Skip Navigation

Cardiovascular Research 2005 65(1):272-282; doi:10.1016/j.cardiores.2004.09.020
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kong, Y.-Z.
Right arrow Articles by Lan, H. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kong, Y.-Z.
Right arrow Articles by Lan, H. Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Evidence for vascular macrophage migration inhibitory factor in destabilization of human atherosclerotic plaques

Yao-Zhong Konga,b, Xiao-Ru Huangb, Xiaosen Ouyangb, Jei-Ju Tana, Gunter Fingerle-Rowsonc, Michael Bacherd, Wei Mub, Larry A. Schere, Lin Lengf, Richard Bucalaf and Hui Y. Lanb,*

aThe First People's Foshan Hospital, Foshan, Guangdong, China
bDepartment of Medicine-Nephrology, Baylor College of Medicine, One Baylor Plaza, Alkek N520, Houston, TX 77030, USA
cGSF/Hämatologikum, Munich, Germany
dInstitute of Immunology, Philipps-University Marburg, Germany
eDivision of Peripheral Vascular Surgery, North Shore Long Island Jewish Health System, Manhasset, NY, USA
fYale University School of Medicine, New Haven, CT, USA

* Corresponding author. Tel.: +1 713 798 1303; fax: +1 713 798 5010. Email address: hlan{at}bcm.tmc.edu

Received 7 April 2004; revised 25 August 2004; accepted 21 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine and has been shown to play a role in pathogenesis of atherosclerosis. The aim of this study is to investigate the potential role of MIF in the destabilization of atherosclerotic plaques by stimulation of vascular MMP-1 expression.

Methods: MIF and matrix metalloproteinase protein-1 (MMP-1) expression in human atherosclerotic plaques were determined by immunohistochemistry. The functional activity of MIF was examined by its ability to induce MMP-1 expression in vascular smooth muscle cells (VSMCs) in vitro.

Results: Two-color immunohistochemistry demonstrated that MIF was strongly upregulated in vulnerable, but not in fibrous plaques. Upregulation of vascular MIF was associated with macrophage accumulation (p<0.01), strong expression of vascular MMP-1 (p<0.001), and collagenolysis in vulnerable atheromatous plaques, but not in the fibrous lesions. Co-expression of MIF and MMP-1 in vulnerable atheromatous plaques appeared to contribute to the weakening of fibrous caps and plaque disruption. The role of MIF in vascular MMP-1 expression was demonstrated by the ability of MIF to directly stimulate VSMCs to express MMP-1 mRNA and protein, and to increase MMP-1 activity in a dose- and time-dependent manner, which was blocked by a neutralizing MIF antibody (p<0.001).

Conclusions: MIF and MMP-1 are markedly upregulated in vulnerable atheromatous plaques and are associated with the weakening of the fibrous cap. The ability of MIF to induce MMP-1 expression and collagenolytic activity in VSMCs suggests that MIF may play a role in the destabilization of human atherosclerotic plaques.

KEYWORDS Atherosclerosis; Cytokines (MIF); Matrix metalloproteinases; Macrophages; Smooth muscle cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Atherosclerosis is an inflammatory process characterized by the accumulation of lipid-rich macrophages, vascular smooth muscle cells (VSMCs), lipids, and extracellular matrix (ECM) [1–4]. Atherosclerosis complicated by plaque rupture or thrombosis is a major cause of potentially lethal acute coronary syndromes and stroke [2–4]. Rupture occurs frequently in plaques containing a soft, lipid-rich core that is covered by a thin and inflamed cap of fibrous tissue [3,4]. It has been demonstrated that the ruptured plaques usually have thinner caps, with less collagen, fewer VSMCs, and more macrophages [3,4]. Therefore, the major determinants of plaque vulnerability and rupture are progressive lipid accumulation, ongoing inflammation, and cap weakening, which are associated with increased collagen degradation and impaired healing and repair process by VSMCs [2–4]. It is generally accepted that matrix metalloproteinases (MMPs) play an essential role in plaque instability [5–7]. Of them, MMP-1 is believed to be important since it is responsible for the initial cleavage of fibrillar collagen type I and III, the major matrix components in atherosclerotic plaques [8,9]. MMP-1 is upregulated in human advanced atherosclerotic plaques and aneurysmal lesions [10]. In addition, MMP-1 is also highly expressed in rabbit atheromatous lesions induced by balloon injury and atherogenic diet, but was downregulated by cholesterol-lowering drugs which are associated with stabilization of the atheromatous lesions [11]. Many inflammatory factors such as oxidized LDL [12], hypercholesterolemia [11], inflammatory cytokines such as IL-1β and TNF-{alpha} [12–14], and C-reactive protein [15], all have been shown to upregulate MMP-1 expression by endothelial cells and VSMCs.

Macrophage migration inhibitory factor (MIF) is a unique pro-inflammatory cytokine and is crucial in regulating immune-mediated diseases including septic shock [16,17], arthritis [18], acute respiratory distress syndrome [19], glomerulonephritis [20,21], and allograft rejection [22]. Recently, we and other investigators also demonstrated that MIF participates in both experimental and human atherosclerosis [23,24]. MIF is expressed by vascular endothelial cells, VSMCs, and macrophages [23,24]. Upregulation of vascular MIF is associated with macrophage adhesion, accumulation, and foam cell transformation during atherogenesis [23]. Blockade of MIF using a neutralizing MIF antibody reduces vascular inflammation, cell proliferation, and neointimal thickening [25]. Furthermore, deficiency of MIF attenuates atherogenesis in low-density lipoprotein receptor-deficient (LDLr–/–) mice [26]. Direct evidence for the role of MIF in destabilization of atheromatous plaques comes from a recent study in Apolipoprotein E-deficient mice [27]. In this study, inhibition of MIF with a neutralizing MIF antibody resulted in a shift in the cellular composition of neointimal plaques toward a stabilized phenotype with reduced macrophage/foam cell content and increased SMC content [27]. These observations underscore a potentially important role for MIF in atherosclerosis and identify a new potential target for modulating atherogenesis. In addition, MIF has been shown to stimulate MMP-1 and MMP-3 mRNA expression in synovial fibroblasts obtained from patients with rheumatoid arthritis and to upregulate MMP-9 and MMP-13 in rat osteoblasts [28,29]. These findings suggest a pathogenic role for MIF in the destruction of joint tissues and cartilage in arthritis. However, the precise mechanism whereby MIF induces the instability of atherosclerotic plaques has not been fully understood. We hypothesize that induction of MMP-1 expression by VSMCs may be a mechanism by which MIF induces the destabilization of atherosclerotic plaques.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Human arterial tissues
Surgical specimens of human atherosclerotic arteries were obtained from 42 patients (31 male, 11 female, mean age 62.3 ± 7.2 years) undergoing femoral bypass (n=29), and repair of an abdominal aortic aneurysm (n=7). Surgical specimens of renal arterial atherosclerosis were obtained from patients undergoing unmatched kidney transplantation (n=6) at the First Foshan Hospital, Guangdong, China, and at the North Shore University Hospital, NY, USA. In addition, five normal renal arteries obtained from unmatched kidney transplantation donors were used as normal controls. The study protocols were approved by the Human Investigation Review Committee at both Institutions and conformed with the Declaration of Helsinki. Atherosclerotic plaques were classified as previously described [9]. Plaques with thickness of fibrous cap ≥ 0.8 mm, fibroblasts/VSMCs and collagen matrix ≥ 10%, and macrophages and lipid content ≥ 10% were designated as fibrous (Fig. 1F), whereas, lesions with fibrous cap thickness ≥ 0.3 mm, fibroblasts/VSMCs and collagen matrix ≥ 10%, and macrophages and lipid content more than ≥ 20% were designated as vulnerable plaques (Fig. 1B). If lesions were complicated by plaque rupture, they were classified as vulnerable plaques regardless of the cap thickness in the intact cap area (Fig. 1D). Of these, 16 cases (6 from patients with abdominal aneurysm, 6 from those with femoral bypass, and 4 from those with renal arterial atherosclerosis) were classified as vulnerable atheromatous plaques including two cases with plaque rupture and 18 cases (all from patients with femoral bypass) as fibrous plaques. Eight cases (one from a patient with abdominal aneurysm, two from renal artery, and five from those with femoral bypass) were between fibrous and vulnerable plaques and were excluded from this study.


Figure 1
View larger version (167K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Double-immunohistochemistry demonstrates that MIF and MMP-1 expression by VSMCs in vulnerable, but not in fibrous plaques. MIF and MMP-1 are labeled as blue and VSMCs labeled with the anti-{alpha}-SMA Ab are brown. Note that co-expression of MIF (or MMP-1) and {alpha}-SMA is labeled as purple. (A, B) A normal human artery. MIF (A) and MMP-1 (B) are weakly expressed by VSMCs. (C, D) A set of serial sections of a vulnerable atheromatous plaque from a patient with abdominal aneurysmal repair. MIF expression (C) is markedly upregulated and is associated with strong expression of MMP-1 (D) by VSMCs, leading to weakening of fibrous cap. (E, F) A set of serial sections of fibrous plaque from a patient with femoral bypass. Note that fibrous cap is thickened and MIF (E) and MMP-1 (F) are not upregulated by VSMCs. *Lipid-core lesions. Magnifications: x 200.

 
2.2. Antibodies and recombinant human MIF
The mouse anti-MIF monoclonal antibody (mAb) [22,23] and rabbit anti-human MMP-1 antibody (Chemicon, Temecula, CA) were used in this study. In addition, polyclonal rabbit antibodies that recognize human collagen type 1 collagen (Santa Cruz Biotech., Santa Cruz, CA) or is reactive to the carboxy-terminal COL2-3/4Cshort neoepitope generated by cleavage of native human collagen by human collagenase MMP-1 (C1, 2C, IBEX Diagnostics, Montreal, Quebec, Canada) were also used. Other antibodies used in this study include: KP-1, a mouse anti-human CD68 mAb (that recognizes monocytes/macrophages); UCHL1, a mouse anti-CD45RO mAb that recognizes mature, activated T-cells and a subset of resting T-cells; a mouse anti-human {alpha}-smooth muscle actin mAb; a rabbit anti-human GAPDH; goat anti-mouse IgG; swine anti-rabbit IgG; mouse or rabbit peroxidase anti-peroxidase complexes (PAP); mouse alkaline phosphatase anti-alkaline phosphatase complexes (APAAP), and DAKO Enversion+Peroxidase System (rabbit). All antibodies were purchased from Dako (Dako, Capinteria, CA). Recombinant human MIF (rhMIF) was cloned, expressed in Escherichia coli, and purified from the soluble fraction of the cell lysate by two-step high-pressure liquid chromatography (HPLC) as follows: (i) size exclusion HPLC on Bio-Sil TSK 250 (Bio-Rad, Munich, Germany) and (ii) ion-exchange HPLC on Ultropac TSK CM-3SW (LKB/Pharmacia, Freiburg, Germany) as described previously [30]. rhMIF contained <10 pg of endotoxin per microgram of recombinant protein as determined by the chromogenic Limulus amoebocyte assay (Chromogenix, Mölndal, Sweden).

2.3. Immunohistochemistry
Collected arteries were cut and serial sections (4 µm) were stained with one- and two-color immunohistochemistry using a previously described microwave-based method [20–23,31]. Briefly, after microwaving, sections were labeled with KP1, UCHL1, MMP-1, or {alpha}-SMA antibodies using a three-layer PAP, and developed with 3,3-diaminobenzidine to produce a brown product. For double immunostaining, a second round of microwave oven heating was used to denature bound immunoglobulins within the tissue, thereby preventing antibody cross-reactivity [28]. Sections then were labeled with the anti-MIF mAb or anti-MMP-1 mAb using a three-layer APAAP method, and developed with Fast Blue BB Salt (Ajax Chemicals, Melbourne, Australia). Sections were mounted in an aqueous medium and examined under microscopy.

A mouse anti-rat CD45 mAb (OX-1) and normal rabbit IgG, which do not react with human tissues, were used as negative controls.

2.4. Cell culture
Characterized rat aorta-derived smooth muscle cells were kindly provided by Dr. Andrew Kahn (University of Texas, Houston, TX, USA) [32]. Cells (passage 8) were cultured in DMEM containing 10% fetal bovine serum (FBS) until sub-confluence. Cells then were serum starved for 24 h and recombinant human MIF (rhMIF) at doses of 0, 10, 25, 50, 100 ng/ml were added into the culture in the presence or absence of a neutralizing MIF mAb (IgG1, 10–20 µg/ml) or an isotype control mAb (73.5 that specifically recognizes the human CD45RO antigen, IgG1, 10–20 µg/ml) for 0, 3, 6, 12, and 24 h. MMP-1 mRNA was examined by real-time PCR and protein expression by Western blot analysis.

2.5. MMP-1 mRNA detection by real-time PCR
RNA isolation for real-time PCR was performed as previously described [23]. Real-time, one step RT-PCR was performed with SYBR Green PCR Reagents (Sigma), the Thermoscript RT-PCR system (Invitrogen) and the Opticon DNA Engine (MJ Research), according to manufacturer's instructions; 100 ng of total RNA was reversibly transcribed prior to PCR as follows: 94 °C for 2 min followed by 40 cycles of denaturation, annealing, and extension at 94 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s each, respectively, and final extension at 72 °C for 10 min. Primers used for detection of MMP-1 mRNA were: forward 5'-ATGTGGATGCTGCATACGAGC-3' and reverse, 5'-GACAGCATCTACTTTGTCGCC-3'. PCR reaction for each sample was done in triplicate for both target genes and for the GAPDH control. Ratios for MMP-1/GAPDH mRNA were calculated for each sample and expressed as the mean ± S.D. At least three independent experiments were performed.

2.6. MMP-1 protein detection by Western blotting
As described previously [9], 20 µg of protein extracts were mixed with SDS-PAGE sample buffer, boiled for 5 min, electrophoresed on a 10% SDS polyacrylamide gel, and electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham International, Buckinghamshire, UK). After blocking, the membrane was incubated with rabbit antibodies to MMP-1, followed by the secondary anti-rabbit antibody (1:20,000) and developed using the ECL detection kit (Amersham) to produce a chemiluminescent signal. Densitometric analysis was performed with NIH Image software. Ratios for MMP-1/GAPDH protein were calculated for each sample and expressed as the mean ± S.D. At least three independent experiments were performed.

2.7. Analysis of MMP-1 activities
Two methods were applied to detect MMP-1 activity. Firstly, in situ collagenase-cleaved interstitial type I collagen in atherosclerotic plaque lesions was detected by immunostaining with a polyclonal rabbit antibody reactive to cleavage of native human collagen by human collagenase MMP-1 (C1, 2C, IBEX Diagnostics). The antibody has been shown to specifically detect collagenase-cleaved type I collagen in human vulnerable atheromatous plaques without immunoreactivity to native or denatured human type I or III collagens [9]. Briefly, after microwaving, paraffin-sections were incubated overnight with either rabbit anti-human collagen I (Santa Cruz) or COL2-3/4Cshort (C1, 2C) antibodies, followed by peroxidase-conjugated goat anti-rabbit antibody and then DAKO Enversion+Peroxidase (rabbit), and developed with DAB to produce brown products. Secondly, MMP-1 activity in cultured medium under various conditions was measured by ELISA in triplicates using The CHEMICON MMP Collagenase Activity Assay Kit (Chemicon International), according to the manufacturer's protocol. MMP-1 collagenase activity was determined using biotinylated bovine native collagen as substrate. The cleaved biotinylated fragments of collagen by MMP-1 were transferred to biotin-binding 96-well microtiter plates and detected by streptavidin-peroxidase complex and enzyme substrate. The optical density of the wells was determined. Collagenase activity was calculated against standards with pre-activated human MMP-1 and data was expressed as the mean ± S.D.

2.8. Quantitation of immunohistochemistry
The number of KP-1, UCHL1 positive cells in atherosclerotic plaques was counted by means of a 0.02 mm2 graticule fitted in the eyepiece of the microscope, and expressed as cells per mm2. Since MIF and MMP-1 were widely expressed in both vulnerable and fibrous plaques, their expressions were determined by quantitative Image Analysis System (Optima 6.5, Media Cybernatics, Silver Springs, MD). Briefly, the examined area of fibrous caps and shoulders was outlined and the positive staining patterns were identified, then the percent positive area in the examined area was measured. The lipid and necrotic core, and vascular lumen space were excluded from the study. All scoring was performed blinded on coded slides and data expressed as the mean ± S.D.

2.9. Statistical analysis
Statistical analysis was performed using GraphPad Prism3.0 (GraphPad Software, San Diego, CA, USA). Differences in MIF and MMP-1 expression, macrophage and T cell accumulation between vulnerable and fibrous plaques were assessed by unpaired t-test, while correlation between MIF expression and macrophage or T cell accumulation, and MMP-1 expression was analyzed by Pearson linear correlation analysis.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. MIF and MMP-1 expression by vascular smooth muscle cells in human vulnerable and fibrous plaques
As shown in Fig. 1A and B, two-color immunohistochemistry demonstrated that MIF and MMP-1 were weakly expressed by {alpha}-SMA+VSMCs in normal human arteries. However, marked upregulation of MIF and MMP-1 was found in vulnerable atheromatous plaques as demonstrated by a large number of MIF+{alpha}-SMA+ (Fig. 1C) or MMP-1+{alpha}-SMA+ (Fig. 1D) cells in the fibrous cap, leading to weakening of the cap. In contrast, expression of MIF and MMP-1 by VSMCs was largely reduced in fibrous plaques with thickening of fibrous caps (Fig. 1E, F). Interestingly, {alpha}-SMA+ cells with strong expression of MIF and MMP-1 were enlarged in size in the vulnerable cap (Fig. 1C, D), while those with weak MIF and MMP-1 expression showed elongated, dense, and small size in the thickened fibrous cap (Fig. 1E, F). These changes may be associated with their functional activities.

3.2. MIF expression and macrophage and T cell accumulation in human vulnerable and fibrous plaques
Two-color immunohistochemistry also showed that MIF was strongly expressed within vulnerable atheromatous plaques by CD68+ macrophages (Fig. 2B). In addition, secreted MIF was also found within the plaque, particularly in the core-lesion (Fig. 2B). Strikingly, upregulation of MIF in vulnerable plaques was associated with the numerous macrophage accumulation (Figs. 2B and 3Go). This was profound in the inflamed shoulder and cap and was associated with weakening of fibrous caps (Fig. 2B). In contrast, stable fibrous plaques had thickened fibrous cap and a few macrophages without significant upregulation of MIF (Figs. 2E and 3Go). Only a few T cells (UCHL1+) within the atheromatous plaques were noted. Semi-quantitation of macrophages and T cells in both vulnerable and fibrous plaques is shown in Fig. 3B.


Figure 2
View larger version (175K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Double-immunohistochemistry demonstrates that MIF and MMP-1 are strongly co-expressed in vulnerable, but not in fibrous plaques. (A) A normal human artery. MIF (blue) and MMP-1 (brown) are weakly expressed by vascular endothelial cells and VSMCs. (B and C) A set of serial sections of a vulnerable atheromatous plaque from a patient with femoral bypass. MIF expression (blue) is markedly upregulated and is associated with numerous CD68+ macrophage (brown) accumulation and weakening of the fibrous cap (B), which is associated with marked upregulation of MMP-1 (brown, C). Note that the secreted MIF (blue) is evident in the lipid-core lesion (B). (D) A ruptured plaque from a patient with abdominal aortic aneurysm. Note that MIF (blue) and MMP-1 (brown) are co-expressed and markedly upregulated by morphologically elongated cells (presumably VSMCs and myofibroblasts, see insert picture) in the fibrous cap and shoulder with plaque rupture (arrow). (E) A fibrous plaque from a patient with femoral bypass. Note that fibrous cap is thickened and MIF (blue) is not upregulated with a few CD68+ macrophages (arrowheads). (F) A fibrous plaque from a patient with femoral bypass. Note that both MIF (blue) and MMP-1 (brown) are not upregulated in a fibrous plaque. *Lipid-core lesions. Magnifications: x 100.

 

Figure 3
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Quantitative analysis of MIF, MMP-1, macrophages, and T cells in vulnerable and fibrous plaques. (A) MIF and MMP-1 expression, (B) CD68+ macrophages and CD45R0+ T cells. Each bar represents the mean value ± S.D. for groups of patients with vulnerable (n=16) or fibrous (n=18) plaques. **p<0.01, ***p<0.001 compared to fibrous plaques.

 
3.3. Co-expression of MIF and MMP-1 in human vulnerable and fibrous plaques
MMP-1 was weakly expressed and co-localized with MIF expression in both VECs and VSMCs in normal human arteries (Fig. 2A). However, two-color immunohistochemistry showed that MMP-1 was markedly upregulated and co-localized with MIF expression in vulnerable atheromatous plaques (Fig. 2C). As indicated in a set of serial sections, CD68+ macrophages and morphologically elongated VSMCs or myofibroblasts were the major cell types for dual expression of MIF and MMP-1 (Fig. 2B vs. C). Importantly, marked upregulation of MIF and MMP-1 in vulnerable plaques was associated with weakening of the fibrous cap (Fig. 2C). Notably, in two cases with progressive plaque disruption, elongated VSMCs and myofibroblasts were a major source of both MIF and MMP-1 in the ruptured fibrous cap (Fig. 2D). In contrast, there was no significant MIF and MMP-1 expression in fibrous plaques and fibrous cap (Fig. 2E, F). Semi-quantitation of MIF and MMP-1 expression in both vulnerable and fibrous plaques was shown in Fig. 3A.

3.4. Upregulation of MIF and MMP-1 is associated with an increase in collagenolysis in vulnerable atheromatous plaques
Since upregulation of immunoreactive MMP-1 in vulnerable atheromatous plaques does not necessarily reflect the activity of MMP-1, we examined MMP-1 collagenase activity by immunostaining for the collagenase-cleaved type I collagen fibrils in sets of serial sections. As shown in Fig. 4, immunohistochemistry revealed that there was a marked increase in immunoreactive cleaved type I collagen (Fig. 4A, C) and reduction in intact type I collagen accumulation (Fig. 4B, D) in the cap and shoulder of vulnerable or ruptured plaques. An increase in collagenolysis in vulnerable and ruptured plaques was associated with upregulation of MIF and MMP-1 as demonstrated in serial sections (Figs. 2B–D). In contrast, fibrous plaques showed little cleavage of type I collagen with abundant intact type I collagen accumulation in the thicken fibrous cap (Fig. 4E, F). This was also associated with reduction of MIF and MMP-1 expression in serial sections (Fig. 2E, F).


Figure 4
View larger version (174K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Increased collagenolysis in human vulnerable and ruptured plaques compared with fibrous plaques. MMP-1 collagenase activity is determined by immunohistochemistry with the antibody that specifically recognizes the cleaved human type I collagen (A, C, E) as described in Methods and compared with the immunoreactive nature human type I collagen (B, D, F) in serial sections. (A, B) Serial sections of a vulnerable plaque obtained from a patient with femoral bypass (see MIF and MMP-1 expression in serial sections in Fig. 2B and C). (C, D) Serial sections of a ruptured plaque from a patient with abdominal aortic aneurysm (see MIF and MMP-1 expression in serial sections in Fig. 2D). (E, F) Serial sections of a fibrous plaque from a patient with femoral bypass (see MIF and MMP-1 expression in serial sections in Fig. 2E and F). Results show that collagenolysis is markedly increased in the cap and shoulder of vulnerable and ruptured plaques, but not in a fibrous plaque. In contrast, type I collagen accumulation is abundant in a thicken fibrous plaque, but not in vulnerable and ruptured plaques. *Lipid-core lesions. Magnifications: x 100.

 
3.5. Correlation of MIF expression and macrophages, T cells, and MMP-1 expression in human atherosclerotic plaques
As shown in Fig. 5, correlation analysis revealed that upregulation of MIF in both vulnerable and fibrous plaques was significantly correlated with macrophage, but not T cell, accumulation (Fig. 5A, B). Further analysis also showed that upregulation of MIF was highly correlated with an increase in MMP-1 expression (Fig. 5C).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Correlation analysis of MIF expression and T cells, macrophages, MMP-1 expression in vulnerable and fibrous plaques. (A) T cells, (B) macrophages, (C) MMP-1. Each point represents one patient.

 
3.6. MIF is able to induce MMP-1 expression and activity by VSMCs in vitro
Since VSMCs and macrophages were the major cell types producing MIF (Figs. 1B and 2BGo) during the development of atherosclerotic plaques and because expression of MMP-1 by VSMCs was tightly associated with the weakening of fibrous cap or plaque rupture (Figs. 1D and 2C, DGo), we postulated that VSMCs and macrophages-derived MIF may be responsible for the upregulation of MMP-1 by VSMCs in vulnerable plaques. Since VSMCs are the major cell type with strong MIF and MMP-1 expression in fibrous caps of vulnerable plaques as shown in Fig. 1C and D and because the functional activities of VSMCs in the cap are a critical determinant of plaque stability [2–4], the functional role of MIF in induction of MMP-1 expression and activity was investigated by stimulating VSMCs with MIF in vitro. As shown in Fig. 6, although real-time PCR demonstrated that constitutive expression of MMP-1 mRNA was low in normal VSMCs (Fig. 6A), addition of MIF strongly induced MMP-1 mRNA expression by VSMCs in a time- and a dose-dependent manner (Fig. 6A, B), being significant at 3 h (Fig. 6A). The specificity of MIF to induce MMP-1 mRNA expression in VSMCs was further demonstrated by the addition of a neutralizing MIF mAb. As shown in Fig. 6B, MIF-induced MMP-1 mRNA expression by VSMCs at 3 h was substantially blocked by a neutralizing MIF mAb, but not by an isotype control Ab.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 MIF induces MMP-1 mRNA expression by VSMCs demonstrated by real-time PCR. (A) MIF (25 ng/ml) induces MMP-1 mRNA expression by VSMCs in a time-dependent manner, being significant at 3 h. (B) MIF induces MMP-1 mRNA expression by VSMCs (at 3 h) in a dose-dependent manner. Note that MIF (25 ng/ml)-induced MMP-1 mRNA expression at 3 h is blocked by a neutralizing MIF mAb (10 µg/ml), but not by an isotype control mAb (CTL Ab, 10 µg/ml). Each bar represents the ratio of MMP-1 mRNA/GAPDH (mean ± S.D.) for at least three independent experiments. *p0.05, ***p<0.001 compared to time 0 (A) or dose 0 (B, open bar); ###p<0.001 compared to MIF (25 ng/ml, solid bar) and MIF (25 ng/ml) +CTL Ab (hatched bar).

 
Next, we further analyzed the ability of MIF to induce MMP-1 protein expression by VSMCs. As shown in Fig. 7, addition of MIF induced MMP-1 protein expression in a time- and a dose-dependent fashion (Fig. 7A, B). Again, addition of a neutralizing MIF antibody, but not an isotype control antibody, was able to block MIF-induced MMP-1 protein synthesis by VSMCs (Fig. 7C), indicating the specificity of MIF to induce MMP-1 protein expression.


Figure 7
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 MIF induces MMP-1 protein expression by VSMCs demonstrated by Western blotting. (A) MIF (25 ng/ml) induces MMP-1 protein expression by VSMCs in a time-dependent manner, being significant at 24 h. (B) MIF, but not control normal mouse IgG1 (nmIgG1, 25 µg/ml), induces MMP-1 protein expression by VSMCs at 24 h in a dose-dependent manner. (C) MIF (25 ng/ml)-induced MMP-1 protein expression at 24 h is blocked by a neutralizing MIF mAb (10 µg/ml), but not by an isotype control mAb (CTL Ab, 10 µg/ml). Each bar represents the ratio of MMP-1 protein/GAPDH (mean ± S.D.) for at least three independent experiments. *p<0.05, ***p<0.001 compared to time 0 (A) or dose 0 and nmIgG1(B), or MIF and CTL Ab.

 
MIF-induced MMP-1 activity measured by The CHEMICON MMP Collagenase Activity Assay Kit was shown in Fig. 8. Indeed, MIF was able to significantly increase MMP-1 collagenolytic activity in a dose-dependent manner, which was completely blocked by the addition of neutralizing MIF antibody.


Figure 8
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 MIF induces MMP-1 activity as determined by The CHEMICON MMP Collagenase Activity Assay Kit. Results show that MIF induces collagenase activity, as determined by cleavage of human type I collagen by MMP-1, in cultured VSMCs at 24 h in a dose-dependent manner. MIF (50 ng/ml)-induced MMP collagenase activity at 24 h is blocked by a neutralizing MIF mAb (20 µg/ml), but not by an isotype control mAb (CTL Ab, 20 µg/ml). Each bar represents the calculated value (mean ± S.D.) against pre-activated human MMP-1 standards for three independent experiments. **p<0.01, ***p<0.001 compared to dose 0, ###p<0.001 compared to MIF (50 ng/ml) and CTL Ab.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Increasing evidence has shown that MIF may play a pathogenic role in atherosclerosis. In experimental rabbit model of atherosclerosis, we first demonstrate that de novo expression of MIF by VECs is associated with monocyte adherence onto endothelial cells and upregulation of vascular MIF by VSMCs is associated with the development of atherosclerotic plaques [23]. The potential importance of MIF in atherogenesis has been further supported by a study in human [24] and by a functional blocking study in atherosclerosis-susceptible mice in which vascular inflammation and neointimal thickening after angioplasty are reduced by a neutralizing MIF antibody [25]. The pathogenic role MIF in atherosclerosis is confirmed by the finding that LDLr–/– mice null for MIF have impaired atherosclerosis [26]. Furthermore, the ability of blocking MIF with a neutralizing MIF antibody to stabilize atherosclerotic plaques in apolipoprotein E-deficient mice indicates that MIF may be involved in the instability of atherosclerotic plaques [27]. In the present study, we extend these previous findings and provide evidence that MIF may play an important, upstream role in the destabilization of human atheromatous plaques by stimulating vascular MMP-1 expression. This is supported by the finding that upregulation of MIF within the vulnerable atheromatous plaques is associated with strong co-expression of MMP-1, which is associated with an increase in collagenolysis and the weakening of fibrous caps or plaque disruption. Furthermore, evidence for a role of MIF in destabilization of atheromatous plaques is also supported by the finding that MIF is able to directly upregulate MMP-1 expression and its functional activity by VSMCs, a key cell type in the production of collagen matrix and determination of the stability of fibrous cap. Thus, MIF may be involved in the destabilization of atheromatous plaques by its chemotatic effect on macrophages and stimulating vascular MMP-1 expression and activity in vulnerable human atheromatous plaques.

It is well documented that the integrity of the fibrous cap is important for the stability of atherosclerotic plaques, which depends largely on the collagenous extracellular matrix produced by VSMCs or myofibroblasts [3,4]. An increased inflammatory response, decreased ECM synthesis, and increased ECM degradation contribute significantly to plaque instability [1–4]. Increased MMP activity appears to be the most important risk factor for the instability of vulnerable atheromatous plaques or plaque rupture [2–8]. Several MMPs including MMP-1, -2, -3, -9, -13, and -14 may be involved [2–8]. Among them, MMP-1 has been considered as a highly specific protease capable of degrading interstitial collagens including Type I and Type III [8,9], the most important components of the ECM in fibrous caps, resulting in the development of vulnerable atheromatous plaques. Because of the functional specificity of MMP-1 and its localization in human vulnerable atheromatous plaques with active collagenolysis [9,33], it has been hypothesized that MMP-1 contributes to the expansion and rupture of the plaque. In the present study, the finding that upregulation of MIF and MMP-1 is associated with marked collagenolysis in vulnerable plaques suggest a causal role of MIF in the destabilization of the plaque. Although macrophages and VSMCs/myofibroblasts are two major cell types expressing MMP-1, the functional activity of this enzyme in atherogenesis and plaque complications may be cell-type specific. Transgenic overexpression of MMP-1 in macrophages has been noted to produce protective effects on the atherogenesis with smaller and less advanced lesions in ApoE knockout mice [34]. This may be associated with the findings that the degradation of type I collagen by MMP-1 prevents the differentiation of monocytes into macrophages and impairs VSMC migration to the lesion, the critical process in atherogenesis [34]. In addition, since oxidized LDL binds collagen types I and III efficiently [35], expression of MMP-1 on macrophages may degrade LDL-bound collagens, thereby reducing neointimal retention of oxidized LDL [34]. Thus, transgenic overexpression of MMP-1 in macrophages delays atherogenesis. In contrast, as shown in the present study, once the atherosclerotic plaque is formed, expression of MMP-1 by VSMCs and myofibroblasts, as well as macrophages, in vulnerable plaques degrade the collagen matrix and impair the healing process, resulting in the weakening of fibrous cap and plaque rupture. Therefore, upregulation of MMP-1 in vulnerable atheromatous plaques may play a critical role in plaque expansion and rupture.

Several inflammatory mediators such as IL-1, TNF-{alpha}, and OxLDL have been shown to increase the expression of MMP-1 by VSMCs, endothelial cells, and macrophages [8–14]. The present study provides the first evidence that MIF, which is considered to be a high, upstream activator of the pro-inflammatory response, also contributes to the pathophysiology of plaque instability by activating MMP-1. Indeed, MIF is constitutively expressed by many cells including macrophages, endothelial cells, and VSMCs [23,24] and it is released immediately from pre-formed pools upon stimulation with endotoxin, exotoxin, TNF-{alpha}, interferon-{gamma}, and OxLDL [16–18,21]. Once released, MIF acts by autocrine and paracrine pathways to further stimulate the expression of cytokines (IL-1, TNF-{alpha}, INF-{gamma}) and adhesion molecules [16–21]. Thus, it is likely that the local production of vascular MIF induced by OxLDL not only causes macrophage adhesion, migration, and foam cell transformation during atherogenesis [23,24], but also contributes to the instability of the plaques by stimulating MMP-1 expression by VSMCs directly, and indirectly by the induction of other inflammatory cytokine cascades.

In summary, this study demonstrates that MIF and MMP-1 are strongly co-expressed in vulnerable human atheromatous plaques and may be associated with the instability or rupture of atherosclerotic plaques. The ability of MIF to activate vascular MMP-1 and may be a key mechanism by which MIF mediates the destabilization of the atherosclerotic plaques.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported in part by grants from the Juvenile Diabetes Foundation (JDRF 1-2001-596) and NIH P50DK064232-01 (HYL) and1RO1AR49610 (RB).


   Notes
 
Time for primary review 36 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Libby P. Inflammation in atherosclerosis. Nature (2002) 420(6917):868–874.[CrossRef][Medline]
  2. Lusis A.J. Atherosclerosis. Nature (2000) 407:233–241.[CrossRef][Medline]
  3. Gronholdt M.L., Dalager-Pedersen S., Falk E. Coronary atherosclerosis: determinants of plaque rupture. Eur. J. Heart (1998) (Suppl. C):C24–C29.
  4. Shah P.K. Pathophysiology of coronary thrombosis: role of plaque rupture and plaque erosion. Prog. Cardiovasc. Dis. (2002) 44:357–368.[CrossRef][ISI][Medline]
  5. Galis Z.S., Khatri J.J. Matrix metalloproteinases in vascular remodeling and atherosclerosis: the good, the bad, and the ugly. Circ. Res. (2002) 90:251–262.[Abstract/Free Full Text]
  6. Loftus I.M., Naylor A.R., Bell P.R., Thompson M.M. Matrix metalloproteinases and atherosclerotic plaque instability. Br. J. Surg. (2002) 89:680–694.[CrossRef][ISI][Medline]
  7. Shah P.K., Galis Z.S. Matrix metalloproteinase hypothesis of plaque rupture: players keep piling up but questions remain. Circulation (2001) 104:1878–1880.[Free Full Text]
  8. Matrisian L.M. The matrix-degrading metalloproteinases. Bioassays (1992) 14:455–463.[CrossRef][ISI][Medline]
  9. Sukhova G.K., Schonbeck U., Rabkin E., Schoen F.J., Poole A.R., Billinghurst R.C., et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable atheromatous plaques. Circulation (1999) 99:2503–2509.[Abstract/Free Full Text]
  10. Orbe J., Fernandez L., Rodriguez J.A., Rabago G., Belzunce M., Monasterio A., et al. Different expression of MMPs/TIMP-1 in human atherosclerotic lesions. Relation to plaque features and vascular bed. Atherosclerosis (2003) 170:269–276.[CrossRef][ISI][Medline]
  11. Aikawa M., Rabkin E., Okada Y., Voglic S.J., Clinton S.K., Brinckerhoff C.E., et al. Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma: a potential mechanism of lesion stabilization. Circulation (1998) 97:2433–2444.[Abstract/Free Full Text]
  12. Rajavashisth T.B., Liao J.K., Galis Z.S., Tripathi S., Laufs U., Tripathi J., et al. Inflammatory cytokines and oxidized low density lipoproteins increase endothelial cell expression of membrane type 1-matrix metalloproteinase. J. Biol. Chem. (1999) 274:11924–11929.[Abstract/Free Full Text]
  13. Galis Z.S., Muszynski M., Sukhova G.K., Simon-Morrissey E., Libby P. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann. N. Y. Acad. Sci. (1995) 748:501–507.[Abstract]
  14. Zhu Y., Hojo Y., Ikeda U., Takahashi M., Shimada K. Interaction between monocytes and vascular smooth muscle cells enhances matrix metalloproteinase-1 production. J. Cardiovasc. Pharmacol. (2000) 36:152–161.[CrossRef][ISI][Medline]
  15. Williams T.N., Zhang C.X., Game B.A., He L., Huang Y. C-reactive protein stimulates MMP-1 expression in U937 histiocytes through Fc[gamma]RII and extracellular signal-regulated kinase pathway: an implication of CRP involvement in plaque destabilization. Arterioscler. Thromb. Vasc. Biol. (2004) 24:61–66.[Abstract/Free Full Text]
  16. Bernhagen J., Calandra T., Mitchell R.A., Martin S.B., Tracey K.J., Voelter W., et al. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature (1993) 365:756–759.[CrossRef][Medline]
  17. Calandra T., Echtenacher B., Roy D.L., Pugin J., Metz C.N., Hultner L., et al. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat. Med. (2000) 6:164–170.[CrossRef][ISI][Medline]
  18. Mikulowska A., Metz C.N., Bucala R., Holmdahl R. Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice. J. Immunol. (1997) 158:5514–5517.[Abstract]
  19. Donnelly S.C., Haslett C., Reid P.T., Grant I.S., Wallace W.A., Metz C.N., et al. Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome. Nat. Med. (1997) 3:320–323.[CrossRef][ISI][Medline]
  20. Lan H.Y., Bacher M., Yang N., Mu W., Nikolic-Paterson D.J., Metz C., et al. The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat. J. Exp. Med. (1997) 185:1455–1465.[Abstract/Free Full Text]
  21. Lan H.Y., Yang N., Metz C., Mu W., Song Q., Nikolic-Paterson D.J., et al. TNF-alpha up-regulates renal MIF expression in rat crescentic glomerulonephritis. Mol. Med. (1997) 3:136–144.[ISI][Medline]
  22. Lan H.Y., Yang N., Brown F.G., Isbel N.M., Nikolic-Paterson D.J., Mu W., et al. Macrophage migration inhibitory factor expression in human renal allograft rejection. Transplantation (1998) 66:1465–1471.[ISI][Medline]
  23. Lin S.G., Yu X.Y., Chen Y.X., Huang X.R., Metz C., Bucala R., et al. De novo expression of macrophage migration inhibitory factor in atherogenesis in rabbits. Circ. Res. (2000) 87:1202–1208.[Abstract/Free Full Text]
  24. Burger-Kentischer A., Goebel H., Seiler R., Fraedrich G., Schaefer H.E., Dimmeler S., et al. Expression of macrophage migration inhibitory factor in different stages of human atherosclerosis. Circulation (2002) 105:1561–1566.[Abstract/Free Full Text]
  25. Chen Z., Sakuma M., Zago A.C., Zhang X., Shi C., Leng L., et al. Evidence for a role of macrophage migration inhibitory factor in vascular disease. Arterioscler. Thromb. Vasc. Biol. (2004) 24:1–8.[Free Full Text]
  26. Pan J.H., Sukhova G.K., Yang J.T., Wang B., Xie T., Fu H., et al. Macrophage migration inhibitory factor deficiency impairs atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation (2004) 109:3149–3153.[Abstract/Free Full Text]
  27. Schober A., Bernhagen J., Thiele M., Zeiffer U., Knarren S., Roller M., et al. Stabilization of atherosclerotic plaques by blockade of macrophage migration inhibitory factor after vascular injury in apolipoprotein E-deficient mice. Circulation (2004) 109:380–385.[Abstract/Free Full Text]
  28. Onodera S., Kaneda K., Mizue Y., Koyama Y., Fujinaga M., Nishihira J. Macrophage migration inhibitory factor up-regulates expression of matrix metalloproteinases in synovial fibroblasts of rheumatoid arthritis. J. Biol. Chem. (2000) 275:444–450.[Abstract/Free Full Text]
  29. Onodera S., Nishihira J., Iwabuchi K., Koyama Y., Yoshida K., Tanaka S., et al. Macrophage migration inhibitory factor up-regulates matrix metalloproteinase-9 and -13 in rat osteoblasts. Relevance to intracellular signaling pathways. J. Biol. Chem. (2002) 277:7865–7874.[Abstract/Free Full Text]
  30. Arndt U., Wennemuth G., Barth P., Nain M., Al-Abed Y., Meinhardt A., et al. Release of macrophage migration inhibitory factor and CXCL8/interleukin-8 from lung epithelial cells rendered necrotic by influenza A virus infection. J. Virol. (2002) 76:9298–9306.[Abstract/Free Full Text]
  31. Lan H.Y., Mu W., Nikolic-Paterson D.J., Atkins R.C. A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J. Histochem. Cytochem. (1995) 43:97–102.[Abstract]
  32. Zhang S., Yang Y., Kone B.C., Allen J.C., Kahn A.M. Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/Calmodulin-dependent protein kinase II. Circulation (2003) 107:1539–1544.[Abstract/Free Full Text]
  33. Nikkari S.T., O'Brien K.D., Ferguson M., Hatsukami T., Welgus H.G., Alpers C.E., et al. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation (1995) 92:1393–1398.[Abstract/Free Full Text]
  34. Lemaitre V., O'Byrne T.K., Borczuk A.C., Okada Y., Tall A.R., D'Armiento J. ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J. Clin. Invest. (2001) 107:1227–1234.[ISI][Medline]
  35. Jimi S., Sakata N., Matunaga A., Takebayashi S. Low density lipoproteins bind more to type I and III collagens by negative charge-dependent mechanisms than to type IV and V collagens. Atherosclerosis (1994) 107:109–116.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kong, Y.-Z.
Right arrow Articles by Lan, H. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kong, Y.-Z.
Right arrow Articles by Lan, H. Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?