FIG. 4.

Apoptosis is the primary fate of endothelial cell death resulting in oxidized HMGB1 release and increases oxidative potential in the media. (A) HPAECs (ScienCell, Carlsbad, CA) were cultured in ECM growth media supplemented with 5% FBS and penicillin–streptomycin in a humidified incubator (21% O₂, 5% CO₂) at 37°C. To induce cell death, cells were treated with Sugen 5416 (20 μM) in 0.5% FBS for 24 h, as published (101). Apoptosis and necrosis were quantified by using Apoptosis and Necrosis Quantification Kit (Biotum, Fremont, CA) according to the manufacturer's protocol, as published (136). Sugen treatment stimulated apoptosis (Early and Late) but not necrosis in HPAEC. (B) Media from untreated HPAEC, HPAEC with apoptosis induced as described in (A) and necrosis induced by three to four cycles of cell freezing and thawing, as published (137) were used for Western blot analysis as previously described (137) to measure reduced and oxidized forms of HMGB1. Necrotic but not apoptotic cells produced a marked extracellular HMGB1 signal and showed an accumulation of the reduced form of HMGB1 in cell culture media. The difference in the total protein in the media related to the difference in FBS amount used in the experiment—negative control (media that were not used for cell culturing) and apoptotic media contain 0.5% FBS, untreated and necrotic media—5% FBS. The difference in total media proteins does not correlate with the HMGB1 signal that is absent in the negative control, and the media collected from untreated cells show a light signal from oxidized HMGB1 in the apoptotic media and a strong but mostly reduced signal in the necrotic media. (C) Same media samples collected for (B) were used to measure plasma redox homeostasis by analyzing ORP and total antioxidant capacity (Cap) electrochemically by using RedoxSys^® Diagnostic System, as reported (137). Apoptotic media showed a significant increase in oxidative potential and a decrease in antioxidant capacity, indicating the development of oxidative stress in apoptotic cells and the release of oxidized cellular content. Necrotic media show a significant reduction in ORP signal, suggesting that necrosis produces release cellular components in a reduced state. N = 4; *p < 0.05 versus untreated; ^#p < 0.05 versus apoptosis. FBS, fetal bovine serum; HMGB1, high mobility group box 1; HPAEC, human pulmonary artery endothelial cell; ORP, oxidation–reduction potential. Color images are available online.