Session Type: Abstract Submissions (ACR)
Synovial fibroblasts (SF) are the most abundant resident stromal cells in the synovium. In rheumatoid arthritis (RA), SF expand and undergo phenotypic changes that contribute to the pathogenesis of chronic arthritis. Among these changes, expression of podoplanin, a membrane protein expressed by normal lymphatic endothelium and cancer cells, has been reported (Ekwall, Arthritis Res Ther 2011). Podoplanin is up-regulated on the invasive front of tumors and participates in tumor cell migration. The only known specific receptor for podoplanin is the platelet membrane signaling protein CLEC-2. We have analyzed the expression and potential functions of podoplanin in rheumatoid arthritis synovial fibroblasts (RASF).
Methods: Podoplanin expression was analyzed by immunohistochemistry (IHC), immunofluorecence labeling (IF), quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry in synovial tissue and SF cultures from RA and osteoarthritis (OA) patients and healthy donors. Podoplanin RASF expression was silenced with podoplanin specific and control siRNA lentiviral expression vectors. Transduced cells were used in RASF-cartilage co-culture experiment to measure cartilage glycosaminoglycan degradation products in supernatants (Blyscan Assay; Biocolor, Northern Ireland, UK). Matrigel invasion and in vitro wound healing assays were performed to evaluate migration capability of silenced fibroblasts. Finally, silenced RASF were co-cultured for 24h with human platelets prepared from healthy donors and RASF RNA was extracted to quantify IL-6, IL-8, MCP-1, MMP-1 and MMP-3 mRNA expression by qRT-PCR. Quantitative data from at least three independent experiments were analyzed by Mann-Whitney U-test or t-test as required and p-value<0.05 was considered significant.
Abundant podoplanin expression was detected by IHC in synovial lining and sublining fibroblasts in RA biopsies (n=40) whereas minimal or absent expression was observed in OA (n=15) or healthy synovium (n=6) respectively. Cultured SF displayed abundant podoplanin membrane expression irrespective of their source (RA, OA or healthy synovium). Treatment of cultured SF with TNF-α induced up-regulation of podoplanin mRNA and protein expression, as determined by qRT-PCR and flow cytometry. Podoplanin expression was effectively down-regulated by specific siRNA lentiviral transduction. Cell migration in matrigel and wound healing assays and ex vivo cartilage degradation were not modified in RASF transduced with podoplanin siRNA compared to RASF transduced with scrambled control siRNA. Co-culture of RASF with platelets induced a significant increase in IL-6 and IL-8 but no MCP-1, MMP-1 or MMP-3 mRNA expression in RASF. siRNA podoplanin silencing in RASF blocked the up-regulation of IL-8 but not IL-6 mRNA expression in response to platelet co-culture.
Conclusion: Our results confirm previous data on the up-regulation of podoplanin in RASF. Functional studies do not support a role for podoplanin on RASF migratory and invasive properties as reported in cancer cells. Podoplanin is potentially involved in the pro-inflammatory response that results from RASF/platelet interaction.
M. J. Del Rey,
J. D. Cañete,
J. L. Pablos,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/podoplanin-mediated-interaction-of-rheumatoid-arthritis-synovial-fibroblasts-with-platelets-modulates-il-8-expression/