Unstable Atheroscleotic Plaques
| Investigators | Collaborators |
|---|---|
|
|
Atherosclerotic plaque rupture
Post mortem clinical investigations into atherosclerotic plaque rupture have provided many insights, and progress has now been accelerated by our development of a small animal model. We have shown that the fat-fed apolipoprotein E knockout mouse is a useful experimental model of plaque destabilisation and rupture. The lesions that develop in the brachiocephalic arteries of these mice progress rapidly to become large, complex and unstable after about 2 months of high-fat feeding. Acutely ruptured plaques are large, lipid-rich, have thin fibrous caps, and are associated with expansive remodelling. All of these are considered to be phenotypic hallmarks of vulnerable plaques in humans. We have shown that plaque rupture in these mice can be prevented by statin treatment at clinically relevant and non-lipid-lowering doses, even when treatment is delayed until plaques are mature and already unstable. We have gone on to use a comprehensive genomic and proteomic screening strategy to identify entirely novel potential pathways of plaque destabilisation. These include the vitamin D receptor, klotho, tissue kallikrein and uteroglobin. We have shown that MMP-12 is involved in plaque destabilisation but MMPs -3 and -9 appear to be more involved with plaque healing. We have also shown that uPA, tPA and cathepsin S are plaque destabilising proteinases and that the major physiological inhibitor of uPA, PAI-1, is protective of plaque stability. We have shown that inhibition of thrombin may be a useful dual-pronged strategy for preventing plaque rupture and its thrombotic consequences. We are working on ways to specifically target therapeutic agents to unstable plaques, and have begun to identify plasma markers that appear just prior to plaque rupture and which may make it possible to screen for patients at risk of sudden cardiac death.
Background
Acute atherosclerotic plaque rupture is the precipitating event in most transmural myocardial infarctions and is a primary cause of sudden cardiac death. Because the lesion core is highly thrombogenic, plaque rupture may also play a role in the pathogenesis of unstable angina and the asymptomatic progression of coronary atherosclerotic lesions. Our development of the fat-fed apolipoprotein E (apoE) knockout mouse model of spontaneous plaque destabilisation (1-3) has facilitated hypothesis testing in this area.
The mechanical integrity of the fibrous cap is vital for preventing sudden catastrophic failure of plaque core containment. Enzymatic degradation of matrix proteins could weaken the fibrous cap and predispose it to mechanical failure: matrix metalloproteinases have long been under suspicion in this regard. We have addressed this hypothesis in three ways. First, we have created a range of double knockout mice where, in addition to apoE, homozygous null mutations have been made to MMPs -2, -3, -7, -9, and -12 and TIMPs -1 and -2 (4). Second, in collaboration with Dr Sarah George, we have over-expressed TIMP-1 and -2 using recombinant adenovirus technology in apoE knockout mice (5). Third, in collaboration with Drs William McPheat and Regina Fritsche-Danielsson at AstraZeneca, we have treated apoE knockout mice with a synthetic broad-spectrum MMP inhibitor (6). The data from these studies have altered our perceptions of the role of MMPs in plaque development and destabilisation, because they show that some MMPs are involved in plaque healing and some in plaque destabilisation. Broad-spectrum MMP inhibition of MMPs is without effect, presumably because both beneficial and deleterious actions of MMPs are lost. Our current hypothesis is that MMP-12 is an important plaque-destabilising proteinase. We are planning to test this using a novel specific inhibitor.
Other proteinase families are also active in the unstable plaque. We have created double knockout animals that as well as apoE also lack uPA, tPA, PAI-1 or cathepsin S. Both uPA and tPA appear to be involved in plaque rupture, with uPA having the more significant effect. Collaborative data obtained with Dr Peter Carmeliet show that PAI-1 double knockouts have significantly more ruptures, so the plasmin system seems to be particularly important to fibrous cap development and destruction.
Cathepsin S activity is increased in the shoulders of human endarterectomy plaques and cathepsin S double knockout mice have significantly fewer plaque ruptures (7), suggesting that this is another plaque destabilising proteinase. We are expanding our work on human plaques by collecting coronary artery samples at post mortem.
We have attempted to unravel the biochemical complexity of plaque rupture by genomic and proteomic screening in the mouse. Several novel pathways have emerged from this, including the vitamin D receptor (VDR), klotho, tissue kallikrein, galectin-3 and uteroglobin. In each case microarray data has been confirmed by RT-PCR and then at the protein level by immunostaining and Western blotting. We have made double knockouts with apoE of each of these.
We are testing the pharmacodynamic suitability of the mouse model by assessing the effects of treatments that show some evidence of increasing plaque stability in humans, such as statins, aspirin, ACE inhibitors and β-blockers. We have shown that pravastatin stabilises mouse plaques despite no change in the plasma lipid profile (3), suggesting that the model is suitable for drug testing and also supporting the concept of statin pleiotropism. Neither aspirin nor clopidogrel appears to have any direct plaque stabilising action, suggesting that their success derives from effects on the blood. We have also examined the role of thrombin using the specific inhibitor ximelagatran. This drug profoundly inhibits plaque initiation and also inhibits the rupture of existing unstable plaques. The drug has failed during development but we are hopeful a successor will prove just as promising.
Drug therapy of unstable atherosclerosis would be greatly facilitated by the ability to identify patients at risk through simple screening, and by targeting agents specifically to vulnerable plaques. The first of these goals is being attacked through a collaboration with Dr Dermot Cox at the RCSI in Dublin. We are using differential proteomic screening to examine plasmas from mice that are about to suffer plaque ruptures. Some candidate proteins have emerged. An extra benefit of this work could be the ability indirectly to monitor drug efficacy in trials of novel plaque rupture inhibitors.
The targeting of vulnerable plaques is the focus of a collaboration with Professor Andrew Baker at the University of Glasgow. Phage display is being used to discover peptide sequences that target unstable plaques, with the hope of targeting drugs and possibly also recombinant viruses to the desired site.
References
- Johnson JL, Jackson CL. The apolipoprotein E knockout mouse: an animal model of atherosclerotic plaque rupture. Atherosclerosis 154:399-406 (2001).
- Williams H, Johnson JL, Carson KGS, Jackson CL. Characteristics of intact and ruptured atherosclerotic plaques in the brachiocephalic arteries of apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 22:788-792 (2002).
- Johnson JL, Carson KGS, Williams H, Karanam S, Newby AC, Angelini GD, George SJ, Jackson CL. Plaque rupture after short periods of fat-feeding in the apolipoprotein E knockout mouse: model characterisation, and effects of pravastatin treatment. Circulation 111:1422-1430 (2005).
- Johnson JL, George SJ, Newby AC, Jackson CL. Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries. Proc Natl Acad Sci USA 102: 15575-15580 (2005).
- Johnson JL, Baker AH, Oka K, Chan L, Newby AC, Jackson CL, George SJ. Suppression of atherosclerotic plaque progression and instability by tissue inhibitor of metalloproteinase-2: involvement of macrophage migration and apoptosis. Circulation 113:2435-2444 (2006).
- Johnson JL, Fritsche-Danielson R, Behrendt M, Westin-Eriksson A, Wennbo H, Herslof M, Elebring M, George SJ, McPheat WL, Jackson CL. Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability. Cardiovasc Res 71:586-595 (2006).
- Rodgers KJ, Watkins DJ, Miller AL, Chan PY, Karanam S, Brissette WH, Long CJ, Jackson CL. Destabilizing role of cathepsin S in murine atherosclerotic plaques. Arterioscler Thromb Vasc Biol 26:851-856 (2006).