Mitochondrial dysfunction has been implicated in the pathogenesis and progression in a wide range of diseases, such as Alzheimer's disease, chronic lung diseases, and various malignancies (26–28). It also plays an important role in PAH pathophysiology (29). Compared with healthy pulmonary artery smooth muscle cells (PASMCs), human PAH PASMCs have hyperpolarized mitochondria and decreased mitochondria-derived reactive oxygen species (30, 31). Increased levels of citric acid cycle intermediates have been found in PAH patients compared with healthy subjects, suggesting possible dysfunction of mitochondrial citric acid cycle in PAH (32). In addition, altered mitochondrial dynamics such as fission/fusion imbalance could promote the proliferation phenotype in pulmonary arterial cells (29). Nevertheless, the exact roles of mitochondrial abnormalities in the progression of PAH remain incompletely understood. Further elucidation of PAH pathogenesis would be beneficial for therapy development. Herein, we employed integrated transcriptomic and metabolomic analyses to explore the key genes and metabolites involved in the development of PAH.
In our analysis of the GEO datasets, KEGG pathway enrichment indicated that the DEGs in calcium signaling and cAMP signaling pathways were significantly enriched in the PAH group. Abnormalities in calcium signaling have been linked with pathological modulation of pulmonary vascular tone and pulmonary artery smooth muscle cell proliferation (33, 34). Moreover, mitochondria served as sensors and regulators of calcium signaling have been shown to affect the Ca2+ feedback regulation of channel activity, and an increased uptake of mitochondrial Ca2+ was observed in pulmonary arterial smooth muscle cells from patients with PAH (35, 36). The cAMP signaling pathway was also implicated in PAH. A recent study revealed that cAMP signaling pathway played an important role in PAH pathogenesis (37). In addition, the cAMP-protein kinase A (PKA) signaling can regulate mitochondrial functions, and alterations of mitochondrial cAMP-PKA signaling have implications in the pathogenesis of various diseases (38). Given the close connections between mitochondria and calcium signaling as well as cAMP signaling pathways, and the growing evidence of the role of mitochondrial dysfunction in PAH, we further examined the alterations of mitochondria-related pathways in the PAH group, and sifted the most critical signals by machine learning. Results revealed that the electron transport chain in mitochondrial oxidative phosphorylation system was significantly down-regulated in PAH, especially the process of electron transport from cytochrome c to oxygen. It was suggested that the main proteins of the electron transport chain involved in mitochondrial dysfunction might contribute to PAH (39). The mitochondrial electron transport chain (also known as respiratory chain) as a major part of mitochondrial oxidative phosphorylation system contains four enzymatic complexes (complex I to IV), and complex IV (cytochrome c oxidases, COX) receives electrons transferred by cytochrome c to reduce oxygen to water for ATP production (40). COX4I2, one of the major gene participating in the process of electron transport from cytochrome c to oxygen, is essential for acute pulmonary oxygen sensing and involved in hypoxia-induced pulmonary hypertension (41, 42). Our findings suggest that dysfunction of the electron transport from cytochrome c to oxygen in mitochondria might contribute to PAH.
To further investigate the findings derived from public datasets, we obtained PAH and non-PAH lung tissue samples from patients for transcriptomic and metabolomic analyses. In the transcriptomic analysis, 14 DEGs were mitochondria-related genes, four of which (KIT, OTC, CAMK2A, and CHRNA1) were highly linked with the differential metabolites identified through subsequent metabolomic profiling. Similar to our findings, quantitative immunohistochemistry in human lung PAH tissues demonstrated an elevation in c-KIT level (43), and c-KIT-positive cells were reported to participate in vascular remodeling in PAH (44). OTC, which had higher gene expression levels in PAH lung tissue samples in the present study, also exhibited a significant increase in PAH gut bacteria (45). As for CAMK2A, it has been reported that alpha1A-adrenoceptor is involved in the proliferation of pulmonary artery smooth muscle cells via CaMKII signaling (46). In addition, variation in CHRNA1 genetics was highly associated with diastolic blood pressure (47). These results indicate that mitochondria-related genes are involved in the development of PAH.
The regulation of gene expression is intricately linked to cell metabolism. Metabolic pathways provide precursor molecules and ATP that are necessary for gene expression (48). Hence, we conducted metabolomic profiling to examine the metabolic alterations in PAH, and elevated levels of ADP were observed. It has been proposed that purinergic signaling activated by an alteration of nucleotides contributes to the pathogenesis of PAH (49). Moreover, activation of purinergic P2Y1-receptor (P2Y1R) and P2Y12R mediated by ADP plays an important role in pulmonary vascular remodeling and inflammation in PAH (49). Our metabolomic analysis also revealed a significant decrease in the levels of citric acid in the PAH samples. Conversely, increased levels of citric acid or citrate (the conjugate base of citric acid) in PAH patients were observed in other studies (50, 51). The discrepancy should be further explored. Citrate is a significant intermediate produced in the initial step of the citric acid cycle, subsequently undergoing conversion into isocitrate (52). Dysfunction of the citric acid cycle has been reported to occur in PAH and might potentially contribute to the pathogenesis of PAH (29). In addition, increased levels of 3-phenyllactic-acid were found in the PAH samples in the current study. 3-phenyllactic acid is a product of phenylalanine catabolism generated by human host and intestinal bacteria (53). It regulates human immune system and acts as a mediator of bacterial-host interactions (53). Phenyllactic acid can be accumulated in the blood and tissues of patients with phenylketonuria (54). It also promotes cell migration and invasion in cervical cancer by up-regulating the expression of the mitochondrial protease MMP9 (55). In fungi, 3-phenyllactic acid could disrupt cell membrane and interfere with mitochondrial energy metabolism (56, 57). Our findings suggest that 3-phenyllactic-acid might potentially be a novel biomarker for PAH.
Certain limitations existed in the current study. Limited lung tissue samples from patients were available for analyses, restricting its broader applicability, and access to larger sample sizes will allow further exploration of the present results. Moreover, future in vitro experiments would provide further insights into the potential pathogenic mechanisms of PAH.