Fig 2: Knocking down lncRNA IGF2-AS down-regulated dNTP metabolization-related enzymes. (a, b) After knocking down lncRNA IGF2-AS, the expressions of RRM2, TK1, and TYMS were measured by qRT-PCR and WB. (c, d) After knocking down HMGA1, qRT-PCR, and WB was applied to detect expression of RRM2, TK1, and TYMS. (e) Proliferation of EPCs was detected by CCK-8 after knocking down HMGA1. (f) After knocking down HMGA1, IF was used to detect the expression of Caspase-1. *P < 0.05 vs. si-NC.

Fig 3: Proteomic and bioinformatics analyses of differentially expressed proteins in vascular smooth muscle cells with or without neutrophil extracellular trap treatment. (a) Heatmap and hierarchical clustering revealed differentially expressed proteins between VSMCs with or without NETs treatment. (b) Hierarchical clustering revealed specific differentially expressed proteins in the two groups. (c) Pathway analyses of common and specific differentially expressed proteins. (d) Hub gene analyses of up-regulated proteins, including both common and specific differentially expressed proteins. In this analysis, node color represents the number of links between a protein and other proteins within the network (where colors from dark gray to light gray indicate more to fewer links). (e) Protein expression levels of CDKN1b in VSMCs with NETs treatment compared with untreated VSMCs. Additionally, VSMCs treated with Ang II were evaluated as a positive control. GAPDH was selected as a reference protein. Histograms show statistical analysis for the relative expression of CDKN1b (f), TOP2a (g), and TK1 (h). (I) Phosphorylated Akt (p-Akt) and total Akt levels in VSMCs, NET-treated VSMCs, and AngII-treated VSMCs. Histograms show statistical analysis for the relative expression of p-Akt (j), the relative expression of Akt (k), and p-Akt/Akt Ratio (l) (*P < 0.05, **P < 0.01). NETs, neutrophil extracellular traps; TK1, thymidine kinase 1; VSMCs, vascular smooth muscle cells.

Fig 4: Exosomes derived from vascular smooth muscle cells with neutrophil extracellular trap treatment accelerate vascular smooth muscle cell proliferation via TK1 and promote vascular smooth muscle cell cycle progression. (a) Proliferation of VSMCs, VSMCs treated with exosomes derived from VSMCs and VSMCs treated with exosomes derived from VSMCs with NETs treatment were assessed by MTS assays, respectively. (b) The histogram shows statistical analysis of cell growth of VSMCs and VSMCs treated with exosomes derived from VSMCs with NETs treatment by OD values. (c) Cell cycle analysis of VSMCs, VSMCs treated with exosomes derived from VSMCs and VSMCs treated with exosomes derived from VSMCs with NETs treatment by PI staining. (d) The histogram shows statistical analysis of cell cycle of VSMCs, VSMCs treated with exosomes derived from VSMCs and VSMCs treated with exosomes derived from VSMCs with NETs treatment. (e) TK1 expression was analyzed in exosomes derived from VSMCs with NET treatment and exosomes derived from VSMCs by western blot analysis. Alix, an exosomal marker, was detected as a reference protein, and GAPDH was included as a control. (f) The histogram shows statistical analysis of TK1 expressed in exosomes derived from VSMCs with NETs treatment and exosomes derived from VSMCs. (g) After TK1 knockdown in VSMCs, western blotting was performed to detect exosomal TK1 expression. (h) The histogram shows statistical analysis of TK1 expression in exosomes derived from VSMCs after TK1 konckdown. (i) Proliferation of VSMCs treated with exosomes derived from VSMCs and VSMCs treated with exosomes derived from VSMCs with TK1 knockdown were assessed by MTS assays, respectively. (j) The histogram shows statistical analysis of cell growth of VSMCs treated with exosomes derived from VSMCs and VSMCs treated with exosomes derived from VSMCs with TK1 knockdown by OD values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). NETs, neutrophil extracellular traps; TK1, thymidine kinase 1; VSMCs, vascular smooth muscle cells.

Fig 5: Thymidine kinase 1 expression was tested in the artery and serum of mice with neutrophil extracellular trap formation in arteries induced by phorbol-12-myristate-13-acetate. (a) Western blot analysis of TK1 expression in the superior mesenteric artery of mice injected with PMA to induce NETs formation in artery and control mice injected with physiological saline. (b) The histogram shows statistical analysis of TK1 expressed in the superior mesenteric artery of mice with NETs induced and control mice. (c) Western blot analysis of TK1 expression in the serum of mice injected with PMA and control mice injected with physiological saline. (d) The histogram shows statistical analysis of TK1 expressed in serum of mice with NETs induced and control mice (*P < 0.05, **P < 0.01). NETs, neutrophil extracellular traps; PMA, phorbol-12-myristate-13-acetate; TK1, thymidine kinase 1.