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. 2023 Mar;82(3):365-373.
doi: 10.1136/ard-2022-223237. Epub 2022 Dec 5.

Biomarkers of haemodynamic severity of systemic sclerosis-associated pulmonary arterial hypertension by serum proteome analysis

Sébastien Sanges #  1   2   3   4   5   6 , Lisa Rice #  7 , Ly Tu  8   9 , Eleanor Valenzi  10 , Jean-Luc Cracowski  11 , David Montani  8   9   12 , Julio C Mantero  7 , Camille Ternynck  13 , Guillemette Marot  13   14   15 , Andreea M Bujor  7 , Eric Hachulla  2   3   4   5   6 , David Launay  2   3   4   5   6 , Marc Humbert  8   9   12 , Christophe Guignabert  8   9 , Robert Lafyatis  16
Affiliations

Biomarkers of haemodynamic severity of systemic sclerosis-associated pulmonary arterial hypertension by serum proteome analysis

Sébastien Sanges et al. Ann Rheum Dis. 2023 Mar.

Abstract

Objectives: To mine the serum proteome of patients with systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH) and to detect biomarkers that may assist in earlier and more effective diagnosis and treatment.

Methods: Patients with limited cutaneous SSc, no extensive interstitial lung disease and no PAH-specific therapy were included. They were classified as cases if they had PAH confirmed by right heart catheterisation (RHC) and serum collected on the same day as RHC; and as controls if they had no clinical evidence of PAH.

Results: Patients were mostly middle-aged females with anticentromere-associated SSc. Among 1129 proteins assessed by a high-throughput proteomic assay (SOMAscan), only 2 were differentially expressed and correlated significantly with pulmonary vascular resistance (PVR) in SSc-PAH patients (n=15): chemerin (ρ=0.62, p=0.01) and SET (ρ=0.62, p=0.01). To validate these results, serum levels of chemerin were measured by ELISA in an independent cohort. Chemerin levels were confirmed to be significantly higher (p=0.01) and correlate with PVR (ρ=0.42, p=0.04) in SSc-PAH patients (n=24). Chemerin mRNA expression was detected in fibroblasts, pulmonary artery smooth muscle cells (PA-SMCs)/pericytes and mesothelial cells in SSc-PAH lungs by single-cell RNA-sequencing. Confocal immunofluorescence revealed increased expression of a chemerin receptor, CMKLR1, on SSc-PAH PA-SMCs. SSc-PAH serum seemed to induce higher PA-SMC proliferation than serum from SSc patients without PAH. This difference appeared neutralised when adding the CMKLR1 inhibitor α-NETA.

Conclusion: Chemerin seems an interesting surrogate biomarker for PVR in SSc-PAH. Increased chemerin serum levels and CMKLR1 expression by PA-SMCs may contribute to SSc-PAH pathogenesis by inducing PA-SMC proliferation.

Keywords: autoimmune diseases; cytokines; systemic sclerosis.

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Conflict of interest statement

Competing interests: SS reports travel grants from Shire, Sanofi-Genzyme, SOBI and Novartis; consulting fees from Novartis; outside the submitted work. J-LC reports grants from United Therapeutic, Bioprojet and Topadur, outside the submitted work. DM reports grants and personal fees from Actelion, grants and personal fees from Bayer, personal fees from GSK, personal fees from Pfizer, personal fees from MSD, personal fees from Chiesi, outside the submitted work. EH reports consulting fees from Actelion, Boehringer Ingelheim, GSK, Roche-Chugai, Sanofi-Genzyme; speaking fees from Actelion, GSK, Roche-Chugai; and research funding from CSL Behring, GSK, Roche-Chugai, outside the submitted work. DL reports personal fees from Actelion, grants and personal fees from Takeda, grants and personal fees from CSL Behring, outside the submitted work. MH reports personal fees from Actelion, grants and personal fees from Bayer, grants and personal fees from GSK, personal fees from Merck, personal fees from United Therapeutics, personal fees from Acceleron, outside the submitted work. RL reports research grants from Formation, Elpidera and Kiniksa, outside the submitted work. RL has served as a consultant for Bristol Myers Squibb, Boehringer-Mannheim, Merck, Magenta and Genentech/Roche, outside the submitted work. Over the last three years, CG, reports grants from Acceleron, ShouTi, and Janssen and grants and personal fees from Merck, outside the submitted work.

Figures

Figure 1.
Figure 1.. Heatmap depicting the differential expression of the 53 candidate biomarkers identified in the discovery cohort:
Standardized expression values of the 53 differentially expressed proteins in the discovery cohort. Red values indicate over-expression and blue values under-expression compared to the mean expression level.
Figure 2.
Figure 2.. Differential expression of chemerin and correlation with PVR in the discovery and the validation cohorts:
PAH: pulmonary arterial hypertension; PVR: pulmonary vascular resistance; RFU: relative fluorescence units; SSc: systemic sclerosis.
Figure 3.
Figure 3.. Differential expression of chemerin in different SSc patient groups:
dc: diffuse cutaneous; ext ILD: extensive interstitial lung disease; HC: healthy controls; lc: limited cutaneous; PAH: pulmonary arterial hypertension; RFU: relative fluorescence units; SSc: systemic sclerosis.
Figure 4.
Figure 4.. Expression of CMKLR1, chemerin and α-smooth muscle actin mRNA in cell populations from SSc-PAH lungs assessed by single-cell RNA-sequencing:
Top panels provide information from all cell populations; Bottom panels provide information on mesenchymal cell populations. α-SMA: α-smooth muscle actin; CMKLR1: chemokine-Like Receptor 1; PAH: pulmonary arterial hypertension; SSc: systemic sclerosis.
Figure 5.
Figure 5.. Representative images of lung sections immunostained with DAPI (blue), α-SMA (green) and CMKLR1 (red) from non-SSc controls (top row), SSc-no PAH (middle row) and SSc-PAH (bottom row):
α-SMA: α-smooth muscle actin; CMKLR1: chemokine-Like Receptor 1; DAPI: 4’,6-diamidino-2-phenylindole; PAH: pulmonary arterial hypertension; SSc: systemic sclerosis. Scale bar = 50 μm in all sections.
Figure 6.
Figure 6.. Proliferation of PA-SMCs from idiopathic PAH patients, after stimulation with serum from SSc-PAH and SSc-no PAH patients, in the presence or absence of α-NETA:
α-NETA: 2-(anaphthoyl)ethyltrimethylammonium iodide; BrdU: 5-bromo-2-deoxyuridine; i: idiopathic; PAH: pulmonary arterial hypertension; PA-SMC: pulmonary arterial smooth muscle cells; SSc: systemic sclerosis.
See this image and copyright information in PMC

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