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Malhi, Naseeb (2021) Use of SRPK1 inhibitors for the treatment of Diabetic Retinopathy. PhD thesis, University of Nottingham.

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Abstract

Microvascular damage results from ischaemia-driven production of pro- angiogenic vascular endothelial growth factor (VEGF). Proximally spliced VEGF is upregulated in the ischemic diabetic retina and has been implicated as the principal driver of the pathological growth and leakage of blood vessels during diabetic retinopathy (DR). Serine-Arginine Rich protein kinase-1 (SRPK1) regulates splicing of VEGF, and inhibition of this kinase with small molecular weight inhibitors has been shown to inhibit choroidal neovascularization (CNV) in mice by decreasing pro-angiogenic and increasing anti-angiogenic VEGF isoforms. These isoforms have previously been described to inhibit increased vascular permeability with protective effects against DR-induced pathology. SRPK1 inhibitors such as SPHINX31 may therefore switch splicing in DR and prevent increased vascular permeability.

Retinal pigment epithelial cells were exposed to hyperglycaemia (HG) and hypoxia (Hx) and treated with SPHINX31. SRSF1 localisation in the nuclear speckles, SRPK1 activity and monolayer permeability were assessed by immunofluorescence and Electrical Cell Impedance Sensing. In a rodent model of type 1 diabetes fluorescein fundus angiography (FFA) and optical coherence tomography (OCT) was performed weekly from day 0 to 28. Animals received twice daily topical eye drops with eye formulation control buffer or SPHINX31. On day 1 animals received a single dose of streptozotocin to induce type I diabetes. FFA was quantified using ImageJ; the intensity of sodium fluorescein in the retinal interstitial area and the retinal vessels were measured and the permeability assessed from this relationship. An FFA and OCT time course was used to determine an estimate of permeability and retinal thickness. Retina petals were stained with IB4 and for junctional proteins to deduce vascular density.

HG and Hx induced a significant increase (p<0.05) in SRSF1 nuclear localisation, which was blocked by SPHINX31. HG induced a release of SRSF1 from the nuclear speckles (p=0.002). Inhibition of SRPK1 decreased RPE monolayer permeability (p<0.05). The increase in retinal permeability on days 14-28 seen in the diabetic eye formulation only control cohort (n=8) was stabilised following topical treatment of diabetic animals with SPHINX31 (n=9) for 28 days (p<0.0001). Mean retinal thickness increased in diabetes (p<0.05) and this increase appeared to be blocked following SPHINX31 treatment.

SPHINX31 protected the retinal barrier from hyperglycaemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. SPHINX31 may therefore be a potential topical therapeutic for DR.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Bates, David Arkill, Kenton
Keywords:	Diabetic retinopathy; Serine-Arginine Rich protein kinase-1; SRPK1 inhibitors; Vascular permeability
Subjects:	W Medicine and related subjects (NLM Classification) > WK Endocrine system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	65527
Depositing User:	Malhi, Naseeb
Date Deposited:	04 Aug 2021 04:42
Last Modified:	04 Aug 2021 04:42
URI:	https://eprints.nottingham.ac.uk/id/eprint/65527

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