Abstract
Objective: Pulmonary hypertension (PH) associated with hypoxia and lung disease (Group 3) is the second most common form of PH and associated with increased morbidity and mortality. This study was aimed to identify hypoxia induced metabolism associated genes (MAGs) for better understanding of hypoxic PH. Methods: Rat pulmonary arterial smooth muscle cells (PASMCs) were isolated and cultured in normoxic or hypoxic condition for 24 h. Cells were harvested for liquid chromatography-mass spectrometry analysis. Functional annotation of distinguishing metabolites was performed using Metaboanalyst. Top 10 enriched metabolite sets were selected for the identification of metabolism associated genes (MAGs) with a relevance score >8 in Genecards. Transcriptomic data from lungs of hypoxic PH in mice/rats or of PH patients were accessed from Gene Expression Omnibus (GEO) database or open-access online platform. Connectivity Map analysis was performed to identify potential compounds to reverse the metabolism associated gene profile under hypoxia stress. The construction and module analysis of the protein-protein interaction (PPI) network was performed. Hub genes were then identified and used to generate LASSO model to determine its accuracy to predict occurrence of PH. Results: A total of 36 altered metabolites and 1,259 unique MAGs were identified in rat PASMCs under hypoxia. 38 differentially expressed MAGs in mouse lungs of hypoxic PH were revealed, with enrichment in multi-pathways including regulation of glucose metabolic process, which might be reversed by drugs such as blebbistatin. 5 differentially expressed MAGs were displayed in SMCs of Sugen 5416/hypoxia induced PH rats at the single cell resolution. Furthermore, 6 hub genes (Cat, Ephx1, Gpx3, Gstm4, Gstm5, and Gsto1) out of 42 unique hypoxia induced MAGs were identified. Higher Cat, Ephx1 and lower Gsto1 were displayed in mouse lungs under hypoxia (all p < 0.05), in consistent with the alteration in lungs of PH patients. The hub gene-based LASSO model can predict the occurrence of PH (AUC = 0.90). Conclusion: Our findings revealed six hypoxia-induced metabolism associated hub genes, and shed some light on the molecular mechanism and therapeutic targets in hypoxic PH.
Highlights
Pulmonary hypertension (PH) is a heterogenous disorder and characterized by high pulmonary artery pressure and elevated pulmonary vascular resistance that usually leads to right heart failure and even death (Hoeper et al, 2013)
We found that distribution of hypoxia exposed rat pulmonary arterial smooth muscle cells (PASMCs) differed from control cells by partial least squares discriminant analysis (PLS-DA) analysis (Figure 2A). 29 up-regulated and 121 down-regulated metabolites were observed in hypoxia challenged PASMCs compared to control cells (FC > 1.5 or
Among those only 36 metabolites were identified to have a Variable important in projection (VIP) score >1 and their relative expressions among each individual sample were visualized in heatmap, of which 7 were upregulated and 29 were downregulated in response to hypoxia (Figure 2C)
Summary
Pulmonary hypertension (PH) is a heterogenous disorder and characterized by high pulmonary artery pressure and elevated pulmonary vascular resistance that usually leads to right heart failure and even death (Hoeper et al, 2013). Growing evidence indicates that the excessive proliferation, apoptosis resistance and a contractile-to-synthetic phenotype switch of PASMCs has emerged as essential mechanisms in the remodeling of the pulmonary vasculature associated with hypoxic PH. Our previous study demonstrated that overexpression of DNMT3B (encoding DNA methyltransferase 3B) mitigated the remodeling of hypoxic PH in mice, suggesting the protective role of DNMT3B on the pulmonary vascular remodeling via the modulation of PASMC proliferation/ migration (Yan et al, 2020). Genetic ablation of CD146 in smooth muscle cells mitigates pulmonary vascular remodeling in chronic hypoxic mice (Luo et al, 2019). Deficiency and pharmacological inhibition of SphK1 mitigated the development of hypoxic PH in vivo and SphK1 deficiency inhibited PASMC proliferation in vitro (Chen et al, 2014)
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