Original ArticleLipid lowering and imaging protease activation in atherosclerosis
Introduction
Lipid-lowering, in conjunction with anti-platelet and anti-hypertensive therapies are the mainstay of modern therapeutic approach to atherosclerosis.1 Part of the beneficial effects of lipid-lowering therapies, such as statins, may be linked to stabilizing effects on plaque biology rather than plaque regression.2 Inflammation is a key determinant of plaque vulnerability. Lipids, such as modified LDL, promote the recruitment and activation of inflammatory cells and trigger vessel wall inflammation in atherosclerosis.3 Macrophages (as well as other vascular cells) produce a number of proteases, including members of the matrix metalloproteinase (MMP) family.4 Activation of these proteases promotes further recruitment of inflammatory cells and directly contributes to plaque rupture through weakening of the fibrous cap.5 Some lipid lowering interventions, especially using statins, reduce vascular events linked to plaque rupture. However, there is little direct information on the effect of these interventions on vessel wall inflammation. More specifically, it is unclear whether lipid lowering, independent of the agent used, is associated with changes in MMP activation in atherosclerosis.
Molecular imaging provides an opportunity to evaluate regional and focal changes in vessel wall biology in vivo. As MMPs mediate vessel wall inflammation in atherosclerosis, MMP-targeted imaging may be a useful tool for assessing inflammation in atherosclerosis.6, 7, 8 Indeed, there is a correlation between macrophage marker expression and MMP activation detected by molecular imaging in atherosclerosis.9,10 Whether this relation persists in the presence of various therapeutic interventions, and hence, MMP-imaging can be used for tracking the effect of specific therapeutic interventions remains to be determined. Here, we investigate whether lipid-lowering, regardless of the agent used, leads to a reduction in plaque inflammation and MMP activation in parallel with changes in blood cholesterol level; and whether the effect of various lipid lowering interventions on plaque inflammation in atherosclerotic mice can be tracked through non-invasive imaging of MMP activation.
Section snippets
Reagents
Reagents were obtained from Sigma (St. Louis, MI), unless otherwise specified. RP805, a 99mTc-labeled tracer with specificity for activated MMPs was provided by Lantheus Medical Imaging (North Billerica, MA).11
Animal Model
Six- to eight-week-old female apoE−/− mice (Jackson Laboratory, Bar Harbor, ME) were fed a high fat diet (1.25% cholesterol, 15.8% fat, TD 90221, Harlan Teklad, Madison, WI) ad libitum for two months to induce atherosclerosis (n = 58). After 2 months the animals were randomized to either
Results
To investigate the effect of lipid lowering on vessel wall inflammation and its imaging in atherosclerosis, apoE−/− mice were fed a high fat diet for 2 months to induce atherosclerotic lesions along the aorta. Next, the animals were randomized to four groups: continuation of the high fat diet (HFD), high fat diet plus simvastatin (Sim), high fat diet plus fenofibrate (Fen), and normal chow [high fat withdrawal (HFW) group, supplemental Figure 1]. High fat withdrawal or fenofibrate treatment led
MMP Imaging of the Effect of Lipid-Lowering Interventions in Atherosclerosis
To address the early and late effects of various lipid-lowering interventions on MMP activation in atherosclerosis, mice underwent serial RP805 (a 99mTc-labeled tracer that specificity targets MMP activation11) microSPECT-CT imaging at 1 and 4 weeks after randomization. There was considerable RP805 uptake in aortic arch on vivo images in the HFD group, which significantly increased from 1 to 4 weeks (0.09 ± 0.01 vs 0.13 ± 0.01 cpv/MBq, respectively, for 1 and 4 weeks, P < 0.01, Figure 2 and
Effect of Lipid Lowering on Plaque Burden and Composition
As expected, oil red O staining of explanted aortas showed considerable development of atherosclerotic lesions along aortic arch in apoE−/−mice fed on the high fat diet for 3 months (relative plaque burden 61.4% ± 3.8%, Figure 4). Switch to normal chow at 2 month (HFW group) led to a significant reduction in aortic arch plaque burden at 3 months (44.2% ± 4.4%, n = 5 in each group, P < 0.05), while treatment with simvastatin or fenofibrate led to a non-statistically significant reduction in
Correlates of MMP Activation In Vivo
MMP activation detected by molecular imaging has been linked to vessel wall inflammation in atherosclerosis.10 To ascertain the validity of this observation in the presence various lipid lowering interventions we investigated the correlation between RP805 uptake and cellular composition of aortic arch atherosclerotic lesions. While there was no significant correlation between RP805 uptake and VE-cadherin (endothelial cell marker) or smooth muscle α-actin (vascular smooth muscle cell marker)
Discussion
Our data demonstrate the feasibility and effectiveness of serial in vivo MMP-targeted molecular imaging for monitoring changes in atherosclerotic plaque inflammation in response to therapeutic interventions, and define the correlates of tracer uptake in the vessel wall. Lipid accumulation and inflammation are hallmarks of atherosclerosis and play a central role in its pathogenesis. Sub-endothelial accumulation of LDL and its oxidative modification promote vessel wall inflammation through
New Knowledge Gained
MMP-targeted molecular imaging can track the effect of lipid-lowering interventions on plaque inflammation in atherosclerosis.
Sources of Funding
This study was supported by National Institutes of Health R01 HL112992, R01 HL114703, R01 HL085093, and Department of Veterans Affairs Merit Award I0-BX001750.
Disclosures
Simon Robinson is employee of Lantheus Medical Imaging. Mehran M. Sadeghi receives experimental tracers from Lantheus Medical Imaging.
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Mahmoud Razavian and Lei Nie contributed equally to this work.