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Circulation. 1996;94:2756-2767

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(Circulation. 1996;94:2756-2767.)
© 1996 American Heart Association, Inc.

Articles

Increased Accumulation of Tissue ACE in Human Atherosclerotic Coronary Artery Disease

Frank Diet, MD; Richard E. Pratt, PhD; Gerald J. Berry, MD; Naoko Momose, MD; Gary H. Gibbons, MD; Victor J. Dzau, MD

the Division of Cardiovascular Medicine, Falk Cardiovascular Center, Stanford University Medical Center, Stanford, Calif.

Correspondence to Richard E. Pratt, PhD, Dept of Medicine, Brigham & Women's Hospital, Thorn-12, 75 Francis St, Boston, MA 02115.


*    Abstract
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 *Abstract
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Background Angiotensin may play a pathophysiological role in experimental and human cardiovascular disease. Clinical studies have shown that ACE inhibitors reduce mortality, recurrent myocardial infarction, and ischemic events in patients with left ventricular dysfunction. Animal studies suggest that tissue ACE, particularly within blood vessels, may be an important target.

Methods and Results To study tissue ACE in human coronary artery disease and to identify potential mechanisms of ACE inhibitor action, we examined ACE expression immunohistochemically in nonatherosclerotic and diseased human coronary arteries. In nonatherosclerotic arteries, ACE immunoreactivity was found in luminal and adventitial vasa vasorum endothelium. In early- and intermediate-stage atherosclerotic lesions, ACE was detected prominently in regions of fat-laden macrophages and in association with T lymphocytes. In advanced lesions, ACE immunoreactivity was also localized to the endothelium of the microvasculature throughout the plaques. Immunoreactive angiotensin II was also detected in these areas. ACE expression in macrophages was further examined by in vitro experiments with a monocytoid cell line. ACE activity was induced threefold after differentiation of the cells into macrophages and was further increased after stimulation with acetylated LDL.

Conclusions These observations demonstrate that significant sources of tissue ACE in human atherosclerotic plaques are regions of inflammatory cells, especially areas of clustered macrophages as well as microvessel endothelial cells. These results suggest that ACE accumulation within the plaque may contribute to an increased production of local angiotensin that may participate in the pathobiology of coronary artery disease. Plaque ACE probably is an important target of drug action.


Key Words: angiotensin • vasculature • endothelium • immunohistochemistry • lipoproteins


*    Introduction
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 *Introduction
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Angiotensin I converting enzyme, a dipeptidyl carboxypeptidase that converts angiotensin (Ang) I to Ang II and inactivates bradykinin, is present on endothelium.1 2 In the rat vascular injury model, smooth muscle cells express high levels of ACE that may influence the local production of Ang II,3 4 which contributes to intimal hyperplasia in this model by stimulating vascular smooth muscle cell (VSMC) growth and migration.5 6 7 8 9 10 Indeed, ACE inhibitors11 and angiotensin receptor antagonists12 block intimal formation in this model. ACE also has been implicated in the pathogenesis of experimental atherosclerosis. Watanabe rabbits, cholesterol-fed rabbits, or cholesterol-fed cynomolgus monkeys treated with ACE inhibitor showed marked reduction in aortic atherosclerosis.13 14 15 Ang II may contribute to the initiation or progression of atherosclerosis by stimulating adhesion molecule expression and increasing oxidative stress16 17 and may participate in ischemic vascular events by stimulating plasminogen activator inhibitor 1 (PAI-1) production and increasing platelet aggregation.18 19 20 Thus, the reduction of Ang II production plus the increase in bradykinin accumulation and nitric oxide production21 may account for the vascular protection effects of ACE inhibitors. The potential clinical significance is suggested by the findings of the Survival and Ventricular Enlargement (SAVE) and Studies Of Left Ventricular Dysfunction (SOLVD) trials showing that ACE inhibitors reduced the incidence of recurrent myocardial infarction.22 23 These data are consistent with the postulate that ACE inhibition may alter the natural history of human coronary atherosclerosis.

Studies suggest that the primary site of ACE inhibitor action is the vasculature and that it is necessary to block tissue ACE24 25 26 for complete inhibitor action. Thus, the purpose of this study was to examine tissue ACE expression in human atherosclerotic coronary arteries, to characterize the relationship of local ACE to vascular pathology,2727 and to examine the role of LDL cholesterol in tissue ACE expression.


*    Methods
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Tissue
Patient characteristics are summarized in the TableDown. A total of 100 coronary artery segments from 61 arteries (right coronary, n=24; left anterior descending coronary, n=23; circumflex branch of the left coronary, n=14) were examined. Epicardial segments (n=31) from 19 arteries were obtained from 7 hearts with ischemic cardiomyopathy, 51 segments from 25 arteries obtained from 11 hearts with dilated cardiomyopathy, and 12 segments from 11 arteries obtained from 4 hearts with congenital heart disease. In addition, 6 directional atherectomy specimens were obtained from 6 patients with ischemic heart disease (1 primary and 5 restenotic lesions). All specimens were obtained at the Stanford University Medical Center, and informed consent for pathological examination of the tissue was obtained.


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  		Table 1. Clinical Patient Characteristics

Artery segments were collected at the time of heart transplantation within 10 minutes of heart explantation. Vessels were perfused with cold PBS, dissected from the myocardium, and kept in ice-cold PBS for 30 to 60 minutes before embedding and freezing in OCT compound (Miles Laboratories). These specimens were collected within 15 months (July 1993 to October 1994) and represent 70% of the total available specimens. Atherectomy specimens, retrieved percutaneously by directional atherectomy (Simpson Athero Cath), were kept in ice-cold saline for 60 to 90 minutes before embedding and freezing in OCT. Specimens were kept at -80°C until further processing. Serial frozen sections (6 µm) were cut on a cryostat (2800 Frigocut, Reichert Jung), placed on 3-amino-propyltriethoxysilane–coated slides, air dried, and stored at -20°C until further processing.

Immunocytochemistry
The antibodies used in this study are as follows (antibody name/number, specificity, source, and working dilution): RIN 7002, human Ang II, Ang III, Peninsula, 1:2000; 9B9, human lung ACE, Biotrack, 15 µg/mL; 5F1, human lung ACE, Biotrack, 20 µg/mL28 29 30 ; EBM11, macrophages, Dako, 1:800; HAM 56, macrophages, some endothelial cells, Dako, 1:200; 1A4, smooth muscle actin, Sigma, 1:800; F8/86, von Willebrand factor (factor VIII), Dako, 1:500; CR3/43, MHC class II molecules on macrophages, endothelial cells, activated T lymphocytes, and subpopulations of smooth muscle cells, Dako, 1:500; RPA 20-10, CD2 antigen on T lymphocytes, thymocytes, and natural killer cells, Zymed, 1:100; Bu20a, control (antibromodeoxyuridine), Dako, 15 or 20 µg/mL; MOPC21, nonspecific control antibody, Sigma, 15 or 20 µg/mL.

Slides were warmed to room temperature, fixed for 10 minutes in cold acetone, dried, and rehydrated in PBS at room temperature. All subsequent steps were performed at room temperature. Sections were pretreated with 10% nonimmune goat serum in PBS (10 minutes), incubated with primary or control antibody for 60 minutes, and washed for 15 minutes in PBS. A biotinylated F(ab')2 fragment of goat anti-mouse immunoglobin (1:100 dilution of the supplied stock solution, Zymed) was incubated for 30 minutes. Endogenous peroxide activity was blocked with 3% H2O2 in PBS at room temperature for 15 minutes. After a brief wash and incubation with streptavidin-peroxidase complex (Zymed) for 20 minutes, color was developed (3 to 6 minutes) with 3-amino-9-ethylcarbazole (AEC) with the use of the Zymed AEC substrate kit. Cell nuclei were counterstained with hematoxylin. Positive staining with AEC appeared as a red color. Negative controls included replacement of the primary antibody by immunoglobins of the same isotype, same species, and same concentration.

Double staining for ACE and macrophages was performed with the use of a modified, commercially available system (Histostain-DS, Zymed) that consists of two sequential immunocytochemical procedures. Sections pretreated with 10% nonimmune rabbit serum in PBS (10 minutes) were incubated with anti-ACE antibody for 60 minutes and washed in PBS plus 0.05% Tween 20 (3x5 minutes). A biotinylated F(ab')2 fragment of rabbit anti-mouse IgG was added (1:100 dilution of the supplied stock) in 1% BSA/PBS. After a wash, the sections were incubated with streptavidin-alkaline-phosphatase. 5-Bromo-4-chloro-3-indolyl phosphate/tetranitro blue tetrazolium was used as a substrate/chromogen, which produces a dark purple stain. Endogenous alkaline phosphate activity was blocked by addition of levamisole (2 mmol/L) to the substrate/chromogen mixture. Macrophages were then identified with HAM 56 as a primary antibody, a biotinylated F(ab')2 fragment of rabbit anti-mouse IgM followed by hydrogen peroxide/AEC/peroxidase as a substrate/chromogen/enzyme system, which produced a red color. Omitting the primary antibody in the second procedure did not produce any staining in the second immunocytochemistry, demonstrating that there was no interaction of the components of the secondary immunocytochemistry with those of the first step.

Immunohistochemical detection of Ang II was performed with antisera (rabbit anti–Ang II, RIN 7002, Peninsula Laboratories, Inc) at a dilution of 1:2000 with paraformaldehyde-fixed, paraffin-imbedded tissue. Antibody-antigen complex was detected with biotinylated goat anti-rabbit IgG followed by streptavidin-peroxidase complex and AEC as described above.

Neutral lipid staining was performed with oil red O. Briefly, cryostat sections were rehydrated in dH2O, rinsed in 60% isopropanol, stained in an aqueous solution of 60% isopropanol containing 0.24% oil red O, rinsed in 60% isopropanol and dH2O, and counterstaining of cell nuclei with hematoxylin.

ACE Activity
ACE enzymatic activity was measured with the use of a fluorometric assay31 measuring the generation of His-Leu from a hippuryl-His-Leu (Sigma Chemical Co). ACE activity was calculated as nanomoles His-Leu generated per minute per 106 cells.

Cell Culture
We used the human monocytoid cell line THP-132 because these cells can differentiate into macrophages. Cells were passaged in RPMI 1640 with L-glutamine, 10% FCS, and 25 mmol/L HEPES. Twenty-four hours after plating, cells were treated with vehicle, PMA (100 nmol/L), acetylated LDL (100 µg/mL), or PMA plus acetylated LDL and harvested 96 hours later. ACE activity was determined as described above. For immunocytochemistry, cells stimulated with PMA (100 nmol/L, 72 hours) were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X and 0.2% gelatin in PBS, and further processed as outlined above.


*    Results
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 *Results
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Classification of Vessel Morphology
Since the severity of atherosclerotic disease exhibits heterogeneity along a vessel, harvested vessels were cut into multiple segments and analyzed independently and classified according to Gown et al.27 The American Heart Association Committee on Vascular Lesions33 has proposed a classification containing nine types and subtypes as reviewed by Fuster.34 Accordingly, we have attempted to relate our finding in this study to this classification. Coronary artery segments were examined and categorized histopathologically as nonatherosclerotic (class I, n=37), fibro-fatty (class II, n=5), fibrous (class III, n=20), or advanced (class IV, n=32) plaques. The distribution of plaque histomorphologies was in agreement with the underlying diagnosis and/or the presence of risk factors. In 31 segments from patients with ischemic heart disease, all were designated as class IV (advanced, n=23) or class III (fibrous, n=8). In contrast, of those with dilated cardiomyopathy, a majority were class I (nondiseased, 37 of 51) or class II (fibro-fatty, 4 of 51). Of the 6 patients with dilated cardiomyopathy exhibiting the presence of class III or IV lesions, 3 had 2 or more cardiovascular risk factors. A majority of the segments (10 of 12) from patients with congenital heart disease exhibited class I morphology.

The atherectomy specimens could not be classified because of the difficulty in orientation of the specimen and the destruction of plaque morphology. Two of the specimens showed areas with many clustered macrophage cells. The other four specimens showed only scattered if any macrophages.

Class I (No Atherosclerosis)
Sections from 37 coronary artery segments were classified as nonatherosclerotic. All segments were obtained from patients with dilated cardiomyopathy or congenital heart disease; no nondiseased segments were found in patients with ischemic heart disease. Nonatherosclerotic segments were characterized by the absence of intima or the presence of concentric intimal thickening with an intact media (Fig 1Down). The medial and intimal layers were composed mainly of actin-positive smooth muscle cells (not shown). Few if any macrophages could be detected in the intima (Fig 1FDown). Immunoreactivity for von Willebrand factor, indicating endothelial cells, could be found both on the luminal surface as well as in the vasa vasorum (Fig 1EDown). This Gown class I morphology corresponds to AHA normal and type I lesions.



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  		Figure 1. Expression of ACE in nonatherosclerotic coronary arteries. Photomicrographs of sections from nonatherosclerotic coronary arteries demonstrating immunocytochemical localization of ACE. ACE immunoreactivity localizes to luminal endothelial cells (A, x100; B, x400) and the tunica media (A and C, x100). The arrowheads point to the internal elastic lamina (IEL). The adventitial vasa vasorum was detected by immunoreactivity for von Willebrand factor (E, x400; arteriole is marked by one asterisk; venule is marked by two). Arterioles, characterized by a thick wall, stain stronger for ACE than adventitial venules (D, x400). Scattered macrophages (F) do not show ACE immunoreactivity (A). An isotype-matched monoclonal control antibody is unreactive with the tissue (G). L indicates the lumen.

ACE immunoreactivity was detected in endothelial cells (Fig 1Up, A, B, and C), but the staining intensity for ACE in the luminal endothelium varied among different segments. In 11 nondiseased segments, no ACE immunoreactivity was found in luminal endothelium. On the other hand, in all segments examined, positive immunoreactivity for ACE could be found in endothelial cells of at least one adventitial vasa vasorum (37 of 37) (Fig 1DUp). These results are consistent with the results of Rogerson et al.35 Adventitial arterioles, characterized by thicker walls than those of venules, stained stronger and more consistently for ACE than did the venules. Luminal endothelial cells stained less intensely in two thirds of the sections or with equal intensity in one third of the sections compared with endothelial cells of adventitial arterioles.

ACE immunoreactivity was variably detected in the medial smooth muscle of the nonatherosclerotic vessels (19 of 37) (Fig 1Up, A and C). The staining was weak but when detected, it was greater than that observed with the two control antibodies for ACE. Although scattered macrophages were occasionally observed in the intima of these sections, they did not stain for ACE (Fig 1FUp).

Class II (Fibro-Fatty Lesions)
A total of 5 segments from 5 patients were classified as having class II, fibro-fatty lesions, characterized by loose connective tissue matrix containing small groups of clustered macrophages (Fig 2Down). The medial and luminal endothelial layers were intact. As in the nonatherosclerotic lesions, ACE immunoreactivity was variably detected on luminal endothelial cells (Fig 2Down, A and B) and vasa vasorum, which exhibited stronger immunoreactivity than did the luminal endothelial cells (2 of 5). This class II lesion likely corresponds to the AHA type II and III.



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  		Figure 2. Expression of ACE in fibro-fatty lesions. Photomicrographs (x100) of serial sections from a fibro-fatty lesion (IEL indicates internal elastic lamina) demonstrating immunocytochemical localization of ACE. In this example, weak ACE immunoreactivity (A) colocalizes with immunostaining for von Willebrand factor, suggesting the presence in endothelial cells (B). In addition, strong immunostaining for ACE is found in regions of strong macrophage immunoreactivity (C). An isotype-matched monoclonal control antibody is unreactive on the tissue (D). L indicates the lumen.

ACE immunoreactivity also could be found in diffuse streaks throughout the intimal regions of all 5 lesions (Fig 2Up, A and C) that were found in regions of clustered, foamy macrophage cells. Invariably, these regions of clustered macrophages exhibited positive ACE immunoreactivity.

Class III (Fibrous Plaque)
Class III lesions are fibrous plaques characterized by a fibrous cap overlaying a hypocellular or acellular core. Macrophages, T lymphocytes, microvessels, and areas of calcification were present in variable amounts, contributing to the heterogeneity of plaques (Fig 3Down). All plaques had macrophage involvement (Fig 3CDown) but were heterogeneous with respect to the presence of clustered, high-density foamy macrophages, with 8 of 20 lesions identified as fibrous lacking significant clustering of macrophages. The medial layers of the vessel walls were intact. According to the AHA classification, these lesions can be defined as types IV, Va, and Vb.



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  		Figure 3. Expression of ACE in fibrous plaques. Photomicrographs (x40) of serial sections from a fibrous plaque. ACE immunoreactivity (A) colocalizes with immunoreactivity for von Willebrand factor (suggesting expression in endothelial cells, B) and immunostaining for macrophages (C). An isotype-matched monoclonal control antibody is unreactive on the tissue (D). Areas with macrophages colocalize with areas of lipid deposition stained with oil red O (E) and with immunostaining for MHC II antigen (F). L indicates the lumen.

In 12 of 20 plaques, ACE immunoreactivity was associated with regions of inflammatory cells, especially in clusters of foamy-appearing macrophages (Fig 3AUp). Indeed, adjacent sections stained with oil red O for lipids showed colocalization of ACE immunoreactivity, foamy macrophages, and areas of lipid staining (Fig 3EUp). In sections with only scattered macrophages, little or no ACE immunoreactivity was detected. Immunostaining of selected sections from fibrous (and advanced) plaques with an antibody for MHC class II antigen HLA-DR revealed strong immunoreactivity associated with macrophage that also stained positive for ACE in sequential sections, suggesting that these macrophages were activated (Fig 3FUp). Staining for MHC class II antigen had a wider distribution than staining for macrophages (Figs 3F and 4UpDown), with endothelial and smooth muscle cells close to infiltrating macrophages also exhibiting MHC II immunoreactivity, suggesting that these cells were activated also. Because it is known that T lymphocytes can induce macrophage ACE expression, we stained serial sections with a T cell–specific marker (Fig 4BDown) or anti-ACE antibody (Fig 4ADown). All plaques with ACE-positive, clustered, foamy macrophages showed a variable number of T lymphocytes colocalizing with areas of macrophages. In the majority of sections the number of T lymphocytes in these plaques was significantly lower than the number of macrophages.



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  		Figure 4. Expression of ACE in fibrous plaques. Photomicrographs (x100) of serial sections from a fibrous plaque demonstrating immunocytochemical localization of ACE (A), T lymphocytes (B), and macrophages (C). L indicates the lumen.

Microvessels were found in 6 of the 20 fibrous plaques. In these, the amount and location of the microvessels varied. In those with few microvessels (3 of 6), the microvessels were found near the intima/media border. However, with greater plaque vascularization, microvessels were observed throughout the plaque. In all 6 plaques, endothelial cells of microvessels exhibited prominent ACE immunoreactivity, with immunoreactivity of endothelial cells from neovessels often stronger than immunoreactivity of the luminal endothelial cells. As in nonatherosclerotic segments (19 of 37) and in fibro-fatty lesions (3 of 5), diffuse ACE immunostaining of medial smooth muscle cell layers could be variably detected in sections from fibrous plaques (16 of 20). (Examples of microvessel staining are shown in Fig 5Down advanced lesions.)



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  		Figure 5. Expression of ACE in advanced plaques. Photomicrographs (x40) of two advanced plaques from two separate patients (first patient, A, C, and E; second patient, B, D, and F) demonstrating immunocytochemical localization of ACE. In serial sections of plaque, ACE immunoreactivity (A and B) colocalizes with an area of high immunoreactivity for macrophages (C and D) and areas of immunoreactive for von Willebrand factor (endothelial cells, F). L indicates the lumen.

Class IV (Advanced Plaque)
Advanced plaques were characterized by a broad spectrum of morphologies including massive intimal thickening, lumen occlusion, extensive plaque calcifications, and reorganization of vessel thrombosis (Fig 5Up). Typically, the media was not intact, with infiltration by macrophages, neovessels, and/or significant distortion of its structure. The internal elastic lamina was disrupted. The luminal endothelial cell lining was variably intact. This class IV morphology encompasses the AHA types Vc and VI lesions.

ACE immunoreactivity, associated with macrophage-rich areas, was detected in all patients (11 of 11) who had plaques of this class and in nearly all segments (27 of 32) (Fig 5Up, A and B).

Plaque vascularization was higher (26 of 32) than in fibrous plaques (Fig 5Up, A, B, E, and F). Similar to those in fibrous plaques, advanced plaque microvessels were found near the intima/media border if only few microvessels were present (7 of 26) but were observed throughout the plaque (19 of 26) if many microvessels were present. Endothelium of microvessels showed prominent ACE immunoreactivity that was greater than or equal to the staining of luminal endothelial cells. Staining intensity for ACE of medial smooth muscle cell layers, luminal endothelial cells, and vasa vasorum was similar to the intensity observed in other lesion classes (I-III).

Localization of ACE in Regions of Clustered Macrophage and T Lymphocytes
ACE immunoreactivity was consistently found to be associated with inflammatory cells, especially in macrophage-rich areas, suggesting that macrophages are a major cell type that contains ACE. To examine this, we performed double immunostaining using the ACE antibody and the macrophage-specific antibody (Fig 6Down). The results suggest that many of the ACE-positive cells also stained positive for the macrophage marker. Indeed, under high power, ACE immunoreactivity appeared associated with membranes of HAM 56–positive cells. However, examination of multiple sections reveals that while in many cases there is good agreement of ACE and macrophage immunoreactivity (Fig 6Down, C through F), the concordance was not perfect, suggesting the presence of ACE immunoreactivity in cells other than the macrophage. However, these areas of ACE immunoreactivity were always found in close proximity to areas of inflammation, especially in areas of clustered macrophages and T lymphocytes (identified by staining with an antibody specific for CD2, Fig 4Up).




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  		Figure 6. Expression of ACE in regions of clustered macrophage cells. Photomicrographs demonstrating the cellular localization of ACE in two high-power views (x400) of two advanced plaques of two different patients. A, Localization of ACE immunoreactivity on the cell surface. B, Section stained in a double staining procedure for ACE (black) and macrophages (red). Note localization of ACE (black) on the cell surface of foamy macrophages. Examination of several regions from different plaques (each row depicts a different plaque) demonstrates that in many cases, there is a strong association between the presence of ACE immunoreactivity (right-hand column) and immunoreactivity for macrophage markers (left-hand column) ( C and D, E and F).

Colocalization of Ang II in Atherosclerotic Plaques
We next performed immunohistochemical analysis of Ang II in selective samples that were fixed in paraformaldehyde and embedded in paraffin. Staining was performed only on specimens from the patient not receiving ACE inhibitors who also had class IV lesions (patient 17), since ACE inhibitors would be expected to affect the levels of Ang II. Our data (Fig 7Down) suggest the presence of immunoreactive Ang II in the atherosclerotic plaque, especially in areas of high macrophage infiltration and ACE localization. However, the data must be considered preliminary because it involves the examination of tissue from only one patient.



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  		Figure 7. Angiotensin (Ang) II in atherosclerotic plaques. Photomicrographs (x100, A and B; x400, C) of serial sections of an advanced plaque demonstrating immunocytochemical localization of Ang II. Ang II immunoreactivity (A) is found diffusely throughout the plaque, especially in areas (C) characteristic of macrophage cells. A nonimmune rabbit serum was negative (B).

Lack of Relation of ACE Immunoreactivity With ACE Inhibitor Treatment
In animals, pulmonary ACE mRNA is decreased by Ang II and increased by converting enzyme inhibitors.36 Since ACE inhibitors are used frequently in heart failure therapy, we performed chart reviews to examine potential relations between ACE inhibitor treatment and tissue ACE immunoreactivity. ACE inhibitors would be expected to decrease ACE activity but not ACE immunoreactivity. A majority of patients with cardiomyopathy were receiving ACE inhibitors at the time of vessel collection (7 of 7 ischemic, 10 of 11 dilated, 1 of 4 with congenital heart disease). Specimens from one patient (patient 17) withdrawn from ACE inhibitors 1 month before vessel harvest had lesions of class II-IV. Of these, 4 of 7 specimens exhibited clustered macrophages that were positive for macrophage-associated ACE immunoreactivity. The remaining 3 specimens did not have clustered macrophage cells. Importantly, all patients with dilated cardiomyopathy were receiving ACE inhibitors, yet the majority had no macrophage ACE staining in their blood vessels. In the patients with congenital heart disease, only 1 of 4 was receiving ACE inhibitors. The 3 not receiving the inhibitors had nondiseased vessels.

We also obtained atherectomy specimens (Fig 8Down) from 6 patients with ischemic heart disease (5 restenotic lesions, 1 primary atherosclerotic lesion) who were not receiving ACE inhibitors. Diffuse ACE immunoreactivity colocalizing with macrophage-rich areas was found in sections of 2 patients. The specimens from the remaining patients did not show any ACE immunoreactivity or macrophage-rich areas. ACE immunoreactivity was not seen in plaque vascular smooth muscle cells. Thus, these data would suggest that the presence of immunoreactive ACE in atherosclerotic plaques was independent of ACE inhibitor therapy.



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  		Figure 8. Expression of ACE in atherectomy samples. Photomicrographs (x100) of serial sections of an atherectomy specimen demonstrating immunocytochemical localization of ACE. ACE immunoreactivity (A) colocalizes with areas of immunoreactivity for macrophages (C). Note the lack of vessels within the specimen (von Willebrand factor immunoreactivity, B). An isotype-matched monoclonal control antibody is unreactive on the tissue (D).

Macrophage ACE Expression and LDL Cholesterol
The data demonstrate the expression of ACE in macrophage-rich regions of the atherosclerotic plaques, especially in those regions in which macrophages appeared to be lipid-laden foam cells. To examine this in more detail, we examined THP-1 cells, a monocytoid cell line of human origin. These cells grow in suspension with characteristics of monocytic cells. Treatment of the cells with phorbol esters (PMA) induced the differentiation of these cells into adherent macrophage-like cells and increased ACE activity threefold37 (Fig 9Down). Treatment of these cells with PMA plus acetylated LDL led to a twofold further increased ACE expression. Cells treated with PMA showed strong ACE immunoreactive staining.




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  		Figure 9. Left, Regulated expression of ACE in cultured macrophage cells. Cultured monocytoid cells, THP-1, were cultured in the presence of vehicle, PMA, acetylated LDL, or PMA plus acetylated LDL for 96 hours. Cells were then harvested, counted, and assayed for ACE activity. Results are expressed as ACE activity per 106 cells (mean±SEM). Statistical analysis was by ANOVA followed by Fisher PLSD. *P<.05 compared with control; +P<.05 compared with PMA. Note that differentiation of the monocytoid cells into macrophage cells results in the induction of ACE activity, which is further enhanced by acetylated LDL. Right, ACE immunoreactivity in PMA-treated THP-1 cells.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Experimental studies in rats, rabbits, and primates suggest a role of Ang II in vascular remodeling and atherosclerosis.3 4 10 11 12 13 14 15 Moreover, clinical trials of ACE inhibitors in patients with ischemic heart disease22 23 demonstrate a reduced incidence of ischemic cardiac events, suggesting a role for ACE in human coronary atherosclerosis. However, there is little information available regarding the expression of tissue ACE in human atherosclerotic coronary arteries. This issue is of interest because ACE inhibitors may exert local effects beyond blood pressure reduction.26 39 40 The purpose of this study was to examine the possibility of whether in human coronary atherosclerotic lesions, tissue ACE expression is significantly increased, and if so, to identify the cell type involved. Our data demonstrate that in human atherosclerotic plaques, in addition to luminal endothelial cells, there is marked ACE accumulation in regions of inflammatory cells, especially in areas of clustered macrophages and T lymphocytes. Furthermore, in advanced lesions, neovasculature within the plaque exhibits enhanced ACE immunoreactivity. The plaque ACE staining in these "hot spots" is substantially more intense than luminal ACE and is colocalized with immunoreactive Ang II in the lesion. In our analysis, we attempted to correlate the presence of ACE within the plaque to a clinical end point or risk factor. However, it appears that ACE immunoreactivity was detected in the vast majority of segments that also contained clustered macrophages, independent of other parameters.

Ang II may participate at many points in the initiation and progression of atherosclerosis. Endothelial cell activation with expression of adhesion molecules and subsequent adhesion of monocytes are thought to be initiating events in atherosclerosis. Ang II induces adhesion molecule expression in human endothelial cells16 and activates human monocytes, resulting in increased adhesion to human endothelial cells.41 Ang II also is chemotactic for T lymphocytes42 and stimulates growth, migration, and matrix production in smooth muscle cells5 6 7 8 9 10 43 44 potentially contributing to plaque growth. Ang II might also affect the plaque microenvironment. For example, Ang II has been shown to induce NADH and NADPH oxidase activity with resulting superoxide production in VSMC.17 Superoxide radicals have numerous effects on cell function, promoting atherosclerotic plaque growth including induction of growth of VSMC, lipid peroxidation, inactivation of nitric oxide, and stimulation of adhesion molecule expression.45 46 47 48 In addition to the generation of Ang II, ACE also inactivates bradykinin, a peptide that also inhibits VSMC growth.49 Thus, there are several possible points at which ACE may contribute to the atherosclerotic plaque.

Several factors may contribute to the increased ACE accumulation within the atherosclerotic plaque. The milieu of the vascular lesion includes cytokines and oxidized LDL cholesterol that may stimulate the macrophage to express ACE. Consistent with this hypothesis, differentiation of THP-1 monocytoid cells into macrophage-like cells is associated with a threefold increase in ACE expression. Further stimulation with acetylated LDL induced an additional twofold increase. The lipoprotein-induced expression of ACE by a human monocytoid cell line, together with the observation that in vivo lipid-laden plaque macrophages express ACE, suggests that oxidized lipids might play a role in activating ACE expression in vivo. In addition, all of these plaques showed varying degrees of staining for T lymphocytes that may also play a role in the increased ACE expression since, in several in vivo and in vitro studies,50 51 52 53 T lymphocytes enhance ACE expression.

We observed differences in ACE immunostaining of endothelial cells in different vessel wall locations and vessel types, perhaps caused by local mechanical forces such as shear stress54 or local paracrine regulation. The novel finding of intense immunostaining for ACE of endothelial cells of plaque microvessels may have pathophysiological implications, since microvessels could act as a paracrine source of Ang II, promoting migration and subsequent proliferation of smooth muscle cells from the media into the plaque. Interestingly, it has been reported that dividing cells within human atheroma, identified by positive staining for the proliferating cell nuclear antigen, localize preferentially to areas of microvascularization, an indication of ongoing growth factor activity in these regions.55 Furthermore, a high plaque ACE concentration caused by numerous microvessels and/or numerous ACE-expressing macrophages might contribute to vasospasm of coronary segments as a result of the inactivation of the vasodilator bradykinin and generate the vasoconstrictor Ang II. Indeed, it has been demonstrated that atherosclerotic coronary artery segments are prone to vasospasm.56

We are intrigued by the observation that ACE inhibition prevents acute ischemic syndromes in patients with ventricular dysfunction. It is postulated that local inflammation, oxidative stress, changes in plaque composition, and hemodynamic stresses may promote plaque rupture and induce an acute coronary event.57 We speculate that the increased ACE expression in coronary vessels may promote the induction of acute ischemic events through the effects of Ang II on platelet aggregation, the inhibition of fibrinolysis, or the increase in oxidative stress or vasospasm. While the precise mechanisms causing plaque rupture are not fully understood,58 a recent autopsy study of patients who died acutely of myocardial infarction reported that the site of plaque rupture or erosion of the thrombosed coronary artery was marked by an inflammatory process59 characterized by abundant expression of HLA-DR antigen on macrophages, T lymphocytes, and smooth muscle cells. It is striking that these areas of plaque instability coincide with the regions in which we have demonstrated colocalization of foamy macrophages and ACE staining, lipid deposition, T lymphocytes, and abundant expression of HLA-DR antigen. Taken together, these findings support the hypothesis that the inhibition of vascular ACE may modify these destabilizing plaque processes and thereby reduce the incidence of ischemic cardiac events.

Summary
Our data demonstrate that besides endothelial cells in diseased and nondiseased human coronary arteries, medial smooth muscle cells and plaque lipid–laden macrophages express ACE. ACE expression in macrophages is induced by oxidatively modified LDL. We hypothesize that ACE is involved in modulation of plaque inflammation by macrophages. These findings might help to explain, in part, the beneficial effects of ACE inhibitor therapy in patients with ischemic heart disease. However, our data should not be overinterpreted with regard to the differentiation between the effects of various ACE inhibitors (ie, penetrant versus nonpenetrant), since this has not been examined in human studies. Future studies are necessary to prospectively examine the incidence of acute ischemic events and the relation to lesion pathology as assessed by new technologies such as quantitative angiography, intravascular ultrasound, and magnetic resonance imaging to further define the mechanisms in which ACE inhibition influences the natural history of human atherosclerosis.


*    Acknowledgments
 
This study was supported by NIH grants HL-42663 and HL-48638. The authors wish to thank Margaret E. Billingham, MD, for her assistance in the plaque classification. The authors also wish to thank Drs Julian Smith, John Conte, John Stevens, Mario Pompili, Chris Heck, and Gregg Ribakove from the Department of Cardiovascular Surgery and Philip Hui (Department of Pathology) for their assistance in obtaining the surgical specimens examined in this study. We also wish to thank Wendy Lee for technical assistance.

Received February 21, 1996; revision received June 25, 1996; accepted July 1, 1996.


*    References
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