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Cardiovascular Research Advance Access first published online on August 14, 2007
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Cardiovascular Research, doi:10.1093/cvr/cvm003
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Mitochondrial uncoupling protein 2 mediates temperature heterogeneity in atherosclerotic plaques

Tim J.L. Van De Parre1, Wim Martinet1, Stefan Verheye2, Mark M. Kockx3, Glenn Van Langenhove2, Arnold G. Herman1 and Guido R.Y. De Meyer1,*

1 Division of Pharmacology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium
2 Antwerp Cardiovascular Institute, Middelheim Hospital, Antwerp, Belgium
3 Department of Pathology, Middelheim Hospital, Antwerp, Belgium

* Corresponding author. Tel: +32 3 820 27 37; fax: +32 3 820 25 67. E-mail address: guido.demeyer{at}ua.ac.be

Time for primary review: 29 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Aims: Rupture-prone atherosclerotic plaques show an elevated temperature, but a molecular explanation for this phenomenon is unknown. Here, we investigated whether mitochondrial uncoupling protein 2 (UCP2) could be involved because this protein is a macrophage homologue of thermogenin in brown fat tissue.

Methods and results: Immunohistochemistry, western blotting, and real-time quantitative polymerase chain reaction were used to detect UCP2 expression in human and rabbit atherosclerotic plaques. Temperature was measured in plaques with thermography catheters and in cultured cells with precision thermometers. UCP2 was abundantly expressed in subendothelial macrophages of atherosclerotic plaques but not in deeper layers of the plaque. Ex vivo temperature measurements in atherosclerotic rabbit thoracic aorta demonstrated a correlation between local plaque temperature, total macrophage mass, and UCP2 expression. In vitro, chemical uncoupling of macrophages with sodium cyanide resulted in heat production ({Delta}T = 0.13 ± 0.04°C vs. controls). Also, overexpression of UCP2 in cultured cells led to a similar increase in temperature.

Conclusion: Our findings provide evidence that temperature heterogeneity in atherosclerotic plaques is at least in part attributed to UCP2 expression in macrophages. The heat generated might be used to detect unstable, macrophage-rich, atherosclerotic plaques via thermography.

KEYWORDS Atherosclerosis; Macrophages; Mitochondria

Received April 27, 2007; revised June 18, 2007; accepted July 17, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Because timely detection of vulnerable atherosclerotic plaques is important to prevent acute coronary events, there is growing interest for the development of new methods to identify unstable lesions by non-invasive as well as invasive means.1 Several ex vivo and in vivo studies, using thermography, showed a relationship between macrophage-rich plaques and the temperature heterogeneity present in these lesions.2–6 Temperature differences within the same patient are significantly increased in plaques from patients with unstable angina and acute myocardial infarction compared with patients with stable angina or age-matched controls without plaques.2 In human carotid artery plaques or plaques from cholesterol-fed rabbits, plaque temperature correlated positively with macrophage density and plaque thickness, but inversely with the distance of the macrophage clusters from the luminal surface.4,6 Decreasing the macrophage content of atherosclerotic plaques without altering plaque area, either by dietary cholesterol withdrawal or by administration of statins, resulted in a reduction of local plaque temperature.6–8 Although the underlying mechanism is unknown, mitochondrial uncoupling proteins (UCPs) could be involved. Indeed, UCPs belong to the mitochondrial anion carrier gene family and, as suggested by their name, can uncouple ATP production from mitochondrial respiration.9–11 The loss of potential energy is hereby converted into heat production. The term ‘uncoupling protein’ was originally used for the mitochondrial membrane protein thermogenin or UCP1, which is uniquely present in mitochondria of brown adipocytes that regulate body temperature in small rodents, hibernators, and mammalian newborns. However, five different orthologues of UCP1 are currently known in mammalian cells. These UCPs differ greatly in tissue distribution and regulation, indicating that they may have distinct physiological roles.11,12 Interestingly, 11 of the 12 amino acid residues critical for the thermogenic function of UCP1 are conserved in UCP2,13 suggesting that data on UCP1 function may also be relevant for UCP2. Moreover, UCP2 is the only uncoupling protein present in macrophages. The aim of the present study was to investigate whether UCP2 expression can explain the underlying mechanisms of temperature heterogeneity in atherosclerosis, particularly in advanced, macrophage-rich lesions.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The institutional ethics committees have approved all experiments. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and with the principles outlined in the Declaration of Helsinki.

2.1. Carotid endarterectomy specimens
Human carotid endarterectomy specimens (n = 5) were obtained from patients with a carotid stenosis of >70%. One-half of the specimens were fixed in 4% formalin within 2 min after surgical removal. The other half was frozen in liquid nitrogen to be used for RNA extraction. Non-atherosclerotic mammary arteries (n = 5) obtained during bypass surgery were used as negative control samples and were manipulated similarly.

2.2. Cholesterol-induced atherosclerosis and ex vivo thermography
Male New Zealand white rabbits (4.0 ± 0.1 kg, Merelbeke, Belgium) were fed a 0.3% cholesterol-supplemented diet (n = 5) for 10 months. Aortic segments were then fixed in 4% formalin and paraffin-embedded. Ex vivo temperature measurements on rabbit aorta were performed as described.6 Briefly, the aorta of each animal was rapidly removed, and the descending aorta was fixed to a corkboard with its dorsal side down. A thermography catheter (Thermocore Medical Systems, Merelbeke, Belgium) was inserted, and before pullback, the immobilized aorta was marked at the start and longitudinally over the complete length with indelible ink. The thermography catheter (accuracy of 0.006°C) is an over-the-wire system that consists of a functional end that can be engaged by retracting a covering sheath. The distal part has four dedicated thermistors at the distal end of four flexible nitinol strips (each at 90°) that, after engagement, ensure endoluminal surface contact of the aorta. The local temperature measurements were plotted against the pullback distance. After pullback, aortas were embedded and prepared for histopathological analysis. Temperature measurements were related to UCP2 immunoreactivity.

2.3. Antibodies
The following mouse monoclonal antibodies were used: anti-CD68 (clone PG-M1, DAKO) and anti-rabbit macrophage (clone RAM11, DAKO), anti-calprotectin (clone MAC387, Serotec), anti-{alpha}-smooth muscle actin (clone 1A4, Sigma), and anti-ß-actin (clone AC-15, Sigma). Polyclonal rabbit anti-inducible nitric oxide synthase was obtained from Biomol. Using in vitro-translated UCP1, UCP2, and UCP3 protein, the specificity of anti-UCP2 antibodies from two different manufacturers [rabbit polyclonal from Alpha Diagnostic and goat polyclonal antibodies raised against an N-terminal (N19) or C-terminal fragment of UCP2 (C20) from Santa Cruz] was tested first on western blot. Only N19 and C20 antibodies were UCP2-specific. Because C20 caused less background staining, this antibody was used in the present study. Peroxidase-conjugated secondary antibodies were obtained from DAKO. For fluorescent double staining, FITC-labelled anti-{alpha}-smooth muscle actin (clone 1A4, Sigma), Alexa Fluor 546-labelled donkey anti-goat (Molecular Probes), and Cy3-labelled donkey anti-mouse (Jackson ImmunoResearch Laboratories) were used. Biotin-conjugated secondary antibodies were detected with a Vectastain ABC kit (Vector Laboratories).

2.4. Cell culture
Murine J774A.1 macrophages were grown in RPMI 1640 medium (Invitrogen). RAW264.7 macrophages and human HEK293T cells were grown in DMEM (Invitrogen). Media were supplemented with 10% heat-inactivated foetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL), gentamycin (50 µg/mL), and polymyxin B (20 U/mL). All cell lines were obtained from the American Type Culture Collection.

Human aortic smooth muscle cells (SMCs) (Cambrex) were grown in SMC basal medium (Clonetics) supplemented with 5% foetal bovine serum, insulin (5 µg/mL), human fibroblastic growth factor (1 µg/mL), human epidermal growth factor (0.5 ng/mL), and antibiotics. Human peripheral blood mononuclear cells (PBMC) were isolated from healthy blood donors via density gradient centrifugation using fresh buffy coats (Blood Transfusion Center, Antwerp, Belgium) and lymphocyte separation medium (ICN Biomedicals). PBMCs were incubated with CD14 microbeads (Miltenyi Biotec) to obtain a highly purified monocyte population (>90% CD14-positive cells). Finally, monocytes were cultured for 7 days in IMDM medium containing 10% serum to allow differentiation into macrophages. Medium was changed every 3 days. Cell lysates were analysed by western blotting as described.14

2.5. cDNA expression array and real-time quantitative polymerase chain reaction
Total RNA was isolated from carotid endarterectomy specimens (n = 5) or non-atherosclerotic mammary arteries (n = 5) using Trizol reagent (Invitrogen). RNA samples were further purified with the Absolutely RNA Microprep Kit (Stratagene). cDNA probes were synthesized and hybridized with a human toxicology array (Clontech) as described.15 To examine the relative abundance of UCP2 mRNA by real-time quantitative polymerase chain reaction (qPCR), TaqMan gene expression assays [Id: Mm00627599_m1 (mouse) or Hs01075225_m1 (human)] were performed on an ABIPrism 7300 sequence detector system (Applied Biosystems) in 25 µL reaction volumes containing 1x Universal PCR Master Mix (Applied Biosystems). The parameters for PCR amplification were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Relative expression of mRNA species was calculated using the comparative threshold cycle method. All data were controlled for quantity of cDNA input by performing measurements on the endogenous reference gene ß-actin [assay Id: Mm00607939_s1 (mouse) or Hs00242273_m1 (human), Applied Biosystems].

2.6. Overexpression of UCP2
cDNA encoding human UCP2 was PCR-amplified from human foetus poly A+ RNA (Clontech) using Pfu DNA polymerase and the UCP2-specific primers 5'-CCGAATTCATCATGGTTGGGTTCAAGGCC-3' and 5'-TAATCTAGACTCAGAAGGGAGCCTCTCGGGA-3'. After sequence verification, the resultant PCR product was EcoRI/XbaI-digested and cloned in the similarly opened plasmid pPICZ-B (Invitrogen). Plasmids were linearized with SacI and transformed to the methylotrophic yeast Pichia pastoris GS115 (Invitrogen) by electroporation according to the recommendations of Invitrogen. Transformants were selected on YPD agar [2% dextrose, 2% peptone (Difco Laboratories), 1% yeast extract (Difco Laboratories)], supplemented with 100 µg/mL zeocine (Invitrogen). For the analysis of UCP2 expression and temperature measurements, P. pastoris transformants were grown under continuous shaking in buffered minimal glycerol (BMGY) medium (containing 1% yeast extract, 2% peptone, 1% glycerol, 1.34% yeast nitrogen base, and 100 mM potassium phosphate, pH 6.0) at 30°C for 24 h. Cultures were harvested by low-speed centrifugation, suspended in buffered minimal methanol medium (same composition as BMGY except that 5 mL methanol/L was added instead of glycerol) and incubated at 30°C for 18 h to induce UCP2 expression.

To overexpress UCP2 in mammalian cells, UCP2 cDNA was inserted into the PmeI site of the lentiviral transfer vector pWPI (Tronolab) containing an eGFP expression cassette. The resulting plasmid, pWPI-UCP2, was co-transfected with the packaging plasmid psPAX2 and envelope plasmid pMD2.G into HEK293T cells using cell line Nucleofector Solution V (Amaxa) and a Nucleofector I device (Amaxa, program Q01). After 2 days, medium containing recombinant lentiviruses was collected and used to infect HEK293T cells, and J774A.1 and RAW264.7 macrophages. In 12-well plates, cells were centrifuged with 1.4 mL viral supernatant containing 5 µM polybrene (Fluka) for 45 min at 180 g and then incubated at 32°C. Twenty-four hours later, macrophages were infected for the second time. After 3 days, infection was confirmed by screening for eGFP expression via flow cytometry.

2.7. In vitro temperature measurements
UCP2-overexpressing cells were collected by centrifugation. Subsequently, a 3000th ‘Precisa’ thermometer (Amarell Electronic) was positioned in the cell pellet. This thermometer has a resolution of 0.001°C and a reproducibility of 0.01°C. A second thermometer was positioned in an empty test tube to monitor temperature fluctuations in the environment. Temperature was measured for 100 s at room temperature. Real-time data acquisition software (Amarell Electronic) allowed us to compare the areas under the curve for 100 s and to calculate temperature differences.

2.8. Statistical analysis
All data are expressed as mean ± SEM. Statistical analysis was performed by SPSS package for Windows (version 12.0). Student's t-test was used for unpaired data and probability levels <0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Expression of UCP2 in human and rabbit atherosclerotic plaques
cDNA expression array experiments showed strong UCP2 and poor UCP3 mRNA expressions in human carotid endarterectomy specimens compared with non-atherosclerotic mammary arteries (Figure 1A). UCP1 mRNA expression was not detectable in either carotid plaques or non-atherosclerotic control vessels. High-level expression of UCP2 mRNA in carotid plaques was confirmed by real-time qPCR (Figure 1B). In addition to UCP2 mRNA, we observed a marked expression of UCP2 protein in the subendothelial layer of the plaque (Figure 2). UCP2-positive cells co-localized with calprotectin, but not with {alpha}-smooth muscle actin (Figure 3), indicating that UCP2 is predominantly expressed in the monocyte/macrophage population of the plaque. Moreover, human peripheral blood monocytes contained a significantly higher amount of UCP2 at both the protein and mRNA levels compared with human aortic SMCs (Figure 4). After differentiation of monocytes to young macrophages, UCP2 expression was even more pronounced (Figure 4). In contrast, co-staining of UCP2-positive cells in carotid plaques with CD68, a marker of fully differentiated macrophages, did not always result in clear co-localization (not shown). Mammary arteries or normal media adjacent to plaque tissue did not contain monocyte/macrophages and were UCP2-negative (Figure 2). Kupffer cells in human liver stained strongly for CD68 but not for UCP2 (Figure 2), indicating that not all tissue macrophages are UCP2-positive.


Figure 1
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  		Figure 1 High-level UCP2 mRNA in human atherosclerotic plaques. (A) cDNA isolated from non-atherosclerotic mammary arteries (n = 5) and carotid endarterectomy specimens (n = 5) was analysed via microarray technology. A detail of the microarray with signals of UCP1, UCP2, and UCP3 gene expression is shown. (B) Confirmation of UCP2 mRNA expression in carotid plaques via real-time quantitative polymerase chain reaction. **P < 0.01 vs. mammary arteries (n = 5, unpaired Student’s t-test).

 


Figure 2
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  		Figure 2 UCP2 is present in the subendothelial layer of human atherosclerotic plaques. Serial sections of human carotid endarterectomy specimens, non-atherosclerotic mammary arteries, and liver were immunohistochemically stained for CD68, UCP2, and {alpha}-SMC actin. UCP2 is not detectable in non-atherosclerotic mammary arteries or liver. Scale bar 20 µm.

 


Figure 3
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  		Figure 3 UCP2 co-localizes with macrophages but not with smooth muscle cells in human carotid endarterectomy specimens. Fluorescent double staining demonstrated that UCP2-positive cells (red) expressed calprotectin (monocytes/macrophages, green) but not {alpha}-SMC actin (smooth muscle cells, green). Scale bar 20 µm.

 


Figure 4
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  		Figure 4 UCP2 is abundantly expressed in monocytes/macrophages, but not in smooth muscle cells. (A) Western blot analysis of UCP2 in crude cell lysates of human peripheral blood monocytes, blood monocyte-derived macrophages, and aortic smooth muscle cells. ß-actin served as a loading control. (B) Confirmation of UCP2 mRNA expression in monocytes and macrophages via real-time quantitative polymerase chain reaction. ***P < 0.001 vs. smooth muscle cells (n = 4), analysis of variance followed by Bonferroni post hoc test; if the variances were not homogenous, data were logarithmically transformed.

 
Similar to human plaques, plaques from cholesterol-fed rabbits showed strong immunoreactivity for UCP2, most notably in the subendothelial layer (Figure 5).


Figure 5
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  		Figure 5 Illustration of the relationship between temperature heterogeneity and UCP2 expression in rabbit atherosclerotic plaques. The thoracic aorta from rabbits fed with a 0.3% cholesterol-rich diet for 10 months was isolated and plaque temperature was measured ex vivo using a thermography catheter. Subsequently, the aorta was formalin fixed, paraffin embedded, and stained for RAM11 (macrophages) and UCP2. Temperature of atherosclerotic plaques in the thoracic aorta from cholesterol-fed rabbits correlates with UCP2 immunoreactivity in macrophages. Scale bar 20 µm.

 
3.2. Ex vivo and in vitro thermography
Ex vivo thermography experiments on thoracic aorta from hypercholesterolaemic rabbits showed a strong correlation between plaque temperature and UCP2 immunoreactivity (R = 0.62, n = 5, P < 0.01). Plaque temperature correlated with the amount of macrophages present (R = 0.79, n = 5, P < 0.01). To evaluate whether UCP2 is responsible for the observed temperature heterogeneity in atherosclerotic plaques, temperature was monitored in cultured cells. Experiments with J774A.1 macrophages treated with sodium cyanide (NaCN), a well-known uncoupler of mitochondrial respiration, showed that these cells produced more heat ({Delta}T = 0.13 ± 0.04°C, n = 8, P = 0.017) compared with non-treated cells (Figure 6). Moreover, transformation of the yeast P. pastoris with human UCP2 cDNA under the control of the methanol-inducible promoter AOX1 resulted in UCP2 overexpression and thermogenesis ({Delta}T = 0.11 ± 0.03°C, n = 12, P = 0.0018) upon growth in methanol-containing medium (Figure 6). Temperature of yeast transformed with an empty vector (i.e. without UCP2) did not differ from untransformed controls ({Delta}T = –0.04 ± 0.02°C, n = 7, P > 0.05). Attempts to induce endogenous UCP2 expression in vitro (by treating macrophages with the oxidative stress inducers SIN1A, H2O2, or 7-ketocholesterol) or to transfect J774A.1 and RAW264.7 macrophages (via either standard transfection agents or a lentiviral method) failed. Therefore, UCP2 could not be overexpressed in these cells (data not shown). However, infection of human HEK293T cells with recombinant lentiviruses containing UCP2 cDNA yielded >95% UCP2-overexpressing cells and stimulated thermogenesis ({Delta}T = 0.16 ± 0.06°C, n = 9, P = 0.02) compared with control HEK293T cells (Figure 6).


Figure 6
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  		Figure 6 Thermogenesis after mitochondrial uncoupling. The following conditions were tested: (i) J774A.1 macrophages treated with 100 µM NaCN for 10 min vs. an untreated control; (ii) the yeast Pichia pastoris transformed with the UCP2 expression plasmid pPICZ-UCP2 and grown in methanol-containing medium for 24 h to induce UCP2 expression; as a control, P. pastoris was transformed with an empty vector and treated similarly; (iii) HEK293T cells 5 days after infection with recombinant lentiviruses containing the UCP2 coding sequence vs. control cells infected with lentiviruses lacking UCP2. Insets represent immunoblot assays for UCP2. *P < 0.05, **P < 0.01 vs. controls (unpaired Student’s t-test).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we demonstrated for the first time that the temperature heterogeneity in atherosclerotic plaques can be attributed, at least in part, to the expression of UCP2 in macrophages. cDNA expression array and real-time qPCR experiments showed strong expression of UCP2 mRNA in advanced human atherosclerotic plaques compared with non-atherosclerotic mammary arteries. Moreover, advanced plaques contained a dense infiltration of macrophages of which a subpopulation strongly expressed the UCP2 protein. UCP2-positive macrophages were localized predominantly in the subendothelial layer of advanced plaques, which is clinically relevant since thermography most likely measures the temperature of the superficial cell layers. Macrophages in deeper layers of the plaque did not express UCP2 and therefore do not seem to influence temperature heterogeneity.6 Importantly, localization of UCP2 in the subendothelial layer of the plaque, but not in aortic SMCs, together with high-level expression of UCP2 in human peripheral blood monocytes strongly indicates that UCP2 is not upregulated in the plaque by locally generated atherogenic factors, but is abundantly present in the plaque owing to infiltration of (UCP2-positive) monocytes from the circulation. Moreover, differentiation of monocytes into macrophages increased UCP2 expression. Therefore, we assume that macrophages in deeper layers of the plaque lose their UCP2 expression because of the presence of pro-apoptotic stimuli. Thus, UCP2 expression in plaques seems to reflect plaque progression and evolution towards an unstable phenotype.

There has been much debate in the literature regarding the thermogenic capacity of UCPs other than UCP1, because UCP2 expression can be up to 1000-fold lower than that of UCP1.16 Therefore, the expected rate of proton conductance controlled by UCP2 would be much less than that produced by UCP1. However, this debate is focusing on thermogenesis, as it pertains to core body temperature rather than energy dissipation in the form of heat at the mitochondrial level. The distinction between these is critical. It has been suggested that UCP2 is not thermogenic, because it does not appear to contribute to the generation of core body temperature.17 Because UCP2 knock-out mice are not cold-sensitive and do not respond to a high-fat diet, an implication of UCP2 in diet- or cold-induced thermogenesis remains controversial.17 Nonetheless, UCP2-positive brain regions have a significantly higher local temperature compared with other sites or with the core body temperature,18 indicating that UCP2 may have a microenvironmental thermogenic function, at least in the brain.19 Indeed, it has been proposed that UCP2-producing neurons have a large impact on thermoregulation, neuronal activity, and regulation of autonomic, endocrine, and metabolic processes.18 For example, the abundant expression of UCP2 may explain the resistance of the brain against fluctuating peripheral temperature or cold exposure. On the other hand, acute heat production in axon terminals could immediately accelerate synaptic transmission, whereas chronic uncoupling may diminish available ATP, thereby compromising neuronal function. In the present study, chemical uncoupling with sodium cyanide as well as UCP2 overexpression in cultured cells revealed heat production compared with appropriate controls. Our findings confirm and extend previous in vitro results in yeast obtained with infrared thermography.20 Although we demonstrated thermogenesis in human HEK239T cells after UCP2 transfection, we could not transfect UCP2 in macrophages. Monocytes and monocyte-derived macrophages are hard to transfect cells. Despite successful transfection-driven eGFP gene expression, monocytes and macrophages fail to express, for unknown reasons, many other gene products upon transfection, as shown before.21,22

Several lines of evidence suggest that UCP2 is involved in the control of reactive oxygen species (ROS) production by decreasing the mitochondrial membrane potential.17,23 Transient overexpression of UCP2 in monocytes reduces steady-state levels of intracellular ROS and the production of ROS in response to H2O2, MCP-1, and TNF-{alpha}.24 Similarly, UCP2 overexpression in human aortic endothelial cells reduces ROS generation.25 In addition, lack of UCP2 in monocytes accelerates atherosclerotic plaque development by impaired handling of oxidative stress.26 Moreover, the common –866G/A single-nucleotide polymorphism in the human UCP2 promoter yields stronger transcriptional activity in monocytes and endothelial cells and associates with less carotid atherosclerosis.27,28 These findings suggest a protective role for UCP2 in early atherosclerosis. Because the amount of macrophages in these lesions is limited, effects on plaque temperature cannot be detected. However, as the macrophage content of the plaque increases and the plaque progresses towards an unstable phenotype, ROS production is overwhelming and probably cannot be counteracted by the antioxidant properties of UCP2. In that case, the increasing number of UCP2-positive macrophages, predominantly in the luminal layer of advanced plaques, correlates with local plaque temperature. This thermogenic property can be detected by intravascular thermography and may be indicative of plaque progression and evolution towards a rupture-prone lesion. The observed heat production is probably a side effect rather than the main function of UCP2 and may have substantial consequences for the structure of the plaque. Heating increases selective macrophage apoptosis, probably by inactivation of NF-{kappa}B and melting of oxidized cholesterol crystals.29 Although some consider these effects as beneficial, the free cholesterol and apoptotic debris are not adequately removed in advanced lesions.30 As a consequence, the increased amount of UCP2-positive macrophages in advanced plaques may stimulate inflammatory responses and enlargement of the necrotic core and may ultimately lead to plaque rupture.

In conclusion, our data showed a correlation between UCP2 expression and plaque temperature. Because unstable (macrophage-rich) plaques have a higher temperature than stable (macrophage-poor) ones and plaque temperature correlates with macrophage density, UCP2 expression in macrophages can be considered a biochemical link between plaque temperature and vulnerability. The heat that is generated by uncoupling mitochondrial UCP2 can be used to detect rupture-prone atherosclerotic plaques via thermography. If vulnerable plaques in coronary arteries can be detected by the generation of thermal maps, it would be possible to identify patients at risk for an acute coronary syndrome. Once identified, these patients can be treated earlier, resulting in risk reduction of acute coronary events.


   Acknowledgements
 
This research was supported by the Fund for Scientific Research (FWO)-Flanders (Belgium) (project no. G.0308.04 and G.0113.06), the University of Antwerp (NOI-BOF), and the Bekales Foundation. The authors are indebted to Rita Van den Bossche and Hermine Fret for their excellent technical assistance. W.M. is a postdoctoral fellow of the FWO-Flanders.

Conflict of interest: none declared.


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

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