TLR4-mediated inflammation promotes foam cell formation of vascular smooth muscle cell by upregulating ACAT1 expression

Vascular smooth muscle cell (VSMC) foam cell formation is an important hallmark, especially in advanced atherosclerosis lesions. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) promotes foam cell formation by promoting intracellular cholesteryl ester synthesis. The present study tests the hypothesis that oxidized low-density lipoprotein (oxLDL) increases the ACAT1 expression by activating the Toll-like receptor 4 (TLR4)-mediated inflammation, and ultimately promotes VSMC foam cell formation. Wild-type, ApoE^−/−, TLR4^−/− and ACAT1^−/− mice on a C57BL/6J background were used. Increased TLR4, proinflammatory cytokines and ACAT1 were observed in high-fat (HF) diet-induced atherosclerotic plaque formation and in oxLDL-stimulated VSMCs. ACAT1 deficiency impeded the HF diet-induced atherosclerotic plaque formation and impaired the TLR4-manipulated VSMC foam cell formation in response to oxLDL. TLR4 deficiency inhibited the upregulation of myeloid-differentiating factor 88 (MyD88), nuclear factor-κB (NF-κB), proinflammatory cytokines and ACAT1, and eventually attenuated the HF diet-induced atherosclerotic plaque formation and suppressed the oxLDL-induced VSMC foam cell formation. Knockdown of MyD88 and NF-κB, respectively, impaired the TLR4-manipulated VSMC foam cell formation in response to oxLDL. Rosiglitazone (RSG) attenuated HF diet-induced atherosclerotic plaque formation in ApoE^−/− mice, accompanied by reduced expression of TLR4, proinflammatory cytokines and ACAT1 accordingly. Activation of peroxisome proliferator-activated receptor γ (PPARγ) suppressed oxLDL-induced VSMC foam cell formation and inhibited the expression of TLR4, MyD88, NF-κB, proinflammatory cytokines and ACAT1, whereas inhibition of PPARγ exerted the opposite effect. TLR4^−/− mice and VSMCs showed impaired atherosclerotic plaque formation and foam cell formation, and displayed no response to PPARγ manipulation. In conclusion, our data showed that oxLDL stimulation can activate the TLR4/MyD88/NF-κB inflammatory signaling pathway in VSMCs, which in turn upregulates the ACAT1 expression and finally promotes VSMC foam cell formation.Atherosclerosis remains the major cause of deaths worldwide, with deteriorated clinical consequence of cardiovascular diseases including myocardial infarction and stroke.¹ In 2008, for example, 17.3 million deaths were caused by cardiovascular diseases, and this number will increase to 23.3 million by 2030.² Therefore, a better understanding of mechanisms involved in atherosclerosis may advance the development of comprehensive therapeutic regimens.Foam cell formation from macrophages or vascular smooth muscle cells (VSMCs) is a crucial event in the development of atherosclerosis. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is an intracellular enzyme that converts free cholesterol into cholesteryl esters for storage in lipid droplets, and promotes foam cell formation in atherosclerotic lesions.^{3, 4, 5} ACAT1 activity is present in a variety of cells and tissues, including the macrophages, neurons, cardiomyocytes, VSMCs, mesothelial cells, alveolar and intestinal epithelial cells and hepatocytes.⁶ In macrophages, the involvement of ACAT1 in foam cell formation has been demonstrated by studies, and multiple molecular mechanisms have been put forward. A well-accepted mechanism is that inflammation increases the expression of ACAT1, promotes the intracellular lipid accumulation and ultimately leads to foam cell formation.⁷ However, in contrast, the mechanisms underlying VSMC foam cell formation, especially the role of ACAT1 in this process, remain largely unelucidated.It is widely accepted that atherosclerosis involves chronic inflammatory reaction.⁸ Toll-like receptor 4 (TLR4), one intensively investigated member of the TLR family, has a critical role in initiating inflammation, and participates in VSMC activation.^{9, 10} Lipopolysaccharide (LPS) is a TLR4-specific ligand that can trigger TLR4-mediated inflammation. A previous study showed that Chlamydia pneumoniae, which contains LPS in its outer membrane, promotes low-density lipoprotein-induced macrophage-derived foam cell formation via upregulation of the expression of ACAT1.¹¹ This further enhanced the association between inflammation and intracellular lipid disorder. However, considering that VSMCs in normal conditions do not have inflammatory properties similar to macrophages, it is unclear whether the TLR4-mediated inflammatory mechanism is also involved in the regulation of ACAT1 in VSMC foam cell formation. Herein, the present study tests the hypothesis that oxidized low-density lipoprotein (oxLDL) increases the ACAT1 expression by activating the TLR4-mediated inflammation, and ultimately promotes VSMC foam cell formation.