Peking Union Medical College Beijing, Beijing, China
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Chenglong Fang1, Lihong Du1, Lili Li2, Yuexin Chen3, Zuoguan Chen4, Yongjun Li4, Jing Li1, Mengtao Li5, xiaofeng Zeng6 and Xinping Tian5, 1Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China, 2The State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China, 3Department of Vascular Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China, 4Department of Vascular Surgery, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China, 5Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China, 6Department of Rheumatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Background/Purpose: Takayasu’s arteritis (TAK) is characterized by persistent vascular inflammation involving aorta and its main branches, which is an important prosenescent factor that in turn amplifies local inflammation via senescence-associated secretory phenotype (SASP). In this study, we aimed to explore the pathogenic role of vascular smooth muscle cells (VSMCs) senescence and the relevant prosenescent pathway in TAK.
Methods: Cellular senescence markers were tested in vascular samples from patients with TAK using immunofluorescence, western blot and SA-β-gal staining. The key prosenescent inflammatory cytokine and its downstream intracellular events were investigated in a series of in-vitro and ex-vivo experiments including coculture experiments, RNA-Seq, gene knockdown, mitochondrial assays, co-immunoprecipitation, and tissue culture.
Results: The features of premature VSMCs senescence, including upregulated p16 expression, more intense SA-β-gal staining, and SASP consisting of increased interleukin-6 (IL-6) and IL-8 expression, were detected in TAK patients, compared with age- and sex-matched control subjects (Figure 1). Treatment with conditioned medium of peripheral blood mononuclear cell (PBMC) from patients (TAK-PBMC CM) and coculture with TAK-PBMC exerted similar prosenescent effects on VSMCs via IL-6 signaling. RNA-Seq suggested that cellular senescence and IL-6-STAT3 pathway were upregulated, while oxidative phosphorylation pathway was downregulated in TAK-PBMC CM-treated VSMCs. TAK-PBMC CM treatment induced multiple senescence-associated mitochondrial dysfunctions that can be significantly improved by IL-6 receptor knockdown (Figure 2). IL-6-induced noncanonical mitochondrial localization of phosphorylated STAT3 (Tyr705) prevented mitofusin 2 (MFN2) from proteasomal degradation, and subsequently promoted mitochondrial elongation and cellular senescence in VSMCs. In addition, coculture with MFN2 activator MASM7-induced senescent VSMCs led to an immunosenescence-like and cytotoxic phenotype switch in TAK-PBMC that can be detected in treatment-naïve patients, including decreased frequencies of naïve T cells, increased frequencies of TEMRA cells, age-associated B cells and CD16brightCD56dim NK cells, and upregulated NKG2D expression in CD4+ T cells (Figure 3). Of clinical relevance, although no significant difference of ESR and CRP levels was observed among patients with various senescent VSMCs proportions, patients with higher percentage of senescent VSMCs were more likely to relapse (Figure 1). However, senescent VSMCs proportions in vascular samples were comparable between patients taking long-term tocilizumab treatment ( >1 year) and those receiving other therapies, which is contradicting our expectation. Instead, treatment with mitoCur-1 (mitochondrial STAT3 inhibitor) or MFI8 ameliorated (MFN2 inhibitor) VSMCs senescence in ex vivo cultured arteries of patients with TAK (Figure 3).
Conclusion: VSMCs of patients with TAK exhibited the features of cellular senescence. IL-6-mitochondrial STAT3-MFN2 signaling is an important driver of VSMCs senescence. This may provide new insights into the mechanisms governing inflammation in TAK.
Figure 1. Premature VSMCs senescence in TAK patients and its association with disease parameters
Figure 2. Prosenescent effects and senescence-associated mitochodrial dysfunctions driven by TAK inflammatory milieu via IL-6 signaling
Figure 3. IL-6-induced prosenescent noncanonical mitochondrial-localized phosphorylated STAT3 (Tyr705) interact with MFN2 and prevented it from proteasomal degradation in VSMCs, resulting mitochondrial elongation in VSMCs. Coculture with senescent VSMCs lead to an immunosenescence-like and cytotoxic phenotype switch in cocultured TAK PBMCs. MitoCur-1 or MFI8 treatment ameliorates VSMCs senescence in ex vivo-cultured arteries from TAK patients.
C. Fang: None; L. Du: None; L. Li: None; Y. Chen: None; Z. Chen: None; Y. Li: None; J. Li: None; M. Li: None; x. Zeng: None; X. Tian: None.