Amanitis, Dimitrios
(2022)
Peripheral contributions to the development and maintenance of inflammation and pain in arthritis.
PhD thesis, University of Nottingham.
Abstract
Vascular endothelial growth factor A (VEGF-A) is a key regulator of vascular growth, permeability, and neuronal function. During articular inflammation in osteoarthritis (OA) and rheumatoid arthritis (RA), there is increased synovial angiogenesis and upregulation of angiogenic growth factors such as VEGF-A. VEGF-A comprises of two splice variant families, VEGF-Axxxa and VEGF-Axxxb (xxx represents the number of amino acids, from 121 to 206), resulting from alternative splice site selection in exon 8. Distal site selection and VEGF-Axxxa expression is controlled by Serine/Arginine Rich Splicing Factor Kinase 1 (SRPK1), which phosphorylates Serine/Arginine Rich Splicing Factor 1 (SRSF1), inducing its translocation to the nucleus. In most normal tissues, VEGF-Axxxb isoforms predominate, with anti-nociceptive and anti-angiogenic functions. In contrast, in pathological conditions such as inflammation and solid tumours SRPK1/SRSF1 activation causes VEGF-Axxxa isoforms to predominate, exerting pro-nociceptive and pro-angiogenic actions. VEGF-A has been proposed as a therapeutic target in OA. To date, a relation between VEGF-A and pain in OA has been reported, but there are no published data on the functionally distinct VEGF-A splice variants inflammation and pain in human OA and RA.
This thesis examines the relationships between the expression of VEGF-A splicing isoforms and the components of the VEGF-A splicing axis, inflammation and pain in human sex, and macroscopic chondropathy-matched OA and RA synovial samples from total knee replacement and post-mortem donors. The expression and regulation of the VEGF-A splicing axis was examined by immunohistochemical staining for activated splicing factor SRSF1, splicing kinase SRPK1, total VEGF-A and the two families of VEGF-A splicing isoforms VEGF-Axxxa and VEGF-Axxxb.
Protein expression was measured as the fractional area of staining (VEGF-A and isoforms, SRSF1, SRPK1 and Dyrk1A. SRSF1 activation was measured by the degree of nuclear localisation of SRSF1 compared to the total cell numbers in superficial synovium.
According to principal component analysis structural abnormalities such as synovial thickening significantly contribute to the VEGF-Axxxb. Similarly, synovitis was positively correlated with the levels of SRSF1 and VEGF-A, but no alterations were documented regarding the two isoforms in relation to the synovitis. Expression of the related splicing kinase Dyrk1A, implicated in RA, was also positively related to the degree of inflammation. 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. OA,PM; ;RA median=0.4, IQR(0.37,0.59); OA median=0.24, IQR(0.19,0.33);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-Axxxb showed no change in expression in OA or RA, although VEGF-Axxxb staining intensity was significantly 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).
Similarly, symptomatic OA was associated with significantly increased SRPK1, SRSF1 and VEGF-A expression, while the VEGF-Axxxb isoform was significantly reduced. 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. OA,PM; ;RA median=0.4, IQR(0.37,0.59); OA median=0.24, IQR(0.19,0.33);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-Axxxb showed no change in expression in OA or RA, although VEGF-Axxxb staining intensity was significantly 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)).
Cultures of human primary fibroblast-like synoviocytes (FLS) were stimulated with tumour necrosis factor-α, and the effects on SRPK1, SRSF1 and VEGF-A expression were determined. An in vitro model of synovial inflammation, using fluorescently labelled THP1 monocytes and was used to determine the effect of VEGF-A, VEGF receptor tyrosine kinase inhibitors, and SRPK1 inhibition on monocyte adherence to an FLS monolayer.
Alteration of splicing kinases CLK1-4 and DYRK1a with T-025 was able to significantly increase SRSF1 mRNA expression, and I also confirmed the presence of VEGF-A mRNA in HFLS, however, no splice variants could be detected.
These results indicate that the VEGF-A expression and splicing axis is altered in relation to both inflammation and pain, but in distinct ways. I present evidence that inhibition of VEGF-A splicing controls could affect both inflammation, such as in OA flares, and pain in OA.
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