Introduction
Systemic sclerosis (SSc) is a rare and complex autoimmune disease characterized by processes that combine immune-mediated inflammation, vasculopathy and fibrosis [
1]. Most organs may be affected, but the fibrotic and vascular pulmonary manifestations, particularly interstitial lung disease (ILD) and pulmonary hypertension (PH), stand as the principal sources of morbidity and mortality [
1‐
3].
Within the context of SSc, the prevalence of PH ranges from 8 to 15% [
4‐
6], and can be included in various classifications. In SSc, the two main categories of PH, are pulmonary arterial hypertension (PAH) (accounting for approximately two-third of patients with SSc and PH) and PH associated to ILD (affecting one-third of patients). In SSc-associated PAH (SSc-PAH, Group 1), progressive pulmonary vascular resistance increase results from pulmonary vascular remodeling due to endothelial dysfunction [
7]. The therapies designed for specific PAH target endothelial dysfunction and pulmonary vascular remodeling, leading to decreased pulmonary vascular resistance.
The coexistence of ILD and PH significantly impacts prognosis, with a 3-year overall survival rate of 35%, with a poorer prognosis than in patients with isolated SSc-PAH [
8]. Furthermore, the efficacy of PAH therapy in patients with SSc-PH-ILD remains uncertain, and certain compounds raise safety concerns [
9].
Classifying SSc patients into group 1 and group 3 PH can be challenging [
10]. PH associated with extensive ILD (> 20% extent of fibrosis on High-Resolution Computed Tomography (HRCT) or forced vital capacity (FVC) < 70% in case of indeterminate HRCT) is typically categorized under group 3. However, for a significant number of patients, precise differentiation between the two groups based on these parameters may be challenging [
11]. The main issue is to accurately separate group 1 and group 3 in patients with concurrent ILD. In certain instances, a patient classified under group 3 of the classification may also present pathophysiological typically associated with group 1. This uncertainty raises questions about the impact of PAH therapies on the outcome in patients with “mixed” disease.
There is a paucity of data regarding the effectiveness of specific PAH treatments in patients with SSc-PH-ILD. While some studies have indicated that PAH-targeted therapies may improve haemodynamics in SSc-PH-ILD but they do not demonstrate any benefit in dyspnoea and survival and there might even be an elevated risk of hypoxia [
12,
13]. The positive hemodynamic response to treatment observed in certain patient cohorts [
13] suggests an overlap between SSc-PAH and SSc-PH-ILD. Consequently, a careful phenotyping of PH in SSc is essential, as it can significantly influence the selection of treatment and prognosis [
9,
14].
Here, we hypothesized that the biological processes underlying the pathophysiology of SSc-PH-ILD might be reflected by perturbations in circulating proteins. Plasma protein level alterations have been previously employed to investigate the molecular drivers of PAH [
15‐
18]. The aim of this exploratory study was to identify specific plasma protein expression patterns associated with survival in patients with SSc-PH-ILD.
Discussion
In a proteomic analysis of 32 patients with SSc-PH-ILD, all included in a prospective multicenter national cohort, we identified 7 plasma proteins associated with haemostasis and fibrosis, and differentially expressed according to patients’ survival. In the long surviving patients, two proteins were increased (ADAMTS13, SERPIND1) and five were decreased (PTGDS, OLFM1, C7, IGFBP7, FBN1) compared to the short surviving patients.
In patients with SSc, two primary mechanisms of PH can coexist: group 1 (SSc-PAH) and group 3 (SSc-PH-ILD). While specific treatment for PAH have shown effectiveness in group 1, their efficacy and safety in group 3 have been a subject of debate [
23]. Furthermore, most of the patients in this study were treated with ERAs and in particular BOSENTAN, which may seem outdated at present. These observation may suggest a lack of comprehension regarding the mechanisms underlying group 3 PH, while also highlighting the heterogeneity of patients. Despite the promising outcomes obtained in PH-ILD from the phase 3 Randomized Control Trial (RCT) of inhaled treprostinil (INCREASE) [
24], these two conditions have distinct prognostic implications and necessitate contrasting therapeutic approaches. Therefore, it is crucial to differentiate between them effectively. Nevertheless, even with careful clinical and radiological evaluation, the classification of PH may be represent challenges in several patients. Indeed, a mixed involvement could be suspected in any patient with an identified ILD, even if minimal. With this perspective in mind, we have chosen to adopt an inclusive approach by including all patients with SSc associated with PH and an ILD regardless of its extent. We hypothesized that the underlying biological processes are likely to be manifested as disturbance in circulating proteins, making proteomic analysis a viable option for unraveling prognosis.
By analyzing the biological processes associated with the identified proteins (using the KEGG database), we found that survival was associated with 7 proteins, involved in blood coagulation and fibrosis pathways.
Indeed, in the group of patients alive at 1500 days (“long surviving patients”), we observed an increase in two pathognomonic biomarkers of blood coagulation, namely ADAMTS13 and Heparin cofactor II. ADAMTS13 is a metalloproteinase present in circulation, responsible for cleaving ultra large vWF, which helps to maintain the balance between thrombosis and haemostasis [
25]. In a recent study, patients with newly diagnosed PAH had lower plasma levels of ADAMTS13 than healthy controls, as well as those with PH due to heart failure and chronic thromboembolic PH (CTEPH) [
26]. Another study found that ADAMTS13 levels were lower in CTEPH patients compared to healthy controls [
27]. However, it is important to note that the plasma ADAMTS13 levels in these two studies were not compared to patients with PH associated with respiratory diseases. Nevertheless, based on these data and our own results, it appears that ADAMTS13 expression may be modulated in PH, and potentially associated with survival.
Heparin cofactor II (HCII) is a serine protease inhibitor (SERPIN) that acts to neutralize thrombin’s activity within subendothelial layer of the blood vessel wall, thereby influencing the blood coagulation cascade. HCII plays a protective role against vascular remodeling, which includes atherosclerosis, and plasma HCII activity could serve as a predictive biomarker and a novel therapeutic target for preventing cardiovascular disease [
28]. However, to the best of our knowledge, no association between this protein and PH has been reported yet. These results suggest that haemostasis may be associated with survival either through the activation of primary haemostasis (ADAMTS 13) or coagulation (HCII), or through the interplay between haemostasis and the vascular endothelium.
In the group of short surviving patients, we observed an increase in 5 proteins, Prostaglandin D2 synthase (PGD2), Noelin, Complement component C7 (C7), insulin-like growth factor-binding protein 7 (IGFBP7) and Fibrillin-1 (FBN1). Interestingly, IGFBP7 and FBN1 upregulated in short surviving patients are recognized as a crucial factors in the pathogenesis of fibrotic diseases [
29,
30]. Specifically, previous studies have shown a significant increase in IGFBP7 expression in the lung tissue of patients with fibrotic lungs condition such as Systemic Sclerosis-associated fibrotic lung disease (SSc-FP) and Idiopathic Pulmonary Fibrosis (IPF), but not in those with SSc-PAH [
31]. In addition, FBN1 has been identified to inhibit endothelial cell proliferation while promoting apoptosis [
29].
Taken together, these results suggest that IGFBP7 and FBN1 may reflect a more pronounced “fibrotic” phenotype and a lesser “vascular” phenotype in short surviving patients compared to long surviving patients SSc-PH-ILD patients.
The biological actions of prostaglandin D2 (PGD2) are associated with vasodilation, bronchoconstriction, platelet inhibition and inflammatory cells recruitment. A significant increase in PGD2 levels was observed in patients with right atrial dysfunction (RVD) compared to those without. In addition, they reported that an increase of L-PGDS suggest an RVD and that this may also constitute a major prognostic factor in predicting mortality in patients with pulmonary thromboembolism [
32]. These finding are in accordance with the results of the INCREASE trial, which demonstrated the beneficial effects of an inhaled prostagladin agonist (Treprostinil) [
24].
Complement component C7 is well-known for its central role in the innate and adaptive immune response, while Noelin is recognized for its regulation of axonal growth in the central nervous system. To date, it is difficult to formally link the pathophysiological application of these two proteins to this research theme.
This study has limitations, related to the biases inherent to ancillary studies. Mainly, despite access to a national multicenter cohort, the relatively small number of patients (n = 32) decreased the power of the statistical analysis. In addition, we have no further information on the course of diffuse interstitial lung disease, nor on the causes of death of the patients included in our study. These data could provide additional clues as to the distinction of phenotypes in these patients. However, proteomic analysis of plasma samples from PH patients continues to be a reliable method for identifying novel biomarkers associated with this disease as recently demonstrated in PAH [
15]. The results of our exploratory study generate potential biomarkers associated with survival, in patients with SSc-PH-ILD and pave the way for further, larger-scale prospective studies.
Acknowledgements
All the authors warmly thank all the investigators of the HYPID and HYPID-2 studies (funded by the Programme Hospitalier de Recherche Clinique of the French Health Ministry), NEUROBIOTEC for the help in blood samples collection.
List of investigators of the HYPID and HYPID-2 studies
Responsible of consortium: Vincent Cottin
Vincent Cottin12, Julie Traclet9, Sabrina Zeghmar9, Lize Kiakouama13, Francois Philit13, M Reynaud-Gaubert7, A. Nieves7, B. Coltey7, H Nunes6, Y Uzunhan6, L Wemeau4, D Launay4, G Lefevre4, E Hachulla4, E Monge4, D Montani2, O Sitbon2, L Savale2, X Jais2, S Quétant10, B Camara10, C Pison10, D Israel Biet8, S Marchand-Adam5, P Magro5, E Gomez14, G Prévot15, L Tetu15, E Bergot16, R Magnier16, C Dromer17, S Dury18, L Bertoletti11
2Service de Pneumologie et Soins Intensifs Thoraciques ; Centre de Référence de l'Hypertension Pulmonaire ; INSERM U999 Centre de compétence maladies pulmonaires rares—OrphaLung Hôpital de Bicêtre, France
3Service de Médecine Vasculaire et Thérapeutique; INSERM, CIC-1408, CHU de Saint-Etienne, France
4Service de Médecine Interne et d'Immunologie Clinique, Centre de Référence des Maladies Auto-Immunes et Systémiques Rares du Nord et Nord-Ouest de France (CeRAINO) CHU Lille, France
5Service de pneumologie et explorations fonctionnaires Respiratoires, Tours France
6Service de Pneumologie, Centre de référence des maladies pulmonaires rares, Hôpital Avicenne, INSERM U1272, Université Sorbonne Paris Nord, Bobigny, France
7Centre de Compétence des maladies pulmonaires rares (OrphaLung), service de Pneumologie et Transplantation Pulmonaire, CHU Nord, AP-HM, Marseille, France
8Service de Pneumologie et Soins Intensifs Centre de Compétence Maladie Pulmonaire Rare (OrphaLung) Hôpital Européen Georges Pompidou, AP-HP, France
9Hospices Civils de Lyon, Louis Pradel Hospital, National Reference Center for Rare Pulmonary Diseases, Department of Respiratory Diseases, F-69677 Lyon, France
10Service de Pneumologie, Grenoble, France.
11Service de Médecine Vasculaire et Thérapeutique; INSERM, UMR1059, Université Jean-Monnet; INSERM, CIC-1408, CHU de Saint-Etienne ; INNOVTE; all in F-42055, Saint-Etienne, France
12Hospices Civils de Lyon, Louis Pradel Hospital, National Reference Center for Rare Pulmonary Diseases, Department of Respiratory Diseases, F-69677 Lyon, France; Univ Lyon, INRA, UMR754, F-69008 Lyon, France
13Service de Pneumologie—Hôpital de la Croix Rousse – HCL – Lyon, France
14Service de pneumologie CHU de Nancy—Hôpitaux de Brabois—Nancy, Franche
15Service de Pneumologie—CHU Larrey—Toulouse, France
16Service de Pneumologie—CHU Caen—Caen, France
17Service de Pneumologie—CHU De Bordeaux-GH Sud—Hôpital Haut-Lévêque, Bordeaux, France
18Service de Pneumologie—CHU de Reims—Reims, France
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