Recent research has increasingly highlighted the link between VTE and the immune system, with confirmed associations to inflammatory factors. DVT arises from complex interactions between enzymes and cellular processes. The endothelium, platelets, and white blood cells collaborate to induce a pro-inflammatory state, ultimately culminating in thrombosis and acute DVT16,33. VTE is associated with a significant mortality rate, particularly from PE. Untreated cases have a mortality rate of 30%. However, prompt diagnosis and treatment can reduce the mortality rate due to PE or treatment-related causes to less than 1%34. VTE also carries the burden of long-term complications and substantial healthcare costs. Approximately 50% of DVT patients may experience post-thrombotic syndrome, a chronic and debilitating condition. Additionally, the expensive treatment of VTE further burdens the healthcare system35. The presence of cardiovascular involvement worsens the prognosis of Budd-Chiari syndrome (BD). Therefore, considering these potential adverse consequences, early prevention, detection, and treatment of VTE are of paramount importance in patients with BD.The precise mechanisms by which Budd-Chiari syndrome and VTE share transcriptional pathogenesis remain to be elucidated. To address this knowledge gap, we conducted a comprehensive analysis of microarray datasets, aiming to identify common biomarkers and biological pathways associated with the development of both diseases. Reliable biomarkers hold significant value in modern medicine. The application of bioinformatics and machine learning methodologies have significantly advanced the investigation of potential mechanisms and biomarker discovery. These methods offer a powerful approach for the accurate identification of disease-associated biomarkers, facilitating research into disease initiation, progression, and exploration of potential pathogenic mechanisms.Assessment of hub gene expression levels in peripheral blood samples from patients diagnosed with BD and VTE offers valuable insights for predicting the risk of VTE development in this specific population. This approach, therefore, emerges as a potentially practical and efficacious clinical tool. Our nomogram facilitates the calculation of both cumulative and total scores for each gene. As a result, this nomogram holds significant promise for clinical application by aiding in the identification and early intervention of BD patients with elevated total scores. Early intervention has the potential to improve the prognosis of this patient population.This study employed the Limma R package, a robust tool for gene expression data analysis, to identify DEGs associated with Budd-Chiari syndrome and VTE. Overlap analysis revealed 160 genes shared between BD- and VTE-associated DEGs. To further explore co-expressed genes potentially related to BD pathogenesis, a gene coexpression network was constructed using WGCNA. This analysis identified significant correlations between gene modules and phenotypes, leading to the discovery of 3552 genes associated with BD. Subsequent analysis of these genes alongside the 160 overlapping DEGs pinpointed 117 DEGs specifically associated with VTE in the context of BD. To elucidate the functional roles of these 117 DEGs, enrichment analyses were performed using the KEGG pathway and GO databases. The results indicated a primary association with cellular senescence, differentiation of Th1 and Th2 cells, platelet activation, negative regulation of cellular metabolism, and leukocyte migration. These findings suggest a strong link between immune system dysregulation, inflammatory responses, and the interplay between BD and VTE. Dysregulations in Th1/Th2 characterize BD’s complex immune responses36. Th1 cell-driven inflammation can compromise vascular endothelium, elevating thrombosis likelihood. Additionally, cellular senescence is intimately linked with both BD and VTE37,38. The exacerbation of senescence-associated secretory phenotype (SASP) during cellular senescence contributes to platelet activation, a critical element in VTE development39. Moreover, leukocyte migration significantly influences the pathology of both BD and VTE. In BD sufferers, aberrant leukocyte migration and activation may intensify vascular inflammation and thrombosis risk40. These aspects support the view that there is a potential association between BD and VTE.Furthermore, the 117 DEGs were mapped onto a PPI network to investigate potential interactions. This analysis identified 23 potential hub DEGs, which may play crucial roles in the underlying biological processes. Machine learning algorithms, including SVM-RFE, LASSO, and Random Forest, are increasingly employed for biomarker discovery due to their effectiveness in identifying promising candidates. These methodologies facilitate the exploration of complex biological processes and aid in the identification of reliable biomarkers. In this study, we utilized three machine learning methods alongside ROC curve analysis to evaluate the diagnostic accuracy of candidate gene expression. This approach led to the identification of four candidate hub genes with potential diagnostic value: E2F1, GATA3, HDAC5, and MSH2. To further validate the diagnostic potential of these genes, an additional dataset (GSE19151) and clinical validation using peripheral blood samples were employed. These analyses confirmed the significant clinical diagnostic value of the identified gene expression signature.E2F1, the first identified member of the E2 promoter binding factor (E2F) family, is believed to play a critical role in cell cycle regulation, differentiation, apoptosis, and the DNA damage response41. Dysregulation of E2F1 can impact downstream transcriptional targets, leading to DNA replication stress. This stress arises when persistent obstacles, both internal and external, hinder DNA replication and ultimately contribute to various diseases, including cancer42. A study suggested a potential link between E2F1 and vascular diseases, as inhibiting its expression protected Human Umbilical Vein Endothelial Cells (HUVECs) from high-glucose-induced injury41. Overexpression of E2F1 has been demonstrably associated with the development and progression of various malignant tumors43. As established, active malignant tumors are a leading cause of VTE from an etiological perspective. Therefore, E2F1 may play a role, albeit potentially limited, in VTE pathogenesis44. VTE is a complex disease influenced by a multitude of factors, including environmental and genetic components. Numerous genes have been identified as conferring susceptibility to VTE. The established association between VTE and inflammation is further supported by the involvement of inflammatory cells like monocytes, platelet aggregation, and C-reactive protein44. Consequently, immune gene mutations linked to inflammation may contribute to VTE pathogenesis. Interestingly, E2F1 upregulation has been observed in both VTE and Behçet’s disease, suggesting a potential connection between E2F1, VTE, and BD.GATA3, a transcription factor belonging to the GATA family, plays a critical role in regulating cell differentiation and proliferation. Studies have shown a potential association between the immune response and low expression of GATA3 (Th2) in patients with Neuro-Behçet’s disease (NBD)45. Interestingly, in active BD with skin lesions, the expression ratio of Th2/Th1 (GATA3/Tbet) in these lesions exhibits a tendency to increase. This suggests a potential bias towards the GATA3(Th2) axis during T cell immune activation within BD lesions46. These findings collectively indicate a close relationship between GATA3 and BD. Additionally, GATA3 has been demonstrated to possess tumor suppressive effects in various cancers, including osteosarcoma47. Nevertheless, the relationship between GATA3 and VTE remains uncertain.Histone deacetylase 5 (HDAC5) is a member of the class IIa histone deacetylase family. Primarily localized within the nucleus of vascular smooth muscle cells (VSMCs), HDAC5 functions as a transcriptional repressor, suppressing the expression of its target genes. Upon phosphorylation, HDAC5 interacts with specific proteins, leading to the de-repression of genes associated with vascular diseases48. Interestingly, HDAC5 expression appears to be dynamically regulated in the endothelium. Studies suggest overexpression of HDAC5 in endothelial cells during venous return, while normal endothelial venous flow conditions are associated with down-regulated HDAC5 expression49. Additionally, HDAC5 has been implicated in impaired angiogenesis, a hallmark of scleroderma (SSc). HDAC5 inhibits pro-angiogenic factors in endothelial cells, contributing to the disease. Silencing HDAC5 in SSc-affected endothelial cells restores normal angiogenic function50. This collective evidence strongly suggests a close link between HDAC5 and vascular diseases. Additionally, research has shown that inhibiting HDAC5 can enhance tissue-type plasminogen activator (t-PA) production, a molecule with antithrombotic properties, thereby ameliorating intravascular thrombosis51. While a study demonstrated that mutant forms of HDAC5 (HDAC5AA) activate the mTOR pathway and promote the survival and regeneration of retinal ganglion cells (RGCs)52, it is important to note that BD can significantly affect patients’ eyes, establishing a connection between BD and vascular-related diseases. However, the precise relationship between BD and VTE remains to be elucidated.MutS homolog 2 (MSH2) is a well-established key component of the mismatch repair (MMR) system. The MMR system plays a critical role in maintaining genomic stability by recognizing and repairing mismatched nucleotides during DNA replication. Mutations in genes encoding components of the MMR system, including MSH2, have been implicated in the pathogenesis of various autoimmune and autoinflammatory diseases. These mutations may disrupt the tightly regulated activity of pattern recognition receptors (PRRs) and their signaling pathways53. Moreover, MSH2 has been demonstrably linked to the initiation and progression of cancer, potentially impacting tumorigenesis, development, and immune regulation54. However, current research on MSH2 function primarily focuses on specific cancer types, with limited investigation into its role in VTE and BD.BD is a chronic, systemic, and inflammatory vascular disorder characterized by vasculitis and endothelial injury. While the precise etiology of VTE associated with BD remains to be fully elucidated, the leading hypothesis suggests a complex interplay between neutrophil activation, endothelial dysfunction, and coagulation abnormalities in the pathogenesis of BD-induced VTE4,5. Neutrophil activation leads to the release of high levels of neutrophil extracellular traps (NETs), which contribute to endothelial cell injury and alterations in coagulation function. Additionally, inflammation triggers the release of proinflammatory factors and chemokines, which in turn activate platelets, leukocytes, and endothelial cells. This activation cascade results in vascular endothelial dysfunction, ultimately promoting thrombosis. BD patients exhibit a pre-thrombotic state characterized by dysregulation of the hemostasis, coagulation, and anticoagulation systems. This dysregulation, potentially arising from alterations in the coagulation mechanism and platelet activation, ultimately contributes to the development of VTE in these patients. The significantly elevated risk of VTE in BD patients presents a substantial clinical challenge. A comprehensive understanding of the relationship between BD and VTE, alongside the identification of reliable disease markers, is crucial for facilitating early diagnosis and effective treatment, thereby improving the prognosis for BD patients.Subsequent analysis of immune cell infiltration in VTE patients revealed a significantly higher prevalence of macrophages compared to the control group. Conversely, VTE samples displayed a lower prevalence of neutrophils. This finding appears to contradict previous literature reporting an increase in neutrophil numbers in VTE55. The observed discrepancy may be attributed to the inclusion of recurrent and chronic VTE samples in the utilized GEO dataset. Neutrophils undergo activation during the acute phase of DVT development; however, their numbers are likely to decline in the chronic phase due to depletion. Existing literature supports the significant role of macrophages in the pathogenesis and progression of both BD and VTE56,57,58. Inflammasome activation in macrophages leads to the release of tissue factor, a critical trigger for thrombosis59. Our findings regarding macrophage prevalence align with these previous studies. Consequently, we investigated the potential interrelationship between the four identified hub genes (E2F1, GATA3, HDAC5, and MSH2) and immune cell infiltration. This analysis revealed a positive correlation between immune-associated macrophages and the expression of all four hub genes (E2F1, GATA3, HDAC5, and MSH2). In conclusion, these findings suggest that modulating macrophage populations may present a promising therapeutic strategy for managing VTE in patients with BD.The ssGSEA analysis revealed significant associations between the four identified hub genes and various biological processes, including hypoxia, heme metabolism, coagulation, cholesterol homeostasis, and angiogenesis. These findings suggest that the four hub genes may be intricately linked to the initiation and progression of VTE in BD patients. Current treatment strategies for VTE in BD primarily focus on immunosuppressants, with anticoagulation therapy potentially unnecessary for managing DVT in this context. However, the high 5-year recurrence rate of 36.5%60 necessitates the exploration of more effective therapeutic options. To address this need, we employed the Enrichr database to screen for potential therapeutic agents targeting the four hub genes. The analysis identified several candidate drugs, including chlorpromazine, dronabinol, trabectedin, vorinostat, and carmustine, which exhibited potential efficacy in targeting the hub genes associated with BD-induced VTE.Our research elucidates potential disease mechanisms and identifies biomarkers related to VTE induced by BD, employing a synthesis of bioinformatics and machine learning methodologies. The nomogram model crafted in our study exhibits significant predictive value for VTE in BD patients. Additionally, this study provides some directions for future studies delving into the molecular mechanisms underlying the pathogenesis of VTE precipitated by BD. However, despite these advantages, the study has limitations. Firstly, despite utilizing validation datasets and clinical samples for diagnostic assessment, further experimental research is essential to validate and explore the underlying mechanisms of VTE induced by BD. Secondly, this study was based on bioinformatics analysis, and the verification of hub genes was constrained by relatively small validation samples due to sample acquisition challenges, so further validation using multicenter studies involving larger sample sizes should be performed. Thirdly, although predicted therapeutic agents have been screened, the absence of concrete experimental corroboration means that significant further research is required to evaluate the efficacy of drug treatments targeting these hub genes for BD with VTE patients.
