Atherosclerosis (AS) is a chronic inflammatory disease involving cell death and inflammatory responses. Pyroptosis, a newly discovered pro-inflammatory programmed cell death process, exacerbates inflammatory responses. However, the roles of cathepsin B (CTSB) in pyroptosis and AS remain unclear. To gain further insight, we fed ApoE^-/- mice a high-fat diet to investigate the effects and mechanisms of CTSB overexpression and silencing on AS. We also explored the specific role of CTSB in vascular smooth muscle cells (VSMCs) in vitro. The study revealed that high-fat diet led to the formation of AS plaques, and CTSB was found to increase the AS plaque lesion area. Immunohistochemical and TUNEL/caspase-1 staining revealed the existence of pyroptosis in atherosclerotic plaques, particularly in VSMCs. In vitro studies, including Hoechst 33342/propidium iodide staining, a lactate dehydrogenase (LDH) release assay, detection of protein indicators of pyroptosis, and detection of interleukin-1β (IL-1β) in cell culture medium, demonstrated that oxidized low-density lipoprotein (ox-LDL) induced VSMC pyroptosis. Additionally, CTSB promoted VSMC pyroptosis. Ox-LDL increased the expression of CTSB, which in turn activated the NOD-like receptor protein 3 (NLRP3) inflammasome and promoted NLRP3 expression by facilitating nuclear factor kappa B (NF-κB) p65 nuclear translocation. This effect could be attenuated by the NF-κB inhibitor SN50. Our research found that CTSB not only promotes VSMC pyroptosis by activating the NLRP3 inflammasome, but also increases the expression of NLRP3.

Background: The heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition. Methods: Weighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria. Results: The CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice. Conclusions: The results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

Background •Atherosclerosis is a chronic inflammatory disease.Pyroptosis triggers and amplifies the inflammatory response and plays an important role in AS.Cathepsin B (CTSB) canpromote AS and activate NLRP3 to mediate pyroptosis.Dapagliflozin (DAPA) can inhibit cell pyroptosis to improve AS.This study aimed to explore the effect of DAPA on oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis of vascular smooth muscle cells (VSMCs)and its underlying mechanism. Methods • VSMCs were transfected with cathepsin B (CTSB)-overexpressing and -silencing lentiviral vectors.Then,VSMCs were treated with different concentrations of ox-LDL ( 0,50,100,150µg/ml ), Hoechst33342/PI double staining and LDH release assay were used to detect cell pyroptosis.After VSMCs were treated with different concentrations of DAPA ( 0.1µM, 1.0µM, 5.0µM, 10µM, 25µM, 50µM ), the proliferation activity of VSMCs was detected by CCK8 method.On the basis of ox-LDL-induced VSMCs pyroptosis, DAPA intervention, overexpression and silencing of CTSB were used to observe the effects of DAPA and CTSB on Ox-LDL-mediated VSMCs pyroptosis. Results • 1) VSMCs stably transfected with CTSB-overexpressing and -silencing lentiviruses were obtained, 150 µg/mL was the optimal concentration of ox-LDL for inducing pyroptosis of VSMCs, and 0.1 µM was the optimal concentration of DAPA for ameliorating pyroptosis of VSMCs. 2) Ox-LDL-induced pyroptosis of VSMCs was worsened by overexpression of CTSB but suppressed by silencing of CTSB. 3) DAPA attenuated ox-LDL-induced pyroptosis of VSMCs through down-regulating CTSB and NLRP3. 4) Overexpression of CTSB based on DAPA intervention aggravated ox-LDL-induced pyroptosis of VSMCs. Conclusion • DAPA attenuates NLRP3/caspase-1 pathway-mediated pyroptosis of VSMCs through down-regulating CTSB.

Cardiovascular disease (CVD) remains a major global health challenge. The cholesterol–high-density lipoprotein–glucose (CHG) index and glucose-