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Pulmonary arterial hypertension (PAH) is a serious disease characterized by elevated pulmonary artery pressure, inflammatory cell infiltration and pulmonary vascular remodeling. However, little is known about the pathogenic mechanisms underlying the disease onset and progression. RNA sequencing (RNA-seq) was used to identify the transcriptional profiling in control and rats injected with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 23200 transcripts and 280, 1342, 908 and 3155 differentially expressed genes (DEGs) were identified at the end of week 1, 2, 3 and 4, of which Svop was the common top 10 DEGs over the course of PAH progression. Functional enrichment analysis of DEGs showed inflammatory/immune response occurred in the early stage of PAH development. KEGG pathway enrichment analysis of DEGs showed that cytokine-cytokine receptor interaction and neuroactive ligand-receptor interaction were in the initiation and progression of PAH. Further analysis revealed impaired expression of cholinergic receptors, adrenergic receptors including alpha1, beta1 and beta2 receptor, and dysregulated expression of γ-aminobutyric acid receptors. In summary, the dysregulated inflammation/immunity and neuroactive ligand receptor signaling pathways may be involved in the onset and progression of PAH.

Pulmonary arterial hypertension (PAH) in humans manifests as a chronic process. However, PAH induced by high-dose monocrotaline (MCT) in animals occurs as a subacute process. To establish a chronic PAH model, rats were randomly divided into three groups, control (ctrl), single injection (SI), and twice injection (TI) groups. Rats in the SI group received a single intraperitoneal injection of 40 mg/kg MCT on day 0. Rats in the TI group received twice injections of 20 mg/kg MCT on days 0 and 7. Survival percentage, characteristic changes of pulmonary arterial variables, and right ventricular features were evaluated. Thirty-five days after the first MCT injection, survival percentage in TI group was higher than that in the SI group. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), pulmonary vascular remodeling, serum tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) were higher either in SI or in TI 28 and 35 days after the first MCT injection. The rats in the SI and TI groups exhibited higher right ventricle end diastolic diameter (RVEDD) and lower adjusted pulmonary artery acceleration time (PAAT/HR), tricuspid annular plane systolic excursion (TAPSE), cardiac output (CO) and right ventricle fractional shortening (RVFS) when compared with controls. However, mPAP, RVHI, TAPSE, PAAT/HR, CO, TNFα, and IL-6 were lower and RVEDD were higher in the TI group than in the SI group. Pulmonary macrophage infiltration and right ventricle (RV) fibrosis were lower in TI than SI groups. The cardiomyocyte cross-sectional area and the beta myosin heavy chain (MYH7) mRNA level of RV were lower in TI than SI, whereas alpha myosin heavy chain (MYH6) was increased. These results show that two intraperitoneal injections of 20 mg/kg MCT with seven days interval could induce a model similar to chronic PAH with increased survival percentage in rats. Impact statement We demonstrated previously that a single intraperitoneal injection of 40 mg/kg MCT produced a subacute, not chronic, PAH model in rats, and the short survival periods of these rats did not represent adequately the chronic PAH seen in humans. To overcome this limitation, in this study, the single dose of 40 mg/kg MCT was divided into twice injections of 20 mg/kg with an interval of seven days. This modified administration of MCT produced an animal model much more similar to chronic PAH with prolonged survival and characteristic changes of structures and function in pulmonary arteries and right ventricles.

Background Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH. Methods A series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. Results Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Conclusions CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH.

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BackgroundPulmonary arterial hypertension (PAH) is a devastating cardio-pulmonary vascular disease in which chronic elevated pulmonary arterial pressure and pulmonary vascular remodeling lead to right ventricular failure and premature death. However, the exact molecular mechanism causing PAH remains unclear.MethodsRNA sequencing was used to analyze the transcriptional profiling of controls and rats treated with monocrotaline (MCT) for 1, 2, 3, and 4 weeks. Weighted gene co-expression network analysis (WGCNA) was employed to identify the key modules associated with the severity of PAH. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore the potential biological processes and pathways of key modules. Real-time PCR and western blot analysis were used to validate the gene expression. The hub genes were validated by an independent dataset obtained from the Gene Expression Omnibus database.ResultsA total of 26 gene modules were identified by WGCNA. Of these modules, two modules showed the highest correlation with the severity of PAH and were recognized as the key modules. GO analysis of key modules showed the dysregulated inflammation and immunity, particularly B-cell-mediated humoral immunity in MCT-induced PAH. KEGG pathway analysis showed the significant enrichment of the B-cell receptor signaling pathway in the key modules. Pathview analysis revealed the dysregulation of the B-cell receptor signaling pathway in detail. Moreover, a series of humoral immune response-associated genes, such as BTK, BAFFR, and TNFSF4, were found to be differentially expressed in PAH. Additionally, five genes, including BANK1, FOXF1, TLE1, CLEC4A1, and CLEC4A3, were identified and validated as the hub genes.ConclusionThis study identified the dysregulated B-cell receptor signaling pathway, as well as novel genes associated with humoral immune response in MCT-induced PAH, thereby providing a novel insight into the molecular mechanisms underlying inflammation and immunity and therapeutic targets for PAH.

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