Radiation-Induced Atherosclerotic Plaque Progression in a Hypercholesterolemic Rabbit: A Prospective Vulnerable Plaque Model? - ScienceDirect

Cardiovascular Radiation Medicine

Volume 4, Issue 3, July–September 2003, Pages 146-151

Cardiovascular Radiation Medicine

https://doi.org/10.1016/S1522-1865(03)00182-3 Get rights and content

Abstract

Purpose

Human observations provide rich soil for making hypotheses, but good animal models are essential for understanding the disease and to test treatment modalities. Currently, there is no standard animal model of vulnerable plaque; therefore, the purpose of this study is to develop a pathophysiologically relevant vulnerable plaque model.

Methods

New Zealand White rabbits were fed with 1% hypercholesterolemic (HC) diet for 7 days, followed by balloon denudation of both the iliac arteries, and continued on 1% HC diet. Four weeks later, in 12 rabbits one of the iliac arteries was radiated (192-Ir, 15 Gy), and in five rabbits both the iliac arteries were sham treated. Following that, rabbits were fed with 0.15% HC diet. Four weeks later, arteries were processed for histomorphometry or immunohistochemistry.

Results

Serum cholesterol levels were similar in all the groups. In radiated arteries, plaque area was significantly larger (32% larger then in sham). Macrophage-positive area in radiated arteries was 2.4 times greater than the macrophage-positive area in the nonradiated arteries. The area positive for macrophages is also positive for metalloproteinases (MMP)-1. The extent of α-actin positive area was significantly less (2.3-fold) in radiated arteries.

Conclusion

The atherosclerotic plaque developed in the current model is predominantly composed of macrophages expressing metalloproteinases with few smooth muscle cells (SMC)—a characteristic of vulnerable plaque. The animal model presented in this study can elucidate at least part of the mechanism of plaque vulnerability and could be used to test treatment modalities to test plaque stability.

Introduction

Prevention of heart attack and stroke depends on the detection of vulnerable plaque and development of plaque stabilizing therapies [1]. Human observations provide rich soil for making hypotheses, but good animal models are essential not only to understand the mechanistic hypothesis in a controlled manner but also to design and test treatments. Currently, there is no standard animal model of vulnerable plaque. Though most researchers focus on mice models [2], [3], [4], [5], [6], mice do not develop atherosclerosis without genetic manipulation. Unlike human arteries, mouse arteries contain only a few layers of smooth muscle cells (SMC), and the lesions developed in the mouse may not mimic human plaque [4] placing a fundamental limit on the applicability of mouse models to test treatment modalities. The currently available vulnerable plaque rabbit model [7] is developed with an aim to evaluate plaque mechanical strength characteristics but not to investigate the pathophysiological mechanisms underlying the development of the vulnerable plaque. Thus, the usefulness of this model is more or less confined to the studies of the mechanical processes of the rupture itself. The atherosclerotic plaque is not a static structure, rather its status at any given moment is the result of the complex and dynamic interplay of its cellular components [8]. Typical atherosclerotic plaque consists of highly thrombogenic lipid/necrotic core and a fibrous cap that separates thrombogenic substances of the core from the blood coagulation system. A plaque with a thin fibrous cap and a large lipid and necrotic core that is prone to rupture is considered vulnerable [9], [10]. The thickness of the cap and the mechanical properties of the plaque are mostly determined by extracellular matrix, specifically fibrillar collagen [9]. Collagen content of the plaque is a result of dynamic balance between its synthesis and degradation. Consequently, presence of collagen-degrading enzymes such as metalloproteinases (MMP) is an indication of plaque vulnerability [11], [12]. Metalloproteinases can be synthesized and secreted by both macrophages and SMC, while their synthesis is stimulated by macrophage-derived cytokines [13], [14]. In general, macrophages are considered as predominantly matrix-degrading cells [15], [16]. Macrophages are also a major source of tissue factor in the plaque [17], [18]. Therefore, macrophage accumulation is considered a predictor of both plaque vulnerability and thrombogenicity. Formation of a new matrix is dependent on SMC [1], and since collagen is responsible for plaque mechanical strength, SMC number also determines the plaque vulnerability [19]. In the current study, we developed a balloon-injured and -radiated hypercholesterolemic (HC) rabbit model. The plaque developed in this model has few SMC with a large number of macrophages expressing MMP-1, some of the criteria for the vulnerable plaque.

Section snippets

Animal preparation

Animal care and procedures were carried out in accordance with the guide for the care and use of laboratory animals. Male New Zealand White rabbits (n=17) were fed with 1% HC diet for 7 days. On the 7th day, animals were anesthetized by intramural injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). During the experimental procedure, anesthesia was maintained by intermittent intramuscular injection of ketamine (17 mg/kg) and xylazine (2.5 mg/kg). A 5F arterial sheath was introduced

Results

Plasma lipids levels were high and identical in the radiated group as compared to the sham group at the time of the denudation (76.3±16.4 vs. 73.7±10.2 mmol/l; P=.81), at the time of radiation (106.15±56.3 vs. 126.43±35.2 mmol/l; P=.43), and at the time of sacrifice (101.4±31.4 vs. 135±28.2 mmol/l; P=.20)

Histological cross sections and quantification of the plaque formation demonstrated more pronounced lesions in the radiated segments as compared to the control segments. In the arteries that

Discussion

The present study demonstrates that radiation accelerates plaque formation in the balloon-denuded HC rabbit. The resultant plaque is primarily composed of monocytes-derived macrophages and very few SMC, expressing MMP-1 a characteristic of vulnerable plaque. To our knowledge, this is the first reproducible vulnerable plaque model that can be used to evaluate pathophysiological mechanisms underlying the development of the vulnerable plaque and also to test various treatment modalities for plaque

Conclusion

The present series of experiments support the hypothesis that ionizing radiation promotes atherogenesis in rabbits fed on high cholesterol diet. On the basis of these results of the current study and available models of atherogenesis, we propose that the atherogenic effects of radiation may involve free radical-mediated promotion of lipoprotein oxidation, leading to increased infiltration and retention of macrophages expressing MMP, thus increased vascular inflammation. Since these plaques are

Limitations

Taking into account the complexity of the pathophysiology of vulnerable plaque, no one-animal model could address all the issues related to plaque vulnerability. Any animal model is based upon a limited set of assumptions and therefore has its own limitations. However, the proposed animal model presented in this study can elucidate at least part of the mechanism of plaque vulnerability and could be used to test treatment modalities to test plaque stability.

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