| Atherosclerosis (AS) is a multifactorial and progressive disease, many complicated factors and biological processes contribute to atherogenesis. The inflammatory reaction and inflammation-related factors play important roles at all stages of atherogenesis. Atherosclerosis involves a large genetic network, not a simple linear pathway. This network extends to interact with many known risk factors such as dyslipidaemia and inflammation for the disease, and involves many cell types and organ systems.Tumor necrosis factor-alpha (TNF-α), a key inflammatory cytokine in the inflammatory cascade, is considered to plays important roles in atherogenesis. TNF-αwas produced by several types of cells, such as macrophages, vascular endothelial cells (ECs) and smooth muscle cells (SMCs). TNF-αcould induce the production of cytokines, chemokines, and increase the expression of adhesion molecules on ECs, monocytes and leukocytes, leading to the recruitment of monocytes / macrophages and infiltration into the subendothelial space of arteries, as well as subsequent activation of T lymphocytes and the migration and proliferation of SMCs. However, its precise characters in the primary stage of the disease remain unclear. Apolipoprotein E-null (apoE-/-) mice which spontaneously develop atherosclerosis with features similar to those observed in humans are the idea model of human atherosclerosis researches. It provides the advantage for the mechanism and intervention of atherosclerosis researches.To assess the function of TNF-αduring atherogenesis in apoE-/- mice, a comparative study on fatty-streak lesion, serum lipid level, the mRNA level of target gene in spleen, aorta and liver of apoE-/- and apoE/TNF-αdouble mutant (AT) mice were performed. By cytokine antibody array, 64 kinds of cytokines expression were detected in aorta with 6-week old mice. The characteristics of expression of inflammatory factors in spleen and aorta, and reverse cholesterol transport (RCT) related genes in liver were analyzed to demonstrate the role of TNF-αin atherogenesis. The results showed that:1. Pathological analysis and lesion quantificationThe results of atherosclerotic lesions in root showed that deficiency of TNF-αled to reduction of atherogenesis in apoE-/- background. Lipid accumulation in the intima of the aorta existed as early as 4-week of age in apoE-/- mice and were progressed with age. There was no observed lesion in the 4-week old AT mice and all ages of TNF-α-/- and WT mice. From 6-week to 24-week old, lipid deposition in the aortic sinus was most prominent in apoE-/- mice, while it tended to be lower in AT mice. The mean lesion area revealed that the promoting effect of TNF-αon AS was more prominent in the early stage. When being 4-week old, the aorta root mean lesion area of apoE-/- mice was 7.81 times of AT mice, but reduced to 2.58 times when at 6-week old. Furthermore, at age of 24-week old, the mean lesion area in apoE-/- mice was only 29% larger than that of AT mice.2. Plasma lipid parametersThe serum total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol ester (LDL-C) and high density lipoprotein cholesterol ester (HDL-C) levels of apoE-/- mice were higher than that of age matched WT mice from 4-week to 24-week. Besides TG level at 4 and 6-week age, TC, TG, LDL-C and HDL-C levels in apoE-/- mice increased significantly when compared with TNF-α-/- and WT mice (P<0.05). The level of plasma lipid between TNF-α-/- and WT showed no significant difference at all stage. TC, TG and LDL-C levels were no statistically different between AT and apoE-/- mice. Compared with apoE-/- mice, the serum lipid level demonstrated that 1) TC and TG were lower in AT mice at the same age, 2) LDL levels were not shown significant differences between the two genotype at all age group. At age of 4-12 weeks, HDL levels of apoE in mice is higher than AT mice, but at 16-week, AT mouse plasma HDL-C level in AT mice increased to a level that was higher than apoE-/- mice. These results showed that, TNF-αinvolved in the apoE-/- mice lipid metabolism, and deletion of TNF-αgene might have impact on the lipid metabolism process through the HDL-C related pathway.3. Variation of the inflammatory factor gene transcription in spleenIn spleen, the expressions of inflammatory markers were dramatically altered in AT mice. Compared with apoE-/ - mice, the transcription level of the six cytokines were decreased, while interleukin (IL)- 1β, IL-4, IL-10 and IL-12 mRNA were significantly reduced (P<0.05) at 6-week old. At 12-week, the detection of cytokines expression between apoE-/- and AT mice showed different pattern. Interferon-γ(IFN-γ) and IL-1βappeared significantly up-regulated (P<0.05), IL-6 and IL-10 still showed a significant downward trend in AT mice (P<0.05), and the IL-4 and IL-12 mRNA expression level showed a minor increase. When it came to 24- week, the trends of cytokine expression were similar with 12-weeks, IFN-γ, IL-1β, IL-4 and IL-12 transcription levels increased in AT mice, while the IL-6 and IL-10 expression level were lower than that in apoE-/- mice, but the IL-1β, IL-4, IL-10 and IL-12 showed no significant difference between the two genotypes. These results indicated that the absence of TNF-αhad impact on the expression of inflammatory factors in spleen, and this influence showed age-dependent characteristics.4. Expression characteristics of inflammatory factors and cytokines in 6-week aorta(1) Inflammatory gene transcription in 6-week aortaAt 6-week, the expression of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γin aorta were decreased in AT mice than those in apoE-/- mice, the gene expression pattern had the same trends with spleen, but only IL-1β, IL-4, IL-12 and IFN-γhad significant difference (P<0.05). The transcription of nuclear factor-κB (NF-κB) level in AT mice was significantly lower, while the expression of inhibitor-κB (IκB) was higher than apoE-/- mice. These results indicated that, the absence of TNF-αmight affect the gene transcription of inflammatory factors gene transcription by NF-κB signaling pathway.(2) Cytokine antibody array in 6-week aortaAmong the 64 cytokines detected by cytokine antibody array, expression levels of 22 cytokines were changed significantly in AT mice compared to apoE-/- mice. Two cytokines,including adhesion-related kinase (ARK), insulin-like growth factor banding protein (IGFBP) expression were significantly increased. Eighteen cytokines expression decreased by 2-fold or more in AT mice compared to apoE-/- mice, including cluster of differentiation (CD)30, CD30 L, CD40, IL-5, IL-6, IL-10, IL-12p40/p70, keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-1γ, MIP-3α, platelet factor(PF)-4, thymus and activation-regulated chemokine (TARC), thymus-expressed chemokine (TECK), tissue inhibitory of metalloproteinase (TIMP-1), soluble TNF receptor I (sTNFRI) and thrombopoietin (TPO). The cytokines which had significant difference were mainly chemokines, receptors, ILs, and a small number of regulatory factors. The cytokine antibody array analysis showed that the deletion of TNF-αimpact cytokine expression pattern in apoE-/- mice aorta. The reduction of cytokines slowed down the monocyte activation and migration, reduced the interaction of endothelial cells, monocytes/macrophages and T-cell, thus slowing the disease process of atherogenesis.5. Expression characteristics of reverse cholesterol transport-related gene in liverTNF-αcould significantly affect the reverse cholesterol transport-related gene expression of apoE-/- mouse liver, and this effect showed different trends at different ages. At 6-week, the transcription level of apolipoprotein A-I (apo A-I), liver X receptor alpha (LXRα), ATP binding cassette transporter A1 (ABCA1),β-hydroxy-β-methyl glutaryl CoA reductase (HMG-CoAR), lecithin-cholesterol acyltrasferase (LCAT) and scavenger receptor class B type I (SR-BI) were decreased in AT mice. In AT mice, expression levels of apo A-I, LCAT and SR-BI started to increase from 12-week compared with apoE-/- mice, and were significantly up-regulated at 24-week (P < 0.01). LXRαand ABCA1 showed similar expression characteristics. The expression of HMG-CoAR in AT mice were lower than those in apoE-/- mice all stage(P<0.05). The experimental results showed that, at different stages of atherogenesis, TNF-αplayed different effects on the reverse cholesterol transport. TNF-αhad promoted the process of the RCT in liver at 6-week, and showed strong inhibitory effects of the RCT at 24-week.In summary, TNF-αdeficiency retarded the atherogenesis in apoE-/- mice. At 6-weeks of age, TNF-αdeficiency inhibited the process of AS by reducing the transcription activity of IL-1β, IFN-γ, IL-12 and other type-I cytokines in spleen and aortic. Cytokine antibody microarray showed that a variety of chemokines, adhesion molecules including MIP-1α, MIP-1β, KC, TARC, MCP-1, VCAM-1 reduced in AT mice, indicated that TNF-αcould affect the expression of these components, and involved in fthe ormation of foam cells and lipid stripes by promoting the interaction between vascular endothelial cells, monocyte/macrophages and T cells. However, along with the increase of age, the effect of TNF-αon the expression of inflammatory factors in the spleen were changed. The expression of IFN-γ, IL-1β, IL-4 and IL-12 showed the opposite trend in 6-week and 24-week, suggesting that TNF-αhad different effect on the expression of inflammatory factors at different age. Lipids HDL-C level suggested that TNF-αmay adopt lipid metabolism by RCT. Real-time RT-PCR results showed that the RCT-related genes transcription level changed significantly after TNF-αdeletion. TNF-αdeficiency inhibit the RCT by down-regulated the expression of apo A-I, LCAT, SR-B1, LXRα, and HMG-CoA. To 24-week, TNF-αhad up-regulated the expression of these gene for RCT. This indicates that TNF-αhad different effects on RCT with age.Based on the present results, it was concluded that the role of TNF-αshowed complex effects and time-dependent characteristics during atherosgenesis. AS was a multi-factor involved pathology process, in which TNF-αextensively involved in the regulation of inflammatory response and lipid metabolism in AS. It was speculated that in fatty-streak period, the dyslipidemia may be just as an important initiator, while inflammation play a dominant role in this process.
|
PDF Full Text Request