Sohail, Roheena
(2024)
Investigation of the contribution of vascular endothelial growth factor and its splicing axis to joint inflammation and damage in arthritis.
PhD thesis, University of Nottingham.
Abstract
Vascular Endothelial Growth Factor-A (VEGF-A) is a key regulator for endothelial cell function, promoting vascular permeability, growth, and angiogenesis. There is an increase in synovial angiogenesis and upregulation of angiogenic growth factors such as VEGF-A during articular joint inflammation in inflammatory arthritis (IA), such as rheumatoid arthritis (RA). This is a major contributor to disease progression.
VEGF-A consists of two splice variant families with each family composed of 9 isoforms of varying length (from 111 – 206 amino acids). The two VEGF-A families, VEGF-Axxxa and VEGF-Axxxb (where xxx denotes the number of amino acids in the isoform) result from the selection of alternative proximal and distal splice sites in exon 8. Selection of the exon 8 proximal splice site results in transcription of pro-angiogenic VEGF-Axxxa and is controlled by the Serine/Arginine Rich Splicing Factor Kinase 1 (SRPK1). When SRPK1 is activated, for example by hypoxia, it phosphorylates Serine/Arginine Rich Splicing Factor 1 (SRSF1) resulting in its nuclear translocation, proximal splice site selection and VEGF-Axxxa production. The CDC-2-dual specificity like protein kinase CLK-1 also contributes to the full activation of SRSF1 and production of VEGF-Axxxa isoforms.
VEGF-Axxxb isoforms predominate in normal tissues with anti-angiogenic functions, whereas VEGF-Axxxa isoforms prevail in pathological angiogenic conditions such as inflammation and solid tumours. The binding of pro-angiogenic VEGF-Axxxa isoforms to endothelial VEGF receptor 2 (VEGFR2) is the main stimulatory signal for angiogenesis. As angiogenesis is a key feature of RA, VEGF-A and its receptors have been suggested as potential therapeutic targets for the condition.
This thesis investigated the effect of systemic inhibition, and endothelial cell-specific genetic knockdown of VEGFR2 on synovitis, cartilage integrity and the expression of two components of the VEGF-A alternative splicing axis, splicing kinase SRPK1 and splice factor SRSF1 in joints, using histology and immunohistochemistry in IA rodent models.
Systemic inhibition of VEGFR2 by PTK787 had no significant effects on inflamed tibiofemoral joint histology in the rat IA model. Inducible endothelial cell-specific genetic VEGFR2 knockdown in mice (VEGFR2ECKO) resulted in greater cartilage damage in inflamed mouse tibio-tarsal joints compared to inflamed mice with no VEGFR2ECKO. There was no effect of VEGFR2ECKO in un-inflamed mice. The greater cartilage damage in inflamed VEGFR2ECKO mice was associated with significantly reduced vascularity in the most superficial layer of subchondral bone (up to 100m) and in synovium. The loss of VEGFR2 in endothelial cells and reduced vascularity could result in increased levels of hypoxia and reduced nutrient supply in the joint, contributing to the increased cartilage damage.
SRPK1 and SRSF1 expression was detected in both synovium and cartilage in rodent joints. There were no significant changes in SRPK1 expression in rat or mouse IA models. SRSF1 activation, as measured by number of cells with SRSF1 nuclear localisation (NL), was increased in inflamed synovium in both mouse and rat IA models. The proportion of synovial cells with nuclear localised SRSF1 was not changed. The mouse and rat models therefore had some similarities to but did not recapitulate published observations in human RA synovium, in which proportions of cells with activated SRSF1 were significantly raised.
PTK787 inhibition of VEGFR2 had no significant effect on SRPK1 or SRSF1 expression or activation. SRSF1 NL was significantly lower in VEGFR2ECKO mice that showed increased cartilage damage and reduced vascularity. Synovial proliferation was still seen in the mouse joints. Endothelial VEGFR2 knock down would reduce angiogenesis and increase hypoxia as even if there is upregulation of VEGF-Axxxa expression this cannot exert a pro-angiogenic action. This would lead to greater hypoxia and increased cartilage damage. The reduced SRSF1 NL in synovium of inflamed VEGFR2ECKO mice suggests that genetic knockdown of endothelial VEGFR2 may also affect VEGF-Axxxa production through an, as yet, unknown mechanism.
The effect of novel inhibitors of the splicing kinases SRPK1 and CLK1 were determined in an in vitro model of chondrocyte hypertrophy. Exposure of chondrocytelike ATDC5 cells to ascorbic acid promotes differentiation (day 8-12) when they express collagen 2A (COL2A1), and transition to a hypertrophic phenotype by day 21 when they express collagen 10A but not COL2A1. Treatment of chondrocyte-like ATDC5 cells in vitro with novel SRPK1 or CLK1 inhibitors for 21 days had no effect on mRNA expression of either collagen 2A1 or 10A1. Despite this, the novel CLK1 inhibitor has very similar properties to the experimental drug lorecivivint, so it may still be worth pursuing this compound for effects on cartilage and / or inflammatory joint pain.
In conclusion, the work presented in this thesis shows that targeting VEGFR2 may not be an ideal therapy for IA due to the potential for reduced subchondral vascularity and increased cartilage damage. Human RA synovium has high levels of activated SRSF1, which is partially recapitulated in the rodent CFA IA models. Targeting VEGF-A alternative splicing mechanisms to switch splicing to VEGF-Axxxb isoforms could therefore be considered as a potential therapeutic target in IA. Further work is needed to fully understand how the different VEGF-A splice families contribute to cartilage maintenance in IA or chondrocyte hypertrophy. In addition, selective CLK inhibitors could also be considered as potential novel therapies in OA given their similarities to drugs in late phase clinical trials.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Donaldson, Lucy |
| Keywords: |
VEGF receptor 2; Synovitis; Cartilage integrity; SRPK1; SRSF1; Histology; Immunohistochemistry |
| Subjects: |
Q Science > QP Physiology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Biomedical Sciences |
| Item ID: |
78112 |
| Depositing User: |
Jacob, Mr Tim
|
| Date Deposited: |
16 Jul 2024 04:40 |
| Last Modified: |
16 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/78112 |
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