Background: VEGF-A is a key regulator of rheumatoid (RA) and osteoarthritis (OA). During articular inflammation in OA and RA there is increased synovial angiogenesis and upregulation of angiogenic growth factors such as VEGF-A. VEGF-A comprises two splice variant families, VEGF-A_xxxa and VEGF-A_xxxb (xxx represents the number of amino acids, from 121 to 206), resulting from alternative splice site selection in exon 8. This site selection is controlled by Serine/Arginine Rich Splicing Factor Kinase 1 (SRPK1), which phophorylates Serine/Arginine Rich Splicing Factor 1 (SRSF1), inducing it to translocate to the nucleus. In most normal tissues, VEGF-A_xxxb isoforms predominate, with anti-nociceptive and anti-angiogenic functions. I contrast, in pathological conditions such as inflammation and solid tumours, VEGF-A_xxxa isoforms predominate, with pro-nociceptive and pro-angiogenic functions. VEGF-A has been proposed as a therapeutic target in RA and OA. To date, there are no published data on the functionally distinct VEGF-A splice variants in either RA or OA.

Objectives: To determine the patterns of, and relationships between, VEGF-A, SRPK1, and SRSF1 expression and activation and synovial inflammation in human RA and OA.

Methods: The study was approved by the Nottingham Research Ethics Committee 1 (05/Q2403/24) and Derby Research Ethics Committee 1 (11/H0405/2). Tissues were selected from age- and sex-matched cases in the University of Nottingham joint tissue repository. Post-mortem (PM) samples of healthy knee synovium (n=14, no history of arthritis or knee pain in the 12 months prior to death, no significant articular or synovial pathology) and arthroplasty-derived synovium samples from OA (n=35) or RA (n=14) patients were compared. OA samples were selected to represent the variety of inflammation levels, from low to high grade (0-3, Haywood et al., 2003). 8um thick sections were stained for SRSF1, SRPK1, total VEGF-A and VEGF_xxxb via immunohistochemistry. Expression was estimated as fractional area, relative staining intensity (VEGF-A_xxxb), and SRSF1 activation quantified by the degree of nuclear localisation. Statistical analyses were performed using Kruskal-Wallis followed by Dunn’s tests and Spearman’s rank correlations.

Results: SRPK1 expression was similar across all conditions. SRSF1 showed significantly higher expression in the OA tissue compared to PM (H(2)= 11.29, p=0.002; OA median=0.2, IQR(0.15, 0.28); PM median=0.09, IQR(0.07, 0.16)), and significantly higher nuclear localisation (indicating activation) in RA vs. OA, and in both RA and OA vs PM (H(2)=37.65, p<0.0001 RA cf. PM; p=0,007 OA cf. PM; RA median=89, IQR(83, 93); OA median=36.1, IQR(29, 42); PM median=19.8, IQR(14,21)). Nuclear SRSF1 was significantly correlated with inflammation score (r= 0.52, p<0.05). Total VEGF-A expression was significantly increased in RA compared to PM and OA (H(2)=23.3, p<0.001 RA cf. PM; RA median=0.4, IQR(0.37,0.59); PM median=0.18, IQR(0.15,0.2)) and was also correlated with the severity of inflammation (r=0.47 p<0.05). VEGF-A_xxxb showed no changed in expression in OA or RA, although VEGF-A_xxxb staining intensity was significant higher in RA samples, compared to controls (H(2)=7.2 p=0.02; RA median=2.3(1, 4); PM median=0.9 (0.7, 1.4)).

Conclusion: Increased levels of SRSF1 activation, and the association of total VEGF-A expression with inflammation score, support the hypothesis that there is activation of alternative splicing in inflamed synovium in RA and OA. Targeting this pathway could be a novel therapeutic strategy in OA & RA.

References: [1]HAYWOOD L., MCWILLIAMS D. F., PEARSON C. I., GILL S. E., GANESAN A., WILSON D. & WALSH D. A. 2003. Inflammation and angiogenesis in osteoarthritis. Arthritis Rheum, 48, 2173-7.

Disclosure of Interests: Dimitrios Amanitis: None declared, Seyed Shahtaheri: None declared, Daniel Daniel McWilliams: None declared, Nicholas Beazley-Long: None declared, David Walsh Grant/research support from: 2016: Investigator-led grant from Pfizer Ltd (ICRP) on Pain Phenotypes in RA; non-personal financial disclosure (payment to University)., Consultant of: DAW has undertaken paid consultancy to Pfizer Ltd, Eli Lilly and Company and GSK Consumer Healthcare., Paid instructor for: 2019: Consultancy to Love Productions; consultancy on programme design, contribution to programme content on self-management of chronic pain (payments to University)

2019: Consultancy to AbbVie Ltd; 13.06.19; presentation on RA pain at EULAR, Madrid, and webinar (payments to University).

2019: Consultancy to Eli Lilly and Company Ltd. 06.06.19 Centre for Collaborative Neuroscience, Windlesham, Surrey, UK (payment to University).

2019: Consultancy to Pfizer (payment to University).

2018: Consultancy to Pfizer. 07.12.18. USA. 1 day. Tanezumab (payment to University).

2018: Consultancy to Pfizer. 23.11.18. Manchester UK. 1 day. Tanezumab (payment to University).

2018: Consultancy to Pfizer. 1.11.18. Skype. 4h. Tanezumab (payment to University).

2018: Consultancy to GlaxoSmithKline Plc. 1 day. Pain in RA and anti-GM-CSF (payment to University).

2018: Consultancy to Pfizer Ltd; Presentation at OARSI; non-personal financial disclosure (payment to University)

2018: Consultancy to Pfizer Ltd; Patient preference study; non-personal financial disclosure (payment to University)

2017: Consultancy to Pfizer Ltd; personal financial disclosure

2017: Consultancy to Pfizer Ltd through Nottingham University; non-personal financial disclosure (payment to University).

2015: Consultancy to GSK Consumer Healthcare; personal financial disclosure., Speakers bureau: 2019: Irish Society of Rheumatology: speaker fees (personal pecuniary), Lucy Donaldson Shareholder of: LFD is a co-inventor on patents protecting alternative RNA splicing control and VEGF-A splice variants for therapeutic application in a number of different conditions. LFD is also a founder equity holder in, and consultant to, Exonate Ltd, and Emenda Therapeutics Ltd. Both companies have with a focus on development of alternative RNA splicing control for therapeutic application in a number of different conditions, including ophthalmology (www.exonate.com), analgesia and arthritis (www.emendatherapeutics.com)., Consultant of:

LFD is a co-inventor on patents protecting alternative RNA splicing control and VEGF-A splice variants for therapeutic application in a number of different conditions. LFD is also a founder equity holder in, and consultant to, Exonate Ltd, and Emenda Therapeutics Ltd. Both companies have with a focus on development of alternative RNA splicing control for therapeutic application in a number of different conditions, including ophthalmology (www.exonate.com), analgesia and arthritis (www.emendatherapeutics.com).