The Effects Of Aerobic Exercise On The Vulnerability Of Atherosclerotic Plaques And The Concentration Of Matrix Metalloproteinase, IL-6 In ApoE Gene Deficient Mice

The effects of aerobic exercise on the vulnerability of carotid atherosclerotic plaques and the concentration of matrix metalloproteinase in ApoE gene deficient miceBackgroundCarotid atherosclerosis is an important reason for ischemic cerebrovascular diseases. Both carotid artery occlusion and atherosclerotic plaque or thrombus ablation can lead to cerebral infarction. The stability of carotid atherosclerotic plaque is closely related to cerebral infarction. Vulnerable plaques are featured by big lipid cores, thin fibrous caps and abundant inflammatory cells, therefore run high risk of rupture and thrombosis. Matrix metalloproteinases, MMPs, are a class of proteolytic enzymes closely relevant to the onset and evolution of carotid atherosclerosis, AS, and the most important enzymes that degrade extracellular matrix, ECM. Increasing evidences demonstrated that MMPs can lead to thrombosis or embolism by degrading unstable fibrous caps and inducing plaque rupture. Exercise plays an active role in AS prevention, which can reduce the mortality of cardiocerebrovascular diseases resulting from AS. Previous researches concerning the mechanism always focus on the effects of exercise on lipid metabolism, function of vascular endothelium, anti-oxidative capacity and on the vascular systolic/relaxant function. Recent studies believed that advisable exercise can improve the anti-inflammatory capacity of body, which should be a new mechanism of exercise on effective AS prevention. The effects of exercise on the vulnerability of AS plaques however remain unknown. The present study attempted to examine the effects of aerobic exercise on the development of carotid AS plaques by constructing models of carotid artery stenosis and vulnerable plaques through carotid cannulation. We also tried to investigate the mechanism of exercise on AS by pathological examination, determination of vulnerability index and the expression of MMP-9 and MMP-14.Objectives1. Elucidate the effects of aerobic exercise on the vulnerability of carotid atherosclerotic plaques and the corresponding mechanisms.2. Elucidate the interaction between aerobic exercise and MMP-9 in the vulnerable plaque and the corresponding mechanisms.3. Elucidate the interaction between aerobic exercise and MMP-14 in the vulnerable plaque and the corresponding mechanisms.Methods1. Animal modelAnimal model with AS vulnerable plaque was established. Ninety-six 10-week-old male ApoE-/- mice were divided into aerobic (48 mice) and control group (48 mice) with Western-type diet (0.25% cholesterol and 15% fat) from the beginning of the study. Carotid atherosclerotic lesions were induced perivascular constrictive collars placement on the right common carotid arteries. Four weeks after surgery, animals in the exercise group were trained on a homemade electric treadmill with a 0% gradient at 15m/min for eight weeks. The frequency of exercise was 30min/d and 6d/week. All mice had a rest on Sunday. Induced stress in the control group was restrained. Four weeks after surgery, the control group mice were caged once a day and one hour per time, for eight weeks.2. Before killed, the mice were anesthetized and blood was collected from the abdominal aorta. Levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum were determined.3.1Histological and Morphology Analyses Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, sirius red staining, V oil red O staining. Immunostaining were performed for detecting MOMA-2,α-actin,MMP-9,MMP-14 expression in the vulnerable plaque. Plaque area, vessel area and cap thickness were measured. Smooth muscle cell, macrophage, lipid and collagen positive areas were quantified and the ratios correlated to the intimae areas were calculated. Plaque rupture rate and vulnerable index were calculated.3.2 RT-PCRThe mRNA levels of MMP-9、MMP-14 mRNA in the fresh carotid lesions were quantified by reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.3.3. Western-blotWestern-blot was performed for examining MMP-9、MMP-14 protein expression in the fresh carotid lesionsResults1. No significant difference was observed in terms of the initial (at the beginning) and final (at the end of the experiment, i.e. kill the animal for sampling) body weight between the aerobic exercise group and the control group (P>0.05 and P>0.05, respectively)2. Blood lipid profileNo statistic differences of serum lipid profiles were found between aerobic group and control.3.1 Histological and Morphology AnalysesAtherosclerotic plaques of 8 animals of each group were analyzed for histology. No statistic difference of plaque area was found between aerobic group and control(81000±9000um2 vs 82000±12000 um2, P>0.05). Compared with control, mean fibrous cap thickness(7.35±1.25um vs 6.63±0.97um, P<0.05), cap/core ratio (0.09±0.01 vs 0.06±0.01, P<0.05) and collagen (16.44%±4.73% vs 11.33%±3.58%, P<0.05) were significantly increased, and lipid content was significantly decreased (12.54%±2.11% vs 21.23%±3.46%, P<0.05) in aerobic group. Plaque rupture rates were 50% in aerobic group significantly higher than in control group(P<0.05).Iimmunohistochemical stainning results showed that there less macrophages in the aerobic group(7.57%±2.67% vs 15.42%±3.11%, P<0.05) than those in control. Moreα-actin expression in the aerobic group (12.97%±1.91% vs 5.87%±1.19%, P<0.05) than those in control. MMP-9 expression decreased in aerobic group compared with that in control (4.19%±0.32% vs 9.89%±0.88%, P<0.05). In addition, MMP-14 expression were significantly decreased in aerobic group than those in control (6.69%±0.56% vs 12.42%±1.54%, P<0.05). Vulnerable index of aerobic group was statistically lower than that of control (0.80±0.53 vs 2.84±1.12, P<0.05).3.2 RT-PCRThe mRNA expression of MMP-9 in the vulnerable carotid plaques decreased 68.4% compared with that in controls (P<0.05). The mRNA expression of MMP-14 in the vulnerable carotid plaques decreased 78.3% compared with that in controls (P<0.05)3.3 Western-blot analysisThe proteins expression of MMP-9 in the vulnerable carotid plaques decreased 62.8%(P<0.05) compared with that in controls. The proteins expression of MMP-14in the vulnerable carotid plaques decreased 55.1%(P<0.05) compared with that in controls. Conclusions1. Aerobic exercise can decrease lipid content and the number of macrophage, increase the expression of smooth muscle cells and collagens significantly in vulnerable plaques. aerobic exercise can decrease the vulnerability of plaques markedly in ApoE-/- mice.2. Aerobic exercise can reduce expression of MMP-9 and MMP-14 in vulnerable plaques in ApoE-/- mice. The effects of aerobic exercise on the inflammation of carotic atherosclerosis in ApoE gene deficient miceBackgroundCarotid atherosclerosis is an important pathology basis for ischemic cerebrovascular diseases. Atherosclerosis is some kind of chronic inflammation. The chronic inflammation is an very important factor in the occurrence and development of atherosclerosis. A number of trials have suggested that inflammatory factors such as the elevated levels of C-reactive protein (CRP) and interleukin-6(IL-6) are closely associated with the occurrence and development of atherosclerosis. C-reactive protein, CRP, is the typical stimulant of inflammatory response that will increase in response to trauma and infection. Recent studies believed that CRP is an independent predictor that indicates coronary heart disease. A great many studies have suggested that cytokines like interleukin 6 (IL-6) and interleukin 8 (IL-8) secreted by inflammatory cells could lead to the activation of endothelial cells, the proliferation, apoptosis, and progressive atherosclerosis of smooth muscle cells, which eventually results in the reduction, thickness and rupture of collagen of fibrous caps. Atherosclerosis (AS) is a multifactorial disease. Transgenic apoprotein E (ApoE) deficient mice are favorable model animal for studying AS. Available results demonstrated that aerobic exercise is an effective measure for preventing AS and its complications because it may reduce the plaque area and delay the evolution of AS. The current study attempted to find out the role of aerobic exercise on the morphology of carotid atherosclerotic plaque in mice, the effects on IL-6 and the anti-atherosclerotic mechanisms of carotid atherosclerosis. Objectives1.Elucidate the effects of aerobic exercise on the histopathological changes ofcarotid atherosclerotic plaques and the corresponding mechanisms.2.Elucidate the interaction between aerobic exercise and IL-6 in the carotid plaque and the corresponding mechanisms.Methods1. Animal modelAnimal model with AS vulnerable plaque was established. Eighty 10-week-old male ApoE-/- mice were divided into aerobic (40 mice) and control group (40 mice) with Western-type diet (0.25% cholesterol and 15% fat) from the beginning of the study. Carotid atherosclerotic lesions were induced by perivascular constrictive collars placement on the right common carotid arteries. Four weeks after surgery, animals in the exercise group were trained on a homemade electric treadmill with a 0% gradient at 15m/min for twelve weeks. The frequency of exercise was 30min/d and 6d/week. All mice had a rest on Sunday. Induced stress in the control group was restrained. Four weeks after surgery, the control group mice were caged once a day and one hour per time, for twelve weeks.2. Before killed, the mice were anesthetized and blood was collected from the abdominal aorta. Levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum were determined. Levels of IL-6 were determined in serum.3.The effect of aerobic exercise on the carotid plaque3.1 Histological and Morphology AnalysesSections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, sirius red staining, V oil red 0 staining. Immunostaining were performed for detecting MOMA-2,α-actin,IL-6 expression in the carotid plaque. Plaque area, and cap thickness were measured. Smooth muscle cell, macrophage, lipid and collagen positive areas were quantified and the ratios correlated to the intimae areas were calculated.3.2 RT-PCRThe mRNA levels of IL-6 mRNA in the fresh carotid lesions were quantified by reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology. 3.3. Western-blotWestern-blot was performed for examining IL-6 protein expression in the fresh carotid lesionsResults1. No significant difference was observed in terms of the initial (at the beginning) and final (at the end of the experiment, i.e. kill the animal for sampling) body weight between the aerobic exercise group and the control group (P>0.05 and P>0.05, respectively)2. Hematological examination2.1 Blood lipid level:statistical difference was observed in serum TC, LDL-C, HDL-C and TG level between the aerobic exercise group and the control group (P<0.05).2.2 IL-6 level:statistical difference was observed in terms of serum IL-6level between the aerobic and control groups (P<0.05)3. Histological and Morphology Analyses3.1 Statistical difference was observed in the plaque area between both groups (P<0.05). Compared to the control group, the thickness of fibrous cap (P<0.05), cap/core ratio (P<0.05) and collagen content (P<0.05) in aerobic group increased, while that of lipid content decreased statistically significant (P<0.05).3.2 The expression of a-SMC-actin in the aerobic exercise group enhanced (P<0.05), while the counts of macrophages decreased statistically significant (P<0.05), as compared to the control group. Similarly, the expression of IL-6 in the plaques in aerobic exercise group decreased statistically significant compared to that in the control group (P<0.05).3.3 RT PCRthe expression of IL-6mRNA in the carotid plaque of the aerobic exercise group decreased statistically significant by 59.2% as compared to that of the control group (P<0.05)3.4 Western-blot analysisThe expression of IL-6 protein in the carotid plaque of aerobic exercise group decreased by 63.9% between the aerobic and control groups. As compared to that of the control group, it was statistically significant (P<0.05).Conclusions1. Aerobic exercise can decrease lipid content and the number of macrophage, increase the expression of smooth muscle cells and collagens significantly in vulnerable plaques. Aerobic exercise can decrease the inflammation of carotid atherosclerosis in ApoE-/- mice.2. Aerobic exercise can reduce expression of IL-6 in carotid plaques in ApoE-/-mice.