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. 2013 Jun;27(6):2220-32.
doi: 10.1096/fj.12-225615. Epub 2013 Feb 13.

D-series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury

Takuya Miyahara  1 , Sara Runge, Anuran Chatterjee, Mian Chen, Giorgio Mottola, Jonathan M Fitzgerald, Charles N Serhan, Michael S Conte
Affiliations

D-series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury

Takuya Miyahara et al. FASEB J. 2013 Jun.

Abstract

Recent evidence suggests that specialized lipid mediators derived from polyunsaturated fatty acids control resolution of inflammation, but little is known about resolution pathways in vascular injury. We sought to determine the actions of D-series resolvin (RvD) on vascular smooth muscle cell (VSMC) phenotype and vascular injury. Human VSMCs were treated with RvD1 and RvD2, and phenotype was assessed by proliferation, migration, monocyte adhesion, superoxide production, and gene expression assays. A rabbit model of arterial angioplasty with local delivery of RvD2 (10 nM vs. vehicle control) was employed to examine effects on vascular injury in vivo. Local generation of proresolving lipid mediators (LC-MS/MS) and expression of RvD receptors in the vessel wall were assessed. RvD1 and RvD2 produced dose-dependent inhibition of VSMC proliferation, migration, monocyte adhesion, superoxide production, and proinflammatory gene expression (IC50≈0.1-1 nM). In balloon-injured rabbit arteries, cell proliferation (51%) and leukocyte recruitment (41%) were reduced at 3 d, and neointimal hyperplasia was attenuated (29%) at 28 d by RvD2. We demonstrate endogenous biosynthesis of proresolving lipid mediators and expression of receptors for RvD1 in the artery wall. RvDs broadly reduce VSMC responses and modulate vascular injury, suggesting that local activation of resolution mechanisms expedites vascular homeostasis.

Keywords: inflammation; intracellular signaling; resolvin.

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Figures

Figure 1.
Figure 1.
RvD1 and RvD2 attenuate proliferation and migration responses of human VSMCs in vitro. A) HVSMC proliferation assay performed in normal growth medium (10% serum) as described in Materials and Methods. Dose-dependent inhibition of VSMC proliferation is shown for RvD1 (n=3). **P < 0.01 vs. control; 2-way ANOVA with Dunnett's post hoc test. B) Proliferation assay performed in 0.5 or 10% serum as noted, with addition of RvD1 (10 nM) and indicated antibodies to GPCRs (n=3). *P < 0.01, **P < 0.0001 vs. 10% FBS + RvD1 + IgG control; #P < 0.05, ##P < 0.0001 vs. 10% FBS + vehicle; 1-way ANOVA with Dunnett's post hoc test. C, D) VSMC migration response to PDGF-AB with RvD1/RvD2 cotreatment, using a transwell assay. Results are expressed as percentage change in migration from unstimulated control (no PDGF). PDGF-neutralizing antibody serves as positive control for inhibition. A dose-dependent inhibition of chemotaxis is demonstrated for both RvD1 and RvD2. Effects of RvD1 on chemotaxis are sensitive to anti-GPR32 blocking antibody, but not to anti-ALX (n=5). NS, not significant. *P < 0.05, **P < 0.01 vs. PDGF-AB; #P < 0.05 vs. PDGF-AB + RvD1; 1-way ANOVA with Dunnett's post hoc test. E, F) Cell shape changes assessed with phalloidin staining and quantitative cytometry. E) RvD1 induces cell shape changes, including an increase decrease of length/width ratio, and inhibits the typical cytoskeletal response to PDGF-AB (n=3). *P < 0.05, **P < 0.01 vs. PDGF-AB; #P < 0.05, ##P < 0.01 vs. vehicle control; unpaired t test. F) Cytoskeletal changes induced by RvD1 are abrogated by PTX and anti-GPR32, but not by anti-ALX (n=3). *P < 0.05, **P < 0.01 vs. isotype control; #P < 0.05 vs. negative control; 1-way ANOVA with Dunnett's post hoc test. Results are means ± se.
Figure 2.
Figure 2.
Modulation of VSMC inflammatory responses by RvD1 and RvD2. A) Monocyte adhesion to VSMCs. HVSMCs were stimulated with TNF-α (10 ng/ml) for 4 h, in the presence or absence of RvD1 or RvD2 at the indicated doses. Labeled U937 monocytes were overlain, and cell adhesion assay was performed as described. Results are shown as relative percentage inhibition, expressed as a percentage of the maximal adhesion for the agonist (n=3). *P < 0.05, **P < 0.01; unpaired t test. B) RvD1 and RvD2 reduce cell adhesion molecule gene expression in VSMCs. VSMCs were treated with TNF-α (10 ng/ml) for 18 h in the presence or absence of RvD1 or RvD2 across a concentration range as shown. Expression of VCAM-1 and ICAM-1 was measured by quantitative RT-PCR (n=3). *P < 0.05, **P < 0.01; unpaired t test. C) Proinflammatory gene expression. VSMCs were stimulated with TNF-α as above in the presence or absence of RvD1 or RvD2 (0.1 nM or 10 nM), and qPCR was performed for multiple proinflammatory gene transcripts. Shown are significant reductions in the expression of TNF-α, IL-1β, MCP-1, IL-6, and IL-1α (n=3). *P < 0.05, **P < 0.01; unpaired t test. D) Effects of RvD2 on TNF-α stimulated gene expression in VSMCs are sensitive to PTX. VSMCs were exposed to TNF-α and RvD2 (10 nM) as described, in the presence or absence of PTX (100 ng/ml). RNA was harvested and analyzed for the expression of ICAM-1, IL-1β, and IL-1α by qPCR (n=3). *P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni's post hoc test). E) RvD1 modulates activity of transcription factors NFκB and AP-1 in TNF-α-stimulated VSMCs. VSMCs were treated with TNF-α (10 ng/ml) with or without RvD1 (10nM for 2 h), and nuclear extracts were prepared. Transcription factor activity was assessed as described in Materials and Methods (n=5). *P < 0.05; unpaired t test. Results are means ± se.
Figure 3.
Figure 3.
Local biosynthesis of lipid mediators in vascular injury. Rabbits underwent unilateral iliofemoral artery balloon injury. Vessels were harvested 3 d after injury for biochemical and histological assays. Metabololipidomics of arterial injury. Representative chromatographic profiles from LC-MS/MS obtained from injured artery showing the presence of proresolving lipid mediators and precursors, including RvD5, MaR1, LXB4, and DHA- and EPA-derived monohydroxy acids. MS-MS spectra are on right.
Figure 4.
Figure 4.
Expression of RvD1 receptors in VSMCs and rabbit artery tissues. A) Identification of GPR32 in human VSMCs. Western blot analysis (50 μg of total cell lysate) of primary cultured VSMCs from 2 donors using anti-GPR32 antibody and anti-β-actin antibodies. A single band is identified of the appropriate size for each protein. B) Identification of GPR32 and ALX in rabbit artery. Western blot analysis (50 μg of total artery lysate) using anti-GPR32 antibody, anti-ALX antibody, and anti-β-actin antibodies. A single band is identified for ALX; GPR32 blot shows band at appropriate size and smaller bands of unknown significance. C–F) Representative GPR32 immunostaining of uninjured (C, D) and injured (E, F) arteries. GPR32 receptor is expressed both in injured and uninjured vessels. Scale bar = 100 μm.
Figure 5.
Figure 5.
RvD2 treatment attenuates the acute response to vascular injury, in vivo. Rabbits (n=6) underwent bilateral femoral artery angioplasty, with immediate local delivery of RvD2 (10 nM) or vehicle control by intraluminal incubation as described. Vessels were harvested 3 d after injury for histological assays. A–C) RvD2 reduces the early proliferative response to angioplasty. Representative Ki-67 immunostaining of vehicle-treated (A) and RvD2-treated (B) arteries, with summary of quantitative analysis (C). D–F) RvD2 treatment attenuates early leukocyte recruitment to the injured vessel. Representative CD45-stained sections of vehicle-treated (D) and RvD2-treated (E) arteries, with summary of quantitation (F). G) RvD2 treatment modulates early inflammatory gene expression in the acutely injured artery in vivo. Shown are fold expression changes of VCAM-1, ICAM-1, TNF-α, IL-1β, MCP-1, IL-6, and IL-1α normalized to uninjured artery. Scale bars = 100 μm. Results are means ± se. *P < 0.05, **P < 0.01; paired t test.
Figure 6.
Figure 6.
RvD treatment modulates superoxide production by VSMCs in vitro and in vivo. A–D, I) RvD1 treatment reduces TNF-α-induced superoxide production in cultured HVSMCs. A–D) Representative merged images of DHE staining, counterstained with DAPI of untreated VSMCs (A), positive control (TNF-α 10 ng/ml for 4 h; B), TNF-α with 10nM RvD1 (C), and TNF-α with 100nM RvD1 (D). I) Quantitative comparison of DHE staining intensity (n=3). *P < 0.05; 1-way ANOVA with Dunnett's post hoc test). E–H, J) RvD2 treatment reduces oxidative stress in the acutely injured rabbit artery (3 d postangioplasty). E–H) Representative images of DHE staining of an uninjured aorta (E), balloon injured and untreated iliac artery (F), vehicle-treated femoral artery (G), and RvD2-treated femoral artery (H). J) Quantitative comparison of staining intensity (n=6). *P < 0.05; paired t test. Scale bars = 200 μm (A–D); 100 μm (E–H). Results are means ± se.
Figure 7.
Figure 7.
RvD2 treatment inhibits neointimal hyperplasia postangioplasty. Angioplasty and local treatment of bilateral rabbit (n=8) femoral arteries with RvD2 vs. vehicle control was performed as described, and vessels were explanted at 28 d by perfusion-fixation. A, B) Histomorphometric analysis was performed on elastin-stained sections of vehicle-treated (A) vs. RvD2-treated (10 nM, B) arteries. Scale bar = 500 μm. C) Summary of morphometric results. Neointima/media (NI/M) ratio and neointimal area were significantly reduced in RvD2-treated vessels. Results are means ± se. NS, not significant. **P <0.01; paired t test.
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