The Characterization of Proteoglycan 4 and Hyaluronan Composition in the Vitreous Humour and Their Potential Contributions to Steady Shear Viscosity
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2020-01-27
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Background and aim: The vitreous humour is a connective tissue in the eye that contributes to several physiological and pathological processes. The full details of the complex vitreous humour composition and the extent of its functionality remain elusive. Proteoglycan 4 (PRG4), a mucin-like O-linked glycosylated protein, has recently been identified in human vitreous humour. PRG4 is known to act synergistically with hyaluronan (HA) in synovial fluids, where it functions as a boundary lubricant, contributes to viscosity, and more. Studying PRG4 in this newly discovered site might advance knowledge about its rheological properties (specifically within the eye) and whether or not PRG4 is involved. The aim of this thesis was to analyze the presence, source and potential function of PRG4 in vitreous humour. The objectives were: 1) to quantify the PRG4 and HA (including its molecular weight (MW) distribution) content, 2) to immunolocalize PRG4 in the eye globe, and examine its local synthesis, and 3) to characterize the viscosity of vitreous humour samples with varying PRG4 and HA concentrations. Methods: Human vitreous humour samples were collected from post-mortem human patients. Alpha amplified luminescent proximity homogeneous assay (AlphaLISA) and enzyme-linked immunosorbent assay (ELISA) tests were performed to measure the concentration of PRG4 and HA, respectively. The MW distribution of HA was determined by agarose gel electrophoresis. Vitreous humour cells were cultured from porcine to examine the synthesis of PRG4. PRG4 was immunolocalized in eye globes from wild type (WT) and PRG4-KO mice. Steady shear viscosity was characterized for human vitreous humour using a rheometer, before and after adding exogenous recombinant human PRG4 (rh PRG4). Results: The mean (± standard deviation) concentration of PRG4 in human vitreous humour (N=36) was 25 ± 32 µg/mL. For HA, the mean concentration in human vitreous humour was 344±317 µg/mL, and 71% of the overall HA in vitreous humour was below 459 kDa. Increasing age was positively correlated with concentration of PRG4. PRG4 was synthesized by cultured porcine vitreous humour cells. Using wild type and PRG4-KO (knockout) mice, PRG4 was immunolocalized in the ciliary body, cornea, sclera, vitreous cortex, and some parts of the retina. Viscosities for human vitreous humour samples showed minor variations, and were negatively correlated with HA MW. Conclusion: This is the first investigation of both the presence and potential function of PRG4 in vitreous humour. The findings serve to expand understanding of vitreous humour composition, and how that might be a factor in ocular pathology and potential therapies.
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Abdulaziz, A. A. (2020). The Characterization of Proteoglycan 4 and Hyaluronan Composition in the Vitreous Humour and Their Potential Contributions to Steady Shear Viscosity (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.