CD163/TWEAK通路对动脉粥样硬化的作用
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摘要
目的:探讨CD163/肿瘤坏死因子样凋亡弱诱导因子(TWEAK)途径对小鼠动脉粥样硬化的影响。方法:以8～10周的载脂蛋白E基因敲除(APOE~(-/-))及野生型(WT)C57BL/6小鼠为研究对象,分为以下4组(n=10):APOE~(-/-)+普食(ND)组、APOE~(-/-)+高脂饮食(WD)组、WT+ND组和WT+WD组。喂养16周后检测血脂水平,主动脉油红O染色测定主动脉斑块面积以明确成功建立动脉粥样硬化模型;Western blot法检测各组小鼠主动脉CD163和TWEAK的蛋白表达水平;免疫组化法明确动脉粥样硬化斑块CD163与TWEAK的表达及空间分布特征;细胞实验研究CD163对1型巨噬细胞(M1)及泡沫细胞TWEAK的调节作用及其可能的下游作用途径。结果:血脂检测及主动脉油红O染色结果显示,APOE~(-/-)+ND及APOE~(-/-)+WD组小鼠成功建立动脉粥样硬化模型。免疫组化可见CD163主要表达于远离脂质核心的部位,而TWEAK可见于动脉粥样硬化斑块的所有部位。主动脉Western blot检测结果显示,与WT小鼠相比,APOE~(-/-)小鼠主动脉CD163的表达水平显著增加(P<0.05),与之相对应的是APOE~(-/-)小鼠主动脉中TWEAK表达水平较WT小鼠亦显著升高(P<0.05)。细胞实验结果显示CD163可显著抑制TWEAK的蛋白表达,明显下调核因子κB(NF-κB)的水平(P<0.05)。结论:CD163可通过抑制TWEAK/NF-κB途径而发挥抗动脉粥样硬化作用。
AIM: To investigate the effect of CD163/tumor necrosis factor-like weak inducer of apoptosis(TWEAK) pathway on atherosclerosis in mice. METHODS: APOE~(-/-)mice and wild-type(WT) C57 BL/6 mice were divided into 4 groups(8～10 weeks, n=10): APOE~(-/-)+normal diet(ND) group, APOE~(-/-)+western diet(WD) group, WT+ND group, and WT+WD group. Detection of blood lipid levels and oil red O staining of aorta artery were performed to confirm whether the atherosclerotic model was well established in 16 weeks after feeding. The aortic tissues were harvested to measure CD163 and TWEAK protein levels by Western blot, and immunohistochemical staining was also performed to localize CD163 and TWEAK protein expression on atherosclerotic plaque in each group. The cell experiments were conducted to study whether CD163 regulated TWEAK expression in M1 macrophages and foam cells, and the possible downstream pathway was investigated. RESULTS: The blood lipid levels and aorta oil red O staining showed that the animal model of atherosclerosis was successfully established in APOE~(-/-)+ND group and APOE~(-/-)+WD group. The protein level of CD163 was significantly increased in the aortic tissue in APOE~(-/-)mice(P<0.05) as compared with C57 BL/6 mice(P<0.05). Consistently, the protein level of TWEAK was also markedly higher in APOE~(-/-)+ND group and APOE~(-/-)+WD group than that in WT+ND group and WT+WD group(P<0.05). Immunohistochemical staining showed that CD163 was mainly expressed in the parts away from the lipid core, and TWEAK was found in all parts of the atherosclerotic plaque. CD163 significantly inhibited the protein expression of TEWAK in the M1 macrophages, and also significantly down-regulated the level of nuclear factor-κΒ(NF-κB) in the M1 macrophages and foam cells(P<0.05). CONCLUSION: The protein levels of CD163 and its ligand TWEAK are significantly increased in atherosclerotic mice. The CD163 positive macrophages are mainly located at the site far away from the lipid core, and CD163 may play an anti-atherosclerotic effect by inhibiting TWEAK/NF-κB pathway.
AIM: To investigate the effect of CD163/tumor necrosis factor-like weak inducer of apoptosis(TWEAK) pathway on atherosclerosis in mice. METHODS: APOE~(-/-)mice and wild-type(WT) C57 BL/6 mice were divided into 4 groups(8～10 weeks, n=10): APOE~(-/-)+normal diet(ND) group, APOE~(-/-)+western diet(WD) group, WT+ND group, and WT+WD group. Detection of blood lipid levels and oil red O staining of aorta artery were performed to confirm whether the atherosclerotic model was well established in 16 weeks after feeding. The aortic tissues were harvested to measure CD163 and TWEAK protein levels by Western blot, and immunohistochemical staining was also performed to localize CD163 and TWEAK protein expression on atherosclerotic plaque in each group. The cell experiments were conducted to study whether CD163 regulated TWEAK expression in M1 macrophages and foam cells, and the possible downstream pathway was investigated. RESULTS: The blood lipid levels and aorta oil red O staining showed that the animal model of atherosclerosis was successfully established in APOE~(-/-)+ND group and APOE~(-/-)+WD group. The protein level of CD163 was significantly increased in the aortic tissue in APOE~(-/-)mice(P<0.05) as compared with C57 BL/6 mice(P<0.05). Consistently, the protein level of TWEAK was also markedly higher in APOE~(-/-)+ND group and APOE~(-/-)+WD group than that in WT+ND group and WT+WD group(P<0.05). Immunohistochemical staining showed that CD163 was mainly expressed in the parts away from the lipid core, and TWEAK was found in all parts of the atherosclerotic plaque. CD163 significantly inhibited the protein expression of TEWAK in the M1 macrophages, and also significantly down-regulated the level of nuclear factor-κΒ(NF-κB) in the M1 macrophages and foam cells(P<0.05). CONCLUSION: The protein levels of CD163 and its ligand TWEAK are significantly increased in atherosclerotic mice. The CD163 positive macrophages are mainly located at the site far away from the lipid core, and CD163 may play an anti-atherosclerotic effect by inhibiting TWEAK/NF-κB pathway.
引文
[1] Ross R. Atherosclerosis: an inflammatory disease[J]. N Engl J Med, 1999, 340(2):115-126.
[2] Guo L, Harari E, Virmani R, et al. Linking hemorrhage, angiogenesis, macrophages, and iron metabolism in atherosclerotic vascular diseases[J]. Arterioscler Thromb Vasc Biol, 2017, 37(4):e33-e39.
[3] Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: a dynamic balance[J]. Nat Rev Immunol, 2013, 13(10):709-721.
[4] Gordon S. Alternative activation of macrophages[J]. Nat Rev Immunol, 2003, 3(1):23-35.
[5] Bouhlel MA, Derudas B, Rigamonti E, et al. PPARγ activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[J]. Cell Metab, 2007, 6(2):137-143.
[6] Kowal K, Silver R, Sawińska E, et al. CD163 and its role in inflammation[J]. Folia Histochem Cytobiol, 2011, 49(3):365-374.
[7] Boyle JJ, Johns M, Kampfer T, et al. Activating transcription factor 1 directs Mhem atheroprotective macrophages through coordinated iron handling and foam cell protection[J]. Circ Res, 2012, 110(1):20-33.
[8] Guo L, Akahori H, Harari E, et al. CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis[J]. J Clin Invest, 2018, 128(3):1106-1124.
[9] Pourcet B, Staels B. Alternative macrophages in atherosclerosis: not always protective![J]. J Clin Invest, 2018, 128(3):910-912.
[10] Patel KV, Pandey A, de Lemos JA. Conceptual framework for addressing residual atherosclerotic cardiovascular disease risk in the era of precision medicine[J]. Circulation, 2018, 137(24):2551-2553.
[11] Bohula EA, Giugliano RP, Leiter LA, et al. Inflammatory and cholesterol risk in the FOURIER trial [J].Circulation, 2018, 138(2):131-140.
[12] Bohula EA, Giugliano R, Leiter L, et al. Residual inflammatory and cholesterol risk in the FOURIER trial[J]. J Am Coll Cardiol, 2018, 71(11 Suppl):A2.
[13] Nielsen MJ, Andersen CB, Moestrup SK. CD163 binding to haptoglobin-hemoglobin complexes involves a dual-point electrostatic receptor-ligand pairing[J]. J Biol Chem, 2013, 288(26):18834-18841.
[14] Valdivielso JM, Coll B, Martín-Ventura JL, et al. Soluble TWEAK is associated with atherosclerotic burden in patients with chronic kidney disease[J]. J Nephrol, 2013, 26(6):1105-1113.
[15] Sastre C, Fernández-Laso V, Madrigal-Matute J, et al. Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice[J]. J Cell Mol Med, 2014, 18(4):721-734.
[16] Pagie S, Gérard N, Charreau B. Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis[J]. Cell Commun Signal, 2018, 16:4.
[17] Moreno JA, Izquierdo MC, Sanchez-Ni MD, et al. The inflammatory cytokines TWEAK and TNFαreduce renal klotho expression through NFκB[J]. J Am Soc Nephrol, 2011,22(7):1315-1325.
[18] Kim SH, Kang YJ, Kim WJ, et al. TWEAK can induce pro-inflammatory cytokines and matrix metalloproteinase-9 in macrophages[J]. Circ J, 2004, 68(4):396-399.
[19] Mǔnoz-GarciaB, Moreno JA, López-Franco O, et al. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) enhances vascular and renal damage induced by hyperlipidemic diet in ApoE-knockout mice[J]. Arterioscler Thromb Vasc Biol, 2009, 29(12):2061-2068.
[20] Bover LC, Cardó-Vila M, Kuniyasu A, et al. A previously unrecognized protein-protein interaction between TWEAK and CD163: potential biological implications[J]. J Immunol, 2007, 178(12):8183-8194.
[21] Fernández-Laso V, Sastre C, Méndez-Barbero N, et al. TWEAK blockade decreases atherosclerotic lesion size and progression through suppression of STAT1 signaling in diabetic mice[J]. Sci Rep, 2017, 7:46679.
[22] 吴莲凤, 陆红, 洪炜龙, 等. M1/M2型巨噬细胞极化参与肾组织炎症损伤和修复进程[J]. 中国病理生理杂志, 2017, 33(12):2245-2251.
[23] Roos C, Wicovsky A, Müller N, et al. Soluble and transmembrane TNF-like weak inducer of apoptosis differentially activate the classical and noncanonical NF-κB pathway[J]. J Immunol, 2010, 185(3):1593-1605.
[24] Stephan D, Sbai O, Wen J, et al. TWEAK/Fn14 pathway modulates properties of a human microvascular endothelial cell model of blood brain barrier[J]. J Neuroinflammation, 2013, 10:9.
[25] Wen J, Doerner J, Weidenheim K, et al. TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice[J]. J Autoimmun, 2015, 60:40-50.
[2] Guo L, Harari E, Virmani R, et al. Linking hemorrhage, angiogenesis, macrophages, and iron metabolism in atherosclerotic vascular diseases[J]. Arterioscler Thromb Vasc Biol, 2017, 37(4):e33-e39.
[3] Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: a dynamic balance[J]. Nat Rev Immunol, 2013, 13(10):709-721.
[4] Gordon S. Alternative activation of macrophages[J]. Nat Rev Immunol, 2003, 3(1):23-35.
[5] Bouhlel MA, Derudas B, Rigamonti E, et al. PPARγ activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[J]. Cell Metab, 2007, 6(2):137-143.
[6] Kowal K, Silver R, Sawińska E, et al. CD163 and its role in inflammation[J]. Folia Histochem Cytobiol, 2011, 49(3):365-374.
[7] Boyle JJ, Johns M, Kampfer T, et al. Activating transcription factor 1 directs Mhem atheroprotective macrophages through coordinated iron handling and foam cell protection[J]. Circ Res, 2012, 110(1):20-33.
[8] Guo L, Akahori H, Harari E, et al. CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis[J]. J Clin Invest, 2018, 128(3):1106-1124.
[9] Pourcet B, Staels B. Alternative macrophages in atherosclerosis: not always protective![J]. J Clin Invest, 2018, 128(3):910-912.
[10] Patel KV, Pandey A, de Lemos JA. Conceptual framework for addressing residual atherosclerotic cardiovascular disease risk in the era of precision medicine[J]. Circulation, 2018, 137(24):2551-2553.
[11] Bohula EA, Giugliano RP, Leiter LA, et al. Inflammatory and cholesterol risk in the FOURIER trial [J].Circulation, 2018, 138(2):131-140.
[12] Bohula EA, Giugliano R, Leiter L, et al. Residual inflammatory and cholesterol risk in the FOURIER trial[J]. J Am Coll Cardiol, 2018, 71(11 Suppl):A2.
[13] Nielsen MJ, Andersen CB, Moestrup SK. CD163 binding to haptoglobin-hemoglobin complexes involves a dual-point electrostatic receptor-ligand pairing[J]. J Biol Chem, 2013, 288(26):18834-18841.
[14] Valdivielso JM, Coll B, Martín-Ventura JL, et al. Soluble TWEAK is associated with atherosclerotic burden in patients with chronic kidney disease[J]. J Nephrol, 2013, 26(6):1105-1113.
[15] Sastre C, Fernández-Laso V, Madrigal-Matute J, et al. Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice[J]. J Cell Mol Med, 2014, 18(4):721-734.
[16] Pagie S, Gérard N, Charreau B. Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis[J]. Cell Commun Signal, 2018, 16:4.
[17] Moreno JA, Izquierdo MC, Sanchez-Ni MD, et al. The inflammatory cytokines TWEAK and TNFαreduce renal klotho expression through NFκB[J]. J Am Soc Nephrol, 2011,22(7):1315-1325.
[18] Kim SH, Kang YJ, Kim WJ, et al. TWEAK can induce pro-inflammatory cytokines and matrix metalloproteinase-9 in macrophages[J]. Circ J, 2004, 68(4):396-399.
[19] Mǔnoz-GarciaB, Moreno JA, López-Franco O, et al. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) enhances vascular and renal damage induced by hyperlipidemic diet in ApoE-knockout mice[J]. Arterioscler Thromb Vasc Biol, 2009, 29(12):2061-2068.
[20] Bover LC, Cardó-Vila M, Kuniyasu A, et al. A previously unrecognized protein-protein interaction between TWEAK and CD163: potential biological implications[J]. J Immunol, 2007, 178(12):8183-8194.
[21] Fernández-Laso V, Sastre C, Méndez-Barbero N, et al. TWEAK blockade decreases atherosclerotic lesion size and progression through suppression of STAT1 signaling in diabetic mice[J]. Sci Rep, 2017, 7:46679.
[22] 吴莲凤, 陆红, 洪炜龙, 等. M1/M2型巨噬细胞极化参与肾组织炎症损伤和修复进程[J]. 中国病理生理杂志, 2017, 33(12):2245-2251.
[23] Roos C, Wicovsky A, Müller N, et al. Soluble and transmembrane TNF-like weak inducer of apoptosis differentially activate the classical and noncanonical NF-κB pathway[J]. J Immunol, 2010, 185(3):1593-1605.
[24] Stephan D, Sbai O, Wen J, et al. TWEAK/Fn14 pathway modulates properties of a human microvascular endothelial cell model of blood brain barrier[J]. J Neuroinflammation, 2013, 10:9.
[25] Wen J, Doerner J, Weidenheim K, et al. TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice[J]. J Autoimmun, 2015, 60:40-50.