Pulmonary hypertension (PH) is mainly identified by exertional dyspnea and fatigue, representing a clinical syndrome influenced by multiple factors16. PH patients display extensive pathological alterations in the intima, media, and adventitia layers of pulmonary blood vessels, involving vascular injury, thickening, proliferation, and fibrotic changes17,18. These modifications progressively narrow and obstruct pulmonary arterial lumens, leading to escalating pulmonary arterial pressure consequent to heightened pulmonary vascular resistance. Ultimately, this cascade of events culminates in heart failure19,20. However, the pathological and biological mechanisms underlying the structural and functional abnormalities of these pulmonary blood vessels have not been fully elucidated21. Current perspectives include changes in gene expression, epigenetic alterations, metabolic dysfunction in the heart and pulmonary vasculature, and inflammatory responses22–24. The main physiological role of mitochondria is to produce ATP via the tricarboxylic acid cycle and oxidative phosphorylation25. Additionally, they generate appropriate levels of mitochondrial reactive oxygen species, regulate calcium ions in both the cytoplasm and mitochondrial matrix, modulate apoptosis, participate in metabolite synthesis and breakdown, and facilitate organelle transport to their proper cellular locations26,27. Any disruptions in these functions are collectively termed mitochondrial dysfunction. Several mechanisms of mitochondrial dysfunction have been linked to the development of PH28,29. But, the role of MRGs in PH remains unclear. Over the past few decades, rapid progress in gene sequencing technologies and related fields of bioinformatics has enabled the utilization of extensive sequencing data for further analysis. In this research, a total of 144 DEGs (78 up-DEGs and 66 down-DEGs) were obtained by intersecting 751 DEGs1 in blood training set and 6,155 DEGs2 in tissue training set. Next, taking the intersection of 144 DEGs and 1,136 MRGs, 7 candidate genes (ALAS2, CPT2, TST, SLC25A39, COQ10B, MRPS30 and MCL1) were acquired. Finally, MRPS30, ALAS2 and TST were recognized as biomarkers through overlapping signature genes by two machine learning algorithms. Bioinformatics methods identified three biomarkers in PH patients that were closely associated with MRGs.
Besides, PPI network construction and functional enrichment analyses were performed to filter key genes and important pathways. The GO annotation analysis revealed that differentially expressed genes (DEGs) are associated with various pathways related to mitochondria. These pathways include mitochondrial transmembrane transport, the mitochondrial inner membrane, and the mitochondrial matrix. KEGG pathway results declared that DEGs were mainly mapped to fatty acid metabolism, cysteine and methionine metabolism, porphyrin metabolism, fatty acid degradation, glycine serine and threonine metabolism ,sulfur metabolism and PPAR signaling pathway. Constructing a protein-protein interaction (PPI) network is advantageous for researchers aiming to explore the underlying molecular mechanisms of PAH. This is because the DEGs can be organized and categorized within the network based on their interactions, facilitating a clearer understanding of their roles30. We constructed a diagnostic graph using the selected MRPS30, ALAS2, and TST, and plotted the ROC curves. The results indicated that their AUC values were all greater than 0.7, demonstrating their good diagnostic value. However, the expression of TST was not validated in the validation set, which may be attributed to sample heterogeneity, insufficient sample size, potential bias introduced by different experimental techniques or platforms.
MRPS30, also known as programmed cell death protein 9 (PDCD9), is responsible for encoding a mitochondrial ribosomal protein that participates in apoptosis31. During the process of apoptosis, mitochondria play a crucial role by releasing factors such as cytochrome C, participating in the regulation of the apoptotic signaling pathway in cells. As a component of the mitochondrial ribosome, MRPS30 may play an important role in regulating these mitochondria-related apoptotic pathways32. Studies have found that knocking out MRPS30 inhibits the proliferation, migration, and invasion of these breast cancer cells, while also inducing apoptosis33. Excessive proliferation and reduced apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs) are the defining characteristics of pulmonary arterial hypertension (PAH), leading to pulmonary vascular remodeling34. This study observed that the expression level of MRPS30 was significantly higher in patients with PH compared to the control group. MRPS30 was highly expressed in PH, suggesting it could serve as a diagnostic or therapeutic target for PAH. It was also found that MRPS30 is associated with ribosomes, spliceosomes, T cell receptor signaling pathways, and other pathways. Protein 5′-Aminolevulinate Synthase 2 of ALAS2 is an erythrocyte-specific mitochondrial localization enzyme and that step one of the heme biosynthetic pathway is catalyzed by this product. Blemishes on ALAS2 will lead to the development of X-linked pyridoxine responsive sideroblastic anemia35,36Sokratis Kariotis et al. discerned through unsupervised transcriptomic profiling of whole blood that the subgroup with a poor prognosis exhibited upregulation of ALAS2 and simultaneous downregulation of multiple immunoglobulin genes37. In previous gene expression investigations encompassing various forms of pulmonary hypertension (PH), such as idiopathic pulmonary arterial hypertension (IPAH), a notable elevation in ALAS2 expression was consistently observed. This pattern was evident not only in systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH) but also in IPAH cases. Within the IPAH cohort specifically, heightened levels of ALAS2 exhibited robust correlations with key hemodynamic parameters including right atrial pressure, pulmonary vascular resistance, pulmonary artery saturation, and cardiac index38,39. Our investigation unveiled a pronounced upregulation of ALAS2 within the pulmonary hypertension cohort. ALAS2 exhibited predominant enrichment in crucial pathways including ribosomal function, T cell receptor signaling, and metabolism of glycine, serine, and threonine. Furthermore, our findings implicated ALAS2 in the modulation of abnormal iron homeostasis, pulmonary vascular remodeling, and the pathogenesis of pulmonary hypertension, aligning seamlessly with earlier research findings40. TST also known as Rhodanese, is a mitochondrial enzyme which catalyzes the transfer of sulfur in several molecular pathways’ gene encodes for thiosulfate sulfurtransferase, an enzyme involved in sulfur metabolism. Thiosulfate sulfurtransferase, a type of enzyme protein, is primarily present in the liver, kidneys, intestines, and blood in the human body, participating in various biological processes. The primary function of thiosulfate sulfurtransferase is to catalyze sulfur metabolism reactions, including the transfer between thiols and sulfur-containing substrates (such as sulfate ions).Thiosulfate sulfurtransferase's roles are pivotal in maintaining sulfur homeostasis and participating in detoxification processes41,42. In recent times, there has been a shift in focus towards elucidating its role in hydrogen sulfide metabolism, antioxidant defense mechanisms, and mitochondrial function, particularly within the framework of safeguarding biological systems against oxidative stress43,44. Following its initial recognition as an enzyme involved in cyanide detoxification, subsequent investigations revealed its involvement in sulfur metabolism, alteration of iron-sulfur clusters, and the reduction of antioxidants such as glutathione and thioredoxin45. Multiple studies have demonstrated that deficiency of iron-sulfur (Fe-S) clusters in pulmonary endothelial cells, which are bioinorganic cofactors necessary for enzymatic redox functions, has been associated with the promotion of pulmonary hypertension46,47. In our investigation, we observed a notable upregulation of TST expression within the pulmonary hypertension cohort, with TST showing predominant enrichment in lysosomal and oxidative phosphorylation pathways, among others.
Through immune function analysis, it was found that monocytes are associated with three genes: MRPS30, ALAS2, and TST. Patients with PH may experience enhanced inflammatory responses, leading to the activation of monocytes and their involvement in regulating inflammation48. It is worth noting that monocytes constituted the highest proportion in the peripheral blood of individuals with PH. Previous studies have indicated that in response to hypoxia, pulmonary monocytes have the ability to sense hypoxic conditions, migrate into pulmonary arterioles, and expedite vascular remodeling. This suggests a direct involvement of the monocyte lineage in the pathogenesis of PAH49. The activation of monocytes may also be associated with thrombus formation and vascular wall thickening. In pulmonary arterial hypertension, thrombus formation and vascular wall thickening are among the key factors leading to increased vascular resistance. Monocyte involvement may impact the structure and function of the vascular wall through promoting thrombus formation, stimulating smooth muscle cell proliferation, and synthesizing collagen, among other mechanisms50. The results above suggest that monocytes may promote the development of PH, which is consistent with our research findings.