Pathogenesis Of Chronic Thromboembolic Pulmonary Hypertension Research Articles

Predominance of M2 macrophages in organized thrombi in chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.

Angiogenesis in Chronic Thromboembolic Pulmonary Hypertension: A Janus-Faced Player?

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare form of pulmonary hypertension characterized by the presence of organized thrombi that obstruct pulmonary arteries, ultimately leading to right heart failure and death. Among others, impaired angiogenesis and inflammatory thrombosis have been shown to contribute to the progression of CTEPH. In this review, we summarize the 2-faced nature of angiogenesis in both thrombus formation and resolution in the context of CTEPH and highlight the dual role of angiogenesis and neovascularization in resolving venous thrombi. Furthermore, we discuss relevant in vitro and in vivo models that support the benefits or drawbacks of angiogenesis in CTEPH progression. We discuss the key pathways involved in modulating angiogenesis, particularly the underexplored role of TGFβ (transforming growth factor-beta) signaling in driving fibrosis as an integral element of CTEPH pathogenesis. We finally explore innovative treatment strategies that target angiogenic pathways. These strategies have the potential to pioneer preventive, inventive, or alternative therapeutic options for patients with CTEPH who may not qualify for surgical interventions. Moreover, they could be used synergistically with established treatments such as pulmonary endarterectomy or balloon pulmonary angioplasty. In summary, this review emphasizes the crucial role of angiogenesis in the development of in fibrothrombotic tissue, a major pathological characteristic of CTEPH.

Therapies of chronic thromboembolic pulmonary hypertension: A review

The article addresses therapies for chronic thromboembolic pulmonary hypertension (CTEPH). The main challenges of patient management at various stages of diagnosis and treatment and the prevalence and pathogenesis of CTEPH are described. Immediate and long-term outcomes after treatment are evaluated. The study aims to present current therapies for CTEPH.

INCREASED LEVELS OF PROINFLAMMATORY CYTOKINES IN BLOOD PLASMA IN PATIENTS WITH CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION

HighlightsIL-8 and MCP-1 have a significant role in the CTEPH pathogenesis, which indicates the importance of nonspecific immunity in the formation and progression of CTEPH. The coupling between cytokines and hemodynamic parameters, cardiac structural changes and plasma biochemical parameters were determined. AbstractBackground. Chronic thromboembolic pulmonary hypertension (CTEPH) pathogenesis is complex and not fully understood. Particular attention to the microvascular damage genesis in CTEPH is given to aseptic inflammation, which in turn could be mediated through various molecular mechanisms. According to the conflicting and incomplete data on changes in the profile of factors controlling inflammation in CTEPH, research in this field would identify new therapeutic targets for the prevention and treatment of CTEPH.Aim. To study the profile of plasma proinflammatory cytokines in patients with chronic thromboembolic pulmonary hypertension (CTEPH) and evaluate the coupling of these cytokines with the main morphofunctional and laboratory values of the disease severity.Methods. 34 patients with CTEPH were included in this study. To characterize the group, the following methods were used: echocardiographic examination, catheterization of the right cardiac chambers. Biomarkers of heart failure, systemic inflammation, as well as erythropoiesis and iron metabolism were assessed in all patients. The control group included 10 donors. To study the proinflammatory cytokine profile in plasma, interleukins (IL) 6, 8, 18, monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase 9 were determined using standard enzyme-linked immunosorbent assay (ELISA) kits.Results. Hemodynamic and morphofunctional changes in the pulmonary circulation specific to pulmonary hypertension were determined with catheterization of the right cardiac chambers and echocardiography. During plasma proinflammatory cytokines analysis, a significant increase in the level of IL-8 (p = 0.030) and MCP-1 (p = 0.031) in CTEPH group compared to the control group was observed. No significant differences for other analyzed markers were found. In the elaboration of the correlation analysis, moderate inverse coupling between proinflammatory markers and hemodynamic parameters characterizing the CTEPH severity were revealed, as well as positive correlations with parameters of remodeling of the right cardiac chambers and iron metabolism.Conclusion. The increased levels of IL-8 and MCP-1 in patients with CTEPH identified in the present study indicate a significant role of nonspecific immunity in the formation and progression of CTEPH. The coupling between cytokines and hemodynamic parameters, structural cardiac changes and plasma biochemical parameters were determined. Based on the obtained data, it is possible to develop new medicinal substances, targeting towards proinflammatory cytokines, their receptors and signaling pathways.

Abstract 220: Post-gwas Functional Genomics Analysis Of Abo Locus To Identify Novel Pathogenic Mechanisms For Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and mysterious complication of pulmonary embolism, characterized by chronic pulmonary arterial thrombosis, occlusions and vasculopathy. Emerging evidence has suggested genetic relevance of CTEPH, particularly to ABO blood type. Genome-wide association study (GWAS) has identified ABO gene as the most significant genetic locus associated with CTEPH. This locus, however, consists only intronic SNPs and there is no mechanistic explanation on how these non-coding SNPs control the risk of CTEPH. Methods: GWAS-identified CTEPH-associated ABO locus was analyzed, and haplotype SNPs were identified based on linkage disequilibrium (LD). Electrophoretic mobility shift assay (EMSA) was applied to screen functional SNPs (fSNPs) based on allele-imbalanced nuclear protein binding. SNP-binding nuclear transcription factors (TFs) were identified by proteomics. Hi-C chromatin-interaction analysis was utilized to determine potential target genes affected by fSNPs. Knockdown of fSNP-associated TFs in human pulmonary artery endothelial cells (hPAECs) was applied to validate the regulation of TFs on target genes. Results: Based on GWAS, the minor allele of tag SNP rs2519093 is associated with higher risk of CTEPH (Odds ratio 2.22). It defines 11 haplotype SNPs in a LD with R 2 > 0.8. Screened by EMSA with hPAEC nuclear proteins, intronic SNPs rs579459 and rs550057 were identified functional given their allele-specific protein binding. Proteomics analysis identified 3 candidate TFs that allele-specifically bind to fSNP rs579459: PBX1, CTCF and SAFB2. Candidate target gene ADAMTS13 is chosen based on its prominent interaction with fSNP revealed by Hi-C analysis, and the potential role in thromboembolism through vWF cleavage. TF knockdown by siRNA downregulates ADAMTS13 in hPAECs, suggesting their regulatory function as activators. Conclusion: Leveraging our post-GWAS functional genomics approach, we identified fSNPs in ABO locus that define a genetic architecture controlling the susceptibility of CTEPH. This genetic architecture, along with the newly identified fSNP-associated TFs and target gene ADAMTS13, provides a biological explanation for the pathogenesis of CTEPH.

Pathophysiology and Treatment of Chronic Thromboembolic Pulmonary Hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is a condition in which an organic thrombus remains in the pulmonary artery (PA) even after receiving anticoagulation therapy for more than 3 months and is complicated by pulmonary hypertension (PH), leading to right-sided heart failure and death. CTEPH is a progressive pulmonary vascular disease with a poor prognosis if left untreated. The standard treatment for CTEPH is pulmonary endarterectomy (PEA), which is usually performed only in specialized centers. In recent years, balloon pulmonary angioplasty (BPA) and drug therapy for CTEPH have also shown good results. This review discusses the complex pathogenesis of CTEPH and presents the standard of care, PEA, as well as a new device called BPA, which is showing remarkable progress in efficacy and safety. Additionally, several drugs are now demonstrating established evidence of efficacy in treating CTEPH.

The alterations in molecular markers and signaling pathways in chronic thromboembolic pulmonary hypertension, a study with transcriptome sequencing and bioinformatic analysis.

BackgroundAt present, the alterations in molecular markers and signaling pathways in chronic thromboembolic pulmonary hypertension (CTEPH) remain unclear. We aimed to compare the difference of molecular markers and signaling pathways in patients with CTEPH and healthy people with transcriptome sequencing and bioinformatic analysis.MethodsWe prospectively included 26 patients with CTEPH and 35 sex- and age-matched healthy volunteers as control. We extracted RNA from whole blood samples to construct the library. Then, qualified libraries were sequenced using PE100 strategy on BGIseq platform. Subsequently, the DESeq2 package in R was used to screen differentially expressed mRNAs (DEmRNAs) and differentially expressed long non-coding RNAs (DElncRNAs) of 7 patients with CTEPH and 5 healthy volunteers. Afterwards, we performed functional enrichment and protein–protein interaction analysis of DEmRNAs. We also performed lncRNA-mRNA co-expression analysis and lncRNA-miRNA-mRNA network construction. In addition, we performed diagnostic analysis on the GSE130391 dataset. Finally, we performed reverse transcription polymerase chain reaction (RT-PCR) of genes in 19 patients with CTEPH and 30 healthy volunteers.ResultsGender and age between patients with CTEPH and healthy controls, between sequencing group and in vitro validation group, were comparable. A total of 437 DEmRNAs and 192 DElncRNAs were obtained. Subsequently, 205 pairs of interacting DEmRNAs and 232 pairs of lncRNA-mRNA relationship were obtained. DEmRNAs were significantly enriched in chemokine signaling pathway, metabolic pathways, arachidonic acid metabolism, and MAPK signaling pathway. Only one regulation pathway of SOBP-hsa-miR-320b-LINC00472 was found through ceRNA network construction. In diagnostic analysis, the area under curve (AUC) values of LINC00472, PIK3R6, SCN3A, and TCL6, respectively, were 0.964, 0.893, 0.750, and 0.732.ConclusionThe identification of alterations in molecules and pathways may provide further research directions on pathogenesis of CTEPH. Additionally, LINC00472, PIK3R6, SCN3A, and TCL6 may act as the potential gene markers in CTEPH.

Sex Differences in Endothelial-to-Mesenchymal Transition in Chronic Thromboembolic Pulmonary Hypertension

<h3>Purpose</h3> Chronic Thromboembolic Pulmonary Hypertension (CTEPH) results from thrombus organization and obstruction of pulmonary arteries. Population studies suggest sex differences in outcomes. Yet, the mechanisms contributing to sex differences in CTEPH remain poorly understood. We propose that endothelial cells present in the organized thrombi undergo endothelial-to-mesenchymal transition (EndoMT); a process in which endothelial cells lose polarity, cell-to-cell contacts, and gradually adopt mesenchymal cells characteristics. Here, we find that markers of EndoMT are significantly higher in female vs male patients, which could contribute to sex differences in CTEPH pathophysiology. <h3>Methods</h3> A retrospective cohort of ten male and ten female CTEPH patients was studied. Pulmonary endarterectomy specimens from these patients were separated into lobar and segmental sections. Two samples (lobar and segmental) from each patient were paraffin embedded and used for Immunohistochemistry (IHC) analysis. IHC staining for markers for EndoMT, including endothelin-1 (ET-1), Transforming growth factor beta-1 (TGF beta-1), vimentin and alpha smooth muscle cell actin (alpha SMA), was performed. Quantitative tissue area analysis for each marker was performed using the HALO image analysis platform. <h3>Results</h3> We found that females have significantly higher total expression of vimentin and TGF beta-1 (Figure A). There was a trend towards increased total expression of ET-1 and alpha SMA in females that is not significantly different from males (Figure B). However, we found that female segmental sections have higher expression levels for vimentin, TGF beta-1, ET-1 and alpha SMA than male segmental sections (Figure C). <h3>Conclusion</h3> Increased expression of EndoMT markers may account for sex differences in the pathophysiology and outcomes of CTEPH. We will validate these findings and further explore EndoMT in the pathogenesis of CTEPH.

Examining the Development of Chronic Thromboembolic Pulmonary Hypertension at the Single-Cell Level.

The mechanism of chronic thromboembolic pulmonary hypertension (CTEPH) is known to be multifactorial but remains incompletely understood. In this study, single-cell RNA sequencing, which facilitates the identification of molecular profiles of samples on an individual cell level, was applied to investigate individual cell types in pulmonary endarterectomized tissues from 5 patients with CTEPH. The order of single-cell types was then traced along the developmental trajectory of CTEPH by trajectory inference analysis, and intercellular communication was characterized by analysis of ligand-receptor pairs between cell types. Finally, comprehensive bioinformatics tools were used to analyze possible functions of branch-specific cell types and the underlying mechanisms. Eleven cell types were identified, with immune-related cell types (T cells, natural killer cells, macrophages, and mast cells) distributed in the left (early) branch of the pseudotime tree, cancer stem cells, and CRISPLD2+ cells as intermediate cell types, and classic disease-related cell types (fibroblasts, smooth muscle cells, myofibroblasts, and endothelial cells) in the right (later) branch. Ligand-receptor interactions revealed close communication between macrophages and disease-related cell types as well as between smooth muscle cells and fibroblasts or endothelial cells. Moreover, the ligands and receptors were significantly enriched in key pathways such as the PI3K/Akt signaling pathway. Furthermore, highly expressed genes specific to the undefined cell type were significantly enriched in important functions associated with regulation of endoplasmic reticulum stress. This single-cell RNA sequencing analysis revealed the order of single cells along a developmental trajectory in CTEPH as well as close communication between different cell types in CTEPH pathogenesis.

Abstract 12433: Distinct Immune and Mesenchymal Populations Identified by Single Cell RNA Sequencing Contribute to Chronic Thromboembolic Pulmonary Hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequelae of acute pulmonary embolism (PE) in which the PE remodels into a chronic scar in the pulmonary arterial system. This results in vascular obstruction, small vessel arteriopathy and pulmonary hypertension. Previous studies have attempted to identify cells that contribute to CTEPH thrombus but have been limited by the use of specific cell markers or phenotypic modulation after cell growth in culture. Here we have used single cell RNA sequencing (scRNAseq) of samples of CTEPH thrombus removed at the time of pulmonary thromboendarterectomy (PTE) surgery to identify clusters of macrophages, T cells, smooth muscle cells and endothelial cells in CTEPH thrombus. We identified that multiple macrophage subclusters, many of which have an inflammatory phenotype. T cells were a heterogeneous population consisting of CD4+ and CD8+ T cells that are predicted to be inflammatory and also promote vascular remodeling. In the smooth muscle cell cluster, a subcluster of myofibroblasts are predicted to arise from smooth muscle cells based on pseudotime analysis. Notably, cultured endothelial, smooth muscle and myofibroblast cells isolated from CTEPH thrombus have distinct phenotypes from control cells. For example, endothelial cells had reduced angiogenic potential, were proliferative and anti-apoptotic. Lastly, gene expression data identified protease-activated receptor 1 (PAR1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH. Together, these studies suggest a model in which chronic inflammation drives vascular remodeling and mesenchymal senescence in CTEPH pathogenesis and suggest new approaches for targeting this disease.

Identification of a Novel Gene Correlated With Vascular Smooth Muscle Cells Proliferation and Migration in Chronic Thromboembolic Pulmonary Hypertension.

Objective: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thrombofibrotic obstruction of the proximal pulmonary arteries, which result in vascular remodeling of the distal pulmonary artery. While the cellular and molecular mechanisms underlying CTEPH pathogenesis remain incompletely understood, recent evidence implicates vascular remodeling. Here, we identify the molecular mechanisms that contribute to vascular remodeling in CTEPH.Methods: Microarray data (GSE130391) for patients with CTEPH and healthy controls were downloaded from the Gene Expression Omnibus (GEO) and screened for differentially expressed genes (DEGs). DEGs were functionally annotated using Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A protein–protein interaction (PPI) network was constructed to identify hub genes. Finally, pulmonary artery samples were harvested from patients with CTEPH (n = 10) and from controls (n = 10) and primary vascular smooth muscle cells (VSMCs) were cultured. Effects of the proto-oncogene FOS on VSMC proliferation and migration were assessed using expression and knockdown studies.Results: We detected a total of 292 DEGs, including 151 upregulated and 141 downregulated genes. GO analysis revealed enrichment of DEGs in biological processes of signal transduction, response to lipopolysaccharide, signal transduction, and myeloid dendritic cell differentiation. Molecular function analysis revealed enrichment in tumor necrosis factor (TNF)-activated receptor activity, transcriptional activator activity, and protein homodimerization activity. The expression of TNF-α and its receptor (sTNFR1 and sTNFR2) were significantly higher in CTEPH group, compared with control group. KEGG pathway analysis revealed enrichment in salmonella infection, pathways in cancer, osteoclast differentiation, and cytokine-cytokine receptor interaction. Hub genes in the PPI included FOS, suggesting an important role for this gene in vascular remodeling in CTEPH. Primary VSMCs derived from patients with CTEPH showed increased FOS expression and high proliferation and migration, which was attenuated by FOS inhibition. In control VSMCs, TNF-α treatment increased proliferation and migration, which FOS inhibition likewise attenuated.Conclusion: TNF-α drives CTEPH pathogenesis by promoting VSMC proliferation and migration via increased FOS expression. These results advance our understanding of the molecular mechanisms of vascular remodeling in CTEPH, and may inform the development of new therapeutic targets.

Pseudo Heparin Resistance After Pulmonary Endarterectomy: Role of Thrombus Production of Factor VIII

Pulmonary endarterectomy (PEA) is the main treatment for chronic thromboembolic pulmonary hypertension (CTEPH). Postoperative unfractionated heparin dosing can be monitored by activated partial thromboplastin time (APTT) or by anti-factor Xa activity (anti-Xa). In pseudo heparin resistance, APTT response to heparin is blunted due to elevated Factor VIII (FVIII) which can underestimate anticoagulation. We examined possible pseudo heparin resistance after PEA and assessed the impact of FVIII. APTT response to heparin before and after operation was determined in 13 PEA patients anticoagulated with unfractionated heparin. APTT and anti-Xa concordance was analyzed from paired postoperative samples, and antithrombin, fibrinogen and FVIII levels were measured. Single-cell RNA sequencing was used to characterize FVIII gene expression in PEA specimens of 5 patients. APTT response to heparin was blunted after PEA. APTT and anti-Xa were discordant in 36% of postoperative samples and most common discordant patterns were subtherapeutic APTT with therapeutic (16%) or supratherapeutic (11%) anti-Xa. Overall, APTT underestimated anticoagulation relative to anti-Xa in one-third of the samples. FVIII levels were elevated before surgery, increased substantially 1 and 3 days (median 4.32 IU/mL) after PEA, and were higher in discordant than concordant samples. Single-cell RNA sequencing showed FVIII gene expression in PEA specimen endothelial cells. Pseudo heparin resistance is common after PEA likely due to highly elevated postoperative FVIII levels indicating that anti-Xa reflects postoperative heparinization better than APTT in these patients. FVIII production by the pulmonary artery endothelium may participate in local prothrombotic processes important for CTEPH pathogenesis.

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1β, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.

Shear stress-exposed pulmonary artery endothelial cells fail to upregulate HSP70 in chronic thromboembolic pulmonary hypertension.

The pathophysiological mechanisms underlying chronic thromboembolic pulmonary hypertension (CTEPH) are still unclear. Endothelial cell (EC) remodeling is believed to contribute to this pulmonary disease triggered by thrombus and hemodynamic forces disbalance. Recently, we showed that HSP70 levels decrease by proatherogenic shear stress. Molecular chaperones play a major role in proteostasis in neurological, cancer and inflammatory/ infectious diseases. To shed light on microvascular responses in CTEPH, we characterized the expression of molecular chaperones and annexin A2, a component of the fibrinolytic system. There is no animal model that reproduces microvascular changes in CTEPH, and this fact led us to isolated endothelial cells from patients with CTEPH undergoing pulmonary endarterectomy (PEA). We exposed CTEPH-EC and control human pulmonary endothelial cells (HPAEC) to high- (15 dynes/cm2) or low- (5 dynes/cm2) shear stress. After high-magnitude shear stress HPAEC upregulated heat shock protein 70kDa (HSP70) and the HSP ER paralogs 78 and 94kDa glucose-regulated protein (GRP78 and 94), whereas CTEPH-ECs failed to exhibit this response. At static conditions, both HSP70 and HSP90 families in CTEPH-EC are decreased. Importantly, immunohistochemistry analysis showed that HSP70 expression was downregulated in vivo, and annexin A2 was upregulated. Interestingly, wound healing and angiogenesis assays revealed that HSP70 inhibition with VER-155008 further impaired CTEPH-EC migratory responses. These results implicate HSP70 as a novel master regulator of endothelial dysfunction in type 4 PH. Overall, we first show that global failure of HSP upregulation is a hallmark of CTEPH pathogenesis and propose HSP70 as a potential biomarker of this condition.

Possible immune regulation mechanisms for the progression of chronic thromboembolic pulmonary hypertension

PurposeThis study aimed to screen key genes significantly associated with chronic thromboembolic pulmonary hypertension (CTEPH) and predicted suitable drugs for the treatment of CTEPH from the perspective of immune cells. MethodsThe dataset GSE130391 was used for this analysis. Differentially expressed genes (DEGs) between the CTEPH and control groups were screened. Abundance of infiltrating immune cells was analyzed and immune-related DEGs were identified. Next, the circular RNA (circRNA)-micro RNA (miRNA)-mRNA network was constructed, followed by functional enrichment analysis. Then, the protein-protein interaction (PPI) network was constructed and drug-gene interactions were predicted. Finally, miRNA and circRNA prediction results were verified by our previously published studies. ResultsFive key immune cell-related DEGs [CD83 molecule (CD83), complement c5a receptor 1 (C5AR1), atypical chemokine receptor 1 (ACKR1), profilin 2 (PFN2), and solute carrier family 2 member 3 (SLC2A3)] were identified. Several circRNA-miRNA-mRNA interactions were obtained, including circ_0022342miR-503-5pSLC2A3 and circ_0002062miR-92b-3p/miR-92a-3pmannosidase alpha class 2A member 1 (MAN2A1). Immune cell for SLC2A3 was eosinophils and for MAN2A1 was regulatory T cells (Tregs). Additionally, Glufosfamide and Kifunensine might be suitable as candidate drugs for CTEPH treatment. ConclusionsSLC2A3 and MAN2A1 may be important genes for the pathogenesis of CTEPH. Possible immune regulation mechanisms in CTEPH may be circ_0022342miR-503-5pSLC2A3 and circ_0002062miR-92b-3p/miR-92a-3pMAN2A1. These results may be helpful for the diagnosis and treatment of CTEPH from the perspective of immunology.

Hsa-miR-106b-5p participates in the development of chronic thromboembolic pulmonary hypertension via targeting matrix metalloproteinase 2.

BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) is characterized by elevated pressure in pulmonary arteries. This study was performed to explore the critical miRNAs and genes affecting the pathogenesis of CTEPH.MethodsGSE56914 dataset (10 CTEPH whole blood samples and 10 control samples) was downloaded from the Gene Expression Omnibus database. Using limma package, the differentially expressed miRNAs (DE-miRNAs) were acquired. After miRNA-target pairs were obtained using miRWalk2.0 tool, a miRNA-target regulatory network was built by Cytoscape software. Using DAVID tool, significantly enriched pathways involving the target genes were identified. Moreover, the protein–protein interaction network and transcription factor-target regulatory network were built by the Cytoscape software. Additionally, quantitative real-time PCR (qRT-PCR) experiments and luciferase assay were conducted to validate miRNA/gene expression and miRNA–target regulatory relationship, respectively.ResultsThere were 25 DE-miRNAs (8 up-regulated and 17 down-regulated) between CTEPH and control groups. The target genes of has-let-7b-3p, has-miR-17-5p, has-miR-3202, has-miR-106b-5p, and has-miR-665 were enriched in multiple pathways such as “Insulin secretion”. qRT-PCR analysis confirmed upregulation of hsa-miR-3202, hsa-miR-665, and matrix metalloproteinase 2 (MMP2) as well as downregulation of hsa-let-7b-3p, hsa-miR-17-5p, and hsa-miR-106b-5p. Luciferase assay indicated that MMP2 was negatively mediated by hsa-miR-106b-5p.ConclusionsThese miRNAs and genes were associated with the pathogenesis of CTEPH. Besides, hsa-miR-106b-5p was involved in the development of CTEPH via targeting MMP2.

Postoperative Heparinization Monitoring by Anti-Xa is More Accurate Than by APTT after Pulmonary Endarterectomy - Role of Increased Systemic and Endothelial Production of Factor VIII

Pulmonary endarterectomy (PEA) is curative for suitable chronic thromboembolic pulmonary hypertension (CTEPH) patients. Careful anticoagulation prevents postoperative bleeding and thromboembolic recurrence. Heparinization is often guided by aPTT but it can be influenced by Factor VIII - a coagulation factor elevated in CTEPH. Anti-Xa-guided anticoagulation is less affected by other factors. We investigated the correlation between aPTT and Anti-Xa after PEA and the impact of coagulation factors. 55 paired perioperative aPTT and Anti-Xa samples from 14 PEA patients were analyzed. aPTT guidance was used for postoperative unfractioned heparin titration. Samples were considered concordant if aPTT (target 45-65sec) and Anti-Xa (target 0.3-0.5UI/ml) were in the same category (subtherapeutic, therapeutic or supratherapeutic) defined by our clinical practice. Antithrombin activity, Factor VIII and fibrinogen were concomitantly measured. Single-cell RNA sequencing of PEA specimens (n=5) was used to screen for coagulation factor transcription. aPTT and Anti-Xa were concordant in 64% of the postoperative sample pairs. Most common discordant pattern was subtherapeutic aPTT with therapeutic (15.6%) or supratherapeutic (11.1%) Anti-Xa. Compared to pre-operative levels, plasma Factor VIII increased on day 1 (p=0.04) and day 3 (p=0.0003), and fibrinogen on day 3 after PEA (p=0.008). Discordant aPTT and Anti-Xa samples had higher antithrombin (p<0.0001), Factor VIII (p=0.0001) and fibrinogen (p=0.0002) levels than concordant samples. Single-cell RNA sequencing showed endothelial Factor VIII transcription in the PEA specimen. aPTT monitoring underestimates anticoagulation after PEA compared to Anti-Xa and is possibly influenced by elevated postoperative Factor VIII levels. Local Factor VIII production by thrombus endothelium may contribute to CTEPH pathogenesis.

A multifaceted investigation into molecular associations of chronic thromboembolic pulmonary hypertension pathogenesis.

PurposeChronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension and right ventricular dysfunction. Conditions such as thrombophilias, dysfibrinogenemias, and inflammatory states have been associated with chronic thromboembolic pulmonary hypertension, but molecular mechanisms underlying this disease are poorly understood. We sought to characterize the molecular and functional features associated with chronic thromboembolic pulmonary hypertension using a multifaceted approach.MethodsWe utilized functional assays to compare clot lysis times between chronic thromboembolic pulmonary hypertension patients and multiple controls. We then performed immunohistochemical characterization of tissue from chronic thromboembolic pulmonary hypertension, pulmonary arterial hypertension, and healthy controls, and examined RNA expression patterns of cultured lymphocytes and pulmonary arterial specimens. We then confirmed RNA expression changes using immunohistochemistry, immunofluorescence, and Western blotting in pulmonary arterial tissue.ResultsClot lysis times in chronic thromboembolic pulmonary hypertension patients are similar to multiple controls. Chronic thromboembolic pulmonary hypertension endarterectomized tissue has reduced expression of both smooth muscle and endothelial cell markers. RNA expression profiles in pulmonary arteries and peripheral blood lymphocytes identified differences in RNA transcript levels related to inflammation and growth factor signaling, which we confirmed using immunohistochemistry. Gene expression data also suggested significant alterations in metabolic pathways, and immunofluorescence and Western blot experiments confirmed that unglycosylated CD36 and adiponectin expression were increased in chronic thromboembolic pulmonary hypertension versus controls.ConclusionsOur data do not support impaired clot lysis underlying chronic thromboembolic pulmonary hypertension, but did demonstrate distinct molecular patterns present both in peripheral blood and in pathologic specimens of chronic thromboembolic pulmonary hypertension patients suggesting that altered metabolism may play a role in chronic thromboembolic pulmonary hypertension pathogenesis.

Analysis of Oxygenation in Chronic Thromboembolic Pulmonary Hypertension Using Dead Space Ratio and Intrapulmonary Shunt Ratio

Current therapeutic methods for chronic thromboembolic pulmonary hypertension (CTEPH) can improve hemodynamic status and are expected to improve prognoses. However, some patients experience dyspnea during effort and continue supplemental oxygenation despite their hemodynamic status being fully improved. Considering the pathogenesis of CTEPH, the dead space and intrapulmonary shunt are assumed to be responsible for hypoxia in CTEPH, but their contributions are unclear. It is also unclear whether they are improved after treatment. The aim of this study was to investigate the implications of the dead space ratio (DSR) and the intrapulmonary shunt ratio (ISR) for hypoxia in CTEPH and treatment for CTEPH.We retrospectively measured the DSR and ISR of 23 consecutive patients with CTEPH. For 11 of these 23 (10 were treated by balloon pulmonary angioplasty, one with riociguat), we also measured these parameters before and after CTEPH treatments. Overall, the DSR and ISR were abnormally elevated (DSR: 0.63 ± 0.06; ISR: 0.20 ± 0.05). After treatment, mean pulmonary artery pressure was improved (from 40.3 ± 8.1 to 25.5 ± 2.7 mmHg). Although atrial oxygen saturation (SaO2), DSR and ISR were improved (SaO2: from 90.2 ± 3.2 to 93.7 ± 1.8%; DSR: from 0.64 ± 0.06 to 0.58 ± 0.05; ISR: from 0.20 ± 0.04 to 0.18 ± 0.02), these improvements were slight compared with that of mean pulmonary artery pressure.The DSR and ISR were abnormally elevated in patients with CTEPH and their improvement by treatment was limited. Only DSR can be a useful marker for normalization of hypoxia in CTEPH.

Potential Involvement of Osteopontin in Inflammatory and Fibrotic Processes in Pulmonary Embolism and Chronic Thromboembolic Pulmonary Hypertension.

Inflammation and incomplete thrombus resolution leading to obstructive fibrotic remodelling are considered critical mechanisms for the development of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism (PE). Osteopontin (OPN) is involved in a variety of biological processes including inflammation and tissue fibrosis. OPN plasma concentrations were measured in 70 CTEPH and 119 PE patients. Tissue material from 6 CTEPH patients removed during pulmonary endarterectomy and murine venous thrombi induced by subtotal ligation of the inferior vena cava in C57BL/6 mice were analysed by (immuno)histochemistry. CTEPH patients had higher OPN plasma concentrations (median, 106.9 [interquartile range, 75.6-155.9]) compared to PE patients (90.4 [53.3-123.9] ng/mL, p = 0.001). OPN- and matrix metalloproteinase (MMP)-9-positive cells were predominantly present in myofibroblast-rich and profibrotic areas of CTEPH tissue material. Early stages of murine thrombus resolution were characterised by high numbers of OPN- and MMP-2-positive cells while OPN was almost absent in fresh thrombi of CTEPH tissue material. PE patients with OPN plasma concentrations of < 55 ng/mL had a 15.2-fold (95% confidence interval, 1.7-135.5, p = 0.015) increased risk for a diagnosis of CTEPH during follow-up. The results of the present observational translational study point to a possible involvement of OPN in the pathogenesis of CTEPH by affecting early inflammatory and late fibrotic processes.