Treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine restores endothelial function in rat adjuvant-induced arthritis

Abstract

Introduction: Endothelial dysfunction (ED) participates to atherogenesis associated to rheumatoid arthritis. We recently reported increased arginase activity/expression in vessels from adjuvant-induced arthritis (AIA) rats. In the present study, we investigated the effects of a curative treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine (nor-NOHA) on vascular dysfunction in AIA rats.

Methods: AIA rats were treated with nor-NOHA (40 mg/kg/d, ip) for 21 days after the onset of arthritis. A group of untreated AIA rats and a group of healthy rats served as controls. ED was assessed by the vasodilatory effect of acetylcholine (Ach) on aortic rings. The role of superoxide anions, prostanoids, endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide synthase (NOS) pathway was studied. Plasma levels of IL-6 and vascular endothelial growth factor (VEGF) were determined by ELISA kits. Arthritis severity was estimated by a clinical, radiological and histological analysis.

Results: Nor-NOHA treatment fully restored the aortic response to Ach to that of healthy controls. The results showed that this beneficial effect is mediated by an increase in NOS activity and EDHF and reduced superoxide anion production as well as a decrease in the activity of cyclooxygenase (COX)-2, thromboxane and prostacyclins synthases. In addition, nor-NOHA decreased IL-6 and VEGF plasma levels in AIA rats. By contrast, the treatment did not modify arthritis severity in AIA rats.

Conclusions: The treatment with an arginase inhibitor has a potent effect on ED in AIA independently of the severity of the disease. Our results suggest that this new pharmacological approach has the potential as a novel add-on therapy in the treatment of RA.

Figure 1

Radiographs and histological aspect of hind limbs at Day 21 after the onset of arthritis. Experiments were performed in controls rats (A, D), AIA rats (B, E) and nor-NOHA treated rats (C, F). On radiographs, both AIA and treated AIA rats exhibited a thickening of the soft tissues of the ankle and foot as well as a joint destruction and periosteal tissue neoformation. Histological slices were stained with HES (enlargement × 20). A severe joint destruction associated to synovial proliferation was present in AIA and treated AIA rats.

Figure 2

Vascular reactivity to vasodilators and vasoconstrictive agents. Experiments were performed on thoracic aortic rings from control rats, AIA rats and nor-NOHA AIA rats 21 days after the onset of arthritis. A, Concentration - response curve for Ach in endothelium-intact rings preconstricted with NE 3 × 10^-7 moles/liter. B, Concentration - response curve for SNP in endothelium-denuded rings preconstricted with NE 3 × 10^-7 moles/liter. C, Concentration - response curve for NE in endothelium-denuded rings, D, Concentration - response curve for ANG-II in endothelium-denuded rings, E, Concentration - response curve for ET-1 in endothelium-denuded rings. Values are the mean ± SEM from 8 to 12 rings. * = P < 0.05. KKCl = Krebs potassium chloride solution.

Figure 3

Effect of L-NAME and the combination of apamin/charybdotoxin on vasodilation response to Ach. Experiments were performed on aortic rings isolated from control rats (A, D), AIA rats (B, E) and nor-NOHA AIA rats (C, F) 21 days after the onset of arthritis. Cumulative concentration-responses curves with Ach were obtained after a 60-minute incubation period with L-NAME at 10^-4 moles/liter (A, B, C) or with apamin-charybdotoxin at 10^-7 moles/liter each (D, E, F). Values are the mean ± SEM from 8 to 12 rings. * = P < 0.05, *** = P < 0.001.

Figure 4

Effect of indometacin, NS398, furegrelate and tranylcypromin on vasodilation response to Ach. Cumulative concentration curves were obtained in aortic rings isolated from AIA, nor-NOHA AIA and control rats 21 days after the onset of arthritis. Cumulative concentration curves with Ach were obtained after a 60-minute incubation period with indometacin at 10^-5 moles/liter or with NS398 at 10^-4 moles/liter in controls (A), AIA (C) and nor-NOHA AIA (E) or with furegrelate at 10^-6 moles/liter or with tranylcypromine at 10^-5 moles/liter in controls (B), AIA (D) and nor-NOHA AIA (F). Values are the mean ± SEM from 8 to 12 aortic rings. * = P <0.05.

Figure 5

Effect of Tempol and apocynin on vasodilation response to ACh. Experiments were performed on aortic rings isolated from control rats (A, B), AIA rats (C, D) and nor-NOHA AIA rats (E, F) 21 days after the onset of arthritis. Cumulative concentration-responses curves with Ach were obtained after a 60-minute incubation period with Tempol at 10^-4 moles/liter (A, C, E) or with apocynin at 3 × 10^-4 moles/liter (B, D, F). Values are the mean ± SEM from 8 to 12 rings. ** = P < 0.01.