OA-NO₂ induces HO-1 mRNA and protein in endothelial and vascular smooth muscle cells. Endothelial cells were incubated with 2.5 and 5 μM OA-NO₂ or the native fatty acid OA for 16 hrs and real time PCR analysis was performed (A). Endothelial cells were treated with the same concentrations of OA-NO₂ or OA for 24 hr and Western blotting was performed (B). VSMCs were treated with the indicated concentrations of OA-NO₂ or OA for 2.5 hr and 24 hr and real time PCR analysis (C) and Western blotting (D) was performed, respectively. The Western blot is a representative of 5–7 separate experiments. The real time PCR analysis results are derived from at least five independent experiments and data are expressed as mean ± SEM.

OA-NO₂ treatment stimulates phosphorylation of eNOS, Akt, ERK, and p38. Confluent ECs were stimulated with 2.5 and 5 μM OA-NO₂ for 7.5 (A) and 30 min (B) and Western blotted for P-eNOS, P-Akt, P-p38, P-ERK, and actin. The results are a representative of 5–7 separate experiments.

OA-NO₂ levels increased in mice treated subcutaneously with OA or OA-NO₂ via osmotic mini pump for three days accompanies enhanced eNOS and HO-1 gene expression. Blood was collected from the treated mice. The serum was analyzed by HPLC ESI MS/MS in the negative ion mode using [¹³C]OA-NO₂ as an internal standard by acquiring MRM transitions consistent with the loss of the nitro functional group (46 amu corresponds to the mass of NO₂): m/z 326/46 and m/z 344/46 for OA-NO₂ and [¹³C]OA-NO₂ respectively. Representative chromatographs of lipids extracted from serum [¹³C₁₈] OA-NO₂ (top panel), OA-NO₂ (middle panel), and OA (bottom panel) are shown (A). Free OA-NO₂ levels (nM) were determined using ANALYST 1.4 quantitation software (B). Real time PCR analysis was performed for eNOS and HO-1 from aortas of mice with the osmotic mini pump for 3 d. Administration of OA-NO₂ (3 mg/kg/d for 3 d) increased eNOS (left) and HO-1(right) mRNA levels compared to OA-treated mice. eNOS and HO-1 mRNA levels were normalized to Actin (C). Data are expressed from 6–8 mice per group and expressed as mean ± SEM.

Endothelial cells increase eNOS following OA-NO₂ treatment. Cells were incubated for 16 hr with indicated concentrations of OA-NO₂ or OA and real time PCR analysis was performed (A) and 24 hr for Western blotting (B). OA-NO₂ treatment for 16 hr stimulates NO release from ECs. NO generation was determined in OA- and 2.5 μM OA-NO₂-treated confluent ECs for 16 hrs using Sievers nitric oxide analyzer (NOA). The levels of nitrite accumulation have been normalized to protein content of the ECs and are reported as a percent of the OA control. Basal levels of NO₂⁻ = 326.5 ± 8.7 pmoles/mg protein. Results are derived from at least five independent experiments and data are expressed as mean ± SEM.

OA-NO₂ treatment stimulates NO release from ECs. NO generation was determined in OA- and 2.5 μM OA-NO₂-treated confluent ECs for the indicated times using Sievers nitric oxide analyzer (NOA). The levels of nitrite have been normalized to protein concentration (basal NO₂⁻ = 4.8 ± 0.2 pmoles/min/mg protein) and are reported as a percent of control at each individual time point (30, 60, and 120 min). Results are derived from at least five independent experiments and data are expressed as mean ± SEM.

OA-NO₂ is rapidly metabolized by ECs. BAEC were grown to confluence and treated with a mixture of 9- and 10-nitro regio-isomers of [¹³C₁₈]-OA-NO₂ (5 μM) in HBSS over a period of 90 minutes. Equimolar distribution of the 9- and 10-nitro regio-isomers of OA-NO₂ is depicted (A, inset) as previously determined ([7], online supplement). The HBSS solution was collected and ECs were scrape harvested at the indicated time points. HBSS, corresponding cell extracts and control HBSS treatments (with [¹³C₁₈]-OA-NO₂ at 37°C at each indicated time point) were extracted using ACN precipitation as described in Materials and Methods and supernatants were analyzed by LC-MS-MS. [¹³C₁₈]-OA-NO₂ was detected following a m/z 344/46 for [¹³C₁₈]-OA-NO₂ and a MRM transition corresponding to the detection of the nitro group (NO₂⁻) with peak at 3.6 min. A continuous decay in peak area was observed for [¹³C₁₈]-OA-NO₂ over the 90 min time course (A). A composite of the chromatograms shows the loss of the [¹³C₁₈]-OA-NO₂ (filled square) and the concomitant formation of [¹³C₁₈]-SA-NO₂ (open triangle) over the period of 90 min (B). [¹³C₁₈]-OA-NO₂ is rapidly saturated to a nitro-stearic acid ([¹³C₁₈]-SA-NO₂). [¹³C₁₈]-SA-NO₂ was detected following a m/z 346/46 MRM transition (C, left panel). Arrow indicates a peak corresponding to the contribution of heavy isotopes from [¹³C₁₈]-OA-NO₂. Fragmentation patterns of product ion analysis (peak at 3.73 min) confirmed the formation of [¹³C₁₈]-SA-NO₂. The major expected fragments from [¹³C₁₈]-SA-NO₂ were detected, corresponding to the formation of 46 (NO₂⁻): m/z 299 (neutral loss of HNO₂,-47 amu) and 328 (the loss H₂O, −18 amu) (C, right panel). Scheme showing the main fragmentation products (C, inset right panel). The two isomers of [¹³C₁₈]-OA-(OH)-NO₂ were detected following a formation of specific breakdown product ions. The MRM transition m/z 362/180 corresponds to the fragmentation of 9-(OH)-10NO₂ (peak at 5.7 and 5.8 min) (D, upper panels), and transition m/z 362/211 corresponds to the fragmentation of 10-(OH)-9NO₂ (peak at 5.8 min) (D, lower panels). Fragmentation patterns of [¹³C₁₈]-OA-(OH)-NO₂ isomers (D, right panels) show characteristic fragmentation for vicinal nitro-hydroxy fatty acids (180 and 193 ions) and formation of NO₂⁻ (46 ion).