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Additional file 1 of The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS Thorben Pape (10181748) Oleg Pak (5748923) Theresa Graalmann (8407923) Christine Ehlers (25135000) Janik Deutscher (25135003) David Spiegelberg (25135006) Paul Horn (297980) Klaus Stahl (5907686) Marius M. Hoeper (9315867) Sascha David (319986) Natascha Sommer (2586913) Benjamin Seeliger (6943853) Biochemistry Space Science Medicine Cell Biology Physiology Pharmacology Biotechnology Immunology Biological Sciences not elsewhere classified Cancer Systemic inflammation ARDS Capillary leakage ECVE ECV Supplementary Material 1: Supplemental Data 1: E-cigarette vapor application in isolated ventilated and perfused mouse lungs. To isolate the lung/heart block, mice were deeply anesthetized with ketamine (100 mg/kg body weight, i.p.) and xylazine (20 mg/kg body weight, i.p.), following anticoagulation with heparin (2,500 U/kg body weight). A tracheostomy was performed for intubation, and the mice were ventilated with a normoxic gas mixture (21% O₂, 5.3% CO₂, balanced with N₂) using a piston pump (Minivent Type 845, Hugo Sachs Elektronik, March-Hugstetten, Germany). Ventilation settings included a tidal volume of 10 μl/g body weight, a respiratory rate of 90 breaths/min (60 breaths/min during direct e-cigarette vapor inhalation), and a positive end-expiratory pressure (PEEP) of 3 cm H₂O. While maintaining ventilation, the chest was opened, and the block of lung and heart was dissected. Perfusion was initiated via a catheter inserted into the pulmonary artery using a peristaltic pump (ISM834A V2.10, Ismatec, Wertheim, Germany). The perfusate consisted of Krebs-Henseleit buffer [120 mM NaCl, 4.3 mM KCl, 1.1 mM KH₂PO₄, 2.4 mM CaCl₂, 1.3 mM MgCl₂, 13.3 mM glucose, and 5% (w/v) hydroxyethyl amylopectin; molecular weight 200,000 Da]. Following removal of the lung-heart block from the thoracic cavity, the preparation was freely suspended from a scale to monitor weight changes. A catheter was inserted into the left atrium to collect the outflow perfusate. The outflow pressure was maintained at 2 cm H₂O. Pulmonary arterial pressure, venous pressure, ventilation pressure, and lung weight were continuously monitored throughout the experiment. Nicotine-containing e-cigarette vapor (ECVnic) was generated using the “eVic Primo Mini” battery pack (Joyetech®) and the “Eleaf Melo 3” clearomizer (0.5 Ω, 30 W; Riccardo®, Riccardo Retail GmbH, Neubrandenburg, Germany). Vapor was drawn into a perfusor syringe via a vacuum pump using normoxic air. The puffing protocol involved four 5-second puffs (20 seconds total). ECVnic was applied to the isolated lung via the inspiratory tube using a syringe driver, mimicking normal ventilation. During each application, the expiratory tube was closed until the target inspiratory volume was reached, then reopened to release the vapor. This process lasted approximately 2 seconds and was repeated 150 times over a 5-minute period. In total, seven ECVnic application cycles were performed at 10-minute intervals. Normoxic ventilation was maintained throughout. Control experiments followed the same protocol but used normoxic gas without ECVnic. 6 µg/ml LPS, 10 µg/ml of the Tie2-agonist PMC-403, or their combination was added to the perfusate 5 minutes before ECVnic inhalation. After ECVnic inhalation, transvascular fluid filtration (endothelial permeability) was assessed under hydrostatic stress by increasing venous pressure from 2 to 12 cm H₂O for 8 minutes (“pressure challenge”), using the capillary filtration coefficient (Kfc) as previously described [23]. Two pressure challenges were performed before the start of PMC-403 application to confirm the quality and stability of the lung preparation. The entire experiment lasted approximately 4 hours. To assess edema formation, net lung weight gain following a hydrostatic challenge was measured as an indicator of fluid retention. Lungs were suspended from a force transducer, and weight gain was calculated as the difference between the baseline and 5 minutes after each pressure challenge. Additionally, wet-to-dry lung weight ratios were determined using the left lungs. Wet weight was recorded immediately after the experiment, and dry weight was measured after a 48-hour drying period at 65°C. 2026-09-26T05:00:00Z Text Journal contribution 10.6084/m9.figshare.34021021.v1 https://figshare.com/articles/journal_contribution/Additional_file_1_of_The_Tie-2_agonist_PMC-403_inhibits_endothelial_damage_induced_by_e-cigarette_vapor_or_LPS/34021021 CC BY