In a previous report 5
, we showed that partial inhibition of ACAT reduced the initiation and progression of early atherosclerosis in apoE-/- mice without evidence of obvious toxicity, in agreement with a recent report in which a specific ACAT 2 inhibitor was used in these mice 45
. In the present study, we have focused on a more clinically relevant scenario- the effects of partial ACAT inhibition on established atherosclerotic plaques that progressed beyond the foam cell stage. Compared to the baseline group, over the next 14 weeks, the plaque size increased, but the progression of increase was significantly retarded in the F1394 group. ACAT inhibition was also associated with beneficial changes in plaque composition. These included fewer macrophages and reductions in the content of tissue factor or neutral lipid in general, and total cholesterol, free cholesterol, and cholesteryl ester in particular, important factors in the pathophysiology of atherothrombosis. Furthermore, these effects were not associated with toxicity that was systemic (e.g., normal weight gain and no dermal pathology) or in the plaques themselves, as assessed by the level of apoptosis, efferocytosis, or signs of necrosis. The lack of an increase in apoptosis is perfectly consistent with the drug-induced decrease in lesional free cholesterol.
As expected, ACAT inhibition tended to have a modest impact on plasma cholesterol levels (), though it was not statistically significant. As in our previous study 5
, there were no statistically significant correlations between the major findings and plasma cholesterol levels. As before, even in the F1394 group, plasma cholesterol levels (>1000 mg/dL) were far in excess of those known to accelerate plaque progression in apoE-/- mice (e.g., 46
). Thus, the benefits of partial ACAT inhibition on plaque progression and composition were in the face of persistent, severe, hyperlipidemia.
Given that plaque macrophage content decreased without an increase in apoptosis, it is interesting to consider the mechanisms for this decrease. The two major kinetic arms of interest would seem to be the recruitment of monocytes to and the egress of macrophages from plaques. Regression of atherosclerosis attributable to both arms has been reported (e.g., 47, 48
). In preliminary studies, we have evidence to support both kinetic possibilities. For example, in the F1394 treated mice, there appeared to be less endothelial, as well as sub-endothelial, expression of a major monocyte recruitment factor, VCAM-1 (Supplemental Figure 4
). VCAM-1 expression on endothelial cells mediates the adhesion of monocytes and promotes their subsequent entry into the intimal space, and its decreased expression results in less atherosclerosis in apoE-/- mice 49
. In addition, we have recently reported 50
that the promoter of the chemokine receptor CCR7, a macrophage egress factor during atherosclerosis regression 48
, is regulated in vitro and in vivo by a sterol responsive element (SRE), which is likely to be stimulated in the F1394-treated plaque macrophages, given the reduction in sterol content we observed ().
The potential of ACAT inhibitors as anti-atherosclerotic agents has been a hotly contested issue. On one hand, ACAT has been considered to be atherogenic by promoting plaque growth by allowing excess macrophage cholesterol to be stored in the form of ester, as well as by increasing the CE content of hepatic lipoproteins, the conveyors of lipids to the arterial wall. Thus, if ACAT were inhibited in macrophages and foam cells, assuming sufficient local acceptors of free cholesterol, excess cholesterol would not accumulate, thereby protecting against foam cell formation, expansion, activation, and apoptosis. Indeed, consistent with the theoretical benefits of ACAT inhibition on atherosclerosis are a number of animal studies in which a variety of inhibitors have delayed progression of disease and favorably altered plaque composition 5, 51, 52
Counterbalancing the potential benefits of ACAT inhibition is the literature that partial or complete deficiency of ACAT activity is pro-atherogenic. For example, mice with complete deficiency of macrophage ACAT (ACAT1-/- mice) have evidence of large, inflamed plaques and skin pathology attributed to the deposition of crystals of free cholesterol 6
. While it is tempting to speculate that the difference between this study and the beneficial outcomes in many pre-clinical inhibitor studies was the degree of toxicity, two clinical studies 52,53
also showed that ACAT inhibition did not reduce plaque volume as measured by intravascular ultrasound (IVUS), although IVUS would not have detected the changes in plaque composition as the histological assays we used can. As recently reviewed 54
, a plausible explanation for these apparent clinical failures includes the accumulation of free cholesterol in macrophages and foam cells, which, in addition to the formation of toxic cholesterol crystals, can also lead to ER stress, activation of the inflammasome, apoptosis, and impaired efferocytosis of dead cells 9-17, 39, 55-57
The mechanisms(s) for the apparent discrepancies between the present results and the clinical trial data will require further study. A key feature of the study here was a decrease in lesional free cholesterol, which is thought to be a key determinant of advanced lesional macrophage death and plaque necrosis. Thus, in settings where plaque free cholesterol increases, such as in the course of normal advanced plaque progression or in the setting of complete ACAT1 inhibition, advanced lesional macrophage apoptosis and plaque necrosis ensue. Accordingly, we propose that under conditions of partial ACAT inhibition by the drug used in this study, either enough HDL particles were available to prevent surpassing a critical threshold level of free cholesterol in the macrophages and foam cells or the residual level of ACAT activity (estimated to be ~50%;5
) was sufficiently protective.
In summary, we have shown that when mice with established plaques are treated with ACAT inhibitor F1394, there are major beneficial results characterized by slower plaque progression and decreased contents of macrophage foam cells, lipids, and tissue factor. Taking into account the previous animal and clinical data, these results argue that the inhibition of ACAT is still a viable clinical target to retard plaque progression, but that the dosing may be the critical issue. Too much or too little inhibition is likely to be undesirable because of cellular toxicity or lack of efficacy, respectively. While this is an important practical issue to resolve, until current imaging techniques are sufficiently sensitive to changes in plaque composition, important potential benefits of ACAT inhibition that are effected through changes in plaque composition, may go undetected, making the determination of efficacy difficult to assess.