Abstract
IntroductionThe present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.MethodsNatriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF4-23. Nitric oxide (NO) production, prostaglandin E2 (PGE2) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data.ResultsThe present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE2 release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF4-23 had the opposite effect and reduced NO and PGE2 release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE2 release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE2 release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression.ConclusionsMechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.
Highlights
The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs
There was a greater abundance of Npr2 expression in diseased cartilage and the number of positive cells significantly increased with age (P < 0.05) (Figure 1B), showing distinct cell membrane localisation (Figure 1B) and expression by western blot analysis
We examined the effect of inhibiting soluble guanylyl cyclase (sGC) with ODQ, as the nitric oxide (NO) and CNP pathways intersect in the intracellular signalling cascade and are both inducers of cyclic guanosine monophosphate (cGMP)
Summary
The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs. More effective intervention, augmented by early diagnosis and integrated biophysical therapies are needed. Progress has been slow due to the wide variety of experimental models that examine the effect of mechanical stimuli and chondroprotective agents on signal transduction pathways. Our understanding of the early mechanopathophysiology is poor, the way in which mechanical stimuli influence cell function and regulate matrix synthesis. This makes it difficult to identify reliable targets and design new therapies for OA treatment
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