High Mobility Group Box 1 Mediates Interferon-γ-Induced Phenotypic Modulation Of Vascular Smooth Muscle Cells

Objective:The effect of IFN-gamma on HMGB11 release are investigated in Rat vascular smooth muscle cells. The role of HMGB1 on SMCs phenotypic modulation is also evaluated.Methods: Male SD Rats were anaesthetized and descending thoracic aorta was removed. Subsequently, Rat primary VSMCs were cultured with the method of attachment-block. And VSMCs were treated with IFN-gamma at various concentrations. The protein levels of HMGB1, SIRT1, the level of HMGB1 in cytoplasmic fraction and nuclear fraction were determined by Western Blotting. The location of cellular HMGB1 was detected by immunofluorescent staining. Total RNA was isolated to determine HMGB1 and SIRT1 transcription using q RT-PCR, and the levels of HMGB1 in the culture medium were measured by ELISA. Then, further study focusing on the effect of SIRT1 on the HGMB1 cytoplasmic translocation and its release, primary VSMCs were treated with either PBS or IFN-γ in the presence or absence of EX-527 or resveratrol. The levels of HMGB1 in cytoplasmic fraction or nuclear fraction were determined by Western blotting and its levels in the culture medium were measured by ELISA. Finally, we observed the effect of IFN-gamma on SMCs differentiation markers such as SM22α and calponin, which were determined by WB. Importantly, we used the neutralizing antibody of HMGB1 to explore the role of HMGB1 on IFN-gamma-induced phenotypic modulation of VSMCs.Results: IFN-γ induces the active release of HMGB1 from VSMCs but has no effect on the expression levels of intracellular HMGB1. Moreover, IFN-γ effectively induces HMGB1 translocation from the nucleus to the cytoplasm. And we also suggested the negative role of IFN-γ in regulation of SIRT1 expression in VSMCs. Thus, EX-527, a selective SIRT1 inhibitor, led to a marked HMGB1 translocation from the nucleus to the cytoplasm, accompanying with a significant increase in the levels of extracellular HMGB1.In contrast, treatment of VSMCs with Resveratrol, a selective SIRT1 activator, profoundly abolished IFN-γ-induced HMGB1 cytoplasmic accumulation and its release to culture medium. Futhermore, IFN-γ caused a marked decrease in the m RNA and protein levels of both SM22α and calponin in VSMCs, and neutralizing anti-HMGB1 markedly abrogated the reduction in the protein levels of both SM22α and calponin induced by IFN-γ.Conclusion: IFN-γ modulates HMGB1 expression and its release through downregulating SIRT1 expression in VSMCs.HMGB1 release is responsible for IFN-γ-induced phenotypic modulation toward a synthetic phenotype of VSMCs. Therefore, HMGB1 plays a critical role in regulating VSMC phenotypic modulation, suggesting that HMGB1 may be a potential therapeutic target to prevent vascular occlusive diseases.