Neutrophil extracellular traps-mediated upregulation of HIF-1α promotes corneal neovascularization

Abstract

Objectives

The objective of this study is to examine the impact of neutrophil extracellular traps (NETs) on angiogenesis, both in vitro and in vivo contexts, as well as to elucidate the regulatory function of hypoxia-inducible factor (HIF-1α).

Methods

The study focused on investigating the regulatory function of HIF-1α in the induction of NETs formation. In vivo, following NaOH stimulation, the formation of NETs was quantitatively assessed through immunofluorescence staining employing specific markers, namely SYTOX Green and PicoGreen. Furthermore, mice were administered with HIF-1α, and seven days post-alkali burn, the formation of NETs in the cornea was evaluated by immunofluorescence staining techniques. Protein immunoblotting analysis validated an increase inHIF-1α expression within human umbilical vein endothelial cells (HUVECs) that were induced to form by NETs. In vitro, human neutrophils were subjected to HIF-1α treatment. Subsequent to NaOH stimulation, the NETs mesh was isolated and co-cultured with HUVECs. The migratory capacity and angiogenic potential of HUVECs were then quantitatively evaluated.

Results

Upon exposure to NaOH, a notable increase in SYTOX fluorescence was observed in neutrophils, indicative of the formation of a prominent network structure. Furthermore, a marked elevation in HIF-1α protein expression was detected in mouse corneal tissue that had undergone NETs formation, hinting at a potential role of NETs in mediating the up-regulation of HIF-1α. The outcomes of hematoxylin and eosin (H&E) staining, coupled with immunofluorescence staining, revealed an augmentation in neutrophils counts, with NETs exhibiting a marked potentiation of corneal neovascularization. In vitro assessments further demonstrated that HIF-1α played a stimulatory role in promoting the migration and tubular morphogenesis of HUVECs.

Conclusion

Exposure to sodium hydroxide serves as a trigger for the induction of NETs formation. NETs mediated an up-regulation of HIF-1α, which subsequently promotes angiogenesis and inflammatory activation in HUVECs. Consequently, this process leads to an enhancement of corneal neovascularization and inflammatory response.