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(Circulation. 1995;92:1393-1398.)
© 1995 American Heart Association, Inc.

Articles

Interstitial Collagenase (MMP-1) Expression in Human Carotid Atherosclerosis

Seppo T. Nikkari, MD, PhD; Kevin D. O'Brien, MD; Marina Ferguson, BS; Thomas Hatsukami, MD; Howard G. Welgus, MD; Charles E. Alpers, MD; Alexander W. Clowes, MD

From the Departments of Surgery (T.H., A.W.C.), Medicine (K.D.O.), and Pathology (M.F., C.E.A.), University of Washington, Seattle; the Department of Medicine, Washington University School of Medicine at the Jewish Hospital, St Louis, Mo (H.G.W.); and the Department of Medical Biochemistry, University of Tampere (Finland) Medical School (S.T.N.).

Correspondence to Alexander W. Clowes, MD, Professor, Department of Surgery, RF-25, University of Washington, Seattle, WA 98195. E-mail clowes@u.washington.edu.

	Abstract

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Background In human atherosclerosis, most clinical events occur when plaque integrity is compromised and hemorrhage and thrombosis result. One mechanism for this might be the release by plaque cells of matrix-degrading proteases, such as interstitial collagenase (matrix metalloproteinase-1, MMP-1), which degrades two major plaque structural proteins, types I and III collagen. This study was undertaken to determine whether MMP-1 is expressed in human atherosclerotic plaques.

Methods and Results To determine the cellular source and location of MMP-1 in human carotid atherosclerotic lesions, in situ hybridization and immunohistochemistry were performed on 20 endarterectomy specimens. Six nonatherosclerotic carotid arteries also were studied. Intense MMP-1 expression (mRNA and protein) was detected in a subset of plaque macrophages located at the borders of the lipid cores adjacent to fibrous caps and shoulder regions. Subsets of plaque smooth muscle cells and endothelial cells also expressed MMP-1. There was a strong correlation between the percentage of the lipid core occupied by hemorrhage and the percentage of the lipid core perimeter positive for MMP-1 (r=.823, P=.0001). MMP-1 was not detected in any cell type in nonatherosclerotic carotid arteries.

Conclusions This study demonstrates that MMP-1 is expressed by several cell types in human carotid atherosclerosis and that there is a correlation between the expression of the protease and histopathological evidence of plaque instability. Since MMP-1 may degrade the major structural collagens of the plaque, expression of the protease by macrophages in regions critical to plaque integrity could contribute to plaque expansion, rupture, and hemorrhage.

Key Words: atherosclerosis • collagenase

	Introduction

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Advanced human atherosclerotic plaques are characterized by a lipid core covered by a fibrous cap composed of smooth muscle cells (SMCs) and extracellular matrix and may obstruct blood flow by protruding into the arterial lumen.¹ These lesions have chronic inflammatory infiltrates of macrophages and T lymphocytes. In the majority of human plaques, microvasculature arising primarily from the capillaries of the adventitial vasa vasorum infiltrates into the plaque base.^{2 3} Plaque instability, manifesting as ulceration of the fibrous cap, plaque rupture, or intraplaque hemorrhage, is largely responsible for the complications of atherosclerosis.⁴ Instability is characteristic of plaques with a high content of extracellular lipid and an excess of macrophages in the cap.^{5 6} Sites of rupture, particularly in the margins of atherosclerotic plaque fibrous caps, are rich in macrophages.^{7 8 9} Release of proteolytic enzymes, in particular matrix metalloproteinases (MMPs), by these cells has been suggested as a mechanism of cap erosion.^{4 5 6 8} Inflammatory cytokines secreted by macrophages also might induce the release of matrix-degrading proteases from neighboring SMCs and endothelial cells.

MMPs are a family of enzymes that, as a group, can degrade a wide variety of extracellular matrix components. They have been implicated in normal tissue remodeling as well as in inflammatory processes, tumor invasion, and wound healing.¹⁰ MMPs are expressed in vitro by a variety of vascular cells, including macrophages, SMCs, and endothelial cells.^{11 12 13} In atherosclerotic plaques, macrophage-derived foam cells and also some SMCs have been shown to express stromelysin (MMP-3),¹⁴ which degrades principally proteoglycan core protein, laminin, and basement membranes.

Interstitial collagenases are the only metalloproteinases that can cleave native collagen types I and III,¹¹ which are major structural components of the fibrous plaque cap.¹⁵ The major human interstitial collagenase, MMP-1, is secreted by a variety of mesenchymal and epithelial cell types.¹¹ MMP-1 might play a significant role in fibrous plaque disruption by contributing to the degradation of interstitial collagens and thinning of the fibrous cap.¹⁶ It is not known whether MMP-1 is expressed in atherosclerotic lesions or whether it might participate in the pathogenesis of plaque instability. The purpose of this study was to determine, by in situ hybridization and immunohistochemistry, the location and cellular source of MMP-1 in human carotid atherosclerotic plaques and the relation of MMP-1 expression to histopathological evidence of plaque instability.

	Methods

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Human Carotid Tissue
Carotid endarterectomy specimens were obtained from 20 consecutive patients (14 men and 6 women; mean age, 71 years; range, 50 to 83 years) treated by endarterectomy for occlusive carotid disease with a >75% occlusion of the common carotid artery. Half of the patients were symptomatic on the ipsilateral side of the carotid sample: 9 had evidence of transient ischemic attack or ocular symptoms, and 1 had had a stroke. The patients were from the University of Washington and the Seattle Veterans' Affairs Medical Centers. As controls, six carotid arteries were obtained from organ donors. Consent was obtained for research use of all carotid specimens. Samples were fixed in 10% neutral buffered formalin and decalcified with formic acid. After being embedded in paraffin, the carotid specimens were cross-sectioned along the entire length at 1-mm intervals. Transverse sections 8 µm thick were cut and used for in situ hybridization and immunohistochemistry.

The control carotid arteries had only diffuse intimal thickening without inflammatory cell infiltrate, as judged by light-microscopic examination of hematoxylin-eosin–stained sections. The presence of extracellular lipid deposition could not be excluded with histological stains for lipid, since any lipid present in these sections would have been extracted during deparaffinization.

In Situ Hybridization
Sections were deparaffinized and rehydrated in graded alcohols, rinsed in 0.5xSSC, and incubated with 50 µg/mL proteinase K (Sigma Chemical Co) for 40 minutes at 37°C. The slides then were washed briefly in 0.5xSSC and prehybridized in 100 mL of hybridization buffer (50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris, pH 8.0, 5 mmol/L EDTA, 1x Denhardt's solution, 10% dextran sulfate, and 10 mmol/L dithiothreitol). The prehybridization was performed in airtight boxes containing blotting paper saturated with 50% formamide and 4xSSC on the bottom at 55°C for 2 hours. ³⁵S-labeled riboprobes were generated as described previously¹⁷ from a 0.55-kb human MMP-1 cDNA cloned into Bluescript vector (Stratagene) and linearized with BamHI for antisense transcriptions and with Sac II for sense transcriptions.¹⁸ Riboprobes were added to the prehybridized slides in 25 µL of fresh hybridization solution at 3.5x10⁵ cpm per slide. Hybridization was continued overnight at 55°C. The slides then were rinsed three times in 0.5xSSC and immersed in RNase A solution (20 mg/mL in 0.5 mol/L NaCl and 10 mmol/L Tris, pH 8.0) for 30 minutes at 37°C. The slides were rinsed in 2xSSC and washed for 2 hours in 0.1xSSC and 0.5% Tween 20 at 37°C. After rinsing in 2xSSC, the slides were dipped in Kodak NTB-2 nuclear track emulsion and exposed at 4°C for 14 days. Positive controls for the MMP-1 antisense riboprobe included human skin burn wounds.¹⁹ Control hybridizations performed with a sense riboprobe for MMP-1 produced nonspecific background signal only (data not shown).

Immunohistochemistry
Immunohistochemical staining was performed as described previously³ by the immunoperoxidase method with 3,3'-diaminobenzidine plus nickel chloride as a chromogen. Cell types were recognized by the following antibodies: human macrophages by the mouse monoclonal antibody HAM-56 (titer, 1:1000) (Dako Corp),²⁰ SMCs by anti–smooth muscle -actin (titer, 1:1000) (Dako),²¹ and endothelial cells by the lectin Ulex europaeus I (Vector Laboratories Inc).²² MMP-1 protein was localized with rabbit polyclonal antiserum to human MMP-1 (1:4000),^{23 24} which was generated to pure interstitial collagenase produced by U937 cells.²⁵ This antiserum has been shown to recognize human MMP-1 and does not cross-react with any other human MMP.²⁶ Slides were counterstained with hematoxylin. Human skin burn wounds were used as positive control tissue for MMP-1 immunohistochemistry.¹⁹ Negative controls included substitution of normal rabbit serum for MMP-1 antiserum or mouse IgG for monoclonal antibodies as well as omission of the primary antibody, antiserum, or lectin. Double-label immunohistochemistry was performed as described previously.³ Briefly, the slides were incubated with the MMP-1 antibody diluted in PBS plus 1% BSA overnight at 4°C. After being washed, sections were incubated with gold-labeled goat anti-rabbit IgG (Amersham) diluted in PBS (1/40) plus 1% BSA and 0.1% gelatin for 1 hour at room temperature. Sections were washed, and the gold was visualized with an IntenSE M silver enhancement kit (Amersham). The sections then were incubated sequentially with (1) anti–-smooth muscle actin or HAM 56, (2) biotinylated horse anti-mouse IgG (Vector), and (3) avidin-biotin-alkaline phosphatase complex (Vector). The alkaline phosphatase was developed with a red substrate kit (Vector), and the slides were counterstained with methyl green. Negative controls included substitution of normal rabbit serum for the MMP-1 antiserum and substitution of normal mouse IgG for the anti–-smooth muscle actin or HAM-56.

Measurement of Lipid Core Parameters
Transverse sections from three different levels of each of the 20 carotid endarterectomy specimens were deparaffinized in xylene and rehydrated in graded alcohols. Sections were immunostained with the MMP-1 antiserum, counterstained with methyl green, and projected onto a computerized digitizing pad with a camera lucida to measure the total lipid core perimeter and the portion of the perimeter occupied by cells staining for MMP-1. Adjacent hematoxylin-eosin–stained sections were digitized for lipid core area and areas in the core representing old hemorrhage, ie, areas of thrombus and areas with fragments of red blood cells. Areas with fragments of red blood cells were further verified because they stained positively with the Ulex europaeus lectin, which binds to a receptor on red blood cell as well as endothelial cell membranes.²² Regions with intact red blood cells were not included in the hemorrhage area, since these most likely represented recent hemorrhage induced by surgical manipulation at the time of endarterectomy. Five plaques were fully analyzed at 1-mm intervals to determine that only three sections around the area of maximal plaque thickness were necessary to evaluate MMP-1 expression.

Statistics
The results are expressed as mean±SD. Least-squares linear regression analysis was performed with the STATVIEW 512+ program (BrainPower Inc). Values of P<.05 were considered statistically significant.

	Results

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Histopathology
All the carotid endarterectomy specimens had a substantial fibrous cap overlying a lipid core. Calcification was identified in 12 of 20 plaques (60%). The lipid cores had widely varying degrees of old intraplaque hemorrhage, ranging from 2% to 73% of core volume (35±21%). Fibrous cap rupture was infrequent, since the zone of hemorrhage connected with the arterial lumen in only 4 of 20 plaques (20%). The lipid core size, as measured in two-dimensional histological sections, varied from 1 to 28 mm² (10±7 mm²).

Localization of Collagenase Expression in Carotid Arteries by In Situ Hybridization and Immunohistochemistry
In situ hybridization and immunohistochemistry demonstrated that MMP-1 mRNA and protein were particularly prominent at the outer edge of lipid cores in regions containing a high density of macrophages (Fig 1). In many carotid lesions, MMP-1 expression also was present in the shoulder regions (Fig 1) as well as beneath the fibrous cap (Fig 2).

View larger version (0K): [in this window] [in a new window]   Figure 1. Localization of matrix metalloproteinase-1 (MMP-1) protein and mRNA by immunohistochemistry and in situ hybridization in the shoulder region of an atherosclerotic plaque. A, Low-power view showing immunostaining with the MMP-1 antiserum (black reaction product); arrow indicates shoulder region, where there also is some artifactual disruption of tissue due to sectioning; B, higher-power view of the MMP-1 expression in the shoulder region; C, in situ hybridization on an adjacent section with the 35S-labeled MMP-1 riboprobe; and D, immunostaining with the macrophage-specific antibody HAM-56 (black reaction product) on an adjacent section localized to the same area as MMP-1 protein (A and B) and mRNA (C). Methyl green nuclear counterstain (A, B, and D), hematoxylin-eosin counterstain (C); magnification x100 (A) or x400 (B through D). Lumen is at the top.

View larger version (0K): [in this window] [in a new window]   Figure 2. Localization of matrix metalloproteinase-1 (MMP-1) protein and mRNA to areas of macrophages beneath a fibrous cap by immunohistochemistry and in situ hybridization. Adjacent arterial sections were stained (black reaction product) with an antiserum to MMP-1 (A), the macrophage-specific antibody HAM-56 (C), or the smooth muscle cell–specific marker anti–smooth muscle -actin (D); in situ hybridization was with the 35S-labeled MMP-1 riboprobe (B). Methyl green nuclear counterstain (A, C, and D), hematoxylin-eosin counterstain (B); magnification x600.

To confirm the presence of MMP-1 protein in subsets of macrophages and SMCs, double-label immunohistochemistry was performed with the MMP-1 antiserum together with either the macrophage marker (HAM-56) or the smooth muscle marker (anti–-actin). The black reaction product identifying MMP-1 protein and the red reaction product of the macrophage marker (HAM-56) are colocalized to a subpopulation of foam cells at the interface of the lipid core and the fibrous cap (thin arrow, Fig 3A). Some regions of SMCs in the fibrous cap showed positive staining with the MMP-1 antibody (thin arrow, Fig 3B) and some did not (Fig 2D). Similar positive MMP-1 staining also was found in subsets of SMCs in areas of intimal thickening adjacent to plaques in all endarterectomy specimens.

View larger version (0K): [in this window] [in a new window]   Figure 3. Localization of matrix metalloproteinase-1 (MMP-1) protein to macrophages and smooth muscle cells (SMCs) by double-label immunohistochemistry. MMP-1 protein (black reaction product) was localized to subsets of macrophages (red reaction product) (light arrow, A) and SMCs (red reaction product) (light arrow, B). Some macrophages and SMCs were negative for MMP-1 (heavy arrows in A and B). Methyl green nuclear counterstain; magnification x1000.

Microvessels were detected frequently near the intima-media border. The majority of the microvessels did not stain for MMP-1 (data not shown), but positive immunostaining for this protease was present in some endothelial cells in small microvessels in the plaque shoulder regions (Fig 4A). Adjacent sections stained with Ulex lectin confirmed the localization of MMP-1 to the endothelium (Fig 4B).

View larger version (0K): [in this window] [in a new window]   Figure 4. Localization of matrix metalloproteinase-1 (MMP-1) to plaque microvessels by immunohistochemistry. A, Positive immunostaining (black reaction product) associated with endothelial cells in a small vessel in a plaque shoulder region; B, an adjacent section stained with Ulex lectin to detect vascular endothelium; and C, normal carotid intima stained for MMP-1. Methyl green nuclear counterstain; magnification x1000 (A and B) or x400 (C).

Although MMP-1 was detected immunohistochemically in macrophages, SMCs, and endothelial cells, as identified by double-label immunohistochemistry performed with the MMP-1 antiserum and, respectively, the HAM-56, -actin, and Ulex markers, the possibility that occasional unlabeled mesenchymal cells such as fibroblasts also might express MMP-1 could not be excluded.

MMP-1 mRNA and protein were found in a similar pattern in each of the 20 diseased carotid arteries examined but were not detected in the plaque periphery at distances of >0.5 mm from the lipid core. In contrast, in 6 carotid arteries removed from organ transplant donor cadavers, areas with diffuse nonatherosclerotic intimal thickening (representing the normal morphology of adult human arteries) did not stain for MMP-1 (Fig 4C).

Comparison of Histopathological and Clinical Data With MMP-1 Expression
Sections obtained from five endarterectomy specimens at 1-mm intervals beginning at the common carotid artery and extending distally to the internal carotid artery were analyzed to determine the relative amounts of MMP-1 expression (percent of lipid core perimeter occupied by MMP-1–positive cells). The mean value of MMP-1 expression for a set of three sections taken at the site of maximal plaque mass was found to be representative of plaque MMP-1 expression, since it fell within the 95% CI for the mean of all levels analyzed in the five specimens. In specimens with more than one discrete plaque, only one plaque was chosen for analysis.

There was a direct linear correlation between the percentage of the lipid core occupied by hemorrhage and the percentage of the lipid core perimeter positive for MMP-1 (r=.823, P=.0001) (Fig 5). Frequently, we observed MMP-1–positive macrophages at the outer edges of lipid cores away from areas of hemorrhage. The correlation of MMP-1 with lipid core size failed to reach statistical significance (r=.4, P=.066), but the percentage of lipid core occupied by hemorrhage did correlate with core size (r=.50, P=.024). There was no correlation of MMP-1 with plaque rupture (r=.25, P=.29) or symptoms (r=.10, P=.66).

View larger version (10K): [in this window] [in a new window]   Figure 5. Graph of lipid core perimeter positive for matrix metalloproteinase-1 (MMP-1) (% of perimeter length) vs hemorrhage in the lipid core (% of core area) in human carotid atherosclerotic lesions. Each dot represents the value of an individual patient and is the mean of three measurements. r=.823, P=.0001.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates that in human carotid arteries, the matrix-degrading enzyme MMP-1 is expressed in advanced atherosclerotic plaques but not in nonatherosclerotic intima. This enzyme, which degrades types I and III collagen, could contribute to plaque expansion, disruption, and thrombosis. The source of MMP-1 is a population of vascular wall cells, in particular a subset of plaque macrophages.

In vitro, macrophages, SMCs, and endothelial cells have been shown to release a number of matrix-degrading proteases, including MMP-1.^{11 12 13} Macrophage-derived foam cells and also some SMCs in atherosclerotic plaques have been shown to express stromelysin (MMP-3),¹⁴ which degrades principally proteoglycan core protein but not types I and III collagen.²⁷ MMP-1 would be required for the degradation of these two matrix proteins,¹¹ and it has been shown that fibrous caps from unstable plaques have collagen fiber disruption.¹⁶ The present study shows that MMP-1 mRNA and protein are expressed primarily by a subset of plaque macrophages located at regions critical to plaque integrity. Thus, the secretion of this protease by macrophages might contribute to plaque instability. MMP-1 protein was associated with macrophages, SMCs, and microvascular endothelial cells both at the boundary between the fibrous cap and the lipid core and in the shoulder region of the plaque. The location of the protease supports the conclusion that it might play a role in fibrous cap disruption.

MMP-1 expression was confined to the atherosclerotic plaques and was not found in control arteries with diffuse intimal thickening, as judged by light-microscopic examination of hematoxylin-eosin–stained, paraffin-embedded sections. This observation raises the possibility that factors present only in the plaque regulate the expression of MMP-1. MMP-1 expression in vascular cells can be stimulated by many growth factors and cytokines, including interleukin-1, tumor necrosis factor-, and platelet-derived growth factor.^{13 28 29} The expression of MMP-1 is controlled at the transcriptional level by the activating protein-1 family of transactivating factors.³⁰ The upregulation of MMPs by cytokines may be important, since platelet-derived growth factor,³¹ interleukin-1,³² and tumor necrosis factor-³³ have been detected in vascular lesions. In addition to being regulated transcriptionally, all metalloproteinases are secreted as latent proenzymes that require activation that is often proteolytic.³⁴ Plasmin and stromelysin together activate MMP-1 in vitro,^{35 36 37 38} and both of these molecules are present in the atherosclerotic plaque.^{14 39} The presence of organizing thrombus in some carotid plaques suggests the presence of sufficient amounts of plasmin to activate latent MMP-1. On the other hand, intramural hemorrhage also might contain other plasma components, such as ₂-macroglobulin, a potent inhibitor of MMP-1.⁴⁰ The tissue inhibitors of metalloproteinases expressed constitutively by vascular wall cells further modulate MMP activity.^{10 13} Thus, MMP-1 activity in carotid atherosclerotic plaques probably is regulated by a number of factors and cannot be correlated directly with the presence of MMP-1 mRNA or protein. However, the strong association of MMP-1 protein with hemorrhage demonstrated in the present study is consistent with the possibility that MMP-1 contributes to instability of human carotid plaques.

This study demonstrates a direct linear relation between hemorrhage in the lipid core of the plaque and MMP-1 expression by cells at the lipid core perimeter. Intraplaque hemorrhage in carotid atherosclerotic plaques has been reported in many studies.^{41 42} The 20 atherosclerotic carotid endarterectomy specimens examined in this study also had a much higher prevalence of hemorrhage than has been reported in carotid arteries examined at autopsy, probably because all 20 patients had a recent increase in the percent diameter stenosis of the carotid segment removed and half had ipsilateral symptoms, either transient ischemic attack or ocular symptoms, consistent with recent plaque disruption. It is important to note that recent hemorrhage, which was identified histologically by the presence of intact red blood cells and which probably was an artifact induced by surgical manipulation, was not included as hemorrhage in our analyses.

In the present study, intraplaque hemorrhages of different histological ages were observed frequently within the lipid core of an individual lesion. Furthermore, although hemorrhage was present in a strikingly large proportion of the plaques, it did not correlate with fibrous cap rupture. The hemorrhages were only occasionally connected with the lumen and therefore might have originated from other sources, such as ruptured intimal microvessels,^{3 39} rather than from fibrous cap disruption.⁴² These results suggest that increased proteolysis in the neighborhood of the microvessels in concert with mechanical shearing forces could lead to intermittent microvascular hemorrhage. This process could be quite dynamic, since atherosclerotic plaques may grow larger and narrow the carotid lumen at a rapid rate.⁴³ In addition, a proteolytically weakened fibrous cap might be more susceptible to mechanical disruption from the increased shear stress present at plaque shoulders.⁴⁴ Thus, increased MMP-1 expression could contribute to both plaque expansion and fibrous cap disruption.

In conclusion, this study demonstrates that several cell types may express MMP-1 in human carotid atherosclerosis but not in histologically normal carotid intima. The expression of this protease by vascular cells could contribute to plaque instability. The increased expression of MMP-1 by macrophages in regions of the plaque identified by others as being critical to plaque integrity has been linked in this study to intraplaque hemorrhage and may represent a pathological event in the progression of complicated atherosclerotic lesions.

	Acknowledgments

 
This study was supported by grants HL-18645, HL-42270, and HL-47151 from the National Institutes of Health. Dr O'Brien was supported in part by a Clinical Investigator Development Award (HL-02788) from the NIH. Dr Nikkari was supported in part by a grant from the Alfred Kordelin Foundation. The authors thank Kelly Hudkins and Randy Small for excellent help with immunohistochemistry and Robert Bergelin for assistance with the statistical analyses.

Received December 21, 1994; revision received March 2, 1995; accepted March 5, 1995.

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