Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice

Abstract

The acute platelet response and chronic smooth muscle cell (SMC) proliferation following aortic injury in apolipoprotein E-deficient mice was investigated. The purpose of this study was to evaluate whether thrombus formation would occur following plaque injury, to determine the type of thrombus that developed, and to evaluate SMC proliferation. Aortic injury was performed by squeezing the aorta between forceps. The response to injury reflects the findings primarily associated with plaque disruption. An attempt was made to exclude the use of injured vascular segments that showed marked injury to the media to minimize the effects that medial SMCs may have in thrombus formation. Acute and chronic experiments following injury were terminated at 30 min and at 2 weeks, respectively. Injury in normal and heterozygous mice and nonplaque injury in apolipoprotein E-deficient mice were accompanied by endothelial denudation. In apolipoprotein E-deficient mice, plaque injury, which released plaque contents, foam cells and fragments of foam cells, was followed by thrombus formation that contained degranulating platelets mixed with fibrin. Large platelet-fibrin aggregates were in close contact with disrupted plaques and were mixed with foam cell debris. In addition, small thrombi were in nonplaque areas following plaque disruptions. These thrombi were not associated with injury to the media and most likely represent a heightened thrombogenicity associated with plaque disruption. At 2 weeks following injury, a thickened neointima was present in both wild type and mutant mice. Lipid filled cells were seen only in the media but not in the intima of apo E−/−vessels at 2 weeks. The results suggest that plaque injury in homozygous apolipoprotein E-deficient mice promotes platelet-fibrin thrombus formation and that these thrombi are primarily associated with disrupted plaque contents. The results also suggest that the platelet response and SMC proliferation induced by aortic injury are not altered by hyperlipidemia caused by apolipoprotein E deficiency.

Introduction

Injury to blood vessels is followed by a series of events that under normal conditions results in complete restitution of the normal structure of the vessel. Superficial injury to nonatherosclerotic vessels promotes the formation of a monolayer of platelets in the area of endothelial damage [1]. The platelets are thought to attach to subendothelial collagen or other structures mediated by the activation of surface receptors for von Willebrand factor (vWF) 2, 3. With deep injury that involves the media of normal vessels, microthrombi form and consist of platelets and other cellular elements enmeshed within a fibrin net [1]. The disruption or fissuring of atherosclerotic plaques is associated with either mural or occlusive thrombi in the area of plaque injury 4, 5, 6, 7, 8. Hypotheses explaining thrombus formation include endothelial dysfunction [6], an inflammatory mediated process [8], contents of the atheromatous core of the atherosclerotic plaque [9], components of the extracellular matrix [10], and procoagulant/antifibrinolytic activity produced by monocytes/macrophages [11]. In hypercholesterolemia, platelets are more reactive and the thrombi that are formed under these conditions are reported to be larger and more compact 12, 13.

Mice with apolipoprotein E (apo E−/−) deficiency have elevated serum cholesterol and develop atherosclerotic lesions in the arterial tree [14]. Plaque size increase with age and the lesions narrow the lumen. Nevertheless, in these mice we have not detected evidence of plaque rupture followed by formation of occlusive thrombi or thromboembolism as had been described in human atherosclerotic lesions. To investigate whether simple endothelial denudation and injury to atherosclerotic plaques in mice promote thrombus formation, aortic injury was performed in normal (+/+), heterozygous (+/−), and to vessels with or without atherosclerotic plaques in homozygous apo E (−/−) mice. We here document platelet deposition, thrombus formation, platelet-platelet interaction, and platelet-plaque interaction immediately after injury, and SMC proliferation 2 weeks after injury.