The significance of interferon-γ on Fas ligand-induced apoptosis of vascular smooth muscle cells

Abstract

Objective To investigate the effect of interferon-γ （IFN-γ on Fas ligand （FasL） -in duced apoptosis of rat vascular smooth muscle cells （VSMCs） and explore the underlying mechanism. Methods VSMCs isolated from rat aorta were cultured in vitro and treated with IFN-γor FasL alone, or both in combination, with or without the pretreatment of Tunicamycin. Hoechst 33342 staining and flow cyto metric analysis of Annexin V-fluoresceine isothiocyanate （FITC）/propidium iodide （ PI ） staining were em ployed to define the effect of IFN-γon FasL-induced VSMCs apoplosis. Protein levels of surface Fas, cyto plasmic Fas and total Fas were detected by Western blotting in order to observe the expression and distribu tion of Fas. Results As shown by both Hoeehst 33342 staining, apoptosis of VSMCs was only slightly in duced by treatment with IFN-γ or FasL alone [ apoptotie index of （7.4 ± 1.9 ） % vs. （6. 8 ± 2. 5 ） % ], but significantly induced by IFN-γ and FasL in combination [ （20. 1 ± 2. 2）%, both P 〈 O. 01 ]. Increased sur face Fas （ relative level of O. 266 ± 0. 042 vs. O. 042 ± O. 017, P 〈 O. O1 ） , decreased cytoplasmic Fas （ O. 193 ±0. 038 vs. O. 426 ±0. 074, P 〈0. O1 ） but unchanged total Fas expression （0. 469 ＋0. 129/0. 383 ±0. 045 for 36 h/72 h vs. 0. 402 ± 0. 031 for 0 h, both P 〉 O. 05 ） were detected by Western blotting after IFN-γ treatment. And pretreatment with membrane trafficking inhibitor Tunicamycin significantly diminished FasL-indueed VSMCs apoptosis promoted by IFN-γ. The apoptotic index [ （6. 1 ± 0. 8）% ] were less than that of control [ （23.0 ± 1.0）% , P 〈0. 011- Flow eytometric analysis also confirmed the results of Ho echst 33342 staining. Conclusion Instead of inducing VSMCs apoptosis directly, IFN-γchanges the Fas distribution by promoting translocation of Fas receptor to the cell membrane, which significantly enhances the sensitivity of VSMCs to FasL-indueed apoptosis. Key words: Interferon-γ; Vascular smooth muscle cells; Fas ligand; Fas; Cell apoptosis