The role of glycosylation in intervertebral disc degeneration: Towards glyco-functionalised therapeutics
Joyce, Kieran
Joyce, Kieran
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Publication Date
2021-09-24
Type
Thesis
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Abstract
Low back pain (LBP) is a common health issue that imposes a significant socio-economic burden on society. Primarily, intervertebral disc degeneration is one of the major contributing causes of LBP. It is initiated by altered mechanobiology and mediated by inflammation causing an imbalance of extracellular matrix homeostasis. This process results in loss of disc integrity, reduced disc height, increased risk of protrusion, extrusion and subsequent nerve compression and impingement resulting in discogenic pain. The molecular processes involved in this disease cascade is made up of glycoproteins. Inflammatory and degenerative processes in IVD degeneration produce altered expression patterns of glycosylation, although the role of glycosylation and localisation on glycoproteins in this process is not well understood. An understanding of IVD pathophysiology, specifically glycomic regulation in the IVD, is necessary to develop clinically relevant treatment strategies. Given the lack of satisfactory outcomes in treatment strategies for IVD degeneration, it is clear that new molecular targets need to be identified and addressed clinically to halt disc degeneration and restore native tissue structure and function. Glycomic profiling is a rapidly emerging field investigating the hierarchal regulation of protein activity and cellular pathways. Insights into the glycome offer key insights into cell activity, tissue homeostasis and mechanisms of degeneration. Small-molecule therapeutics have demonstrated efficacy in modulating glycomic-based targets. Next-generation biomaterials will likely encompass multi-omics functionalisation to address multiple disease targets, increasing the specificity and efficacy of regenerative therapies. The altered glycomic profile of the IVD degeneration may be targeted by specific inhibitors of glycosylation. The use of hyaluronic acid (HA) as a delivery system for therapeutic agents offers promising results in tissue regeneration as it can, itself, reduce inflammation. The overall aim of this study was to characterise the altered N-glycome of the IVD in degeneration and subsequently develop an optimally cross-linked high molecular weight of HA hydrogel, functionalised with a small molecule inhibitor of glycosylation to investigate the role of glycosylation in degeneration. In Phase I, the N-glycan profile of human intervertebral disc tissue was characterised by UPLC-MS. The characterisation of the N-glycan profile of the IVD has uncovered the altered expression of glycans in degeneration. The change in the expression of glycans demonstrates the dynamic regulation of glyco-enzymes and highlights the role of glycans as a biomarker for cell behaviour and disease status as well as their functional role in the disease process. This highlights the need for multi-omics approaches to biomaterial design and functionalisation. Future studies may include the localisation of modulated glycan expression. For example, the postulated increase in outer arm fucosylation of TIMP1 may be investigated by protein purification from human IVD and further glycan isolation and characterisation by UPLC-MS. In Phase II, the glycosignature of the NP cell in healthy and diseased tissues has been characterised and a validated inflammatory model of IVD degeneration has been developed. The role of sialylation and fucosylation have been investigated in the NP cell in an in vitro model of IVD degeneration and potential glycomic based mechanisms of degeneration and disease targets have been identified. When introducing a biomaterial into the intervertebral disc, tissue integration and material resorption are important parameters that can be influenced by modulated sialylation. 3Fpereacetyl Neu5Ac (Neu5Ac-inhib) may have a potential beneficial therapeutic effect in a hydrogel-loaded system in treating IVD degeneration. In Phase III, a HA-based hydrogel was optimally cross-linked with PEG-free amine and DMTMM, exerted hydrolytic stability and resistance to enzymatic degradation. No cytotoxic effect of NP cells was marked after treatment with HA hydrogels for ten days. These findings indicate that optimally stabilised cross-linked HA hydrogel is an ideal delivery vehicle to trial glycosylation inhibitors in vivo. The efficacy of Neu5Ac-inhib release from a HA hydrogel was determined in vitro in canine NP cells. Cytokine induced inflammation also increased sialylation expression in canine NP cells and sialylation inhibition effectively restored the glycosylation expression to physiological levels. Furthermore, a canine pre-clinical model, using beagles, was validated for determining the efficacy of glyco-functionalised materials by demonstrating a similar modulation in the glycome of the canine IVD compared to humans in degeneration. These findings pave the way for a pre-clinical trial using a sialyltransferase inhibitor, working synergistically with HA hydrogel, to regenerate the IVD in canine IVD degeneration, suggesting that glyco-modulation and specifically sialylation inhibition has a potential therapeutic application for the treatment of IVD degeneration.
Publisher
NUI Galway