Selective inhibition of Lp-PLA2 with darapladib reduced development of coronary atherosclerosis and, more notably, inhibited the subsequent progression to advanced lesions, resulting in a more stable plaque phenotype. As such, the major finding of this study was the marked reduction in necrotic core development and the change in arterial lesion composition resulting from Lp-PLA2 inhibition. To our knowledge, this is the first study in a large-animal model of advanced, human-like coronary artery disease to show that inhibition of vascular inflammation in the absence of an effect on cholesterol abundance reduces development of coronary lesions with high-risk phenotypic characteristics. This antiatherogenic effect was associated with a marked decrease in macrophage content and the expression of various proinflammatory genes, the majority of which are crucial for macrophage and T lymphocyte recruitment and functioning. Hence, darapladib decoupled the effect of hypercholesterolemia on inflammation, resulting in stabilization of the potential vulnerable coronary lesions thought to be responsible for unstable ischemic syndromes and myocardial infarction.
Individuals with both diabetes mellitus and hypercholesterolemia have an increased risk of macrovascular atherosclerotic complications35
, an observation replicated in the DM-HC porcine model24,25
. Such individuals have an increased percentage of coronary artery lesion area occupied by lipid-rich atheroma, macrophages and T lymphocytes and have larger necrotic cores compared to individuals without diabetes36,37
. These differences are noteworthy, as inflammation and necrotic core size are indicative of atherosclerotic plaque progression to a more vulnerable phenotype. Increased oxidative stress seems to be important for diabetic cardiovascular disease38
hydrolyzes oxidatively-modified PC, such that Lp-PLA2
inhibitors would be expected to reduce the generation of two proinflammatory lipids, LPC and oxNEFA11
. Thus, conditions that are associated with high oxidative stress, as seen in the DM-HC–induced pig with greatly upregulated coronary NADPH oxidase expression, represent an ideal scenario for studying the involvement of Lp-PLA2
in the development of complex coronary atherosclerosis. The finding that Lp-PLA2
expression was elevated in both circulating apolipoprotein B–containing lipoproteins and in complex coronary lesions in DM-HC–induced pigs provides further support for the clinical relevance of this animal model, as both observations mirror what has been reported in humans14,22
Darapladib treatment was associated with a decrease in elevated arterial LPC abundance together with a marked decrease in the coronary expression of proinflammatory genes. The LPC species influenced by darapladib treatment in the current in vivo
study bore close resemblance to those identified during the in vitro
oxidation of LDL, namely LPC 16:0, LPC 18:1 and LPC 18:0 (ref. 16
). It should be noted that our analysis of LPC abundance does not distinguish between species generated intracellularly and extracellularly, with the latter more likely to be associated with Lp-PLA2
activity. Additionally, this static analysis provides no information on the relative turnover rates of these different intracellular and extracellular pools of LPC in arterial samples, which is likely to be a key determinant of the effectiveness of Lp-PLA2
inhibition in reducing LPC abundance. Nonetheless, these data provide mechanistic support for the proposal that Lp-PLA2
, generated predominately by intimal macrophages, contributes to atherosclerosis through the generation of proinflammatory mediators, such as LPC12,13,17
, that activate pathways promoting leukocyte infiltration and activation. Of note was the striking near normalization of arterial CCR2
expression by darapladib, consistent with recent findings showing that the subset of circulating monocytes that express the MCP-1 chemokine receptor CCR2 preferentially accumulate within atherosclerotic lesions32,39
. A similar decrease in arterial expression was observed for the gene encoding the IP-10 chemokine receptor, CXCR3, which is a recognized marker of T helper type 1 lymphocytes, the principal T cell type detected within atheroma31
. Taken together, these data show that darapladib was able to influence the recruitment, activation or both of the principal inflammatory cell types present within atheromas, namely monocyte-derived macrophages and T lymphocytes.
The observation that the arterial oxidized PC content was not influenced by inhibition of Lp-PLA2
, nor were oxidized PC species elevated in atherosclerotic lesions, is of considerable interest. The low values detected of these species were not due to methodological limitations, as mass spectrometry analyses performed at the same time readily detected a considerably higher abundance of oxidized PC species in lipid extracts of human carotid artery plaques16
. Blockade of Lp-PLA2
activity would be predicted to result in the accumulation of short chain oxidized PC substrates (molecular mass 594–666 kDa), as was shown with in vitro
oxidation of LDL16
. The observation that these substrates did not accumulate suggests they are alternatively metabolized. In addition, the observation that the arterial concentration of oxidized PC did not change upon DM-HC induction casts doubt on previous suggestions that these phospholipids are centrally involved in promoting disease progression10,40
. A limitation of our findings is that we measured the abundances of oxidized PC species at a single time point, which may overlook their potential role in the initiation of the disease process and does not provide insight into the dynamics of their clearance. As these are reactive molecules, it is probable that they could form a number of protein adducts41
. Nevertheless, DM-HC induction provides favorable circumstances to address the importance of these molecules because the fat-feeding regimen used led to a decreased content of oleate (18:1)-containing PC species and an increased content of 18:2-containing PC species, the net effect of which results in a greater percentage of PC species prone to oxidative modification.
The diet used in this study contained sodium cholate to increase cholesterol levels and the development of atherosclerotic lesions. In mouse models, the addition of sodium cholate to the diet is associated with pleiotropic effects that may lead to chronic inflammation42
. No such effects have been reported in swine models of atherosclerosis, although it is possible that some of the effects observed on inflammatory gene expression may have resulted from an exaggerated effect of sodium cholate on inflammation rather than from coronary atherosclerosis per se
. However, both the control and treated DM-HC–induced groups were subjected to similar cholate doses, and, therefore, the results reflect the impact of Lp-PLA2
inhibition. Also, lesion area did not correlate significantly with cumulative cholesterol or cholate doses. Of note, the lesions shown in and were obtained from pigs that had received the same diet.
In summary, the results of this study support the hypothesis that Lp-PLA2 is causally involved in the development of coronary atherosclerosis and formation of an unstable lesion phenotype. Selective inhibition of Lp-PLA2 resulted in a marked decrease in progression to complex coronary artery lesions. The treated lesions were less severe, contained fewer macrophages and showed smaller necrotic cores. These morphological observations were paralleled by a reduction in inflammatory gene expression. As this effect was independent of cholesterol abundance or the severity of dysglycemia, these results demonstrate the crucial and independent role for vascular inflammation in the development of complex coronary artery disease. Ongoing and planned clinical studies will test the hypothesis that selective inhibition of Lp-PLA2 can reduce necrotic core formation and the incidence of myocardial infarction and death in humans.