NKX2-5 expression. TEER measurements evaluated endothelial barrier function. Pharmacological inhibition was performed to determine the pathways that lead to NKX2-5 activation. Casein kinase II (CKII)- inhibited mice in a chronic hypoxia mouse model of PAH was used to assess EndoMT and right ventricular systolic pressure (RVSP). Results: Immunofluorescence showed a strong expression of NKX2-5 in the endothelium of SSc-PAH human lungs (p<0.0001). Western blot analysis demonstrated a 5.3-fold downregulation of CD31 (p<0.001) and of p-YAP/YAP (2.3-fold, p < 0.05), and increased production of NKX2-5 (5.6-fold, p<0.0001), Procollagen I (12-fold, p¼0.0009), and of p-STAT3/STAT3 (3.8-fold, p<0.05) after 5 days of cytokine stimulation on HPAECs. Relative mRNA expression has shown a 3-fold gene downregulation of CD31 (p ¼ 0.0002) and a 2.3-fold reduction of VE- Cadherin (p¼0.0008) in EndoMT, whereas gene expression of COL1a2 (8.5-fold, p<0.0001), and NKX2-5 (1.5-fold, p¼0.003) were upregu- lated. Immunofluorescence has revealed a decreased VE-Cadherin expression concomitant with upregulation of NKX2-5 in EndoMT cells. Forced expression of NKX2-5 downregulated endothelial markers, whereas these markers were preserved in shNKX2.5 HPAECs. Endothelial barrier function was impaired and proliferation rate was increased in cells forcing expression of NKX2-5. Inhibition of PI3K, ERK5, ALK5 and CKII decreased NKX2-5 protein expression. CKII- inhibited mice normalised RVSP and EndoMT occurrence. Conclusion: Activated HPAECs undergoing EndoMT express NKX2-5 in vitro and in vivo, via mediation of CKII, TGF-b, ERK5 and PI3K signalling. NKX2-5 downregulates adherence junctional proteins via YAP and p-STAT3, disrupting the endothelial barrier function and increasing the proliferation rate of HPAECs. This study highlights a new pathway associated to EndoMT and to SSc-PAH. Disclosures: J. Santos Cade: None. I. Papaioannou: None. Y. Siddiqui: None. A. Holmes: None. M. Loizidou: None. C. Denton: None. D. Abraham: None. M. Ponticos: None. 014 APPA INHIBITS NEUTROPHIL PRO-INFLAMMATORY FUNCTIONS WITHOUT IMPAIRING HOST DEFENCE: IS THIS A POTENTIAL NEW THERAPY FOR ARTHRITIS? Andrew L. Cross 1 , Jennifer J. Hawkes 1 , Helen L. Wright 1 , Robert J. Moots 1 and Steven W. Edwards 2 1 Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UNITED KINGDOM, and 2 Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UNITED KINGDOM Background: APPA (a fixed dose combination of synthetic apocynin, AP and paeonol, PA) has shown efficacy in several animal models of osteoarthritis (OA) and is currently in Phase I evaluation for the treatment of OA. Its efficacy is thought to be mediated through synergistic effects of constituent components, AP and PA, on the regulation of NF-kB transcription factor activation. NF-kB plays a key role in both constitutive and inducible gene expression, particularly in immune cells, and is therefore an attractive therapeutic target. Neutrophils are key to innate host defence via phagocytosis of pathogens and activation of granule enzymes and reactive oxygen species (ROS). Neutrophil function during inflammation is regulated by the activities of a number of cytokines, including TNFa and GM-CSF. Neutrophils are potent producers of ROS (activators of NF-kB) via NADPH oxidase (NOX2) and many inflammatory functions, including delayed apoptosis, are regulated by NF-kB activation. The purpose of this study was to investigate the effects of APPA on key neutrophil functions and explore its potential use in treating inflammation. Methods: Healthy subject blood neutrophils (n ¼ 8) were used to assay a variety of neutrophil functions. All tests were performed in the absence and presence of APPA (600 mM). Inflammatory signalling pathways were activated using TNFa, GM-CSF & IL-6 and the effects of APPA on signal transduction measured using Western blotting. Formation of neutrophil extracellular traps (NETs), phagocytosis of latex beads, killing of S. aureus, chemotaxis of neutrophils, cell surface receptor expression (integrins and FcgRs) and ROS production were also measured. Expression of cytokine/chemokine genes was mea- sured using qPCR. Results: APPA did not significantly affect neutrophil host defence functions including apoptosis, receptor expression, phagocytosis and bacterial killing, but was a potent ROS scavenger. APPA significantly (p < 0.05) inhibited NET formation and decreased neutrophil degranu- lation. TNFa-induced NF-kB signalling was inhibited by APPA (600 mM and above), as was GM-CSF and IL-6 signalling via ERK1/2 and STAT3, respectively. APPA decreased TNFa-activated expression of IL-8 and TNFa mRNA but upregulated NRF2, an anti-inflammatory regulator of antioxidant proteins. APPA was also an effective inhibitor of IL-6 and chemokine expression (CCL3, CCL4) induced by the TLR8 agonist and chromatin re-modelling agent, R848 (p < 0.05). This agonist triggers the expression of these genes via endogenous TNFa secretion and activation of neutrophils; APPA was as effective as the biologic Infliximab in this inhibition. Conclusion: APPA does not significantly impair host defence neutrophil functions and may have significant anti-inflammatory potential in diseases characterised by dysregulation of cytokine expression or oxidative stress, such as rheumatoid arthritis. These data also describe the novel finding that APPA is as effective as biologic drugs in inhibiting the effects of endogenous TNFa on immune cell activation. Thus it may have therapeutic potential in TNFa-driven inflammatory conditions. Disclosures: A.L. Cross: None. J.J. Hawkes: None. H.L. Wright: None. R.J. Moots: Grants/research support; RJM’s group at the University of Liverpool has received research grants from AKL pharma for investigator-initiated phase I and II trials of APPA in osteoarthritis. S.W. Edwards: None. 015 DUAL INHIBITION BY PHOSPHODIESTERASE-5 AND 5-HT2B INHIBITORS LEADS TO NEAR COMPLETE AMELIORATION OF FIBROTIC POTENTIAL OF HUMAN DERMAL FIBROBLASTS FROM SCLERODERMA PATIENTS Vikas Agarwal 1 , Saurabh Chaturvedi 1 , Harshit Singh 1 , Mohit K. Rai 1 , Kritika Singh 1 , Durga P. Misra 1 , Narayan Prasad 2 and Vinita Agrawal 3 1 Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, INDIA, 2 Nephrology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, INDIA, and 3 Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, INDIA Background: Conversion of quiescent resident fibroblasts to acti- vated myofibroblasts (MFBs) by transforming growth factor beta 1 (TGF-b1) is the hallmark of the pathogenesis of fibrosis in scleroderma. MFBs are characterised by increased alpha-smooth muscle actin (a- SMA) expression and production of extracellular matrix (ECM) proteins. Previous literature has demonstrated the potential role of 5- hydroxy tryptamine (5-HT) in driving fibrosis. Our prior work demon- strated the effectiveness of 5-HT 2B inhibitor in ameliorating fibrosis induced by TGF-b1 in human adult dermal fibroblasts derived from scleroderma patient. Considering the widespread use of phospho- diesterase 5 inhibitors in the management of Raynaud’s phenomenon and pulmonary hypertension associated with scleroderma, we evaluated the anti-fibrotic potential of dual inhibition by PDE5i (sildenafil or zaprinast) plus 5-HT 2B inhibitor, SB204741 in human dermal fibroblasts (HDF) isolated from mid-forearm skin of sclero- derma patients. Methods: In a strategy mimicking disease in vitro, HDF were incubated with TGF-b1 (10ng/ml) for 1 hour, followed by TGF-b1 (10ng/ml) along with sildenafil (10mM) plus SB204741 (1mM) or with Zaprinast (10mM) plus SB204741 (1mM) for 24 hours. In the pre- treatment strategy, HDF were pretreated with [sildenafil (10mM) plus SB204741 (1mM) or zaprinast (10mM) plus SB20474 (1mM)] for 1 hour, and thereafter with only TGF-b1 (10ng/ml) for 24 hours. Real time quantitative polymerase chain reaction (qPCR) for pro-fibrotic (COL1A1, COL1A2, ACTA2, CTGF, FN1 and TIMP1) and anti-fibrotic genes (MMP2) was performed. Immunoblotting was performed for a- SMA. The study was approved by the institutional ethics committee, and written, informed consent obtained from all participants. Results: In TGF-b1 stimulated HDF, upregulated expression of pro- fibrotic genes was observed (p < 0.05). Expression of pro-fibrotic genes in HDF cells stimulated with TGF-b1 was significantly (p < 0.05) reduced by dual inhibition strategy with complete amelioration of ACTA2 in comparison to their respective individual treatments (p < 0.05), whereas, the ratio of MMP2/TIMP1 (suggesting anti-fibrotic efficacy) was restored significantly (p < 0.05). In TGF-b1stimulated HDF, dual inhibition strategy decreased expression of a-SMA protein significantly (p < 0.05). Conclusion: Dual inhibition strategy with inhibitors of PDE5 plus 5- HT 2B receptors leads to near complete amelioration of conversion of fibroblasts to MFBs, and thus may have potential to treat fibrosis in scleroderma. Disclosures: V. Agarwal: None. S. Chaturvedi: None. H. Singh: None. M.K. Rai: None. K. Singh: None. D.P. Misra: None. N. Prasad: None. V. Agrawal: None. 016 TARGETING COMBINED TREATMENT TO ARTHRITIC BY ANTIBODY SPECIFIC TO DAMAGED CARTILAGE Nissim 1 Mary University, William Harvey Research Institute, London, KINGDOM iii46 Tuesday 30 April 2019 INVITED SPEAKERS ABSTRACTS Downloaded from https://academic.oup.com/rheumatology/article/58/Supplement_3/kez106.014/5444723 by guest on 08 December 2021