Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Abstract

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO(2)) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO(2) via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO(2)-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO(2)-FAs stimulated the phosphorylation of eNOS at Ser(1179). These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO(2)-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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).