Systemic sclerosis
Chemerin/CMKLR1 axis and pulmonary vascular remodelling Study population Patients were recruited from Le Kremlin-Bicêtre and Pittsburgh reference centres for PAH. Lung specimens were obtained during transplantation in patients diagnosed with SSc-PAH and/or ILD or idiopathic PAH (iPAH), 4 and during lobectomy or pneumo- nectomy for localised lung cancer in control subjects (non-SSc controls). Lung specimens from controls were collected at a distance from the tumour foci. Preoperative echocardiography was performed in controls to rule out PAH. Lung samples from four SSc-PAH patients and four non-SSc controls were transported in Perfadex and processed within 30 min of explantation as previously described. 21 Of note, three SSc patients had extensive ILD. Following mechanical and enzy- matic digestion by Liberase DL and DNase I, resulting single- cell suspensions were loaded into 10 x Genomics Chromium instrument (Pleasanton, California, USA), with scRNA-seq library preparation per manufacturer’s protocol. Sequencing was performed on an Illumina NovaSeq 6000 instrument through the UPMC Genome Centre. Single-cell RNA sequencing in explanted human lungs Data generation Data analysis Data analysis was performed with R (V.4.0.4) package Seurat (V.4.1.0). Samples were merged with batch correction by Harmony for individual sample, followed by normalisation, identification and visualisation of cell populations, and differen- tial expression testing. Cell populations were identified by gene markers and visualised by uniform manifold and approximation projection plot. Confocal immunofluorescence analyses in explanted human lungs Immunofluorescent staining for chemerin and chemokine-like receptor 1 (CMKLR1) was performed on lung paraffin sections from SSc-PAH patients, SSc-no PAH patients and non-SSc controls as previously described. 22 Of note, both SSc patient groups had extensive ILD. Briefly, lung sections were deparaf- fined and incubated with retrieval buffer. Then, sections were saturated with blocking buffer and incubated overnight with specific antibodies (sc-398769, CliniSciences, France), followed by corresponding secondary fluorescent-labelled antibodies (Thermo-Fisher Scientific, France). Nuclei were labelled using 4',6-diamidino-2-phénylindole (DAPI) (Thermo-Fisher Scien- tific). Mounting was done using ProLong Gold antifade reagent (Thermo-Fisher Scientific). Images were taken using LSM700 confocal microscope (Zeiss, France). PA-SMCs were isolated from distal PA of lung explants from iPAH patients and non-SSc controls, and cultured as previously described. 23 24 The isolated PA-SMCs were strongly positive for α -smooth muscle actin ( α -SMA), smooth muscle-specific SM22 protein and calponin, and negative for von Willebrand factor and CD31. Cells were used at passage <5. The mRNA expression of CMKLR1 was measured by real- time quantitative PCR using TaqMan gene expression assay (assay ID: Hs01081979_s1) as previously described. 22 25 Relative quantification was calculated by normalising the Ct (threshold cycle) of the gene of interest to the Ct of 18S in the same sample, according to the comparative CT method (ΔΔCT method). Role of CMKLR1 in the proliferation of human PA-SMCs PA-SMC proliferation experiments
PA-SMCs were cultured with 5% serum from 5 SSc-PAH and 5 SSc-no PAH patients included in the proteomic validation cohort, in the presence or absence of 2-(alpha-naphthoyl)ethyl- trimethylammonium iodide ( α -NETA) at 1 µ M. Proliferation was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation and direct cell counting. Descriptive analysis Figures were created using GraphPad Prism V.9.1.0 (GraphPad Software, California, USA). Differences were assessed visually without statistical analysis due to small sample size. RESULTS Serum levels of chemerin are elevated and correlate with PVR in SSc-PAH patients In a first step, serum expression of 1129 biomarkers was assessed by SOMAscan in 15 cases (SSc-PAH patients) and 16 controls (SSc-no PAH patients) from a discovery cohort (table 1; for Table 1 Characteristics of SSc-PAH patients from the discovery and the validation cohorts Discovery cohort Validation cohort N N Sex (female) 15 14 (93%) 24 17 (71%) Age (years) 15 65 (±7) 24 68 (±9) RP duration (years) 10 19 (±13) 8 18 (±12) SSc duration (years) 15 1.25(0.05 ;6) 9 0.5(0 ;9) BMI (kg/m 2 ) 15 28 (±4) 16 28 (±6) NYHA functional class 15 18 Class I 0 (0%) 0 (0%) Class II 7 (47%) 6 (33%) Class III 8 (53%) 10 (56%) Class IV 0 (0%) 2 (11%) BNP (pg/mL) 12 147(69 ;505) 14 84(39;264) or Nt-pro-BNP (pg/mL), if BNP not available / / 9 878(374;1124) eGFR (mL/min) 12 73 (±16) 15 81 (±28) ANA 13 13 (100%) 17 17 (100%) ACA 12 9 (75%) 8 7 (88%) ATA 12 0 (0%) 8 0 (0%) Anti-U1RNP 12 1 (8%) 8 0 (0%) LVEF (%) 12 60 (±9) 14 65 (±9) FVC (% predicted) 13 85 (±10) 20 88 (±20) TLC (% predicted) 13 90 (±12) 14 87 (±15) DLCO (% predicted) 15 35(8;52) 10 41(33;65) ILD on chest CT-scan 15 6 (40%) 24 5 (21%) mPAP (mm Hg) 15 44 (±10) 24 37 (±8) PAWP (mm Hg) 15 10 (±2) 24 10 (±3) CO (L/min) 15 4.9 (±1.6) 24 4.6 (±1.2) CI (L/min/m 2 ) 15 2.7 (±1.0) 18 2.5 (±0.5) PVR (WU) 15 8.5 (±5.0) 24 6.2 (±2.3) ACA, anticentromere antibodies; ANA, antinuclear antibodies; Anti-U1RNP, anti-U1- ribonucleoprotein antibodies; ATA, anti-topoisomerase I antibodies; BMI, body mass index; BNP, brain natriuretic peptide; CI, Cardiac Index; CO, cardiac output; DLCO, diffusing capacity of the lung for carbon monoxide; eGFR, estimated glomerular filtration rate; FVC, forced vital capacity; ILD, interstitial lung disease; LVEF, left ventricular ejection fraction; mPAP, mean pulmonary arterial pressure; Nt-pro-BNP, N-terminal pro-BNP; NYHA, New York Heart Association; PAH, pulmonary arterial hypertension; PAWP, pulmonary arterial wedge pressure; PVR, pulmonary vascular resistance; RP, Raynaud phenomenon; SSc, systemic sclerosis; TLC, total lung capacity; WU, wood unit.
Sanges S, et al . Ann Rheum Dis 2023; 82 :365–373. doi:10.1136/ard-2022-223237
367
Powered by FlippingBook