Blocking TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in apolipoprotein E-deficient mice

Acta Pharmacologica Sinica, Jul 2013

Aim: Toll-like receptor 2 (TLR2) signaling plays a critical role in the initiation of atherosclerosis. The aim of this study was to investigate whether blocking TLR2 activity could produce therapeutic effects on advanced atherosclerosis. Methods: Forty-week old apolipoprotein E-deficient (ApoE−/−) mice fed on a normal diet were intravenously injected with a TLR2-neutralizing antibody or with an isotype-matched IgG for 18 weeks. Double-knockout ApoE−/−Tlr2−/− mice were taken as a positive control. At the end of the treatments, the plasma lipid levels were measured, and the plaque morphology, pro-inflammatory cytokines expression and apoptosis in arteries were analyzed. In the second part of this study, 6-week old ApoE−/− and ApoE−/−Tlr2−/− mice fed on a high-cholesterol diet for 12 to 24 weeks, the expression levels of TLR2 and apoptotic markers in arteries were examined. Results: Blockade of TLR2 activity with TLR2-neutralizing antibody or knockout of Tlr2 gene did not alter the plasma lipid levels in ApoE−/− mice. However, the pharmacologic and genetic manipulations significantly reduced the plaque size and vessel stenosis, and increased plaque stability in the brachiocephalic arteries. The protective effects of TLR2 antagonism were associated with the suppressed expression of pro-inflammatory cytokines IL-6 and TNF-α and the inactivation of transcription factors NF-κB and Stat3. In addition, blocking TLR2 activity attenuated ER stress-induced macrophage apoptosis in the brachiocephalic arteries, which could promote the resolution of necrotic cores in advanced atherosclerosis. Moreover, high-cholesterol diet more prominently accelerated atherosclerotic formation and increased the expression of pro-apoptotic protein CHOP and apoptosis in ApoE−/− mice than in ApoE−/−Tlr2−/− mice. Conclusion: The pharmacologic or genetic blockade of TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in ApoE−/− mice. Thus, targeting TLR2 signaling may be a promising therapeutic strategy against advanced atherosclerosis.

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Blocking TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in apolipoprotein E-deficient mice

Abstract Aim: Toll-like receptor 2 (TLR2) signaling plays a critical role in the initiation of atherosclerosis. The aim of this study was to investigate whether blocking TLR2 activity could produce therapeutic effects on advanced atherosclerosis. Methods: Forty-week old apolipoprotein E-deficient (ApoE−/−) mice fed on a normal diet were intravenously injected with a TLR2-neutralizing antibody or with an isotype-matched IgG for 18 weeks. Double-knockout ApoE−/−Tlr2−/− mice were taken as a positive control. At the end of the treatments, the plasma lipid levels were measured, and the plaque morphology, pro-inflammatory cytokines expression and apoptosis in arteries were analyzed. In the second part of this study, 6-week old ApoE−/− and ApoE−/−Tlr2−/− mice fed on a high-cholesterol diet for 12 to 24 weeks, the expression levels of TLR2 and apoptotic markers in arteries were examined. Results: Blockade of TLR2 activity with TLR2-neutralizing antibody or knockout of Tlr2 gene did not alter the plasma lipid levels in ApoE−/− mice. However, the pharmacologic and genetic manipulations significantly reduced the plaque size and vessel stenosis, and increased plaque stability in the brachiocephalic arteries. The protective effects of TLR2 antagonism were associated with the suppressed expression of pro-inflammatory cytokines IL-6 and TNF-α and the inactivation of transcription factors NF-κB and Stat3. In addition, blocking TLR2 activity attenuated ER stress-induced macrophage apoptosis in the brachiocephalic arteries, which could promote the resolution of necrotic cores in advanced atherosclerosis. Moreover, high-cholesterol diet more prominently accelerated atherosclerotic formation and increased the expression of pro-apoptotic protein CHOP and apoptosis in ApoE−/− mice than in ApoE−/−Tlr2−/− mice. Conclusion: The pharmacologic or genetic blockade of TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in ApoE−/− mice. Thus, targeting TLR2 signaling may be a promising therapeutic strategy against advanced atherosclerosis. Introduction Atherosclerosis is a common cause of cardiovascular disease. Cardiovascular disease is the leading cause of death and poses an increasing threat to human health worldwide1. Epidemiological and experimental studies have established a predominant role for inflammation in all stages of atherosclerosis, from the formation of early stable atherosclerotic plaques to advanced plaques that are prone to rupture2,3,4,5. Recently, several studies have focused on the effects of pharmacological interventions on the stability of advanced plaques because the rupture of unstable lesions contributes to acute coronary syndrome4,6,7. Unstable plaques are characterized by large, lipid-filled necrotic cores encapsulated by a thin fibrous cap, high levels of pro-inflammatory mediators and matrix proteases, and apoptosis. The development of unstable plaques is associated with the involvement of both the innate and adaptive immune systems. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that play a key role in innate immunity and initiate inflammatory responses. The ligation of these receptors initiates the activation of nuclear factor-κB (NF-κB), resulting in the expression of a wide array of inflammatory genes8,9. Among the characterized TLRs, TLR2 is unique by virtue of its ability to heterodimerize with TLR1 or TLR6, resulting in relatively broad ligand specificity, including both endogenous and exogenous ligands10,11. It is noteworthy that all of the potential endogenous TLR2 agonists, including biglycan, high-mobility box chromosomal protein 1 (HMGB1) and components of oxidized lipoproteins, are found in atherosclerotic lesions12,13,14. In addition, the TLR2 signaling pathway is associated with the endoplasmic reticulum (ER) stress response. ER stress can increase TLR2 expression in vitro and in vivo, and TLR2 contributes to apoptosis in macrophages undergoing lipid-triggered ER stress15. Thus, TLR2 may be a potential target for therapeutic interventions in the treatment of atherosclerosis. Indeed, TLR2 ligands and increased TLR2 expression have been observed in human atherosclerotic plaques, and the important role of TLR2 in inflammation and matrix degradation is indicated by the cells found in human carotid atherosclerotic lesions16,17. Moreover, TLR2 gene deficiency in a mouse model significantly inhibited the progression of early atherosclerosis18,19. However, there are limited data regarding the effects of TLR2 on advanced lesions in animal models. Using pharmacological and genetic inhibition of TLR2 activity, we investigated the therapeutic and prophylactic effects of TLR2 blockade on advanced atherosclerotic plaques in the brachiocephalic artery of chow-fed apolipoprotein E (ApoE)-deficient mice, which have never been evaluated before. We found that blocking TLR2 activity reduces and stabilizes advanced atherosclerotic lesions in ApoE-deficien (...truncated)


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Xiao-xing Wang, Xiao-xi Lv, Jia-ping Wang, Hui-min Yan, Zi-yan Wang, Han-zhi Liu, Xiao-ming Fu, Zhuo-wei Hu. Blocking TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in apolipoprotein E-deficient mice, Acta Pharmacologica Sinica, 2013, pp. 1025-1035, Issue: 34, DOI: 10.1038/aps.2013.75