Neutrophil Extracellular Traps Are Formed In Response To Hypertensive Stretch And Contribute To Vascular Dysfunction In Hypertension

Background: Neutrophils play a crucial role in the innate immune response, in part by the release of neutrophil extracellular traps (NETs), which consist of decondensed chromatin and proteins including citrullinated histone 3 (H3-Cit). The process of NETosis involves the disassociation of DNA from the nucleosome catalyzed by the enzyme peptidyl arginine deiminase 4 (PAD4). TRPV4 is a mechanosensitive calcium channel expressed in neutrophils. Studies of human subjects and rodent models have described the accumulation of NETs in renal and vascular tissue in hypertension (HTN), however the causative role of NETs has not been described. We hypothesized that NETs form in response to hypertensive stretch and contribute to endothelial cell (EC) dysfunction and HTN. Methods: Mice deficient for Padi4 lack the PAD4 enzyme and cannot efficiently form NETs. Padi4 -/- and C57Bl/6J mice were treated with 490 ng/kg/min angiotensin (Ang) II for 4 weeks. Blood pressure was measured by radiotelemetry. Vascular reactivity studies were performed on mesenteric arteries. Vascular infiltration was measured by flow cytometry. Murine neutrophils and aortic ECs were co-cultured and subjected to uniaxial stretch under normotensive (5%) and hypertensive (10%) conditions. Neutrophils were treated with a TRPV4 agonist (GSK1016790A) and NETs were quantified by immunofluorescence. Results: Padi4 -/- mice exhibit attenuated HTN and improved vascular reactivity following Ang II treatment (systolic BP 159.4 ± 1.4 mmHg in C57Bl/6J mice vs 147.9 ± 2.6 mmHg in Padi4 -/- mice, n= 7-8, p= 0.007). Flow cytometry revealed a marked reduction in aortic infiltration of immune cells in Padi4 -/- mice compared to C57Bl/6J mice. Co-culture of murine neutrophils with ECs improves neutrophil survival and augments NETosis. Hypertensive stretch of co-cultured neutrophils and ECs augmented NETosis compared to normotensive stretch. Immunoblot revealed increased H3-Cit in neutrophils exposed to hypertensive stretch. Neutrophils treated with GSK1016790A exhibit augmented NETosis. Conclusions: These findings suggest a vital role of stretch-induced NETosis as a mediator of EC dysfunction and HTN. Mechanistically, both agonism of neutrophil TRPV4 and hypertensive stretch of ECs augment NETosis.