NETosis Drives Blood Pressure Elevation and Vascular Dysfunction in Hypertension

Abstract

Background: Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction.

Methods: NETosis-deficient Padi4^-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence.

Results: Padi4^-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis.

Conclusions: These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

Keywords: extracellular traps; histones; hypertension; neutrophils; protein-arginine deiminase type 4.

Figure 1.

Neutrophil extracellular trap (NET)-deficient mice exhibit attenuated hypertension. Padi4^−/− and C57Bl/6 mice were treated with Ang II (angiotensin II) infusion for 4 weeks. Radiotelemetry was used to measure (A) systolic blood pressure, (B) diastolic blood pressure, (C) mean arterial pressure (MAP), and (D) heart rate. Single day variability represents day/night cycles. Data were analyzed by repeated-measures 2-way ANOVA (5 animals per group). BP indicates blood pressure.

Figure 2.

Neutrophil extracellular trap (NET)-deficient mice exhibit improved endothelial cell function and reduced vascular inflammation in response to Ang II (angiotensin II). Padi4^−/− and C57Bl/6J mice were treated with Ang II for 4 weeks. Wire myography was used to measure vascular relaxation of mesenteric vessels in the presence of (A) sodium nitroprusside (SNP) and (B) acetylcholine (ACH). Data were analyzed by repeated-measures 2-way ANOVA (4 animals per group). C, Urine volume was quantitated following a saline challenge (4 C57Bl/6 animals and 5 Padi4^−/− animals). Aortas were harvested and quantitation of (D) neutrophils, (E) dendritic cells (DCs), and (F) CD8⁺ cytotoxic T cells was performed by flow cytometry (10 animals per group). Data were analyzed by 2-tailed Student t test (urine volume and neutrophils/aorta) and 2-tailed Mann-Whitney U test (DCs/aorta and CD8⁺ T cells/aorta).

Figure 3.

Hypertensive stretch augments NETosis in the presence of endothelial cells. Murine neutrophils were cultured with and without endothelial cells (ECs) under normal (5%) and hypertensive (10%) stretch for 18 hours. A, Representative flow cytometry plots of neutrophil extracellular trap (NET) formation. B, Quantitation of NETs. Data were analyzed by 2-way ANOVA with Šidák multiple comparison test (−EC+5%, n=8; −EC+10%, n=8; +EC+5%, n=6; +EC+10%, n=8). MPO indicates myeloperoxidase.

Figure 4.

Endothelial cells (ECs) prolong neutrophil survival. Murine neutrophils were cocultured with or without ECs and subjected to 5% and 10% stretch for 36 hours. Neutrophils were stained with live/dead cell stain that indicates cell viability. A, Representative histograms of live/dead in neutrophils. B, Quantitation of live neutrophils as a percent of total seeded neutrophils. Data were analyzed by 2-way ANOVA with Šidák multiple comparison test (n=12 for each group).

Figure 5.

Long-term hypertensive stretch augments suicidal NETosis in the presence of endothelial cells. Murine neutrophils were cocultured with endothelial cells and subjected to normotensive (n=12) and hypertensive (n=12) stretch for 36 hours. A, Representative flow cytometry plots of neutrophil extracellular trap (NET) formation. B, Quantitation of NETs. C, Representative histograms of live/dead staining of NETs. D, Quantitation of vital NETs. E, Quantitation of suicidal NETs. Data were analyzed by 2-tailed Student t test. MPO indicates myeloperoxidase.

Figure 6.

Hypertensive stretch increases neutrophil histone H3 citrullination and H3-Cit exposure disrupts endothelial cell function. A, Immunoblot for H3-Cit and histone H3 on stretched neutrophils. B, Quantification of the relative expression of H3-Cit normalized to histone H3 in 5% (n=4) or 10% (n=4) stretched neutrophils. Data were analyzed by 2-tailed Mann-Whitney U test. Endothelial cells were treated with vehicle (n=4), histone H3 (n=4), or H3-Cit (n=4), and real-time PCR (RT-PCR) was used to quantify the mRNA expression of (C) Icam1, (D) Vcam1, (E) Sell, and (F) Sele normalized to the expression of Gapdh and expressed as relative expression. Data were analyzed by 1-way ANOVA with Tukey post hoc test. F, Vascular reactivity of aortic rings in response to acetylcholine (Ach) was measured following treatment with control, histone H3, and H3-Cit. Data were analyzed by a repeated-measures 2-way ANOVA with Tukey post hoc test (4 animals per group). Endothelial cells were exposed to 10% stretch and treated with vehicle (H and I, n=6; J, n=4) or H3-Cit (H–J, n=6), and RT-PCR was used to quantify mRNA expression of (H) Il6, (I) Tgfβ, and (J) Tnfα normalized to the expression of “Gapdh” and expressed as relative expression. Data were analyzed by 2-tailed Student t test (Il6 and Tgfβ) or 2-tailed Mann-Whitney U test (Tnfα).

Figure 7.

TRPV4 (transient receptor potential cation channel subfamily V member 4) activation in neutrophils augments intracellular calcium and NETosis. Murine neutrophils were treated with control (n=5), 1 nM (n=5), 10 nmol/L (n=5), and 100 nM (n=5) of the TRPV4 agonist GSK1016790A. Flow cytometry was used to detect intracellular calcium with the calcium sensitive Fluo-4 am dye. A, Representative histograms of Fluo-4 am. B, Quantitation of mean fluorescence intensity (MFI) of Fluo-4 am. Neutrophils were stained for H3-Cit, MPO (myeloperoxidase), and DNA to determine NETosis. C, Representative images of co-localized area of H3-Cit, MPO, and DNA representing neutrophil extracellular traps (NETs) in cultured neutrophils. Scale bar=100 µm. D, Quantitation of NET area per field. E, Quantitation of H3-Cit/cell count. Data were analyzed by 1-way ANOVA with Tukey post hoc test.