B-cells in pulmonary arterial hypertension: friend, foe or bystander?

There is an unmet need for new therapeutic strategies that target alternative pathways to improve the prognosis of patients with pulmonary arterial hypertension (PAH). As immunity has been involved in the development and progression of vascular lesions in PAH, we review the potential contribution of B-cells in its pathogenesis and evaluate the relevance of B-cell-targeted therapies. Circulating B-cell homeostasis is altered in PAH patients, with total B-cell lymphopenia, abnormal subset distribution (expansion of naïve and antibody-secreting cells, reduction of memory B-cells) and chronic activation. B-cells are recruited to the lungs through local chemokine secretion, and activated by several mechanisms: 1) interaction with lung vascular autoantigens through cognate B-cell receptors; 2) costimulatory signals provided by T follicular helper cells (interleukin (IL)-21), type 2 T helper cells and mast cells (IL-4, IL-6 and IL-13); and 3) increased survival signals provided by B-cell activating factor pathways. This activity results in the formation of germinal centres within perivascular tertiary lymphoid organs and in the local production of pathogenic autoantibodies that target the pulmonary vasculature and vascular stabilisation factors (including angiotensin-II/endothelin-1 receptors and bone morphogenetic protein receptors). B-cells also mediate their effects through enhanced production of pro-inflammatory cytokines, reduced anti-inflammatory properties by regulatory B-cells, immunoglobulin (Ig)G-induced complement activation, and IgE-induced mast cell activation. Precision-medicine approaches targeting B-cell immunity are a promising direction for select PAH conditions, as suggested by the efficacy of anti-CD20 therapy in experimental models and a trial of rituximab in systemic sclerosis-associated PAH.

Signal Transduction during Metabolic and Inflammatory Reprogramming in Pulmonary Vascular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.

Plasma markers in pulmonary hypertension subgroups correlate with patient survival.

BACKGROUND: Recent studies have provided evidence for an important contribution of the immune system in the pathophysiology of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). In this report, we investigated whether the inflammatory profile of pulmonary hypertension patients changes over time and correlates with patient WHO subgroups or survival. METHODS: 50 PAH patients (16 idiopathic (I)PAH, 24 Connective Tissue Disease (CTD)-PAH and 10 Congenital Heart Disease (CHD)-PAH), 37 CTEPH patients and 18 healthy controls (HCs) were included in the study. Plasma inflammatory markers at baseline and after 1-year follow-up were measured using ELISAs. Subsequently, correlations with hemodynamic parameters and survival were explored and data sets were subjected to unbiased multivariate analyses. RESULTS: At diagnosis, we found that plasma levels of interleukin-6 (IL-6) and the chemokines (C-X3-C) motif legend CXCL9 and CXCL13 in CTD-PAH patients were significantly increased, compared with HCs. In idiopathic PAH patients the levels of tumor growth factor-ß (TGFß), IL-10 and CXCL9 were elevated, compared with HCs. The increased CXCL9 and IL-8 concentrations in CETPH patients correlated significantly with decreased survival, suggesting that CXCL9 and IL-8 may be prognostic markers. After one year of treatment, IL-10, CXCL13 and TGFß levels changed significantly in the PAH subgroups and CTEPH patients. Unbiased multivariate analysis revealed clustering of PH patients based on inflammatory mediators and clinical parameters, but did not separate the WHO subgroups. Importantly, these multivariate analyses separated patients with < 3 years and > 3 years survival, in particular when inflammatory mediators were combined with clinical parameters. DISCUSSION: Our study revealed elevated plasma levels of inflammatory mediators in different PAH subgroups and CTEPH at baseline and at 1-year follow-up, whereby CXCL9 and IL-8 may prove to be prognostic markers for CTEPH patients. While this study is exploratory and hypothesis generating, our data indicate an important role for IL-8 and CXCL9 in CHD and CTEPH patients considering the increased plasma levels and the observed correlation with survival. CONCLUSION: In conclusion, our studies identified an inflammatory signature that clustered PH patients into WHO classification-independent subgroups that correlated with patient survival.

Discovery of Distinct Immune Phenotypes Using Machine Learning in Pulmonary Arterial Hypertension.

RATIONALE: Accumulating evidence implicates inflammation in pulmonary arterial hypertension (PAH) and therapies targeting immunity are under investigation, although it remains unknown if distinct immune phenotypes exist. OBJECTIVE: Identify PAH immune phenotypes based on unsupervised analysis of blood proteomic profiles. METHODS AND RESULTS: In a prospective observational study of group 1 PAH patients evaluated at Stanford University (discovery cohort; n=281) and University of Sheffield (validation cohort; n=104) between 2008 and 2014, we measured a circulating proteomic panel of 48 cytokines, chemokines, and factors using multiplex immunoassay. Unsupervised machine learning (consensus clustering) was applied in both cohorts independently to classify patients into proteomic immune clusters, without guidance from clinical features. To identify central proteins in each cluster, we performed partial correlation network analysis. Clinical characteristics and outcomes were subsequently compared across clusters. Four PAH clusters with distinct proteomic immune profiles were identified in the discovery cohort. Cluster 2 (n=109) had low cytokine levels similar to controls. Other clusters had unique sets of upregulated proteins central to immune networks-cluster 1 (n=58; TRAIL [tumor necrosis factor-related apoptosis-inducing ligand], CCL5 [C-C motif chemokine ligand 5], CCL7, CCL4, MIF [macrophage migration inhibitory factor]), cluster 3 (n=77; IL [interleukin]-12, IL-17, IL-10, IL-7, VEGF [vascular endothelial growth factor]), and cluster 4 (n=37; IL-8, IL-4, PDGF-ß [platelet-derived growth factor beta], IL-6, CCL11). Demographics, PAH clinical subtypes, comorbidities, and medications were similar across clusters. Noninvasive and hemodynamic surrogates of clinical risk identified cluster 1 as high-risk and cluster 3 as low-risk groups. Five-year transplant-free survival rates were unfavorable for cluster 1 (47.6%; 95% CI, 35.4%-64.1%) and favorable for cluster 3 (82.4%; 95% CI, 72.0%-94.3%; across-cluster P<0.001). Findings were replicated in the validation cohort, where machine learning classified 4 immune clusters with comparable proteomic, clinical, and prognostic features. CONCLUSIONS: Blood cytokine profiles distinguish PAH immune phenotypes with differing clinical risk that are independent of World Health Organization group 1 subtypes. These phenotypes could inform mechanistic studies of disease pathobiology and provide a framework to examine patient responses to emerging therapies targeting immunity.

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

KCNK3 Mutation Causes Altered Immune Function in Pulmonary Arterial Hypertension Patients and Mouse Models.

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we exposed kcnk3fl/fl animals to hypoxia, metabolic diet and low dose lipopolysaccharide (LPS) and performed molecular characterization of their tissue. We also used tissue samples from KCNK3 patients (skin fibroblast derived inducible pluripotent stem cells, blood, lungs, peripheral blood mononuclear cells) and performed microarray, immunohistochemistry (IHC) and mass cytometry time of flight (CyTOF) experiments. Although a hypoxic insult did not alter vascular tone in kcnk3fl/fl mice, RNASeq study of these lungs implied that inflammatory and metabolic factors were altered, and the follow-up diet study demonstrated a dysregulation of bone marrow cells in kcnk3fl/fl mice. Finally, a low dose LPS study clearly showed that inflammation could be a possible second hit driving PAH in kcnk3fl/fl mice. Multiplex, IHC and CyTOF immunophenotyping studies on human samples confirmed the mouse data and strongly indicated that cell mediated, and innate immune responses may drive PAH susceptibility in these patients. In conclusion, loss of function KCNK3 mutation alters various physiological processes from vascular tone to metabolic diet through inflammation. Our data suggests that altered circulating immune cells may drive PAH susceptibility in patients with KCNK3 mutation.

Transcriptomic analysis identifies Toll-like and Nod-like pathways and necroptosis in pulmonary arterial hypertension.

Inflammation and immunity play a causal role in the pathogenesis of pulmonary vascular remodelling and pulmonary arterial hypertension (PAH). However, the pathways and mechanisms by which inflammation and immunity contribute to pulmonary vascular remodelling remain unknown. RNA sequencing was used to analyse the transcriptome in control and rats injected with monocrotaline (MCT) for various weeks. Using the transcriptional profiling of MCT-induced PAH coupled with bioinformatics analysis, we clustered the differentially expressed genes (DEGs) and chose the increased expression patterns associated with inflammatory and immune response. We found the enrichment of Toll-like receptor (TLR) and Nod-like receptor (NLR) pathways and identified NF-κB-mediated inflammatory and immune profiling in MCT-induced PAH. Pathway-based data integration and visualization showed the dysregulated TLR and NLR pathways, including increased expression of TLR2 and NLRP3, and their downstream molecules. Further analysis revealed that the activation of TLR and NLR pathways was associated with up-regulation of damage-associated molecular patterns (DAMPs) and RIPK3-mediated necroptosis was involved in the generation of DAMPs in MCT-induced PAH. Collectively, we identify RIPK3-mediated necroptosis and its triggered TLR and NLR pathways in the progression of pulmonary vascular remodelling, thus providing novel insights into the mechanisms underlying inflammation and immunity in the pathogenesis of PAH.

Can intestinal microbiota and circulating microbial products contribute to pulmonary arterial hypertension?

Pulmonary arterial hypertension (PAH) is a fatal disease with a median survival of only 5-7 yr. PAH is characterized by remodeling of the pulmonary vasculature causing reduced pulmonary arterial compliance (PAC) and increased pulmonary vascular resistance (PVR), ultimately resulting in right ventricular failure and death. Better therapies for PAH will require a paradigm shift in our understanding of the early pathophysiology. PAC decreases before there is an increase in the PVR. Unfortunately, present treatment has little effect on PAC. The loss of compliance correlates with extracellular matrix remodeling and fibrosis in the pulmonary vessels, which have been linked to chronic perivascular inflammation and immune dysregulation. However, what initiates the perivascular inflammation and immune dysregulation in PAH is unclear. Alteration of the gut microbiota composition and function underlies the level of immunopathogenic involvement in several diseases, including atherosclerosis, obesity, diabetes mellitus, and depression, among others. In this review, we discuss evidence that raises the possibility of an etiologic role for changes in the gut and circulating microbiome in the initiation of perivascular inflammation in the early pathogenesis of PAH.

Pulmonary arterial hypertension associated with protein kinase inhibitors: a pharmacovigilance-pharmacodynamic study.

The pathophysiology of pulmonary arterial hypertension (PAH) induced by protein kinase inhibitors (PKIs) remains unclear. To gain knowledge into this rare and severe pathology we performed a study combining a pharmacovigilance approach and the pharmacodynamic properties of PKIs.A disproportionality analysis on the World Health Organization pharmacovigilance database VigiBase using the reporting odds ratio (ROR) and 95% confidence interval was first performed. Then, we identified the most relevant cellular targets of interest through a systematic literature review and correlated the pharmacovigilance signals with the affinity for the different PKIs. We further performed a hierarchical cluster analysis to assess patterns of binding affinity.A positive disproportionality signal was found for dasatinib, bosutinib, ponatinib, ruxolitinib and nilotinib. Five non-receptor protein kinases significantly correlate with disproportionality signals: c-Src (r=0.79, p=0.00027), c-Yes (r=0.82, p=0.00015), Lck (r=0.81, p=0.00046) and Lyn (r=0.80, p=0.00036), all belonging to the Src protein kinase family, and TEC (r=0.85, p=0.00006). Kinases of the bone morphogenetic protein signalling pathway also seem to play a role in the pathophysiology of PKI-induced PAH. Interestingly, the dasatinib affinity profile seems to be different from that of other PKIs in the cluster analysis.The study highlights the potential role of the Src protein kinase family and TEC in PAH induced by PKIs. This approach combining pharmacovigilance and pharmacodynamics data allowed us to generate some hypotheses about the pathophysiology of the disease; however, the results have to be confirmed by further studies.

Therapeutic Monoclonal Antibody Antagonizing Endothelin Receptor A for Pulmonary Arterial Hypertension.

Endothelin receptor A (ETA) is a G protein-coupled receptor and a major therapeutic target for pulmonary arterial hypertension (PAH). We took a novel approach and developed an antagonistic monoclonal antibody, getagozumab, specifically against ETA. Getagozumab displayed a K d value of 8.7 nM and an IC50 value of 37.9 nM in the cell-based assays. Getagozumab could significantly lower pulmonary arterial pressure in both hypoxia-induced and monocrotaline (MCT)-induced PAH monkey models and further attenuate the pulmonary arterial and right ventricular hypertrophy in MCT-induced PAH monkeys. The preclinical studies demonstrated that getagozumab is safe, long lasting, and efficacious. Getagozumab may provide a new and effective treatment for PAH patients.

Idiopathic and connective tissue disease-associated pulmonary arterial hypertension (PAH): Similarities, differences and the role of autoimmunity.

Pre-capillary pulmonary arterial hypertension (PAH) is hemodynamically characterized by a mean pulmonary arterial pressure (mPAP) ≥ 20 mmHg, pulmonary capillary wedge pressure (PAWP) ≤15 mmHg and pulmonary vascular resistance (PVR) > 2. PAH is classified in six clinical subgroups, including idiopathic PAH (IPAH) and PAH associated to connective tissue diseases (CTD-PAH), that will be the main object of this review. The aim is to compare these two PAH subgroups in terms of epidemiology, histological and pathogenic findings in an attempt to define disease-specific features, including autoimmunity, that may explain the heterogeneity of response to therapy between IPAH and CTD-PAH.

Endothelin B Receptor Immunodynamics in Pulmonary Arterial Hypertension.

Introduction: Inflammation is a major pathological feature of pulmonary arterial hypertension (PAH), particularly in the context of inflammatory conditions such as systemic sclerosis (SSc). The endothelin system and anti-endothelin A receptor (ETA) autoantibodies have been implicated in the pathogenesis of PAH, and endothelin receptor antagonists are routinely used treatments for PAH. However, immunological functions of the endothelin B receptor (ETB) remain obscure. Methods: Serum levels of anti-ETB receptor autoantibodies were quantified in healthy donors and SSc patients with or without PAH. Age-dependent effects of overexpression of prepro-endothelin-1 or ETB deficiency on pulmonary inflammation and the cardiovascular system were studied in mice. Rescued ETB-deficient mice (ETB-/-) were used to prevent congenital Hirschsprung disease. The effects of pulmonary T-helper type 2 (Th2) inflammation on PAH-associated pathologies were analyzed in ETB-/- mice. Pulmonary vascular hemodynamics were investigated in isolated perfused mouse lungs. Hearts were assessed for right ventricular hypertrophy. Pulmonary inflammation and collagen deposition were assessed via lung microscopy and bronchoalveolar lavage fluid analyses. Results: Anti-ETB autoantibody levels were elevated in patients with PAH secondary to SSc. Both overexpression of prepro-endothelin-1 and rescued ETB deficiency led to pulmonary hypertension, pulmonary vascular hyperresponsiveness, and right ventricular hypertrophy with accompanying lymphocytic alveolitis. Marked perivascular lymphocytic infiltrates were exclusively found in ETB-/- mice. Following induction of pulmonary Th2 inflammation, PAH-associated pathologies and perivascular collagen deposition were aggravated in ETB-/- mice. Conclusion: This study provides evidence for an anti-inflammatory role of ETB. ETB seems to have protective effects on Th2-evoked pathologies of the cardiovascular system. Anti-ETB autoantibodies may modulate ETB-mediated immune homeostasis.

Bioinformatic exploration of the immune related molecular mechanism underlying pulmonary arterial hypertension.

This study aimed to explore the molecular mechanisms related to immune and hub genes related to pulmonary arterial hypertension (PAH). The differentially expressed genes (DEGs) of GSE15197 were identified as filters with adjusted P value <0.05, and |Log2 fold change|> 1. Biofunctional and pathway enrichment annotation of DEGs indicated that immunity and inflammation may play an important role in the molecular mechanism of PAH. The CIBERSORT algorithm further analyzed the immune cell infiltration characteristics of the PAH and control samples. Subsequently, 16 hub genes were identified from DEGs using the least absolute shrinkage and selection operator (LASSO) algorithm. An immune related gene CX3CR1 was further selected from the intersection results of the 16 hub genes and the top 20 genes with the most adjacent nodes in the protein-protein interaction (PPI) network. GSE113439, GSE48149, and GSE33463 datasets were used to validate and proved CX3CR1 with a remarkable score of AUC to distinguish PAH samples caused by various reasons from the control group.

Identification of key genes and immune profile in limited cutaneous systemic sclerosis-associated pulmonary arterial hypertension by bioinformatics analysis.

AIMS: Limited cutaneous systemic sclerosis-associated pulmonary arterial hypertension (lcSSc-PAH) is a complex multi-system disease with high morbidity and mortality. The purpose of this study is to identify the hub genes and immune characteristics of limited cutaneous systemic sclerosis (lcSSc) and lcSSc-PAH through bioinformatics. MAIN METHODS: LcSSc-PAH raw data were obtained from the GEO database (GSE19617). Weighted gene Co-expression Network analysis (WGCNA) was used to evaluate key modules. Then, we performed Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis with R software and verified the diagnostic value of the hub genes. Finally, Immune Cell Abundance Identifier (ImmuCellAI) was used to analyze the immune characteristics of the normal subjects, lcSSc and lcSSc-PAH patients, the results were displayed graphically. KEY FINDINGS: Enrichment of two important modules by GO and KEGG identified key biological processes and pathways related to pathogen infection and immune function. Three hub genes (BID, IFNGR1, ZAP70) related to immune function were identified. The analysis of immune characteristics showed that the correlation and abundance of immune cells such as inducible regulatory T (iTreg) cells, B cells, macrophages, natural killer (NK) cells, CD8T cells, mucosal-associated invariant T(MAIT) cells and dendritic cells(DCs) were significantly different in the normal subjects, lcSSc and lcSSc-PAH patients. SIGNIFICANCE: Pathogen infection, changes in the number and function of immune cells, and interactions among immune cells may preliminarily reveal the pathological mechanism of lcSSc-PAH. The hub genes, pathways and immune characteristics identified in this research remains to be further studied.

Candidate Genes Identified in Systemic Sclerosis-Related Pulmonary Arterial Hypertension Were Associated with Immunity, Inflammation, and Cytokines.

BACKGROUND: Pulmonary complications of systemic sclerosis (SSc), including pulmonary arterial hypertension (PAH), are the leading causes of patient death. However, the precise molecular mechanisms of its etiology are unclear. This study's objective was to identify the candidate genes involved in the progression of SSc-PAH and investigate the genes' function. METHODS: The gene expression profiles of GSE33463 were obtained from the Gene Expression Omnibus (GEO) database. A free-scale gene coexpression network was constructed using the weighted gene coexpression network analysis (WGCNA) to explore the association between gene sets and clinical features and identify candidate biomarkers. Then, gene ontology analysis was performed. A second dataset was used, GSE19617, to validate the hub genes. The verified hub genes' potential function was further explored using gene set enrichment analysis (GSEA). RESULTS: Through average link-level clustering, a total of seven modules were classified. A total of 938 hub genes were identified in the key module, and the key module's function mainly enriched was related to chemokine activities. Subsequently, four candidate genes, BTG3, CCR2, RAB10, and TMEM60, were filtered. The expression levels of these four hub genes were consistent in the GSE19617 and GSE33463 datasets. We plotted the ROC curve of the hub genes (all AUC > 0.70). Furthermore, the results of the GSEA for hub genes were correlated with complement and inflammatory responses. CONCLUSIONS: The hub genes (BTG3, CCR2, RAB10, and TMEM60) performed well in distinguishing the SSc-PAH patients from controls, and some biological functions, related to immunity, inflammation, and cytokines, might pave the way for follow-up studies on the diagnosis and treatment of SSc-PAH.

Antisynthetase syndrome and pulmonary hypertension: report of two cases and review of the literature.

Antisynthetase Syndrome (ASS) is a subset of idiopathic inflammatory myopathies characterised by specific clinical features such as interstitial lung disease (ILD), fever, myositis, Raynaud's phenomenon, cutaneous involvement and arthritis related to the presence of anti-aminoacyl-tRNA-synthetase (anti-ARS) autoantibodies. Moreover, Pulmonary arterial hypertension (PAH) is a life-threatening complication associated with connective tissue diseases mainly systemic sclerosis (SSc-PAH). It has been suggested that PAH can complicate ASS patients but little is known about the prevalence and risk factors to develop this complication. Here we report on two patients with ASS and PH. The first one represents a complete picture of ASS anti-Jo-1 positive, the second an amyophatic ASS anti-PL-12 positive. In one of our ASS-PAH patients, specific treatment lead to improvement of PAH. There are no specific recommendations on current guidelines regarding either PAH screening or treatment in ASS, but performing echocardiogram, ECG, pulmonary function test and prompt initiation of specific therapies seems to improve right heart catheterisation (RHC) parameters and survival.

The Role of Regulatory T Cells in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a chronic, incurable condition characterized by pulmonary vascular remodeling, perivascular inflammation, and right heart failure. Regulatory T cells (Tregs) stave off autoimmunity, and there is increasing evidence for their compromised activity in the inflammatory milieu of PAH. Abnormal Treg function is strongly correlated with a predisposition to PAH in animals and patients. Athymic Treg-depleted rats treated with SU5416, an agent causing pulmonary vascular injury, develop PAH, which is prevented by infusing missing CD4+CD25highFOXP3+ Tregs. Abnormal Treg activity may also explain why PAH disproportionately affects women more than men. This mini review focuses on the role of Tregs in PAH with a special view to sexual dimorphism and the future promise of Treg therapy.

Screening of key biomarkers and immune infiltration in Pulmonary Arterial Hypertension via integrated bioinformatics analysis.

This study aimed to screen key biomarkers and investigate immune infiltration in pulmonary arterial hypertension (PAH) based on integrated bioinformatics analysis. The Gene Expression Omnibus (GEO) database was used to download three mRNA expression profiles comprising 91 PAH lung specimens and 49 normal lung specimens. Three mRNA expression datasets were combined, and differentially expressed genes (DEGs) were obtained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the protein-protein interaction (PPI) network of DEGs were performed using the STRING and DAVID databases, respectively. The diagnostic value of hub gene expression in PAH was also analyzed. Finally, the infiltration of immune cells in PAH was analyzed using the CIBERSORT algorithm. Total 182 DEGs (117 upregulated and 65 downregulated) were identified, and 15 hub genes were screened. These 15 hub genes were significantly associated with immune system functions such as myeloid leukocyte migration, neutrophil migration, cell chemotaxis, Toll-like receptor signaling pathway, and NF-κB signaling pathway. A 7-gene-based model was constructed and had a better diagnostic value in identifying PAH tissues compared with normal controls. The immune infiltration profiles of the PAH and normal control samples were significantly different. High proportions of resting NK cells, activated mast cells, monocytes, and neutrophils were found in PAH samples, while high proportions of resting T cells CD4 memory and Macrophages M1 cell were found in normal control samples. Functional enrichment of DEGs and immune infiltration analysis between PAH and normal control samples might help to understand the pathogenesis of PAH.

Severe Pulmonary Arterial Hypertension Is Characterized by Increased Neutrophil Elastase and Relative Elafin Deficiency.

BACKGROUND: Preclinical evidence implicates neutrophil elastase (NE) in pulmonary arterial hypertension (PAH) pathogenesis, and the NE inhibitor elafin is under early therapeutic investigation. RESEARCH QUESTION: Are circulating NE and elafin levels abnormal in PAH and are they associated with clinical severity? STUDY DESIGN AND METHODS: In an observational Stanford University PAH cohort (n = 249), plasma NE and elafin levels were measured in comparison with those of healthy control participants (n = 106). NE and elafin measurements were then related to PAH clinical features and relevant ancillary biomarkers. Cox regression models were fitted with cubic spline functions to associate NE and elafin levels with survival. To validate prognostic relationships, we analyzed two United Kingdom cohorts (n = 75 and n = 357). Mixed-effects models evaluated NE and elafin changes during disease progression. Finally, we studied effects of NE-elafin balance on pulmonary artery endothelial cells (PAECs) from patients with PAH. RESULTS: Relative to control participants, patients with PAH were found to have increased NE levels (205.1 ng/mL [interquartile range (IQR), 123.6-387.3 ng/mL] vs 97.6 ng/mL [IQR, 74.4-126.6 ng/mL]; P < .0001) and decreased elafin levels (32.0 ng/mL [IQR, 15.3-59.1 ng/mL] vs 45.5 ng/mL [IQR, 28.1-92.8 ng/mL]; P < .0001) independent of PAH subtype, illness duration, and therapies. Higher NE levels were associated with worse symptom severity, shorter 6-min walk distance, higher N-terminal pro-type brain natriuretic peptide levels, greater right ventricular dysfunction, worse hemodynamics, increased circulating neutrophil levels, elevated cytokine levels, and lower blood BMPR2 expression. In Stanford patients, NE levels of > 168.5 ng/mL portended increased mortality risk after adjustment for known clinical predictors (hazard ratio [HR], 2.52; CI, 1.36-4.65, P = .003) or prognostic cytokines (HR, 2.63; CI, 1.42-4.87; P = .001), and the NE level added incremental value to established PAH risk scores. Similar prognostic thresholds were identified in validation cohorts. Longitudinal NE changes tracked with clinical trends and outcomes. PAH PAECs exhibited increased apoptosis and attenuated angiogenesis when exposed to NE at the level observed in patients' blood. Elafin rescued PAEC homeostasis, yet the required dose exceeded levels found in patients. INTERPRETATION: Blood levels of NE are increased while elafin levels are deficient across PAH subtypes. Higher NE levels are associated with worse clinical disease severity and outcomes, and this target-specific biomarker could facilitate therapeutic development of elafin.