Phenylephrine

The thoracic aorta was dissected and placed in cold physiological buffer: 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂ × 2H₂O, 1.17 mM MgSO₄ × 7H₂O, 20 mM NaHCO₃, 1.18 mM KH₂PO₄, 0.027 mM EDTA, 10.5 mM glucose; bubbled with a mixture of 95% O₂ and 5% CO₂, resulting in pH 7.4. Aortic rings (2–3 mm) were mounted onto a multichamber isometric myograph system (Model 620M, Danish Myo Technology) and equilibrated at 37°C. The resting tension was reached by the normalization process using the ADInstuments Normalization module, where the segments of aorta were normalized to their length and diameter. At the beginning of each experiment, the ability of the preparation to develop a contraction was assessed by exposing the aorta segments to a high KCl solution (124 mM). Endothelium-dependent relaxations were determined by administration of cumulatively increasing concentrations of acetylcholine (10^–9 to 10^–5 M, MilliporeSigma) to aortas precontracted with phenylephrine (10^–10 to 10^–6 M, MilliporeSigma). The aorta segments were then washed 3 times every 10 minutes for 30 minutes. The endothelium-independent relaxation was tested by sodium nitroprusside (SNP, 0.1 nM to 10 μM, Merck), following precontraction with phenylephrine (10 μM, MilliporeSigma). Data were recorded and analyzed with the LabChart Pro evaluation program (ADInstruments).

Healthy mice were sacrificed by pentobarbital administration (100 mg/kg i.p.), and the thoracic aorta and mesenteric arteries were immediately collected. The vessels were placed in 6-well culture plates with RPMI and incubated with 10 μg of isolated mouse NETs with or without mLR12 (25 mg/ml). Three hours after stimulation, the vessels were placed in an ice-cold physiological salt solution composed of 130 mM NaCl, 14.9 mM NaHCO₃, 3.7 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, and 5.5 mM glucose. Two-millimeter-long rings were cut and mounted on a 40 μm stainless-steel wire in a small vessel myograph (EMKA Technologies, France). The preparation was supplied with a carbogen gas mixture (95% O₂, 5% CO₂). After an equilibration period (at least 20 min) under optimal passive tension, two successive contractions in response to the combination of KCl depolarization and phenylephrine (Sigma, St. Louis, MO, USA) were used to measure the maximal contractile capacity of the vessels. Concentration–response curves to phenylephrine (1 nM to 100 μM) were obtained. After a washout period of 20 min, the vessels were precontracted with phenylephrine (1 μM) to 80% of the maximum contraction, and the acetylcholine (Sigma, St. Louis, MO, USA) concentration–response curve was obtained (1 nM to 100 μM).

The thoracic aortas were removed and placed in cold Krebs solution, cleaned of fat and loose connective tissue, and sectioned into 4-mm-long rings. The sectioned rings were mounted on stainless steel hooks and suspended in 10-mL tissue baths. The bath was filled with Krebs solution (at 37°C, pH 7.4, with 95% O₂/5% CO₂) of the following compositions (mm): NaCl 120, KCl 5.5, MgCl₂∙6H₂O 1.2, NaH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 20, and glucose 10. The preload was 0.75 g, and the rings were equilibrated for 60 min. The Krebs buffer solution in the tissue bath was replaced every 15 min. At the end of the equilibration period, the maximal force generated by addition of 3 × 10⁻⁶m phenylephrine (Sigma-Aldrich) was determined. To evaluate endothelium-dependent relaxations, the rings were preconstricted with phenylephrine (3 × 10⁻⁶m) to obtain a stable plateau and then a cumulative dose–response curve to acetylcholine (10⁻⁹ to 10⁻⁴m; Sigma-Aldrich) was obtained. The NO-donor sodium nitroprusside (10⁻¹⁰ to 10⁻⁵m; Sigma-Aldrich) was added to examine endothelium-independent relaxations.