Additional file 1 of The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS

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.