2.5 Nitric oxide, peroxynitrite, and superoxide production and measurement of endothelial cell viability

The details of the methodology have previously been reported.10 Frozen primary culture human umbilical vein endothelial cells (HUVECs, Lonza, Walkersville, MD) at passage 1 isolated from pooled donors by the corporate source were cultured and frozen down for future experiments as per the source’s recommended protocols to form a working stock culture to draw from. Fresh cultures were seeded from these stocks and cells (passages 5–8) were allowed to grow to full confluence before treatment. Individual experimental treatments were in separate seeded cultures from the frozen stocks. HUVECs seeded into 10 cm² conical culture tubes each with a glass cover slip at the bottom were treated with vehicle control, medin 5 µM without or with PEG-SOD (300 U/ml), FPS–ZM1 (100 μM), or BH4 (100 μM) for 1 h and medin 5 µM without or with FPS–ZM1 (100 µM) for 20 h.

·NO, superoxide, and peroxynitrite production were measured in endothelial cells exposed to treatment for 1 and 20 h while endothelial cell viability was measured following exposure to treatment for 20 h. ·NO production was assessed by directly measuring ·NO head gas production for cells treated for 20 h, and using 4,5-diaminofluorescein diacetate (DAF-2 DA) fluorescence for cells and arterioles treated for 1 h, a well-validated fluorescence method to indirectly measure ·NO production.15 Head gas was measured by piercing the seal of the conical tube and using Sievers 280 nitric oxide Analyzer (General Electric Analytical Instruments, Waukesha, WI) and normalized to cell count. For fluorescence microscopy measurements, endothelial cell-containing glass coverslips were removed from the culture tubes, transferred into 12-well plates, washed, and then stained for 15 min with 5 µmol/l dihydroethidium (Thermo Fisher Scientific, Waltham, MA), a marker of superoxide production16 and 20 µmol/l coumarin boronate pinacolate ester (Cayman Chemical, Ann Arbor, MI), a marker of peroxynitrite production17 in HEPES buffer containing 10⁻⁴ M acetylcholine. ·NO was assessed in HUVECs treated for 1 h by exposing cells to treatment (vehicle, medin ± PEG-SOD or BH4 or FPS–ZM1) for 1 h, administration of acetylcholine (10⁻⁴ M) and staining with DAF-2 DA (10 μM, Life Technologies, Carlsbad, CA) 45 min into treatment exposure allowing 15 minutes of staining. Cells were then washed and fixed in 4% formaldehyde in PBS followed by cold methanol and mounted on glass slides for imaging. Using EVOS FL Auto (Life Technologies), reacted dihydroethidium products were imaged using the RFP light cube (excitation 531/40 nm; emission 593/40 nm), reacted coumarin boronate were imaged using the DAPI light cube (excitation 357/44 nm; emission 447/60 nm) and reacted DAF-2 DA were imaged using the GFP light cube (excitation 470/22 nm; emission 510/42 nm). Cell fluorescent signals were measured using ImageJ 1.49 analysis software and results expressed as values relative to control. For 20-h treated cells, the remaining cells from the culture tubes were lifted with trypsin into flow cytometry tubes, washed, and resuspended in 10 nM calcein acetyoxymethyl (Life Technologies) HEPES buffer containing 1.6 mM calcium for 15 min and then washed. This non-fluorescent compound is a marker of cell viability as presence of intact cellular endogenous esterases will hydrolyze the compound and release fluorescent calcein anion.18 Cellular fluorescent signals were measured using Beckman-Coulter FC500 flow cytometer (Indianapolis, IN) reading with excitation at 488 nm on the FL-1 channel. Results are expressed as values relative to control. In separate experiments, HUVECs were treated for 20 h with vehicle, medin 5 μM without or with α,β,γ,δ-tetrakis(4-N-methylpyridyl) porphine (FeTMPyP, 50, 100, and 200 μM), a peroxynitrite decomposition catalyst,19 and cell viability was assessed using calcein acetyoxymethyl fluorescence as described.

Adipose arterioles were exposed to 1 h treatment with vehicle control, medin 5 μM ± PEG–SOD (300 U/ml) or FPS–ZM1 (100 μM) and ·NO was measured using DAF-2 DA staining while superoxide was measured using dihydroethidium staining similar to methods described above.