Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Séguin, Cheryle A.

Abstract

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.

Summary for Lay Audience

Diffuse idiopathic skeletal hyperostosis (DISH) is the second most common form of arthritis and characterized by abnormal mineral formation along the spine. DISH results in mineralization of spinal tissues including ligaments, and the 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 trouble swallowing and loss of sensation due to spinal cord compression. However, the molecular mechanism responsible for DISH is unknown so there are no treatments to slow or prevent mineral formation in DISH; treatment is limited to surgical removal of mineralized tissue often requiring revision surgery. To identify the molecular mechanism of DISH, we studied the gene expression, protein expression, and small molecule (metabolite) composition of the AF from wild-type (WT) and mice lacking ENT1 (mouse model of DISH), but first needed to establish the baseline gene expression of a healthy, aging IVD. We found that cartilage marker genes Prg4,Cilp, Ibsp, and Comp were enriched >50-fold in the AF, making these potential tissue-specific markers in the mouse IVD (Chapter 2). We then found that loss of ENT1 is associated with increased cell proliferation in the AF (Chapter 3). We then wanted to explore protein and small molecule (metabolite) changes, but first needed to establish protocols to do so. In developing protocols, we were able to increase our quantification of proteins greater than 2-fold, avoid bias of certain highly abundant proteins in the IVD, and identify >300 metabolites in the blood and AF of mice which had not been accomplished before (Chapter 4). We then used these protocols to find that abnormal mineralization in mice lacking ENT1 is associated with altered molecular signaling, fatty acid metabolism, and that certain fatty acids may serve as biomarkers of early-stage DISH (Chapter 5). Finally, we explored symptoms in mice lacking ENT1 using behavioral assays and protein stains to find that these mice exhibit increased pain and stiffness associated with abnormal mineralization (Chapter 6). In summary, this work uncovered molecular mechanisms associated with DISH, DISH biomarkers, and examined DISH symptoms using a mouse model.

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