Targeting vascular endothelial growth factor-A in an inflammatory model of arthritis affects the development of mechanical secondary hyperalgesia

Rheumatoid arthritis (RA) affects ~1% of the UK population, is severely painful and significantly disrupts the quality of life for those affected. Joint swelling, inflammation, damage and pain, the latter being extremely debilitating, characterize the disease. Current treatments target pro-inflammatory cytokines including tumour necrosis factor-α and have significantly improved RA therapy yetstillonly ~50% of patients respond to therapy. Vascular endothelial growth factor-Axxxa (VEGF-Axxxa) isoforms are pro-inflammatory, pro-angiogenic and pro-nociceptive and VEGF-A protein is upregulated in the serum and synovial fluid of RA patients.

Aim: test the hypothesis that targeting VEGF-A, either systemically or locally, will affect pain behaviours in a model of inflammatory arthritis and investigate the underlying mechanism.

Methods: all animal procedures met the requirements dictated by the Animals (Scientific Procedures) Act 1986 / ASPA Amendment Regulations 2012. VEGF-A receptor inhibitors (DC-101 12mg/kg; PTK787 15mg/kg; VEGF-A165b 20μg/kg) were administered systemically (intraperitoneal) or locally (intra-articular) to complete Freund’s adjuvant (CFA)-treated rats (100μg CFA, intra-articular, under brief isoflurane anaesthesia). In this model CFA causes joint swelling, inflammation and angiogenesis with a concomitant development of pain-associated behaviours: a shift in weight bearing and the development of mechanical secondary hyperalgesia through central sensitization, a process that underpins many states of chronic pain. The percentage of weight borne on theipsilateral hind paw to CFA was measured using an in capacitance tester (Linton). Mechanical stimulus withdrawal thresholds were measured by scoring withdrawals following application of von Frey monofilaments (1, 2, 4, 8, 15, 26, 60g) to the plantar surface of the hind paws. Immunofluorescence was performed on PFA perfuse-fixed 20μm spinal cord sections to investigate the level of microgliosis, astrocytic activation and blood vessel activation in the dorsal horn. The following primary antibodies were added in PBS containing 10%FBS, 5%BSA & 0.2% TritonX-100: anti-CD11b (OX42, Bio-Rad MCA275, 2μg/mL), anti-glial fibrillary acidic protein (GFAP, Abcam, ab7260 antiserum used 1 in 1000), vascular cellular adhesion molecule-1(VCAM-1, Bio-Rad MCA4633, 2μg/mL) and intercellular cellular adhesion molecule-1 (ICAM-1, MCA532, 1μg/mL). Biotinylated secondary antibodies (Thermo Scientific Pierce) and AlexaFluor-conjugated streptavidin (Life Technologies) were used to detect and amplify the signal. Species and concentration-matched IgG was used as a negative control.

Results: systemic, but not local, delivery of VEGF-A receptor inhibitors significantly inhibited the development of secondary hyperalgesia, with no significant effecton the shift in weight bearing or joint swelling. Day 7 mechanical thresholds: control, 4.71±1.0g; DC-101, 13.9±2.1g**; PTK787, 13.9±2.0g***; VEGF-A165b 10.5±1.3g*; mean±SEM, 2-way ANOVA+ multiple comparisons; *p<0.05, **p<0.01***p<0.001. In the ipsilateral dorsal horn of the spinal cord, mechanical stimulus withdrawal threshold significantly correlated (Spearman) with the intensity of OX42 (r=-0.567, p=0.012),the number of VCAM-1-positive cells(r=-0.614, p= 0.007) and ICAM-1-positive vessels (r=-0.538, p=0.021). Interestingly the VEGFR2 antibody DC-101 significantly reduced the number of VCAM-1-positive cells in the ipsilateral dorsal horn compared with control, p<0.05, 1-way ANOVA+ Dunnett’s multiple comparisons test.

Conclusion: VEGF-A-mediated signalling contributes to development of secondary hyperalgesia by promoting central sensitization. In our model of CFA-induced inflammatory monoarthritis, microgliosis, and astrocytic and blood vessel activation in the dorsal horn of the spinal cord correlated with pain behaviour. Targeting VEGF-A may be able to alleviate pain experienced by patients of inflammatory conditions including RA. Funded by Arthritis Research UK