Regulators of Ectopic Calcification in a Mouse Model of Dish: A Multi-Omics Perspective

• Main Author: Veras, Matthew A
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2020
• Subjects: Adenosine; Age; Aging; Antigens; Arthritis; Back pain; Bioinformatics; Biology; Cartilage; Cell cycle; Cell growth; Cellular biology; Hemoglobin; Hypoxia; Ligaments; Medical research; Medicine; Metabolites; Pathogenesis; Physical therapy; Physiology; Plasma; Proteomics; Spinal cord; Therapy
• ISBN: 9798351493695

Diffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory spondyloarthropathy and the second most common form of arthritis characterized by formation of ectopic mineral along the spine. Pathological findings in DISH include regional calcification of the anterior longitudinal ligament, paraspinal connective tissues, and annulus fibrosus (AF) of the intervertebral disc (IVD). Clinical symptoms of DISH include increased spine stiffness, decreased spinal range of motion, and in severe cases dysphagia and spinal cord/nerve root compression. The molecular pathways responsible for DISH have not been delineated and as such, there are no disease-modifying treatments. Clinical treatment for DISH is limited to surgical resection of mineralized tissue often requiring revision surgery. To identify the molecular pathology of DISH, we sought to characterize the transcriptome, proteome and metabolome of the AF from wild-type (WT) and ENT1-/- mice (mouse model of DISH). To do so, we needed to establish the baseline gene expression of a healthy, aging IVD. We found that canonical cartilage markers Prg4, Cilp, Ibsp, and Comp were enriched >50-fold in the AF compared to the NP, making these potential tissue-specific markers of the murine IVD (Chapter 2). We then used microarrays and showed that loss of ENT1 is associated with increased cell proliferation in the AF, implicating the MAPK signaling pathway (Chapter 3). To explore proteomic and metabolomic changes in ENT1-/- mice, we first established and validated the necessary protocols. The protocols we developed increased our quantification of proteins greater than 2-fold over previous methods, avoided bias of ECM proteins, and identified >300 metabolites in the plasma and AF of mice which had not been accomplished before (Chapter 4). Using these protocols, we showed that ectopic calcification in the ENT1-/- mouse is associated with altered PI3K/Akt signaling, fatty acid metabolism, and that lysophosphatidylcholine isoforms may serve as biomarkers of early-stage DISH (Chapter 5). Finally, we explored the symptoms associated with ectopic calcification in the ENT1-/- mouse using behavioral assays and immunohistochemistry to find that these mice exhibit increased pain and stiffness (Chapter 6). In summary, this work uncovered molecular pathways associated with DISH, DISH biomarkers, and examined DISH symptoms using a mouse model.