Mechanism(s) by which pigment epithelium-derived factor regulate angiogenesis

Zhang, Yadan 2007. Mechanism(s) by which pigment epithelium-derived factor regulate angiogenesis. PhD Thesis, Cardiff University.

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Abstract

Pigment Epithelium-derived Factor (PEDF), a natural protein possessing both neuroprotective and anti-angiogenic properties, is a very unique and attractive candidate as a therapeutic agent in the management of pathological neovascular diseases, such as tumours, age-related macular degeneration (AMD) and diabetic retinopathy. While it is well-known that PEDF can exert powerful effects on various tissues and cells, the underlying mechanism of PEDF's action is not well understood. This study investigated the relationship between vascular endothelial growth factor (VEGF)/PEDF and VEGFR-1 /VEGFR-2 by exploring Presenilin-l (PS-l) dependent regulated intramembrane proteolysis (RIP). Work on this non-classical pathway was initiated by Cai et al., (2006) using in vitro models of bovine retinal microvascular endothelial cells (BRMECs). Current study used BRMECs and human retinal pigment epithelial (HRPE) cells. In this study, BRMECs and HRPE cells were isolated and cultured. BRMECs were used as an angiogenic cell type while HRPE cells were used as an angiogenic regulator cell type. The characteristics of endothelial and epithelial cells and the localisation of VEGFR-1, VEGFR-2 and PS in BRMECs and HRPE cells were determined using immunocytochemistry techniques. The effects of VEGF and PEDF on VEGFR-1, VEGFR-2 and PS were assessed using immunocytochemistry and Western blotting, y-secretase activity in BRMECs and HRPE cells treated with various growth factors were analysed using a y-secretase activity kit. The role of VEGF on the production of PEDF and the expression of VEGFR-1, VEGFR-2 and PS in HRPE cells was investigated at both the transcriptional and translational levels. The techniques, VEGF-small interfering ribonucleic acid (VEGF-siRNA), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and Enzyme-linked immunosorbent assay (ELISA) were used for the investigation. Results obtained from the project showed that PEDF had a regulatory role in the counterbalance of VEGFR-1 and VEGFR-2 expression in cultured BRMECs. PEDF upregulated y-secretase activity and PS-1 expression in BRMECs while VEGF acted as an antagonist of the effect of PEDF. In contrast, in HRPE cells, VEGF upregulated y-secretase activity and PEDF acted as an antagonist of the effect of VEGF. VEGF-siRNA induced a reduction of PEDF at both transcriptional and protein levels and a reduction of VEGFR-1 at the protein level. The effects of VEGF and PEDF on VEGFR-1 and VEGFR-2 may be cell type dependent. This study strengthens the view that PEDF can exert different regulatory effects on the same molecule (s) in different cell types. PEDF acts either antagonistically to VEGF or synergistically dependent upon the target molecule. Deciphering the cellular and molecular mechanisms underlying these interactions will not only contribute to our understanding of PEDF's action but also provide the foundation to maximise the therapeutic potential of this protein.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Optometry and Vision Sciences
Subjects:	R Medicine > RE Ophthalmology
ISBN:	9781303209932
Date of First Compliant Deposit:	30 March 2016
Last Modified:	10 Oct 2017 15:24
URI:	https://orca.cardiff.ac.uk/id/eprint/54652

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