized by a concentric proliferation within the intima of cells with the histological features of smooth muscle cells. The second consisted of complex intimal lesions containing spindle-shaped mesenchymal cells, macrophages, lipid-filled foam cells, extracellular lipid, areas of calcification, and the appearance of neovasculature within the lesion. Such diversity of histological appearance in transplant vascular disease has been reported previously.3 4 5 6
No immunohistochemical evidence for 15-LO was found in transplanted coronary arteries that did not have lipid-rich lesions and exhibited only concentric intimal hyperplasia of smooth muscle cells. 15-LO was absent even when the proliferative process was so advanced as to produce luminal obstruction. The causes of graft arteriosclerosis are not well understood. Current theories suggest that chronic low-level immunological rejection in response to foreign human lymphocyte antigens expressed on endothelial and other cells of the graft is associated with the release of cytokines. This in turn promotes the expression of growth factors that initiate smooth muscle cell migration and proliferation in the intima.31 40 41 The absence of 15-LO staining in lesions of this type of TCAD suggests that the enzyme and possibly cytokines such as IL-4 (known to induce the expression of 15-LO) released by T lymphocytes do not participate to a major extent in the lesions' pathogenesis. The causes of the proliferative lesions of TCAD are currently unknown and the subject of active investigation.31 41
Abundant and intense 15-LO immunostaining was observed in the
transplanted coronary arteries that exhibited complex lipid-rich
atheromatous lesions. By the use of serial sections and specific
antibodies, it was apparent that 15-LO immunostaining occurred in the
cytoplasm of both spindle cells (positive for –smooth muscle actin)
and macrophages (positive for HAM-56). The 15-LO staining of
lipid-laden foam cells was very prominent. Although some of the
15-LO–positive foam cells were of macrophage origin, it appeared that,
in contrast to previous reports of native vessel
atherosclerosis,18 19 the majority were of smooth muscle
cell origin. Endothelial cells also stained positively for 15-LO in
this type of lesion. Although there was variability in the amount of
15-LO staining of endothelial cells from location to location, the
intensity in some regions was striking, and exceeded that observed
occasionally in atherosclerosis of native coronary vessels. Because
endothelial cells of normal vessels, smooth muscle cells of the tunica
media, and normal human monocytes did not manifest 15-LO
immunostaining, the data suggest that 15-LO expression is induced in
endothelial cells, intimal smooth muscle cells, and macrophages in the
atheromatous form of TCAD.
The nature of the signal for 15-LO expression in a subset of lesions in
TCAD is unknown. Conceivably, IL-4 could be involved; it is released in
increased amounts from cardiac allografts even in patients receiving
immunosuppressive therapy sufficient to prevent overt graft
rejection.33 Conrad and coworkers32 reported
that, of a large number of cytokines tested, only IL-4 induced 15-LO
expression in human monocytes in vitro, and that this expression was
inhibited by coincubation with interferon gamma. The cytokine milieu in
TCAD in vivo is probably more complex,31 33 40 41 and is
only partially known. It is possible that synergistic interactions
between different cytokines promote intense stimulation of 15-LO
expression exceeding that reported in in vitro
experiments.32 Finally, the finding that 15-LO expression
was only noted in lipid-rich lesions suggests a role for hyperlipidemia
in the pathogenesis of TCAD. Indeed, this is a common problem in
transplant patients,10 and has been linked in some studies
to the development of the disease.11 12 Of note, all 3
patients in the present study with immunohistochemical expression
of 15-LO had markedly elevated cholesterol levels (Table 2).
In the complex atherosclerotic lesions of native coronary arteries, 15-LO immunostaining occurred predominantly in lipid-filled foam cells, macrophages, myointimal cells, and, to a lesser extent, endothelial cells. In the previous reports of atherosclerosis of native coronary arteries, macrophages positive for 15-LO by in situ hybridization and immunocytochemistry also had positive immunostaining for epitopes of oxidized LDL and for the scavenger receptor involved in the cellular uptake of oxidized LDL.18 19 Slight 15-LO staining of mesenchymal smooth muscle cells and endothelial cells within lesions was also noted. The present observations that the atheromatous type of TCAD manifests prominent expression of 15-LO, as well as abundant intracellular and extracellular lipids, suggest that lipid peroxidation, possibly involving 15-LO, may be involved in the formation of these lesions.
Steinberg, Witztum, and Parthasarathy and their coworkers14 15 16 have reviewed the possible involvement of lipoxygenases, particularly 15-LO, in the oxidative modification of LDL. Their work indicated that LDL is oxidized in vitro by a combination of phospholipase A2 and soybean lipoxygenase and that the oxidative modification of LDL by endothelial cells or monocytes/macrophages can be significantly reduced by lipoxygenase inhibitors such as nordihydroguaiaretic acid or 5,8,11,14-eicosatetraynoic acid.25 29 Although others have challenged the inhibitor studies,42 Belkner and coworkers28 recently demonstrated that recombinant 15-LO is capable of directly oxidizing LDL. They subsequently found that 12-LO from porcine leukocytes also directly oxygenates human lipoproteins in vitro, whereas the human platelet 12-LO and the human 5-LO were less effective.30 Oxidative modification of LDL involves the formation of lipid hydroperoxides, fatty acid fragmentation, conjugation of aldehydes to apo B and phospholipids, and modification of lysine residues on apo B so it is no longer recognized by the LDL receptor and is recognized by the scavenger receptor.16 17 20 The mechanisms by which 15-LO may initiate oxidation of LDL are unclear; it could be a direct effect of release of 15-LO into the extracellular space or, alternatively, an indirect effect with initial oxidation of cell membranes by 15-LO followed by exchange of cell lipids with lipids of the lipoprotein particle.16 17 20 Because the initial products of the oxygenation of arachidonic acid or linoleic acid by 15-LO are hydroperoxy acids, it is also possible that hydroperoxide formation may contribute to LDL oxidation.43
In the lipid-rich lesions of TCAD, as in native atherosclerosis, the presence of 15-LO and oxidized LDL may initiate a series of cellular events and gene expression that lead to additional migration and proliferation of smooth muscle cells and macrophages in the intima of the vessel, with subsequent cellular uptake of lipids to form foam cells, immobilization of the cells, cell death, and extravasation of lipids into the matrix.14 15 16 Study of these phenomena in atherosclerosis of native vessels has led to current investigations of the use of antioxidant drugs to modify the process of atherogenesis. Antioxidant drugs such as probucol (a compound that lowers cholesterol but is also a powerful antioxidant) have been shown to reduce LDL oxidation and slow the progression of atherosclerotic lesions in Watanabe hypercholesterolemic rabbits21 44 and in nonhuman primates fed a diet containing moderately increased amounts of cholesterol.45 Other antioxidants such as N,N'-diphenyl-phenylenediamine or BHT have also slowed the progression of atherosclerosis in rabbit models.24 Interestingly, lower levels of the antioxidant vitamin E and higher levels of lipid peroxides have been found in cardiac transplant patients with graft atherosclerosis than in normal subjects.46 It is therefore possible that, if a role for increased 15-LO expression in promoting TCAD is confirmed by future studies, investigations of antioxidants or specific lipoxygenase inhibitors could provide new insights into the treatment of TCAD.
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Acknowledgments |
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This study was supported in part by National Heart, Lung, and Blood Institute grant HL-21006. The authors wish to thank Llewellyn Ward for his invaluable technical assistance with histological and immunohistochemical techniques.
Received June 3, 1994; accepted December 28, 1994.
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