This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lin, S.-G. Articles by Lan, H. Y. Search for Related Content PubMed PubMed Citation Articles by Lin, S.-G. Articles by Lan, H. Y. Related Collections Animal models of human disease Pathophysiology

(Circulation Research. 2000;87:1202.)
© 2000 American Heart Association, Inc.

Molecular Medicine

De Novo Expression of Macrophage Migration Inhibitory Factor in Atherogenesis in Rabbits

Shu-Guang Lin, Xi-Yong Yu, Yong-Xiong Chen, Xiao R. Huang, Christine Metz, Richard Bucala, Chu-Pak Lau, Hui Y. Lan

From the Guangdong Provincial Cardiovascular Institute (S.-G.L., X.-Y.Y.), Guangzhou, China; Department of Medicine (Y.-X.C., X.R.H., C.-P.L., H.Y.L.), The University of Hong Kong, China; and Picower Institute for Medical Research (C.M., R.B.), Manhasset, NY.

Correspondence to Hui Y. Lan, MD, PhD, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong. E-mail hylan{at}hkucc.hku.hk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Macrophage migration inhibitory factor (MIF) has been shown to play an important role in macrophage-mediated diseases. We investigate the potential role of MIF in atherogenesis using a hypercholesterolemic rabbit model. New Zealand White rabbits fed with a 2% cholesterol diet developed hypercholesterolemia and early fatty streaks at 1 month. The lesions became advanced at 3 months and were associated with de novo MIF expression by vascular endothelial cells (VECs) and smooth muscle cells (SMCs), as demonstrated by immunohistochemistry, reverse transcriptase–polymerase chain reaction, and in situ hybridization. By contrast, there was no increase in MIF levels in rabbits fed a normal diet. In early atherogenesis, marked upregulation of MIF mRNA and protein by VECs and some intimal cells were closely associated with CD68⁺ monocyte adhesion onto and subsequent migration into subendothelial space. Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, which may be associated with the macrophage-rich fatty streak lesion formation. Upregulation of MIF by SMCs is transient during atherogenesis. Importantly, strong MIF expression by activated macrophages may be responsible for the development of foam cell-rich lesions. Finally, the ability of MIF to induce intercellular adhesion molecule-1 expression by VECs implicates its pathogenic role in atherogenesis. In conclusion, the present study provides the first demonstration that MIF is markedly upregulated during atherogenesis. Upregulation of MIF by VECs and SMCs may play a role in macrophage adhesion, transendothelial migration, accumulation, and, importantly, transformation into foam cells. Furthermore, strong MIF expression by macrophages may both initiate and amplify the atherogenesis process.

Key Words: atherosclerosis • migration inhibitory factor • macrophages • foam cells • smooth muscle cells • vascular endothelial cells

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocytes and macrophages have been shown to be key cells involved in the development of atherosclerotic plaques.¹ An early event in atherogenesis is monocyte adherence to activated endothelial cells.² After transmigrating across the endothelial cell layer, monocytes mature into macrophages, which phagocytose lipids to become macrophage foam cells forming the early fatty streak.³ Monocyte chemoattractant protein-1 (MCP-1) and a group of adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], vascular adhesion molecule-1 [VCAM-1], and P-selectin) have been shown to be important in the attachment of monocytes to activated vascular endothelial cells (VECs) in the initial step of atherosclerotic plaque formation.^{4 5 6 7 8 9 10} However, there are several fundamental questions remaining in the development of macrophage-rich atherosclerotic plaque, including why the infiltrating macrophages persist for a long time within the lesions and cease migrating within the vessel wall after their transendothelial migration and what factors regulate the infiltrating macrophages to become activated, phagocytose lipids and transform into lipid-laden macrophages (foam cells).

Migration inhibitory factor (MIF) was first described more than 30 years ago as a product of activated T cells, which inhibits the migration of macrophages in vitro and promotes macrophage accumulation in the skin delayed–type hypersensitivity reaction.^{11 12} The recent cloning and characterization of MIF has led to the recognition that this molecule plays a pivotal role in regulating the inflammatory and immune responses,^{13 14 15 16 17} although there is no receptor for MIF identified to date. It has now recognized that MIF is constitutively expressed in a variety of tissues and cells and is a potent macrophage activator.^{18 19 20} Upregulation of MIF has been demonstrated to be responsible for the recruitment and localization of macrophages and T cells to areas of severe tissue damage in both experimental and human glomerulonephritis and allograft rejection.^{19 20 21 22 23 24 25} The use of neutralizing antibodies has confirmed the central role of MIF in the cutaneous response to tuberculin and endotoxic shock.^{14 15 16 26} In addition, anti-MIF treatment significantly inhibits macrophage-dependent progressive renal injury and arthritis.^{22 27 28} This inhibition was associated with the suppression of blood monocyte recruitment and subsequent macrophage-mediated tissue injury, confirming the important role of MIF in controlling cell-mediated inflammatory and immune responses.

On the basis of the known role of MIF, we postulate that MIF may participate in atherogenesis by promoting macrophage recruitment and localization to form macrophage-rich fatty streaks and their subsequent transformation into lipid-laden foam cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal Model
A rabbit model of atherosclerosis was induced in New Zealand White rabbits fed a standard (control, n=12) or 2% cholesterol diet for 4 (n=6) and 12 (n=6) weeks. The animals were killed at 1 and 3 months. A piece of aortic arch (0.5 cm) was collected from each animal for histology, and thoracic and abdominal aortas (5 cm) and carotid and femoral arteries (2 cm) were collected for reverse transcriptase–polymerase chain reaction (RT-PCT). Serum total cholesterol, triglycerides, HDL, and LDL cholesterol were measured using radioimmunoassay kits (Wako Pure Chemical Industries). Data are expressed as mean±SD for groups of animals.

Antibodies and Probes
The mouse anti-MIF monoclonal antibody (mAb) III.D.9 was used in this study. The specificity of the antibody has been determined previously.^{19 24 25} Other mAbs used in this study include RAM11, mouse anti-rabbit CD68 (monocytes and macrophages), mouse anti–-smooth muscle actin (-SMA), CD31 (VEC), and ICAM-1, goat anti-mouse IgG, mouse peroxidase antiperoxidase complexes, and mouse alkaline phosphatase antialkaline phosphatase complexes. All antibodies were purchased from Dakopatts.

A 420-bp fragment of mouse MIF cDNA was used to prepare a digoxigenin-labeled antisense and sense cRNA probes for in situ hybridization.¹⁹ The specificity of the antisense probe was confirmed as described previously.^{19 24 25} In addition, a rat MIF-specific primer (5'-CCATGCCTATGTTCATCGTG-3' and 5'-GAACAGCGG-TGCAGGTAAGTG-3') and a human ICAM-1 primer (5'-TATG-GCAACGACTCCTTCT AND 5'-CATTCAGCGTCACCTTGG-3') were used.²⁹

Histopathology and Immunohistochemistry
Collected arteries were fixed in 4% formalin and stained with hematoxylin and eosin or periodic acid–Schiff reagent. Atherosclerotic lesions were assessed using an update standard of histological classification.³⁰ One- and two-color immunohistochemical staining was used as previously described microwave-based method.^{19 20 21 22 23 24 25 31} An isotype-matched mAb (73.5) against human CD45R was used as the negative control.¹⁹

Cell Culture
The endothelial-derived cell line EA.hy926 (generously provided by Dr S.H. Lin, Baker Institute, Melbourne, Australia) was cultured in DMEM containing 20% FCS until subconfluence. Cells were then serum-starved for 24 hours, and rhMIF 0, 10, 25, and 50 ng/mL in the presence or absence of the neutralizing antibody (III.D9, 25 µg/mL) was added into the culture for 0, 3, 6, 12, and 24 hours. Cellular RNA and protein were extracted for RT-PCR and Western blot analysis for ICAM-1 expression.²¹

MIF and ICAM-1 mRNA Detection by RT-PCR and In Situ Hybridization
RNA isolation and RT-PCR for MIF and ICAM-1 were performed as previously described.²⁹ In situ MIF mRNA expression was detected by a digoxigenin-labeled antisense MIF cRNA probe on 4-µm paraffin sections using a microwave-based protocol.^{19 20 21 22 23 24 25 32} Controls used a sense MIF cRNA or the probe was omitted completely. No staining was seen in either normal or diseased arteries using the sense probe or with no probe at all.

Quantitation of MIF Expression
The number of MIF⁺ VECs in the areas with or without lesions was counted under high-power field (x400) and expressed as percent positive cells (%). The number of MIF⁺ smooth muscle cells (SMCs) in the medial areas immediate to the early or advanced atherosclerotic lesions or in the areas without lesions was counted by means of a 0.02-mm² graticule fitted in the eyepiece of the microscope and expressed as cells per mm². All counting was performed on blinded slides, and data are expressed as mean±SD.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 3.00 (GraphPad Software, Inc). Differences in MIF expression and macrophage accumulation were assessed by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypercholesterolemia
All animals fed with a 2% cholesterol diet developed hypercholesterolemia at 1 month (Figure 1), with up to a 10-fold increase in total serum cholesterol (Figure 1A) and a 14-fold increase in LDL (Figure 1B) compared with the normal-diet control rabbits. However, there was only a 4-fold increase in serum HDL (Figure 1C), resulting in a 3-fold increase in the LDL/HDL ratio compared with the normal control animals (Figure 1D). In contrast, serum levels of triglycerides remained within the normal range throughout the entire experimental time course (0.99 to 1.44 mmol/L).

View larger version (18K): [in this window] [in a new window]   Figure 1. Figure 1. Serum lipid levels in normal-diet and cholesterol-fed rabbits. A, Total cholesterol (CHOL); B, LDL; C, HDL; and D, the ratio of LDL/HDL. Open bars represent the values from normal-diet control rabbits (n=6). Solid bars represent the values for groups of 6 rabbits on the cholesterol diet. ***P<0.001 compared with time-matched control.

Constitutive MIF Expression in the Normal Artery
Examination of arteries (aortas and carotid and femoral arteries) from normal-diet control animals showed undetectable fatty streak grossly. In situ hybridization and immunohistochemistry demonstrated that a few VECs exhibited weak positive expression for MIF mRNA (Figures 2A and 3A) but undetectable MIF protein expression and macrophage accumulation (Figures 2B and 3B). In addition, there was undetectable or very little MIF mRNA and protein expression by SMCs (Figures 2A, 2B, 3A, and 3B). Similarly, measurement of arterial MIF expression by RT-PCR demonstrated constitutive but weak MIF mRNA expression by all normal arteries examined (Figure 4).

View larger version (111K): [in this window] [in a new window]   Figure 2. Figure 2. In situ hybridization (left) and double immunohistochemistry (right) of the serial sections demonstrate MIF mRNA and protein expression by VECs, SMCs, and macrophages during atherogenesis. Sets of serial sections of aorta from normal sections (A and B) and atherosclerotic lesions (C through L) were stained for MIF mRNA (purple, left) and double-stained for MIF protein (blue) and CD68+ macrophages (brown, right). A and B, Serial sections of normal aorta show weak MIF mRNA expression by some VECs but not by SMCs. There is undetectable MIF protein expression by both VECs and SMCs. C and D, Serial sections show strong MIF mRNA and protein expression by both VECs and intimal cells (arrows in C) in the initial step of atherogenesis. Note that many CD68+ macrophages strongly express MIF (blue-brown) and adhere onto MIF+ VECs (blue) or migrate into the subendothelial space (arrows), where strong MIF expression is apparent. There is no evidence for MIF upregulation by SMCs. E and F, MIF mRNA and protein expression in the early fatty streak of the serial sections. Note that all macrophages within the early fatty streak strongly express MIF mRNA and protein, and de novo expression of MIF mRNA and protein by SMCs is apparent. G and H, Marked upregulation of MIF mRNA and protein by macrophages and SMCs in the progressive fatty streak. I and J, Foam cell lesions. Note that few SMCs express MIF and all foam cells are CD68+ and remain MIF+, although the intensity is reduced. K and L, Serial sections of the advanced plaque demonstrate that the decreased MIF expression is associated with the reduction of macrophage accumulation. Note that many macrophages are MIF-negative. Sections are counterstained with periodic acid-Schiff or methyl green (A through C, E, G, and K). Magnifications: x250 (A through F, I through L); x100 (G and H).

View larger version (18K): [in this window] [in a new window]   Figure 3. Figure 3. Semiquantitation of MIF mRNA and protein expression by VECs (A) and SMCs (B). Each bar represents the mean value±SD for groups of 6 animals. NC indicates normal-diet control; 1M, 1 month; and 3M, 3 months after cholesterol diet. *P<0.05, **P<0.01, and ***P<0.001 compared with NC.

View larger version (30K): [in this window] [in a new window]   Figure 4. Figure 4. MIF mRNA expression demonstrated by RT-PCR. A, Carotid artery; B, aorta; and C, Femoral artery. Each bar represents the ratio of MIF/GAPDH (mean±SD) for groups of 6 animals. *P<0.05, **P<0.01, and ***P<0.001 compared with normal control.

De Novo MIF Expression by VECs and SMCs During Atherogenesis
Macroscopically, the fatty streak lesion was apparent in cholesterol-fed rabbits, accounting for 45% to 55% of the aortic surface areas with profound lesions at the branch sites of abdominal aorta. In situ hybridization and immunostaining of the serial sections demonstrated that there was marked upregulation of MIF mRNA and protein by all VECs in the initial microscopic lesions (the earliest stage of fatty streak) in aortas from the hypercholesterolemic rabbits at 1 month after starting the cholesterol diet (Figures 2C and 2D), which was confirmed by double immunostaining with the anti-MIF and CD31 mAb (Figures 5A and 5B). Indeed, de novo MIF expression was associated with many CD68⁺ macrophages adherent onto the MIF⁺ VECs (Figures 2D and 5B). In the areas without lesions, few VECs expressed MIF (Figure 6A) and no macrophage adherence to VECs was seen. Importantly, marked upregulation of MIF mRNA and protein also was detected in the subendothelial space, which may be associated with the initiation of CD68⁺ macrophage transendothelial migration and subendothelial accumulation (Figures 2C, 2D, 5A, and 5B), ultimately leading to the macrophage-rich early fatty streak formation (Figures 2E through 2H and 5E). Within the early fatty streak, marked upregulation of MIF mRNA and protein was found to be associated with macrophage accumulation, as illustrated in the serial sections (Figures 2E through 2H and 5E). De novo expression of MIF mRNA and protein by SMCs was first evident in the medial areas beneath the macrophage-rich early fatty streak (Figures 2E and 2F), which was additionally identified by combined anti-MIF and -SMA mAb staining (Figure 5C), whereas MIF expression by SMCs in the media without fatty streak lesions remained low (Figure 6B). Interestingly, strong MIF expression by medial SMCs was transient and virtually disappeared as the macrophage-rich early fatty streak developed into foam cell–rich or advanced lesions (Figures 2I through 2L and 5D). In advanced atherosclerotic plaques (Figures 2K and 2L), positive cells for MIF mRNA and protein were reduced, and this was associated with a marked reduction in the number of CD68⁺ macrophages (Figure 2L). Overall, there are at least 50% of the CD68⁺ macrophages less in advanced lesions than in the early fatty streak (Figures 2C through 2H). Few SMCs expressed MIF in media in advanced lesions (Figures 2K, 2L, and 5D). In contrast, the number of -SMA⁺ cells with characteristic myofibroblast morphology within the advanced lesions were markedly increased compared with the early fatty streak (Figure 5C). Double immunostaining demonstrated that all -SMA⁺ cells within the lesions also highly expressed MIF (Figure 5D).

View larger version (158K): [in this window] [in a new window]   Figure 5. Figure 5. Double-immunostaining demonstration of MIF expression by VECs, SMCs and myofibroblasts, macrophages, and foam cells during atherogenesis. A and B, Marked MIF (blue) expression by CD31+ VECs (brown). Note that strong MIF expression is associated with monocyte (*) adhesion onto the VEC and subendothelial accumulation (B). C and D, Strong MIF (blue) expression by -SMA+ SMCs (brown) in the media underneath the macrophage-rich early streak (C). In contrast, many MIF-expressing -SMA+ cells with characteristic myofibroblast morphology (see insert) are found within the advanced lesion (D). E and F, Strong MIF (blue) expression by monocytes and macrophages (brown) in the early atherogenesis (E). Note that MIF expression by foam cells (FCs) is less apparent. Magnifications: x400 (C and D) and x1000 (A, B, E, and F).

View larger version (17K): [in this window] [in a new window]   Figure 6. Figure 6. Comparison of MIF mRNA and protein expression by VECs and SMCs in the areas with or without atherosclerotic lesions. A, MIF expression by VECs. B, MIF expression by SMCs. Each bar represents the mean value±SD for groups of 6 animals. NL indicates non–lesion-associated areas; EL, early fatty streak lesions; and AL, advanced lesions. ***P<0.001 compared with NL.

MIF mRNA expression by the atherosclerotic artery was also semiquantitated by RT-PCR. Figure 4 demonstrated that weak constitutive expression of the expected a 0.6-kb MIF mRNA species in normal aortas and carotid and femoral arteries was markedly upregulated during atherogenesis, which was consistent with the results seen in situ hybridization.

Expression of MIF by Macrophages and Foam Cells During Atherogenesis
In situ hybridization and double immunohistochemistry showed that all CD68⁺ monocytes and macrophages that adhered onto the MIF⁺ VECs and accumulated within the early fatty streak were strongly positive for MIF expression (Figures 2C through 2L), which was additionally illustrated in Figure 5E. Macrophages within the early fatty streak showed stronger MIF mRNA and protein expression (Figures 2C through 2H and 5E) compared with those with foam cell morphology in the fatty streak (Figures 2I, 2J, and 5F) or with those in the advanced plaque (Figures 2K and 2L). These data suggest that MIF expression by macrophages may be a good indicator of macrophage functional activity within atherosclerotic lesions.

MIF Upregulates ICAM-1 Expression By Endothelial Cells In Vitro
We next tested the potential role of MIF in atherogenesis. Because it is difficult to block MIF with the neutralizing anti-MIF mAb in long-term experiments of atherosclerosis in rabbits, an alternative in vitro study was performed to investigate the functional importance of MIF in upregulation of endothelial ICAM-1 during atherogenesis. RT-PCR and Western blot analysis showed that MIF strongly induced ICAM-1 mRNA and protein expression by endothelial cells (ECs) in both time- and dose-dependent manners, which was abrogated by the neutralizing anti-MIF mAb (Figures 7 and 8).

View larger version (38K): [in this window] [in a new window]   Figure 7. Figure 7. RT-PCR demonstrates that MIF induces ICAM-1 expression by ECs. A, MIF (50 ng/mL) induces ICAM-1 mRNA expression in a time-dependent fashion. B, MIF induces ICAM-1 mRNA expression (6 hours) in a dose-dependent manner. Note that the ability of MIF- (50 ng/mL) induced upregulation of ICAM-1 mRNA expression at 6 hours is abrogated by a neutralizing anti-MIF mAb. Each bar represents the mean±SD for 3 experiments. *P<0.05 and **P<0.01 compared with control (0).

View larger version (13K): [in this window] [in a new window]   Figure 8. Figure 8. MIF induces ICAM-1 protein expression by ECs. Western blot analysis demonstrates that MIF markedly upregulates ICAM-1 expression at 24 hours in a dose-dependent manner. Note that MIF- (50 ng/mL) induced upregulation of ICAM-1 by ECs is abrogated by the use of a neutralizing anti-MIF antibody. This is one of 3 experiments that produced similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study has demonstrated de novo expression of MIF mRNA and protein by intrinsic VECs, SMCs, and inflammatory macrophages and foam cells during atherogenesis in hypercholesterolemic rabbits. Upregulation of vascular MIF may play an important role in the initiation and progression of atherosclerosis.

There are several novel findings in the present study. First, de novo expression of MIF mRNA and protein by VECs was found in the earliest stage of fatty streak formation, and this was associated with monocyte adherence onto the VEC. It is now well-known that the adherence of monocytes onto activated endothelial cells is an initial step of atherogenesis. The expression of VCAM-1, ICAM-1, and P-selectin has been shown to be upregulated on endothelial cells in regions overlying atheromatous lesions, and these molecules play a role in the earliest steps of atherogenesis.^{4 7 8 9 10} The new finding of marked MIF expression and monocyte adherence onto VECs indicates that MIF may play a role in the interaction between monocytes and VECs during early atherogenesis. Furthermore, MIF is a proinflammatory cytokine and is constitutively expressed by a variety of cells, including VECs and monocytes and macrophages.^{19 33} It is possible that hypercholesterolemia or modified LDL can cause the release of MIF from VECs and monocytes. Once released, MIF may be able to induce ICAM-1 and VCAM-1 upregulation by VECs directly or indirectly via MIF-induced tumor necrosis factor (TNF)-, interleukin-1 mechanism (Reference 33³³ and C. Metz, unpublished data, September 2000). In the present study, the ability of MIF to induce ICAM-1 mRNA expression by ECs as early as 3 hours strongly indicates that MIF, as a key mediator, directly causes local inflammatory response and thus plays an important role during atherogenesis. This is supported by the recent in vivo study that immunoneutralization of MIF produces a significant inhibition of interleukin-lß, ICAM-1, and VCAM-1 expression and macrophage accumulation in an experimental rat model of glomerulonephritis.²⁷

Second, marked upregulation of MIF mRNA and protein was found within the intima in the earliest step of atherogenesis. This may contribute to monocyte and macrophage transendothelial migration and subsequent accumulation to form the macrophage-rich early fatty streak. MCP-1, a member of the CC family and a potent chemokine for monocytes, has been found to be highly expressed in macrophage-rich atherosclerotic lesions in both human and animal models^{5 6} and postulated to be central in monocyte recruitment into the arterial wall. This was confirmed by the recent study that the absence of MCP-1 and its receptor causes a dramatic protection from macrophage recruitment and atherosclerotic lesion formation.^{34 35 36}

Questions that remain are why macrophages are able to persist within the atherosclerotic lesions and what factors regulate their accumulation, activation for lipid phagocytosis, and transformation into lipid-laden foam cells. Upregulation of MIF within the intima may explain certain of these questions. First, MIF may drive macrophage transendothelial migration via its macrophage activating properties. It is also likely, but not yet proven, that MIF could induce MCP-1 expression, thereby recruiting macrophages via a MCP-1–dependent mechanism. Second, consistent with the known role of MIF, upregulation of MIF within the intima may function to inhibit macrophage movement, ultimately resulting in macrophage-rich fatty streak formation. The ability of MIF to inhibit monocyte and macrophage chemotaxis and random migration induced by MCP-1³⁷ suggests the importance of MIF in causing macrophage localization in the early fatty streak. Furthermore, MIF is also a potent macrophage activator, inducing expression of many proinflammatory mediators by macrophages³³ and strongly promoting inflammatory and immune response. It is very likely that MIF can activate macrophages to phagocytose lipids, regulating in the macrophage–foam cell transformation. Third, the transient expression of MIF by SMCs after macrophage subendothelial accumulation may be temporally responsible for the additional recruitment of macrophages into the arterial wall, leading to the progressive development of macrophage-rich atherosclerotic lesions. In normal artery, there are undetectable or very few, if any, SMCs that express MIF, which is consistent with our previous finding in normal human vessels.²⁴ De novo MIF expression by SMCs is pronounced within the macrophage-rich atherosclerotic lesion, whereas it becomes minimal or undetectable when the advanced lesions develop. This indicates that macrophage-derived cytokines from the early fatty streak may contribute to the upregulation of MIF by SMCs, which, in turn, recruit more macrophages to the lesions and promote macrophages to uptake lipids and transform to foam cells. This is in contrast to our previous finding that the upregulation of vascular MIF causes macrophage and T-cell accumulation, thereby producing transmural vasculitis in human renal allograft rejection.²⁴ The transient expression of MIF by SMCs observed in the present study may help to limit macrophage-mediated injury and prevent the occurrence of unstable atherosclerotic lesions. In contrast, marked expression of MIF by -SMA⁺ cells with characteristic myofibroblast morphology was found within the advanced lesions, indicating that MIF may also be involved in the SMC migration and differentiation during advanced plaque formation. Therefore, SMC-derived MIF may play a role in both progression and regression of atherogenesis.

Finally, an important observation made by this study is that macrophages are an important source of MIF, which may act as a key mediator governing the atherogenesis. Indeed, macrophages have been identified to be a rich source of MIF that is released after stimulation with endotoxin, exotoxin, and cytokines such as TNF- and interferon-.^{33 38} In macrophage-mediated renal disease, including human glomerulonephritis and allograft rejection,^{19 20 21 22 23 24 25 27} strong MIF expression was found in activated macrophages within areas of severe tissue injury. Only weak or undetectable MIF expression was observed in resting macrophages present in the uninjured areas. All macrophages within the early fatty streak exhibited strong MIF expression, suggesting that they are likely to be activated and play a role in the progression of atherogenesis. In contrast, MIF expression by macrophages was reduced in the advanced lesions. This may reflect their relatively low functional activities in the regressive phase of atherosclerotic lesions.

Local production of MIF by both intrinsic vascular cells and macrophages may be a central mechanism to cause macrophage accumulation, activation, and transformation to foam cells in atherosclerotic plaque. However, the mechanisms responsible for the upregulation of MIF expression by VECs, SMCs, and macrophages remain to be addressed. The accumulation of lipids within the arterial wall, especially cholesterol and LDL, is a distinguishing characteristic of the atherosclerotic lesion and has been shown to be responsible for all stages of plaque development. Indeed, LDL can induce VEC expression of ICAM-1, VCAM-1, and MCP-1.^{39 40 41} VLDL has been shown to activate nuclear factor-B, which induces TNF-, ICAM-1, and VCAM-1 expression by VECs and SMCs.⁴² Accordingly, it is likely that LDL may also be a key inducer for MIF expression by VECs, SMCs, and macrophages during atherogenesis.

In summary, this is the first study to identify that VECs, SMCs, and macrophages are a major source of MIF expression in the atherosclerotic vessels of hypercholesterolemic rabbits. Upregulation of MIF expression by intrinsic vascular cells may contribute to local macrophage accumulation, ultimately resulting in macrophage-rich atherosclerotic lesion formation. In addition, MIF production by macrophages within atherosclerotic lesions may initiate autokine mechanisms, which cause and amplify the inflammatory response, promoting macrophage–foam cell transformation during atherogenesis. However, the pathogenic role of MIF in atherogenesis needs to be additionally investigated in vivo by MIF-blocking studies.

	Acknowledgments

 
This work was supported in part by grants from the Research Grants Council, Hong Kong (RGC, HKU 7325/99M), a CRCG (Committee on Research and Conference Grants) grant from The University of Hong Kong (33704/00), and a grant aid from the Institute of Cardiovascular Science and Medicine (HKU).

Received June 26, 2000; revision received October 25, 2000; accepted October 25, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–808.[Medline] [Order article via Infotrieve]
2. Joris IZT, Nunnari JJ, Krolikowski FJ, Majno G. Studies on the pathogenesis of atherosclerosis, I: adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats. Am J Pathol. 1983;113:341–358.[Abstract]
3. Gerrity RG. The role of the monocyte in atherogenesis, I: transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1973;103:181–191.[Abstract]
4. Cybulsky MI, Gimbone MA. Endothelial expression of a mononuclear adhesion molecule during atherogenesis. Science. 1991;251:788–791.[Abstract/Free Full Text]
5. Reape TJ, Groot PH, Rollins BJ. Chemokines and atherosclerosis. Atherosclerosis. 1999;147:213–225.[Medline] [Order article via Infotrieve]
6. Nelken NA, Coughlin SR, Gordon D, and Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest. 1991;88:1121–1127.
7. Ramos, CL, Huo Y, Jung U, Ghosh S, Manka DR, Sarembock IJ, Ley K. Direct demonstration of P-selectin– and VCAM-1–dependent mononuclear cell rolling in early atherosclerotic lesions of apolipoprotein E–deficient mice. Circ Res. 1999;84:1237–1244.[Abstract/Free Full Text]
8. Dong ZM, Brown AA, Wagner DD. Prominent role of P-selectin in the development of advanced atherosclerosis in ApoE-deficient mice. Circulation. 2000;101:2290–2295.[Abstract/Free Full Text]
9. Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, Beaudet AL. P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med. 2000;191:189–194.[Abstract/Free Full Text]
10. Reckless J, Rubin EM, Verstuyft JB, Metcalfe JC, Grainger DJ. Monocyte chemoattractant protein-1 but not tumor necrosis factor- is correlated with monocyte infiltration in mouse lipid lesions. Circulation. 1999;99:2310–2316.[Abstract/Free Full Text]
11. David JR. Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction. Proc Natl Acad Sci U S A. 1966;56:72–77.[Free Full Text]
12. Bloom BR, Bennett B. Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science. 1966;153:80–82.[Abstract/Free Full Text]
13. Bernhagen J, Calandra T, Bucala R. Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features. J Mol Med. 1998;76:151–161.[Medline] [Order article via Infotrieve]
14. Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, Manogue KR, Cerami A, Bucala R. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature. 1993;365:756–759.[Medline] [Order article via Infotrieve]
15. Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M, Donnelly T, Cerami A, Bucala R. MIF as a glucocorticoid-induced modulator of cytokine production. Nature. 1995;376:68–71.
16. Bernhagen J, Bacher M, Calandra T, Metz CN, Doty SB, Donnelly T, Bucala R. An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction. J Exp Med. 1996;183:277–282.[Abstract/Free Full Text]
17. Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, David JR. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J Exp Med. 1999;189:341–346.[Abstract/Free Full Text]
18. Bacher M, Meinhardt A, Lan HY, Mu W, Metz CN, Chesney JA, Calandra T, Gemsa D, Donnelly T, Atkins RC, Bucala R. Migration inhibitory factor expression in experimentally induced endotoxemia. Am J Pathol. 1997;150:235–246.[Abstract]
19. Lan HY, Mu W, Yang N, Meinhardt A, Nikolic-Paterson DJ, Ng YY, Bacher M, Atkins RC, Bucala R. De novo renal expression of macrophage migration inhibitory factor during the development of rat crescentic glomerulonephritis. Am J Pathol. 1996;149:1119–1127.[Abstract]
20. Tesch GH, Nikolic-Paterson DJ, Metz CN, Mu W, Bacher M, Bucala R, Atkins RC, Lan HY. Rat mesangial cells express macrophage migration inhibitory factor in vitro and in vivo. J Am Soc Nephrol. 1998;9:417–424.[Abstract]
21. Lan HY, Yang N, Metz C, Mu W, Song Q, Nikolic-Paterson DJ, Bacher M, Bucala R, Atkins RC. TNF- up-regulates renal MIF expression in rat crescentic glomerulonephritis. Mol Med. 1997;3:136–144.[Medline] [Order article via Infotrieve]
22. Yang N, Nikolic-Paterson DJ, Ng YY, Mu W, Metz C, Bacher M, Meinhardt A, Bucala R, Atkins RC, Lan HY. Reversal of established rat crescentic glomerulonephritis by blockade of macrophage migration inhibitory factor (MIF): potential role of MIF in regulating glucocorticoid production. Mol Med. 1998;4:413–424.[Medline] [Order article via Infotrieve]
23. Brown FG, Nikolic-Paterson DJ, Metz C, Bucala R, Atkins RC, Lan HY. Up-regulation of macrophage migration inhibitory factor in acute renal allograft rejection in the rat. Clin Exp Immunol. 1999;118:329–336.[Medline] [Order article via Infotrieve]
24. Lan HY, Yang N, Brown FG, Isbel NM, Nikolic-Paterson DJ, Mu W, Metz CN, Bacher M, Atkins RC, Bucala R. Macrophage migration inhibitory factor expression in human renal allograft rejection. Transplantation. 1998;66:1465–1471.[Medline] [Order article via Infotrieve]
25. Lan HY, Yang N, Nikolic-Paterson DJ, Yu XQ, Mu W, Isbel NM, Metz CN, Bucala R, Atkins RC. Expression of macrophage migration inhibitory factor in human glomerulonephritis. Kidney Int. 2000;57:499–509.[Medline] [Order article via Infotrieve]
26. Calandra T, Echtenacher B, Roy DL, Pugin J, Metz CN, Hultner L, Heumann D, Mannel D, Bucala R, Glauser MP. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat Med. 2000;6:164–170.[Medline] [Order article via Infotrieve]
27. Lan HY, Bacher M, Yang N, Mu W, Nikolic-Paterson DJ, Metz C, Meinhardt A, Bucala R, Atkins RC. 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]
28. Mikulowska A, Metz CN, 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]
29. Bacher M, Meinhardt A, Lan HY, Dhabhar FS, Mu W, Metz CN, Chesney JA, Gemsa D, Donnelly T, Atkins RC, Bucala R. MIF expression in the rat brain: implications for neuronal function. Mol Med. 1998;4:217–230.[Medline] [Order article via Infotrieve]
30. Stary HC. Natural history and histological classification of atherosclerotic lesions: an update. Arterioscler Thromb Vasc Biol. 2000;20:1177–1178.[Free Full Text]
31. Lan HY, Mu W, Nikolic-Paterson DJ, Atkins RC. 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. Lan HY, Mu W, NG YY, Nikolic-Paterson DJ, Atkins RC. A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology. J Histochem Cytochem. 1996;44:281–287.[Abstract]
33. Calandra T, Bernhagen J, Mitchell RA, Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med. 1994;179:1895–1902.[Abstract/Free Full Text]
34. Gu L, Okada Y, Clinton SK Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor deficient mice. Mol Cell. 1998;2:275–281.[Medline] [Order article via Infotrieve]
35. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2^-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998;394:894–897.[Medline] [Order article via Infotrieve]
36. Gosling J, Slaymaker S, Gu L, Tseng S, Zlot CH, Young SG, Rollins BJ, Charo IF. MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B. J Clin Invest. 1999;103:773–778.[Medline] [Order article via Infotrieve]
37. Hermanowski-Vosatka A, Mundt SS, Ayala JM, Goyal S, Hanlon WA, Czerwinski RM, Wright SD, Whitman CP. Enzymatically inactive macrophage migration inhibitory factor inhibits monocyte chemotaxis and random migration. Biochemistry. 1999;38:12841–12849.[Medline] [Order article via Infotrieve]
38. Calandra T, Spiegel LA, Metz CN, Bucala R. Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of gram-positive bacteria. Proc Natl Acad Sci U S A. 1998;95:11383–11388.[Abstract/Free Full Text]
39. Smalley DM, Lin JH, Curtis ML, Kobari Y, Stemerman MB, Pritchard KAJ. Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol. 1996;16:585–590.[Abstract/Free Full Text]
40. Lin JH, Zhu Y, Liao HL, Kobari Y, Groszek L, Stemerman MB. Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Atherosclerosis. 1996;127:185–194.[Medline] [Order article via Infotrieve]
41. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ, Fogelman AM. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990;87:5134–5138.[Abstract/Free Full Text]
42. Dichtl W, Nilsson L, Goncalves I, Ares MP, Banfi C, Calara F, Hamsten A, Eriksson P, Nilsson J. Very low-density lipoprotein activates nuclear factor-B in endothelial cells. Circ Res. 1999;84:1085–1094.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Zernecke, J. Bernhagen, and C. Weber Macrophage Migration Inhibitory Factor in Cardiovascular Disease Circulation, March 25, 2008; 117(12): 1594 - 1602. [Abstract] [Full Text] [PDF]

				M. Kwaijtaal, A. J. van der Ven, R. van Diest, C. A. Bruggeman, F. W. H. M. Bar, T. Calandra, A. Appels, and F. C. G. J. Sweep Exhaustion is Associated With Low Macrophage Migration Inhibitory Factor Expression in Patients With Coronary Artery Disease Psychosom Med, January 1, 2007; 69(1): 68 - 73. [Abstract] [Full Text] [PDF]

				A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]

				M. A. Amin, C. S. Haas, K. Zhu, P. J. Mansfield, M. J. Kim, N. P. Lackowski, and A. E. Koch Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NF{kappa}B Blood, March 15, 2006; 107(6): 2252 - 2261. [Abstract] [Full Text] [PDF]

				V. A. Korshunov, T. A. Nikonenko, V. A. Tkachuk, A. Brooks, and B. C. Berk Interleukin-18 and Macrophage Migration Inhibitory Factor Are Associated With Increased Carotid Intima-Media Thickening Arterioscler. Thromb. Vasc. Biol., February 1, 2006; 26(2): 295 - 300. [Abstract] [Full Text] [PDF]

				Y.-Z. Kong, X.-R. Huang, X. Ouyang, J.-J. Tan, G. Fingerle-Rowson, M. Bacher, W. Mu, L. A. Scher, L. Leng, R. Bucala, et al. Evidence for vascular macrophage migration inhibitory factor in destabilization of human atherosclerotic plaques Cardiovasc Res, January 1, 2005; 65(1): 272 - 282. [Abstract] [Full Text] [PDF]

				C. Weber, A. Schober, and A. Zernecke Chemokines: Key Regulators of Mononuclear Cell Recruitment in Atherosclerotic Vascular Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2004; 24(11): 1997 - 2008. [Abstract] [Full Text] [PDF]

				F. M. H. van Dielen, W. A. Buurman, M. Hadfoune, J. Nijhuis, and J. W. Greve Macrophage Inhibitory Factor, Plasminogen Activator Inhibitor-1, Other Acute Phase Proteins, and Inflammatory Mediators Normalize as a Result of Weight Loss in Morbidly Obese Subjects Treated with Gastric Restrictive Surgery J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4062 - 4068. [Abstract] [Full Text] [PDF]

				J.-H. Pan, G. K. Sukhova, J.-T. Yang, B. Wang, T. Xie, H. Fu, Y. Zhang, A. R. Satoskar, J. R. David, C. N. Metz, et al. Macrophage Migration Inhibitory Factor Deficiency Impairs Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice Circulation, June 29, 2004; 109(25): 3149 - 3153. [Abstract] [Full Text] [PDF]

				Z. Chen, M. Sakuma, A. C. Zago, X. Zhang, C. Shi, L. Leng, Y. Mizue, R. Bucala, and D. I. Simon Evidence for a Role of Macrophage Migration Inhibitory Factor in Vascular Disease Arterioscler. Thromb. Vasc. Biol., April 1, 2004; 24(4): 709 - 714. [Abstract] [Full Text] [PDF]

				A. Schober, J. Bernhagen, M. Thiele, U. Zeiffer, S. Knarren, M. Roller, R. Bucala, and C. Weber Stabilization of Atherosclerotic Plaques by Blockade of Macrophage Migration Inhibitory Factor After Vascular Injury in Apolipoprotein E-Deficient Mice Circulation, January 27, 2004; 109(3): 380 - 385. [Abstract] [Full Text] [PDF]

				C.-M. Yu, K. W.-H. Lai, Y.-X. Chen, X.-R. Huang, and H. Y. Lan Expression of Macrophage Migration Inhibitory Factor in Acute Ischemic Myocardial Injury J. Histochem. Cytochem., May 1, 2003; 51(5): 625 - 631. [Abstract] [Full Text] [PDF]

				D. N. Muller, E. Shagdarsuren, J.-K. Park, R. Dechend, E. Mervaala, F. Hampich, A. Fiebeler, X. Ju, P. Finckenberg, J. Theuer, et al. Immunosuppressive Treatment Protects Against Angiotensin II-Induced Renal Damage Am. J. Pathol., November 1, 2002; 161(5): 1679 - 1693. [Abstract] [Full Text] [PDF]

				J. Fukuzawa, J. Nishihira, N. Hasebe, T. Haneda, J. Osaki, T. Saito, T. Nomura, T. Fujino, N. Wakamiya, and K. Kikuchi Contribution of Macrophage Migration Inhibitory Factor to Extracellular Signal-regulated Kinase Activation by Oxidative Stress in Cardiomyocytes J. Biol. Chem., July 5, 2002; 277(28): 24889 - 24895. [Abstract] [Full Text] [PDF]