Background/Purpose:   We have previously investigated the interplay between TGFβ, BMP, VEGF and endothelin in SSc using the TβRIIΔk-fib strain, a transgenic mouse model in which there is balanced upregulation of TGFβ signalling. The pivotal role of endothelin within this multifaceted pathology was confirmed using macitentan, a licensed endothelin receptor antagonist of proven efficacy, to prevent and treat pulmonary hypertension in this model. We have extended these studies to examine whether cellular pathways targeted by modern therapies currently undergoing clinical trials in SSc are altered in this model of constitutive skin fibrosis and pulmonary vasculopathy.

Methods:  RNA was isolated from cultured skin and lung fibroblasts and aortic smooth muscle cells (SMCs) explanted from adult transgenic and wildtype littermate control mice and subjected to gene expression analysis using the Illumina microarray platform (n=3 each group). Arrays were analysed for differential expression of genes involved in inflammatory, vascular and fibrotic cellular pathway components targeted by therapeutic agents undergoing multicentre clinical trial evaluation in SSc. Differential expression was defined as a 2-fold average change from wildtype with p<0.05.

Results: There were between 50 and 400 differentially expressed genes from the cell lines described. These gene lists were then examined to identify genes involved in the nitric oxide synthase pathway and downstream mediators (riociguat); IL-6 receptor and downstream pathways (tocilizumab) and PDGFR, FGFR and VEGFR axes (nintedanib). Several genes involved in nitric oxide metabolism were altered in lung fibroblasts, including NOS2 (inducible nitric oxide synthase), PTGIS (prostaglandin I2 synthase) and GUCY1A3 (guanylate cyclase isoform alpha 3). Genes also involved in vasoconstriction were differentially expressed in SMCs, including upregulation of multiple phosphodiesterases and PTGS-1 (prostaglandin endoperoxide synthase 1; COX-1). IL-6 gene expression was upregulated in transgenic skin and lung fibroblasts, as was STAT1, STAT3 (fold change 36:1, p<0.004 in skin fibroblasts) and JAK1 expression. In contrast, gp130, STAT3 and PIAS3 (protein inhibitor of STAT3) were all downregulated in SMCs. Pathways related to the mechanism of action of nintedanib are more complex since several of these downstream mediators form the non-canonical signalling pathways of TGFβ and are known to be altered in this strain. We identified multiple genes involved in these axes, for instance, upregulation of PDGFRA (fold change 137:1, p<0.02), FGF10, FGFR1 and downregulation of Ras and related proteins in SMCs.

Conclusion:  Identification of multiple alterations in pathways targeted by potential therapeutics for SSc underpins the utility of this strain in pre-clinical investigation of candidate treatments and demonstrates the importance of selecting the most appropriate model for in vivo assessment of emerging molecular therapies in SSc. Pathways involved in vasoconstriction and inflammation are altered in relevant tissues in this TGFβ dependent strain. It is validated as a platform for experimental therapeutic studies particularly in pulmonary complications of SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/multi-tissue-gene-expression-pathway-analysis-of-emerging-therapeutics-in-a-tgf%ce%b2-dependent-mouse-model-of-systemic-sclerosis/