Session: (2387–2424) Vasculitis – Non-ANCA-Associated & Related Disorders Poster III
2390: Characterization of Senescent Cells in Temporal Arteries of Patients with Giant Cell Arteritis Reveal an Inflammatory Phenotype and Strong Dependence from IL-6
Dept. of Pathophysiology, School of Medicine, University of Athens Athens, Greece
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Dimitrios Veroutis1, Ourania D Argryropouou1, Andreas Goules2, Konstantinos Kambas3, Dimitris Anastasios Palamidas1, Konstantinos Evangelou1, Sophia Havaki1, Aikaterini Polyzou1, Evangelia Xingi3, Elli Karatza4, Kyriaki Boki5, alberto cavazza6, Christos Kittas1, Dimitris Thanos7, caterina Ricordi6, chiara marvisi6, Francesco Muratore8, Elena Galli6, Stefania Croci6, Carlo Salvarani9, Vassilis G Gorgoulis1 and Athanasios Tzioufas2, 1National and Kapodistrian University of Athens, Athens, Greece, 2Dept. of Pathophysiology, School of Medicine, University of Athens, Athens, Greece, 3Hellenic Pasteur Institute, Athens, Greece, 4Laikon General Hospital, Athens, Greece, 5Sismanoglion Hospital, Athens, Greece, 6Azienda Unità Sanitaria Locale-IRCCS Di Reggio Emilia, Reggio Emilia, Italy, 7Biomedical Research Foundation of the Academy of Athens, Athens, Greece, 8IRCCS di Reggio Emilia, Reggio Emilia, Italy, 9Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy
Background/Purpose: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. So far, no studies have investigated adequately this question. The current study aims to identify the various senescent cell types in temporal artery biopsies (TABs) of GCA and polymyalgia rheumatica (PMR) patients by applying the novel multi-marker algorithm, define the secretory associated senescent phenotype (SASP) key molecules and explore possible implications of senescence in GCA pathogenesis.
Methods: Seventy five positive TABs from GCA patients and 22 negative from PMR patients were retrospectively analyzed. Senescent cells and their histologic origin were identified after staining for specific cellular markers including GL13, p21, vimentin, CD68, CD3 and aSMA, following the established multi-marker algorithm(1,2); IL-6 and MMP-9 were investigated as components of the (SASP) by triple co-staining. Twenty-four hour GCA or PMR artery culture supernatants were applied to primary skin fibroblasts with or without IL-6 blocking agent to explore the induction of IL-6 associated cellular senescence.
Results: Senescent cells were present in GCA arteries at higher proportion compared to PMR (9.50% vs 2.66% respectively, p< 0.0001) adjacent to the inflammatory cells and were mainly originated from fibroblasts (29.6%), macrophages (16.2%) and endothelial cells (14.3%) (Figure 1). IL-6 was expressed mainly by senescent fibroblasts and macrophages while MMP-9 by fibroblasts only (Figure 2). IL-6 positive senescent cells were associated with the extension of vascular wall inflammation (adventitial limited disease vs transmural inflammation: 10.02% vs 4.37% respectively, p< 0.0001) (Figure 3). Giant cell arteritis but not PMR artery culture supernatant induced IL-6-associated senescence that was partially inhibited by IL-6 blockade.
Conclusion: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the inflammatory burden of the vascular wall. These findings suggest a potential implication of senescent cells in disease pathogenesis by perpetuating inflammation and affecting vascular remodeling via IL-6 dependent mechanisms.
Figure 1. Detection of senescent cells in tissue artery biopsies of GCA patients. (A) Representative images for single GL13 staining with immunohistochemistry (upper panel) and immunofluorescence (lower panel) in TABs of GCA and PMR patients, showing higher proportion of GL13 positive cells adjacent to the inflammatory cells in GCA arteries compared to PMR. (B) Graphical representation of the proportion of GL13 positive senescent cells, after immunohistochemical evaluation by two independent readers and quantification analysis in 75 GCA and 22 PMR TABs (GCA vs PMR): 9.50% vs 2.66%, p<0.001. (C) Confirmation of senescent cells by co-staining for GL13 and p21WAF1/Cip1 in a TAB of a GCA compared to a PMR patient (representative image). (D) Electron micrographs of senescent cells in artery of GCA patients showing Lipofuscin (LF) granules in their cytoplasm (Di,ii). (E) Electron micrographs of cells in artery of PMR patient without lipofuscin granules (Ei,ii). Higher magnification of the area in the black box of (Ei). N: nucleus. Staining: uranyl acetate/lead citrate. Objectives (A) 20x, (C) 63x. Scale bars: 50μm (A) 10μm (C). 1μΜ (Di,ii, Eii), 2μM (Ei). ****p < 0.0001
Figure 2. Characterization of senescent cells per cell type in tissue artery biopsies of GCA patients. Representative images of double positive senescent cells after staining for GL13 and (A) Vimentin(+) (fibroblasts), (B) CD68(+) (macrophages), (C) CD34(+) (endothelial cells) or (D) aSMA(+) (smooth cells) in a TAB of one GCA and one PMR patient (double staining immunofluorescence). Squares depict magnified cells with presence of double positive staining. (E) Quantification analysis by two independent observers, showing significantly higher proportion of senescent fibroblasts (GL13(+)/Vimentin(+)), macrophages (GL13(+)/CD68(+)) and endothelial cells (GL13(+)/CD34(+)) in 13 GCA compared to 13 PMR biopsies. Objectives 20x. Scale bars: 50 μm. n.s. not significant, *p < 0.05, **p < 0.01, p < 0.001
Figure 3. Senescent cells in GCA express SASP that includes IL-6 and MMP-9. Representative images of IL-6 expression by senescent cells of different origin: (A) GL13(+)/Vimentin(+) (senescent fibroblasts, (B) GL13(+)/CD68(+) (senescent macrophages), (C) GL13(+)/CD34(+) (senescent endothelial cells)) and (D) GL13(+)/aSMA(+) (senescent smooth muscle cells) in a TAB of n=10 GCA and n=10 PMR patients (triple staining immunofluorescence). (E) Graphical representation of the proportion of IL-6 positive senescent cells in 10 GCA and 10 PMR biopsies, showing significantly higher proportion of IL- 6 positive senescent fibroblasts (GL13(+)/Vimentin(+)) and macrophages (GL13(+)/CD68(+)). Representative images of MMP-9 expression by GL13/vimentin double positive (senescent fibroblasts) cells in a TAB of one GCA and one PMR patient (triple stainingimmunofluorescence). (F-G) Graphical representation showing higher proportion of MMP-9 positive senescent fibroblast (GL13(+)/vimentin(+)) in the 3 GCA compared to 3 PMR specimens. Objectives (A), (D) 20x, (B), (C), 63x. Scale bars: (A), (D) 50 μm, (B), (C), 10μm. n.s. not significant, *p < 0.05, **p < 0.01
D. Veroutis: None; O. Argryropouou: None; A. Goules: None; K. Kambas: None; D. Palamidas: None; K. Evangelou: None; S. Havaki: None; A. Polyzou: None; E. Xingi: None; E. Karatza: None; K. Boki: None; a. cavazza: None; C. Kittas: None; D. Thanos: None; c. Ricordi: None; c. marvisi: None; F. Muratore: None; E. Galli: None; S. Croci: None; C. Salvarani: CSL Vifor, 1, 2, 6, Eli Lilly, 1, 2, 6; V. Gorgoulis: None; A. Tzioufas: None.
