Establishment And Mechanism Research Of Atherosclerosis In Guinea Pig Model

BackgroundAtherosclerosis is a condition in which patchy deposits of fatty material (atheromas of atherosclerosis plaques) develop in the walls of medium-sized and large arteries, leading to reduced or blocked flow. In the USA and most other developed countries, atherosclerosis is one of the main life-threatening diseases and the leading cause of illness and death, prediction and prevention of atherosclerosis is strongly required. The development of atherosclerosis is extremely complicated, and increasing body evidences indicate that serious involvement of risk factors in atherosclerosis including abnormal metabolism of cholesterol, inflammation, oxidative stress, and other emerging risk factors. Now, the fundamental underlying cause of atherosclerosis has not fully established and the mechanisms related to atherosclerotic events in the aorta have not been explored in detail due to the lack of a suitable animal model.The use of appropriate animal models is every important to study the cholesterol and lipoprotein metabolism, the pathogenesis of atherosclerosis and effects of drug therapy. Over the past decade, significant advances have been made in the development of animal models of human atherosclerosis, including rabbits, rodents (mouse, rats, hamster), avian (pigeons, quail), swine, anon-human primates, transgenic or gene knock-out animals. However, limitations of these animal models of atherosclerosis have been discovered gradually. For example, although transgenic/knock-out mice have been used to study a specific pathway involved in development of atherosclerosis, these data are of limited use because other metabolism pathways and responses to interventions may differ from the human condition. Cholesterol metabolism in certain animal models such as rats and mouse is completely different from that of human, therefore these animal models may not be appropriate for studying the mechanism of cholesterol metabolism and atherosclerosis. To select an appropriate animal model, the efforts must be made to optimize the parameters and to maximize the similarities with human physiology and atherosclerotic development.Recent studies show that guinea pig is one of the few species that carry the majority of cholesterol in LDL, which is the most striking similarity to humans, whereas other rodents present major differences in lipoprotein cholesterol distribution and even genetic manipulations result in dissimilar lipoprotein profiles when compared to humans. Guinea pig has other main similarities to human, including:1) Higher concentrations of free compared to esterified cholesterol in the liver; 2) In contrast to other rodents, guinea pig posses plasma CETP activity; 3) Guinea pigs also have LCAT and LPL activities that contribute to modify plasma HDL subclasses; 4) Guinea pigs exhibit comparable moderate rates of hepatic cholesterol synthesis and catabolism; 5) Guinea pigs respond to dietary fat saturation, dietary cholesterol and dietary fiber by alterations in LDL-C; 6) It has also been shown that guinea pigs are good models for studying the mechanisms of statins, cholestyramine, ACAT inhibitions and Fibric acids. It is clear that guinea pigs also respond to diet factors, drug treatment, oxidative stress, exercise, gender and hormonal status undoubtedly mimic the human situation. Guinea pigs were used as the experimental animal model because their lipoprotein profile and response to dietary cholesterol factors are similar to human. We hypothesized that guinea pigs maybe a suitable and appropriate model to evaluate fatty steak accumulation and atherosclerotic lesions resulting from intake of hypercholesterolemic diet.ObjectiveThe aim of this paper is to explore methods of successful establishment of experimental guinea pig atherosclerosis model induced by high-cholesterol diets, which has the typical pathological features of atherosclerosis, and elucidate the mechanisms underlying development of atherosclerosis with comparison to rats, in order to clarify the advantages of this new atherosclerosis model in guinea pigs.Methods1. Male guinea pigs and rats were random divided into normal control (common forage), model 1 (common forage contained 0.5% cholesterol and 10% lard) and model 2 (common forage contained 1% cholesterol and 10% lard) groups, respectively, and the study lasted 10 weeks for the establishment of atherosclerosis.2. Body weight, food intake and condition were monitored every week.3. After induction, blood samples were drawn from femoral artery to separate serum for quantitation of serum lipids.4. The lipids levels in serum and liver were measured by the enzyme methods.5. Levels of serum CETP, LCAT, hs-CRP, and Ox-LDL were detected using Enzyme-linked immunosorbent assay (ELISA).6. The whole aorta was dissected and cut into segments for pathological detection. The section from arch of aorta to bottom of thoracic aorta was cut open longitudinally and stained with Sudan IV, lipids deposition in arterial wall was observed and the percent of plaque area (PA%) was analysis.7. 4μl thick paraffin slices from aortic root to aortic arch were mounted on masked slides and stained with hematoxylin and eosin (HE) for light microscopy to observe the atherosclerotic lesion. Aortic intima-media thickness (IMT) was measured by analyzing the percent initima area and the ratio of intima area/ media area. Then, the characteristic of lipid plaque and infiltration inflammatory cells were evaluated under microscope.8. Masson's trichrome staining was performed to observe the collagen deposition in atherosclerostic plaques.9. The protein expressions of ICAM-1, CD36 and ABCA1 in aortic wall of atherosclerosis were evaluated by immunohistochemistry with specific monoclonal antibodies, then area of positive staining (Area Sum), integral optical density (IOD) and mean optical density (OD) of the stained sections were measured with a computer-assisted image-processing and analytical system for the semi-quantitative analysis.10. In addition, the relative expressions of PPARa, LCAT, LDL-R and SR-BI mRNA in liver were detected by real-time PCR.11. Guinea pig SR-BI gene cloned and sequenced.12. Statistical analysis: all data were expressed as mean±standard deviation and analysis using statistical package SPSS15.0. One-way ANOVA was used to determine statistically significant differences. Data of real-time PCR was measured by Relative Expression Software Tool (REST) and data of pathologic detection were analyzed by professional image processing software (Image Pro Plus 6.0). Values of P<0.05 were considered statistically significant.Results1. Changes in serum and hepatic lipidsCompared with normal control groups, serum TC, TG and LDL-C levels were remarkably increased in guinea pigs fed with high-cholesterol diets for 10 weeks, which indicate that typical hyperlipidemia occurred, whereas fed with the same diet, the rats han only LDL-C increased in model 1 group and TC, LDL-C increased in model 2 group, however, their increased amplitudes were much lower than that of guinea pigs. Both the hepatic TC and TG concentrations were increased in rat and guinea pig models, and their increased amplitudes were approximately equal.Serum HDL-C concentrations in guinea pigs fed with high cholesterol diets were 9.9 and 10.8 times higher than that of control group, furthermore, two major HDL subclasses HDL3/ HDL2 ratio increased, the distribution and composition of HDL changed. However, no significant changes of HDL-C level and HDL3/ HDL2 ratio were found in rats.2. Identification of atherosclerosis modelThe aortic walls in normal guinea pigs and rats were thin and flat; endothelial cells intact, connected tightly and adhering to inner elastic plates; the vascular smooth muscle cells (VSMC) in arranged regularly; few collagenous fibers were scattered in extra cellular matrix; without inflammatory cells aggregation and plaque formation. In the model groups of guinea pigs, gross examinations revealed that white or light yellow lesions (fatty streaks) of various sizes and thickness located on the lining endothelium of the aortas; under microscope (×400), histopathological result exhibited pathological intimal thickening, intimal lipid deposition, the infiltration and aggregation of inflammatory cells (monocytes, macrophages, etc.), plaques consisting of massive lipid-laden foam cells accumulation, the formation of extracellular lipid pool, proliferation and migration of VSMC which arranged irregularly, some lipid pool and even fibrous cap could found in more severe samples, without any calcification, plaque rupture, bleeding or thrombosis.Atherosclerotic lesions were characterized using a classification scheme based on the recommendations of the American Heart Association (AHA). Guinea pig fed with high cholesterol diets developed atherosclerosis, the lesion was predominantly of AHA classⅡ-Va lesion, and between early and late stage of atherosclerosis. Semi-quantitative analysis on pathological changes showed that incidence of plaque was 70% (7/10) and 75% (6/8) in guinea pig model 1 and model 2 group, respectively. In comparison with control group, the plaque area %, collagen fiber area%, intimal area% and intimal area/media area ratio were significantly increased. No such significant differences were observed in rats fed with the same high-cholesterol diets.3. Molecular mechanisms studiesCompared with control group, hepatic LDL-r mRNA expression significantly decreased and serum Ox-LDL concentration increased in both of the model groups of guinea pigs, indicating that the normal physiological process of LDL-C uptake and metabolism mediated by LDL receptor were blocked, and excessive oxidative modification of LDL resulting in the formation of oxidized LDL. Meantime, CD36 expression in aorta was markedly enhanced suggested that massive Ox-LDL uptake by scavenger receptor CD36 which existed in cell surface of macrophages can, therefore, lead to accumulation of cholesterol and development of them into form cells. The serum CETP concentration was significantly increased in guinea pig model groups, hence the enhancement of CETP- mediated the exchange of CE /TG between VLDL, LDL and HDL contributes significantly to the formation of more atherogenic small dense LDL and TG-rich HDL leading to a relative increase of the small dense HDL3 particles. Moreover, serum LCAT and hepatic LCAT mRNA expression were apparently declined in guinea pig model groups, which exhibited severely decreased LCAT-mediated esterification of cholesterol in nascent HDL and reduced the rate of HDL maturation, resulting in HDL3/HDL2 ratio increase. Aortal ICAM-1 adhesion molecules protein expression greatly elevated in guinea pig model groups, resulting in adhesion and aggregation of inflammatory cells. The increase in serum hs-CRP observed in guinea pig model groups in response to vascular inflammation, promoting the development of atherosclerosis. Whereas, only serum Ox-LDL, hs-CRP concentrations and aortal protein expression of CD36 were increased in rats model 2 group induced by the same diet with guinea pigs.In addition, the compensatory increase in hepatic SR-BI mRNA and aortal ABCA1 protein expression in response to high cholesterol diet in guinea pig model groups, and no such changes were observed in rat model group.Conclusion1. Guinea pigs develop typical atherosclerosis with hypercholesterolemic diets containing 0.5% cholesterol and 10% lard for 10 weeks.2. Compared with rats, guinea pigs are more sensitive to dietary cholesterol, it is easy and reliable to establish a atherosclerosis model induced by high cholesterol diets.3. The mechanisms of susceptibility to diet-induced atherosclerosis in guinea pigs are closely associated to abnormal metabolism of serum lipid and lipoprotein, changes in key enzymes or protein which regulate the lipid metabolism and development of atherosclerosis, such as CETP, LCAT, CD36, LDL-R, Ox-LDL and ICAM-1.4. Guinea pigs develop the characteristic features of atherosclerosis in a relatively short period of time and this method is inexpensive, easy available, cheap to maintain and easy to manipulate. As an easy and accessible experimental model, it is suitable for various atheroscleorotic investigations, including the screening and evaluation of therapeutic drug in lipid metabolic disorders and diseases related to atherosclerosis.