In Vitro Characterization of Doxorubicin-Mediated Stress-Induced Premature Senescence in Human Chondrocytes

Abstract

Accumulation of senescent chondrocytes is thought to drive inflammatory processes and subsequent cartilage degeneration in age-related as well as posttraumatic osteoarthritis (OA). However, the underlying mechanisms of senescence and consequences on cartilage homeostasis are not completely understood so far. Therefore, suitable in vitro models are needed to study chondrocyte senescence. In this study, we established and evaluated a doxorubicin (Doxo)-based model of stress-induced premature senescence (SIPS) in human articular chondrocytes (hAC). Cellular senescence was determined by the investigation of various senescence associated (SA) hallmarks including β-galactosidase activity, expression of p16, p21, and SA secretory phenotype (SASP) markers (IL-6, IL-8, MMP-13), the presence of urokinase-type plasminogen activator receptor (uPAR), and cell cycle arrest. After seven days, Doxo-treated hAC displayed a SIPS-like phenotype, characterized by excessive secretion of SASP factors, enhanced uPAR-positivity, decreased proliferation rate, and increased β-galactosidase activity. This phenotype was proven to be stable seven days after the removal of Doxo. Moreover, Doxo-treated hAC exhibited increased granularity and flattened or fibroblast-like morphology. Further analysis implies that Doxo-mediated SIPS was driven by oxidative stress as demonstrated by increased ROS levels and NO release. Overall, we provide novel insights into chondrocyte senescence and present a suitable in vitro model for further studies.

Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue

“…Similar to our study, Dezfouli et al described lengthening of the G2 phase 14 days after Dox treatment of different aged mouse MSCs [ 47 ]. In contrast, others have found that Dox treatment arrested cell proliferation in G1 phase in human ASCs and chondrocytes [ 32 , 45 ]. To the fact that senescent hallmarks are neither exclusively nor universally expressed in senescent cells, we performed a multi-level characterization to confirm the senescent state of our models.…”

“…Stress-induced premature senescence (SIPS) is commonly triggered by exposure to sub-lethal stress in terms of oxidative stress [ 28 ], irradiation [ 29 ] or DNA damaging agents. The cytostatic drug and chemotherapeutic agent Doxorubicin (Dox) induces either apoptosis or senescence in a dose- and time-dependent manner [ 30 , 31 ] and is frequently applied for induction of SIPS [ 32 ].…”

Knee Osteoarthritis: Epidemiology, Pathogenesis, and Mesenchymal Stem Cells: What Else Is New? An Update

“…SASPs can be produced not only by chondrocytes, but also by other cells within the OA joint, such as osteoblasts, synovial fibroblasts, and macrophages [ 44 ]. For example, TGF-β and IL-6 are both SASP factors that can contribute to chondrocyte aging by activating p15, p21, and p27, therefore, promoting its senescence through the SMAD complex or STAT3 pathway [ 45 , 46 ]. Additionally, the release of SASPs by senescent chondrocytes can produce a chemotactic effect on surrounding immune cells; thus, establishing an inflammatory environment further stimulates cartilage degradation [ 43 ].…”

CircGNB1 drives osteoarthritis pathogenesis by inducing oxidative stress in chondrocytes

“…5 Accumulating evidence suggests that an imbalance in the redox state, leading to oxidative stress in chondrocytes, is a key event that perturbs cartilage homeostasis during OA development. 6,7 Oxidative stress activates proinflammatory pathways 8 and can trigger an inflammatory response 9 and chondrocyte senescence, 10 thereby accelerating the degradation of cartilage ECM during OA pathogenesis. 11 Previous clinical studies have illustrated that oxidative stress, aging, and OA are interrelated.…”

“…Thus, the progress of OA is associated with biological factors that disrupted the well‐being of chondrocytes and lead to an abnormal state 5 . Accumulating evidence suggests that an imbalance in the redox state, leading to oxidative stress in chondrocytes, is a key event that perturbs cartilage homeostasis during OA development 6,7 …”