The present study shows, for the first time, that cIAP2 reduces atherosclerotic lesions at early and advanced stages of the disease in an apoE-/- mouse model. Using a cIAP2 KO mouse model, we were able to elucidate the effect of cIAP2 deletion on the progression of the disease at different stages. We also show that not only are the atherosclerotic lesions smaller in size in the KO model, but contain a smaller number of macrophages when compared to WT mice. This data suggests an important role for cIAP2 in the pathogenesis of the disease and unveils potential opportunities for drug therapies.
6.1 The role of apoptosis
Accumulating in vivo data published to date show that decreased early lesion macrophage apoptosis in mice increases lesion area. It is also postulated that secondary necrosis of macrophages, after inefficient phagocytosis and clearance in vivo, may promote inflammation. Previous studies suggest that the promotion of apoptosis in more advanced atherosclerotic plaques would promote further lesion development which could spawn clinical events. However, evidence underlying these processes has been controversial.
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The present study examines the role of a novel anti-apoptotic protein (cIAP2) in both early and late stage atherosclerosis by using a KO model lacking the cIAP2 protein. As represented in Figures 9, 9.1, 10 and 10.1, the KO model reduces lesion size in different vascular regions (aortic root, aortic arch and ascending aorta) in mice on a 4wk HFD. More importantly, this trend is continued in the later more advanced stages as the mice are fed a HFD for 12 wks as is represented in Figures 11, 11.1, 12 and 12.1.
6.2 The role of early macrophage apoptosis in lesion development
A number of genetic alterations in mouse models result in an increase or decrease in early lesion macrophage apoptosis, and the combined results of these studies suggest an inverse relationship between early lesion macrophage apoptosis and early lesion area. A BMT study by Van Vlijman et al. reconstituted an apoE-3 atherosclerotic mouse model with bone marrow from mice deficient in the pro-apoptotic protein p53 (Van Vlijmen BJ. et al., 2001). The recipient mice transplanted with p53-/- bone marrow had a tendency towards decreased macrophage apoptosis compared to the WT bone marrow recipient group. These findings were associated with a 2.3-fold increase in early atherosclerotic lesion area indicating that inhibition of early lesion macrophage apoptosis promotes the growth of early atherosclerotic lesions. However, these findings initially appeared controversial as a similar study using p53-BMT into LDLr-/- mouse models found a decrease in macrophage apoptosis to correlate with an increase in early atherosclerotic lesion development. In this particular study, the decrease in macrophage apoptosis was due to an increase in macrophage cell proliferation which likely explains the controversial findings (Merched AJ. et al., 2003). More evidence regarding the importance of lesion macrophage apoptosis in early atherogenesis was obtained from a study using LDLr-/- mice transplanted with bone marrow lacking the Bax gene - a pro-apoptotic protein (Liu JJ. et al., 2005). As predicted, early lesion macrophage apoptosis was reduced in the mice receiving Bax-/- bone marrow vs. WT marrow. This data suggests that macrophage apoptosis is normally occurring in early atherosclerosis perhaps via one or more pathways that involve p53 and Bax. Work by Arai et al. examined the role of inhibitory factor AIM, an apoptosis inhibitor expressed by mature tissue macrophages. AIM belongs to the macrophage SR cysteine-rich domain super family (Arai S. et al., 2005). In this study, early lesions in AIM-/- x LDLr-/- double KO mice were found to be dramatically reduced when compared to AIM+/+ x LDLr-/- controls. In summary, the studies conclude that macrophage apoptosis in early foam cell lesions in mice appears to limit lesion cellularity and progression. In the present study, cIAP2-/- x apoE-/- double KO mice show reduced lesion cellularity at early stages when compared to their wild type mice as represented in Figures 9 and 10. As delineated in Figure 11, the lesions in the KO mice also show reduced macrophage content when compared to the WT group.
cIAP2 is believed to inhibit apoptosis by binding to the TRAF receptor complex (Wu H. et al., 2007). By deleting the cIAP2 gene, the inhibitory effect of the cIAP2 protein is alleviated in all cell lines. TRAF2 freed from cIAP2 facilitates recruitment of the kinase Rip1 to TNFR1. This results in the activation of the noncanonical and canonical NF-ÎºB pathways, causing TNFÎ± production in a substantial number of macrophage cells. Unlike the Fas Ligand, TNFÎ± does not readily induce cell death in most circumstances. Rather, it activates NF-ÎºB and MAP kinases, leading to cell survival and cell activation. Previous studies have shown that TNFÎ±-induced cell death is accomplished via a secondary TRADD-RIP1-FADD-caspase- 8 complex devoid of TNFR1 (Micheau O. and Tschopp J., 2003). In keeping with this notion, Rip1 is also required for cell death induced by a Smac mimetic, even in the presence of exogenous TNFÎ± for cells that are resistant to Smac mimetics alone but do respond to co-stimulation by TNFÎ± (Petersen et al., 2007).
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In a macrophage under stress from lipid-infiltration, receptor mediated apoptosis is induced driving the macrophage towards self destruction. With the absence of the inhibitory effect of cIAP2, macrophage cells are destroyed and in effect the apoptotic bodies are rapidly and safely removed by phagocytes. The fact that we observe reduced macrophage content in the KO model suggests that their apoptotic clearance is efficient as represented in Figures 9 and 10 by reduced plaque size. These findings mirror the work from Arai et al. examining the role of inhibitory factor AIM and show that its removal reduces atherosclerotic plaque size (Arai S. et al., 2005). The effect of increased early lesion macrophage apoptosis resulted in reduced fatty streak formation as was measured by en face analysis in the KO model compared to the WT.
The present study also examined the extent of macrophage content in early atherosclerotic plaque. Figure 13 represents aortic root macrophage content in KO and WT mice on a 4wk HFD. The total lesion area filled with macrophages is higher in WT vs. KO mice. This supports the notion that cIAP2 deletion increases macrophage apoptosis and results in effective phagocytic clearance of the apoptotic bodies leading to reduced plaque cellularity and size. This is reflected in Figure 14 and 14.1, which summarizes the results of both Sudan IV and CD68 macrophage staining. The graph represents the relation correlation between macrophage content and plaque size in the early lesions. Figure 15 further emphasizes this the strong correlation between the CD68 stained area and Sudan IV. In the KO group, the lesion sizes at different levels from the coronary ostia overlap from both analysis. A similar observation is made with the WT mice, where both Sudan IV and CD68 macrophage staining curves overlap and both measurements are well correlated. There is a decrease in lesion size as well as macrophage infiltration size in all the groups. Analysis was not carried out for the 12wk HFD mice due to technical difficulties leading to a small sample size. However, there is sufficient evidence from the 4wk CD68 staining data to demonstrate a trend that is consistent with the significant Sudan IV findings. That it did not reach statistical significance likely reflects the sample size tested with CD68. Even so, the trend in the CD68 staining data lends support for the notion regarding the role that cIAP2 plays a role in early lesion development and progression. Moreover, it is important to outline the effects of cIAP2 on early lesions since the outcomes of this stage predict its later effect on advanced late stages. For effective drug applications, a pharmaceutical agent that targets early lesion progression may help prevent later clinical manifestations of the disease
6.3 The role of macrophage apoptosis in advanced lesions
By feeding mice a HFD for 12wks, we were able to examine the effect of cIAP2 deletion on advanced atherosclerotic lesions. As expected, the extent of atherosclerotic plaque in the group of mice fed a 12wk HFD was greater than the group fed a HFD for 4 weeks as indicated by the en face analysis (4wk: Fig. 9; 12wk: Fig 11) and the aortic root analysis (4wk: Fig. 10; 12wk: Fig 12).
Despite differences in lesion stages (early for 4wk and advanced for 12wk), the same trend was observed regarding the size of the atherosclerotic plaque. Figures 11 and 11.1 shows that cIAP2-/- x apoE-/- mice on 12wk HFD had significantly reduced lesion area when compared to the WT controls through en face analysis. The reduction in atherosclerotic lesions was also seen in aortic root lesion analysis, represented in Figures 12 and 12.1. As mice are put on a prolonged HFD, the atherosclerotic plaque increases in size. However, the cIAP2-/- x apoE-/- mice still maintains a reduced lesion size when compared to the WT control group. This implies that finding the right target for reducing atherosclerosis progression at an early stage could help reduce advanced lesions from developing. Ultimately, this may be expected to reduce the chance of plaque rupture and thrombus formation that leads to strokes and myocardial infarctions.
Schrijvers et al. showed that human carotid atherosclerotic lesions contained a substantial number of apoptotic cells that were not engulfed by phagocytes (Schrijvers DM. et al., 2005). In addition, a number of studies have shown that late lesion apoptotic macrophages are more numerous in areas surrounding the necrotic core of these late lesions (Geng YJ. et al., 1995; Hegyi L. et al., 1996; Akishima Y. et al., 2005). The necrotic core is made primarily of dead macrophages rich in inflammatory cytokines and this is consistent with the prediction that defective phagocytosis would lead to post apoptotic macrophage necrosis and a heightened state of inflammation. Studies recently established a relationship between defective clearance of apoptotic macrophages and enhanced inflammation in atherosclerosis, as has been reported in previously studied chronic inflammatory components (Pickering MC. et al., 2000; Vandivier RW. et al., 2002). Defective phagocytic clearance of apoptotic macrophages in advanced plaques could promote a number of processes that are thought to be important in plaque disruption and acute atherothrombotic vascular occlusion. Although late inhibition of cIAP2 may in fact be detrimental, this notion was not the focus of our investigation.
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This study suggests a role for cIAP2 in early disease progression. However, more in-depth examination of the role that cIAP2 plays in the advanced lesions is necessary to understand its implication on disease manifestations. In other words, how cIAP2 inhibition might change lesion cellularity, fibrous cap formation, plaque content and ultimately its vulnerability. As argued in several reviews, macrophage apoptosis at later more advanced stages of the disease could be detrimental. Therefore, administering Smac mimetics at a later time point may potentially have a negative impact on disease pathogenesis. However, this requires further study.
6.4 cIAP2 and lipoprotein profile.
There are a variety of risk factors that are known to be associated with the initiation and progression of atherosclerosis. While it was encouraging that no differences in plasma cholesterol or triglycerides were observed between any of the groups in this study, it was also essential to examine total lipid profile levels in the mice. It is possible that even though there is no difference in total lipids, lipid profiles may be altered between groups and manifesting itself, for instance, as a shift towards pro-atherogenic particles such as VLDL. To confirm that the KO model was not advantaged by reductions in cholesterol and lipoprotein levels, we separated plasma lipoproteins by size using FPLC methods and determined that there were no differences in the distribution of lipoproteins between any groups. This ensures that deletion of cIAP2 does not modulate cholesterol levels in the apoE-/- model. Therefore, any observed differences in atherosclerosis in the experimental mouse model can be attributed solely to the participatory role of cIAP2 in lesion development.
6.5 cIAP2 and lesion formation
It can be observed, while performing the lesion analysis on all the study groups, that cIAP2 does in fact play a role in the stages of the disease at different time points. However, it must be re-iterated that atherosclerosis is a dynamic inflammatory disease and that there could be multiple factors affecting the lesion stage at one point in time. When studying early atherosclerotic lesions, the group lacking cIAP2 (KO) appeared to have fewer fatty streaks than the control groups. Furthermore, even at a later stage of the disease, the KO group had fewer advanced lesions (stage IV and V) when compared to the WT group. This observation ties in with the finding that the KO have reduced atherosclerotic lesion area and fewer macrophage numbers when compared to the control group.
A possible explanation could be made with regards to the role cIAP2 plays in TNF-Î± activation. Loss of cIAPs due to Smac mimetic treatment appear to assist in TNFÎ±- induced cell death. A study by Boesten L. assessed the role that TNF-Î± plays in advanced atherosclerotic lesion development using an apoE-3 KO mouse model (Boesten L. et al., 2005). The study shows that TNF-Î± promotes advanced lesion formation, and increases the extent of necrosis in these lesions. The observation that TNF-Î± increases the ratio of necrosis vs. apoptosis suggests that TNF-Î± modulates the lesion towards a more unstable phenotype. Moreover, in vitro studies have shown that TNF-Î± stimulates both macrophages and SMC to synthesize matrix proteases contributing to plaque instability by degrading the fibrous cap (Galis ZS. et al., 1994). The reduction in lesion necrosis upon TNF-Î± deletion, coincides with an increase in lesion apoptosis. TNF-Î± is known to exert its action, at least partially, through the activation of the pro-inflammtory transcription factor, NF-ÎºÎ² (Ashkenazi A. and Dixit VM. 1998). In the case where cIAP2 binds to TRAF2 complex and thus inhibits the activation of NF-ÎºÎ², TNF- Î± production is halted. Deletion of cIAP2 should in term alleviate the inhibitory effect it has on NK-ÎºÎ² and induce TNF- Î± production. However, TNF- Î± induced apoptosis has also been demonstrated. In the case of early atherosclerotic lesions, deletion of cIAP2 is beneficial because it induces early lesion apoptosis through TNF- Î± activated pathway. This leads to more macrophage apoptosis, enhanced apoptotic body clearance and reduced plaque size and cellularity. Reduced lesion size and cellularity is still observed in the late stage atherosclerotic lesions, but to a lesser degree. KO lesions are less necrotic than the WT lesions, however, the difference is not significant. This could possibly mean that cIAP2 deletion is more beneficial at early stages of the disease than at later stages, where macrophage apoptosis is reduced and TNF- Î± secretion increases the rate of necrosis.
6.6 Proposed mechanism of action of cIAP2
It was thought that cIAP2 inhibits apoptosis by directly binding to caspases in vitro, however, this idea has been eliminated as it has later been shown that cIAP2 is involved in various transduction pathways including NF-kB activation in response to TNFÎ± (Rothe M. et al., 1995; Samuel T. et al., 2006).
When a macrophage cell (in an atherogenic milieu) ingests lipids and becomes a foam cell, different environmental cues and stimuli induce receptor-mediated apoptosis. In a normal environment (e.g. in WT control mice), cIAP2 binds to the TRAF2 complexes through its RING domain by poly- or trans-ubiquitylating the Rip1 protein, targeting it for either degradation or other localization in the cell. This series of actions destruct the TRAF2 complex and essentially inhibits its effect on the effecter caspases which lead to apoptosis. However, in the cIAP2 KO model, where cIAP2 protein is absent, this inhibitory effect of cIAP2 is eliminated and the receptor induced apoptotic stimuli can ultimately lead to macrophage apoptosis. Indeed, studies have demonstrated that cIAP1/2 proteins regulate TNF-Î±-mediated canonical NF-ÎºB activation by acting as critical ubiquitin E3 ligases for Rip1, and non-canonical NF-ÎºB signaling by promoting ubiquitination and proteasomal degradation of the NF-ÎºB-inducing kinase (Blankenship JW. et al., 2009). Induction of the cIAP1/2 E3 ubiquitin ligase activity results in rapid autoubiquitylation and proteasomal degradation of the cIAP proteins. Because the cIAP proteins also regulate the non-canonical NF-ÎºB pathway through ubiquitylation of NIK, their loss results in activation of the non-canonical pathway through NIK stabilization. NF-ÎºB activation leads to induction of NF-ÎºB target genes, including TNF-Î±. In the absence of the cIAP proteins, TNF- Î± can activate caspase-8 through engagement of TNFR1.
6.7 The role of Smac mimetic drugs.
It has well been established that over-expression of the IAP proteins confers protection against a variety of pro-apoptotic stimuli, including chemotherapeutics, and are markers for poor prognosis in a variety of solid tumors and hematologic malignancies (Salvesen GS. et al. 2002; Liston P. et al. 2003; Nachmias B. et al. 2004). Conversely, suppression of IAP protein levels can sensitize cancer cells to functionally diverse pro-apoptotic therapeutics (Yang L. et al., 2003; Kasof GM. et al., 2001). As mentioned earlier, all IAPs share at least one BIR domain and antagonism of IAP-mediated inhibition of caspases is required for efficient caspase-dependant cell death. This can be achieved by endogenous Smac/DIABLO that is released into the cytoplasm in response to pro-apoptotic stimuli (Du C. et al., 2000; Verhagen AM. et al., 2000). Extensive studies have examined the particular electrostatic interaction between the Smac/DIABLO proteins and select BIR domains of the IAP proteins (Wu G. et al., 2000; Franklin MC. et al., 2003). In summary, Smac/DIABLO blocks caspase interaction by competing directly for the same peptide-binding site on the BIR domains of the IAPs. Smac-derived peptides have, accordingly, been shown to sensitize a number of different tumor cell lines to apoptosis induced by a variety of pro-apoptotic molecules both in vitro and in vivo. Figure 18 illustrates the proposed pathways for the action of Smac-mimetics on cIAP2. One may hypothesize that, in addition to these potential cancer applications, Smac-mimetic drugs could be administered in order to reduce early atherosclerotic lesion development through IAP inhibition via promotion of apoptosis in plaque macrophages.
6.8 Methodological and technical considerations
As mentioned earlier, the sample sizes in some experiments did not add up to the 15 mice per group as had been initially planned. This was due to technical difficulties during sample collection and processing. Moreover, aortic root section collection can be challenging. The inside temperature of the cryosectioning machine is very critical and in some occasions, when the temperature is not set correctly by previous users, samples were not sectioned and collected properly and/or did not adhere to the slides adequately. Furthermore, there are many washing steps during staining (both for CD68 and Sudan IV) and this occasionally results in the sections separating from the slide and dissolving into the solution.
Some of the mice on a 12wk HFD underwent cardiac perfusion with 4% PFA in order to fix the animal for further analysis (for imaging purposes discussed in the "future directions" section). This fixation method was performed before deciding to stain for macrophages using CD68 antibody. Cardiac fixation of the whole mouse "fixes" the tissues and interferes with staining techniques where the antibody can no longer bind the desired target. Therefore, a significant amount of 12wk macrophage aortic root analysis could not be performed. However, the group of mice on 4wk HFD was not all fixed with 4% PFA cardiac perfusion and so could be stained and quantified.
Variability within the group, especially in an animal model, is inevitable. Despite all the mice being from the same gene pool, each mouse in this study ingests a different amount of food, metabolizes it differently, have different energy levels and expenditures. This translated into different disease rate progression and/or differences in lesion sizes. These differences were observed during analysis where some mice displayed lesion sizes very differently from the other mice in the study group.
6.9 Study Limitations
The current study used cIAP2-/- x apoE-/- male mice to study the effect of cIAP2 on atherosclerotic lesion development. Only male mice were used because several studies have reported a wide variation in lesion size in female mice attributed mainly to fluctuating estrogen levels believe to play a protective role in atherosclerosis (Nakagami F. et al., 2010; Thomas CM and Smart EJ., 2007). However, it would be helpful to examine how female mice would react to cIAP2 deletion. This would clarify whether cIAP2 action is gender related or not.
In order to fully understand the mechanism of action of cIAP2, in vitro data using peritoneal macrophages derived from the different study groups could be performed. Caspase activity assays (in different macrophage cells (4wk/12wk KO and WT)) would help understand to what extend cIAP2 affects the effector caspases-3 and/or -7.
Measuring apoptosis in the plaque could certainly have been a relevant component of the analysis considering that we were examining the role of an inhibitor of apoptosis protein and its effect on lesion progression. Accordingly, we have started staining using fluorescent TUNEL assays on aortic root sections. The aim was to asses both CD68 staining along with TUNEL staining using dual fluorescent staining methods. This could enable us to quantify the apoptotic macrophages in the lesions. The selected aortic root sections were 10um distant from the sections previously stained for lipids using Sudan IV. However, this fluorescent TUNEL (as well as CD68 fluorescent staining) assay was problematic as the fluorescent stains decayed very quickly and restricted us from quantifying the data properly. Furthermore, the rate of apoptosis in the study sections did not seem to be well-reflected by this staining method. However, it is not clear how much of the apoptotic process is detectable using these systems in these early lesions. If cleaved caspase-3 antigen is only detectable for part of the time the cell is undergoing apoptosis, measurement of the cleaved enzyme alone will underestimate the frequency (Clarke CH. and Bennet MR. CH., 2008). Similarly, the presence of TUNEL-positive bodies after apoptosis has finished may grossly overestimate the frequency (as may have been the case in the 12wk HFD mouse group). Of note,it is frequencies of apoptosis that can be measured not the rates of apoptosis. We do not know the duration of the apoptosis process in vivo, and most studies have not examined 2 or more time points to calculate these rates. We also do not know the time it takes for apoptotic body clearance in vivo. Previous studies identifying "delayed clearance" have only studied one time point. It is also extremely difficult to determine whether an apoptotic body is inside or outside a cell in a histological section given the multiple planes of view. However, some forms of "programmed" cell death appear to be caspase-independent, and TUNEL staining can yield false-positive results. Other features are often used to identify apoptotic cells in vivo including externalized phosphatidylserine, as assessed by staining with labeled annexin V, and distinct morphological features of apoptotic cells, notably condensed nuclei.
CHAPTER 7: Conclusion and future directions
In summary, we show for the first time, using an atherosclerotic apoE-/- mouse model, that cIAP2 deletion reduces atherosclerotic lesion development at an early and later disease stage. We also show trends for reduction of macrophage infiltration in the early disease stage in the KO mouse model (cIAP2 -/- x apoE -/-) compared to WT mice consistent with a role for cIAP2 in macrophage longevity. This study presents new evidence for the role of cIAP2 protein in atherosclerosis progression and outlines its possible mechanism of action.
Using a total body KO model for cIAP2 as well as a WT model, aided us in identifying the role which cIAP2 plays in the progression of atherosclerotic disease. This was done using standard atherosclerosis quantification techniques such as en face and aortic root lesion analysis techniques. Measuring total serum cholesterol and lipoprotein profiling was employed to eliminate this risk factor as a cause for differences in disease progression. Analysis indicates that cIAP2 deletion was linked with reduced lesion size in both early and late animal models of the disease when compared to the wild type littermates. cIAP2 is hypothesized to inhibit macrophage apoptosis in the atherosclerotic plaque. This was supported by quantifying the extent of active macrophage infiltration in the atherosclerotic plaque by using CD68 antibody specific to active macrophages. Analysis using mice on a 4wk HFD indicated reduced macrophage infiltration in the KO model, which goes hand in hand with reduced atherosclerotic lesions. In conclusion, cIAP2 deletion helps protect mice from developing atherosclerotic lesions. The data suggest that this occurs by reducing the extent of macrophage infiltration in the plaque.
An issue in the literature in this field (as well as in the current project) is determination of cause and effect rather than association. While many processes accompany apoptosis, only direct manipulation of apoptosis alone in a single cell type can reliably assume to determine a direct consequence of that cell type. It is hard to interpret many studies that show changes in plaque composition and apoptosis with drugs or genetic manipulations that affect multiple cell lines or have multiple effects other than apoptosis.
Future studies will need to explore the early apoptosis concept by using cIAP2-/- BMT experiments to detect the specific role of cIAP2 in macrophage of apoE-/- mice. This experimental approach will ensure that only hematopoietic cells deficient in cIAP2 protein are responsible for the decrease in atherosclerosis. This will pin-point the reduction in atherosclerosis to macrophage cells alone as opposed to vascular smooth muscle cells or other cell types found in the atherosclerotic vessel milieu that may be affected by a total body KO of cIAP2. This BMT concept has been proposed to test in apoE-/- mice fed an atherosclerosis-promoting diet for a period known to induce early lesions in control mice (Whitman SC., 2000). The study should further explore the role of cIAP2 in both sexes, male and female.
If the hypotheses are supported, such findings would then indicate that cIAP2 may be a specific therapy target. Smac-mimetic drugs, specific cIAP2 antagonists, could delay or limit atherosclerosis at early stages. On the other hand,, smac-mimetics may be detrimental to late complex atherosclerotic plaque. Future in vitro studies will also need to explore the specific interaction of cIAP2 protein with caspases in peritoneal derived macrophages. Despite evidence in this study, more experiments should be conducted in cIAP1 KO models to understand if it also plays a role in atherosclerosis. This is important since cIAP1 and 2 are very homologous and may share many pathways and mechanisms of action.
Appreciation for the cellular and molecular mechanisms of atherosclerosis, thrombosis, and vascular inflammation opens the way for sophisticated approaches to disease characterizations through imaging. The common goal for molecular imaging approaches is to accelerate and refine diagnostics, provide insights to understand disease, guide and monitor the effects of those therapies. As a future direction, the possibility to apply imaging to quantify changes over time in atherosclerotic lesion size will be explored. F-18-fluorodeoxyglugose (F18-FDG) can be used as a glucose analogue that is taken up by active macrophages in the atherosclerotic plaque (Rudd JH. et al., 2008). Using this marker of monocyte metabolism and plaque inflammation, we aim to image these mice using positron imaging tomography (PET) as well as computed tomography (CT) in order to monitor disease progression and therapy affects in humans. Our laboratory shows promising preliminary results from mice on HFD showing FDG uptake in the atherosclerotic plaques.
Ultimately, the progression and/or regressions of the disease could be followed via such plaque imaging techniques. These imaging techniques will, for example, target different molecules that are characteristic of different disease stages and thereby act a as biomarker to monitor the effectiveness of the treatment. Various methods, including ultrasonography, CT, and MRI have been employed for this purpose. However, most of these structural imaging techniques have major limitations. SPECT and PET/CT combined functional and structural whole-body imaging modality hold the most potential for this purpose.
The data from the current project set the stage for future interventions to prevent inhibition of apoptosis in early atherosclerosis. They also set the platform for developing means to serially evaluate disease progression and response to therapy in vivo. In the future, these approaches and treatments may be translatable to humans. The data from this proposal help to identify a potential target for therapy that may reduce atherosclerotic burden in patients and thereby reduce long term adverse clinical outcomes