AD is a life-threatening cardiovascular disease. Data from a recently updated study have shown that the mortality rate among non-surgical patients who present with type A acute aortic dissection was 0.5% per hour within the first 48 hours and 5.8% in the 48 hours[15]. However, the pathogenesis of AD is still unclear at present, which can affect and delay the diagnosis of AD; thus, it is necessary to find indicators to ensure quick and accurate diagnosis of AD. Anoikis is a form of apoptosis induced by the detachment of anchorage-dependent cells from adjacent cells and/or surrounding matrix, which leads to their incompatibility with the microenvironment[16, 17]. Anoikis resistance has been extensively studied in the field of oncology due to the ability of invasive cancer cells to survive away from the primary site[18], while degradation of ECM components, oxidative stress, and apoptosis of smooth muscle cells are closely related to the functions of ARGs in the pathogenesis of AD, therefore, we speculated that certain ARGs might be early diagnostic indicators for AD.
First, we used two cohorts of the GEO database to conduct differential analysis of ARGs expression between cases of AD and healthy controls and obtained significant genes; the findings suggested that most of the genes were up-regulated and only a small number of them were down-regulated in the cases of AD. We then performed GO, KEGG, and Metascape enrichment analyses of DEGs associated with anoikis, which indicated that the functions of ARGs were mainly enriched in cellular response to oxygen levels, regulation of cellular stress response, components of the extracellular matrix, and lipids and atherosclerosis, which might be mechanisms underpinning the association between anoikis and AD.
Studies have shown that intermittent hypoxia (IH) in obstructive sleep apnea syndrome (OSAS) is associated with the pathogenesis of AD, and IH-induced reactive oxygen species (ROS) and hypoxia-inducible transcription factor-1 (HIF-1) can contribute to the harmful consequences of cardiovascular disease[19]. Experiments have demonstrated that IH can promote the progression of AD through the ROS-HIF-1α-MMP pathway[20]. Furthermore, macrophages can mediate the HIF1α-ADAM17 pathway through metabolic reprogramming, which promotes the pro-inflammatory response and destruction of elastic fibers, ultimately leading to aggravation of AD[21]. A recent study suggested that the proportion of M1 macrophages, lipid metabolism indicators such as low-density lipoprotein, high-density lipoprotein, and apolipoprotein, and inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-10 were significantly associated with the development of AD[22]. Lipid metabolism disorders can eventually lead to thickening and sclerosis of arterial wall, and rupture of atherosclerotic plaques can lead to intimal tears, leading to the formation of AD[23]. The aortic intima is composed of elastic fibers and vascular smooth muscle cells (VSMCs), which bind to collagen fibers, proteoglycans, glycosaminoglycans, and various adhesion proteins to form ECM and play a crucial role in the elasticity and tensile strength of the aorta[24]. Studies have shown that a variety of metabolic abnormalities can eventually result in the development of AD via ECM degradation[25, 26]. Thus, the oxidative stress of cells, changes in components of the extracellular matrix, and lipids and atherosclerosis may play an important role in AD, further supporting our hypothesis that anoikis might lead to the development of AD.
With the use of two machine learning algorithms (LASSO, SVM-RFE), we finally identified three most related genes: GRSF1, TP53, and TUBB3, and verified the function of these genes using the ROC curve, PPI network, GSEA, and GSVA enrichment analyses. G-rich RNA sequence binding factor 1 (GRSF1) is a high-affinity RNA-binding protein to G-rich sequences and plays a key role in RNA post-transcriptional regulation[27]. Studies have shown that miR-G1 can promote the expression of TMED5 mediated by GRSF1, thereby activating the WNT-CTNB1/β-catenin pathway and promoting the onset and development of human cervical cancer cells. Studies have also demonstrated that GRSF1 can mediate PI3K/AKT/NF-κB and TIMP3/ADAM17/MMP9 pathways involved in the metastasis of cervical cancer[28]. Meanwhile, GRSF1 has been found to promote the expression of LMNB1 and regulate the response of cells to genomic damage and nucleophagy[29]. Loss of GRSF1 can lead to increases in ROS, and increased oxidative stress leads to DNA damage and activation of mTOR, which initiates the inflammatory responses and promotes IL-6 production[30]. The PPI results showed that GRSF1 played a important role in the action of glutathione peroxidase 4 (GPX4), and related studies suggested that GRSF1 could inhibit the production of mitochondrial ROS by regulating the expression of GPX4, thereby maintaining cellular redox homeostasis[31]. The above studies support our validation of the role of GRSF1 in cell adhesion, oxidative stress response, and the inflammatory response pathway.
Tumor suppressor p53 (TP53) is an important tumor suppressor gene that plays an important regulatory role in apoptosis induction, DNA damage and abnormal proliferation[32]. Studies have shown that p53 can mediate the protection of acute lung injury by inhibiting the iron death Nrf2/HIF-1/TF signaling pathway[33]. TP53-induced glycolysis and apoptosis regulator (TIGAR) can reduce the level of fructose-2,6-bisphosphate in cells, leading to glycolysis inhibition and reduction of intracellular ROS, thereby inhibiting microglial pyroptosis and protecting newborns from hypoxic-ischemic brain injury[34]. The above studies have demonstrated that TP53 can mediate iron death, pyroptosis, and apoptosis of cells through oxidative stress pathways. βIII-tubulin (TUBB3) is a major microtubule (MT) protein; it is a cytoskeletal protein involved in many cell pathophysiological processes, such as maintenance of shape, intracellular transport, mitosis, carcinogenesis, and chemoresistance[35], and has been found to inhibit apoptosis and promote the growth and invasion of gallbladder cancer cells through the Akt/mTOR signaling pathway[36].
The above experimental studies have indicated that the three genes identified in the present study can promote cell death in different forms through a variety of pathways, with GRSF1 being the most significant one, which is closely related to the pathogenesis of AD. VSMCs play a critical role in the pathogenesis of AD, and their phenotypic transformation promotes pro-inflammatory responses and MMP production, eventually leading to degradation of extracellular matrix and weakening of the aortic wall[37]. A growing body of evidence suggests that programmed cell death pathways, including apoptosis, necroptosis, pyroptosis, and iron death, play a key role in VSMC loss[38]; however, there has been few in-depth studies on the anoikis of VSMCs. Based on our current bioinformatics analysis, differentially expressed ARGs have been demonstrated in both cases of AD and healthy controls, and various programmed cell death pathways might jointly promote the development of AD, which might provide new ideas and insights for the diagnosis of AD.
Some limitations to the present study also need to be mentioned. First, this study was conducted primarily using bioinformatic methods, thus, laboratory-based experiments are needed to verify these findings. To offset the above limitation, this study performed rigorous data analysis with reference to a large body of literature to ensure the reliability of results. Secondly, anoikis is a relatively new concept; although research on anoikis in the field of oncology has made significant progress, the specific mechanism of anoikis in the development of AD needs further investigation. To this end, the present study used bioinformatic methods to identify potential biomarkers for AD, which provided a strong basis for further experiments.