Dephosphorylated uncarboxylated Matrix-Gla-Protein and vascular remodeling in pulmonary hypertension: an immunological connection

Dephosphorylated uncarboxylated Matrix-Gla-Protein and vascular remodeling in pulmonary hypertension: an immunological connection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dephosphorylated uncarboxylated Matrix-Gla-Protein and vascular remodeling in pulmonary hypertension: an immunological connection R. Tobal, J. Potjewijd, F. Vries, D. P.C. Doorn, A. Jaminon, R. Bittner, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4329956/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling. Since dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP) is associated with cardiovascular mortality in systemic sclerosis, a disease associated with PAH, and immune-system involvement in PAH is increasingly recognized, we investigated the relationship between dp-ucMGP, vascular remodeling and soluble immune-checkpoint proteins in PAH. This prospective cohort study included patients with idiopathic (I)PAH, connective tissue disease (CTD)-PAH, chronic thrombo-embolic PH (CTEPH) and CTD patients without PAH. Patients with IPAH and CTD-PAH were stratified by clinical signs of immune-mediated inflammatory disease (IMID). We measured dp-ucMGP plasma levels, soluble immune-checkpoint proteins (sICPs), and vascular smooth muscle cell (iVSMC) calcification. We found elevated dp-ucMGP levels in all PAH subtypes and CTD patients compared to healthy controls. PAH patients showed increased iVSMC calcification, but no direct correlation was found with dp-ucMGP. IMID-PAH patients had higher dp-ucMGP levels than non-IMID PAH patients. dp-ucMGP correlated with several sICPs in both IPAH and CTD patients; multiple sICPs were elevated in IMID PAH patients. High dp-ucMGP levels in IPAH patients were associated with worse survival. Our findings suggest dp-ucMGP as a potential biomarker of immune-mediated vascular remodeling in PAH. Hence, dp-ucMGP, could help identify PAH patients who might benefit from immunosuppressive therapies. Biological sciences/Biochemistry Biological sciences/Immunology Health sciences/Medical research/Biomarkers Health sciences/Medical research/Translational research Pulmonary hypertension vascular remodeling dp-ucMGP inflammation immune checkpoint proteins biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Pulmonary arterial hypertension (PAH) is a complex and debilitating condition characterized by elevated blood pressure within the pulmonary arteries, leading to increased strain on the right side of the heart 1 . This chronic condition has a great impact on patient well-being and life expectancy 2 , 3 . The increase in pulmonary arterial pressure parallels structural changes in the pulmonary vasculature, which is characterized by increased muscularization, fibrosis, and gradual narrowing of blood vessels 4 . This pulmonary vascular remodeling involves the complex and incompletely understood interaction of endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and fibroblasts 4 . The immune system is an important driver of this interaction, which is best illustrated by the elevated risk of developing PAH in patients with systemic autoimmune diseases such as systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) 5 . Interestingly, even in idiopathic PAH (IPAH) there is ample evidence for immune activation and dysregulation during the disease course 6 – 8 . T -and B-cell infiltrates have been found around prototypical pulmonary vascular lesions in PAH patients and in both preclinical models and in numerous patients, autoantibodies such as anti-endothelial cell antibodies, immune complexes, or increased cytokine levels have been observed 8 – 12 . IgG from a subset of IPAH patients induces increased vascular cellular adhesion molecule-1 (VCAM-1), integrin cellular adhesion molecule-1 (ICAM-1), monocyte chemo-attractant protein-1 MCP-1 and interleukin-6 (IL-6) production in cultured endothelial cells 13 . However, the diagnostic and therapeutic approach with focus on the role of the immune system in the clinical practice of PAH patients is ill-defined and non-standardized. The strongest benefit from immunosuppression is shown in SLE-PAH, whereas this treatment is less or not effective in limited SSc-PAH 14 . This may indicate differences in pathogenesis and disease progression but also highlights the diagnostic delay in many PAH patients. Therefore, novel biomarkers are necessary to improve a more detailed understanding of the role and stage of immune activation related to the process of pulmonary vascular remodeling leading to PAH. We hypothesize that dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP) could be such a novel marker. We recently showed that plasma levels were increased in early systemic sclerosis patients, as compared to age-matched healthy controls 15 , 16 . Notably, dp-ucMGP levels exhibited a positive correlation with disease severity and the incidence of cardiovascular events in these patients. These findings were not explained by traditional cardiovascular risk factors. MGP is mostly known as a protein (14 kD) predominantly secreted by VSMCs and ECs, which plays a pivotal role as a vitamin K-dependent protein in inhibiting both intimal and medial vascular calcification 16 . dp-ucMGP represents the fully inactive form, lacking the ability to bind calcium or extracellular matrix, and is consequently released into the circulation. SSc patients have a high risk of developing PAH over time, so we questioned whether dp-ucMGP may provide a link between inflammation and vasculopathy in PAH. Such a connection has also been suggested between the increased signaling pathways of TGF- β and bone morphogenetic protein 4 (BMP-4) that is observed in both systemic sclerosis and PAH 17 – 21 . In the present study we first measure plasma MGP in patients with idiopathic PAH (IPAH), connective tissue disease-associated PAH (CTD-PAH), chronic thromboembolic pulmonary hypertension (CTEPH), and in patients suffering from connective tissue disease (CTD) alone. We subsequently classify all patients as either immune-mediated inflammatory PAH (IMID-PAH) or non-IMID PAH according to the strict but non-validated criteria we use in our clinical practice. In addition, we measure soluble immune checkpoint proteins (sICPs), once thought to be solely cell membrane-bound regulators of immune cell function, but now recognized as biologically active molecules similar to stimulatory or inhibitory cytokines 22 , 23 . We aim to explore whether sICPs are a valuable tool for detecting immune profiles within PAH and whether these profiles support the concept of IMID-PAH. Lastly, we investigated the potential variations in dp-ucMGP levels across distinct subtypes of pulmonary hypertension in relation to circulating levels of sICPs and functional vascular calcification as a readout of vascular damage in these patients. Altogether, we hypothesize that plasma dp-ucMGP may serve as a valuable biomarker for both immune-mediated disease severity and survival prognosis in PAH. 2. Methods Study design and participants This study cohort consists of participants from the volatile organic compound-PAH (VOC-PAH) study, an observational prospective cohort study in IPAH, CTD-PAH, CTEPH, and CTD patients conducted between 2018 and 2023 at the Maastricht University Medical Center, Center of Expertise for PAH, in the Netherlands. The study was performed in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice, and approval was obtained from the local ethics committee. Written informed consent was obtained from each participant. This study is registered with clinicaltrials.gov number NCT03819777. Adults with IPAH, CTD-PAH, CTEPH, or CTD classified according to current criteria 24 – 30 were included. The exclusion criteria were not being treatment naïve, age < 18 years, or incapable of understanding or signing informed consent. All patients were clinically analyzed for the presence of an underlying auto-immune disease and received a comprehensive immunological assessment by a clinical immunologist. In this study, IPAH patients were subcategorized according to clinical signs of immune-mediated inflammatory disease (IMID). This was the case when there was the presence of auto-antibodies and increased inflammatory parameters, i.e., increased soluble-interleukin-2 receptor (sIL-2R) with low C-reactive protein (CRP) and/or signs of immune activation based on increased IgM rheumatoid factor or cryoglobulinemia. (Sub)acute infection was ruled out as an underlying cause based on clinical evaluation. CTD-PAH patients were also categorized as IMID due to the autoimmune nature of their underlying connective tissue disease. CTD patients without PAH were used as a disease control cohort. The disease duration of the CTD patients was defined as the time from the first non-Raynaud phenomenon manifestation; the IPAH patients were included within one month of diagnosis by right heart catheterization. Oral corticosteroids and hydroxychloroquine were permitted if the patient was on a stable dose regimen for more than three months prior to the baseline visit. No other background immunomodulatory therapies were allowed. Demographic data, as well as data on inflammatory parameters in the blood (C-reactive protein (CRP), soluble interleukin-2 receptor (sIL2r)), pulmonary function tests (percentage of predicted forced vital capacity (%pFVC), and percentage of predicted diffusing capacity for carbon monoxide (%pDLCO)), 6-minutes walking test (6-MWT), New-York Heart Association (NYHA) class, echocardiography and if appropriate, right-sided heart catheterization, were recorded. Collection and processing of samples Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) vials and centrifuged at 1300g and room temperature for 10 minutes. The supernatant was carefully obtained and subsequently stored at -80 o C. Measurement of plasma dp-ucMGP Circulating dp-ucMGP levels were determined in EDTA plasma in a single run by the Laboratory of Coagulation Profile (Maastricht, the Netherlands) using the commercially available IVD CE marked chemiluminescent InaKtif MGP assay on the IDS-iSYS system (IDS, Boldon, UK) 31 , 32 . Patient samples were incubated with magnetic particles coated with murine monoclonal antibodies against dpMGP, acridinium-labeled murine monoclonal antibodies against ucMGP, and assay buffer. The magnetic particles were captured using a magnet and washed to remove any unbound analyte. Trigger reagents were added, and the resulting light emitted by the acridinium label was directly proportional to the concentration of dp-ucMGP in the sample. This assay's within-run and total precision were 0.8–6.2% and 3.0–8.2%, respectively. The assay measuring range was between 300 and 12,000 pmol/L and was found to be linear up to 11,651 pmol/L. Vascular smooth muscle cell calcification assay Calcification experiments were conducted with induced pluripotent stem cell (iPSC)-derived iVSMCs. iVSMCs were seeded in culture well plates at 1.0x10 4 cells/well. After 24h, iVSMCs were cultured in calcification medium (DMEM 31966, 5% IPAH, CTD-PAH, CTEPH or CTD patient plasma, 1% penicillin-streptomycin solution, and 3.6 mM Ca 2+ ) for up to 9 days. Plasma samples were supplemented with 14.3 nM Hirudin to prevent coagulation upon re-calcifying the plasma. 5% healthy control pooled plasma was used as a negative control due to the lower magnitude of calcification. For the BioHybrid assay, Fetuin-A AlexaFluor-546 (1µg/mL; prepared in-house) and Hoechst 33,342 (0.5 µg/mL, Invitrogen, Waltham, MA, USA) were supplemented at the start of the experiment 33 , 34 . At various time points after calcification induction, fluorescent images were taken in the RFP and DAPI channel (for cell count) were imaged for up to 9 days. Imaging was done with the Cytation 3 system (BioSPX, Abcoude, the Netherlands) and analyzed using Gen5 software v.3.10. (BioTek, Abcoude, the Netherlands). As readout, fluorescent RFP area/well was normalized against cell count. Measurement of soluble immune checkpoints The plasma concentrations of the sICPs were quantified using a commercially available multiplex immunoassay (Life Technologies; ProcartaPL HU-IMM96T: EPX140-15803-901), which measures the soluble concentrations of B- and T-lymphocyte attenuator (BTLA), glucocorticoid-induced TNFR-related protein (GITR), herpesvirus entry mediator (HVEM), indoleamine 2,3-dioxygenase (IDO), Lymphocyte-activation gene 3 (LAG3), programmed death protein-1 (PD1), programmed death ligand-1 (PDL1), programmed death ligand-2 (PDL2), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), cluster of differentiation (CD)27, CD28, CD80, CD137 and CD152 (CTLA4/cytotoxic T-lymphocyte-associated antigen 4). The manufacturer’s protocols were followed by running patient samples in duplicates with 25 µL of plasma each and were measured using Magpix (Darmstadt, Germany). Data were analyzed using the xPONENT 4.2 software (Luminex). The given values represent the mean of the duplicates given in pg/mL. For checkpoint PDL1, many values were found to fall below the standard curve, and these results were excluded. Statistical analysis Categorical baseline characteristics were expressed as numbers and percentages. Numerical baseline characteristics were expressed as mean and standard deviation (SD) or median and interquartile range (IQR) based on normal distribution estimated by Shapiro/Wilk test and data visualization. Statistics were performed using the ANOVA, Chi-Square, Mann-Withney and Fisher’s Exact tests. The Kaplan-Meier curves were analyzed by the Log-Rank test to estimate differences in survival. The statistical analysis was performed using the IBM SPSS software version 24.0. A p-value less than 0.05 was considered statistically significant. Figures were made with GraphPad Prism version 9.5.0. 3. Results Clinical characteristics of the study cohort Forty-five PAH patients and 28 CTD disease controls without PAH were included from the prospective VOC-PAH cohort for dp-ucMGP and calcification analysis at baseline. PAH patients were classified in IPAH ( n = 26), CTD-PAH ( n = 8), and CTEPH ( n = 11). The clinical characteristics at the time of presentation have been depicted in Table 1 . Clinical characteristics of PAH patients with IMID and without IMID have been depicted in Supplemental Table 1 . Sex, age and body mass index (BMI) did not differ significantly between the groups. Hemodynamic parameters did not differ significantly among PAH groups. Patients with IPAH were most likely to have heart failure class III or IV ( n/N = 16/26, 62%), according to the New York Heart Association. Patients with CTEPH had the lowest forced expiratory volume after one second (FEV1), with a median percentage of 66%. Other pulmonary function parameters did not differ significantly among groups. The CTD disease control group mostly consisted of patients diagnosed with SSc ( n/N = 27/28, 96%). sIL2R concentrations were the highest in patients with CTD-PAH, with a median of 910 U/mL. CRP concentrations, however, did not differ significantly among groups. dp-ucMGP plasma levels in treatment-naïve IPAH, CTD-PAH, and CTEPH patients at diagnosis Plasma dp-ucMGP levels of 26 IPAH, 8 CTD-PAH and 11 CTEPH patients were compared with a control cohort of 28 age-matched CTD patients. Median dp-ucMGP levels were 795 (525.3;1027.0), 1029 (646.3;1336.0), 847 (593.0;1090.0), and 542 (374.3;757.8) pmol/L, respectively (Figure 1) . This is all greatly increased as compared to a median dp-ucMGP level of 375 (285.0;469.0) pmol/L in 400 age-matched Dutch HCs (p<0.001) . CTD-PAH patients had significantly higher dp-ucMGP levels than our control CTD patients (p=0.017)(Figure 1A) . Subsequently, we stratified IPAH and CTD-PAH patients based on clinical signs of immune-mediated inflammatory disease (IMID). dp-ucMGP plasma levels had a median of 646 (519.8;758.3) in the non-IMID group and 911 (619.0;l166.0) in the IMID group which was significantly different (p=0.030)(Figure 1B) . Plasma of PAH and CTD patients can induce calcification of vascular smooth muscle cells (iVSMCs) in vitro To investigate the calcification potential of the same patients, plasma-induced calcification of iVSMCs was analyzed in the Biohybrid assay. Median calcification levels were 7.8 (4.2;12.2) in IPAH, 7.5 (3.5;16.4) in CTD-PAH, 6.4 (4.5;12.0) in CTEPH, and 6.8 (4.9;11.3) RPF mcm 2 /cell count in CTD patients, which were significantly increased compared to a median of 1.9 (0.07;3.2) in aged matched healthy controls (Figure 2A) . When IPAH and CTD-PAH patients were stratified based on IMID, no significant difference was observed with a median of 7.8 (10.4;3.9) in non-IMID PAH and 7.5 (4.;12.6) mcm 2 /cell in IMID-PAH patients (Figure 2B) . No statistical correlation was found between dp-ucMGP plasma levels and the amount of calcification (Figure 4) . Soluble immune checkpoint protein levels and correlation with dp-ucMGP sICPs were measured in plasma of 24 (92%) of the IPAH and 27 (96%) of the CTD patients. Interestingly, no significant differences were observed between these groups of patients. However, both IPAH and CTD patients showed higher sBTLA, sCD137, sCD152, sLAG3 and sTIM3 levels when compared with healthy controls ( Figure 3) . sPD-L2 levels were significantly lower in CTD patients. dp-ucMGP plasma levels were correlated in IPAH patients with sBTLA, sCD137, sCD152, sCD27, sCD28, sGITR, sHVEM, and sIDO with a mean Spearman r of 0.56 (Figure 4A) . In CTD patients, dp-ucMGP plasma levels were correlated with all soluble ICs with a mean Spearman r of 0.52 (Figure 4B) . Plasma levels of the sICPs in IMID and non-IMID PAH patients are depicted in Figure 5 . Compared to non-IMID PAH patients, IMID-PAH patients exhibited significantly higher levels of sICPs, including sPD-1, sPD-L2, sBTLA, sCD137, sGITR, sHVEM, sIDO, and sLAG3 ( Figure 5). dp-ucMGP as a new biomarker for prognosis of survival in IPAH patients We aimed to explore whether dp-ucMGP levels correlate with survival in IPAH, CTD-PAH and CTEPH. Groups were stratified based on high or low dp-ucMGP plasma levels accordingly. Cut-off values were estimated based on the 50 th percentile, which was 795 in IPAH, 1029 in CTD-PAH and 847 pmol/L in CTEPH respectively. In IPAH (n=26) 1, 3 and 5 year survival was 100% in the low dp-ucMGP group, compared to 51.3%, and both 42.7% in the high dp-ucMGP group. The difference in survival based on dp-ucMGP stratification was statistically significant (p=0.003)(Figure 6). Median survival time for the high dp-ucMGP group was 1.42 years. Survival was higher than 50% in the low dp-ucMGP group and median survival time was therefore undefined. Both CTD-PAH (n=8) and CTEPH (n=11) did not show a significant difference in survival when stratified based on high or low dp-ucMGP levels (Suppl. Figure 1). When survival was analyzed based on high or low sICPs in IPAH patients, only sCD137 showed a significant difference in survival (p=0.003)(Suppl. Figure 2). Confounding factors were analyzed and showed a significant difference in DLCO and sIL-2 levels between high and low dp-ucMGP patients (Suppl. Figure 3). 4. Discussion In this single-center prospective cohort study we found significantly increased dp-ucMGP plasma levels in all PAH subtypes (IPAH, CTD-PAH, CTEPH) and CTD patients compared to age-matched healthy controls. All PAH patients demonstrated significantly higher iVSMC calcification compared to age-matched controls. Furthermore, while plasma dp-ucMGP levels of IPAH patients did not differ from CTD-PAH patients, IMID-PAH patients had significantly higher levels than non-IMID-PAH patients. IMID-PAH patients also exhibited higher levels of sPD-1, sPD-L2, sBTLA, sCD137, sGITR, sHVEM, sIDO, and sLAG3 compared to non-IMID PAH patients. Importantly, dp-ucMGP levels correlated with several sICPs (sBTLA, sCD137, sCD152, sCD27, sCD28, sGITR, sHVEM, and sIDO) but not with iVSMC calcification. Finally, our analysis revealed that high baseline dp-ucMGP levels were associated with significantly worse survival in IPAH patients. While no confounding factors were immediately apparent, these patients did exhibit higher s-IL2 levels and lower DLCO. In recent years, dp-ucMGP emerged as a biomarker of vascular vitamin K status 35 . Epidemiological data has highlighted an inverse association between dp-ucMGP levels and vitamin K status 36 , 37 . Earlier investigations involving patients with diabetic kidney disease and systemic sclerosis (SSc) have also documented elevated plasma levels of dp-ucMGP 38 , 39 . This plasma concentration is notably associated with heightened risks of cardiovascular mortality and vascular calcification as has been shown in hemodialysis patients as well as individuals with diabetes and heart failure 40 . However, the reason for the elevated dp-ucMGP levels has remained unclear. MGP is mostly studied for its protective role in vascular calcification. Vascular calcification triggers increased MGP transcription and, upon exhaustion of vitamin K, produces the inactive dp-ucMGP, which is released into the systemic circulation 41 . MGP contains five γ-carboxyglutamate amino-acid residues requiring post-translational carboxylation to be activated 42 . In addition to carboxylation, phosphorylation of the serine residues is important for the role of MGP in inhibiting vascular calcification 42 . MGP can bind directly to calcium-crystals thereby inhibiting further crystal growth 43 . Interestingly, there are similarities between the underlying mechanisms causing vascular calcification and the pathophysiological mechanisms involved in the development of PAH. MGP also binds to BMP-2 and 4 inhibiting their binding to the BMPR-2 43 . Recently, the research group led by Ten Dijke proposed a mechanism wherein pro-inflammatory cytokines IL-1b and TNF induce endothelial-to-mesenchymal transition (endoMT), sensitizing newly formed VSMCs to BMP-9 44 . This induction promotes vascular calcification, with downregulation of the BMPR2 receptor identified as a crucial event in this process. Given that these pathophysiological mechanisms coincide with the dysfunctional pathways observed in PAH, particularly the downregulation of BMPR2, which is currently recognized as an effective therapeutic target influenced through Sotatercept, a potential common mechanism is conceivable 17 , 18 , 21 , 45 . However, vascular calcification has not yet been demonstrated in the context of PAH and is currently not acknowledged as a prominent histopathological characteristic 46 . This indicates that BMP2R may be involved through a different mechanism. In PAH, features such as intimal fibrosis, arterial muscularization, and progressive VSMC proliferation are more evident 4 , 47 . Despite the pathophysiological connection between calcification and MGP, this correlation does not seem to extend to the inactive form 48 . While we also did not find a direct correlation between dp-ucMGP levels and calcification in this study, all PAH and CTD patients demonstrated increased calcification signals in the Biohybrid assay. This finding, rather than solely indicating calcification, may suggest a shift toward a more synthetic VSMC phenotype. Such a phenotypic change is associated with the extensive vascular remodeling observed in both PAH and CTD. As many of these mechanisms are multifaceted, certain features may instigate VSMC calcification or processes more commonly observed in PAH, such as VSMC proliferation or, potentially, VSMC senescence or inflammatory phenotype transition 4 . Increased dp-ucMGP levels in this cohort of patients with extensive pulmonary or systemic vascular remodeling have led to questions by which mechanisms these levels are increased. The precise triggers of increased dp-ucMGP plasma levels in our patients, as well as the primary cell types responsible, remain to be elucidated. It is necessary to determine whether there is inhibition in the conversion to active MGP, increased mechanisms that could lead to MGP deactivation, the potential impact of endoplasmic reticulum stress, and the contribution of inflammatory processes in these mechanisms. Additional in-vitro experiments, particularly utilizing material from PAH patients, should be directed toward elucidating these mechanisms. Considering prior evidence indicating that a subset of IPAH patients may display greater immunological involvement than others, the importance of a biomarker capable of distinguishing these patients has greatly increased 7 , 11 – 13 , 49 . This is particularly important as current therapeutic strategies for both IPAH and mostly CTD-PAH patients, with the exception for SLE-PAH, do not involve immunosuppressive treatment 14 . Instead, the focus currently remains on vasodilative therapy, which is not curative 1 . In this study, we have shown a strong connection between increased dp-ucMGP levels and inflammation. Both PAH and CTD patients with higher sICP levels and/or clinical signs of an immune-mediated disease have shown increased levels of dp-ucMGP. Besides, plasma levels of dp-ucMGP were significantly elevated in patients with CTD-PAH compared to those with CTD alone. This difference may be attributed to the various underlying systemic diseases present in our CTD-PAH patients. As SSc patients often exhibit subtle systemic immunological activity after the early inflammatory phase in comparison to conditions like SLE or rheumatoid arthritis, there is a potential for increased inflammation in the CTD-PAH group compared to our predominantly SSc based CTD patient cohort 50 – 52 . sICPs, far from being mere degradation by-products, actively modulate immune responses both locally and systemically. Compared to traditional markers like CRP, they offer a more nuanced view of inflammation. While the proteins responsible for co-stimulation or inhibition between antigen-presenting cells and T-cells function directly on the cell surface, their soluble forms may operate differently. Our findings of elevated sPD1 in IMID-PAH patients, alongside increased sTIM3, sLAG3, and sCD152 in both IPAH and CTD patients, point towards T-cell exhaustion 23 . This suggests a long-term ongoing inflammatory response within these patient populations. Our study, suggests that immune involvement in PAH extends beyond CTD-PAH alone. Since dp-ucMGP is correlated with sICPs and clinical signs of immune-mediated disease, this could be a valuable biomarker for distinguishing IMID and non-IMID PAH. Further research with a larger cohort and additional immunological markers, such as interleukins, chemokines, and comparison with immune-cell surface checkpoint proteins, is needed to provide a more detailed perspective of immune activation in specific IPAH patient subtypes who might benefit from immunosuppressive treatment. Most notably, high plasma dp-ucMGP levels demonstrated poorer survival outcomes in IPAH patients. Given its correlation with inflammation and the frequently poorer survival observed in patients with CTD-PAH compared to IPAH patients, inflammation may contribute to inferior survival in the high dp-ucMGP group 53 . Elevated sIL-2 levels, without increased CRP, further supports the presence of a selective T-cell-mediated immune response. Additionally, the substantially lower DLCO levels in these patients, without underlying parenchymal lung disease, suggest extensive vascular remodeling and inflammation as a cause for higher mortality. These findings highlight the urgent need to identify PAH patients with distinct immunological profiles, as they may benefit from targeted immunosuppressive therapy to potentially improve disease outcomes. dp-ucMGP did not exhibit efficacy as a survival marker in CTEPH patients, potentially due to a different underlying pathophysiological mechanism. While the study of dp-ucMGP as a survival marker in CTD-PAH patients did not yield significant results, it is important to note the limited sample size in this group. Limitations of this study include the small number of patients in the CTD-PAH and CTEPH groups, as well as variability in disease duration before presentation, a common challenge in diagnosing PAH. Future investigations should delve into the clinical role of dp-ucMGP as a biomarker for inflammation and in-vitro experiments in PAH to establish a compelling rationale for incorporating immunosuppressive treatment in these patients. In conclusion, this study highlights the potential significance of dp-ucMGP as a biomarker in PAH, particularly in IPAH, where elevated levels are associated with immune-mediated disease and poorer survival outcomes. The observed correlations with immune-mediated markers and the lack of correlation with calcification levels indicate the need for further research to elucidate the complex molecular mechanisms involved in vascular remodeling underlying these associations. These findings contribute to our understanding of the pathophysiology of PAH and may pave the way for the development of novel diagnostic and therapeutic strategies. Declarations Acknowledgements We gratefully thank Lucienne Debrus-Palmans and Nele Bijnens (Laboratory of Clinical Immunology, Maastricht University Medical Center, Maastricht, The Netherlands) and Lisette Unghethum, Petra Lux, and Cecile Maassen for their technical assistance (Laboratory of Biochemistry, Maastricht University, Maastricht, The Netherlands). We also thank Leon Frenken, MD, for his contribution to the collection of clinical data for this cohort. Author contributions RT wrote the concept of this research article which was adapted to the final version by the other authors. RT and DvD performed the statistical analysis and RT prepared the figures. The final version was approved by all authors. All authors contributed to the concept of the research article. Conflict of Interest The authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest. Data Availability Data of this research article is provided within the manuscript or supplementary information files References Humbert, M. , et al. 2022 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Respir J 61 (2023). Rawlings, G.H. , et al. Adults' experiences of living with pulmonary hypertension: a thematic synthesis of qualitative studies. BMJ Open 10 , e041428 (2020). Chang, K.Y. , et al. Mortality in Pulmonary Arterial Hypertension in the Modern Era: Early Insights From the Pulmonary Hypertension Association Registry. J Am Heart Assoc 11 , e024969 (2022). Tobal, R. , et al. Vascular Remodeling in Pulmonary Arterial Hypertension: The Potential Involvement of Innate and Adaptive Immunity. Front Med (Lausanne) 8 , 806899 (2021). Thoreau, B. & Mouthon, L. Pulmonary arterial hypertension associated with connective tissue diseases (CTD-PAH): Recent and advanced data. Autoimmun Rev , 103506 (2023). Koudstaal, T., Boomars, K.A. & Kool, M. Pulmonary Arterial Hypertension and Chronic Thromboembolic Pulmonary Hypertension: An Immunological Perspective. J Clin Med 9 (2020). Heukels, P. , et al. Loss of immune homeostasis in patients with idiopathic pulmonary arterial hypertension. Thorax 76 , 1209-1218 (2021). Funk-Hilsdorf, T.C., Behrens, F., Grune, J. & Simmons, S. Dysregulated Immunity in Pulmonary Hypertension: From Companion to Composer. Front Physiol 13 , 819145 (2022). Arends, S.J. , et al. Prevalence of anti-endothelial cell antibodies in idiopathic pulmonary arterial hypertension. Eur Respir J 35 , 923-925 (2010). Marsh, L.M. , et al. The inflammatory cell landscape in the lungs of patients with idiopathic pulmonary arterial hypertension. Eur Respir J 51 (2018). Koudstaal, T. , et al. Plasma markers in pulmonary hypertension subgroups correlate with patient survival. Respir Res 22 , 137 (2021). Koudstaal, T. & Boomars, K.A. Inflammatory biomarkers in pulmonary arterial hypertension: ready for clinical implementation? Eur Respir J 61 (2023). Arends, S.J. , et al. Functional implications of IgG anti-endothelial cell antibodies in pulmonary arterial hypertension. Autoimmunity 46 , 463-470 (2013). Ding, Y. , et al. Immunosuppressive therapy in patients with connective tissue disease-associated pulmonary arterial hypertension: A systematic review. Int J Rheum Dis 25 , 982-990 (2022). Potjewijd, J. , et al. Plasma Dephosphorylated-Uncarboxylated Matrix Gla-Protein in Systemic Sclerosis Patients: Biomarker Potential for Vascular Calcification and Inflammation. Diagnostics (Basel) 13 (2023). Schurgers, L.J., Cranenburg, E.C. & Vermeer, C. Matrix Gla-protein: the calcification inhibitor in need of vitamin K. Thromb Haemost 100 , 593-603 (2008). Xu, B., Xu, G., Yu, Y. & Lin, J. The role of TGF-beta or BMPR2 signaling pathway-related miRNA in pulmonary arterial hypertension and systemic sclerosis. Arthritis Res Ther 23 , 288 (2021). Tielemans, B., Delcroix, M., Belge, C. & Quarck, R. TGFbeta and BMPRII signalling pathways in the pathogenesis of pulmonary arterial hypertension. Drug Discov Today 24 , 703-716 (2019). Derk, C.T. Transforming growth factor-beta (TGF-beta) and its role in the pathogenesis of systemic sclerosis: a novel target for therapy? Recent Pat Inflamm Allergy Drug Discov 1 , 142-145 (2007). Lomeli-Nieto, J.A. , et al. Transforming growth factor beta isoforms and TGF-betaR1 and TGF-betaR2 expression in systemic sclerosis patients. Clin Exp Med 23 , 471-481 (2023). Tatius, B., Wasityastuti, W., Astarini, F.D. & Nugrahaningsih, D.A.A. Significance of BMPR2 mutations in pulmonary arterial hypertension. Respir Investig 59 , 397-407 (2021). Riva, A. Editorial: Soluble immune checkpoints: Novel physiological immunomodulators. Front Immunol 14 , 1178541 (2023). Blank, C.U. , et al. Defining 'T cell exhaustion'. Nat Rev Immunol 19 , 665-674 (2019). van den Hoogen, F. , et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthritis Rheum 65 , 2737-2747 (2013). Humbert, M. , et al. 2022 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Heart J 43 , 3618-3731 (2022). Petri, M. , et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 64 , 2677-2686 (2012). Cappelli, S. , et al. "To be or not to be," ten years after: evidence for mixed connective tissue disease as a distinct entity. Semin Arthritis Rheum 41 , 589-598 (2012). Aletaha, D. , et al. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 62 , 2569-2581 (2010). Jennette, J.C. , et al. 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum 65 , 1-11 (2013). Lundberg, I.E. , et al. 2017 European League Against Rheumatism/American College of Rheumatology Classification Criteria for Adult and Juvenile Idiopathic Inflammatory Myopathies and Their Major Subgroups. Arthritis Rheumatol 69 , 2271-2282 (2017). Dai, L. , et al. Functional vitamin K insufficiency, vascular calcification and mortality in advanced chronic kidney disease: A cohort study. PLoS One 16 , e0247623 (2021). Rapp, N. , et al. Hepatic and Vascular Vitamin K Status in Patients with High Cardiovascular Risk. Nutrients 13 (2021). Jaminon, A.M.G. , et al. Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity. Cells 10 (2021). Jaminon, A.M.G., Akbulut, A.C., Rapp, N., Reutelingsperger, C.P. & Schurgers, L.J. The BioHybrid Assay: A Novel Method for Determining Calcification Propensity. Methods Mol Biol 2664 , 317-331 (2023). Roumeliotis, S., Dounousi, E., Eleftheriadis, T. & Liakopoulos, V. Association of the Inactive Circulating Matrix Gla Protein with Vitamin K Intake, Calcification, Mortality, and Cardiovascular Disease: A Review. Int J Mol Sci 20 (2019). Boxma, P.Y. , et al. Vitamin k intake and plasma desphospho-uncarboxylated matrix Gla-protein levels in kidney transplant recipients. PLoS One 7 , e47991 (2012). Caluwe, R., Vandecasteele, S., Van Vlem, B., Vermeer, C. & De Vriese, A.S. Vitamin K2 supplementation in haemodialysis patients: a randomized dose-finding study. Nephrol Dial Transplant 29 , 1385-1390 (2014). Roumeliotis, S. , et al. The Association of dp-ucMGP with Cardiovascular Morbidity and Decreased Renal Function in Diabetic Chronic Kidney Disease. Int J Mol Sci 21 (2020). Griffin, T.P. , et al. Plasma dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP): reference intervals in Caucasian adults and diabetic kidney disease biomarker potential. Sci Rep 9 , 18452 (2019). Dahlberg, S. , et al. Desphospho-Uncarboxylated Matrix-Gla Protein Is Increased Postoperatively in Cardiovascular Risk Patients. Nutrients 10 (2018). Houben, E., Neradova, A., Schurgers, L.J. & Vervloet, M. The influence of phosphate, calcium and magnesium on matrix Gla-protein and vascular calcification: a systematic review. G Ital Nefrol 33 (2016). Schurgers, L.J. , et al. Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification. J Thromb Haemost 5 , 2503-2511 (2007). Schurgers, L.J., Uitto, J. & Reutelingsperger, C.P. Vitamin K-dependent carboxylation of matrix Gla-protein: a crucial switch to control ectopic mineralization. Trends Mol Med 19 , 217-226 (2013). Sanchez-Duffhues, G. , et al. Inflammation induces endothelial-to-mesenchymal transition and promotes vascular calcification through downregulation of BMPR2. J Pathol 247 , 333-346 (2019). Hoeper, M.M. , et al. Phase 3 Trial of Sotatercept for Treatment of Pulmonary Arterial Hypertension. N Engl J Med 388 , 1478-1490 (2023). Liu, S.F. , et al. Pulmonary hypertension: Linking inflammation and pulmonary arterial stiffening. Front Immunol 13 , 959209 (2022). Stacher, E. , et al. Modern age pathology of pulmonary arterial hypertension. Am J Respir Crit Care Med 186 , 261-272 (2012). Jaminon, A.M.G. , et al. Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease. Sci Rep 10 , 6586 (2020). Price, L.C. , et al. Inflammation in pulmonary arterial hypertension. Chest 141 , 210-221 (2012). Cossu, M. , et al. Earliest Phase of Systemic Sclerosis Typified by Increased Levels of Inflammatory Proteins in the Serum. Arthritis Rheumatol 69 , 2359-2369 (2017). Potjewijd, J. , et al. Favorable long term effects of intensified immunosuppression combined with therapeutic plasma exchange in patients with early-onset progressive systemic sclerosis-related interstitial lung disease. J Transl Autoimmun 5 , 100174 (2022). Bellocchi, C., Chung, A. & Volkmann, E.R. Predicting the Progression of Very Early Systemic Sclerosis: Current Insights. Open Access Rheumatol 14 , 171-186 (2022). Distler, O. , et al. Treatment strategies and survival of patients with connective tissue disease and pulmonary arterial hypertension: A COMPERA analysis. Rheumatology (Oxford) (2023). Table Table 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 10 Jul, 2024 Reviews received at journal 09 Jun, 2024 Reviewers agreed at journal 27 May, 2024 Reviews received at journal 22 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviewers invited by journal 03 May, 2024 Editor assigned by journal 03 May, 2024 Editor invited by journal 30 Apr, 2024 Submission checks completed at journal 30 Apr, 2024 First submitted to journal 26 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4329956","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":299217033,"identity":"91afb137-0d01-453d-b3d1-2b67616e6a44","order_by":0,"name":"R. Tobal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACNoYENiAlwcB44ANEhPEAMVokgJDh4AyoEGEtDFAth3mI0WLefvjZgwdlDHX80s0PDtvmHI7mb+A9gFeLzJk0c4OEcwwSknOOGRzO3XY4d8YBvgS8WiQYctgkEtsYJAxuJEC0bGDgMcCvhf8NTEv6h8OWRGmRgNuSY3CYkTgtz8wkEs5JSM6cc6bgYO+29NwZhwk6LPmZ5I8yG35+6faND35us87tb+8xfIBPC0wnEpuZCPWjYBSMglEwCvADAFXYSt4nBpOZAAAAAElFTkSuQmCC","orcid":"","institution":"Maastricht University Medical Center","correspondingAuthor":true,"prefix":"","firstName":"R.","middleName":"","lastName":"Tobal","suffix":""},{"id":299217034,"identity":"b0b5d795-e1a7-4052-bd97-338a1128d072","order_by":1,"name":"J. Potjewijd","email":"","orcid":"","institution":"Maastricht University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"","lastName":"Potjewijd","suffix":""},{"id":299217035,"identity":"5352fbb3-165f-4c8e-8f73-7e1df62808f2","order_by":2,"name":"F. Vries","email":"","orcid":"","institution":"CARIM, Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"F.","middleName":"","lastName":"Vries","suffix":""},{"id":299217036,"identity":"001326f7-f768-4fab-a9d1-f91f2c644f86","order_by":3,"name":"D. P.C. Doorn","email":"","orcid":"","institution":"Maastricht University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"P.C.","lastName":"Doorn","suffix":""},{"id":299217037,"identity":"78916681-2d10-4a0f-84a9-ef9011e03e4c","order_by":4,"name":"A. Jaminon","email":"","orcid":"","institution":"CARIM, Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Jaminon","suffix":""},{"id":299217038,"identity":"60db247a-cd04-49bb-a945-280697b5dce5","order_by":5,"name":"R. Bittner","email":"","orcid":"","institution":"CARIM, Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"","lastName":"Bittner","suffix":""},{"id":299217039,"identity":"fa188136-8dd5-426c-b7e7-7697bd780737","order_by":6,"name":"C. Akbulut","email":"","orcid":"","institution":"CARIM, Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"","lastName":"Akbulut","suffix":""},{"id":299217040,"identity":"6d399a87-23ee-4026-8ace-aaaf2a0b6530","order_by":7,"name":"V. Empel","email":"","orcid":"","institution":"Maastricht University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"V.","middleName":"","lastName":"Empel","suffix":""},{"id":299217041,"identity":"8079ca75-d2e1-40a4-a5b4-5fea5b40dc42","order_by":8,"name":"P. Heeringa","email":"","orcid":"","institution":"University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"","lastName":"Heeringa","suffix":""},{"id":299217042,"identity":"9288e882-ee8f-44d2-8c5c-c04e9469a0d4","order_by":9,"name":"J. Damoiseaux","email":"","orcid":"","institution":"Maastricht University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"","lastName":"Damoiseaux","suffix":""},{"id":299217043,"identity":"c5dc1e0e-68ec-4373-abaf-4235848165ab","order_by":10,"name":"L. Schurgers","email":"","orcid":"","institution":"CARIM, Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"L.","middleName":"","lastName":"Schurgers","suffix":""},{"id":299217044,"identity":"46c830ce-1bb4-43e4-90b9-8c325c4f6a2c","order_by":11,"name":"P. Paassen","email":"","orcid":"","institution":"Maastricht University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"","lastName":"Paassen","suffix":""}],"badges":[],"createdAt":"2024-04-26 13:46:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4329956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4329956/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-77000-w","type":"published","date":"2024-11-04T15:58:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56122175,"identity":"db05e8fa-dd3b-4430-aecd-01c3b652fc96","added_by":"auto","created_at":"2024-05-08 20:31:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1374845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma levels of dp-ucMGP in IPAH, CTD-PAH, CTEPH, and CTD patients.\u003c/strong\u003e A) Plasma levels of dp-ucMGP in IPAH (n=26), CTD-PAH (n=8), CTEPH (n=11) and CTD (n=28) patients. B) Plasma levels of dp-ucMGP in non-IMID-PAH (n=8) and IMID-PAH (n=26) patients\u003cstrong\u003e. \u003c/strong\u003eData are shown as symbols for individual patients or HCs. Patients depicted as red dots are categorized as CTD-PAH (n=8). The horizontal dotted line represents the mean value of dp-ucMGP in aged-matched healthy controls (393pmol/L). Horizontal gray bars represent median values with interquartile range. \u003cem\u003eStatistical analysis was performed using the Kruskal-Wallis and Mann-Whitney test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. dp-ucMGP, dephosphorylated-uncarboxylated Matrix-GLA-Protein; IPAH, idiopathic pulmonary hypertension; CTD-PAH, connective tissue associated pulmonary arterial hypertension; CTEPH, chronic thrombo-embolic pulmonary hypertension; CTD, connective tissue disease; IMID, immune mediated inflammatory disease.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/85bf16d3bd2dfc1ae87c4b68.png"},{"id":56122177,"identity":"b740f3e1-5a86-4798-aa1a-5fda07886771","added_by":"auto","created_at":"2024-05-08 20:31:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1453056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmount of plasma-induced calcification of induced vascular smooth muscle cells \u003c/strong\u003eA) Plasma-induced iVSMC calcification in IPAH (n=26), CTD-PAH (n=8), CTEPH (n=11), and CTD (n=28) patients. B) plasma-induced iVSMC calcification in IPAH non-IMID-PAH (n=8) and IMID-PAH (n=26) patients. Horizontal bars represent median values with interquartile range. \u003cem\u003eStatistical analysis was performed using the Kruskal-Wallis and Mann-Whitney test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. RFP, red fluorescent protein; HC, healthy controls; IPAH, idiopathic pulmonary hypertension; CTD-PAH, connective tissue associated pulmonary arterial hypertension; CTEPH, chronic thrombo-embolic pulmonary hypertension; CTD, connective tissue disease; IMID, immune-mediated inflammatory disease.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/d5f76fbf28241469ea56139c.png"},{"id":56122178,"identity":"26e6b300-202f-4294-94e0-331b09480f12","added_by":"auto","created_at":"2024-05-08 20:31:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2137948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma levels of different soluble immune checkpoint proteins (sICPs) in IPAH, CTD patients and healthy controls.\u003c/strong\u003e The indicated soluble ICs were measured in plasma samples of 24 IPAH patients and 27 CTD patients. sICPs are classified as either co-stimulatory or co-inhibitory and are found on either T-cells or antigen-presenting cells. Horizontal bars represent median values with interquartile range. \u003cem\u003eStatistical analysis was performed using the Kruskal-Wallis test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.. sPD1, programmed death protein-1; sPDL2, programmed death ligand-2; sBTLA, soluble B- and T-lymphocyte attenuator; cluster of differentiation (CD)27, CD28, CD80, CD137 and CD152 (CTLA4/cytotoxic T-lymphocyte-associated antigen-3; sGITR, glucocorticoid-induced TNFR-related protein; sHVEM herpesvirus entry mediator; sIDO, indoleamine 2,3-dioxygenase; sLAG3, Lymphocyte-activation gene 3; sTIM3, T-cell immunoglobulin and mucin-domain containing-3\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/4e10a5a8b9c3ce281c73c95d.png"},{"id":56122176,"identity":"6c7940bb-f640-4f02-af14-07a170048c31","added_by":"auto","created_at":"2024-05-08 20:31:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation matrices for different soluble immune checkpoint proteins (sICPs) with dp-ucMGP and calcification in IPAH (n=26) and CTD (n=28) patients.\u003c/strong\u003e The c\u003cem\u003eorrelation coefficient was calculated using nonparametric Spearman pairwise correlation.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/98d2c7fce91e3b1a3a9abc8c.png"},{"id":56122179,"identity":"b065b482-66b7-4644-94e5-2fcadac54932","added_by":"auto","created_at":"2024-05-08 20:31:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1823439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma levels of different soluble immune checkpoint proteins (sICPs) in non-IMID-PAH and IMID-PAH patients.\u003c/strong\u003e The indicated soluble ICs were measured in plasma samples of non-IMID-PAH (n=8) patients and IMID-PAH (n=19) patients. Horizontal bars represent median values with interquartile range. \u003cem\u003eStatistical analysis was performed using the Mann-Whitney test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/8b62f4943c21a65232851b25.png"},{"id":56122180,"identity":"6bdc6fb5-d907-4c52-970b-6f74a7418d52","added_by":"auto","created_at":"2024-05-08 20:31:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":266544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan-Meyer survival analyses for dp-ucMGP high/low of IPAH patients (n=26).\u003c/strong\u003e \u003cem\u003eHigh/low cut-off values were based on the 50\u003c/em\u003e\u003csup\u003e\u003cem\u003eth\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e percentile of dp-ucMGP plasma levels. Statistical analysis was performed using the log-rank test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/82827b20ba1255c48ed1c018.png"},{"id":68750092,"identity":"715eb531-10dc-4224-b8ea-5a5b880d4074","added_by":"auto","created_at":"2024-11-11 16:09:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7707177,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4329956/v1/fe1b2339-b320-48c2-97da-11c093cdb52e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dephosphorylated uncarboxylated Matrix-Gla-Protein and vascular remodeling in pulmonary hypertension: an immunological connection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePulmonary arterial hypertension (PAH) is a complex and debilitating condition characterized by elevated blood pressure within the pulmonary arteries, leading to increased strain on the right side of the heart \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This chronic condition has a great impact on patient well-being and life expectancy\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The increase in pulmonary arterial pressure parallels structural changes in the pulmonary vasculature, which is characterized by increased muscularization, fibrosis, and gradual narrowing of blood vessels\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This pulmonary vascular remodeling involves the complex and incompletely understood interaction of endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and fibroblasts\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The immune system is an important driver of this interaction, which is best illustrated by the elevated risk of developing PAH in patients with systemic autoimmune diseases such as systemic sclerosis (SSc) and systemic lupus erythematosus (SLE)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Interestingly, even in idiopathic PAH (IPAH) there is ample evidence for immune activation and dysregulation during the disease course\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. T -and B-cell infiltrates have been found around prototypical pulmonary vascular lesions in PAH patients and in both preclinical models and in numerous patients, autoantibodies such as anti-endothelial cell antibodies, immune complexes, or increased cytokine levels have been observed\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. IgG from a subset of IPAH patients induces increased vascular cellular adhesion molecule-1 (VCAM-1), integrin cellular adhesion molecule-1 (ICAM-1), monocyte chemo-attractant protein-1 MCP-1 and interleukin-6 (IL-6) production in cultured endothelial cells\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the diagnostic and therapeutic approach with focus on the role of the immune system in the clinical practice of PAH patients is ill-defined and non-standardized. The strongest benefit from immunosuppression is shown in SLE-PAH, whereas this treatment is less or not effective in limited SSc-PAH\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This may indicate differences in pathogenesis and disease progression but also highlights the diagnostic delay in many PAH patients. Therefore, novel biomarkers are necessary to improve a more detailed understanding of the role and stage of immune activation related to the process of pulmonary vascular remodeling leading to PAH. We hypothesize that dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP) could be such a novel marker. We recently showed that plasma levels were increased in early systemic sclerosis patients, as compared to age-matched healthy controls\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Notably, dp-ucMGP levels exhibited a positive correlation with disease severity and the incidence of cardiovascular events in these patients. These findings were not explained by traditional cardiovascular risk factors. MGP is mostly known as a protein (14 kD) predominantly secreted by VSMCs and ECs, which plays a pivotal role as a vitamin K-dependent protein in inhibiting both intimal and medial vascular calcification \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. dp-ucMGP represents the fully inactive form, lacking the ability to bind calcium or extracellular matrix, and is consequently released into the circulation. SSc patients have a high risk of developing PAH over time, so we questioned whether dp-ucMGP may provide a link between inflammation and vasculopathy in PAH. Such a connection has also been suggested between the increased signaling pathways of TGF- β and bone morphogenetic protein 4 (BMP-4) that is observed in both systemic sclerosis and PAH\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the present study we first measure plasma MGP in patients with idiopathic PAH (IPAH), connective tissue disease-associated PAH (CTD-PAH), chronic thromboembolic pulmonary hypertension (CTEPH), and in patients suffering from connective tissue disease (CTD) alone. We subsequently classify all patients as either immune-mediated inflammatory PAH (IMID-PAH) or non-IMID PAH according to the strict but non-validated criteria we use in our clinical practice. In addition, we measure soluble immune checkpoint proteins (sICPs), once thought to be solely cell membrane-bound regulators of immune cell function, but now recognized as biologically active molecules similar to stimulatory or inhibitory cytokines\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We aim to explore whether sICPs are a valuable tool for detecting immune profiles within PAH and whether these profiles support the concept of IMID-PAH. Lastly, we investigated the potential variations in dp-ucMGP levels across distinct subtypes of pulmonary hypertension in relation to circulating levels of sICPs and functional vascular calcification as a readout of vascular damage in these patients. Altogether, we hypothesize that plasma dp-ucMGP may serve as a valuable biomarker for both immune-mediated disease severity and survival prognosis in PAH.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e \u003cb\u003eStudy design and participants\u003c/b\u003e \u003c/p\u003e \u003cp\u003e This study cohort consists of participants from the volatile organic compound-PAH (VOC-PAH) study, an observational prospective cohort study in IPAH, CTD-PAH, CTEPH, and CTD patients conducted between 2018 and 2023 at the Maastricht University Medical Center, Center of Expertise for PAH, in the Netherlands. The study was performed in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice, and approval was obtained from the local ethics committee. Written informed consent was obtained from each participant. This study is registered with clinicaltrials.gov number NCT03819777. Adults with IPAH, CTD-PAH, CTEPH, or CTD classified according to current criteria \u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e were included. The exclusion criteria were not being treatment na\u0026iuml;ve, age\u0026thinsp;\u0026lt;\u0026thinsp;18 years, or incapable of understanding or signing informed consent. All patients were clinically analyzed for the presence of an underlying auto-immune disease and received a comprehensive immunological assessment by a clinical immunologist. In this study, IPAH patients were subcategorized according to clinical signs of immune-mediated inflammatory disease (IMID). This was the case when there was the presence of auto-antibodies and increased inflammatory parameters, i.e., increased soluble-interleukin-2 receptor (sIL-2R) with low C-reactive protein (CRP) and/or signs of immune activation based on increased IgM rheumatoid factor or cryoglobulinemia. (Sub)acute infection was ruled out as an underlying cause based on clinical evaluation. CTD-PAH patients were also categorized as IMID due to the autoimmune nature of their underlying connective tissue disease. CTD patients without PAH were used as a disease control cohort. The disease duration of the CTD patients was defined as the time from the first non-Raynaud phenomenon manifestation; the IPAH patients were included within one month of diagnosis by right heart catheterization. Oral corticosteroids and hydroxychloroquine were permitted if the patient was on a stable dose regimen for more than three months prior to the baseline visit. No other background immunomodulatory therapies were allowed. Demographic data, as well as data on inflammatory parameters in the blood (C-reactive protein (CRP), soluble interleukin-2 receptor (sIL2r)), pulmonary function tests (percentage of predicted forced vital capacity (%pFVC), and percentage of predicted diffusing capacity for carbon monoxide (%pDLCO)), 6-minutes walking test (6-MWT), New-York Heart Association (NYHA) class, echocardiography and if appropriate, right-sided heart catheterization, were recorded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollection and processing of samples\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBlood samples were collected in ethylenediaminetetraacetic acid (EDTA) vials and centrifuged at 1300g and room temperature for 10 minutes. The supernatant was carefully obtained and subsequently stored at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of plasma dp-ucMGP\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCirculating dp-ucMGP levels were determined in EDTA plasma in a single run by the Laboratory of Coagulation Profile (Maastricht, the Netherlands) using the commercially available IVD CE marked chemiluminescent InaKtif MGP assay on the IDS-iSYS system (IDS, Boldon, UK) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Patient samples were incubated with magnetic particles coated with murine monoclonal antibodies against dpMGP, acridinium-labeled murine monoclonal antibodies against ucMGP, and assay buffer. The magnetic particles were captured using a magnet and washed to remove any unbound analyte. Trigger reagents were added, and the resulting light emitted by the acridinium label was directly proportional to the concentration of dp-ucMGP in the sample. This assay's within-run and total precision were 0.8\u0026ndash;6.2% and 3.0\u0026ndash;8.2%, respectively. The assay measuring range was between 300 and 12,000 pmol/L and was found to be linear up to 11,651 pmol/L.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVascular smooth muscle cell calcification assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCalcification experiments were conducted with induced pluripotent stem cell (iPSC)-derived iVSMCs. iVSMCs were seeded in culture well plates at 1.0x10\u003csup\u003e4\u003c/sup\u003e cells/well. After 24h, iVSMCs were cultured in calcification medium (DMEM 31966, 5% IPAH, CTD-PAH, CTEPH or CTD patient plasma, 1% penicillin-streptomycin solution, and 3.6 mM Ca\u003csup\u003e2+\u003c/sup\u003e) for up to 9 days. Plasma samples were supplemented with 14.3 nM Hirudin to prevent coagulation upon re-calcifying the plasma. 5% healthy control pooled plasma was used as a negative control due to the lower magnitude of calcification. For the BioHybrid assay, Fetuin-A AlexaFluor-546 (1\u0026micro;g/mL; prepared in-house) and Hoechst 33,342 (0.5 \u0026micro;g/mL, Invitrogen, Waltham, MA, USA) were supplemented at the start of the experiment \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. At various time points after calcification induction, fluorescent images were taken in the RFP and DAPI channel (for cell count) were imaged for up to 9 days. Imaging was done with the Cytation 3 system (BioSPX, Abcoude, the Netherlands) and analyzed using Gen5 software v.3.10. (BioTek, Abcoude, the Netherlands). As readout, fluorescent RFP area/well was normalized against cell count.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of soluble immune checkpoints\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe plasma concentrations of the sICPs were quantified using a commercially available multiplex immunoassay (Life Technologies; ProcartaPL HU-IMM96T: EPX140-15803-901), which measures the soluble concentrations of B- and T-lymphocyte attenuator (BTLA), glucocorticoid-induced TNFR-related protein (GITR), herpesvirus entry mediator (HVEM), indoleamine 2,3-dioxygenase (IDO), Lymphocyte-activation gene 3 (LAG3), programmed death protein-1 (PD1), programmed death ligand-1 (PDL1), programmed death ligand-2 (PDL2), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), cluster of differentiation (CD)27, CD28, CD80, CD137 and CD152 (CTLA4/cytotoxic T-lymphocyte-associated antigen 4). The manufacturer\u0026rsquo;s protocols were followed by running patient samples in duplicates with 25 \u0026micro;L of plasma each and were measured using Magpix (Darmstadt, Germany). Data were analyzed using the xPONENT 4.2 software (Luminex). The given values represent the mean of the duplicates given in pg/mL. For checkpoint PDL1, many values were found to fall below the standard curve, and these results were excluded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCategorical baseline characteristics were expressed as numbers and percentages. Numerical baseline characteristics were expressed as mean and standard\u003c/p\u003e \u003cp\u003edeviation (SD) or median and interquartile range (IQR) based on normal distribution estimated by Shapiro/Wilk test and data visualization. Statistics were performed using the ANOVA, Chi-Square, Mann-Withney and Fisher\u0026rsquo;s Exact tests. The Kaplan-Meier curves were analyzed by the Log-Rank test to estimate differences in survival. The statistical analysis was performed using the IBM SPSS software version 24.0. A p-value less than 0.05 was considered statistically significant. Figures were made with GraphPad Prism version 9.5.0.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003eClinical characteristics of the study cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty-five PAH patients and 28 CTD disease controls without PAH were included from the prospective VOC-PAH cohort for dp-ucMGP and calcification analysis at baseline. PAH patients were classified in IPAH (\u003cem\u003en\u003c/em\u003e= 26), CTD-PAH (\u003cem\u003en\u003c/em\u003e= 8), and CTEPH (\u003cem\u003en\u003c/em\u003e= 11). The clinical characteristics at the time of presentation have been depicted in \u003cstrong\u003eTable 1\u003c/strong\u003e. Clinical characteristics of PAH patients with IMID and without IMID have been depicted in \u003cstrong\u003eSupplemental Table 1\u003c/strong\u003e. Sex, age and body mass index (BMI) did not differ significantly between the groups. Hemodynamic parameters did not differ significantly among PAH groups. Patients with IPAH were most likely to have heart failure class III or IV (\u003cem\u003en/N\u003c/em\u003e= 16/26, 62%), according to the New York Heart Association. Patients with CTEPH had the lowest forced expiratory volume after one second (FEV1), with a median percentage of 66%. Other pulmonary function parameters did not differ significantly among groups. The CTD disease control group mostly consisted of patients diagnosed with SSc (\u003cem\u003en/N\u003c/em\u003e= 27/28, 96%). sIL2R concentrations were the highest in patients with CTD-PAH, with a median of 910 U/mL. CRP concentrations, however, did not differ significantly among groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003edp-ucMGP plasma levels in treatment-na\u0026iuml;ve IPAH, CTD-PAH, and CTEPH patients at diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma dp-ucMGP levels of 26 IPAH, 8 CTD-PAH and 11 CTEPH patients were compared with a control cohort of 28 age-matched CTD patients. Median dp-ucMGP levels were 795 (525.3;1027.0), 1029 (646.3;1336.0), 847 (593.0;1090.0), and 542 (374.3;757.8) pmol/L, respectively \u003cstrong\u003e(Figure 1)\u003c/strong\u003e. This is all greatly increased as compared to a median dp-ucMGP level of 375 (285.0;469.0) pmol/L in 400 age-matched Dutch HCs \u003cstrong\u003e(p\u0026lt;0.001)\u003c/strong\u003e. CTD-PAH patients had significantly higher dp-ucMGP levels than our control CTD patients \u003cstrong\u003e(p=0.017)(Figure 1A)\u003c/strong\u003e. Subsequently, we stratified IPAH and CTD-PAH patients based on clinical signs of immune-mediated inflammatory disease (IMID). dp-ucMGP plasma levels had a median of 646 (519.8;758.3) in the non-IMID group and 911 (619.0;l166.0) in the IMID group which was significantly different \u003cstrong\u003e(p=0.030)(Figure 1B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasma of PAH and CTD patients can induce calcification of vascular smooth muscle cells (iVSMCs) in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the calcification potential of the same patients, plasma-induced calcification of iVSMCs was analyzed in the Biohybrid assay. Median calcification levels were 7.8 (4.2;12.2) in IPAH, 7.5 (3.5;16.4) in CTD-PAH, 6.4 (4.5;12.0) in CTEPH, and 6.8 (4.9;11.3) RPF\u0026nbsp;mcm\u003csup\u003e2\u003c/sup\u003e/cell count in CTD patients, which were significantly increased compared to a median of 1.9 (0.07;3.2) in aged matched healthy controls \u003cstrong\u003e(Figure 2A)\u003c/strong\u003e. When IPAH and CTD-PAH patients were stratified based on IMID, no significant difference was observed with a median of 7.8 (10.4;3.9) in non-IMID PAH and 7.5 (4.;12.6)\u0026nbsp;mcm\u003csup\u003e2\u003c/sup\u003e/cell in IMID-PAH patients \u003cstrong\u003e(Figure 2B)\u003c/strong\u003e. No statistical correlation was found between dp-ucMGP plasma levels and the amount of calcification \u003cstrong\u003e(Figure 4)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoluble immune checkpoint protein levels and correlation with dp-ucMGP\u003cbr\u003e\u0026nbsp;\u003c/strong\u003esICPs were measured in plasma of 24 (92%) of the IPAH and 27 (96%) of the CTD patients.\u0026nbsp;\u003cbr\u003eInterestingly, no significant differences were observed between these groups of patients. However, both IPAH and CTD patients showed higher sBTLA, sCD137, sCD152, sLAG3 and sTIM3 levels when compared with healthy controls (\u003cstrong\u003eFigure 3)\u003c/strong\u003e. sPD-L2 levels were significantly lower in CTD patients. dp-ucMGP plasma levels were correlated in IPAH patients with sBTLA, sCD137, sCD152, sCD27, sCD28, sGITR, sHVEM, and sIDO with a mean Spearman r of 0.56 \u003cstrong\u003e(Figure 4A)\u003c/strong\u003e. In CTD patients, dp-ucMGP plasma levels were correlated with all soluble ICs with a mean Spearman r of 0.52 \u003cstrong\u003e(Figure 4B)\u003c/strong\u003e. Plasma \u0026nbsp;levels of the sICPs in IMID and non-IMID PAH patients are depicted in\u003cstrong\u003e\u0026nbsp;Figure 5\u003c/strong\u003e. Compared to non-IMID PAH patients, IMID-PAH patients exhibited significantly higher levels of sICPs, including sPD-1, sPD-L2, sBTLA, sCD137, sGITR, sHVEM, sIDO, and sLAG3 (\u003cstrong\u003eFigure 5).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003edp-ucMGP as a new biomarker for prognosis of survival in IPAH patients\u0026nbsp;\u003cbr\u003e We aimed to explore whether dp-ucMGP levels correlate with survival in IPAH, CTD-PAH and CTEPH. Groups were stratified based on high or low dp-ucMGP plasma levels accordingly. Cut-off values were estimated based on the 50\u003csup\u003eth\u003c/sup\u003e percentile, which was 795 in IPAH, 1029 in CTD-PAH and 847 pmol/L in CTEPH respectively. In IPAH (n=26) 1, 3 and 5 year survival was 100% in the low dp-ucMGP group, compared to 51.3%, and both 42.7% in the high dp-ucMGP group. The difference in survival based on dp-ucMGP stratification was statistically significant (p=0.003)(Figure 6). Median survival time for the high dp-ucMGP group was 1.42 years. Survival was higher than 50% in the low dp-ucMGP group and median survival time was therefore undefined. \u0026nbsp;Both CTD-PAH (n=8) and CTEPH (n=11) did not show a significant difference in survival when stratified based on high or low dp-ucMGP levels (Suppl. Figure 1). When survival was analyzed based on high or low sICPs in IPAH patients, only sCD137 showed a significant difference in survival (p=0.003)(Suppl. Figure 2). Confounding factors were analyzed and showed a significant difference in DLCO and sIL-2 levels between high and low dp-ucMGP patients (Suppl. Figure 3).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this single-center prospective cohort study we found significantly increased dp-ucMGP plasma levels in all PAH subtypes (IPAH, CTD-PAH, CTEPH) and CTD patients compared to age-matched healthy controls. All PAH patients demonstrated significantly higher iVSMC calcification compared to age-matched controls. Furthermore, while plasma dp-ucMGP levels of IPAH patients did not differ from CTD-PAH patients, IMID-PAH patients had significantly higher levels than non-IMID-PAH patients. IMID-PAH patients also exhibited higher levels of sPD-1, sPD-L2, sBTLA, sCD137, sGITR, sHVEM, sIDO, and sLAG3 compared to non-IMID PAH patients. Importantly, dp-ucMGP levels correlated with several sICPs (sBTLA, sCD137, sCD152, sCD27, sCD28, sGITR, sHVEM, and sIDO) but not with iVSMC calcification. Finally, our analysis revealed that high baseline dp-ucMGP levels were associated with significantly worse survival in IPAH patients. While no confounding factors were immediately apparent, these patients did exhibit higher s-IL2 levels and lower DLCO.\u003c/p\u003e \u003cp\u003eIn recent years, dp-ucMGP emerged as a biomarker of vascular vitamin K status\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Epidemiological data has highlighted an inverse association between dp-ucMGP levels and vitamin K status\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Earlier investigations involving patients with diabetic kidney disease and systemic sclerosis (SSc) have also documented elevated plasma levels of dp-ucMGP \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This plasma concentration is notably associated with heightened risks of cardiovascular mortality and vascular calcification as has been shown in hemodialysis patients as well as individuals with diabetes and heart failure \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. However, the reason for the elevated dp-ucMGP levels has remained unclear.\u003c/p\u003e \u003cp\u003eMGP is mostly studied for its protective role in vascular calcification. Vascular calcification triggers increased MGP transcription and, upon exhaustion of vitamin K, produces the inactive dp-ucMGP, which is released into the systemic circulation\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. MGP contains five γ-carboxyglutamate amino-acid residues requiring post-translational carboxylation to be activated\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In addition to carboxylation, phosphorylation of the serine residues is important for the role of MGP in inhibiting vascular calcification\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. MGP can bind directly to calcium-crystals thereby inhibiting further crystal growth\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Interestingly, there are similarities between the underlying mechanisms causing vascular calcification and the pathophysiological mechanisms involved in the development of PAH. MGP also binds to BMP-2 and 4 inhibiting their binding to the BMPR-2\u003csup\u003e43\u003c/sup\u003e. Recently, the research group led by Ten Dijke proposed a mechanism wherein pro-inflammatory cytokines IL-1b and TNF induce endothelial-to-mesenchymal transition (endoMT), sensitizing newly formed VSMCs to BMP-9\u003csup\u003e44\u003c/sup\u003e. This induction promotes vascular calcification, with downregulation of the BMPR2 receptor identified as a crucial event in this process. Given that these pathophysiological mechanisms coincide with the dysfunctional pathways observed in PAH, particularly the downregulation of BMPR2, which is currently recognized as an effective therapeutic target influenced through Sotatercept, a potential common mechanism is conceivable\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, vascular calcification has not yet been demonstrated in the context of PAH and is currently not acknowledged as a prominent histopathological characteristic\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This indicates that BMP2R may be involved through a different mechanism. In PAH, features such as intimal fibrosis, arterial muscularization, and progressive VSMC proliferation are more evident\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Despite the pathophysiological connection between calcification and MGP, this correlation does not seem to extend to the inactive form\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. While we also did not find a direct correlation between dp-ucMGP levels and calcification in this study, all PAH and CTD patients demonstrated increased calcification signals in the Biohybrid assay. This finding, rather than solely indicating calcification, may suggest a shift toward a more synthetic VSMC phenotype. Such a phenotypic change is associated with the extensive vascular remodeling observed in both PAH and CTD. As many of these mechanisms are multifaceted, certain features may instigate VSMC calcification or processes more commonly observed in PAH, such as VSMC proliferation or, potentially, VSMC senescence or inflammatory phenotype transition \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Increased dp-ucMGP levels in this cohort of patients with extensive pulmonary or systemic vascular remodeling have led to questions by which mechanisms these levels are increased. The precise triggers of increased dp-ucMGP plasma levels in our patients, as well as the primary cell types responsible, remain to be elucidated. It is necessary to determine whether there is inhibition in the conversion to active MGP, increased mechanisms that could lead to MGP deactivation, the potential impact of endoplasmic reticulum stress, and the contribution of inflammatory processes in these mechanisms. Additional in-vitro experiments, particularly utilizing material from PAH patients, should be directed toward elucidating these mechanisms.\u003c/p\u003e \u003cp\u003eConsidering prior evidence indicating that a subset of IPAH patients may display greater immunological involvement than others, the importance of a biomarker capable of distinguishing these patients has greatly increased\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. This is particularly important as current therapeutic strategies for both IPAH and mostly CTD-PAH patients, with the exception for SLE-PAH, do not involve immunosuppressive treatment\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Instead, the focus currently remains on vasodilative therapy, which is not curative\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In this study, we have shown a strong connection between increased dp-ucMGP levels and inflammation. Both PAH and CTD patients with higher sICP levels and/or clinical signs of an immune-mediated disease have shown increased levels of dp-ucMGP. Besides, plasma levels of dp-ucMGP were significantly elevated in patients with CTD-PAH compared to those with CTD alone. This difference may be attributed to the various underlying systemic diseases present in our CTD-PAH patients. As SSc patients often exhibit subtle systemic immunological activity after the early inflammatory phase in comparison to conditions like SLE or rheumatoid arthritis, there is a potential for increased inflammation in the CTD-PAH group compared to our predominantly SSc based CTD patient cohort\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. sICPs, far from being mere degradation by-products, actively modulate immune responses both locally and systemically. Compared to traditional markers like CRP, they offer a more nuanced view of inflammation. While the proteins responsible for co-stimulation or inhibition between antigen-presenting cells and T-cells function directly on the cell surface, their soluble forms may operate differently. Our findings of elevated sPD1 in IMID-PAH patients, alongside increased sTIM3, sLAG3, and sCD152 in both IPAH and CTD patients, point towards T-cell exhaustion\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This suggests a long-term ongoing inflammatory response within these patient populations. Our study, suggests that immune involvement in PAH extends beyond CTD-PAH alone. Since dp-ucMGP is correlated with sICPs and clinical signs of immune-mediated disease, this could be a valuable biomarker for distinguishing IMID and non-IMID PAH. Further research with a larger cohort and additional immunological markers, such as interleukins, chemokines, and comparison with immune-cell surface checkpoint proteins, is needed to provide a more detailed perspective of immune activation in specific IPAH patient subtypes who might benefit from immunosuppressive treatment.\u003c/p\u003e \u003cp\u003eMost notably, high plasma dp-ucMGP levels demonstrated poorer survival outcomes in IPAH patients. Given its correlation with inflammation and the frequently poorer survival observed in patients with CTD-PAH compared to IPAH patients, inflammation may contribute to inferior survival in the high dp-ucMGP group\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Elevated sIL-2 levels, without increased CRP, further supports the presence of a selective T-cell-mediated immune response. Additionally, the substantially lower DLCO levels in these patients, without underlying parenchymal lung disease, suggest extensive vascular remodeling and inflammation as a cause for higher mortality. These findings highlight the urgent need to identify PAH patients with distinct immunological profiles, as they may benefit from targeted immunosuppressive therapy to potentially improve disease outcomes. dp-ucMGP did not exhibit efficacy as a survival marker in CTEPH patients, potentially due to a different underlying pathophysiological mechanism. While the study of dp-ucMGP as a survival marker in CTD-PAH patients did not yield significant results, it is important to note the limited sample size in this group.\u003c/p\u003e \u003cp\u003eLimitations of this study include the small number of patients in the CTD-PAH and CTEPH groups, as well as variability in disease duration before presentation, a common challenge in diagnosing PAH. Future investigations should delve into the clinical role of dp-ucMGP as a biomarker for inflammation and in-vitro experiments in PAH to establish a compelling rationale for incorporating immunosuppressive treatment in these patients.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights the potential significance of dp-ucMGP as a biomarker in PAH, particularly in IPAH, where elevated levels are associated with immune-mediated disease and poorer survival outcomes. The observed correlations with immune-mediated markers and the lack of correlation with calcification levels indicate the need for further research to elucidate the complex molecular mechanisms involved in vascular remodeling underlying these associations. These findings contribute to our understanding of the pathophysiology of PAH and may pave the way for the development of novel diagnostic and therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eWe gratefully thank Lucienne Debrus-Palmans and Nele Bijnens (Laboratory of Clinical Immunology, Maastricht University Medical Center, Maastricht, The Netherlands) and\u0026nbsp;Lisette Unghethum,\u0026nbsp;Petra Lux, and Cecile Maassen for their technical assistance (Laboratory of Biochemistry, Maastricht University, Maastricht, The Netherlands). We also thank Leon Frenken, MD, for his contribution to the collection of clinical data for this cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT wrote the concept of this research article which was adapted to the final version by the other authors. RT and DvD performed the statistical analysis and RT prepared the figures. The final version was approved by all authors. All authors contributed to the concept of the research article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Data of this research article is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHumbert, M.\u003cem\u003e, et al.\u003c/em\u003e 2022 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. \u003cem\u003eEur Respir J\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e(2023).\u003c/li\u003e\n\u003cli\u003eRawlings, G.H.\u003cem\u003e, et al.\u003c/em\u003e Adults\u0026apos; experiences of living with pulmonary hypertension: a thematic synthesis of qualitative studies. \u003cem\u003eBMJ Open\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e041428 (2020).\u003c/li\u003e\n\u003cli\u003eChang, K.Y.\u003cem\u003e, et al.\u003c/em\u003e Mortality in Pulmonary Arterial Hypertension in the Modern Era: Early Insights From the Pulmonary Hypertension Association Registry. \u003cem\u003eJ Am Heart Assoc\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e024969 (2022).\u003c/li\u003e\n\u003cli\u003eTobal, R.\u003cem\u003e, et al.\u003c/em\u003e Vascular Remodeling in Pulmonary Arterial Hypertension: The Potential Involvement of Innate and Adaptive Immunity. \u003cem\u003eFront Med (Lausanne)\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 806899 (2021).\u003c/li\u003e\n\u003cli\u003eThoreau, B. \u0026amp; Mouthon, L. Pulmonary arterial hypertension associated with connective tissue diseases (CTD-PAH): Recent and advanced data. \u003cem\u003eAutoimmun Rev\u003c/em\u003e, 103506 (2023).\u003c/li\u003e\n\u003cli\u003eKoudstaal, T., Boomars, K.A. \u0026amp; Kool, M. Pulmonary Arterial Hypertension and Chronic Thromboembolic Pulmonary Hypertension: An Immunological Perspective. \u003cem\u003eJ Clin Med\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e(2020).\u003c/li\u003e\n\u003cli\u003eHeukels, P.\u003cem\u003e, et al.\u003c/em\u003e Loss of immune homeostasis in patients with idiopathic pulmonary arterial hypertension. \u003cem\u003eThorax\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 1209-1218 (2021).\u003c/li\u003e\n\u003cli\u003eFunk-Hilsdorf, T.C., Behrens, F., Grune, J. \u0026amp; Simmons, S. Dysregulated Immunity in Pulmonary Hypertension: From Companion to Composer. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 819145 (2022).\u003c/li\u003e\n\u003cli\u003eArends, S.J.\u003cem\u003e, et al.\u003c/em\u003e Prevalence of anti-endothelial cell antibodies in idiopathic pulmonary arterial hypertension. \u003cem\u003eEur Respir J\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 923-925 (2010).\u003c/li\u003e\n\u003cli\u003eMarsh, L.M.\u003cem\u003e, et al.\u003c/em\u003e The inflammatory cell landscape in the lungs of patients with idiopathic pulmonary arterial hypertension. \u003cem\u003eEur Respir J\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e(2018).\u003c/li\u003e\n\u003cli\u003eKoudstaal, T.\u003cem\u003e, et al.\u003c/em\u003e Plasma markers in pulmonary hypertension subgroups correlate with patient survival. \u003cem\u003eRespir Res\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 137 (2021).\u003c/li\u003e\n\u003cli\u003eKoudstaal, T. \u0026amp; Boomars, K.A. Inflammatory biomarkers in pulmonary arterial hypertension: ready for clinical implementation? \u003cem\u003eEur Respir J\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e(2023).\u003c/li\u003e\n\u003cli\u003eArends, S.J.\u003cem\u003e, et al.\u003c/em\u003e Functional implications of IgG anti-endothelial cell antibodies in pulmonary arterial hypertension. \u003cem\u003eAutoimmunity\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 463-470 (2013).\u003c/li\u003e\n\u003cli\u003eDing, Y.\u003cem\u003e, et al.\u003c/em\u003e Immunosuppressive therapy in patients with connective tissue disease-associated pulmonary arterial hypertension: A systematic review. \u003cem\u003eInt J Rheum Dis\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 982-990 (2022).\u003c/li\u003e\n\u003cli\u003ePotjewijd, J.\u003cem\u003e, et al.\u003c/em\u003e Plasma Dephosphorylated-Uncarboxylated Matrix Gla-Protein in Systemic Sclerosis Patients: Biomarker Potential for Vascular Calcification and Inflammation. \u003cem\u003eDiagnostics (Basel)\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e(2023).\u003c/li\u003e\n\u003cli\u003eSchurgers, L.J., Cranenburg, E.C. \u0026amp; Vermeer, C. Matrix Gla-protein: the calcification inhibitor in need of vitamin K. \u003cem\u003eThromb Haemost\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 593-603 (2008).\u003c/li\u003e\n\u003cli\u003eXu, B., Xu, G., Yu, Y. \u0026amp; Lin, J. The role of TGF-beta or BMPR2 signaling pathway-related miRNA in pulmonary arterial hypertension and systemic sclerosis. \u003cem\u003eArthritis Res Ther\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 288 (2021).\u003c/li\u003e\n\u003cli\u003eTielemans, B., Delcroix, M., Belge, C. \u0026amp; Quarck, R. TGFbeta and BMPRII signalling pathways in the pathogenesis of pulmonary arterial hypertension. \u003cem\u003eDrug Discov Today\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 703-716 (2019).\u003c/li\u003e\n\u003cli\u003eDerk, C.T. Transforming growth factor-beta (TGF-beta) and its role in the pathogenesis of systemic sclerosis: a novel target for therapy? \u003cem\u003eRecent Pat Inflamm Allergy Drug Discov\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 142-145 (2007).\u003c/li\u003e\n\u003cli\u003eLomeli-Nieto, J.A.\u003cem\u003e, et al.\u003c/em\u003e Transforming growth factor beta isoforms and TGF-betaR1 and TGF-betaR2 expression in systemic sclerosis patients. \u003cem\u003eClin Exp Med\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 471-481 (2023).\u003c/li\u003e\n\u003cli\u003eTatius, B., Wasityastuti, W., Astarini, F.D. \u0026amp; Nugrahaningsih, D.A.A. Significance of BMPR2 mutations in pulmonary arterial hypertension. \u003cem\u003eRespir Investig\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 397-407 (2021).\u003c/li\u003e\n\u003cli\u003eRiva, A. Editorial: Soluble immune checkpoints: Novel physiological immunomodulators. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1178541 (2023).\u003c/li\u003e\n\u003cli\u003eBlank, C.U.\u003cem\u003e, et al.\u003c/em\u003e Defining \u0026apos;T cell exhaustion\u0026apos;. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 665-674 (2019).\u003c/li\u003e\n\u003cli\u003evan den Hoogen, F.\u003cem\u003e, et al.\u003c/em\u003e 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. \u003cem\u003eArthritis Rheum\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 2737-2747 (2013).\u003c/li\u003e\n\u003cli\u003eHumbert, M.\u003cem\u003e, et al.\u003c/em\u003e 2022 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. \u003cem\u003eEur Heart J\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 3618-3731 (2022).\u003c/li\u003e\n\u003cli\u003ePetri, M.\u003cem\u003e, et al.\u003c/em\u003e Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. \u003cem\u003eArthritis Rheum\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 2677-2686 (2012).\u003c/li\u003e\n\u003cli\u003eCappelli, S.\u003cem\u003e, et al.\u003c/em\u003e \u0026quot;To be or not to be,\u0026quot; ten years after: evidence for mixed connective tissue disease as a distinct entity. \u003cem\u003eSemin Arthritis Rheum\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 589-598 (2012).\u003c/li\u003e\n\u003cli\u003eAletaha, D.\u003cem\u003e, et al.\u003c/em\u003e 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. \u003cem\u003eArthritis Rheum\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 2569-2581 (2010).\u003c/li\u003e\n\u003cli\u003eJennette, J.C.\u003cem\u003e, et al.\u003c/em\u003e 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. \u003cem\u003eArthritis Rheum\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 1-11 (2013).\u003c/li\u003e\n\u003cli\u003eLundberg, I.E.\u003cem\u003e, et al.\u003c/em\u003e 2017 European League Against Rheumatism/American College of Rheumatology Classification Criteria for Adult and Juvenile Idiopathic Inflammatory Myopathies and Their Major Subgroups. \u003cem\u003eArthritis Rheumatol\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 2271-2282 (2017).\u003c/li\u003e\n\u003cli\u003eDai, L.\u003cem\u003e, et al.\u003c/em\u003e Functional vitamin K insufficiency, vascular calcification and mortality in advanced chronic kidney disease: A cohort study. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, e0247623 (2021).\u003c/li\u003e\n\u003cli\u003eRapp, N.\u003cem\u003e, et al.\u003c/em\u003e Hepatic and Vascular Vitamin K Status in Patients with High Cardiovascular Risk. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e(2021).\u003c/li\u003e\n\u003cli\u003eJaminon, A.M.G.\u003cem\u003e, et al.\u003c/em\u003e Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e(2021).\u003c/li\u003e\n\u003cli\u003eJaminon, A.M.G., Akbulut, A.C., Rapp, N., Reutelingsperger, C.P. \u0026amp; Schurgers, L.J. The BioHybrid Assay: A Novel Method for Determining Calcification Propensity. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e2664\u003c/strong\u003e, 317-331 (2023).\u003c/li\u003e\n\u003cli\u003eRoumeliotis, S., Dounousi, E., Eleftheriadis, T. \u0026amp; Liakopoulos, V. Association of the Inactive Circulating Matrix Gla Protein with Vitamin K Intake, Calcification, Mortality, and Cardiovascular Disease: A Review. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e(2019).\u003c/li\u003e\n\u003cli\u003eBoxma, P.Y.\u003cem\u003e, et al.\u003c/em\u003e Vitamin k intake and plasma desphospho-uncarboxylated matrix Gla-protein levels in kidney transplant recipients. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e47991 (2012).\u003c/li\u003e\n\u003cli\u003eCaluwe, R., Vandecasteele, S., Van Vlem, B., Vermeer, C. \u0026amp; De Vriese, A.S. Vitamin K2 supplementation in haemodialysis patients: a randomized dose-finding study. \u003cem\u003eNephrol Dial Transplant\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1385-1390 (2014).\u003c/li\u003e\n\u003cli\u003eRoumeliotis, S.\u003cem\u003e, et al.\u003c/em\u003e The Association of dp-ucMGP with Cardiovascular Morbidity and Decreased Renal Function in Diabetic Chronic Kidney Disease. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e(2020).\u003c/li\u003e\n\u003cli\u003eGriffin, T.P.\u003cem\u003e, et al.\u003c/em\u003e Plasma dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP): reference intervals in Caucasian adults and diabetic kidney disease biomarker potential. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 18452 (2019).\u003c/li\u003e\n\u003cli\u003eDahlberg, S.\u003cem\u003e, et al.\u003c/em\u003e Desphospho-Uncarboxylated Matrix-Gla Protein Is Increased Postoperatively in Cardiovascular Risk Patients. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e(2018).\u003c/li\u003e\n\u003cli\u003eHouben, E., Neradova, A., Schurgers, L.J. \u0026amp; Vervloet, M. The influence of phosphate, calcium and magnesium on matrix Gla-protein and vascular calcification: a systematic review. \u003cem\u003eG Ital Nefrol\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e(2016).\u003c/li\u003e\n\u003cli\u003eSchurgers, L.J.\u003cem\u003e, et al.\u003c/em\u003e Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification. \u003cem\u003eJ Thromb Haemost\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 2503-2511 (2007).\u003c/li\u003e\n\u003cli\u003eSchurgers, L.J., Uitto, J. \u0026amp; Reutelingsperger, C.P. Vitamin K-dependent carboxylation of matrix Gla-protein: a crucial switch to control ectopic mineralization. \u003cem\u003eTrends Mol Med\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 217-226 (2013).\u003c/li\u003e\n\u003cli\u003eSanchez-Duffhues, G.\u003cem\u003e, et al.\u003c/em\u003e Inflammation induces endothelial-to-mesenchymal transition and promotes vascular calcification through downregulation of BMPR2. \u003cem\u003eJ Pathol\u003c/em\u003e \u003cstrong\u003e247\u003c/strong\u003e, 333-346 (2019).\u003c/li\u003e\n\u003cli\u003eHoeper, M.M.\u003cem\u003e, et al.\u003c/em\u003e Phase 3 Trial of Sotatercept for Treatment of Pulmonary Arterial Hypertension. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e388\u003c/strong\u003e, 1478-1490 (2023).\u003c/li\u003e\n\u003cli\u003eLiu, S.F.\u003cem\u003e, et al.\u003c/em\u003e Pulmonary hypertension: Linking inflammation and pulmonary arterial stiffening. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 959209 (2022).\u003c/li\u003e\n\u003cli\u003eStacher, E.\u003cem\u003e, et al.\u003c/em\u003e Modern age pathology of pulmonary arterial hypertension. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e \u003cstrong\u003e186\u003c/strong\u003e, 261-272 (2012).\u003c/li\u003e\n\u003cli\u003eJaminon, A.M.G.\u003cem\u003e, et al.\u003c/em\u003e Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 6586 (2020).\u003c/li\u003e\n\u003cli\u003ePrice, L.C.\u003cem\u003e, et al.\u003c/em\u003e Inflammation in pulmonary arterial hypertension. \u003cem\u003eChest\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 210-221 (2012).\u003c/li\u003e\n\u003cli\u003eCossu, M.\u003cem\u003e, et al.\u003c/em\u003e Earliest Phase of Systemic Sclerosis Typified by Increased Levels of Inflammatory Proteins in the Serum. \u003cem\u003eArthritis Rheumatol\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 2359-2369 (2017).\u003c/li\u003e\n\u003cli\u003ePotjewijd, J.\u003cem\u003e, et al.\u003c/em\u003e Favorable long term effects of intensified immunosuppression combined with therapeutic plasma exchange in patients with early-onset progressive systemic sclerosis-related interstitial lung disease. \u003cem\u003eJ Transl Autoimmun\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 100174 (2022).\u003c/li\u003e\n\u003cli\u003eBellocchi, C., Chung, A. \u0026amp; Volkmann, E.R. Predicting the Progression of Very Early Systemic Sclerosis: Current Insights. \u003cem\u003eOpen Access Rheumatol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 171-186 (2022).\u003c/li\u003e\n\u003cli\u003eDistler, O.\u003cem\u003e, et al.\u003c/em\u003e Treatment strategies and survival of patients with connective tissue disease and pulmonary arterial hypertension: A COMPERA analysis. \u003cem\u003eRheumatology (Oxford)\u003c/em\u003e (2023).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary hypertension, vascular remodeling, dp-ucMGP, inflammation, immune checkpoint proteins, biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-4329956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4329956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling. Since dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP) is associated with cardiovascular mortality in systemic sclerosis, a disease associated with PAH, and immune-system involvement in PAH is increasingly recognized, we investigated the relationship between dp-ucMGP, vascular remodeling and soluble immune-checkpoint proteins in PAH. This prospective cohort study included patients with idiopathic (I)PAH, connective tissue disease (CTD)-PAH, chronic thrombo-embolic PH (CTEPH) and CTD patients without PAH. Patients with IPAH and CTD-PAH were stratified by clinical signs of immune-mediated inflammatory disease (IMID). We measured dp-ucMGP plasma levels, soluble immune-checkpoint proteins (sICPs), and vascular smooth muscle cell (iVSMC) calcification. We found elevated dp-ucMGP levels in all PAH subtypes and CTD patients compared to healthy controls. PAH patients showed increased iVSMC calcification, but no direct correlation was found with dp-ucMGP. IMID-PAH patients had higher dp-ucMGP levels than non-IMID PAH patients. dp-ucMGP correlated with several sICPs in both IPAH and CTD patients; multiple sICPs were elevated in IMID PAH patients. High dp-ucMGP levels in IPAH patients were associated with worse survival. Our findings suggest dp-ucMGP as a potential biomarker of immune-mediated vascular remodeling in PAH. Hence, dp-ucMGP, could help identify PAH patients who might benefit from immunosuppressive therapies.\u003c/p\u003e","manuscriptTitle":"Dephosphorylated uncarboxylated Matrix-Gla-Protein and vascular remodeling in pulmonary hypertension: an immunological connection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 20:31:10","doi":"10.21203/rs.3.rs-4329956/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-10T04:07:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-09T18:30:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201027796856281006578951924076080732606","date":"2024-05-27T13:55:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T13:44:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128180878331067843051818339816500667892","date":"2024-05-15T14:52:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-03T15:50:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-03T15:49:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-30T11:17:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-30T11:14:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-26T13:25:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4abcbbf9-53d1-497a-817a-a2609e360984","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31564978,"name":"Biological sciences/Biochemistry"},{"id":31564979,"name":"Biological sciences/Immunology"},{"id":31564980,"name":"Health sciences/Medical research/Biomarkers"},{"id":31564981,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2024-11-11T16:04:15+00:00","versionOfRecord":{"articleIdentity":"rs-4329956","link":"https://doi.org/10.1038/s41598-024-77000-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-04 15:58:22","publishedOnDateReadable":"November 4th, 2024"},"versionCreatedAt":"2024-05-08 20:31:10","video":"","vorDoi":"10.1038/s41598-024-77000-w","vorDoiUrl":"https://doi.org/10.1038/s41598-024-77000-w","workflowStages":[]},"version":"v1","identity":"rs-4329956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4329956","identity":"rs-4329956","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}