Temporal transcriptomic profiling of pulmonary thromboembolism reveals persistent NETosis- and ferroptosis-associated gene signatures and enhanced thrombolysis with adjunctive DNase I

Abstract

Objective To investigate the dynamic pathophysiology of pulmonary thromboembolism (PTE), focusing on the roles of neutrophil extracellular traps (NETs) and ferroptosis, and to evaluate intravenous DNase I as an adjunct to recombinant tissue plasminogen activator (rt-PA). Methods A rabbit autologous thrombus PTE model was established using 62 animals for the time-course and therapeutic-intervention experiments. Disease progression was assessed at days 1, 3, 7, and 14 using histology (collagen volume fraction, CVF), serology (e.g., MPO-DNA, PAI-1, t-PA), and RNA-sequencing. Bioinformatics identified NETs- and ferroptosis-related genes, validated by qRT-PCR. Based on peak NETs activity at day 7, an intervention study (n = 12) compared rt-PA monotherapy versus rt-PA plus intravenous DNase I, with outcomes assessed 7 days post-treatment. In addition, an independent four-group mechanistic validation cohort (Control, PTE, PTE + DNase I, and PTE + ferrostatin-1 [Fer-1]; n = 5 per group) was used to evaluate NETs, TLR9, phospho-p65, total p65, and GPX4 by ELISA and qRT-PCR. Results The PTE model demonstrated progressive pulmonary fibrosis (increasing CVF, P < 0.05) and a sustained hypofibrinolytic state. The NETs marker MPO-DNA peaked at day 7 (P < 0.01). Transcriptomic analysis revealed persistent activation of neutrophil degranulation and iron homeostasis pathways, with early ferroptosis and late collagen metabolism enrichment. Therapeutically, rt-PA plus DNase I was superior to rt-PA alone, yielding greater improvements in CVF (P < 0.01), pulmonary artery acceleration time (P < 0.01), oxygen saturation (P < 0.001), left ventricular function (P < 0.05), and serological markers of endothelial injury and cardiac strain (P < 0.05). In the independent validation cohort, PTE increased circulating MPO-DNA complexes, TLR9, phospho-p65 signaling, and the phospho-p65/total p65 ratio while decreasing GPX4; DNase I attenuated upstream MPO-DNA-associated changes in TLR9-NF-kB signaling, whereas Fer-1 more prominently restored GPX4 expression. Conclusion These findings suggest sustained NETs-associated activity and ferroptosis-related processes during PTE progression, identify stage-specific molecular signatures, and support DNase I as a potential adjunct to enhance thrombolysis, while mechanistic relationships require further validation.