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 status35. Epidemiological data has highlighted an inverse association between dp-ucMGP levels and vitamin K status36,37. 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 circulation41. MGP contains five γ-carboxyglutamate amino-acid residues requiring post-translational carboxylation to be activated42. In addition to carboxylation, phosphorylation of the serine residues is important for the role of MGP in inhibiting vascular calcification42. MGP can bind directly to calcium-crystals thereby inhibiting further crystal growth43. 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-243. 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-944. 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 conceivable17,18,21,45. However, vascular calcification has not yet been demonstrated in the context of PAH and is currently not acknowledged as a prominent histopathological characteristic46. 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 evident4,47. Despite the pathophysiological connection between calcification and MGP, this correlation does not seem to extend to the inactive form48. 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 increased7,11–13,49. 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 treatment14. Instead, the focus currently remains on vasodilative therapy, which is not curative1. 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 cohort50–52. 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 exhaustion23. 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 group53. 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.