Introduction:
Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator (SPM), attenuates migration in vascular smooth muscle cells (VSMC), which is critical to the development of neointimal hyperplasia. SPM are known to interact with G-protein coupled receptors (GPCR). We sought to investigate the pathways by which RvD1 influences VSMC migration.
Methods:
VSMC were harvested from human saphenous veins. cAMP levels were measured via ELISA in the absence or presence of RvD1 receptor (ALX, GPR32) blockers. NF449, a G
s
-protein inhibitor, was also used. PDGF-BB (10ng/ml) was used as an agonist in a VSMC scratch assay as well as the receptor blockers. PDGF-BB-induced cytoskeletal changes were measured as aspect ratio after actin staining, and a scratch assay was used to assess migration. PDGF-induced Rac1 activity was measured via ELISA; VASP phosphorylation was assessed by Western blot and Paxillin translocation by Immunofluorescence. PKA inhibitor Rp-8-Br-cAMP (10μM) was used in the phenotypic and downstream signaling studies.
Results:
RvD1 treatment (10nM) of VSMC induced a significant acute flux in cAMP levels at 5 minutes (Fig. 1A; n≥3); this increase was abolished by an ALX antagonist (WRW4), the anti-GPR32 blocking Ab, and NF449. RvD1 (10nM) attenuated PDGF-induced VSMC migration (Fig. 1B-C; n≥3), cytoskeletal rearrangements (n=4), Rac1 activation (n≥3) and increased phosphorylation of VASP (n=3). These effects were negated by the addition of Rp-8-Br-cAMP, suggesting cAMP involvement. RvD1’s anti-migratory effect was reversed by blocking ALX or GPR32 (Fig. 1C; n≥8).
Conclusion:
Our results suggest that RvD1 attenuates VSMC migration by increasing levels of cAMP through ALX and GPR32 via a G
s
-protein-mediated action. Interference with Rac1, VASP and Paxillin function appear to be important for the anti-migratory activity of RvD1.
Unidirectional and sustained delivery of the proresolving lipid mediator resolvin D1 from a biodegradable thin film device