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Alpha-lipoic acid inhibits fractalkine expression and prevents neointimal hyperplasia after balloon injury in rat carotid artery Kyeong-Min Lee, Keun-Gyu Park, Yong-Deuk Kim, Hyo-Jung Lee, Hyoung Tae Kim, Won-Hyun Cho, Hye-Soon Kim, Seong-Wook Han, Gou Young Koh, Joong-Yeol Park, Ki-Up Lee, Jung-Guk Kim, In-Kyu Lee Atherosclerosis Volume 189, Issue 1, Pages (November 2006) DOI: /j.atherosclerosis Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 1 Effect of α-lipoic acid on neointimal formation after balloon injury. Cross-sections of the left common carotid artery of a control rat (A) and rats 14 days after balloon injury without α-lipoic acid (B) and with 25 (C), 50 (D) and 100mg/kg/day (E) of α-lipoic acid. Average ratio of the intimal to medial layer of the left carotid artery in groups treated with or without α-lipoic acid (F). The bars represent the neointima/media ratio of the common carotid arteries after injury from each group of animals studied (n=6 for each group). Statistical significance was determined as *P<0.05, **P<0.01, #P<0.005 compared with balloon-injured artery without α-lipoic acid. All figures are depicted at 25× magnification. The scale bar represents 200μm. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 2 Immunohistochemical analysis of the time course of TNF-α and fractalkine expression in the medial region after balloon injury: (A) Immunhistochemical staining of TNF-α. TNF-α staining of a control vessel (C) and balloon-injured vessels (1, 3 and 24h) are shown. TNF-α positive cells appear brown. (B) Immunohistochemical staining of fractalkine. Fractalkine staining of a control vessel (C), balloon-injured vessels without α-lipoic acid (1, 3, 6, 12 and 24h) and with pretreatment with α-lipoic acid (100mg/kg) for 3 days (24h/A) are shown. Fractalkine positive cells appear brown. All figures are depicted at 200× magnification. The scale bar represents 50μm. Semi-quantification of fractalkine expression in the medial region (right lower). For each section, three to five medial portions of artery were graded on a scale from 1 (no staining) to 5 (very strong). C, control; A, balloon-injured vessel with α-lipoic acid pretreatment. Bars represent the mean±S.E.M. for each of group (n=5). Statistical significance was determined as *P<0.05, **P<0.01 and #P<0.005 compared with C and ##P<0.005 compared to 24h after injury without α-lipoic acid. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 3 Effect of α-lipoic acid on fractalkine expression in the neointimal region after balloon injury. Fractalkine staining of balloon-injured vessels without α-lipoic acid (A, 14 days after balloon injury) and with 25 (B), 50 (C) and 100mg/kg/day (D) of α-lipoic acid treatment (14 days after balloon injury) are shown. Fractalkine positive cells appear brown. TNF-α staining (E) and α-smooth muscle actin staining (F) of balloon-injured vessels are shown. TNF-α and smooth muscle specific α-smooth muscle actin positive cells appear brown and red, respectively. Nuclear sections were counter stained with DAPI. All figures are depicted at 200× magnification. The scale bar represents 50μm. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 4 TNF-α-induced fractalkine expression in cultered VSMCs: (A) Western blot analysis of the time-course expression of fractalkine in TNF-α-stimulated VSMCs. VSMCs were incubated for the indicated times with TNF-α (5ng/mL) after 48h serum starvation. (B) Western blot analysis of the dose response expression of fractalkine in TNF-α-stimulated VSMCs. VSMCs were incubated with the indicated amounts of TNF-α for 24h after 48h of serum starvation. Data are expressed as mean±S.E.M. of three separate measurements (lower). Statistical significance was determined as *P<0.05, **P<0.01 and #P<0.005 compared with basal expression. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 5 Effects of α-lipoic acid and anti-fractalkine antibody on VSMC proliferation. α-Lipoic acid (ALA, 1, 2 and 4mmol/L), anti-fractalkine antibody (FKN-Ab, 1, 10 and 20μg/mL) and control antibody (IgG, 20μg/mL) were incubated with VSMCs for 1h after 48h of serum starvation. NF-κB decoy ODN (D, 1, 10 and 100nmol/L) and mismatched NF-κB decoy ODN (MD, 100nmol/L) were transfected into VSMCs for 5h after 48h of serum starvation. An index of cell proliferation was determined by using a WST cell counting kit 48h after either treatment or transfection. Proliferation activities are calculated as the mean±S.E.M. of six separate measurements. Statistical significance was determined as *P<0.005 compared with control, **P<0.05, #P<0.01 and ##P<0.005 compared with TNF-α only. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 6 Effect of α-lipoic acid on TNF-α-stimulated fractalkine expression. VSMCs were incubated with TNF-α (5ng/mL) for 24h with or without pretreatment with α-lipoic acid at the indicated dose for 1h. (A) Northern blot analysis of fractalkine, VCAM-1 and MCP-1 mRNA expression. (B) Western blot analysis of fractalkine and VCAM-1 protein expression. Data are expressed as the mean±S.E.M. of three separate measurements (lower). Statistical significance was determined as *P<0.05, **P<0.01 and #P<0.005 compared with TNF-α only. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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Fig. 7 Effect of α-lipoic acid on TNF-α-stimulated NF-κB DNA binding activity: (A) Western blot analysis of VSMCs treated with either NF-κB inhibitor or decoy ODN. VSMCs were incubated with TNF-α (5ng/mL) for 24h in the presence of either PDTC (P, 100μmol/L), NF-κB decoy ODNs (D, 100nmol/L) or mismatched NF-κB decoy ODN (MD, 100nmol/L). Data are presented as the mean±S.E.M. of three separate measurements (lower). Statistical significance was determined as *P<0.005 compared with basal expression and **P<0.005 compared with TNF-α only. (B) Typical gel shift assay of α-lipoic acid treated VSMC lysates. VSMCs were treated with TNF-α (5ng/mL) for 24h with or without pretreatment of the indicated dose of α-lipoic acid for 1h. Data are presented as the mean±S.E.M. of three separate measurements (lower). Statistical significance was determined as *P<0.005 compared with control, **P<0.05, #P<0.01 and ##P<0.005 compared with TNF-α only. Atherosclerosis , DOI: ( /j.atherosclerosis ) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions
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