Identification of extracellular matrix-based targets for the prevention of age-related macular degeneration (AMD), a laboratory medicine diagnostic based approach

Algwairi, O 2017, Identification of extracellular matrix-based targets for the prevention of age-related macular degeneration (AMD), a laboratory medicine diagnostic based approach, Doctor of Philosophy (PhD), Health and Biomedical Sciences, RMIT University.

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Collection: Theses

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Title Identification of extracellular matrix-based targets for the prevention of age-related macular degeneration (AMD), a laboratory medicine diagnostic based approach
Author(s) Algwairi, O
Year 2017
Abstract Age related macular degeneration (AMD) is a major cause of blindness in human beings. AMD negatively impacts on the quality of life of patients and has severe economic implications because of impaired or loss of sight. Globally, eye diseases cost the health system about three trillion USD with eye diseases in Australia costing the health system 9.85 billion AUD in 2004. Macular degeneration diseases were estimated to cost 5 billion AUD in 2010. In Australia, at the age of 50 and over, one in seven people are affected by AMD due to the influence of risk factors including ageing, smoking, and family history. There are two stages of AMD, early and late. In the early stage, there are changes in retinal endothelial and retinal epithelial cells (RPE) which lead to the thickening of Bruch’s membrane (BM) and a loss of central vision. Understanding the processes and mechanisms involved in the regulation of PG expression is critical to understanding the various functions of endothelial and epithelial cells. BM constitutes the boundary between retina and choroid and the thickening of BM is associated with the deposition of proteoglycans (PGs) and glycosaminoglycan (GAG) in the tissue. Moreover GAG can bind to lipids and form hyperelongated GAG. PGs with elongated GAG are long molecules that if too large to pass through BM, can result in accumulation of PG and elongated GAG and may start to form drusen.

The pathophysiology underlying the early AMD is not fully understood and there are currently no therapies targeting this stage. Early AMD is more prevalent, most likely representing the chronological progression of the disease. Therefore, the aim of this study was to investigate the role of selected growth factors on PG synthesis and GAG elongation in retinal endothelial and epithelial cells assessing their potential impacts on AMD.

The results obtained from this study showed that TGFβ, thrombin, PDGF and IGF dose-dependently stimulated the retinal endothelial cells to synthesize PGs with elongated GAG chains. However, VEGF did not stimulate PG synthesis nor GAG elongation which was surprising as anti VEGF is widely used in the treatment of late AMD. This study therefore investigated the effect of VEGF on both the phosphorylation of the poly functional ERK and ubiquitous serine/threonine protein kinase AKT. There are several pathways involving ERK to GAG hyperelongation which are activated by VEGF.

Investigations of the effects of these growth factors on the genes involved in mRNA expression of PGs synthesis and GAG elongation enzyme XT-1, ChGn-2, ChPF, ChSy-1, C4ST-1 and C4ST- showed that TGFβ, thrombin and PDGF treatment stimulated the endothelial cells to express the mRNA of enzymes required to synthesize elongated GAGs. These findings supported the study hypothesis; growth factors affect PG synthesis and GAG elongation and consequently may impact early changes in AMD and alter ECM composition of RF/6A cells.

The observed effect of TGFβ, thrombin, PDGF, IGF and VEGF on RPE was unexpected as none of these growth factors (except thrombin) stimulated the synthesis of PGs. Indeed there was no lipid binding PGs secreted by RPE cells. Thrombin treatment generated a PG band approximately 260-340 kDa on SDS-PAGE gel. This band was subjected to further investigations to determine its release mechanism. Through a series of experiments, it was determined that thrombin treatment resulted in cell surface PG shedding via thrombin proteolytic activity rather than a PAR-1 mediated shedding process. The cell surface shed PG could be playing vital roles in binding growth factors, influencing growth factor activities and transportation. The shed PGs could be of heparin sulfate (HS) which are highly sulfated soluble molecules that kept their HS moiety. They would be very reactive with a high potential of changing the extracellular matrix and consequently impacting on AMD.

The roles of the growth factors were evaluated especially with regards to causing alterations in ECM composition. Changes in ECM composition have direct impact on cellular metabolism and waste accumulation which could lead to morphological changes to retinal endothelial and epithelial cells. These morphological alterations cause the gradual thickening BM and accumulation of sulfated PGs which at a certain point, could trigger the secretion of growth factors. Associated with this secretion stage (deemed to be the late disease change) was the breakage of retinal blood barrier and exposure of cell surface PGs to the direct effect of thrombin. This would result in excessive shedding of cell surface PGs and the accumulation of shed PGs.

Growth factors can regulate PGs synthesis and GAG elongation in the retinal endothelial cells, could directly affect waste accumulation and alter ECM composition causing BM thickening in early stage AMD. In the retinal epithelial cells, thrombin was observed to play a crucial role in cell surface PGs shedding which would allow the disease to progress to the late stage.

Future research should explore the role of growth factors in regulating PG metabolism in the retina with an established role of GAG elongation and lipid binding. Our interesting finding of PG shedding requires investigation of the structure and role of shed PGs in AMD.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health and Biomedical Sciences
Subjects Clinical Chemistry (diagnostics)
Keyword(s) Age related macular degeneration (AMD)
Bruch’s membrane (BM)
retinal epithelial cells (RPE)
proteoglycans (PGs)
glycosaminoglycan (GAG)
retinal endothelial cells
heparin sulfate (HS)
PG shedding
extracellular matrix (ECM)
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Created: Thu, 21 Dec 2017, 09:52:29 EST by Denise Paciocco
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