Role of DNA damage and cellular senescence in osteoarthritis pathophysiology

DNA 损伤和细胞衰老在骨关节炎病理生理学中的作用

基本信息

批准号：
10801026
负责人：
Brian O Diekman
金额：
$ 65.93万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-30 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10801026
关键词：
Age Aging Alleles Antibodies Apoptosis Automobile Driving Biological Assay Cadaver Cartilage Cell Aging Cell Cycle Cell Cycle Arrest Cell physiology Cells Chondrocytes Chronic Disease Clustered Regularly Interspaced Short Palindromic Repeats Comet Assay Computer Models DNA Damage DNA Repair DNA strand break Data Degenerative polyarthritis Development Disease Elderly Endonuclease I Flow Cytometry Functional disorder Future Goals Harvest Hip region structure Histologic Human Image Indirect Immunofluorescence Inflammatory Intervention Intra-Articular Injections Joints Knowledge Link Measures Medial meniscus structure Mediating Mediator Methods Methyl Methanesulfonate Modeling Mus Operative Surgical Procedures Outcome Pain Pathologic Phenotype Physiological Play Predisposition Prevalence Prevention Production Proteins Public Health Replacement Arthroplasty Reporter Research Risk Factors Role Signal Pathway Sirtuins Site Societies Source Stains Stimulus Stress Response Signaling Synovial Cell Testing Therapeutic Therapeutic Intervention Time Tissues United States National Institutes of Health Work age effect arthropathies cartilage cell disability effective therapy ellipticine experimental study improved in vivo innovation irradiation joint injury novel prevent repaired response senescence small molecule stressor targeted treatment

项目摘要

PROJECT SUMMARY A key priority for the NIH is to limit disability caused by osteoarthritis (OA) and other chronic diseases that emerge with age. Senescent cells within joint tissues contribute to OA, but there is a knowledge gap regarding the triggers by which decades of aging initiate cellular senescence. One key mediator of senescence in other contexts is persistent DNA damage and the subsequent activation of a set of signaling pathways known as the DNA damage response (DDR). The DDR can drive the production of inflammatory and matrix-degrading molecules collectively known as the senescence-associated secretory phenotype (SASP), which has strong overlap with catabolic molecules known to contribute to OA. As demonstrated through the use of a single-cell gel electrophoresis “comet” assay, chondrocytes accumulate significant levels of DNA damage throughout aging and during OA. This damage is mostly in the form of single-strand breaks (SSBs) but a subset of cells also harbor double-strand breaks (DSBs). These distinct forms of damage can be initiated in cells from young cadaveric donors and mice to mimic the levels found in older donors/mice, with methyl methanesulfonate (MMS) for SSBs, ellipticine for DSBs, and irradiation to generate both SSBs and DSBs. Conversely, the burden of DNA damage in older cadaveric donors and older mice can be reduced by boosting DNA repair with activation of Sirtuin 6 (SIRT6) using the small molecule MDL-800. The long-term goal of this work is to catalyze more effective treatments for OA by determining the mechanisms by which joint cells become senescent. The central hypothesis is that the accumulation of DNA damage in joint tissues plays a causal role in driving senescence, the SASP, and subsequent OA. The first aim is to establish the contribution of SSBs and DSBs to senescence by applying distinct forms of DNA damage (irradiation, MMS, ellipticine) to cadaveric human chondrocytes and synovial cells. The second aim is to determine the extent to which DNA damage drives senescence and OA in mice. We also use intra-articular injection of agents to increase damage (MMS or ellipticine) or decrease damage (MDL-800) in the joints of p16tdTom reporter mice to assess senescence and functional/histologic OA. The third aim is to define the protein signatures that contribute to progression towards the SASP using a multiplex antibody staining method known as iterative indirect immunofluorescence imaging (4i) to track the signaling pathways that are activated in response to DNA damage. The expected outcomes of this work include a better understanding of the types of DNA damage that lead to senescence in joint tissues and the signaling pathways that link the DDR to SASP. This work is innovative in that tailored interventions are employed to alter the levels of DNA damage, with sophisticated readouts of senescence, including 4i for assessing protein signatures and senescence reporter mice. These contributions are expected to have a positive impact on society by stimulating more effective strategies to target senescent cells for the prevention and treatment of OA.

项目概要 NIH 的首要任务是限制骨关节炎 (OA) 和其他慢性疾病造成的残疾随着年龄的增长，关节组织内的衰老细胞会导致骨关节炎，但目前在这方面还存在知识差距。数十年的衰老引发细胞衰老的触发因素。背景是持续的 DNA 损伤和随后激活的一组信号通路，称为 DNA 损伤反应 (DDR) DDR 可以驱动炎症和基质降解的产生。统称为衰老相关分泌表型（SASP）的分子，具有很强的与已知有助于 OA 的分解代谢分子重叠，如通过使用单细胞所证明的。凝胶电泳“彗星”测定，软骨细胞在整个衰老过程中积累了显着水平的 DNA 损伤在 OA 期间，这种损伤主要以单链断裂 (SSB) 的形式出现，但也有一部分细胞出现这种损伤。这些不同形式的损伤可以在年轻细胞中引发。尸体供体和小鼠，以模仿老年供体/小鼠中发现的甲磺酸甲酯 (MMS) 水平对于SSB，玫瑰树碱对于DSB，以及照射以离线产生SSB和DSB，DNA的负担。通过激活DNA修复来减少老年尸体供体和老年小鼠的损伤 Sirtuin 6 (SIRT6) 使用小分子 MDL-800 这项工作的长期目标是更有效地催化。通过确定关节细胞衰老的机制来治疗 OA。假设是关节组织中 DNA 损伤的积累在驱动衰老中起着因果作用， SASP 和随后的 OA 的首要目标是确定 SSB 和 DSB 对衰老的贡献。通过对尸体软骨细胞施加不同形式的 DNA 损伤（辐射、MMS、玫瑰树碱），第二个目标是确定 DNA 损伤在多大程度上导致滑膜细胞衰老和 OA。我们还使用关节内注射药物来增加损伤（MMS 或玫瑰树碱）或减少损伤。 (MDL-800) 在 p16tdTom 报告小鼠的关节中评估衰老和功能/组织学 OA。目的是使用多重抗体定义有助于 SASP 进展的蛋白质特征称为迭代间接免疫荧光成像 (4i) 的染色方法可追踪信号通路被激活以应对 DNA 损伤这项工作的预期成果包括更好地理解。导致关节组织衰老的 DNA 损伤类型以及连接关节组织的信号通路 DDR 到 SASP 的创新之处在于采用定制干预措施来改变 DNA 水平。损伤，具有复杂的衰老读数，包括用于评估蛋白质特征的 4i 和衰老报告小鼠的这些贡献预计将通过刺激对社会产生积极影响。针对衰老细胞预防和治疗 OA 的更有效策略。