Background/Purpose: Pulmonary arterial hypertension (PAH) is prominent as a vascular involvement of systemic sclerosis (SSc), which remains a leading cause of death in spite of current best treatments. As the pulmonary vascular disease (PVD) can be well compensated for, more than a half of the pulmonary circulation is impaired before PAH is detected. So far, the underlying molecular mechanisms have not been fully elucidated, especially at the early stage of SSc.In this study, we focused on the stage of subclinical PVD, and try to detect candidate genes involved in the pathogenesis of exercise-induced pulmonary hypertension (exPH) at the early stage of SSc.

Methods: Total of 88 patients who had not met PAH criteria with Raynaud phenomenon (n=75), skin sclerosis (n=58) or SSc-related autoantibody(n=59) was enrolled. To detect the early PVD, exercise Doppler echocardiography was carried out. The exPH group was segregated from normal response group (exN) with using the reported definition.¹⁾For gene expression analysis, total RNAs from whole peripheral blood cells were extracted by PAXgene system and multiplex sequencing was done. To identify changes of transcriptomes for developing exPH, hierarchical clustering, weighted gene co-expression network analysis (WGCNA), pathway enrichment analysis (PathVisio) and volcano plots were performed with using differentially expressed genes (DEGs) between exPH and exN group.

Results: After applying 1204 DEGs to hierarchical clustering analysis, 5 major clusters were identified and 93% of exPH samples were segregated into 2^ndcluster. WGCNA and pathway analysis revealed 6 co-expression modules and they weresignificantly enriched with genes of several pathways, such as Wnt, endothelin, IL-1, TNF-alpha, prostaglandin, toll-like receptor, EGF, VEGF-A, type3 interferon and integrin-mediated cell adhesion. Volcano plots were scatter plots calculated to visualizefold-changes and p-values of DEGs. It indicated that expressions of TMEM176A and TMEM176B (log₂fold-change >1.25 and –log₁₀ p-value >3.5) were prominent in exPH patients. We also found that the expressions of these genes were highly correlated with each other (R²=0.9724, p<0.01).

Conclusion: The paradigm of SSc-PAH management should ideally be aimed at starting treatment before development of PAH. Although elucidating the mechanisms for progression of PVD in the early stage of SSc remains a major challenge, (1) we found exPH patients were segregated from exN group by gene expression profiles of peripheral blood; (2) DEGs were enriched with genes of several important pathways for pathogenesis of SSc, which reported previously; (3) up-regulation of TMEM176A and TMEM176B were prominent in exPH group. The trans-membrane protein TMEM176A and TMEM176B were found initially to be a regulator for DC maturation in mice²⁾, but functions of human ortholog of these genes remain still unclear. As our findings revealed active changes of transcriptomes in peripheral blood prior to fulfilling PAH criteria in SSc patients, it will encourage the therapeutic intervention at early stage of SSc to prevent PVD.

References:¹⁾R. Naeijeet al., Am J resp and critical care med187, 576-583 (2013). ²⁾Condamine, T.et al., J of leuko biol88, 507-515 (2010).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/gene-expressions-of-tmem176a-and-tmem176b-were-prominent-at-the-stage-of-subclinical-pulmonary-vascular-disease-in-systemic-sclerosis/