ABSTRACT
Over the past 20 years, there have been a number of significant advances in our knowledge about the superfamily of PLA2enzymes. PLA2 hydrolyzes the fatty acid from the sn-2 position of
phospholipids. In vivo, the sn-2 position of phospholipids frequently contains polyunsaturated fatty acids such as arachidonic acid, and when released, these can be metabolized to form various eicosanoids and related bioactive lipid mediators (Funk, 2001). The metabolism of arachidonic acid, of course, is a very familiar signaling pathway to cardiologists as it represents the target of the class of drugs known as non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, that exert their effects through inhibition of the enzyme cyclooxygenase (Vane and Botting, 2003). The remaining products of PLA2 action, lysophospholipids, can also have a number of important roles in biological processes (Rivera and Chun, 2008). The secreted PLA2s were the first type of PLA2 enzymes discovered (Lambeau and Gelb, 2008) with the human genome containing nine sPLA2 genes (i.e., groups IB, IIA, IID, IIE, IIF, III, V, X, and XIIA). These proteins are disulphide-rich and have molecular weights of around 16 kDa except for the group III sPLA2, whose cDNA predicts a protein with a molecular weight of ~55 kDa. These enzymes share a common aspartic acid/histidine catalytic dyad that cleaves substrate in a calcium-dependent manner via interfacial kinetics, i.e., when substrate is presented as a large lipid aggregate (Scott et al., 1990). Varespladib is a broad inhibitor of these sPLA2 enzymes with IC50values in the low nanomolar range (e.g., group IIA IC50: 9−14 nM; group V IC50: 77 nM; and group X IC50: 15 nM) (Snyder et al., 1999; Mihelich and Schevitz, 1999). Although varespladib has the ability to inhibit all of the sPLA2 enzymes listed above, this article will focus primarily on groups IIA, V, and X, since there is evidence that these enzymes are implicated in the pathogenesis of atherosclerosis. Lp-PLA2, also known as platelet-activating factor acetylhydrolase (PAF-AH) or PLA2 type VIIA (Schaloske and Dennis, 2006), is also secreted but is a calcium-independent PLA2. Lp-PLA2 is different from other secreted PLA2s in that it utilizes a distinct Ser/His/Asp triad active site (Tjoelker et al., 1995; Burke and Dennis, 2009). Unlike the sPLA2s described above, which contain a His/Asp dyad active site, Lp-PLA2 accesses its substrate in the aqueous phase (Min et al., 1999). This property of Lp-PLA2 enables a broad substrate specificity that is governed primarily by aqueous phase solubility. Since the active site of Lp-PLA2 is not related to those of the other secreted PLA2s, it is not surprising that although darapladib is an extremely potent inhibitor of Lp-PLA2 (IC50 270 pM), it is a relatively poor inhibitor of the sPLA2s listed above demonstrating IC50s > 1 µM
2(Wilensky et al., 2008). Darapladib is also a significantly less potent inhibitor of the structurally related but intracellular enzyme, PLA2type VIIB (IC50 600 nM, Macphee et al., unpublished data) (Schaloske and Dennis, 2006). 7.3 Substrates Preferences, Lipoprotein
7.3.1 Lp-PLA2Lp-PLA2 was first discovered and originally named for its ability to cleave the acetyl group from the sn-2 position of PAF in vitro, for example, when micromolar concentrations of PAF were added to isolated plasma (Blank et al., 1983). This observation led naturally to the assumption that this enzyme was responsible for the inactivation of PAF in vivo and that Lp-PLA2 inhibition could enhance PAF-mediated biology (Prescott et al., 2000; Stafforini, 2009). However, little to no direct evidence exists implicating a role for Lp-PLA2 in the cleavage and inactivation of physiologically relevant concentrations of PAF in vivo. Although the Km of Lp-PLA2 for PAF is reported to be around 10 µM (Tew et al., 1996), PAF elicits responses at nanomolar concentrations (Snyder, 1995). Furthermore, it has been reported that the half-life for PAF in plasma or whole blood (which contains secreted Lp-PLA2) from healthy individuals is approximately 5 minutes (Yoshida et al., 1996). These enzyme characteristics tend to question a role for Lp-PLA2 in hydrolyzing and inactivating physiologically meaningful concentrations of PAF in vivo, since this phospholipid mediator is biologically active in the low nM range, having a calculated Kd for its receptor of around 1 nM (Hwang, 1990), a concentration of PAF that is closer to levels found circulating in humans (Vadas et al., 2008). Indeed, the most direct study conducted thus far has suggested a lack of a prominent role of Lp-PLA2 in modifying PAF-mediated responses. Naoki et al. (2004) made use of a natural deficiency in Lp-PLA2 (Stafforini et al., 1996) amongst the Japanese population to explore PAF responsiveness. This clinical study demonstrated no statistically significant differences in either pulmonary function or transient neutropenia following PAF inhalation in individuals deficient in Lp-PLA2 when compared with age-and sex-matched controls. These data are entirely consistent
with observations made in a very recent article by Liu et al. (2011), suggesting that rapid clearance of PAF by endothelial cell-rich organs such as the liver represents the major route of in vivo PAF catabolism rather than Lp-PLA2 activity. The observation that the catalytic site of Lp-PLA2 is uniquely exposed to the aqueous phase allows broad substrate recognition. Thus, in addition to hydrolyzing PAF, Lp-PLA2 notably catalyzes oxidized phospholipids that can be both short (i.e., fragmented) or long chain in nature, including even F2-isoprostane esterified phospholipids (Stremler et al., 1991; Tew et al., 1996; Min et al., 2001; Stafforini et al., 2006; Kriska et al., 2007; and Davis et al., 2008). It is exactly this unique capability of Lp-PLA2 to cleave oxidized phospholipids that has implicated it as a potential key player in atherosclerosis (Zalewski and Macphee, 2005). One process, therefore, that Lp-PLA2 is definitely not involved in is the release of unmodified arachidonic acid for eicosanoid formation. Unlike sPLA2s, Lp-PLA2, as its name suggests, associates with lipoproteins in human plasma (Stafforini et al., 1987; Caslake et al., 2000). The vast majority of Lp-PLA2 is found associated with LDL due primarily to a specific interaction between two domains on human Lp-PLA2 and the carboxy terminus of apolipoprotein B-100 Stafforini, D. M., Tjoelker, L. W., McCormick, S. P. A., Vaitkus, D., McIntyre, T. M., Gray, P. W., . . . Prescott, S. M. (1999). Molecular basis of the interaction between plasma platelet-activating factor acetylhydrolase and low density lipoprotein. Journal of Biological Chemistry, 274(11), 7018-7024.. Intriguingly, it has been demonstrated that Lp-PLA2 is enriched on two highly atherogenic apolipoprotein B-100 containing particles, small dense LDL and lipoprotein(a) (Lp(a)) (Tselepsis et al., 1995; Blencowe et al., 1995). Recent findings have shown a more pronounced association of Lp-PLA2 with allele-specific levels of small size apo(a) (Enkhmaa et al., 2010), which is interesting as several studies have demonstrated an association between small size apo(a) and elevated cardiovascular risk (Clarke et al., 2009). Critically, these apo B-100 containing lipoproteins are viewed to be especially atherogenic once they become oxidized (Tabas et. al., 2007; Libby et al., 2009), thus providing Lp-PLA2 with a ready supply of substrate, in this instance, oxidized phosphatydylcholine. In short, Lp-PLA2 remains latent until these lipoproteins undergo oxidative modification, thereafter Lp-PLA2 rapidly generates biologically meaningful quantities of lysophosphatidylcholine (LPC) and oxidized
2free fatty acids, both of which have been demonstrated to be causal agents in promoting atherosclerosis (Wilensky and Macphee, 2009) (Wilensky, R. L., & Macphee, C. H. (2009). Lipoprotein-associated phospholipase A2 and atherosclerosis. Current Opinion in Lipidology, 20(5), 415-420). Cleavage of oxidized phosphatidylcholines by Lp-PLA2 has been demonstrated for oxidized LDL, oxidized small dense LDL as well as oxidized Lp(a) (Tselepis et al., 1995; Karabina et al., 1996; Macphee et al., 1999). The situation for Lp(a) is even more interesting as this lipoprotein particle appears to be a preferential carrier of oxidized phospholipids (oxPLs) in human plasma (Tsimikas et al., 2005) and therefore potentially provides a continuous supply of substrate for Lp-PLA2. Evidence exists for a regulatory role for LPC, in particular, in promoting atherosclerotic plaque development that could ultimately lead to the formation of a necrotic core. These steps include recruitment and activation of leukocytes, induction of apoptosis, and impaired removal of dead cells (Matsumoto et al., 2007; Wilensky and Macphee, 2009; Schmitz and Ruebsaamen, 2010). Thus, through both its lipoprotein association and substrate preferences, Lp-PLA2 is uniquely placed amongst PLA2s to directly influence oxidized lipoprotein-mediated inflammatory responses. Oxidized phospholipids have been proposed by several groups to directly promote inflammatory responses with much of the data originating from in vitro studies using exogenously added oxPLs (McIntyre et al., 1999; Berliner and Watson, 2005). Thus, inhibition of Lp-PLA2 may lead to an accumulation of oxPLs, its substrate, and potentially augment putative processes directly dependent upon intact oxPLs. Accumulation of oxPLs does not appear to be an issue with chronic Lp-PLA2 inhibition, since longterm darapladib treatment did not influence atherosclerotic lesion oxidized phosphatidylcholine content in a porcine model while reducing significantly the elevated lesion LPC content (Wilensky et al., 2008). This observation suggests that Lp-PLA2 substrates can be alternatively metabolized or, perhaps, even actively removed through specific transport mechanisms such as Lp(a). It should be noted that more recent studies have demonstrated that oxPLs can induce anti-inflammatory effects through inhibition of Toll-like receptors, an action that could aid resolution of chronic inflammatory states (Bochkov, 2007; Von Schlieffen et al., 2009; Feige et al., 2010). Another feature worth considering is the role of the various secreted PLA2s in host defense, in particular in modulating
bacterial infection, and how this could impact atherosclerosis. Recent evidence indicates that Lp-PLA2 can degrade lipoteichoic acid (LTA), a polyphosphate attached to the cell membrane via a diacyl glycolipid that is an abundant component of the envelopes of gram-positive bacteria (Ho and Nahm, 2009). LTA is a ligand for the Toll-like receptor, TLR2, a pattern recognition receptor that is a component of the innate immune system that acts as a defense mechanism against pathogen invasion (O’Neill et al., 2010). Diacylation of LTA is required for TLR2 signaling, thus deacylation by Lp-PLA2 leads to inactivation of LTA (Ho and Nahm, 2009), which potentially could lead to a reduction in the detection of LTA-containing bacteria. It is interesting in this respect to note that Lp-PLA2-deficient mice displayed decreased mortality compared to wild-type mice in a model of septic enterocolitis (Lu et al., 2010). Clearly, much more work is needed to elucidate a role for Lp-PLA2in bacterial infection, but such a connection could be relevant for atherosclerosis, since compelling data exists indicating that infection contributes to atherogenesis and the acute complications caused by plaque rupture (Epstein et al., 2009). A recent study of human carotid plaques showed a significant association of plaque Lp-PLA2with both macrophages and chlamydia pneumoniae that prompted the authors to suggest an interactive role of infection and Lp-PLA2 in accelerating inflammation in atherosclerosis (Atik et al., 2010). 7.3.2 sPLA2While group V and X sPLA2s hydrolyzes both anionic (e.g., phosphatidylglycerol) and zwitterionic (e.g., phosphatidylcholine, PC) phospholipids, group IIA sPLA2 has extremely low activity on vesicles of high PC content when compared to anionic vesicles (Singer et al., 2002; Gora et al. (2006) Lambeau and Gelb, 2008). These properties have significant physiological consequences particularly when considering hydrolysis of lipoproteins that are PC rich. There is accumulating evidence, however, linking these three sPLA2s to arachidonic acid release from cellular phospholipids for the biosynthesis of eicosanoids (Lambeau and Gelb, 2008). However, the precise mechanisms remain unclear but appear to require the concomitant participation of cytosolic PLA2s to fully maximize eicosanoid production (Ghosh et al., 2006; Ni et al., 2006; Henderson
et al., 2007). Thus, unlike Lp-PLA2, these sPLA2s are directly linked with inflammatory pathways resulting from eicosanoid formation. Of specific interest to atherosclerosis, considerable effort has been invested in evaluating the role of groups IIA, V, and X sPLA2s in the hydrolysis of lipoproteins. It is important to point out that these enzymes, unlike Lp-PLA2, do not directly associate with human lipoproteins in vivo and much of the evidence has been derived from in vitro studies where sPLA2s have been added to lipoprotein preparations. Further evidence has been derived from overexpression studies in preclinical animal models that have very different lipoprotein profiles to humans. The relevance of these studies to the human situation could, therefore, be questioned. For instance, the relative ability of groups IIA, V, and X sPLA2s to hydrolyze the major phospholipid within lipoproteins, PC, confirmed previous substrate studies listed above in that only group V and X sPLA2s significantly hydrolyzed LDL and HDL in vitro (Gesquiere et al., 2002; Hanasaki et al., 2002; Pruzanski et al., 2005; Jönsson-Rylander et al., 2008). These studies indicated that Groups V and X sPLA2s are potentially physiologically more important than group IIA sPLA2 in lipoprotein metabolism. An important consequence of this lipoprotein remodeling was the finding that LDL modified by either group V and X sPLA2 was demonstrated to induce lipid accumulation in macrophages and activate endothelial cells in culture (Wooten-Kee et al., 2004; Karabina et al., 2006), two proatherogenic activities. It is also speculated that sPLA2-modified LDL could transit the vessel wall more readily than unmodified particles and promote retention of these atherogenic particles in the vessel wall (Rosenson and Gelb, 2009). The finding that lipolysis of LDL by sPLA2 results in a conformational change in apolipoprotein B that enhances LDL binding to intimal proteoglycans certainly supports such a notion (Sartipy et al., 1999; Hakala et al., 2001). Overexpression studies in mice with group IIA sPLA2 resulted in profound alterations in HDL metabolism, which is somewhat of a surprise given the findings described above (Tietge et al., 2000; Menschikowski et al., 2000). These transgenic mice had significantly lower total cholesterol, which was explained primarily by a reduction in HDL cholesterol due to its enhanced catabolism. Overexpression of group V sPLA2 in mice, in contrast to the above, had no influence on plasma lipoprotein levels (Bostrom et al., 2007). It should be noted that mice have very different lipoprotein profiles to humans, meaning that it is difficult to extrapolate these findings to
the human situation. This appears to be borne out by observations made with varespladib in the clinic (section 7.7.2) that consistently demonstrate a reduction in LDL cholesterol levels with no alteration in HDL cholesterol, a result not predicted by the overexpression studies in mice. The most recognized physiological function of group IIA sPLA2 is its antibacterial activity mediated by its selective and potent ability to attack and degrade the phosphatidylglycerol-rich membranes of bacteria, thereby contributing to the first line of host defense (Koduri et al., 2002; Nevalainen et al., 2008). Importantly, group IIA sPLA2 transgenic mice show decreased mortality following infection by both Gram-positive and Gram-negative bacteria with improved clearance from infected organs. Interestingly, the polyanionic properties of LTA (a possible substrate of Lp-PLA2 – see above 7.3.1) in the gram-positive bacterial cell wall facilitate the hydrolysis of membrane phospholipids by binding group IIA sPLA2 (Koprivnjak et al., 2002). Several other sPLA2s also exhibit bactericidal activity, with the rank order against Gram-positive bacteria being IIA > X > V > XIIA > IIE > IB (Koduri et al., 2002; Nevalainen et al., 2008). Thus, it appears that sPLA2s and Lp-PLA2 may have opposite activities against bacteria, which could have implications for varespladib and darapladib in the treatment of coronary artery disease, since infection is thought to contribute to the acute complications caused by atherosclerotic plaque rupture (Epstein et al., 2009). 7.4 Cellular Sources, Expression Within
7.4.1 Lp-PLA2Lp-PLA2 is expressed and secreted by the following hematopoietic cells, monocytes, macrophages, T-lymphocytes, and mast cells (Zalewski and Macphee, 2005; McIntyre et al., 2009). Lp-PLA2 is not considered to be an acute phase protein but can be upregulated in vitro by lipopolysaccharide, certain inflammatory cytokines (e.g., IL-1β and TNFα), oxLDL but not PAF (Wu et al., 2004; Shi et al., 2007; De Keyzer et al., 2009; Wang et al., 2010). With this leukocyte expression profile, it is not surprising, therefore, to find that Lp-
2PLA2 expression is greatly upregulated in rabbit, pig, and human atherosclerotic lesions (Hakkinen et al., 1999; Kolodgie et al., 2006; Papaspyridonos et al., 2006; Wilensky et al., 2008; De Keyzer et al., 2009). The critical finding that Lp-PLA2 is highly upregulated in macrophages undergoing apoptosis within the necrotic core and fibrous cap of human coronary vulnerable and ruptured plaques, but not within stable lesions, supports the notion that Lp-PLA2 products could be crucial in determining plaque instability (Kolodgie et al., 2006). Consistent with this notion is the observation from patients undergoing carotid endarterectomy that Lp-PLA2 expression along with its product, LPC, was higher in plaques from patients with cardiovascular events than those without suggesting that the enzyme is a key component of a causal pathway for plaque vulnerability (Herrmann et al., 2009). Pathological studies have shown that thrombotic coronary occlusion after rupture of a lipid-rich atheroma with only a thin fibrous layer of intimal tissue covering the necrotic core (the socalled thin-cap fibroatheroma, TCFA) is the most common cause of myocardial infarction and death from cardiac causes (Virmani et al., 2006). Before discussing preclinical model overexpression studies, it is important to point out that the majority of preclinical animal models, including mice, do not develop complex, humanlike, TCFAs. Liver specific overexpression of human Lp-PLA2 in apolipoprotein E-deficient mice was shown to inhibit injury-induced neointimal formation (Quarck et al., 2001). How such ectopic expression of Lp-PLA2 by hepatocytes in this mouse model relates to human atherosclerosis is difficult to ascertain but may point to an important interaction between apolipoprotein E and Lp-PLA2. This study, however, is not consistent with a later study demonstrating a reduction in complex coronary lesion development with darapladib in a porcine model of atherosclerosis with normal apolipoprotein E content (Wilensky et al., 2008; see later section 7.6.1). 7.4.2 sPLA2Group IIA sPLA2 is an acute phase protein whose expression is greatly upregulated by endotoxin, which should come as no surprise given its potent in vivo antimicrobial action. Indeed, its expression is markedly induced by proinflammatory stimuli in a wide variety
of tissues (Jönsson-Rylander et al., 2008; Murakami et al., 2010). Group V sPLA2 transcripts are also widely expressed, for example high levels have been located in the heart and placenta as well as certain leukocytes, including mouse bone marrow-derived mast cells (Balestrieri and Arm, 2006). Expression of group X sPLA2 has been demonstrated in thymus, spleen, and leukocytes, which suggests an involvement in the immune system (Murakami et al., 2010). Microarray gene expression studies of human carotid plaques versus non-diseased human vessels failed to show an upregulation of either group IIA or V sPLA2 in disease while clearly demonstrating a large upregulation of Lp-PLA2 (Zalewski and Macphee, 2005). Indeed, in this study group IIA sPLA2 expression was much lower in the endarterectomy samples when compared with control vessels highlighting the contribution of the smooth muscle-rich media to its expression as this was missing from the tissue sample. These findings were replicated in an analysis of plaque from hypercholesterolemic pigs (De Keyzer et al., 2009). Thus, the only one of the three considered sPLA2 genes that is expressed in atherosclerotic lesions to any great extent is group X sPLA2, which, like Lp-PLA2, reflects its macrophage expression profile (Karabina et al., 2006; De Keyzer et al., 2009). However, although gene expression studies have been somewhat neutral for Group IIA and V sPLA2s, immunohistochemical analyses have detected their presence within lesions (Hurt-Camejo et al., 1997; Rosengren et al., 2006; Kimura-Matsumoto et al., 2008). Considerable effort has been invested in genetically altering the expression of sPLA2s in mice to elucidate a role in atherosclerosis (Boyanovsky and Webb, 2009; Murakamo et al., 2010). Interestingly, the common mouse strain used to study atherosclerosis, C57BL/6, is a natural knockout of group IIA sPLA2 due to a frame shift that interferes with gene translation. Thus, group IIA sPLA2 expression is not critical for lesion formation in the mouse. However, overexpession studies in mice, either broadly or ectopically in macrophages, have demonstrated an increased susceptibility to atherosclerosis (Ivandic et al., 1999; Webb et al., 2003; Webb, N. R., Bostrom, M. A., Szilvassy, S. J., Van der Westhuyzen, D. R., Daugherty, A., & De Beer, F. C. (2003). Macrophage-expressed group IIA secretory phospholipase A2 increases atherosclerotic lesion formation in LDL receptor-deficient mice. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(2), 263-268; Tietge et al., 2005). Similarly, as for group IIA, transplantation of bone marrow-derived cells that overexpressed group V sPLA2 in LDLr−/− mice also increased atherosclerotic lesion area. Moreover,
2group V sPLA2 deficiency in bone-marrow derived cells caused a reduction in atherosclerosis in LDLr−/− (Bostrom et al., 2007) but not in apoE−/− mice (Boyanovsky et al., 2009). Unfortunately, although group X sPLA2 demonstrates perhaps the most convincing profile for a role in atherosclerosis, both in substrate preferences and leukocyte expression, no direct evidence is currently available to support a role in atherosclerotic lesion formation. However, mice deficient in group V sPLA2 are available, with recent data showing that the incidence and severity of angiotensin II-induced abdominal aortic aneurysms were significantly reduced in apoE−/− mice also deficient in group X sPLA2 (Zack et al., 2011). In summary, the accumulated data for sPLA2 involvement in murine atherosclerosis is certainly much stronger than for Lp-PLA2. 7.5 Human Epidemiology and Genetics
The first study to highlight the role of Lp-PLA2 as an independent risk predictor for cardiovascular disease was the West of Scotland Coronary Prevention Study (Packard et al., 2000) Packard, C. J., O'Reilly, D. S. J., Caslake, M. J., McMahon, A. D., Ford, I., Cooney, J., . . . Burczak, J. D. (2000). Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. New England Journal of Medicine, 343(16), 1148-1155. This study not only showed a positive association between elevated circulating levels of Lp-PLA2 and risk of coronary events but, critically, demonstrated that the increased risk was not confounded by classical cardiovascular risk factors such as lipids, as well as C-reactive protein (CRP). This observation has now been replicated in many studies covering both primary and secondary prevention and has been summarized in several recent reviews (Caslake and Packard, 2005; Garza et al., 2007; Epps and Wilensky, 2011). In a recent meta-analysis incorporating over 79,000 participants from 32 prospective studies, the risk of developing cardiovascular disease was increased by 11% for each standard deviation unit increase in Lp-PLA2 activity, after correction for other cardiovascular risk factors (Thompson et al., 2010). The finding that Lp-PLA2 retains its independence as a predictor of future cardiovascular events following adjustment for classical risk factors is somewhat surprising given that it is consistently found to
be significantly associated with LDL (positively), HDL (negatively), and triglycerides (positively). Although the clinical epidemiology around Lp-PLA2 levels is relatively consistent in demonstrating an independent association between the enzyme and CHD, the same cannot be said for Lp-PLA2genetics that has provided a somewhat confusing story primarily due to mixed reports on a loss-of-function variant of PLA2G7 (V279F) first discovered in Japanese individuals (Stafforini et al., 1996). Various reports have since confirmed that homozygous carriers of this variant are lacking Lp-PLA2 in plasma and that heterozygous carriers have around 50% the activity of individuals carrying both copies of the wild-type allele. Moreover, while the 279F null allele is relatively frequent in Japan, with approximately 25% and 2% of the population carrying one or two copies, respectively, its prevalence shows a declining gradient towards the West, with intermediate frequencies in China and Korea and almost complete absence in Europeans (Yamada et al., 2000; Jang et al., 2006). The first studies published reported an association of the loss-of-function variant with an increased risk for myocardial infarction and stroke (Yamada et al., 1998; Yamada et al., 2000), whereas a subsequent study showed only a male-and high cholesterol-specific increased risk (Shimokata et al., 2004). Others reported no association in either Japanese or Chinese subjects (Yamada et al., 2002; Hou et al., 2009). Lastly, a decreased risk of CHD was observed in male carriers of the 279V allele from Korea (Jang et al., 2006), which has very recently been confirmed in a much larger analysis comparing 3,767 male cases with CAD with 4,358 male controls without CAD (Jang et al., 2011). The data from this latest Korean study demonstrated that carriage of one copy of the 279F null allele (approx. 50% reduced plasma activity) conferred a 20% reduction in risk for CHD, which is in line with epidemiological findings on plasma Lp-PLA2 activity. Clearly, further studies are needed to fully understand the association between PLA2G7 genetic variations and coronary risk. 7.5.2 sPLA2To date, only a few studies have addressed the utility of plasma sPLA2as a biomarker of cardiovascular risk. Moreover, when sPLA2 activity is monitored, the contribution of each individual sPLA2 isozyme is unknown. The significant but weak correlation between group IIA
2sPLA2 mass and sPLA2 activity (r = 0.20) made in one study highlights this issue particularly as a better predictive value was observed for the latter (Mallat et al., 2007). The epidemiology surrounding sPLA2has been summarized in a recent review by Mallat et al. (2010). Two of the larger studies are worth mentioning, EPIC-Norfolk and KAROLA. For EPIC-Norfolk, baseline group IIA sPLA2 mass and sPLA2 activity were shown to be significantly associated with the occurrence of a first coronary event at follow up (Boekholdt et al., 2005; Mallat et al., 2007), with both significantly associated with traditional CV risk factors, including CRP. In KAROLA, which studied stable coronary disease, group IIA sPLA2 mass and sPLA2 activity showed a good correlation (r = 0.63) with both demonstrating a significant association with adverse outcomes following multivariate analyses (Koenig et al., 2009). These data support a role for sPLA2 in the prediction of cardiovascular events, although larger studies are needed to further substantiate initial findings. Several studies have attempted to use a genetic approach to determine whether polymorphisms in the genes for sPLA2s were able to support an etiological role in atherosclerosis. Wootton and colleagues (2006) reported a strong impact of haplotypic variation in the group IIA sPLA2 gene on circulating group IIA sPLA2 levels, although the study was not adequately powered to detect an association between genotype and CHD risk. The same group also found a unique association of group V sPLA2 gene haplotypes with total cholesterol and oxidized LDL, although their association with CHD risk remains to be determined (Wootton et al., 2007). Finally, a polymorphism in the group X sPLA2 gene leading to a R38C substitution demonstrated no detectable impact on cardiovascular risk, even though functional studies found the change resulted in profound reductions in catalytic activity (Gora et al., 2009). 7.6 Pharmacological Intervention: Darapladib
The effect of darapladib on atherosclerotic plaque composition was demonstrated in a diabetic and hypercholesterolemic porcine model of accelerated coronary atherosclerosis (Wilensky et al., 2008). Four weeks after induction of diabetes and hypercholesterolemia, plasma and vascular Lp-PLA2 activity increased, while 24 weeks of
darapladib treatment significantly reduced plasma and vascular Lp-PLA2 activity to levels nearly equivalent to age-matched non-diabetic, non-hypercholesterolemic controls. Importantly, the administration of darapladib (10 mg/kg/day) not only inhibited coronary artery lesion development but more profoundly reduced progression to advanced coronary lesions. In this model of complex atherosclerosis, macrophage content, plaque area, necrotic core area, and tunica media destruction were all significantly reduced compared to controls. Darapladib has not been studied in mouse models of atherosclerosis primarily, because Lp-PLA2 associates with different lipoprotein fractions (Wilensky et al., 2008) rendering the species inadequate for studying the effects of Lp-PLA2 inhibition. 7.6.2 ClinicDarapladib is currently being studied in two large Phase III trials: STABILITY (Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy Trial, NCT00799903), a fully enrolled trial involving 15,828 patients with CHD (White et al., 2010), and SOLID-TIMI 52 (the Stabilization of Plaques Using Darapladib - Thrombolysis in Myocardial Infarction 52 Trial, NCT01000727), which is estimated to include 11,500 patients with ACS. Both are event-driven trials, using a single daily dose of 160 mg (enteric coated) darapladib, with similar primary end points of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke. The 160 mg daily dose of darapladib was selected based upon data obtained from several Phase II clinical trials. Firstly, in a small Phase II trial, treatment with darapladib for two weeks prior to carotid endarterectomy significantly and dose-dependently reduced elevated Lp-PLA2 activity within the atherosclerotic plaque, demonstrating that the drug could access the critical target site (Johnson et al., 2004). Secondly, a multicenter dose ascending Phase II study was conducted where 959 stable, atorvastatin-treatment CHD patients (with achieved LDL cholesterol concentrations of less than 115 mg/dl) were randomized and treated with darapladib at 40, 80, and 160 mg for 12 weeks. In this study, Lp-PLA2 activity was reduced by 43, 55, and 66%, respectively at the three dose levels administered (Mohler et al., 2008). Treatment in this trial was not accompanied by changes in lipids, but a reduction in both CRP and Il-6 was noted. Finally, the IBIS-2 (Integrated Biomarker
and Imaging Study 2) trial compared the effects of placebo with 12 months of darapladib (160 mg) treatment on plasma CRP levels, coronary atheroma composition, and deformability in 330 patients with angiographically documented CAD (Serruys et al., 2008). Although the primary endpoints of the study were not met, nor did darapladib affect total atheroma volume, treatment did halt the increase in necrotic core volume as assessed by intravascular ultrasound radiofrequency suggesting a stabilization of the overall plaque. These clinical observations of an effect of darapladib on necrotic core formation appear consistent with results obtained in a diabetic pig model of coronary atherosclerosis (Wilensky et al., 2008). Whether these changes translate into differences in clinical outcomes awaits the results of the previously mentioned ongoing Phase III trials, STABILITY and SOLID-TIMI 52. 7.7 Pharmacological Intervention: Varespladib
The effect of sPLA2 inhibition with varespladib on atherosclerosis has been investigated using apo E knockout mice fed a high fat diet (Shaposhnik et al., 2009; Fraser et al., 2009). In these studies, varespladib was shown to significantly reduce atherosclerosis by 40−75% in addition to attenuating the development of angiotensin II-induced aortic aneurysms. In one study (Fraser et al., 2009), plasma total cholesterol that could have contributed to the atheroprotective effect of varespladib was also decreased. Interestingly, a similar reduction in plasma cholesterol in response to varespladib was also observed in the clinic (see below 7.7.2). The fact that varespladib was able to exert an antiatherogenic effect in a mouse strain that does not express group IIA sPLA2 suggests that the benefit is derived through inhibition of one or both of group V and X sPLA2s. 7.7.2 Clinic
Promising preclinical findings were sufficiently encouraging for Eli Lilly to initially explore the potential benefit of LY315920 (varespladib) in a Phase II clinical trial for severe sepsis (Reid, 2005). Although elevated baseline group IIA sPLA2 activity was
ablated by LY315920, treatment offered no overall survival benefit to patients and development of this compound for the treatment of sepsis was terminated (Abraham et al., 2003). A later clinical study with LY333013, a more bioavailable methyl ester prodrug of LY315920, was similarly found to be ineffective in rheumatoid arthritis (Bradley et al., 2005). This methyl prodrug of varespladib is now in clinical development for the treatment of atherosclerosis by Anthera Pharmaceuticals (Rosenson et al., 2010 ref 2010b). Enrollment has already commenced for a pivotal Phase III clinical study named VISTA-16 (Vascular Inflammation Suppression to Treat Acute Coronary Syndrome for 16 Weeks; NCT01130246) designed to evaluate a 16 week therapy with varespladib in combination with statins for the prevention of secondary major adverse coronary events in patients who have recently experienced an acute coronary syndrome. Up to 6500 subjects will be randomized to receive either varespladib 500 mg once daily or placebo in addition to atorvastatin and standard of care. The VISTA-16 study is expected to enroll patients with similar characteristics to those who participated in the Phase IIb FRANCIS (Fewer Recurrent Acute Coronary Events with NearTerm Cardiovascular Inflammatory Suppression; NCT00743925) acute coronary syndrome clinical study. The first Phase II results for varespladib in cardiovascular patients were from a randomized, double-blind, placebo-controlled parallel arm dose-ranging study that enrolled 393 patients with stable CHD (Rosenson et al., 2009). The primary endpoint was the change in Group IIA sPLA2 mass or activity from baseline to week 8. The authors reported a significant dose-dependent decrease in group IIA sPLA2mass levels, which reached a 95% reduction in the highest, 500 mg, dose group. However, they could not evaluate the difference in sPLA2activity, since the enzyme mass was below the limits of detection for the assay. Interestingly, plasma LDL was also significantly lowered by varespladib, attaining a 10% reduction in the highest dose compared with a 1.7% increase in the placebo group. A significant varespladib-mediated reduction in LDL cholesterol beyond standard of care was also observed in the Phase IIb FRANCIS clinical study, which enrolled 624 acute coronary syndrome patients randomized to varespladib 500 mg daily or placebo for a minimum of 6 months (Rosenson et al., 2010 ref 2010a). In addition to the expected large reduction in group IIA sPLA2 mass of 78.5%, trends were also noted for a reduction in CRP with patients treated with varespladib.
