| Background and Objectives:Atherosclerosis is a chronic vascular disease which involves the progressive occlusion of blood vessels. With clinical consequences such as acute coronary syndrome and stroke, atherosclerosis remains the biggest cause of deaths worldwide. The World Health Organization estimates that 17.3 million adults have cardiovascular diseases in 2008, and this number will increase to 23.3 million by 2030. Therefore, understanding the pathogenesis of atherosclerosis is very important for preventing the atherosclerotic disease.Foam cells are formed by lipid uptake and storage of macrophages or vascular smooth muscle cells(VSMCs), which plays a very important role in the development of atherosclerosis. Intracellular cholesterol esterification is a critical p rocess in the foam cell formation, and Acyl-coenzyme A : cholesterol acyltransferase 1(ACAT1) is the key enzyme that converts free cholesterol into cholesteryl esters in this process. Previous studies have demonstrated that ACAT1 is widely expressed in the endoplasmic reticulun of a variety of cells and tissues, such as macrophages and VSMCs. In macrophages, the molecular mechanisms of foam cell formation involving ACAT1 have been widely studied. One of the major mechanisms is the inflammation that promotes the expression of ACAT1 and that leads to the foam cell formation. Compared with macrophages, the detailed mechanisms that how VSMCs become foam cells have not been well elucidated, and the potential role of ACAT1 in the VSMC foam cell formation remains unclear.An inflammatory response is widely accepted as an essential event in atherosclerosis [8]. Toll-like receptor 4(TLR4), as a transmembrane protein, plays a critical role in initiating inflammation, and participates in the development of atheroscler osis. Most recently, inhibition of the TLR4-mediated inflammation by peroxisome proliferator-activated receptor γ(PPARγ) has been reported to inhibit platelet-derived growth factor-induced VSMC proliferation and migration. Meanwhile, new evidence demonstrates inhibition of PPARγ by Chlamydia pneumoniae promotes low-density lipoprotein-induced macrophage-derived foam cell formation via up-regulating the expression of ACAT1. Chlamydia pneumoniae is a Gram-negative bacterium with lipopolysaccharide(LPS) as a major constituent of its outer membrane. LPS has been demonstrated to be a specific ligand of TLR4, induces TLR4-mediated inflammation, and increases the production of pro-inflammatory factors, including interleukin(IL)-1, IL-6, monocyte chemoattractant protein-1, and tumour necrosis factor α(TNF-α). The above results suggested the important role of PPARγ, TLR4 and ACAT1 in the process of the foam cell formation. Along these findings, in the present study we test the hypothesis that PPARγ inhibits VSMC foam cell formation via suppressing the TLR4-mediated inflammation and ACAT1 expression.Materials and Methods:In the present study, in vivo and in vitro studies were performed simultaneously. C57BL/6J wide type(WT) mice, Apo E gene knockout(Apo E-/-) mice, TLR4 gene knockout(TLR4-/-) mice and ACAT1 gene knockout(ACAT1-/-) mice were used in vivo studies, and VSMCs derived from WT mice, ACAT1-/- mice and TLR4-/- mice were used in vitro studies.1. Hematoxylin and eosin(HE) staining was applied to identify the formation of the atherosclerotic plaque in aortas.2. Oil red O staining was applied to detected the VSMC foam cell formation.3. Enzymatic assay was applied to detected the intracellular total cholesterol.4. Adenoviral vectors containing mouse ACAT1 c DNA was applied to produce ACAT1 overexpression cells.5. TLR4 ligand LPS and TLR4 inhibitor eritoran were used to respectively upregulate and downregulate the expression of TLR4 in vitro studies.6. Myeloid differentiation factor 88(My D88) and Nuclear factor-kappa B(NF-κB) small interfering RNA(si RNA) were used to knock down My D88 and NF-κB.7. Peroxisome proliferator-activated receptor γ(PPARγ) ligand Rosiglitazone(RSG) and PPARγ inhibitor GW9662 were used to upregulate and downregulate the expression of PPARγ in vivo and in vitro studies, respectively.8. Enzyme-linked Immunosorbent Assay(ELISA) kits was applied to detected the expression of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α).9. Western blot was applied to detected the expression of TLR4, My D88, NF-κB p65, phosphorylated IκBα(p-IκBα) and ACAT1. Iimmunofluorescence was also applied to detected the expression of ACAT1.Results:1. The role of ACAT1 in atherosclerotic plaque formation and in ox LDL-induced VSMC foam cell formation.⑴. High fat(HF) diet significantly up-regulated the ACAT1 expression in the aortas of Apo E-/- mice. Meanwhile, HF diet promoted the atherosclerotic plaque formation in the aortas of Apo E-/- mice. However, for the Apo E/ACAT1-/- mice, we did not observe the atherosclerotic plaque formation in response to HF diet.⑵. ox LDL significantly up-regulated the ACAT1 expression in a time-dependent manner in VSMC, and the maximum effect appeared at 48 h. Meanwhile, ox LDL stimulation promoted the VSMC foam cell formation. Moreover, ACAT1 overexpression further promoted ox LDL-induced foam cell formation of VSMC, and ACAT1 deficiency markedly inhibited the ox LDL-induced foam cell formation.2. The role of TLR4 in atherosclerotic plaque formation and in ox LDL-induced VSMC foam cell formation.⑴. HF diet significantly up-regulated the expression of TLR4 and cytokines(IL-1β, IL-6 and TNF-α) in the Apo E-/- mice. Meanwhile, HF diet promoted the atherosclerotic plaque formation in the aortas of Apo E-/- mice. However, for the Apo E/TLR4-/- mice, HF diet did not induce the atherosclerotic plaque formation effectively. In addition, HF diet also failed to induce the expression of cytokines.⑵. ox LDL significantly up-regulated the expression of TLR4 in a time-dependent manner in VSMC, and the maximum effect appeared at 24 h. Besides, ox LDL together with LPS further up-regulated the TLR4 expression. Meanwhile, the expression of cytokines were also up-regulated by ox LDL and/or LPS stimulation. In addition, ox LDL stimulation promoted the VSMC foam cell formation in WT VSMCs, which was enhenced by adding LPS stimulation. However, for the TLR4-/- VSMCs, ox LDL and/or LPS stimulation failed to up-regulate the expression of cytokines. Furthermore, the VSMC foam cell formation was also inhibited by TLR4 deficiency accordingly.3. Association between TLR4 and ACAT1 in regulating atherosclerotic plaque formation and VSMC foam cell formation.⑴. TLR4 deficiency abolished the HF diet-induced ACAT1 expression in the aortas of Apo E-/- mice.⑵. Activation of TLR4 further increased, while inhibition of TLR4 impeded the ox LDL-induced ACAT1 expression in WT VSMCs. As for TLR4-/- VSMCs, neither ox LDL alone nor ox LDL together with LPS/eritoran could change the ACAT1 expression.⑶. Activation of TLR4 further promoted, while inhibition of TLR4 suppressed the ox LDL-induced foam cell formation in WT VSMCs. By comparison, ACAT1 deficiency inhibited the ox LDL-induced VSMC foam cell formation in WT VSMCs. In addition, neither TLR4 activation nor inhibition could affect the foam cell formation in ACAT1-/-VSMCs. Moreover, ACAT1 deficiency did not affect the expression of TLR4 in WT VSMCs.⑷. ox LDL alone or together with LPS stimulation significantly up-regulated the expression of My D88, NF-κB p65(nuclei) and p-IκBα in WT VSMCs but not TLR4-/-VSMCs. In addition, Knock down of My D88 abolished the ox LDL- and/or LPS-induced expression of NF-κB p65(nuclei), p-IκBα and ACAT1. Besides, knock down of NF-κB p65 also abolished the ox LDL- and/or LPS-induced ACAT1 expression.4. The role of PPARγ in atherosclerotic plaque formation and in ox LDL-induced VSMC foam cell formation.⑴. Activation of PPARγ markedly inhibited the HF diet-induced atherosclerotic plaque formation in the Apo E-/- mice, as well as the expression of TLR4, cytokines and ACAT1. In contrast, PPARγ activation displayed undetectable effect on the atherosclerotic plaque formation in response to HF diet in the Apo E/TLR4-/- mice. In addition, the expression of cytokines and ACAT1 were not affected by PPARγ activation. Moreover, TLR4 deficiency did not affect the expression of PPARγ in the Apo E-/- mice.⑵. Activation of PPARγ dramatically inhibited, whereas inhibition of PPARγ further promoted, the ox LDL-induced foam cell formation in WT VSMCs, as well as the expression of TLR4, My D88, NF-κB p65(nuclei), p-IκBα, cytokines and ACAT1. However, for the TLR4-/- VSMCs, neither PPARγ activation nor PPARγ inhibition could affect the foam cell formation. Furthermore, PPARγ activation or inhibition also exerted undetectable effect on the TLR4-mediated inflammation and ACAT1 expression in the TLR4-/- VSMCs. Moreover, TLR4 deficiency did not affect the expression of PPAR γ in the WT VSMCs.Conclusions:The present study provided evidence that ox LDL stimulation can activate the TLR4/My D88/NF-κB inflammatory signaling pathway in VSMCs, which in turn up-regulates the ACAT1 expression and finally promote VSMC foam cell formation. In addition, PPARγ can inhibit the ox LDL-induced VSMC foam cell formation via suppressing the above pathway. Thus, our study shows novel pathological role of PPARγ, TLR4 and ACAT1 in VSMC foam cell formation, which may provide therapeutic target in atherosclerosis. |
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