Christopher L. Jackson

Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries

Matrix metalloproteinases (MMPs) are thought to be involved in the growth, destabilization, and eventual rupture of atherosclerotic lesions. Using the mouse brachiocephalic artery model of plaque instability, we compared apolipoprotein E (apoE)/MMP-3, apoE/MMP-7, apoE/MMP-9, and apoE/MMP-12 double knockouts with their age-, strain-, and sex-matched apoE single knockout controls. Brachiocephalic artery plaques were significantly larger in apoE/MMP-3 and apoE/MMP-9 double knockouts than in controls. The number of buried fibrous layers was also significantly higher in the double knockouts, and both knockouts exhibited cellular compositional changes indicative of an unstable plaque phenotype. Conversely, lesion size and buried fibrous layers were reduced in apoE/MMP-12 double knockouts compared with controls, and double knockouts had increased smooth muscle cell and reduced macrophage content in the plaque, indicative of a stable plaque phenotype. ApoE/MMP-7 double knockout plaques contained significantly more smooth muscle cells than controls, but neither lesion size nor features of stability were altered in these animals. Hence, MMP-3 and MMP-9 appear normally to play protective roles, limiting plaque growth and promoting a stable plaque phenotype. MMP-12 supports lesion expansion and destabilization. MMP-7 has no effect on plaque growth or stability, although it is associated with reduced smooth muscle cell content in plaques. These data demonstrate that MMPs are directly involved in atherosclerotic plaque destabilization and clearly show that members of the MMP family have widely differing effects on atherogenesis.

Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury

The process of intimal thickening after de-endothelializing injury to the rat carotid artery is dependent on the migration of smooth muscle cells from the media. Recent reports have suggested that platelet-derived growth factor may be an important mediator of migration after injury. We have addressed this issue by directly determining smooth muscle cell migration in injured arteries of animals depleted of platelets and after administration of an antibody that blocks platelet-derived growth factor. Because there is a reported association between plasminogen activator synthesis and smooth muscle cell migration, we assayed the activity levels of plasminogen activators after arterial injury and also assessed the effect of a plasmin inhibitor on migration. The data suggest that platelet-derived growth factor, released by platelets at sites of arterial injury, is an endogenous mediator of smooth muscle cell migration; that plasmin generation, catalyzed by tissue-type plasminogen activator, is necessary for migration; and that one way in which platelet-derived growth factor may act is by stimulation of the synthesis of tissue-type plasminogen activator by smooth muscle cells.

Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse.

The rupture of an atherosclerotic plaque is the main underlying cause of coronary artery thrombotic occlusion and subsequent myocardial infarction, but research into the causes and treatment of plaque rupture is hampered by the lack of a suitable animal model. Although complex atherosclerotic plaques can be induced in a number of experimental animal systems, in none of these is plaque rupture an established feature. We have surveyed branch points in the carotid arteries and aortas of apolipoprotein E knockout mice fed a diet supplemented with 21% lard and 0.15% cholesterol for up to 14 months. Six male and five female mice were used. Four of the male mice and four of the female mice died, after 46+/-3 weeks of feeding (range 37-59 weeks). Lumenal thrombus associated with atherosclerotic plaque rupture was observed in three male and all four female mice. In six of these seven mice, an atherosclerotic plaque rupture was found where the brachiocephalic artery branches into the right common carotid and right subclavian arteries. The ruptures were characterised by fragmentation and loss of elastin in the fibrous caps of relatively small and lipid-rich plaques overlying large complex lesions, with intraplaque haemorrhage. Immunocytochemical analysis revealed loss of smooth muscle cells from ruptured caps. These data suggest that long-term fat-feeding of apolipoprotein E knockout mice is a useful and reproducible model of atherosclerotic plaque rupture, and that these ruptures occur predominantly in the brachiocephalic artery.

Suppression of Atherosclerotic Plaque Progression and Instability by Tissue Inhibitor of Metalloproteinase-2. Involvement of Macrophage Migration and Apoptosis

Background— Matrix metalloproteinase (MMP)–associated extracellular matrix degradation is thought to contribute to the progression and rupture of atherosclerotic plaques. However, direct evidence of this concept remains elusive. We hypothesized that overexpression of tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2 would attenuate atherosclerotic plaque development and instability in high fat–fed apolipoprotein E–knockout (apoE−/−) mice. Methods and Results— Seventy male apoE−/− mice (n=10/group) fed a high-fat diet for 7 weeks were injected intravenously with first-generation adenoviruses expressing the gene for human TIMP-1 (RAdTIMP-1) or TIMP-2 (RAdTIMP-2) or a control adenovirus (RAd66) and were fed a high-fat diet for a further 4 weeks. Analysis of brachiocephalic artery plaques revealed that RAdTIMP-2 but not RAdTIMP-1 infection resulted in a marked reduction (48±13%, P<0.05) in lesion area compared with that in control animals. Markers associated with plaque instability, assessed by smooth muscle cell and macrophage content and the presence of buried fibrous caps, were significantly reduced by RAdTIMP-2. Effects on lesion size were not sustained with first-generation adenoviruses, but murine TIMP-2 overexpression mediated by helper-dependent adenoviral vectors exerted significant effects on plaques assessed 11 weeks after infection. In an attempt to determine the mechanism of action, we treated macrophages and macrophage-derived foam cells with exogenous TIMP-2 in vitro. TIMP-2 significantly inhibited migration and apoptosis of macrophages and foam cells, whereas TIMP-1 failed to exert similar effects. Conclusions— Overexpression of TIMP-2 but not TIMP-1 inhibits atherosclerotic plaque development and destabilisation, possibly through modulation of macrophage and foam cell behavior. Helper-dependent adenovirus technology is required for these effects to be maintained long term.

Inhibitory effect of calcium antagonists on balloon catheter-induced arterial smooth muscle cell proliferation and lesion size

Calcium antagonists inhibit atherogenesis in the cholesterol-fed rabbit without producing hypolipidaemia, suggesting a direct action on the arterial wall. In this study, the effects of several calcium antagonists on the myoproliferative response to balloon catheter injury of the aorta have been investigated in normolipidaemic rats and rabbits. The incorporation of [3H]thymidine into rat aortic DNA 48 h after balloon injury was markedly reduced by twice daily oral administration of nifedipine, verapamil, diltiazem or lanthanum. DNA synthesis in other proliferating tissues was unaffected. Twice daily oral administration of prazosin or minoxidil, antihypertensive agents that are not calcium antagonists, also selectively reduced arterial DNA synthesis. In balloon catheterised rabbits twice daily oral administration of nifedipine (10 mg/kg) caused a 39% reduction in the cross-sectional area of the neo-intima 14 days after injury. These results show that nifedipine and other antihypertensive agents inhibit smooth muscle cell proliferation.

Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice

Clinical complications of atherosclerosis arise primarily as a result of luminal obstruction due to atherosclerotic plaque growth, with inadequate outward vessel remodeling and plaque destabilization leading to rupture. IL-1 is a proinflammatory cytokine that promotes atherogenesis in animal models, but its role in plaque destabilization and outward vessel remodeling is unclear. The studies presented herein show that advanced atherosclerotic plaques in mice lacking both IL-1 receptor type I and apolipoprotein E (Il1r1⁻/⁻Apoe⁻/⁻ mice) unexpectedly exhibited multiple features of plaque instability as compared with those of Il1r1⁺/⁺Apoe⁻/⁻ mice. These features included reduced plaque SMC content and coverage, reduced plaque collagen content, and increased intraplaque hemorrhage. In addition, the brachiocephalic arteries of Il1r1⁻/⁻Apoe⁻/⁻ mice exhibited no difference in plaque size, but reduced vessel area and lumen size relative to controls, demonstrating a reduction in outward vessel remodeling. Interestingly, expression of MMP3 was dramatically reduced within the plaque and vessel wall of Il1r1⁻/⁻Apoe⁻/⁻ mice, and Mmp3⁻/⁻Apoe⁻/⁻ mice showed defective outward vessel remodeling compared with controls. In addition, MMP3 was required for IL-1-induced SMC invasion of Matrigel in vitro. Taken together, these results show that IL-1 signaling plays a surprising dual protective role in advanced atherosclerosis by promoting outward vessel remodeling and enhancing features of plaque stability, at least in part through MMP3-dependent mechanisms.

Assessment of Unstable Atherosclerosis in Mice

There is an urgent need for representative animal models where prospective examination of the events leading up to plaque rupture and the rupture process itself can be performed. Recently, reports have begun to emerge that apolipoprotein E and low density lipoprotein receptor knockout mice may spontaneously develop unstable atherosclerosis, with plaques in certain parts of the arterial tree showing features suggestive of plaque rupture. Here we discuss the problems inherent in applying definitions of plaque rupture as seen in human arteries to mice; the anatomic locations in mice where unstable plaques do and do not occur; methods of inducing plaque instability in mice; and how to assess plaque stability in mice. These considerations lead us to a number of general recommendations.

A Selective Matrix Metalloproteinase-12 Inhibitor Retards Atherosclerotic Plaque Development in Apolipoprotein E–Knockout Mice

Objective—Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. Methods and Results—A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. Conclusion—Our data demonstrate that a selective MMP-12 inhibitor retards atherosclerosis development and results in a more fibrous plaque phenotype in mice. Our study provides proof of principle to motivate translational work on MMP-12 inhibitor therapy in humans.

The Role of Plasminogen Activation in Smooth Muscle Cell Migration after Arterial Injury

The migration of smooth muscle cells from the media to the intima that occurs after balloon catheter injury to the rat common carotid artery has been quantified by an electron microscopic surveying technique. These vessels have also been assayed for plasminogen-activator activity, which was found to rise sharply 4 days after balloon injury. At this time point smooth muscle cells begin to migrate in appreciable numbers. In order to investigate whether there is a causal relationship between plasminogen-activator activity and smooth muscle cell migration, animals were dosed with tranexamic acid. This synthetic inhibitor of plasmin activity reduced smooth muscle cell migration by 73% (p < 0.05), indicating that plasmin activity is necessary for migration after balloon injury. Lisinopril, an inhibitor of angiotensin-converting enzyme, inhibited smooth muscle cell migration after balloon injury by 78% (p < 0.01) but did not influence plasminogen-activator activity. Taken together, these results show that plasmin is a necessary but not sufficient component in the pathway that leads to smooth muscle cell migration after balloon catheter injury in the rat.

Destabilizing Role of Cathepsin S in Murine Atherosclerotic Plaques

Objective—Lysosomal proteinases have been implicated in a number of pathologies associated with extracellular matrix breakdown. Therefore, we investigated the possibility that the lysosomal proteinase cathepsin S may be involved in atherosclerotic plaque destabilization. Methods and Results—Atherosclerotic plaques in the brachiocephalic arteries of fat-fed apolipoprotein E/cathepsin S double knockout mice had 73% fewer acute plaque ruptures (P=0.026) and were 46% smaller (P=0.025) than those in age-, strain-, and sex-matched apolipoprotein E single knockout controls. When the incidence of acute plaque rupture was normalized for plaque size, the reduction in the double knockouts was 72% (P=0.039). The number of buried fibrous layers, indicative of an unstable plaque phenotype, was reduced by 67% in the double knockouts (P=0.008). The cysteine proteinase inhibitor, egg white cystatin, was biotinylated and used as an active-site-directed probe for cathepsins. Biotinylated cystatin selectively detected cathepsin S in extracts of human carotid atherosclerotic plaque. Active cathepsin S was detectable in extracts of human atherosclerotic plaque but not in nondiseased carotid arteries. Active cathepsins were especially prominent in macrophages in the shoulder regions of plaques, areas considered to be vulnerable to rupture. Cathepsin S protein colocalized with regions of elastin degradation in human coronary plaques. Conclusion—These data provide direct evidence that an endogenous proteinase, cathepsin S, plays an important role in atherosclerotic plaque destabilization and rupture.