Abstract
Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.
Highlights
Peripheral artery disease (PAD) affects >200 million people globally, and it currently represents a major cause of morbidity as well as healthcare expenditures in the United States
Treatment of Vascular smooth muscle cells (VSMC) with aspirin-triggered epimer 17R-Resolvin D1 (RvD1) (AT-RvD1) inhibited platelet-derived growth factor (PDGF)-stimulated Rac1 activity, increased vasodilator stimulated phosphoprotein (VASP) phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-Cyclic adenosine monophosphate (cAMP)
Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/ FPR2 and cAMP/protein kinase A (PKA)
Summary
Peripheral artery disease (PAD) affects >200 million people globally, and it currently represents a major cause of morbidity as well as healthcare expenditures in the United States. Intimal hyperplasia (IH) is a prototypic response to vascular injury that, when excessive, leads to restenosis [10,11]. Vascular smooth muscle cells (VSMC) and their phenotypic alterations are central to the pathophysiology of IH. VSMCs naturally reside in the tunica media; in response to vascular injury they migrate into the tunica intima and proliferate, causing thickening of the vessel wall and narrowing of the lumen [12,13]. Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration
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