The pro-resolving lipid mediator maresin 1 (MaR1) attenuates inflammatory signaling pathways in vascular smooth muscle and endothelial cells

Abstract

Objective: Inflammation and its resolution are central to vascular injury and repair. Maresins comprise a new family of bioactive lipid mediators synthesized from docosahexaenoic acid, an ω-3 polyunsaturated fatty acid. They have been found to exert anti-inflammatory and pro-resolving responses in macrophages, neutrophils and bronchial epithelial cells and impart beneficial actions in murine models of peritonitis and colitis. We investigated the impact of maresin-1 (MaR1) on tumor necrosis factor alpha (TNF-α) induced inflammatory responses in human vascular endothelial (EC) and smooth muscle cells (VSMC).

Methods: Primary cultures of human saphenous vein EC and VSMC were employed. We tested the naturally occurring MaR1 as modulator of TNF-α effects, with examination of monocyte adhesion, oxidant stress, and intracellular inflammatory signaling pathways.

Results: MaR1 attenuated TNF-α induced monocyte adhesion and reactive oxygen species (ROS) generation in both EC and VSMC, associated with down-regulated expression (cell surface) of the adhesion molecule E-selectin (in EC) and NADPH-oxidases (NOX4, NOX1, NOX2). MaR1 attenuated TNF-α induced release of pro-inflammatory mediators by EC and VSMC. MaR1 caused an attenuation of TNF-α induced NF-κB activation in both cell types associated with inhibition of I-κ Kinase (IKK) phosphorylation, IκB-α degradation and nuclear translocation of the NF- κB p65 subunit. MaR1 also caused a time-dependent increase in intracellular cyclic AMP (cAMP) in both naive and TNF-α stimulated VSMC and EC.

Conclusions: MaR1 has broad anti-inflammatory actions on EC and VSMC, which may be partly mediated through up-regulation of cAMP and down-regulation of the transcription factor NF-κB. The results suggest that the pro-resolving lipid mediator MaR1 exerts homeostatic actions on vascular cells that counteract pro-inflammatory signals. These findings may have direct relevance for acute and chronic states of vascular inflammation.

Conflict of interest statement

Competing Interests: The authors would also like to declare no competing interests related to the funding agency "American Heart Association." This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1. MaR1 attenuates TNF-α induced monocyte adhesion.

(A, B, C) Endothelial cells grown in 24-well plates were treated with vehicle (A, B) or 100 nM MaR1 (C) for 30 min, followed by TNF-α at 1 ng/ml (B, C) for 4 hr and adhesion of labeled U937 monocytes were visualized under a microscope. (D) EC were grown in 96-well plates and adhesion of labeled U937 monocytes (4 hr post TNF-α) were quantified using a fluorescence plate reader. Treatment protocol of MaR1 was same as (Fig 1A, B, C). N≥4. N =  each well of a 96-well plate. ANOVA (oneway) with Dunnett's posthoc test (p = 0.003). *:p≤0.05 compared to vehicle control, †: p≤0.05 compared to TNF-α alone (t-test). (E) EC grown in 12-well plates were treated with vehicle or 100 nM MaR1 as (Fig 1A, B, C) and after 4 hr of TNF-α, analyzed for cell surface E-selectin expression by flow cytometry. N≥5. N =  each well of 12-well plate. *:p≤0.05 compared to TNF-α alone (t-test). Error bar  =  SEM. (F) Representative histogram of (E) for TNF-α at 20 ng/ml.

Figure 2. MaR1 attenuates TNF-α induced ROS production in vascular smooth muscle and endothelial cells.

(A–C) Representative images of dihydroethidium/DHE stained (red) vascular smooth muscle cells counterstained with DAPI (nucleus). VSMC were grown in 8-well chamber slides and received vehicle (A, B) or 100 nM of MaR1 (C) for 30 min, followed by TNF-α, 10 ng/ml for 4 hr (B, C). (D) Quantitative analysis of mean DHE intensity (nucleus RFU/area of nucleus) of vascular smooth muscle cells, N≥3 per group. N =  each well of an 8-well chamber slide. (E) EC grown in 96-well plates, received 100 nM of MaR1, 30 min prior to TNF-α and ROS production was measured 2 hr post TNF-α, using a ROS specific dye CellRox Deep Red reagent, N≥3 per group. N =  each well of a 96-well plate. *:p≤0.05 compared to vehicle control, †: p≤0.05 compared to TNF-α alone (t-test). Error bar  =  SEM.

Figure 3. MaR1 attenuates TNF-α induced expression of NADPH-oxidases.

VSMC (A, B) and EC (C, D) were treated with 100 nM of MaR1 for 30 min, followed by TNF-α for 6 hr. After TNF-α treatment, cells were lysed and probed for NOX-4, NOX-1 and NOX-2. (A) N = 14 per group, (B) N = 17 per group, (C) N≥7 per group, (D) N = 3 per group. *:p≤0.05 compared to vehicle control, †: p≤0.05 compared to TNF-α alone (t-test). N = each well of a 6-well plate. Error bar  =  SEM.

Figure 4. MaR1 attenuates TNF-α induced inflammatory pathways.

EC were treated with 100 nM MaR1 for 30 min followed by 18 hr of TNF-α (1 ng/ml), after which the conditioned medium was analyzed for the presence of 40 different inflammatory mediators using antibody arrays. (A, B) Representative images of three independent experiments done on EC. (C) After densitometric analysis of individual spots (in duplicate) normalized to protein content (cell lysates), inflammatory mediators that were down-regulated significantly in TNF-α+ MaR1 (t-test: p≤0.05), compared to TNF-α alone are shown in the bar-graph, N = 3. (D) Graphical representation of down-regulated inflammatory proteins found in the media from VSMC that underwent same treatment protocol as (Fig 4 A–C) but with TNF-α at 10 ng/ml. N = 3. *:p≤0.05 compared to TNF-α alone (t-test). N =  each well of a 6-well plate. Error bar  =  SEM.

Figure 5. MaR1 attenuates TNF-α induced NF-κB activation in endothelial cells.

(A–C) Representative images of nuclear translocation of p65 NF-κB subunit in EC treated with vehicle (A), vehicle + TNF-α, 2 hr (B), 100 nM of MaR1 (30 min) + TNF-α, 2 hr (C). (D) Quantitative analysis of p65 translocation (ratio of nuclear to cytoplasmic fluorescence) in EC. N≥3 where N =  each well of an 8-well chamber slide. (E) Endothelial whole cell extracts were analyzed for phospho- and total-IKK, 15 min post TNF-α addition that received 100 nM MaR1, 30 min prior to TNF-α. N = 3 where N =  one 10 cm plate. *:p≤0.05 compared to vehicle control, †: p≤0.05 compared to TNF-α alone (t-test). (F) EC received vehicle or 100 nM MaR1 for 30 min and TNF-α for 1 hr, after which they were lysed and probed for IκB-α. *:p≤0.05 compared to vehicle control, †: p≤0.05 compared to TNF-α alone (t-test) Error bar  =  SEM.

Figure 6. MaR1 and Resolvin-D1 attenuates TNF-α induced NF-κB reporter activity in vascular smooth muscle cells.

VSMC transfected with firefly luciferase (NF-κB) and renilla luciferase vectors were treated with MaR1 and resolvin-D1, followed by TNF-α for 6 hr and were analyzed for firefly and renilla luciferase activity. Net NF-κB activity is shown as a ratio of firefly luciferase activity normalized to renilla luciferase for transfection efficiency. N≥6 per group. *:p≤0.05 compared to vehicle control, †: p≤0.05 compared to TNF-α alone (One way ANOVA with Dunnett's post hoc test). #: p = 0.01 (t-test) compared to MaR1, 100 nM +TNF-α. N =  each well of a 96-well plate. Error bar  =  SEM.

Figure 7. MaR1 increases cAMP levels in vascular smooth muscle and endothelial cells.

(A) VSMC and EC were seeded to confluency in 24-well plates and were treated with 100 nM MaR1 for the indicated time-points and cAMP levels were determined and normalized to mg protein. N = 3 per time-point (B) VSMC and EC were treated with vehicle or MaR1 (100 nM) for 30 min, followed by TNF-α (10 ng/ml for VSMC, 1 ng/ml for EC) for 120 min and assayed for cAMP. N = 4. *:p≤0.05 compared to vehicle control (0 min) (t-test); †: p≤0.05 compared to TNF-α + vehicle (t-test). N =  each well of a 24-well plate. Error bar  =  SEM.