Nitro-fatty acid inhibition of neointima formation after endoluminal vessel injury

Abstract

Rationale: Fatty acid nitroalkenes are endogenously generated electrophilic byproducts of nitric oxide and nitrite-dependent oxidative inflammatory reactions. Existing evidence indicates nitroalkenes support posttranslational protein modifications and transcriptional activation that promote the resolution of inflammation.

Objective: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury.

Methods and results: The in vivo administration (21 days) of nitro-oleic acid (OA-NO(2)) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO(2) treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals,P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO(2) in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO(2)-induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO(2) in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1(-/-) mice (OA-NO(2)-treated wild-type versus HO-1(-/-) mice, P=0.016).

Conclusions: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury.

Fig. 1

Quantitation of OA-NO₂ in serum following administration of V, OA, or OA-NO₂. (A) Serum from treatment groups was analyzed by HPLC ESI MS/MS in the negative ion mode using [¹³C]OA-NO₂ as an internal standard (dashed tracing) and by acquiring MRM transitions consistent with the loss of the nitro functional group: m/z 326/46 and m/z 344/46 for OA-NO₂ and [¹³C]OA-NO₂ respectively. (B) Free OA-NO₂ levels (nM) were quantitated using ANALYST 1.4 quantitation software. Data are expressed as mean ± SD of 6–7 mice per group where *p ≤ 0.001.

Fig. 2

Nitro-oleic acid decreases neointimal formation. Femoral artery tissue sections from (A) V, (B) OA, (C) OA-NO₂ treatment groups, as well as (D) control mice were labeled with anti-smooth muscle actin (red) and anti-CD31 (blue), with autofluorescence used to visualize the inner and outer elastic membrane (green) 21d after wire-induced endoluminal injury (Magnification 20X, Olympus Provis I fluorescence microscope; bar indicates 100 μm). (E–G) Quantitative morphometric analysis of artery remodeling 21d after injury. Data are expressed as mean ± SEM of 6–7 mice per group.

Fig. 2

Nitro-oleic acid decreases neointimal formation. Femoral artery tissue sections from (A) V, (B) OA, (C) OA-NO₂ treatment groups, as well as (D) control mice were labeled with anti-smooth muscle actin (red) and anti-CD31 (blue), with autofluorescence used to visualize the inner and outer elastic membrane (green) 21d after wire-induced endoluminal injury (Magnification 20X, Olympus Provis I fluorescence microscope; bar indicates 100 μm). (E–G) Quantitative morphometric analysis of artery remodeling 21d after injury. Data are expressed as mean ± SEM of 6–7 mice per group.

Fig. 3

Nitro-oleic acid induces HO-1 expression in vitro and in vivo. (A) HO-1 and HO-2 (B) expression in vitro. RASMC were treated with OA-NO₂, OA, or vehicle (MeOH) at the indicated concentrations for 2h [real-time PCR analysis, *P<0.01 (ANOVA)] or 18h (immunoblot analysis). Real time PCR data are expressed as mean ± SD of 3–4 independent experiments. (C) HO-1 expression in vivo. Injured femoral arteries were labeled with anti-smooth muscle actin (green) and anti HO-1 (red), 21d after injury. Representative images are shown for V (top 3 panels) and OA-NO₂ (bottom 3 panels) treated mice (Magnification 40X; Zeiss confocal microscope; bar indicates 10 μm). (D) HO-1 mRNA levels are increased in isolated femoral arteries 3d following OA-NO₂ administration [compared to vehicle, mean ± SD, *P<0.01 (ANOVA)], where HO-2 mRNA levels remain unchanged (E).

Fig. 3

Nitro-oleic acid induces HO-1 expression in vitro and in vivo. (A) HO-1 and HO-2 (B) expression in vitro. RASMC were treated with OA-NO₂, OA, or vehicle (MeOH) at the indicated concentrations for 2h [real-time PCR analysis, *P<0.01 (ANOVA)] or 18h (immunoblot analysis). Real time PCR data are expressed as mean ± SD of 3–4 independent experiments. (C) HO-1 expression in vivo. Injured femoral arteries were labeled with anti-smooth muscle actin (green) and anti HO-1 (red), 21d after injury. Representative images are shown for V (top 3 panels) and OA-NO₂ (bottom 3 panels) treated mice (Magnification 40X; Zeiss confocal microscope; bar indicates 10 μm). (D) HO-1 mRNA levels are increased in isolated femoral arteries 3d following OA-NO₂ administration [compared to vehicle, mean ± SD, *P<0.01 (ANOVA)], where HO-2 mRNA levels remain unchanged (E).

Fig. 4

Nitro-oleic acid inhibits vascular smooth muscle cell proliferation in vitro and in vivo. (A, B) RASMC were serum starved for 24h, stimulated to proliferate with DMEM (2% serum), and treated with OA (2.5 μM) or OA-NO₂ (1, 2.5 μM), and with SnPP (50 μM) or HO-1 siRNA (50μM). Concentrations for HO-1 siRNA experiments were 2.5 μm for OA and OA-NO₂. After 24h cell proliferation was assessed using Cyquant NF proliferation assay. Data are presented as mean ± SEM of 3 independent experiments (*P<0.001). (C) Western blot analysis confirmed OA-NO₂ (2.5 μM)-induced HO-1 gene expression is knocked-down in cells transfected with siRNA for HO-1 (50 μM), where HO-2 expression is unaffected. Control siRNA (50 μM) did not change OA-NO₂ (2.5 μM) -induced HO-1 gene expression. (D) OA-NO₂ significantly inhibited smooth muscle cell proliferation in vivo. Proliferative cells were visualized by staining femoral arterial sections with Ki67. Color threshold was used for quantification of Ki67 positive cells in 6 different fields of view per section (3 sections per animal, 6–7 mice per group). Magnification 40X; Olympus Provis I fluorescence microscope; Bar indicates 10 μm.

Fig. 4

Nitro-oleic acid inhibits vascular smooth muscle cell proliferation in vitro and in vivo. (A, B) RASMC were serum starved for 24h, stimulated to proliferate with DMEM (2% serum), and treated with OA (2.5 μM) or OA-NO₂ (1, 2.5 μM), and with SnPP (50 μM) or HO-1 siRNA (50μM). Concentrations for HO-1 siRNA experiments were 2.5 μm for OA and OA-NO₂. After 24h cell proliferation was assessed using Cyquant NF proliferation assay. Data are presented as mean ± SEM of 3 independent experiments (*P<0.001). (C) Western blot analysis confirmed OA-NO₂ (2.5 μM)-induced HO-1 gene expression is knocked-down in cells transfected with siRNA for HO-1 (50 μM), where HO-2 expression is unaffected. Control siRNA (50 μM) did not change OA-NO₂ (2.5 μM) -induced HO-1 gene expression. (D) OA-NO₂ significantly inhibited smooth muscle cell proliferation in vivo. Proliferative cells were visualized by staining femoral arterial sections with Ki67. Color threshold was used for quantification of Ki67 positive cells in 6 different fields of view per section (3 sections per animal, 6–7 mice per group). Magnification 40X; Olympus Provis I fluorescence microscope; Bar indicates 10 μm.

Fig. 5

Nitro-oleic acid inhibits vascular smooth muscle cell migration and neointima formation via HO-1-dependent mechanisms. (A, B) OA-NO₂ inhibition of RASMC migration. After inducing monolayer wounding, cells maintained in serum free media were treated with PDGF (20 ng/ml) and OA (250 nM) or OA-NO₂ (25, 50, 100 and 250 nM), with SnPP (50 μM) or HO-1 siRNA (50 μM). Concentrations for HO-1 siRNA transfected experiments were as follows: OA (250 nM), OA-NO₂ (250 nM) and PDGF (20 ng/ml). Quantitative image analysis was conducted 18h later to reveal extents of migration of RASMC into the denuded area. Data are presented as mean ± SEM of 6 independent experiments (*P<0.01).

Fig. 6

Protective actions of OA-NO₂ are inhibited in HO-1^−/− mice. Femoral artery tissue sections (HO-1^−/− mice) from (A) V and (B) OA-NO₂ treatment groups were labeled with anti-smooth muscle actin (red) and anti-CD31 (blue), with autofluorescence used to visualize the inner and outer elastic membrane (green) 21d after wire-induced endoluminal injury (Magnification 20X; Olympus Provis I fluorescence microscope; bar indicates 100 μm). (C) Quantitative morphometric analysis of the intima to media ratio wild-type and HO-1^−/− mice treated with or without OA-NO₂ for 21d. Data are expressed as mean ± SEM of 4–6 mice per group.