Transcriptional changes of the extracellular matrix in chronic thromboembolic pulmonary hypertension governs right ventricle remodeling and recovery

Abstract

Chronic thromboembolic pulmonary hypertension (CTEPH) is a complex and multifactorial disease with progressive right ventricular (RV) failure. CTEPH, which is associated with high morbidity and mortality, is classified as group 4 pulmonary hypertension (PH) according to the WHO classification. Pulmonary endarterectomy (PEA) is a potentially curative treatment for operable CTEPH patients, leading to a reduction in RV afterload and improvement in interventricular septum (IVS) asynchrony. The effect of PEA in operable CTEPH patients on the RV and the transition from chronic impairment to normal RV function makes it a unique model for study. The molecular mechanism and RV dynamics were investigated in different subgroups of patients with CTEPH before and after PEA. In addition, RNA-sequencing (RNA-seq) was performed on the RV tissue of both the exploratory (A-prePEA) and confirmatory cohorts (B-prePEA). In addition, patients in both the exploratory and confirmatory cohorts were classified as moderate (A-prePEAm, n = 4; B-prePEAm, n = 30) and severe-risk (A-prePEAs, n = 5; B-prePEAs, n = 18) based on their clinical parameters and ERS/ESC guidelines before PEA surgery. Furthermore, a group in the confirmatory cohort was classified as intermediate-risk (B-prePEAi, n = 23). The gene expression patterns of the septum before (B-pre-septal-PEA, n = 3) and after PEA (B-postPEA, n = 21) were compared with RV (B-prePEA, n=3 and B-prePEA, n=21) in pairwise comparisons in the same patients to determine the reversal effect of PEA. Pathway Enrichment Analysis was performed to determine the differences between prePEA subgroups. In addition, molecular and biological approaches were used to determine the role of differentially regulated genes. The functional roles of Ankyrin Repeat Domain 1 (ANKRD1), Activating Transcription Factor 3 (ATF3), Interleukin 7 Receptor (IL-7R), Inhibin Subunit Beta B (INHBB), and Serpin Family E Member 1 (SERPINE1) were determined in various experimental models including in vitro, ex vivo, and in vivo. The results of this study showed that first, the RNA profile of prePEA separates prePEAm from postPEAs in both the exploratory and confirmatory cohorts, confirming the classification of patients based on their clinical parameters and ESC guidelines. Second, the results of the RNA profile in the exploratory cohort can be validated in the confirmatory cohort with a large number of patients. Third, ECM-related terms were significantly activated in prePEAm versus prePEAs of both cohorts. Adipogenesis was detected in all the subgroups of prePEA whereas, oxidoreductase activity was not observed in prePEAm versus prePEAi. The TGF-beta signaling pathway, BMP signaling pathway, and Hippo signaling pathways were identified in B-prePEAm versus B-prePEAs and B-prePEAm versus B-prePEAi. Fourth, pathway enrichment analysis in B-prePEA and B-postPEA in the same patients revealed the following signaling pathways: MAPK, PI3K-Akt, and AGE-RAGE are common pathways between moderate, intermediate, and severe risk groups. Fifth, bioinformatics analysis showed that the high expression of SERPINE1 in prePEAs compared with prePEAm plays an important role in triggering the disease in CTEPH. Furthermore, activation of the AGE-RAGE pathway in prePEAs compared to prePEAm with SERPINE1 involvement confirmed the importance of SERPINE1 in the pathogenesis of CTEPH. Sixth, the comparison of the gene expression profile of septum (pre and post PEA) with that of RV (prePEA), revealed that in addition to ECM changes, simultaneous alterations in the myocardium, including sarcomere organization, and contractile fiber, were induced and ANKRD1 was associated with actin filament organization. ATF3, INHBB, and IL-7R are the genes related to the PI3K-Akt signaling pathway. Seventh, investigation of the functional role of ANKRD1, AFT3, IL-7R, INHBB, and SERPINE1 molecules in in vitro and in vivo models related to these signaling pathways highlights the crucial role of these molecules in modulating cardiac cell proliferation, migration, angiogenesis, and wound healing. Overall, these results demonstrate that before PEA, altered gene expression profile and signaling pathways together with changes in ECM and myocardium play an important role in the pathophysiology of CTEPH. In contrast, the reverse RV remodeling after PEA due to reverse ECM changes led to improved pulmonary hemodynamics and exercise capacity.