RESUMO
Biological therapies such as tumor necrosis factor-alpha inhibitors have advanced the treatment of rheumatoid arthritis, but one-third of patients do not respond to such therapy. Furthermore, these inhibitors are now usually administered in combination with conventional disease-modifying antirheumatic drugs, suggesting they have not achieved their early promise. This study investigates a novel therapeutic target, proteinase-activated receptor (PAR)-2, in joint inflammation. Intra-articular carrageenan/kaolin (C/K) injection in mice resulted in joint swelling that was associated with synovial PAR2 up-regulation. Inhibiting receptor up-regulation using small interfering RNA technology, as confirmed by immunoblotting, substantially reduced the inflammatory response in the joint. Serine proteinase-induced joint swelling was mediated primarily via PAR2 activation, since the response to exogenous application of trypsin and tryptase was absent in PAR2 knockout mice. Furthermore, serine proteinase inhibitors were effective anti-inflammatory agents in this model. Disrupting proteolytic activation of PAR2 using antiserum (B5) directed to the receptor cleavage/activation site also attenuated C/K-induced inflammation, as did the similarly targeted PAR2 monoclonal antibody SAM-11. Finally, we report the activity of a novel small molecule PAR2 antagonist, N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068), that dose dependently attenuated joint inflammation. Our findings represent a major advance in collectively identifying PAR2 as a novel target for the future treatment of arthritis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite/tratamento farmacológico , Receptor PAR-2/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Piperazinas/farmacologia , RNA Interferente Pequeno/farmacologiaRESUMO
Using physiological, pharmacological, and gene disruption approaches, we demonstrate that proteinase-activated receptor-2 (PAR-2) plays a pivotal role in mediating chronic inflammation. Using an adjuvant monoarthritis model of chronic inflammation, joint swelling was substantially inhibited in PAR-2-deficient mice, being reduced by more than fourfold compared with wild-type mice, with virtually no histological evidence of joint damage. Mice heterozygous for PAR-2 gene disruption showed an intermediate phenotype. PAR-2 expression, normally limited to endothelial cells in small arterioles, was substantially upregulated 2 weeks after induction of inflammation, both in synovium and in other periarticular tissues. PAR-2 agonists showed potent proinflammatory effects as intra-articular injection of ASKH95, a novel synthetic PAR-2 agonist, induced prolonged joint swelling and synovial hyperemia. Given the absence of the chronic inflammatory response in the PAR-2-deficient mice, our findings demonstrate a key role for PAR-2 in mediating chronic inflammation, thereby identifying a novel and important therapeutic target for the management of chronic inflammatory diseases such as rheumatoid arthritis.
Assuntos
Artrite/metabolismo , Receptores de Trombina/biossíntese , Receptores de Trombina/fisiologia , Regulação para Cima , Alelos , Animais , Cartilagem/lesões , Endotélio/metabolismo , Éxons , Fêmur/lesões , Vetores Genéticos , Heterozigoto , Hibridização In Situ , Inflamação , Camundongos , Modelos Químicos , Modelos Genéticos , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Fenótipo , Receptor PAR-2 , Receptores de Trombina/agonistas , Recombinação Genética , Fatores de TempoRESUMO
The role of different isoforms of cyclo-oxygenase (COX) in mediating the acute (0-6 h) and late (24 h) phases of inflammation was investigated in the rat knee joint following intra-articular injection of carrageenan. The hyperaemic response was assessed transcutaneously using laser Doppler imaging (LDI). Samples were taken at corresponding time points for detection of synovial COX-1, COX-2 and inducible nitric oxide synthase (iNOS) mRNA, and measurement of urinary prostaglandin (PG) and nitric oxide metabolites (NO(x)). A non-selective COX inhibitor (indomethacin, 15 mg kg(-1) I.P.), a selective COX-2 inhibitor (SC-236, 16.8 mg kg(-1) I.P.) or vehicle were administered 1 h prior to carrageenan in the acute phase study. LDI scans were taken hourly for 4 h post-induction. Inflammatory hyperaemia in the vehicle group was attenuated in the indomethacin- (P < 0.001, two-way ANOVA) and SC-236-treated groups (P < 0.0001), with no difference between these treatments. At 24 h, I.V. infusion of indomethacin (0.1 mg min(-1)), increased vascular resistance (24 +/- 7.1 %; P < 0.05) compared to vehicle infusion, whereas SC-236 (0.11 mg min(-1)) did not. Resistance changes to indomethacin also differed from SC-236 (P < 0.05). Knee joint diameter progressively increased over 24 h (P < 0.0001, one-way ANOVA). Urinary PG levels increased by 6 h (P < 0.05), but returned to baseline by 24 h. COX-1 mRNA was detectable at all time points; COX-2 mRNA only at 3 h. Urinary NO(x) levels increased progressively over 24 h (P < 0.05), paralleled by induction of iNOS in the 3 and 24 h samples. Prostaglandin production via COX-2 appears to mediate the development of acute inflammatory hyperaemia, but nitrergic mechanisms may supervene subsequently. COX-1 but not COX-2 contributes to the maintenance of basal blood flow in the hyperaemic joint at 24 h.