Background:We have previously shown that decreased expression of the deacetylase sirtuin-1 (SIRT1) contributes to the proliferative, activated and proangiogenic profile of endothelial cells (EC) in rheumatoid arthritis (RA) (1). The matricellular protein CCN1, characterized by proangiogenic and immunomodulatory properties, may be directly implicated in these processes, since its expression is negatively regulated by SIRT1 (1).Objectives:To study the implication of CCN1 in RA pathogenesis.Methods:CCN1 expression was assessed in ECs (25 RA and 10 controls) by quantitative RT-PCR, western blot and ELISA, in the synovial tissue (5 RA and 5 controls) by immunohistochemistry and immunofluorescence, and in the serum (205 RA and 20 controls) by ELISA. Invalidation of CCN1 in RA ECs was achieved through the use of shRNA and neutralizing monoclonal antibodies. The functional consequences of CCN1 invalidation in RA ECs were studied i) in vitro by the analysis of proliferation (cell impedance), tube formation in Matrigel and migration in Boyden chambers; and ii) in vivo in the murine model of tumor neoangiogenesis.Results:CCN1 mRNA and protein expression were increased by 1.72- (p = 0.012) and 7.2-fold (p=0.008) in RA ECs compared to controls, respectively. CCN1 concentrations were significantly increased in RA EC culture supernatants (930±153 vs. 359±199 pg/mL, p=0.007). CCN1 was overexpressed in the synovial tissue of RA patients (Figure 1A) and confocal microscopy analyses revealed a prominent CCN1 expression in the vascular endothelium (CD31 +) and T cells (CD3 +) (Figure 1B).In vitro, recombinant TNF-α and IL-17 induced the mRNA and protein expression of CCN1 in RA ECs. CCN1 invalidation was associated with reduced proliferative capacities, delayed capillary tube formation and decreased migration of RA ECs (Figure 1E). In vivo, subcutaneous transplantation of CT26 tumor cells combined with RA ECs transfected with CCN1 shRNA to CB17 SCID mice was associated with a 51% reduction in tumor volume (p=0.008) and a 27% reduction in tumoral vascular density (p=0.032) compared with mice transplanted with MOCK transfected RA-ECs (Figure 1F).Serum concentrations of CCN1 were significantly reduced in the serum of RA patients compared to controls (233±118 vs. 279±75 pg/mL, p=0.045) (Figure 1C). However, serum CCN1 concentrations were significantly higher in the presence of bone erosions (253±139 vs. 202±7 pg/mL, p=0.002) (Figure 1D) and correlated with radiographic Larsen score (r=0.3, p=0.001) and HAQ (r=0.25, p=0.012).Conclusion:CCN1 is overexpressed in ECs and the synovial tissue of patients with RA. CCN1 also regulate the functional properties of RA ECs and their angiogenic potential in vivo. CCN1 could represent a new therapeutic target, which is being evaluated in experimental models of erosive arthritis.CCN1 may be a reliable biomarker of structural damages given the association between its serum concentrations and the extent of radiographic lesions. The performance of CCN1 serum levels to predict structural progression is under investigation.References:[1]Leblond et a, Ann Rheum Dis 2020.Figure 1.Implication of CCN1 in the pathogenesis of rheumatoid arthritis (RA). A, Representative immunohistochemistry staining for CCN1. B, Representative confocal microscopy analyses. C-D, CCN1 serum concentrations; statistical test: Student t test, ** p<0.01. E, Representative images of RA endothelial cell (EC) migration; Y-axis shows the number of migrated cells, statistical test: Wilcoxon test, * p<0.05. F, Representative subcutaneous tumors, Y-axis shows the fluorescence area in %, statistical test: Wilcoxon test, * p<0.05.Disclosure of Interests:None declared
Can Metabolic Pathways Be Therapeutic Targets in Rheumatoid Arthritis?
