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Atherosclerotic cardiovascular disease (CVD) is one of the leading causes of death in the Western world and has always been high on the public health priority list (Lusis J. 2000). It is the foremost underlying cause of myocardial infarction, peripheral artery disease and stroke (Lucas AD. and Greaves DR., 2001). Yet, despite the outstanding progress in clinical management, the frequency of CVD is always increasing (Singhal A., 2009). Atherosclerosis is by no means a simple or straightforward disease, but rather a very complex and progressive disease characterized by the build up of lipid rich plaques in the walls of larger arteries (Lusis J. 2000). Generally, those plaques are referred to as "atheromas" and are composed of a thick lesion with a soft, yellow core of lipids consisting mainly of cholesterol and cholesterol esters covered by a white fibrous cap (Ross R., 1999). As the atheromas grow in size, they obstruct blood flow and weaken the underlying media causing it to rupture which leads to vessel thrombosis, strokes and MIs (Lusis J. 2000). With the general recognition of atherosclerosis as a chronic inflammatory disease, there has been rejuvenation of efforts to examine the role played by regulatory mechanisms of macrophages in the progression of atherosclerotic disease and is a focus of this project.
2.1.1 Working model of atherosclerosis
Extensive studies have shown that, during atherogenesis, the vessels undergo a series of changes whereby the blood-derived inflammatory cells (such as monocytes and macrophages) are heavily involved (reviewed in Lusis J., 2000). Furthermore, cell culture studies have well established that disease initiation and progression in the endothelium is largely impacted by the accumulation of oxidized LDLs in the intima hence contributing to monocyte recruitment and foam cell formation (reviews in Lusis J., 2000).
Generally, the endothelium functions as a barrier between the blood and tissues where it is also able to generate molecules that regulate vascular tone, vascular remodeling, inflammation and thrombosis (Gimbrone MA., 1999). However, endothelial cells morphology is affected in areas where there is high fluid shear stress - endothelial cells in regions of arterial branching or curvatures show signs of increased permeability to molecules, such as circulating LDLs, which enter the subendothelial space through transcytosis (Gimbrone MA., 1999). Once in the subendothelial space, "trapped" LDLs are exposed to matrix proteoglycans and undergo modifications such as oxidation, lipolysis, proteolysis and aggregation (Goldstein JL., 2979). Injury to the endothelium leads to the over-expression of certain molecules that are responsible for the adherence, migration and accumulation of monocytes and T cells into the endothelial space (Ross R., 1999). Such molecules include platelet-endothelial-cell adhesion molecules and chemo-attractant molecules (such as MCP-1 or monocyte chemoattractant molecule -1 as well as modified LDLs) and act collectively to attract monocytes and T cells into the endothelial space where they multiply (Ross R., 1999). If these responses continue, the arterial wall thickens and compensates by gradually dilating where the radius of the lumen maintains the same size and dimensions - this is also known as the remodeling phenomena (Stastny J. et al., 1986). Upon activation, the inflammatory cells induce additional damage and ultimately result in focal necrosis by means of release of cytokines, chemokines, hydrolytic enzymes and growth factors (reviewed in Lusis J., 2000). An outline of the events leading to the initiation of atherosclerosis is represented in Figure 1.
Clinical manifestations of atherosclerosis are often only visible very late in the process when lesions reach stage VI (Whitman SC., 2004). However, type I lesions constitute the primary lipid deposits that are microscopically and chemically detectable in the intimal space (Wissler RW., 1992). Type I lesions eventually lead to Type II lesions or fatty streaks which mainly consist of microscopically evident macrophage foam cells (Stary HC. et al., 1994). More advanced Type II lesions with a larger number of macrophages (+7 layers thick) are termed Type III lesions. These continuous events of macrophage recruitment and lipid deposition eventually lead to the enlargement and restructuring of the lesions until they develop a core of lipids with necrotic tissue that is covered with a thick fibrous cap (Whitman SC, 2004).However, the first lesions to be considered advanced are stage IV which are also the first to develop a widespread and yet well defined area of the tunica intima filled with extracellular lipids (Whitman SC, 2004). An increase in fibrous tissue accumulation on the lumen side of the lipid core results in "cap" formation which labels the lesion as stage V (Whitman SC., 2004). Complicated lesions, or stages IV and V, sometimes contain thrombotic deposits and have been shown to form in the lumen of apoE-/- mice fed an atherogenic diet for a long period (14 to 20 weeks) (Whitman SC, 2004). An outline of the events leading to lesion progression is represented in Figure 2.
The late forms of lesions (or type VI lesions) are also known as advanced, complicated lesions. MIs and strokes are usually a result of plaque rupture as opposed to the progressive narrowing of the lumen (reviewed in Libby P. et al., 1997). Thrombosis and platelet adherence are products of a coagulation cascade that starts with the exposure of lipids from the ruptured plaque to blood components and tissue factor (reviewed in Libby P. et al., 1997). The shoulder region of the plaque has a relatively high concentration of lipid-laden macrophages compromising a large necrotic core, and once exposed to high fluid shear stress is more likely to rupture and lead to acute cardiovascular events (Davies MJ. et al., 1993). However, the dynamic nature and complexity of the mechanisms leading to atherosclerosis make it difficult to pin point the cause and effect relationship especially when trying to examine the role of a specific molecule whether it is a protein or a gene of interest.
In order to better understand how atherosclerotic disease is initiated, the mechanisms leading to foam cell formation need to be fully examined. Much research in the field has led to a somewhat complex picture of these events as described in the next section.
2.1.2 Foam cell formation
As previously mentioned, lipoproteins travel from the plasma to the sub-endothelium by either transcytosis or by escaping through transient gaps between endothelial cells (Weinbaum S. and Chien S., 1993). Once the lipoproteins are in the sub-endothelial space, they either return to the circulation or become "trapped" in the sub-endothelial matrix mainly through binding to proteoglycans and collagen (Williams KJ. and Tabas I., 1995). Native LDLs can be taken up by macrophages via their classical LDL receptor (LDLr-mediated endocytosis). Transcription of the LDLr gene is regulated by the amount of cholesterol within the cell whereby LDLr expression decreases as cholesterol becomes more abundant (Vainio S. and Ikonen E., 2003). Since the uptake of cholesterol via the LDLr is down regulated by increasing amounts of cholesterol, this route does not lead to excessive lipid accumulation within the cell. Therefore, macrophage uptake of native LDL does not contribute largely to the formation of foam cells during the early stages of atherosclerosis.
Endothelial cells, SMCs, as well as macrophages are all able to oxidizing LDL into oxidized LDL (oxLDL) (Tiwari RL. et al., 2008). oxLDL's uptake is regulated by mechanisms different than those of native LDL uptake (Vianio S. and Ikonen E., 2003). Modified LDLs are taken up by scavenger receptors (SR) which are a different class of receptors specific to macrophages (Vianio S. and Ikonen E., 2003). Scavenger receptor A (SR-A) expressed on the surface of macrophages has been demonstrated to recognize and internalize oxLDL (Vianio S. and Ikonen E., 2003). In this cholesterol uptake pathway, oxLDL stimulates its own uptake by up-regulating the expression of surface receptors. This continuous uptake of oxLDL by macrophages generates a lipid-laden foam cell. However, macrophage apoptosis in the plaque has been attributed to high concentrations of oxLDL, amongst other factors in atherosclerotic lesions.
The section below discusses the different factors affecting macrophage apoptosis in atherosclerotic lesions and the impact this has on lesion stability.
2.2 Atherosclerosis and Apoptosis
As the plaque becomes more advanced, macrophages apoptosis increases and is found to be triggered by several upstream signaling pathways (Lutgens E. et al., 1999). The most studied of the signaling pathways are ones involving the cell surface death receptors as well as those generated through disruption of the mitochondrial and endoplasmic reticulum (Tabas I., 2005). Atherosclerotic lesions are infiltrated with lipid-laden macrophages where macrophage cell death in this setting is typically found to be influenced by multiple factors (ibid.). A few pro-apoptotic factors have been recognized on the basis of in vitro studies that try to mimic molecular and cellular conditions taking place in atherosclerotic lesions. Some of these pro-apoptotic factors are tumor necrosis factor-alpha (TNF-Î±), Fas ligand, growth factor withdrawal, hypoxia/adenosine triphosphate (ATP) depletion, nitric oxide and intracellular accumulation of un-esterified cholesterol (Stoneman VE and Bennet MR., 2004). It has been suggested that early and late atherosclerotic lesions are influenced, at least in part, by different death inducers (ibid.).
The final outcome of macrophage apoptosis is largely dependant on how well the phagocytes can clear the apoptotic bodies whether it be at early or later stages (Savill J. and Fadok V., 2000).Generally, apoptotic cells are recognized and internalized by phagocytes in a quick and efficient way. The interaction of phagocytes with apoptotic cells leads to inhibition of inflammatory signaling and activation of anti-inflammatory pathways (Savill J. and Fadok V., 2000).
Apoptosis in early atherosclerotic lesions
In early atherosclerotic lesions, the clearance of macrophage apoptotic bodies is generally thought to be efficient and greatly beneficial. In vitro studies have demonstrated that cholesteryl ester-loaded macrophages are able to effectively recognize and engulf apoptotic macrophages (Schrijvers DM. et al., 2005). The beneficial effect of early lesion macrophage apoptosis on atherosclerosis appears to be largely due to the ability of phagocytes to efficiently clear dead macrophages. This results in reduced macrophage content in the plaque and hence a reduced plaque size. On the other hand, effective macrophage phagocytosis could also promote plaque progression by signaling to circulating macrophages and recruiting them to the site of injury thereby increasing macrophage content in the plaque. This likely explains why increasing early lesion macrophage apoptosis has been considered to have both anti- and pro-atherogenic consequences (Schrijvers DM. et al., 2005). Overall, however, it is now generally accepted that early macrophage apoptosis has a beneficial effect on plaque progression and size.
Apoptosis in advanced and late atherosclerotic lesions
Defects in both Acyl CoA: cholesterol acyl transferase (ACAT)-mediated cholesterol esterification and cholesterol efflux in late atherosclerotic lesions, lead to the buildup of large amounts of free cholesterol (FC) in the plaque causing it to rupture and ultimately result in strokes and MIs (reviewed in Tabas I., 2005). Studies using cell cultured macrophages show that the recruitment, proliferation and survival of macrophages can be altered when the apoptotic cells are phagocytosed. Therefore, the alteration of phagocytic clearance of the apoptotic macrophages is high (reviewed in Tabas I., 2005). For example, direct tissue damage can result from intracellular proteases being released from secondarily necrotic apoptotic cells causing coagulation and thrombosis (reviewed in Tabas I., 2005). The endothelial repair process is altered as a result of defective phagocytes, leading to the recruitment of monocytes to the site of injury and decreased survival of any remaining phagocytic cells (reviewed in Tabas I., 2005).
It has been observed that macrophages are more abundant in advanced lesions, which leads one to think that phagocytic clearance of the apoptotic macrophages in the late lesions is faulty or defective. A review by Ira Tabas discusses that "promotion of late lesion progression results in a number of consequences that lead to the accumulation of non-cleared apoptotic cells and post-apoptotic necrotic cells: (a) tissue factors can be secreted from non-cleared apoptotic cells; (b) the emigration of macrophages from atherosclerotic lesions may be inhibited by necrotic core formation; (c) macrophages may secrete matrix-degrading proteases when induced by inflammatory factors in the plaques necrotic core (d) finally, failure of phagocytes to internalize apoptotic cells may make them more susceptible to apoptosis leading to a reduced number of phagocytes in lesions which amplifies the events mentioned above" (reviewed in Tabas I., 2005).
There are several pro- and anti- apoptotic proteins that can be targeted for studying whether macrophage apoptosis is more beneficial in early or advanced atherosclerosis. Clever pro-apoptotic strategies, such as the ones discussed later in section 2.3.3, reveal some of the potential targets. This study examines the role of an anti-apoptotic or inhibitor of apoptosis protein in the development of the atherosclerotic plaque.
2.2.1 Mechanism of apoptosis
Mechanisms of cell death can be activated by various stimuli with the aid of a central group of proteolytic enzymes known as caspases (cystein aspartate-specific proteases) (Creagh EM and Martin SJ., 2001). All caspases are found within the cell as inactive precursor enzymes known as a zymogen. Studies have revealed that caspases are initially synthesized as pro-enzymes consisting of a large internal domain with a large catalytic active site, a small C-terminal domain, and an NH2-terminus pro-domain known as the death domain (DD) (Creagh EM and Martin SJ., 2001). The transmission of apoptotic signals is the responsibility of the large N-terminal pro-domains also called "the death domain superfamily" (Martinon F. et al., 2001). The DD is further made up of two subdomains; the "death effector domain" and the "caspase-recruitment domain" (CARD) (Creagh EM. and Martin SJ., 2001). These two subdomains constitute the downstream portion of the signaling pathway and their main task is to recruit caspases to the plasma membrane before they become activated (Fesik SW., 2000). Activated caspases then produce controlled cellular destructions grossly known as apoptosis. Caspases involved in apoptosis activation are documented as caspases -3, -4, -5, -6, -7, -8, -9, -10 and -12 (Creagh EM and Martin SJ., 2001).
Caspases are divided into two sub-groups; effector caspases and initiator caspases.
The caspases responsible for the activation of the signaling cascade are known as the upstream or initiator caspases and the caspases responsible for the actual destruction of the cell are known as the downstream effector caspases (Creagh EM and Martin SJ., 2001). In order to understand apoptosis, one should first understand caspase activation and regulation mechanisms.
2.2.2 Intrinsic and Extrinsic Apoptotic Pathways.
Intense research over the past 30 years has revealed details of two highly regulated central apoptosis pathways; (1) intrinsic pathway, regulated by mitochondria and endoplasmic reticulum and (2) extrinsic pathway, regulated by cell surface death receptors (reviewed in Movassagh M. and Foo R., 2008).
The extrinsic pathway involves death ligands such as the Fas-L or the CD95L, tumor-necrosis factors alpha (TNF-Î±), TNF-related apoptosis inducing ligand or Apo2 L, and TNF ligand superfamily member 10 or TNFSF10. These death receptors interact or bind to their respective receptors, being; Fas receptor or CD95, TNF-receptor 1 (TNFR1), death receptor 4 (DR4 )or TRAILR1 and DR5 or TRAILR2 (reviewed in Movassagh M. and Foo R., 2008). In case of the Fas receptor pathway, the binding of the ligand to the receptor results in the re-organization of the receptor complex and stimulating the recruitment of adaptor proteins such as FADD or Fas associated death domain (reviewed in Movassagh M. and Foo R., 2008). This in turn recruits procaspase-8 and procaspase-10 into another complex called death inducing signaling complex or DISC which results in the activation of the caspases.
The activation and processing of the downstream caspases can depend on multiple factors. In some cells, processed caspase-8 is enough to directly activate other caspases and execute apoptosis (reviewed in Choudhury I. et al., 2008). In other cells, however, it requires further amplification whereby caspase 8 mediates the cleavage and binding to the Bcl-2 pro-apoptotic family members (ibid.). This then leads to the release or pro-apoptotic mitochondrial factors and hence tying in with the intrinsic apoptotic pathway.
While extrinsic apoptosis is more involved in transmitting death signals from the plasma membrane, a wide variety of extracellular and intracellular signals are transduced via the intrinsic pathway (ibid.). Stimuli can include loss of survival, toxins, DNA damage, radiation, hypoxia/reperfusion, and oxidative stress (ibid.). All these stimuli, in the end, result in the activation of the apoptotic cascade through downstream signaling leading to death machinery on the mitochondria and ER (ibid.). Briefly, once the death signaling cascades reach the mitochondria, they result in what is known as mitochondrial outer membrane permeabilization (MOMP) which is a crucial event during apoptosis (reviewed in Movassagh M. and Foo R., 2008). Following those events, cytochrome c is released from the intermembrane mitochondrial space and is a very important apoptogen (ibid.). Once released into the cytosol, cytochrome c binds to an adapter molecule Apaf-1. The nucleotide binding site on Apaf-1 is exposed and allows for cytochrom c to binds to it which induces a conformation change that oligomerizes Apaf-1 into an apoptosome (ibid.). This apoptosome activates caspase-9 which then activates many downstream effector caspases leading to the execution of apoptosis (ibid.).
2.2.3 Regulation of Apoptosis
Different molecules carefully work on regulating the expression, processing and activation/inactivation of caspases in healthy cells. Furthermore, different mammalian cells are controlled by different transcriptional and post-transcriptional regulators of pro-capsase genes (Shi Y., 2004). Since caspase function is trivial to a cell's fait, it is tightly regulated by many molecules whose function ranges from blocking caspase activation at the DISC to the inhibition of caspase enzymatic activity (ibid.). There are at least three distinct types of caspase regulators, but the present study examines the inhibitory effects of one specific IAP, cIAP2, and its role in early and late atherosclerosis development.
2.3 Inhibitors of Apoptosis
As mentioned earlier, apoptosis must be tightly regulated in order to prevent any unnecessary cell death and disease. Any unregulated apoptotic mechanisms usually lead to uncontrollable cell death, which results in the pathogenesis seen in many diseases and injury models (Lotocki G. et al., 2002). A group of proteins known as IAPs, first recognized in insect cells infected by a baculovirus, have been key in the regulation of the caspase cascade and thus in the apoptotic signaling pathway (Salvesen GS., 2002; Cheng EH. et al., 1996; Stennicke HR. et al., 2002). These proteins share at least 1 cystein-rich domain of around seventy amino acids known as the baculoviral IAP repeat or BIR (ibid). Despite all the IAPs having a BIR domain, some of them do not necessarily act as inhibitors of apoptosis. Eight human IAPs have been discovered to date, and include; BIRC1/NAIP, BIRC2/IAP1/HIAP2, BIRC3/IAP2/HIAP1, BIRC5/survivin, BIRC4/XIAP/hILP, BIRC6/Apollon, BIRC7/ML-IAP and BIRC8/ILP2 (reviewed in Deveraux and Reed, 1999). Figure 4 illustrates the structure of the mammalian IAPs.
IAP-deleted mice, in spite of normal development, reveal the importance of these proteins in survival, proliferation and some differentiation processes. NAIP, cIAP2 and XIAP have been involved in the survival of neurons, cardiomyocytes and macrophages in stress conditions. BIRC4/XIAP and BIRC6/Apollon seem important for mammary glands and placenta normal development, respectively (Perrelet D. et al., 2002; Pots MB. et a., 2005; Conte D. et al., 2006; Olayioye MA. et al., 2005).
A somewhat recent review published in Molecular Cell by Srinivasula SM and colleagues discusses in great details the structural and functional features of IAPs (Srinivasula SM and Ashwell JD, 2008). The authors discuss that most IAPs have at least 1-3 BIR repeats, as well an additional functional domains such as RING-regions and/or capsapse-associated recruitement domains (CARD) closer to the C-terminal of the IAP molecule ((Srinivasula SM and Ashwell JD, 2008). IAPs that contain multiple BIR regions usually have highly conserved properties where the BIR2 and BIR3 are involved in binding with proteins containing IBMs (IAP-binding motifs), whereas the BIR1 usually interacts with several other signaling intermediates (ibid.). RING domains are involved in ubiquitylation and hence express E3 or ubiquitin ligase activity which, along with E1 or ubiquitin activating enzyme and E2 or ubiquitin conjugating enzyme, which works on catalyzing the transfer of ubiquitin molecules to end proteins (Lorick KL. et al., 1999). Ubiquitylation is modification process that is post-translational and is carried out by three ubiquitin enzymes, E1, E2 and E3 (reviewed in Wilkinson AD., 1987). Target proteins that are labelled with ubiquitin can either be poly- or mono-ubiquitylated (ibid.). Polyubiquitylation acts as a tag that signals the protein-transport machinery to ferry the protein to the proteaseome for degradation (ibid.). Mono-ubiquitylation can alter the fate of the protein in a less terminal fashion, potentially affecting its cellular sub-location, function or degradation though lysosomes (ibid.).
The identification of interacting partners of IAPs also brings to light the involvement of IAPs in TNF-Î± and Transforming Growth Factor-Î² (TGFÎ²) signaling pathways (Geisbrecht ER. and Montell DJ., 2004). This study focuses on the role of one member of the IAP family, namely cIAP2, in atherosclerosis development. The section below delineates the mechanism of action of cIAP2 and its role in apoptosis inhibition and cell proliferation.
2.3.1 Cellular inhibitor of apoptosis 2 (cIAP2)
Research has revealed the involvement of cIAP2 in different diseases such as pneumonia, bronchopulmonary dysplasia, and Hodgkin's disease (Dong Z. et al., 2003; Esposito I., et al., 2007; Seidelin JB. et al., 2007). cIAP2 is also found to be over-expressed early in the development of cervical and pancreatic cancer but, on the other hand, helps prevent hypoxic cell death (Espinosa M. et al., 2006; Christie LA. et al., 2007). Among mammalian IAPs, XIAP, cIAP1 and cIAP2 are the most studied and have been found to be associated with modulating the TNF superfamily members along with other death receptor complexes (Silke J and Brink R., 2010). Studies aiming to reveal components of the TNFR signaling complex first discovered cIAP1/2 to be recruited by the TNFR associated factor-2 (TRAF2) (rothe M. et al., 1995; Uren AG., et al., 1996). cIAP1 and cIAP2 share many structural similarities including three BIR domains, a CARD domain, and a RING domain near the C-terminal (Mace PD. et al., 2010). The BIR1 domain of cIAP1 and 2 have been shown to function in several signaling pathways via oligomerization of several binding partners (Mace PD. et al., 2010). The only domain that binds to TRAF2 and is responsible for the recruitment of cIAP1 and 2 to the TNFR complex is the BIR1 region (Samuel T. et al., 2006).
Chung JY et al. demonstrate that mammalian TRAF proteins recruit other proteins to the TNFR and have been shown to regulate many processes such as immune function and regulated cell death (Chung JY. et al., 2002). TNF signaling is regulated by cIAP1 and cIAP2 's E3 ubiquitin ligase function, and the recruitment of cIAP2 to the TNFR1 by TRAF2 is necessary for the activation of NF-kB by the TNFR1 (Vince JE et al., 2009). Studies have demonstrated that despite their wide expression, cIAP1 and 2 have a normally low abundance in tissues (Reviewed in Mace PD. et al., 2010). In vitro studies have shown that cIAP2 expression is regulated by the E3 ubiquin ligase activity of cIAP1, thus resulting in its degradation and low abundance in cells (Wu CJ. et al., 2005). However, cIAP1 knock-out cells did not exhibit an elevation in cIAP2 mRNA suggesting that the physiological levels of cIAP1 regulate cIAP2 post-transcriptionally via ubiquitylation and subsequent degradation (ibid.). cIAP1 and 2 have been involved in many signal transduction pathways which some of them will be discussed in the section below.
A study by DJ. Mahoney and colleagues carefully examined the role that cIAP1 and 2 play in the regulation of NF-ÎºÎ² via TNF-Î± (Mahoney DJ. et al., 2007). The group used a genetic knock-down method using siRNA techniques and demonstrated that the loss of cIAP2 diminishes NF-ÎºÎ² signaling via TNF- Î± (Mahoney DJ. et al., 2007). The study also demonstrated the important role that cIAP2 plays in proper Rip1 poly-ubiquitylation and aids in the activation of NF-ÎºÎ² once stimulated by TNF- Î± (ibid.). TNF-Î± is a cytokine that is involved in many processes such as development and correct functioning of the immune system and regeneration of tissue once a response to injury is triggered (Wajant H. et al., 2003).
A study by Conte D. and colleagues further examined the role the cIAP2 plays in macrophage survival (Conte D. et al., 2006). Among other things, NF-ÎºÎ² regulates the degree that a cytokine responds to an inflammatory factor such as lipopolysachcharides or LPS (Conte D. et al., 2006). When a macrophage is activated by an inflammatory factor such as LPS, it induces the production of TNF- Î± which in turns helps the macrophage function in this "aggressive" environment by increasing its resistance to apoptosis ( Kumar A. et al., 1996). However, the study was able to demonstrate that once macrophages have been depleted of cIAP2, their resistance to cell death has increased (Conte D. et al., 2006). This leads to the assumption that cIAP2 helps active macrophages survive when under stressful pro-apoptotic environment or milieu (Conte D. et al., 2006). What is most striking about this finding is that cIAP1 and cIAP2, despite their high similarity and structure and function, could actually be working independent of each other and do not substitute for one another.
Although IAPs have been scrutinized in cancer research, investigators' attention to their role in atherosclerosis has been limited. A study by Blanc-Brude and colleague examined the role that survivin (an IAP family member) plays in the formation of atherosclerotic plaques (Blanc-Brude OP. et al., 2007). Western blot analysis of excised human plaque revealed an increase in XIAP as well as cIAP2 protein expression levels when compared to regular healthy aorta (Blanc-Brude OP. et al., 2007). A representative diagram of the proposed mechanism of action of cIAP1/2 in cells, is shown in Figure 5.
Despite being regulators of apoptosis, IAPs (including cIAP2) are themselves regulated by at least three different mechanisms; transcriptional and post-transcriptional control, stability regulation, and IAP activity control by regulatory molecules. Understanding the regulation of IAPs (and ultimately cIAP2) helps in the development of potential pharmaceutical agents that will target specific IAPs and mimic the action of their regulators/inhibitors.
2.3.2 Regulation of inhibitors of apoptosis
IAP interacting molecule Smac/DIABLO (direct IAP binding protein with low pI), is a nucleus-encoded protein composed of 239-residues; however, in non-apoptotic cells it is localized to the inter-membrane space of the mitochondria (Du C. et al., 2000; Verhagen AM. et al., 2000). During the apoptotic process, a cell releases smac/DIABLO proteins into the into the cytosolic space which allows for the displacement of the IAPS from the IBM of caspases, thus inhibiting the apoptotic process (Du C. et al., 2000). As a result, smac/DIABLO enhance apoptosis and such are negative regulator of IAPs (Du C. et al., 2000). Today's advanced studies have revealed the mechanism by which smac/DIABLO regulate XIAP. However, given that the BIRs and other structures are highly conserved across most IAPs, one can assume a similar biochemical pathway within the group. However, little is known about the triggers of ubiquitylation, how it is achieved and whether or not caspase activation is involved.
It has been found that treatment with smac-mimetic compounds encourages the auto-ubiquitylation and thus further degradation of IAP proteins (Varfolomeev E. et al., 2007; Vince JE. et al., 2007). The loss of cIAPs by either treatment with smac-mimetic compounds or by gene knock-out studies have lead to apoptotic death of cells when stimulated with TNF (Bertrand MJ. et al., 2008; Varfolomeev E. et al. 2007). This re-iterates the importance of cIAPs in the TNFR-complex, suggesting that apoptosis can be induced by perhaps upsetting the recruitment of cIAPs by TRAF2.
Despite having understood most of the complex mechanisms leading to atherosclerosis lesion development and the pathways involved in the process, the majority of the studies are conducted in vitro. To better understand how foam cells lead to the pathogenesis and progression of the disease, animal disease models mimicking human disease models must be closely examined. For example, in atherosclerosis research, genetic alteration in mouse lipoproteins results in an atherosclerotic disease model that closely resembles the human hyperlipidemic/hypercholesterolemic patients and enables the study of the disease in mice with more confidence.
2.4 Mouse models for atherosclerosis
Atherosclerosis in humans is usually detectable during the advanced or complex stages but often not detected until after the patient has suffered an adverse atherosclerotic related event. As such, currently, the early events leading to the development of atherosclerotic lesions cannot be studied effectively in humans. Experimental models of the disease have enhanced the understanding of the patho-physiological process leading to vascular obstruction in both spontaneous and accelerated atherosclerosis and thrombosis (Fuster V. et al., 1991).
Mice have been invaluable in giving insight into many human disease patho-physiological processes and for studying the genetic contributions to diseases. The ability to perform genetic manipulations using transgenic and gene-targeting technologies is one of the many advantages for using the mouse model. Other advantages of mouse models are that tey are easy to breed, can be generated within 3 weeks from mating, inbred strains are widely available. Despite the use of many animal models for studying atherosclerosis, the mouse model has become a powerful tool for investigating biology and molecular pathways involved in the earliest stage lesions.
By nature, mice are very resistant to developing atherosclerosis except for the C57BL/6 mouse strain known to be spontaneously atherogenic. When this strain of mice is put on a an atherogenic diet composed of 1.25% cholesterol , 15% fat, , and 9.5% cholic acid (more than the regular human cholesterol diet intake by 10-20 times), the C57BL/6 mice build up atherogenic plaques in the intima of the ascending aorta (Paigen B., 1990). However, in order to make mice even more susceptible to atherosclerosis, their lipid metabolism must be genetically altered. This study uses the apoE KO model which is discussed in more details below.
2.4.1 Apolipoprotein E and the apoE null mouse model
Being the primary ligand for the LDLr, the main function of apoE is to mediate LDLr-mediated lipoprotein removal from the circulation. ApoE is implicated in 3 lipoprotein metabolism pathways: fat transport from the diet, endogenous transport of fat, and reverse cholesterol transport (Rall SC Jr. et al., 1989). Despite being recognized by two different receptors, apoE remains to be the most potent physiological ligand for the LDLr. ApoE, as a ligand, mediates the clearance of apoE-containing lipoprotein particles (chylomicrons, VLDL). If apoE is absent or has binding defects, atherosclerosis is likely to develop.
There are many reviews delineating the importance of the apoE KO mouse model as the most extreme of the viable phenotypes observed in lipoprotein transport (Zhong S., 1994; Rubin EM., 1994). The apoE-null mice become severely hyper-lipidemic because their capability to clear plasma lipoproteins from the blood is damaged (Rubin EM., 1994). ApoE KO mice have increased levels of VLDL and intermediate density lipoprotein (IDL) particles in the plasma. When put on a regular chow diet, mice exhibit plasma cholesterol levels of around 500mg/dl (5.64 mmol/L), as opposed to control WT mice with cholesterol plasma levels of 75mg/dl (0.84 mmol/L) (Plump AS. et al., 1992; Zhang SH. et al., 1992).
When peritoneal macrophages obtained from apoE KO mice were incubated with the lipoprotein fraction containing both VLDL and IDL (isolated from plasma of apoE KO mice by ultracentrifugation), the level of cellular cholesteryl esters increased with the concentration of VLDL/IDL lipoprotein fraction (Hakamata H. et al., 1998). The cellular cholesteryl ester mass was also significantly increased indicating that the VLDL and IDL of apoE KO mice produced a significant cholesteryl ester accumulation in macrophages through an apoE independent pathway (Zhang C. et al., 1999). This pathway may explain, at least partly, the mechanisms of foam cell formation in the arterial wall and the succeeding development of atherosclerotic plaques in apoE KO mice (Hakamata H. et al., 1998).
Despite sharing a significant number of genes with mice, many processes in the human body are different. Therefore, atherosclerotic disease models are not expected to be exactly the same between humans and mice. Considerable differences in atherosclerotic lesions have been and continue to be studied (Allayee H et al., 2003). This study examines atherosclerosis lesion development in a mouse model keeping in mind that the mechanisms involved mimic the ones in the human disease model.
2.4.2 Comparison between atherosclerosis in humans and mice
As noted in section 2.2.1, plaque development is a multifaceted and active process. Based on morphological criteria, the atherosclerotic lesion will pass through predicted stages of development as the disease proceeds. These stages have been thoroughly reviewed by Stewart Whitman (Whitman SC., 2004). The morphological features of the apoE null mouse and humans are very comparable (Stary HC., 1990; Stary HC., 1992). Mouse atherosclerotic lesions, similar to those found in humans, are made up of an irregular buildup of lipoproteins and a "cellular gathering" consisting mainly of macrophages and T-cells (Zhou X. and Hansson GK., 1999). Representative images of atherosclerotic lesions stage I, II, III and V can be found in the review by Dr. Stewart Whitman. The review also explains the cellular and molecular processes involved with each step (Whitman SC., 2004).
Whether or not advanced lesions (stage IV and V) develop in mice, the cellular and molecular progression of early atherosclerosis in mice mirrors the development of atherosclerosis in humans. This is very helpful not only in understanding the mechanism but also potentially for treating the disease with specific pharmaceutical agents. In order to decide if a certain pharmaceutical agent or genetic loss of function of even an enhancement of function effects atherosclerosis, there has to be a predefined method for measuring lesion size. The section below briefly describes a well developed method for quantifying atherosclerotic lesions that is used in this study.
2.5 Preliminary studies
Dr. Stewart Whitman's laboratory carried out preliminary experiments that acted as the foundations of this project. Studies used the technique of bone-marrow transplantation (BMT) as a way to examine whether hematopoietic-specific deficiency of two proteins, cIAP2 and caspase-3, would modulate lesion development in LDL receptor null (LDLr -/-) mice. In these studies, LDLr -/- mice (C57BL/6) underwent lethal irradiation followed by the injection of marrow cells isolated from the femurs and tibias of wild type control mice (C57BL/6 strain) or experimental mice (also C57BL/6 strain) that were either cIAP2-/- or caspase-3 -/-. Following sufficient time to enable full marrow repopulation, the chimeric LDLr -/- mice were put on an atherogenic diet for 8 wks. When the dietary period ends, the study mice we culled and then underwent cholesterol analysis as well as atherosclerotic lesion analysis.
cIAP2 BMT study - hematopoietic-specific deficiency of cIAP2 lead to a major reduction in the size lesions of plaque in the ascending aorta of chimeric LDLr -/- mice compared to control wilt-type mice with no significant change in serum total cholesterol concentrations as a result of hematopoietic-specific deficiency of cIAP2. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was utilized as a method to detect apoptosis in frozen sections. More apoptotic cells (+ve TUNEL staining) were detected in mice that were regenerated with cIAP2 -/- bone marrow.
Caspase-3 study - hematopoietic-specific deficiency of caspase-3 lead to a significant reduction in the size of the atherosclerotic size in the ascending aorta of chimeric LDLr -/- mice compared to mice that have hematopoietic cells expressing competent caspase-3. No significant change in serum total cholesterol was found as a result of this hematopoietic-specific deficiency of caspase-3.
The fact that cIAP2 deficiency has a significant effect on lesion development, despite the fact that there would have been a functional cIAP1 protein present in cIAP2-/- cells, indicates that a unique function can be attributed to cIAP2 during the promotion of atherosclerosis and therefore justifies the focus of this research project. The fact that casp-3 deficiency lead to decreased atherosclerosis suggests that cIAP2 exerts its effect in a casp-3-independent manner.